Cell, Division 2 Course Packet 2011-12

The Ohio State University College of Medicine
Med 1 Integrated Pathway
The Cell, Division 2

Lecture Handouts
December 6, 2011 January 17, 2012
Block Leader:
Charles Hitchcock, M.D.,Ph.D.
Department of Pathology
084 Heart & Lung Research Institute
247-7469
charles.hitchcock@osumc.edu
Cell, Division 2
Med 1 Integrated Pathway 2011- 2012
Table of Contents
Lecture Handouts- Title
Introduction to Cytogenetics- Dr. Westman
RNA: Properties- Dr. Hai
Gene Structure- Dr. Westman
RNA: Transcription- Dr. Hai
Mutation and Gene Repair- Dr. Westman
RNA: Processing and Translation- Dr. Wang
Mendelian Genetics- Dr. Westman
RNA: Gene Regulation- Dr. Wang
Developmental Genetics- Dr. Westman
Nucleotide Metabolism- Dr. Jacob
Cell Injury Part 1- Dr. Hitchcock
DNA- Dr. Wu
Apoptosis- Dr. Vandre
Cell Injury Review Questions- Dr. Hitchcock
Neoplasia Part 1- Dr. Hitchcock
Cell Cycle- Dr. Vandre
Oncogenes- Dr. Vandre
Counseling and ELSI- Dr. Westman
Introduction to TBL- Dr. Westman
Pathology Review Questions- Dr. Hitchcock
Genetics of Complex Diesase- Dr. Westman
Angiogenesis- Dr. Vandre
Pharmacogenetics and Gene Therapy
Biomarkers- Dr. Hitchcock
Antineoplastics- Dr. Briesewitz
Proteomics- Dr. Vandre
Imaging in Cancer- Dr. Murrey
MicroRNA- Dr. Piper
Clinical Case: Solid Tumor Presentation- Dr. Otterson
Page(s)
Carmen
1-15
Carmen
16-29
Carmen
30-49
Carmen
50-65
Carmen
66-84
85-90
91-112
113-117
118-122
123-128
129-135
136-138
139-146
147-153
154-157
158-165
166-172
Carmen
Carmen
173-176
177-193
Carmen
194-197
Carmen
198-203
Carmen
204-210
Carmen
211-217
Carmen
Med 1, Cell Block (Division 2)
Dr. Hai, Handout cover page
MED 1. CELL BLOCK

Lectures by Dr. Hai
Lectures
Lecture 1
Lecture 2:
Title
Properties of RNA
Transcription
Date and Time

December 6, 2011, 10:30AM-11:20AM
December 7, 2011, 10:30AM-11:20AM
Reading assignments:
Marks Essentials of Medical Biochemistry: A Clinical Approach, by Lieberman, Marks, and Smith, 2007
Lecture 1 - Properties of RNA: Chapter on Structure of the nucleic acids
Lecture 2 - Transcription: Chapter on Transcription: Synthesis of RNA
Objective and Content Overview:
The objective of my lectures is to cover the basic concepts in RNA and transcription. These are the topics
listed under the Biochemistry and molecular biology section in the Step 1 Content Outline published by
the United States Medical Licensing Exam Board. All handouts are based on the contents in the recommended
textbook. Important concepts will be emphasized during the lectures.
Contact Information:
Tsonwin Hai, Ph.D.
Professor
Department of Molecular and Cellular Biochemistry
Office: 174 Rightmire Hall
Tel: 292-2910, Email: hai.2@osu.edu
The Cell, Division 2 2011-12
Dr. Hai, Handout cover page
Practice Exam Questions for Dr. Hais Lectures

(1) RNA molecule is unstable, because of its
(A) 2'-OH group
(B) Modified bases
(C) Hairpin loop structure
(D) Unusual base pairing
(E) U residues
(2) The figure shows the base pairing of a short duplex DNA molecule; circles represent the phosphorous
molecules, and the letters represent each of the four common bases present in DNA. An enzyme cuts
symmetrically within the six base-pair palindrome of dyad symmetry. Which of the following portions
of the DNA molecule has the enzyme cutting site?
(A) CCCGGT
(B) TGATCA
(C) CGGTTG
(D) CCGGTT
(E) TTGATC
GGGCCA
ACTAGT
GCCAAC
GGCCAA
AACTAG
(3) If the transcriptional machinery travels along the double stranded DNA below at the direction from
your left to your right, which of the following RNA will be produced?
3
A
T
G
C
G
C
C
G
T
A
T
A
A
T
C
G
G
C
C
G
C
G
A
T
(A) 5-AGGCUUACGCCA-3
(B) 5-TGGCGTAAGCCT-3
(C) 5-AGGCTTACGCCA-3
(D) 5-UGGCGUAAGCCU-3
(E) 5-ACCGCAUUCGGA-3
Answers
(1) A, (2) B, (3) A
Note: If a question is ambiguous, chose the best answer.
ii
Dr. Hai lecture 1, RNA Property
PROPERTIES OF RNA
Reading assignment: Marks, Chapter on Structure of the nucleic acids
Outlines:
(1) Different types of RNA
(2) Features of RNA
(3) mRNA
(4) tRNA
(5) rRNA
(6) miRs
(7) Other types of RNAs: snRNA, 7SL RNA, RNA as genomes
(8) Clinical discussions
Learning Objectives:
1. Name the physical properties of RNA.
2. Compare mRNA, tRNA and rRNA.
3. Identify palindromic sequences.
4. Learn nomenclature related to RNAs (intron, exon, splicing, open reading
frame, CAP, polyA).
(1) Different types of RNA

- Messenger RNA (mRNA)
- Transfer RNA (tRNA)
- Ribosomal RNA (rRNA)
- microRNA (miRNA, miR)
- Other types of RNA: small RNAs (such as snRNA, only in eukaryotes)
All RNA molecules are synthesized by RNA polymerases using DNA as templates, a
process called "transcription". The mRNA is then used as templates to synthesize
proteins. That is, mRNA is the intermediate to carry the "message" to synthesize
proteins. tRNA and rRNA also participate in protein synthesis; they do not carry the
"message", but they are part of the protein-synthesizing machinery. Therefore,
these three major RNA molecules, mRNA, tRNA and rRNA, are all involved in
synthesizing proteins, a process called "translation". (mRNA can be viewed as the
software, while tRNA and rRNA can be viewed as the "hardware".) Protein
synthesis will be discussed later.
Flow of genetic information: (central dogma)
transcription
translation
DNA ------------------ RNA ----------------------
* DNA stores the genetic information to make proteins, but it is not used directly
as templates to synthesize proteins.
miRNAs regulate the level of protein production by inhibiting translation or
targeting mRNA for degradation.
(2) Features of RNA
(a) RNA is a long chain of nucleotides linked by phosphodiester bonds.
DNA
5- End
RNA
5- End
Phosphodiester
bond
(b) RNA is made of four bases: adenine (A), uracil (U), guanine (G) and cytosine
(C). But some RNA molecules may contain modified bases.
Some modified bases:
(c) The sugar in RNA is ribose, not deoxy ribose: 2' position is OH.
(d) The 2'-OH is susceptible to hydrolysis by base, making RNA molecules more
unstable than DNA molecules. RNA is cleaved in alkaline solutions.
nucleophilic
attack
(e) RNA is single stranded. The only exception is that some viral RNAs are double
stranded.
(f) RNA forms secondary structures: some segments of RNA can form base pairs
with other segments of the same RNA molecules (intra-chain base pairing) due to
the palindromic sequence. A palindrome is a word or a number reads the same
backward or forward. For example, RADAR and 12821 are palindromes. An
example of a palindromic sequence is:
Important!
A palindromic sequence
5'-------->
5'-UCCUAxxxUAGGA-3'
The sequence reads backward is: (on the complementary strand, from 5' to 3',
** DNA or RNA can't be read from 3 to 5'.)
3'-AGGAUxxxAUCCU-5'
<-----5'
The palindromic sequence can form base pairs, resulting in a stem and loop structure
or a hairpin loop structure. The double stranded region is called the helical region.
In most cases, the pairing is still complementary and anti-parallel.
X
A
U
C
C
5'- U
X
U
A
G
G
A -3'
The proportion of helical regions in different types of RNA varies over a wide range.
5'
3'
(g) RNA molecules may form unusual base pairing: non-Watson-Crick types of
base paring.
Watson-Crick base pairing (DNA)
Adenine
Thymine
Some unusual base pairing (non-Watson Crick base pairing):
Wobble base pair

A hydrogen donor and a hydrogen acceptor can form hydrogen bond as long as they
are close enough and in a proper orientation.
(1) hydrogen donor and hydrogen acceptor
(2) distance
(3) orientation
These usual base pairings allow some RNA to form tertiary structures. For
example, the tRNA clover leaf secondary structure can fold back on itself to form
the L-shape tertiary structure.
* The ability of RNA to form secondary and tertiary structures is the hallmark
of RNA.
Clover leaf secondary structure
L-shape tertiary structure
5'
5'
(h) When first synthesized, many eukaryotic RNAs contain segments of RNA that
will be removed later. These segments are called the intervening sequences or
introns. The remaining segments are called exons. The process of removing the
introns is called splicing. Newly synthesized mRNA is called pre-mRNA; after
processing, the mRNA is called mature mRNA. Only exons remain in the mature
mRNA and are used as templates to synthesize proteins.
Splicing occurs in mRNA, tRNA, and rRNA.
(3) mRNAs: templates for protein synthesis
Only a segment of the (mature) mRNA is used to make proteins. This region is
called the "open reading frame" (ORF). ORF starts from an initiation codon and
ends at a termination codon. The region upstream from the initiation codon is called
the 5' untranslated region (5' UTR) and the region downstream from the termination
codon is called the 3' untranslated region (3' UTR).
As an example:
2
3
splicing
start
stop
2
3
4
Open Reading Frame
5'UTR
3'UTR
5'UTR, 3'UTR and ORF - All from exons.
Prokaryotic mRNA
Many prokaryotic mRNAs encode for more than one protein. These RNAs are
called polycistronic RNAs.
5'UTR
ORF
3'UTR
ORF1
Monocistronic
ORF2
Polycistronic
Eukaryotic mRNA
The 5' end of the eukaryotic mRNAs is usually modified to form a so called "CAP"
structure: 7-methyl-guanosine attached to the 5' end of mRNA by a triphosphate
linkage (7mGppp).
7- Methlyguanosine
5, 5-Triphosph ate
linkag e
Som etimes
meth ylated
Som etimes
meth ylated
The 3-end of the most eukaryotic mRNAs is the poly (A) tail, a stretch of adenine
residues (50-300 long). This poly (A) is not encoded in the DNA; rather it is added
after the mRNA is synthesized.
AUG
m7Gppp
5'UTR
Added
UAG
ORF
All from exons
AAAAnAA OH
3'UTR
Added
(4) tRNA: Adapter molecule
10
tRNAs serve as adapter molecules to bring amino acids to the protein synthesis
machinery. All tRNAs have the same secondary (clover leaf) and tertiary structures
(L shape). In addition, they all contain unusual bases (modified bases) which are
formed by enzymatic modifications of a standard ribonucleotide in a tRNA
precursor. That is, they are modified after the RNA molecules are synthesized. A
tRNA molecule can be divided into several regions:
acceptor stem
D loop
anticodon loop
TC loop
the stem to accept the amino acids

important for forming the tertiary structure
to base pair with the codon on mRNA
important for forming the tertiary structure
The 3' end sequence of all tRNAs is CCA. This CCA end is added after tRNA is
synthesized. The 2' or 3' OH group of "A" forms ester bond with the carboxyl group
of the amino acids.
5'
Amino Acid
11
(5) rRNA: Component of ribosomes (most abundant RNA)

rRNAs are the RNA components of the ribosomes.
Structure provides functions.

Mitochondrial ribosomes are similar to those of the 70S ribosomes of bacteria.
Mitochondria can be thought as "bacterial residents" in eukaryotic cells.
10
12
Each rRNA has specific structure conferred by intra-chain base pairing. Below is a
postulated representation of 5S rRNA and 16S rRNA.
miRNA gene
(6) microRNAs (miRNAs, miRs)

The miRs do not encode proteins. They are regulatory
RNAs that modulate the level of protein. They do so by
targeting mRNAs for degradation or inhibiting translation.
After synthesis, miRNAs are processed to short products,
approximately 21-22 nucleotides in length. By
recognizing mRNAs that contain complementary
sequences or partially complementary sequences, these
short RNAs then direct the cellular machinery to degrade
the mRNA or to inhibit translation.
Pri-miRNA
Microprocessor
Nucleus
Cytoplasm
miR:miR*
duplex
Helicase
miRNP
miR
mRNA degradation or
Translational repression
(from a review by Leung
and Sharp, 2006 )
11
13
(7) Other types of RNAs

snRNA: Small nuclear RNA such as U1, U2, U3 U4, U5 and U6 form
ribonucleoprotein complexes with proteins. These ribonucleoprotein complexes
are involved in splicing (see Processing).
7SL RNA: It is involved in protein targeting. This RNA forms a ribonucleoprotein
complex with six proteins. The resulting complex, called signal recognition
particle (SRP), delivers the newly synthesized protein molecules containing the
signal sequences to the ER (endoplastic reticulum) membrane. From here, the
proteins will be transported to their destinations.
RNA as genomes: Certain types of viruses, known as retroviruses, use RNA
(instead of DNA) as their genomes. To replicate their genomes in the host cells, the
RNA is first transcribed into DNA. The viral DNA is then integrated into
chromosomal DNA of the host to form a DNA provirus, which is transcribed to yield
progeny virus RNA. The process of making DNA from RNA is called "reverse
transcription", hence the name "retrovirus". The discovery of reverse transcription
revolutionized our understanding on how genetic information can be transmitted and
proved that the central dogma of information flow from DNA to RNA is not always
correct. The human immunodeficiency virus (HIV) is a retrovirus. It infects T
lymphocytes and causes acquired immunodeficiency syndrome (AIDS).
A schematic representation of HIV:

(a) HIV infects T lymphocytes and destroys the host's immune system. In an
uninfected person, the helper T lymphocytes usually number about 1,000/mL. When
the number of T lymphocytes drops below 200/mL, opportunistic infections occur.
Therefore, what kills AIDS patients at the end is usually the opportunistic
infections by agents such as fungi, viruses and bacteria. Certain neoplasms also
frequently develop in AIDS.
(b) Mr. Appleman recently had a bacterial infection and was treated with
erythromycin, an antibiotic that kills bacteria by interfering with the function of its
12
14
ribosomes. Because mitochondrial ribosomes are similar to the bacterial ribosomes,

this agent may also affect Mr. Appleman's mitochondrial ribosomes, leading to such
side effects as epigastric distress, diarrhea and infrequently cholestatic jaundice.
Summary of RNA properties

Different types of RNA: three major RNAs for protein synthesis (mRNA,
tRNA and rRNA), miRs for regulation of protein levels.
Features of RNA: phosphodiester bond, 2'-OH, secondary structure,
palindromic sequence, tertiary structure, exons, introns
mRNA: open reading frames, 5' CAP, poly(A), polycistronic RNA
tRNA: cloverleaf, L shape, CCA
rRNA: most abundant RNA
miRs
Other types of RNAs: snRNA, 7SL RNA, RNA as genomes
13
15
Dr. Hai lecture 2, Transcription

TRANSCRIPTION
Reading assignment: Marks, Chapter on Transcription: Synthesis of RNA

Outlines:
(1) DNA directed RNA synthesis (transcription)
(1. A) Prokaryotic transcription
(i) Promoter
(ii) Promoter activity
(iii) Direction of transcription: 5' to 3'
(iv) RNA polymerase
(v) Other features
(vi) Reaction
(1. B) Eukaryotic transcription
(i) Eukaryotes versus prokaryotes
(ii) Pol II promoter and promoter activity
(iii) Proteins involved in Pol II transcription
(iv) Enhancer and silencer
(1.C) Inhibitors of transcription
(2) RNA directed DNA synthesis (reverse transcription)
(3) RNA directed RNA synthesis
1. Learn features of transcription.
2. Predict transcription product.
3. Learn gene structure and transcription factors.
4. Differentiate inhibitors of transcription.
5. Relate reverse transcriptase to anti-HIV treatment.
16
(1) DNA directed RNA synthesis

(1.A) Prokaryotic Transcription:
(i) Promoter:
Promoter is a segment of DNA that specifies the binding of RNA polymerase and
controls the transcription of a specific gene. The promoter can be positively or
negatively regulated. Many prokaryotic promoters contain two common motifs:
the -10 and -35 sequences. They are centered around 10 and 35 nucleotides
upstream of the transcriptional start site. Each motif (site) is about 6 base pair long
and the consensus sequences are:
TTGACA
-35
TATAAT
-10
+1
Pribnow Box
(ii) Promoter activity:

Promoters differ in their efficiency: Strong promoters cause more frequent initiation
of transcription than the weak promoters. The activity is determined by several
factors:
(1) The sequence of the -10 and -35 sitesStrong promoters have the sequences
that correspond closely to the consensus sequences.
(2) The distance between these two sites.
(3) Regulatory sequences near the promoter sites. These sequences provide
binding sites for specific trans-acting protein factors.
-35
Regulatory
sequence
-10
RNA Polymerase
17
Terminology: cis-acting element and trans-acting factor

A cis-acting element refers to a short stretch of DNA sequence that constitutes a
signal for specific protein(s) to bind. Protein factor that bind to the cis-acting
elements is called the trans-acting factor. These trans-acting proteins interact with
RNA polymerase. How strong these factors bind to the DNA, and how well they
work with RNA polymerase are important factors that determine the activity of the
promoter.
(iii) Direction of transcription is from 5' to 3':
Needs double stranded DNA to function as a promoter, but only one of the two
DNA strands is used as the template.
5' promoter
3'
3'
5'
tanscription
from 5' to 3'
non-template=(+) strand=mRNAlike=coding stran d

DNA template=(-) strand=anti-sense strand
3' mRNA
5'
coding strand: similar to mRNA,

encodes protein
5'-CGCTATAGCGTTT-3'
3'-GCGATATCGCAAA-5'
tanscription
from 5' to 3'
5' -CGCUAUAGCGUUU-3'
Promoter
Exon 1
Exon 2
Exon 4
Exon 3
Transcription
Exon 1
Exon 2
Exon 4
Exon 3
RNA Processing
m7Gppp
Exon 1
Exon 2
Exon 3 Exon 4
5UTR
Poly A
3UTR
ORF
18
(iv) RNA polymerase:

RNA polymerase from E. coli is a large enzyme composed of four types of
subunits: , , ' and .
(v) Other features of prokaryotic transcription:
In prokaryotes, all three RNA molecules mRNA, tRNA, and rRNA are
synthesized by the same RNA polymerase. Sometimes, one promoter directs the
synthesis of more than one gene (polycistronic RNA). These RNAs can then be
cleaved to give rise to separate RNA molecules. One example is the polycistronic
transcript for -galactosidase, galactoside permease and thiogalactoside
transacetylase. All three protein products are engaged in lactose metabolism; thus, it
makes sense to express the genes simultaneously. These three genes plus the
promoter region are called the lactose operon.
(vi) Reaction:
(1) Binding and unwinding-The RNA polymerase binds to DNA, slides along the DNA, recognizes the -10
and -35 sequences. The DNA and RNA polymerase complex is called the "close
promoter complex". The polymerase then unwinds the DNA so that they can
form Watson-Crick base pairing with the incoming ribonucleotide
triphosphates. This stage is called the "open promoter complex"
19
close complex
open complex
(2) de novo synthesis--The 5' end of a new RNA chain starts with pppG or pppA, no primer is
required. (In contrast to DNA replication)
Schematic representation:
(3) Elongation--After the formation of the first phosphodiester bond, the factor leaves. The
core polymerase moves along the DNA template, adds new nucleotides to the
nascent RNA strand using the information from the template DNA. The newly
synthesized RNA forms base pairs with the template DNA. The region
containing DNA, RNA polymerase and the nascent RNA is called the
20
transcription bubble. The DNA is unwound in front of the transcription bubble

and rewound behind the bubble.
Coding
Strand
RNA polymerase does not contain nuclease activity. Conventionally, RNA

synthesis is thought to not have proofreading capability. However, more recent
findings indicate that proofread indeed occur during RNA synthesis, albeit with
much lower efficiency (than that in DNA replication). The current understanding
is that accessory proteins are required to remove wrong nucleotides when
mistakes occur. Therefore, the fidelity of transcription is much lower than that
of DNA replication.
DNA replication:
High fidelity to preserve the accuracy of genetic information
(4) Termination
Right after the core polymerase encounters the termination site, it stops
transcription and falls off the DNA. The signal for termination is in the RNA,
not the DNA. Immediately after the polymerase synthesizes this stretch of RNA,
the RNA forms a hairpin structure and signals the polymerase to stop
transcription. This process can be either rho-dependent or rho-independent.
Rho: a hexamer protein.
RNA polymerase
UUUUU
GC-rich, stable
hairpin structure
weak base pairing

between rU and dA
21
(1. B) Eukaryotic transcription

(i) Prokaryotic versus eukaryotic RNA synthesis:
(1) Similarity--Similar to the prokaryotic RNA polymerase, the eukaryotic polymerases synthesize
RNA in a 5' to 3' direction, do not require primer and do not edit the RNA (no
nuclease activity) (lower fidelity than DNA replication)
(2) Differences---(a) In prokaryotes, the same RNA polymerase makes mRNA, tRNA and rRNA. In
contrast, in eukaryotes, different polymerases make these RNA molecules.
RNA polymerase I (Pol I):
RNA polymerase II (Pol II):
RNA polymerase III (Pol III):
Mitonchondrial polymerase
5.8S, 18S and 28S rRNAs

mRNA and snRNA
tRNA and 5S rRNA
Mitochondrial RNAs
(b) In prokaryotes, translation and transcription occur in the cytoplasm and are
coupled: Translation begins while the mRNA is being synthesized. In eukaryotes,
transcription occurs in the nucleus, while translation occurs in the cytoplasm.
nucleus
(c) Prokaryotic transcription is controlled by only one or two proteins in addition to

RNA polymerase. Eukaryotic transcription, on the other hand, is controlled by
many protein factors in addition to RNA polymerase.
22
(d) Prokaryotic mRNAs are usually not processed (modified) after they are
synthesized. Eukaryotic mRNAs, on the other hand, are extensively modified (see
RNA processing).
(ii) The eukaryotic Pol II promoter and promoter activity: (mRNA synthesis)
Most eukaryotic Pol II promoters contain an AT rich sequence called TATA box at
around -25 from the transcriptional start site. The TATA box (similar to the
Pribnow box in prokaryotes) is not sufficient for strong promoter activity.
* Some promoters are TATA-less.
Many upstream activating sequences are required for strong promoter activity.
Therefore, a given promoter is composed of a combination of DNA binding sites.
* Promoter is modular!
TATA
(iii) Proteins involved in transcribing the Pol II promoter:

Multiple transcription factors interact with eukaryotic promoters. They can be
classified into two groups: the general transcription factors and the sequence specific
transcription factors.
(1) The general transcription factors-General transcription factors are required for the transcription of all promoters.
RNA polymerase II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH form a large protein
complex called the initiation complex or the basal transcription apparatus. This
complex binds to the DNA around the transcriptional start site. In the absence of
any upstream sequence specific transcription factors, the initiation complex gives
rise to a basal, low level of transcription.
23
*TF stands for transcription factor, II refers to RNA polymerase II. TFIID is
composed of the TATA binding protein (TBP) and several other proteins.
TFIID: TBP plus many factors

TBP is the heart of the initiation complex. The crystal structure of TBP has been
solved. It is a saddle-shaped protein composed of two similar domains.
The concave surface of TBP binds to DNA at the TATA box, and induces a sharp
kink (bending). The AT rich nature of the TATA box allows the DNA to be more
flexible and bend more easily.
TATA Box
Plus TBP
TATA Box
24
(2) The sequence specific transcription factors:

Transcription factors are composed of functional domains. Usually, they contain a
DNA binding domain and a transcriptional regulatory domain (either an activation
domain or repressor domain). These domains are modular; that is, they can be
moved from one protein to another protein and are still functional.
* Transcription factor is modular!
By convention, the upstream factors have been classified into groups on the basis
of their DNA binding domains. Table below shows three groups of DNA-binding
proteins in eukaryotes.
Structural motif
Helix-turn-helix
Structural features
two -helices separated
by a -turn
Examples
homeodomain proteins
(regulate development)
Zinc finger
contains zinc bound to

Cys and His side chains
steroid hormone receptors
Leucine zipper
one domain with

Fos and Jun
basic residues to bind to
(growth regulators)
DNA, the other domain with
regularly spaced leucines
for dimerization
(coil-coil
structure)
Basic region
(coil-coil)
(alphahelix)
leucine zipper
(on DNA)
zinc finger
10
25
Each transcription factor recognizes a specific consensus DNA sequence. Table

below shows the consensus recognition sequences of a few eukaryotic sequence
specific transcription factors.
Transcription factor
Consensus recognition sequence
Fos and Jun
TGACGTCAC
SP1
GCGCC
HSTF
CNNGAANNTCCNNG
The recognition site for a given transcription factor is usually located in a variety of
promoters in different tissues. (Remember, as described above, a given promoter is
composed of a combination of DNA binding sites.) As an example, a liver
specific-gene and a pancreas-specific gene may share some common binding sites
in their promoters. This raises an important question: How is tissue-specific
transcription achieved? We will discuss this issue in the lecture on Regulation of
Gene Expression.
Promoter is modular.
A liverspecifc gene
TATA
A pancreasspecifc gene
TATA
(3) Communication:
How do the sequence-specific factors transmit information (up or down regulation)
to the basal transcription apparatus?
Transcription factors transmit the information to the basal transcription apparatus by
direct or indirect interaction with the basal transcription factors.
co-fact
ors
TAF
IIE
IIF
IIJ
IIA
IIB
RNA Polymerase IIIIH
TBP
Direct interaction
(iv) Enhancer and silencer:
TAF
IIE
IIF
IIJ
IIA
IIBRNA Polymerase IIH
TBP
Indirect interaction
11
26
A segment of DNA that can greatly increase (enhancer) or decrease (silencer) the
transcriptional activity in the following manner: (operational definition)
(1) It regulates transcription independent of its orientation.
(2) It regulates transcription even at a distance of several thousand base pairs
away from the transcriptional start site.
(3) It can be upstream, downstream or even in the midst of the gene.
The enhancers are usually composed of clusters of binding sites for the sequence
specific transcription factors. Therefore, they can be thought as DNA segments with
very strong activity, making them orientation- and position-independent.
(1. C) Inhibitors of transcription:

Actinomycin D:
an antibiotic, planar, intercalates into the double helical DNAs,

thereby preventing it from being an effective template for RNA
synthesis. It inhibits RNA synthesis in both prokaryotes and
eukaryotes; therefore it is not used in human.
Acridine
A planar compound, similar to actinomycin D in activity
Rifamycin
an antibiotic, interferes with the formation of the first

phosphodiester bond, therefore, it inhibits the initiation of RNA
synthesis, inhibits prokaryotic but not eukaryotic transcription
because it binds to the prokaryotic RNA polymerase.
Rifampicin
a synthetic derivative of rifamycin
-amanitin:
a toxin from a poisonous mushroom, a cyclic octapeptide, binds

to RNA polymerase II tightly and therefore blocks eukaryotic
RNA synthesis. It does not block prokaryotic RNA synthesis It
causes more than a hundred deaths each year in the world.
12
27
(2) RNA dependent DNA synthesis (Reverse transcription):
Retrovirus contains reverse transcriptase which uses RNA as templates to make

DNAs. tRNA is used as a primer in this process. Some retroviruses cause cancer
because they carry the cancer causing genes called oncogenes. The concept of
oncogene will be discussed by other lecturers.
Retrovirus: such as HTLV (causes T cell lymphoma and Tropical Spastic
Paraparesis), HIV (causes AIDS)
(3) RNA dependent RNA synthesis
RNA-directed RNA polymerase (RNA replicase)
(4) Clinical discussions:
(a) Mr. Allen ingested 8 wild mushrooms that contains -amanitin. He developed
gastrointestinal disturbances, electrolyte imbalance, fever, then liver and renal
dysfunction. An average-size mushroom weights about 50 g and contains about 7
mg of -amanitin. The LD50 (the oral dose that kills 50% of those who ingest the
toxin) of -amanitin is 0.1 mg/kg body weight. Mr. Allen weighs about 90 kg. Is he
likely to survive the poisoning?
No, he ingested too much toxin (8x7=56 mg; LD50 for him is 0.1x90=9 mg).
Between 40% and 90% patients ingested -amanitin die within a few days.
There is no effective antidote now, and the treatment is primarily supportive.
13
28
(b) Reverse transcriptase of HIV is about tenfold less accurate in replication than
other known reverse transcriptases, resulting in high mutation rate in this virus. This
high mutation rate causes changes in the envelope protein, evading the immune
surveillance and creating problems in developing effective vaccines. This high
mutation rate also results in mutations in reverse transcriptase itself, causing the
viruses to become resistant to drugs such as AZT which targets the reverse
transcriptase. combination therapy
A small percent of HIV-infected patients never develops AIDS. That is, they are
resistant to HIV infection. It is hoped that by understanding how these individuals
develop resistance to HIV, scientists may be able to design prevention or therapeutic
agents for AIDS. Because the targets of treatment are in the hosts (the patients),
instead of in the virus, the high mutation rate of HIV may not pose a problem.
Summary of transcription:
DNA directed RNA synthesis (transcription)
Prokaryotic transcription
Promoter: TATA (-10) and -35 consensus
Promoter activity: sequence, distance
Direction of transcription: 5' to 3'
Other features: polycistronic RNA
Reaction: close complex, open complex (unwind), de novo synthesis
(do not need primer), no proof reading, and transcription termination
(RNA provides the information)
Eukaryotic transcription
Eukaryotes versus prokaryotes:
Difference--three different RNA polymerases, in nucleus, more
complex, extensively modified after synthesized
Similarity-- 5' to 3', de novo synthesis, no proof reading
Pol II promoter and promoter activity: TATA, promoter (upstream
sequences)
Proteins involved in Pol II transcription: general transcription
factors, basal transcription apparatus, sequence-specific transcription
factors, modular functional domains, communication (directly or
indirectly through co-factors)
two terms -- enhance, silencer
Inhibitors of transcription
RNA directed DNA synthesis (reverse transcription)
RNA directed RNA synthesis
14
29
Med 1, Cell Block (Division 2) Dr. Wang lecture 1, Processing and Translation
RNA PROCESSING AND TRANSLATION
RNA PROCESSING
Reading assignment: Marks, Chapter on Transcription: Synthesis of RNA
Outlines:
(1) Eukaryotic RNA processing
(1.A) mRNA processing
(1.B) tRNA processing
(1.C) rRNA processing
(2) One gene, multiple products
1. Recognize the major steps in RNA processing.
2. Associate each processing step with its function.
30
(1) Eukaryotic RNAs
(1.A) mRNA processing:
5' cap addition:
During transcription, the 5' end of the
phosphohydrolase
nascent eukaryotic mRNA is
modified: A phosphate is released by
guanylyltransferase
hydrolysis, and a guanine nucleotide
is a guanine nucleotide is attached
through a "5', 5'-triphosphate
linkage", and then the N-7 of the
guanine-7-methyltransferase
guaine is mehtylated. That is, a 7methylguanosine is linked to the 5'end of a message through a
2-O-methlytransferase
triphosphate linkage. This structure
is called a cap structure. The cap
structure protects the 5' end from
phosphatases and nuclease and
therefore increase the stability of
mRNA. In addition, cap structure facilitates the binding of ribosome to mRNA
and thereby enhances translation.
Splicing:
Introns in the primary mRNAs (pre-mRNA) are removed by a process called
splicing. The splicing signals reside in the primary transcript. They are:
(a) the 5' splice site and the 3' splice site: the sequence in the intron and exon
junction (GU------AG)
(b) the branch site: an internal site located between 20 to 50 nucleotides upstream
of the 3' splice site.
Splicing complexes (splicesomes) bring the exons close together. The
RNA components of the splicing complexes play the directive roles.
Splicesomes (snRNPs) are ribonucleoprotein complexes composed of snRNAs and

proteins.
31
poly(A) addition:
Most eukaryotic mRNAs contain a run of A residues at their 3' end called the
poly(A) tail. These A residues are not encoded in the genome; instead, they are
added after the RNA is synthesized. Before the addition of poly(A), an
endonuclease recognizes the sequence AAUAAA and cleaves the RNA.
Polyadenylate polymerase then adds 20-250 adenylate residues. The function of
poly(A) is to inhibit mRNA degradation and facilitate translation.
AAUAAA: Signal for

Cleavage
Promoter
Exon 1
Exon 2
Exon 4
Exon 3
Transcription
Exon 1
Exon 2
Exon 4
Exon 3
RNA processing
m7Gppp
Exon 1
Exon 2
Exon 3 Exon 4
5UTR
Poly A
3UTR
ORF
32
Practice Exam Q:
Failure of an endonuclease to recognize the sequence AAUAAA in the 3

end of mRNA will cause a defect in which of the following processes?
(A) capping
(B) hybridization
(C) splicing
(D) transport
(E) polyadenylation
(See the bottom of this page for answer.)
(Answer for the practice exam question: (E) polyadenylation)
33
RNA editing:
The peptide coding regions (open reading frames) of some mRNAs are edited after
transcription. An example: ApoB mRNA.
Cholesterol metabolism:
Defective Apo B-100 leads
to hypercholesterolemia.
One
Polypeptide
(Liver)
UAA:
Termination
codon
Digestion: for assembly of

intestinal lipoproteins.
A shorter
Polypeptide
(Intestine)
C U deamination:
Summary: mRNA processing involves cap addition, splicing, poly(A) addition and
editing for some mRNA.
34
(1.B) tRNA processing:
5' cleavage: cut by RNase P to generate a mature 5' end. RNase P is an enzyme
composed of both protein and RNA, called ribonucleoprotein.
Surprisingly, the RNA component is the catalytic part of the enzyme!
3' cleavage: cut by RNase D endonuclease
Splicing: removes the intron, involves cleavage by endonucleases and joining by
RNA ligase
CCA addition
Base modification
5 and 3
cleavage
splicing
base
modification
CCA
addition
Ribonucleoprotein complexes:
Ribosome (for translation)
snRNPs (for splicing)
Signal recognition particles (7SL RNA, for protein targeting)
RNase P (for tRNA processing)
In ribosome and snRNPs, RNAs play the directive roles; in RNase P, RNAs play
the catalytic roles!
35
(1.C) rRNA processing:
In eukaryotes, three of the four rRNAs are produced by RNA polymerase I in the
nucleolus as a polycistronic RNA: a 45 S transcript is made and then cleaved to
produce the 18S, 28S and 5.8S rRNAs. About 1,000 copies of rRNA gene units are
present in human genome. They are linked in tandem, separated by spacer regions
that contain the termination signal for one unit and the promoter for the next unit.
spacer
tandem repeats
of rRNA genes
rRNA gene
rRNA gene
rRNA gene
rRNA gene
polycistronic
rRNA transcript
(5S RNA is produced by RNA polymerase III.)

rRNA: Most abundant RNA
36
(2) One gene, multiple products:
A series of related proteins can be generated by processing a nascent RNA in
different ways, such as alternative splicing and alternative polyadenylation. This
is a more economic way to utilize the genetic information. (The central dogma "one
gene, one product" is not always correct.)
Alternatively splicing: As an example, calcitonin (a calcium-regulating hormone)
in the thyroid is different from calcitonin in brain. They are derived from the same
gene, but are from alternatively spliced mRNA.
2
Alternative polyadenylation: As an example, the immunoglobulin heavy chain

mRNA has two poly(A) sties: A1 and A2 (below). Cleavage at these two sites
results in two proteins that differ from each other only at their C' terminal ends: One
contains the membrane-anchoring domain and is membrane bound; the other does
not contain the membrane-anchoring domain and is soluble.
A1
A2
AAAAA
AAAAA
membrane-anchoring
domain
Note #1: RNA editing, alternative splicing, and alternative polyadenylation allow
the genome to make more than one product from one gene.
efficient utilization of genome
Note #2: DNA as genetic blueprint and mRNAs as intermediates to make proteins:
This strategy tolerates mistakes and allows flexibility (in the amount of proteins
produced and the variation of products produced).
37
(3) Clinical discussion:
Systemic leupus erythematosus (SLE) is an auto-immune disease caused by
antibodies against a number of nuclear and cytoplasmic antigens. snRNPs are one
of the targets of these antibodies. In fact, snRNPs were discovered as a result of
studies using antisera from patients with SLE.
Summary of RNA processing:

Eukaryotic RNA processing
mRNA processing: capping, splicing, poly(A) addition and editing
tRNA processing: 5' and 3' cleavage, splicing, CCA addition, modifications
rRNA processing: cleavage of polycistronic RNA and splicing
One gene, multiple products
Alternative splicing and alternative poly(A) addition
Clinical discussions
38
TRANSLATION
(Protein Synthesis)
Reading assignments: Marks, Chapter on Translation: Synthesis of proteins
Outlines:
(1) Components for translation
(1.A) mRNA
(1.B) aminoacyl-tRNA
(1.C) ribosome: rRNAs and proteins
(1.D) protein factors
(2) Protein synthesis: General features
(3) Protein synthesis inhibitors
1. Define codon degeneracy and various mutations that affect codons.
2. Identify mutations.
3. List features of translation.
4. Distinguish inhibitors of translation.
5. Differentiate different forms of anemia.
10
39
(1) Components for translation: mRNA, aminoacyl-tRNA, ribosomes and some
protein factors
amino acid
activated
tRNA
5'
3'
mRNA
ribosome
(1.A) mRNA
mRNA carries the genetic information: the genetic codes.
Three nucleotides encode one amino acid. These three nucleotides together is
called a "codon". The initiation codon is AUG which codes for methionine. There
are three termination codons (also called stop codons or nonsense codons): amber,
ochre and opal. The region between the initiation codon to the termination codon is
called an open reading frame (ORF).
Codon Table
Codon is degenerate:
There are total 64 (4x4x4)
different codons. Three of
them code for termination
codons. Therefore, 61
codons code for 20 different
amino acids. That is, more
than one codon can code
for the same amino acid.
For example, AUU, AUC
and AUA all code for
isoleucine. Codons that
code for the same amino
acid are called "synonyms".
Most synonyms differ only
in their 3rd position.
11
40
Mutations that affect codons: (Important!)
Non-sense mutation: mutation that introduces a termination codon, such as
UAU to UAA (changes tyrosine to termination codon). This mutation shortens
the protein. The shorten (truncated) protein is usually not functional.
Therefore, this mutation may cause significant phenotypes.
Mis-sense mutation: mutation that introduces a different codon, such as AAU
to AAG (changes asparagine to lysine). Sometimes, a single amino acid change
does not affect the function of the protein. But, sometimes it does, and some
diseases can be caused by a single amino acid mutation (see sickle cell anemia,
below).
Silent mutation: mutation that does NOT result in amino acid change, such as
ACU to ACG (both encode threonine).
Q: How can silent mutation exist?
Frame shift mutation: mutation that shifts the reading frame. Insertion or
deletion of nucleotides can cause frame shift mutation, if the number of
nucleotides is not divisible by 3.
Reading frames:
** In exam questions with any given sequence, if the reading frame is not
specified, one can not decide how to decode the sequence.
Insertion mutation (at protein level): An addition of amino acids (resulted
from the insertion of nucleotides at a number divisible by 3)
Deletion mutation (at protein level): A deletion of amino acids (resulted from
the deletion of nucleotides at a number divisible by 3)
* Insertion (deletion) of nucleotides can result in frame shift or insertion
(deletion) of extra amino acids, depending on the number of nucleotides.
12
41
** Important note:
Sometimes a mutation may not cause any phenotypes. As an example, a Ser to Thr
mutation in a certain protein at a certain position may not affect the function of the
protein. However, it is NOT a silent mutation; it is a mis-sense mutation. No
phenotype does not mean "silent mutation". "Silent mutation" is a very specific
term referring to no change of amino acid sequence.
Q: Can a mistake at transcription (due to low RNA polymerase fidelity) that results
in amino acid changes cause deleterious effects?
Mistakes due to transcription versus Mutations at the DNA level.
A sample question:
If an open reading frame contains the changes indicated below, what kind of mutation
does it result (at the protein level)? 5- GTTAACCTTG -> changed to 5GTTAACCAGTTG
(A) nonsense mutation
(B) mis-sense mutation
(C) frame shift mutation
(D) silent mutation
(E) insertion mutation
(See the bottom of the page for answer.)
Answer for the sample question: (C) frame shift mutation.

(1.B) aminoacyl-tRNA
13
42
tRNA carries amino acids.

tRNA versus aminoacyl-tRNA (activated or charged tRNA): Aminoacyl-tRNA
refers to tRNA that carries an amino acid
tRNAphe: denotes a tRNA that its function is to carry phe.
phe-tRNAphe: denotes tRNAphe that is "charged" with phe, a reaction catalyzed by
specific aminoacyl-tRNA synthetase. Charged with Phe
*The amino acid in charged tRNA does not play a role in selecting a codon.
The tRNA determines the selection of codon.
For an example, tyr-tRNAphe is a tRNAphe that carries tyr instead of phe. It will
result in a point mutation in the protein.
(A) Phe -> Tyr mutation, or
(B) Tyr -> Phe mutation
What kind of mutation is this called?
Non-sense
Mis-sense
Silent
Frame shift
Answer: (A) Phe to Tyr, a mis-sense mutation.

(1.C) Ribosomes (composed of a small and a large subunit)
14
43
As described in RNA Properties, ribosome is a ribonucleoprotein that carries out

protein synthesis (translation). There are two subunits of ribosome: the large and
small subunits. Each subunit is composed of multiple proteins and some RNA
molecules.
The rRNAs fold into specific structures and play directive roles in protein
synthesis. Different parts of rRNAs base pair with mRNA and tRNA. As an
example, the prokaryotic mRNA contains a sequence called the Shine-Dalgarno
sequence in front of its initiation codon. This sequence base pairs with the 16S
rRNA and this pairing plays an important role in determining the translational start
site in mRNA. Proteins in the ribosome play a more supportive role. Most
antibiotics that interfere with protein synthesis by interacting with an rRNA rather
than a ribosomal protein.
(1.D) Protein factors

Initiation factors (eIFs), elongation factors, translocase, and release factors
(2) Protein synthesis: General features
15
44
Proteins are synthesized in the amino to carboxyl direction (N' >C'). mRNA is
translated in the 5' to 3' direction. Therefore, newly synthesized mRNA, before it is
completely synthesized, can be translated in prokaryotes (coupled transcription and
translation). If the mRNA were transcribed from 3' to 5', then only fully synthesized
mRNA could be translated. In eukaryotes, the mRNA needs to be completely
synthesized, processed and transported from the nucleus to the cytoplasm before it
can be translated.
An mRNA molecule can be translated simultaneously by multiple ribosomes. As a
group, these ribosomes are called polysome. The ribosomes in a polysome function
independently.
(3) Protein synthesis inhibitors:

Protein synthesis is the primary target for many antibiotics and toxins. As described
above, most antibiotics that interfere with protein synthesis by interacting with a
rRNA rather than a ribosomal protein.
Prokaryotic protein synthesis inhibitor:
Tetracycline
Chloramphenicol
Erythromycin
Streptomycin
Neomycin
Kanamycin
Gentamycin
blocks the A site and inhibits binding of aminoacyl-tRNA.

inhibits peptidyl transferase activity
binds to 50S subunit and inhibits translocation
causes misreading and inhibits initiation
interferes with 16S rRNA
Eukaryotic protein synthesis inhibitors:

Cycloheximide Blocks peptidyl transferase activity
16
45
Diphtheria toxin A toxin secreted by the bacterium Corynebacterium
diphtheriae, composed of 2 polypeptides
inactivates translocation by covalently modifying the
translocase. Immunization against this toxin is universal
in the US.
Ricin
Inactivates the 60S subunit of the eukaryotic ribosomes
Prokaryotic and eukaryotic inhibitors:
Puromycin
Structure similar to the 3' end of an aminoacyl-tRNA, binds to
the empty A site on ribosome, resulting in peptide with
puromycin at its C'
(4) Clinical discussion

(a) Thalassemias: Any mutation that affects the synthesis of either or chains of
globin can cause a decreased production of hemoglobin and consequently anemia.
Table on the right shows some
examples of mutations in thalassemia. Because so many
mutations can cause
thalassemia, it is not surprising
that thalassemias represent the
single most common gene
disorder in the world (carrier
rate of about 7%). The disease
is most prevalent in countries
around the Mediterranean Sea.
Some examples of mutations in -thalassemia

Type of mutation
Pheotype
Origin
Decreased production of
wild type globin, OR
Production of mutant globin
Decreased production of
hemoglobin
Thalassemias
A sample exam question:

DNA analyses of a patient with
17
46
-thalassemias showed three mutations indicated by arrows in the figure below. The
three nucleotides of the wild type sequence are required for which of the following?
(A) Binding to snRNPs

(B) Binding to elongation factor
(C) Binding to transcriptional activators
(D) Binding to polyA polymerase
(E) Binding to transcriptional repressors
(See the bottom of next page for answer.)
(b) Sickle cell anemia: It is caused by a mis-sense mutation in the -globin chain
of the hemoglobin. The mutant protein functions properly under high oxygen
conditions and displays the phenotypes only under low oxygen conditions. A
specific amino acid change
normal red blood cell
sickled red blood cell
The frequency of sickle gene is as high as 40% in some parts of Africa. The reason
that the frequency is so high is that the heterozygotes are protected against the most
lethal form of malaria. Therefore, in areas with high incidence of malaria, the sickle
gene has an advantage over the normal gene and is evolutionarily favored.
(c) Iron deficiency anemia: It is caused by the lack of iron in the diet. Iron is part
of the heme. When iron is low, heme concentration is low. Without heme, the
18
47
translation initiation factor elF2 is not active and protein synthesis in the red blood
cells is halted. Consequently, globin is not synthesized and the blood level of
hemoglobin is low and an anemia results.
Heme = iron + porphyrin (organic rings)
Hemoglobin = Heme + globin (carries 8 molecules of oxygen)
Heme
Hemoglobin
Q: Does iron deficiency inhibit synthesis of all proteins, since eIF2 is a factor
required for translation initiation?
A: The regulator that mediates the effect of iron deficiency is primarily expressed in
red blood cellsnot in other cells. Thus, only protein synthesis in red blood cells is
affected by iron concentration. Globin is the major protein produced by the mature
red blood cells (see next lecture notes).
* HRI kinase (Heme-regulated inhibitor kinase): When iron/heme is low, HRI kinase
phosphorylates eIF2 and inhibits its activity.
Answer to the question on the previous page: (C)

(d) Many antibiotics work by inhibiting protein synthesis. The major concerns of
using antibiotics are as follows. First, antibiotics may also affect human
mitochondria and cause side effects. This is because the protein synthesis system in
19
48
mitochondria is similar to that in bacteria (see RNA property). Second, the bacteria
may develop resistance to antibiotics. Bacteria can do so by mutating their genes
to make mutated ribosomes that are no longer sensitive to the antibiotics, or by
picking up plasmids carrying genes that inactivate the antibiotics.
Summary of translation
Components for translation
mRNA:
codon degeneracy, mutations
tRNA:
adapter (to bring amino acids)
ribosome:
rRNAs and proteins -- RNA has the directive roles
protein factors:
Protein synthesis General features: 5' to 3' on mRNA, N' to C' on protein
Protein synthesis inhibitors: targets for many antibiotics, interfering with rRNA
Clinical Discussions
20
49
Dr. Wang lecture 2, Gene Regulation
REGULATION OF GENE EXPRESSION

Reading assignment: Marks, Chapter on Regulation of gene expression
Outlines:
(1) Eukaryotes versus prokaryotes
(2) Eukaryotic gene regulation
(2.A) Alteration in genes
(2.B) Chemical modification of genesDNA methylation
(2.C) Transcriptional regulation (by proteins)
(2.D) Post-transcriptional regulation
(2.E) Translational regulation
(2.F) Post-translational regulation
1. Describe the functions of different steps in gene regulation.
2. Relate abnormalities in gene regulation to diseases.
3. Distinguish prokaryotes from eukaryotes.
50
(1) Eukaryotes versus prokaryotes:

The significance of gene regulation
The process of turning on a specific gene to make protein is called "gene
expression". All organisms regulate their gene expression in biological processes
such as development and responses to environmental changes. In addition, many
diseases including cancer are the consequences of mis-regulation of gene
expression.
Eukaryotes versus prokaryotes
In eukaryotes, this process involves many steps: transcription, processing, and
export of mRNA to the cytoplasm, translation, post-translational modifications and
protein targeting (delivery of the proteins to the correct locations). The control of
gene expression can occur at any of these steps. A large part of the control,
however, occurs in transcription.
Promoter
Enhancer
Silencer
co-factors
Transcriptional
machinery
TATA
Transcription
exon
(mRNA
precursor)
intron
RNA processing:
capping, splicing
poly(A) addition,
and editing
poly(A)
5'cap
(mature
mRNA)
Transport to
cytoplasm
mRNA
5'cap
Protein
5'
UTR
poly(A)
AUG
UAA
Open reading frame
3'
UTR
UTR: untranslated
region
Translation
Protein:
post-translational
modifications and
protein targeting
51
Prokaryotes, in contrast, are much simpler: they are single-celled organisms; they do
not have nucleus; their DNAs are not complexed with histones; their mRNAs do not
have cap, introns or poly(A). Therefore, many control steps available in eukaryotes
are NOT present in prokaryotes. The following table lists the differences between
prokaryotes and eukaryotes.
Differences between Eukaryotes and Prokaryotes

Eukaryotes
Prokaryotes
A sample exam question:

A new opportunistic pathogen was isolated from the stool of HIV-infected patients.
Molecular analyses of this new isolate was carried. Which of the following features
suggest that this is a eukaryotic microorganism?
(A) DNA
(B) RNA
(C) Cell membrane
(D) Intron
(E) Ribosome
(See the bottom of next page for answer.)
52

Eukaryotic gene expression can be regulated at many steps.
(2.A) Alteration in genes (physical modification)
(1) Gene loss: If genes are deleted or partially deleted, the corresponding functional
proteins are not made. An extreme example is the gene loss during red blood cell
development. Immature red blood cells produce globin mRNAs; during maturation,
they lose their nuclei. Therefore, mature red blood cells no longer contain any
genes to produce mRNA. The proteins that mature red blood cells will make are
the globin proteins, because the corresponding mRNAs are deposited during
maturation.
(2) Gene amplification: Many copies of certain genes may be generated in order to
make large amount of the corresponding proteins. One example is the DHFR gene
(see Clinical discussions).
(3) Gene rearrangement: Segment of DNA may move from one location to another
so that different proteins are made. One example is the antibody gene rearrangement
during B cell maturation.
Answer to the practice exam question on the previous page: (D) Intron
53
(2.B) Chemical modification of genes

DNA methylation: Certain genes may be methylated at the cytosine residues and
become less transcribed. Note that the methyl group does not interfere with the
normal base pairing.
Most of the methylcytosine is present in the sequence 5 CG -3. Some regions of the genome contain
disproportionally high content of CG sequence. These
regions are called CpG islands. Highly methylated areas
of DNA are not transcribed
Below are some examples.

(a) Globin genes are more methylated in non-erythroid than in erythroid.
(b) Transcriptionally inactive X chromosome is highly methylated. In females,
only one X chromosome is transcribed. The transcriptionally inactive X
chromosome is more methylated than the
transcriptionally active X chromosome
(called X chromosome inactivation).
(c) Genomic imprinting - (will be
discussed in more detail by another
lecturer)
Although in most cases both the paternal
and maternal alleles of a gene are
expressed, a few genes are imprinted their expression depends on whether they
are from the father or the mother. In some
cases, only the paternal allele of the gene
is expressed; in other cases, only the
maternal allele is expressed. As an
egg
sperm
H19
fertilization
active
maternal
allele
inactive
paternal
allele
54
example, the H19 gene is methylated during the development of the male germ
cells. Therefore, sperm contain a methylated H19 allele. However, eggs contain
an unmethylated allele. Following fertilization, the methylated paternal allele
remains inactive, and only the unmethylated maternal allele is expressed.
(Maternal imprinting means the maternal allele is methylated and thus off.)
The methylation pattern can be maintained after DNA replication for the
following reason: The CG sequence is a palindrome. Therefore, both strands of
DNA will have the
CpG sequence.
CH3
The methylating
Methylated
5
CpG
3
enzyme systems
GpC
3
5
parental DNA
CH3
work preferentially
on the
Hemi-methylated
DNA replication
hemimethylated
CH3
CpG sequences.
5
CpG
3
5
CpG
3
GpC
3
5
GpC
3
5
Therefore, after
CH3
Methylation
DNA replication,
the information is
CH3
CH3
preserved and the
5
CpG
3
5
CpG
3
GpC
3
5
GpC
3
5
cells know which
CH3
CH3
Methylated daughter DNA
CpG sequence to
methylate.
Maintenance of imprinted DNA:
Maternal
(un-methylated)
Remember, in each allele, the DNA is

double-stranded. Therefore, the
methylated allele in the genomic imprinting
example can maintain its methylation status
after cell division.
Paternal
(Methylated)
One allele:
ds DNA
Methylation is maintained
after DNA replication.
55
(2.C) Transcriptional regulation (by proteins)

(1) Chromatin condensation: (regulation by histones)
Genes in condensed chromatin (called heterochromatin) are not transcribed. As
an example, as red blood cells mature, their chromatin becomes more condensed and
RNA synthesis decreases. Remember, the nucleus is eventually extruded from the
mature red blood cell an extreme example of inactivating the gene. In the
condensed region, histones bind to the DNA and inhibit transcription. One way to
look at this is that histones and sequence-specific transcription factors compete for
binding to the DNA. In order for sequence-specific transcription factors to activate
transcription, they must replace the histones that non-specifically bind to the
promoter or enhancer regions.
A light micrograph shows the
de-condensed crhomosome
regions in Drosophila.
(indicated by arrows)
This figure shows the replacement

of nucleosomes from the
promoter by a transcriptional
activator.
Activator
DNA wrapped around

nucleosome cores
Nucleosome remodeling factor
Active
Nucleosome displaced from promoter,
Binding of basal transcription factors
56
(2) Regulation by transcriptional factors:

The major differences between prokaryotic and eukaryotic transcriptional regulation
are: (1) The eukaryotic polymerases are not active on their own. A multi-subunit
transcriptional apparatus must be assembled first (the basal transcription machinery).
(2) Most eukaryotic genes are controlled by multiple proteins rather than by just one
or two proteins.
Sequence-specific transcription factors play a major role in regulating eukaryotic
gene expression. This is an extensively researched area and we will only discuss
three points here: (a) activator versus repressor, (b) regulation by ligands, and (c)
tissue-specific transcription.
(a) activator versus repressor: Sequence-specific transcription factors can be either
an activator or a repressor. As shown above, activators can replace the nucleosome
and activate transcription by either direct or indirect contacting of the general
transcription machinery contact (also see handout Transcription). Several modes
of repression have been demonstrated; here we will only discuss two modes: active
versus passive repression. In active repression, the repressors repress transcription
by communicating (directly or indirectly) with the basal transcription machinery.
In passive repression, repressors compete with activators for binding to DNA, but do
not interact with the transcription machinery. Therefore, their effect on transcription
is passive. Some repressors use the active mode and some use the passive mode to
repress transcription.
Active repression
Passive repression
Activator
Repressor
domain
DNA binding
domain
Repressor
TATA
TATA
Repressor binds DNA and inhibits

transcription by protein-protein interaction
Repressor competes with

activator for binding to DNA
(b) regulation by ligands: The activity of some sequence-specific transcription

factors can be regulated by ligands. As described in Transcription, most
transcription factors contain a DNA binding domain, and a transcriptional activation
57
(or repression) domain. However, some transcription factors contain a third

domain, called the ligand binding domain or the regulatory domain. Binding of
ligands to the domain alters the activity of these transcription factors. We will only
discuss one group of transcriptional activators here - the steroid hormone receptors.
(modular domains)
Note: Although they are called steroid hormone "receptor", they are NOT
membrane proteins; they are transcription factors.
Cytosolic receptor superfamily:
Many steroid hormone receptors are cytosolic. Steroid hormones such as estrogens,
progesterone and glucocorticoids, traverse the plasma membrane and bind to specific
receptors; the receptor-hormone complexes then migrate to the nucleus, bind to
DNA and regulate transcription.
Nuclear receptor superfamily:
Some steroid receptors locate in the nucleus already. The ligands need to traverse
both the plasma membrane and the nuclear membrane. Ligand binding results in
DNA binding and transcriptional activation. Examples are the vitamin D3 receptors.
(Vitamin D3 is a steroid derivative.) Some nuclear receptors bind to ligands that are
unrelated to steroids, such as thyroxin (a derivative from tyrosine) and retinoic acids
(vitamin A derivative).
(c) tissue-specific transcription: As described in Transcription, the recognition site

for a given transcription factor is usually located in a variety of promoters in
different tissues. As an example, a liver specific-gene and a pancreas-specific gene
may share some common binding sites in their promoters. This raises an important
58
question: How is tissue-specific transcription achieved? The current model is that

this is achieved by transcription factors through a "combinatorial mechanism:
Different promoters may share some common cis-acting elements, but these
elements are combined with different binding sites in different promoters. Binding
of the corresponding transcription factors to these sites determines the activity of the
given promoter. The reason that different cells express different gene products is
because different cells have different sets of transcription factors (different cellular
contexts). One way to think about this is that the transcription factors in a given cell
type constitute a combination of passwords that cooperatively open the lock.
Another way to think about this is that each promoter is like a jigsaw puzzle. Only
when a given cell contains the correct pieces (the correct transcription factors) can it
solve the puzzle (switch on the gene).
One example for tissue-specific transcription is that immunoglobulin enhancer
functions in B lymphocytes but not in other cells. This is because only the correct
D
A TATA
D
A TATA
cells contain the "cognate activating proteins". Another example is the restricted
host ranges of viruses. For example, the mouse mammary tumor virus (MMTV)
contains the glucocorticoid enhancer in their genome. Therefore, MMTV thrives in
cell that contains the "cognate proteins", that is, the cell that responds to
glucocorticoid.
* Alternative promoters: (a special example of tissue-specific promoter)
Some genes contain more than one promoter, and only a given promoter is active in
a certain cell type. One example is the glucokinase gene: It has one promoter that is
used in the liver and another one used in the pancreas. This can be viewed as a
special example of tissue-specific gene expression: Instead of two genes regulated
by two promoters, this is one gene regulated by two alternative promoters.
Pancreatic RNA
Promoter 1 Promoter 2
GENE
Hepatic RNA
10
59
(2.D) Post-transcriptional regulation

Alternative splicing: (see RNA processing, the calcitonin example)
Alternative polyadenylation: (see RNA processing, the immunoglobulin
example)
mRNA editing: (see RNA processing, the apoB example)
mRNA transport: In eukaryotes, mRNA must travel from the nucleus to the
cytoplasm to be translated. One example is the HIV RNA. The un-spliced HIV
RNA (the viral genome) requires a viral protein, rev, to be transported to the
cytoplasm.
mRNA stability: One example is the transferrin receptor mRNA. When
cellular iron concentration is low, a protein binds to the 3' end of the mRNA and
prevents it from being degraded. Therefore, more protein (transferrin receptor) is
made. The receptor is a membrane protein and allows cells to pick up more
transferrin, an iron carrying protein.
(2.E) Translational regulation
Most eukaryotic translational control affects the initiation of protein synthesis. As
an example, the translation of globin mRNA is regulated by the heme concentration.
When heme concentration is high, the synthesis of globin protein increases. In iron
deficiency anemia, lack of iron in the diet results in a low concentration of heme (an
iron containing molecule), leading to a decreased synthesis of globin protein.
Consequently, the concentration of hemoglobin is low and the red blood cells are
pale. Also see the lecture notes on Translation.
Red blood cells: (has HRI kinase)

Low iron -> low heme -> activates the heme-regulated inhibitor (HRI) kinase ->
phosphorylates eIF2 and inhibits its activity -> low protein synthesis (low globin
synthesis).
11
60
** We have discussed the following regulations in red blood cells: chemical

modification (Globin genes are more modified, therefore less active, in nonerythroid.), chromatin condensation (As red blood cells mature, DNA becomes
condensed and less active.), gene loss (Eventually red blood cell extrudes its
nucleus.) and translational regulation (In mature red blood cells, there is no DNA to
transcribe). The only possible control is at the translational or post-translational
level. It turned out to be at the translational level: initiation of globin protein
synthesis is regulated by heme concentration.).
Globin Gene:
Non-red blood cells: DNA methylated
Immature:
Synthesize
Globin mRNA
Red blood cells
Mature: Only globin mRNA

deposited earlier
DNA
condensation
Extrude
Nuclei
12
Regulated by iron
concentration
61
(2.F) Post-translational regulation

Proteins have different half-lives. Some last for hours or days and others last for
months or even years. In addition, proteins need to be properly modified and
targeted to the correct locations.
A summary of regulation of gene expression:
Physical Modifications:
Rearrangement
Amplification
Gene loss
TATA
Transcription
Chemical Modifications:
Methylation
RNA processing:
capping, splicing
poly(A) addition,
and editing
Transcription Regulation:
Histone
Transcription factors (activator
vs. repressor, ligand, tissuespecific regulation)
poly(A)
5'cap
Transport to
cytoplasm
mRNA 5'cap
Alternative splicing
Alternative pA
mRNA editing
mRNA stability
RNA transport
poly(A)
ORF
Protein
Translation
Translational regulation
Protein:
post-transnational
modifications and
protein targeting
Post-Translational regulation
Protein half-life
Modifications
Targeting
13
62
(3) Clinical discussions:

Mis-regulation of gene expression in any regulatory steps we discussed above can
have profound effects on human health. Below are some examples.
(a) Methotrexate is a drug for cancer treatment, but sometimes patients become
resistant to it. One mechanism for methotrexate resistance is gene amplification.
Methotrexate inhibits the enzyme dihydrofolate reductase (DHFR). When DHFR is
inhibited, cells cannot produce adequate amount of thymine for DNA synthesis.
Therefore, cell proliferation is inhibited. Sometimes, cells treated with methotrexate
amplify their DHFR gene and become resistant to methotrexate.
(b) A DNA rearrangement (translocation) between chromosomes 9 and 22 results in
chronic myelogenous leukemia (CML). This translocated chromosome is called
"Philadelphia chromosome", because it was first observed in Philadelphia. To
date, many cancers have been shown to be caused by DNA translocation.
(c) Patients suffering testicular feminization have the male karyotype (XY
chromosome), and produce androgens (the male hormones). However, their cells
fail to respond to the hormones, because the appropriate intracellular receptors are
lacking. Therefore, they can not turn on the genetic program that is normally
activated by androgens and is responsible for masculinization.
Summary:
(1) Eukaryotes versus prokaryotes: nucleus, histones, cap, introns, poly(A)
(2.A) Alteration in genes: gene loss, gene amplification, gene rearrangement
(2.B) Chemical modification of genes: DNA methylation
(2.C) Transcriptional regulation (by proteins):
chromatin condensation, activators, repressors, regulation by ligands,
tissue-specific transcription, combinatorial mechanism
(2.D) Post-transcriptional regulation: alternative splicing, alternative
polyadenylation, RNA editing, RNA transport, RNA stability
(2.E) Translational regulation: mostly at translational initiation
(2.F) Post-translational regulation: protein degradation, modification, and
targeting
More details on X-inactivation and imprinting
14
63
(not on Dr. Hais exam)

X-inactivation: The entire X chromosome is inactivated (to the first
approximation). But X-inactivation is mosaic at the tissue levels. During sperm
and egg production, the methylation is erased!! During female embryogenesis (~ 4
cell stage or a little later), X-inactivation occurs (there is no X-inactivation in
males, which has one copy of X). The process of X-inactivation is stochastic:
some cells inactivate the maternal X; some cells inactivate the paternal X. Figure
below explains the effects of X-inactivation on color blind trait.
Red X indicates the affected allele. When transmitted to the sons from the
mothers, the sons show the
X-Inactivation
phenotype of color blindness.
If the affected allele (X) is
Erased during gametogenesis &
transmitted from the mothers to
Re-established at 4-cell stage or later
the daughters, the daughters
Example: Color blind
will have XX at the one cell
stage. During embryogenesis,
Mother
Father
X
X-inactivation occurs: either
XY
XX
the wild type allele of the Xchromosome (the paternal
Daughters
Sons
allele) is inactivated (indicated
XX
XX
XY
XY
by the grey X) or the affected
allele (the maternal allele) is
X-inactivation
inactivated (indicated by the
In the affected XX XX
XY XY
light red X). These clusters of
daughter
(a stochastic
mosaic cells contribute to the
XX XX
XY XY
event)
formation of all tissues,
including retina. Depending on
Entire
Mosaic
the percentage of cells with XX
Retina
Retina,
(functional) versus XX (nonas XY,
Not
functional) in the retina, the
Color Blind
Color Blind
daughters will display different
degrees of color blindness.
X-inactivation results in mosaic tissues;
Thus, unlike the affected sons
thus, even the daughter, who inherited the
who will certainly display
bad allele, is not completely color blind.
color blindness the affected
daughters do not (despite of
having only one active X chromosome).
15
64
Genomic imprinting: Imprinted allele

is the inactive allele. Thus, paternal
imprinting means the fathers allele is
inactive. Genomic imprinting is reprogrammed during gametogenesis,
depending on the whether the allele is
passed via the male or female. For a
paternally imprinted gene (such as
H19), it is methylated during the sperm
production. As shown in the figure, the
son received an un-methylated allele
from his mother and a methylated allele
from his father (indicated by red dots
on the double stranded DNAs). When
he generates sperms, both alleles will
be methylated. Thus, the alleles he
passes on are imprinted, no matter
where he received the original allele
from his mother (un-methylated) or his
father (methylated). The case for the
daughter is reversed: neither allele is
methylated in the eggs.
16
Imprinting
Erased and re-established
during gametogenesis
Example: Paternal Imprinting
Eggs
Daughter
Eggs
Sperms
Son
Sperms
Because of the re-programming

during gametogenesis, the
imprinting for this gene is always
paternal!
65
NUCLEOTIDE METABOLISM
Samson T. Jacob, Ph.D.
Friday :
Monday :
12/12/11
12/13/11
10:30-12:00
8:30-10:30
OBJECTIVES
The main objectives are to study:
(a)
How purine and pyrimidine nucleotides, the building blocks of RNA and DNA) are
synthesized from small molecules such as amino acids, carbon dioxide and formyl
groups ( de novo pathway ) and by salvage pathways using preformed purines and
pyrimidines (derived from nucleic acid-rich diet).
(b)
Key enzymes involved in this process.
(c)
How purines and pyrimidines are degraded
(d)
Clinical disorders associated with the nucleotide metabolism
(e)
Utilization of Purine and pyrimidine analogs as antiviral and anticancer drugs
OVERVIEW
Ribo- and deoxyribonucleotides are the building blocks of RNA and DNA, respectively. (Fig. 1)
Nucleotides also serve as intermediates in the synthesis of some carbohydrates, lipids and
proteins, as energy donors in the cell and as regulatory molecules in many intermediary
metabolic pathways.
66
PURINE NUCLEOTIDE BIOSYNTHESIS

The sources of 5 Carbon atoms and 4 nitrogen atoms are shown in Fig 2A. Liver is the major
site of purine synthesis.
De novo Pathway
FIGURE 2A
O RIG IN S OF TH E ATO MS OF THE PURINE RIN G
GLY CIN E
CO 2
C6
ASPARTIC
ACI D
N7
N1
C5
C2
C4
C8
(from N 10
"FORM YL "
formyl THFA)
"FORM YL "
(from N 10 formyl THFA)
N9
N3
AM IDE - N OF GLUTAM IN E
This pathway is most active in the liver. The first step is the production of PRPP (the source of
ribose and phosphate) from ribose-5-phosphate (a product of the Pentose Phosphate Pathway
in the carbohydrate metabolism) and ATP providing the pyrophosphate group. The first ratelimiting reaction is that catalyzed by PRPP amidotransferase, which produces 5phosphoribosylamine from PRPP and glutamine. The parent purine nucleotide IMP (inosinic
acid) is produced after a series of reactions beginning with PRPP (Fig. 2B, 2C). One glycine,
two glutamines and one arpartic acid are utilized in the synthesis of purines. Five reactions
utilize ATP for energy, and formyl tetrahydrofolate (THF) is utilized in two reactions- an
extremely energy consuming process.
GTP is a substrate for AMP synthesis whereas ATP functions as a substrate in the GMP
synthesis. The reciprocal relationship of the two substrates appears to balance the synthesis
of adenine and guanine ribonucleotides.
IMP is converted to AMP and GMP (Fig. 2B, 2C). This pathway that produces purine
nucleotides from simple molecules is called de novo biosynthesis of purine nucleotides.
67
RIBOSE-5'-PHOSPHATE
1
ATP
FIGURE 2B
AMP
PHOSPHORIBOSYL PYROPHOSPHATE (PRPP)

2
GLUTAMINE
RATE LIMITING
PHOSPHORIBOSYLAMINE
3
GLYCINE
ATP
ADP
GLUTAMINE
FORMYL TETRAHYDROFOLATE (THF)

THF
ATP
1
ADP
FIRST RING CLOSURE
ATP
6
7
8
ASPARTIC ACID
ADP
10
PRPP AMIDOTRANSFERASE
(AMIDO PHOSPHORIBOSYLTRANSFERASE)
& 10
FORMYLTRANSFERASE
CO 2
ONE GLYCINE (REACTION #3)
ATP
TWO GLUTAMINE (REACTIONS #2&5)
ADP
ONE ASPARTIC ACID (REACTION #8)
FUMARIC ACID
PRPP SYNTHETASE
TWO FORMYL THF (REACTIONS #4&10)

FIVE ATP (REACTIONS # 1,3,5,6&8)
FORMYL THF
THF
SECOND RING CLOSURE

11
H2 O
INOSINE-5'-MONOPHOSPHATE (IMP)
(PARENT PURINE NUCLEOTIDE)

5 molecules of ATP required for each mole of IMP synthesized. Additional 1 mole of ATP for
subsequent reaction to produce GMP, and 1 mole of GTP for AMP synthesis (see Fig. 2C)
ADENINE - NH2
HYPOXANTHINE
HYPOXANTHINE + RIBOSE
INOSINE + P
ADENOSINE + P
INOSINE
IMP ( INOSINE MONOPHOSPHATE OR INOSINIC ACID )
AMP (ADENOSINE MONOPHOSPHATE OR ADENYLIC ACID)
Purine nucleoside
phosphorylase
Base + Ribose-1-P
Base - Ribose + Pi
(Nucleoside)
68
FIGURE 2C
De Novo Pathway
P-O-CH2
ATP
H OH
H H
OH
OH
OH
PHOSPHORIBOSYL
PHOSPHATE (PRPP)
D-RIBOSE5-PHOSPHATE
Glutamine
phosphoribosyl
2
amidotransferase
OH
N
HNN
H O-P -O-P
H H
PRPP
synthase
1
OH
P-O-CH 2
AMP
P -O-CH2
OH
H H
OH
OH
INOSINE MONOPHOSPHATE
(IMP)
PARENT PURINE NUCLEOTIDE
H H
H H
Reactions 3-11
H H
IMP Dehydrogenase XANTHYLIC ACID

(XMP)
NAD
NADH
GMP
synthase
ATP
NH2
R-P
ADENYLIC ACID OR
ADENOSINE-5-MONOPHOSPHATE
(AMP)
Glutamate
N
N
N
Glutamine
(provides NH
OH
SAMP (ADENYLO SUCCINATE)
OH
5-PHOSPHORIBOSYLAMINE
GTP Adenylo
Aspartic Acid Succinate
(provides NH ) Synthase
Glutamate
NH 3+
P-O-CH2
N
Glutamine
H2 N
R-P
GUANYLIC ACID OR
GUANOSINE-5-MONOPHOSPHATE
(GMP)
69
NH
N
O
CH
N
N
C OH
C NH CH CH CH
NH
Folic Acid
H
NH
N
OH
C OH
O
OH
NH 2
CH
N
5
10
NH
N
10
CH 2 N
CH
OH
CH 2
(source of Methyl
group in Thymine)
10
N 5,N - Methylene THF
N 5-Methyl THF
H
NH
2
N
10
N
N
OH
CH
CHO
THF (source of carbons 2 and 8 in purine structure; Reactions 4 and 10

N10 -FORMYL
in de novo synthesis of purine nucleotides)
The other pathway is called Salvage Pathway. The predominant pathway utilizes preformed
purines (from the degradation of diet rich in purines) and utilizes either the enzyme APRT or
the enzyme HGPRT to convert hypoxanthine/guanine to IMP/GMP (See Fig. 3A &3B for
details). Another pathway (not as significant as the former) converts purine bases sequentially
to the nucleotide, first to nucleoside (adds ribose) by the enzyme purine nucleoside
FIGURE 3 A
SALVAGE PATHWAY FOR PURINE BIOSYNTHESIS
(MORE SIGNIFICANT)
1
AMP + PPi
ADENINE + PRPP
2
HYPOXANTHINE + PRPP
IMP + PPi
GMP + PPi
GUANINE + PRPP
1 ADENINE PHOSPHORIBOSYLTRANSFERASE
(APRT)
2
HYPOXANTHINE - GUANINE PHOSPHORIBOSYLTRANSFERASE (HGPRT)
3
70
phosphorylase, and then to the nucleotide by the enzyme nucleoside kinase (adds a
phosphate). These reactions reduce the requirement for the energetically costly de novo
biosynthesis. Also, the salvage pathway provides the purine nucleotides for those
tissues (most notably, the brain) in which the de novo pathway is absent or partially
Figure 3B
NH2
NH2
N
N
H
PRPP
APRT
RIBOSE-P
ADENINE
ADENOSINE 5'- MONOPHOSPHATE

OR
ADENYLIC ACID
(AMP)
ATP
ADP
developed. HGPRT is the most important salvage pathway enzyme.

In the absence of Adenine phosphoribosyl transferase, Adenine is converted to 8-OH and 2,8,
dihydoxy adenosine. The latter can be deposited as kidney stones.
FIGURE 3C
NH2
NH2
OH
N
N
H
HO
ADENINE
N
H
2, 8-Dihydroxyadenine
NH2
N
OH
N
N
H
8-Hydroxyadenine
71
FEEDBACK CONTROL OF PURINE NUCLEOTIDE BIOSYNTHESIS

The most important metabolic control of purine nucleotide biosynthesis is the inhibition
of PRPP amidotransferase (The enzyme that converts PRPP to phosphoribosylamine)
by AMP and GMP.
The enzyme PRPP synthetase that synthesizes PRPP from ribose-5-phosphate is feedback regulated by AMP, ADP, GMP and GDP (Fig. 4).
FIGURE 4
FEEDBACK CONTROL OF PURINE

NUCLEOTIDE BIOSYNTHESIS
RIBOSE - 5 -PHOSPHATE
ATP
ADP
PRPP
AMP
PHOSPHORIBOSYLAMINE
IMP
XMP
GMP
GTP
GDP
= FEEDBACK INHIBITION
CATABOLISM OF PURINE BASES (occurs mainly in the liver)

GMP is metabolized to guanosine (end product in intestine) by nucleotidase. Guanosine is
then converted to the base guanine in some tissues by nucleoside phosphorylase. Guanine
then enters the salvage pathway and is utilized in the production of GMP (Fig. 5A).
PURINE CATABOLISM
FIGURE 5A
GUANOSINE
GMP
(end point in intestine)
NUCLEOTIDASE
PURINE
NUCLEOSIDE
PHOSPHORYLASE
(in some tissues)
DEGRADED
GUANINE
SALVAGE PATHWAY
ALSO CONVERTS
INOSINE TO
HYPOXANTHINE
+
AMP
DEAMINASE
AMP
5 NUCLEOTIDASE
ADENOSINE
NUCLEOTIDASE
IMP
ADENOSINE *
KINASE
PURINE
NUCLEOSIDE
PHOSPHORYLASE
INOSINE
ADENOSINE
DEAMINASE
HYPOXANTHINE
DEGRADED
SALVAGE PATHWAY
+ ADENOSINE IS NOT A SUBSTRATE FOR THIS ENZYME

* GUANOSINE OR INOSINE CANNOT BE CONVERTED TO GMP OR IMP DIRECTLY
72
AMP is metabolized first to IMP by adenylate deaminase. IMP is then metabolized to inosine
(Base + Ribose) and to hypoxanthine (Base) by nucleotidase and nucleoside phosphorylase,
respectively (Fig. 5A). Note that nucleoside phosphorylase is a reversible enzyme; it can either
add a ribose to the base to form the nucleoside or convert the nucleoside back to the base,
depending upon the reaction conditions.
Why should GMP be converted to G that is converted back to GMP by salvage pathway? GMP
conversion to GDP and then to GTP (synthetic reactions) is not as efficient as its conversion to
Guanosine and then to G (degradative reactions) in cells, particularly in intestines. Intestines
have very high levels of degradative/digestive enzymes that function optimally at acidic pH.
Further, Guanosine is more permeable than GMP to cells.This would facilitate its transport
from the intestines to other tissues such as liver for its conversion to GMP in these tissues by
salvage pathway. Similar reactions apply to hypoxanthine. If excess of G is produced, some of
it will be converted to uric acid (See Fig.5B).
Guanine (from GMP) and hypoxanthine (from AMP) are then metabolized to Xanthine and
subsequently to uric acid by the enzyme Xanthine oxidase. Uric acid is the end product of
purine catabolism in humans and is produced primarily in the liver and excreted by the kidney
into the urine (Fig. 5B). Xanthine oxidase is an important target for pharmacologic
intervention in patients with Hyperuricemia and Gout.
FIGURE 5B
HYPOXANTHINE
Xanthine
oxidase
Guanine deaminase
(Abundant in liver
and Brain)
Xanthine
XANTHINE
GUANINE
GUANOSINE
GMP
oxidase
(end product
in humans) URIC ACID (Primarily in the liver and excreted by the kidney into the urine)
Xanthine oxidase contains FAD, non-heme iron and molybdenum.

Uric acid is also an antioxidant (like Vitamin C, -tocopherol , GSH).
73
CLINICAL SIGNIFICANCE OF PURINE METABOLISM

Folic acid consists of PABA (Para Amino Benzoic Acid) as part of its structure. Sulfonamides,
the antimicrobial agents, are structural analogs of PABA that competitively inhibit folic acid
synthesis in bacteria.
Since purine synthesis pathway (de novo) requires THF
(Tetrahydrofolate) as a coenzyme, the sulfa drugs suppress this pathway (Fig. 6).
Because humans are unable to synthesize folic acid and rely on external sources for this
vitamin, sulfa drugs do not interfere with the purine synthesis in humans.
FIGURE 6
PTERIDINE PRECURSOR
COOH
H2N
p - AMINOBENZOIC ACID (PABA)

DIHYDRO PTEROATE
SYNTHETASE
SULFANILAMIDES
FOLIC ACID
SULFANILAMIDES AND ITS DERIVATIVES ARE STRUCTURAL ANALOGS OF PABA.
These drugs competitively inhibit folic acid synthesis, and thereby

decrease synthesis of critical nucleotides needed for the replication
of DNA and RNA . Do not affect human DNA or RNA
synthesis because mammalian cells cannot synthesize folic acid.
Abnormal purine metabolism ranges from mild to severe and even fatal
disorders (Table 1).
TABLE 1: CLINICAL DISORDERS-PURINE METABOLISM

DISORDER
GOUT
SITE OF ACTION
PRPP SYNTHETASE
BIOCHEMICAL TARGET CLINICAL SYMPTOMS

INCREASED ENZYME
ACTIVITY (V max up)
HYPERURICEMIA
INCREASED AFFINITY
HYPERURICEMIA
OF THE ENZYME FOR
RIBOSE-5-PHOSPHATE
(low Km for substrate)
LESCH NYHAN
SYNDROME
SCID
(Severe
Combined
Immunodeficiency
Syndrome)
LOSS OF FEED BACK

CONTROL
HYPERURICEMIA
HGPRT
PARTIALLY DEFECTIVE
ENZYME
HYPERURICEMIA
HGPRT
LACK OF ENZYME
HYPERURICEMIA
ADA
LACK OF ENZYME
HYPOURICEMIA
74
Excess uric acid accumulation leads to hyperuricemia, more commonly known as GOUT, a
condition characterized by precipitation of sodium urate crystals in the synovial fluid of the
joints that leads to severe inflammation and arthritis. Urate salts can coprecipitate with calcium
salts and form stones in kidney or bladder. The incidence of Gout (that is accompanied in all
patients by a very high level of blood uric acid) is 3/1000.
Most forms of Gout result from excess purine production due to altered activities of PRPP
About 3 individuals in 1000 suffer from hyperuricemia.--either increased synthesis
(overproduction) of purine nucleotides or impaired uric acid excretion through the kidneys.
HGPRT deficiency could lead to increase in PRPP level due to lack of its utilization in the
reaction H/G to IMP/GMP. This, in turn, causes increase in PRPP amidotransferase acitivity
through mass action. Also incomplete conversion of H or G to respective nucleotides causes
higher levels of uric acid due to increased catabolism of H/G.
Gout is treated by the antimetabolite, Allopurinol, a structural analog of Hypoxanthine, which
inhibits Xanthine oxidase, resulting in the accumulation of hypoxanthine and Xanthine that are
much more soluble than uric acid (Fig. 7). Alcohol should be avoided in gouty patients, as it
may impair uric acid excretion. Acute attack of gouty arthritis is often triggered by an alcoholic
binge. Purine-rich food (e.g caviar-fish eggs or lentils rich in nucleic acids) may also
exacerbate the condition.
FIG U R E 7
O
O
N
HN
HN
N
H
H YP OX AN TH INE
HY PO X AN TH IN E
(Com petitive
inhibitor of XO.
M etabolizes to
Ox ypurinol that
tightly binds to
XO, inactivating
it.)
N
H
A LLO P UR IN O L
XO
X AN TH IN E
XO
UR IC AC ID
Total absence of the enzyme HGPRT can lead to devastating consequences. The most
striking is the Lesch-Nyhan Syndrome, a compulsive self-mutilation behavior and mental
retardation-X-linked recessive disorder. The structural gene for HGPRT is located on X
chromosome, and the disease is a congenital, recessive, sex-linked trait manifested only in
males. The deficiency leads to a marked increase in the de novo pathway and uric acid levels
are dramatically elevated. The increase in the latter pathway is probably due to increase in
PRPP levels (not utilized in salvage pathway) that are utilized in the de novo pathway. These
explanations do not, however, account for the neurological disorders characteristic of the
disease. Formation of urate stones early in life, followed by Gout at later stage . Patients do
not normally live beyond their 20th year. Since brain is deprived of de novo pathway, absence
of salvage pathway deprives the brain of purine nucleotides, which may have caused the
10
75
mental retardation and other behavioral problems -- not caused by excess uric acid
accumulation. PRPP accumulates (underutilization) in the absence of HGPRT, resulting in
stimulation of PRPP amidotransferase and
increased production of purines and
hyperuricemia.
Severe combined immunodeficiency (SCID) is a group of related inherited disorders
characterized by the absence of an immune response to infectious disease. This results in the
inability of B and T lymphocytes to proliferate and produce antibodies in response to antigenic
stimuli. Approximately one third of the patients exhibit lack of adenosine deaminase (ADA) that
causes hypouricemia. This enzyme deficiency also implicated in other diseases, including
AIDS, anemia, and various lymphomas and leukemias. The very first clinical trial using gene
therapy technology to repair a genetic disorder was conducted on a patient with SCID due to
ADA deficiency. The recombinant version of the gene was introduced to the patient to restore
the functional enzyme. Overall, no adverse effect or toxicity.
In the absence of ADA, deoxyadenosine is not degraded, but converted to dAMP and then
into dATP , a potent feedback inhibitor of ribonucleotide reductase.
The affected patients die before 2 years of age. Lack of conversion of adenosine to inosine
results in the accumulation of dATP (see Fig. 8):
FIGURE 8
ADENOSINE KINASE
ADENOSINE
Nucleotidase
ADENOSINE
DEAMINASE
DEFICIENCY
STATE
IMP
AMP
SCID
Adenosine
deaminase
INOSINE
AMP Kinase
Purine Nucleoside
Phosphorylase
ADP
SALVAGE PATHWAY
HYPOXANTHINE
dADP
dATP
Adenosine is a poor substrate for the

phosphorylase. It should, therefore, be
deaminated first to inosine.
RIBONUCLEOTIDES
Reductase
dADP
DEOXYRIBO
NUCLEOTIDES
dAMP
DeoxyadenosineKinase
DEOXYADENOSINE
Adenosine
Deaminase
DEOXYINOSINE
B- and T-lymphocytes could accumulate as much as 50-fold more dATP than normal cells. High levels
of dATP inhibit the ribonucleotide reductase (that converts ribonucleoside diphosphates to
deoxyribonucleoside diphosphates). This causes inhibition of formation of all dNTPs (i.e., dATP, dGTP,
dCTP, dUTP) and consequently of DNA polymerase (DNA synthesis) (Fig. 9). Rapidly proliferating
cells such as lymphocytes are particularly susceptible to reduced DNA replication. Accumulated
Deoxyadenosine is particularly toxic to immature lymphocytes that fail to mature. Deoxyadenosine
derived from degradation of purine-rich diets is also accumulated in lymphocytes.
11
76
FIGURE 9
*dATP (INHIBITOR)
DEOXYRIBONUCLEOSIDE
DIPHOSPHATE
RIBONUCLEOSIDE
DIPHOSPHATE
RIBONUCLEOTIDE
REDUCTASE
* This explains the toxicity of increased levels of

dATP in Adenosine Deaminase Deficiency
It has been well established that there are three major cell types involved in acquired immunity
and complex interactions among these cell types are required for the expression the full range
of immune responses. Two of these cell types come from a common lymphoid precursor cell
but differentiate along different developmental lines. One line matures in the thymus and is
referred to as T cell; the other matures in the bone marrow and is referred to as B cell. Cells
of the B and T-lymphocyte series differ in many functional aspects but share one of the
important properties of the immune response, namely, they exhibit specificity toward an
antigen. Thus the major recognition and reaction functions of the immune response are
contained within the lymphocytes.
PURINE ANALOGS AS ANTIVIRAL AND ANTICANCER DRUGS
ARA-A (VIDARABINE): ARA-A, an antiviral agent is an analog of Adenosine. It competes
with dATP in the DNA polymerase reaction (Fig. 10).
F IG U R E 1 0
NH 2
NH 2
CH
HC
N
C
HOCH 2
H H
HO
OH
A R A -A
(V ID A R A B IN E )
( A D E N IN E - A R A B IN O S E )
A R A -A
AR A H y p o x a n t h in e
(In e f fe c t iv e )
HOCH 2
A D E N O S IN E
D E A M IN A S E
CH
HC
N
C
A R A -A M P
OH
OH
A D E N O S IN E
(A D E N IN E - R IB O S E )
A R A -A D P
A R A -A T P
d ATP
DNA
D N A P O L Y M E R AS E
A r a - a i s t a k e n u p r a p i d l y b y c e r t a in v ir u s e s s u c h a s h e r p e s s im p l e x
T y p e s 1 a n d 2 a n d v a r i c e l l a - z o s t e r . K i ll s t h e s e D N A v ir u s e s b y
in h i b it i n g D N A r e p l i c a t i o n . A t o p i c a l o p t h a l m i c p r e p a r a t io n is a l s o
e f f e c t i v e i n H e r p e s s i m p le x k e r a t it i s .
12
77
Ara-ATP is a selective inhibitor of DNA polymerases encoded by Herpes viruses. Thus Ara A
selectivley interferes with viral DNA replication. Since Ara-A is susceptible to degradation by
adenosine deaminases, its effectiveness can be increased when administered with an inhibitor
of the latter enzyme. The ratio of kinase to deaminase will determine its effectiveness.
6MP (6 Mercaptopurine)
6MP (an analog of Adenine) is a potent anticancer drug. It is converted to 6MP-ribose
phosphate (6MP-nucleotide) which in turn inhibits PRPP amidotransferase, the rate-limiting
enzyme in purine biosynthesis (Fig 11).
FIGURE 11
OH
SH
HN
C
N
N
H
HYPOXANTHINE
N
C
C
HC
N
CH
N
H
6-MERCAPTO PURINE
(6MP)
6MP RIBOSE-PHOSPHATE (6MP-RP)
6MP
HGPRT
FEEDBACK INHIBITION
OF
PURINE BIOSYNTHESIS
PRPP
PHOSPHORIBOSYLAMINE
PRPP AMIDOTRANSFERASE
AMP AND GMP SYNTHESIS ARE INHIBITED. WHEN USED ALONE, 6MP CAN
CAUSE REMISSION OF ACUTE LYMPHOCYTIC LEUKEMIA (25% IN CHILDREN
AND 10% ADULTS). IN COMBINATION WITH OTHER DRUGS, ALMOST 100%
REMISSION CAN BE ACHIEVED.
PYRIMIDINE NUCLEOTIDE BIOSYNTHESIS

de novo synthesis
The sources of the carbon and nitrogen atoms in the pyrimidine ring are glutamine, CO2, and
aspartic acid. Carbamoyl phosphate is the first key molecule, which is converted to carbamoyl
aspartate by the enzyme carbamoyl aspartate synthetase (Aspartate Trancarbamylase). Two
distinct carbamoyl Phosphate Synthetase (CPS I and CPS II) exist in the cell. CPS 1 is located
in the mitochondria that produces Carbamoyl Phosphate which is a precursor of urea. CPS 11
is a cytosolic enzyme involved in pyrimidine biosynthesis.
In animal cells, carbamoyl phosphate synthase is the rate-limiting enzyme, which is inhibited
by the product UTP, and is activated by ATP. The reactions leading to the synthesis of UMP
(uridylic acid) are depicted in Fig. 12. Unlike the purine biosynthesis, the ring structure is
13
78
assembled as a free base, not built upon PRPP. PRPP is added to the first fully formed
pyrimidine base (orotic acid) that results in the formation of OMP (orotidylic acid) which is then
decarboxylated to form UMP.
FIGURE12
CARBAMOYL PHOSPHATE
H3N
C
O
(Rate-limiting)
O
O-
-O
H2 N
CH 2
CH 2
H
C
CH
C
N
CO O O
H3 N
COOH
ASP. ACID
CARBAMOYL ASPARTATE
CO 2 + GLUT + ATP
O
CYCLIZATIO N
Oxidation
HN
3
O
C
N
COOH
OROTIC ACID
PRPP NADH+
The six activities of de novo pyrimidine

synthesis are coded by three structural
genes. The first gene codes for a large
multifunctional protein that consists of
the first three enzymes. The second
gene codes for enzyme 4 whereas the
third gene codes for enzymes 5 and 6.
1.
2.
3.
4.
5.
6.
4
HN 3
5
2 1 6
N
O
6
N
R-P
OMP
COO
CH
N
COONA O
H
DIHYDRO OROTIC ACID
O
C
PP
HN
CH 2
HN
CO
R-P
UMP
CARBAMOYL PHOSPHATE SYNTHETASE II (CSPII)

ASPARTATE TRANSCARBAMYLASE (Carbamoyl Aspartate Synthatase)
DIHYDRO-OROTASE
DIHYDRO-OROTATE DEHYDROGENASE
OROTATE PHOSPHORIBOSYL TRANSFERASE
OROTIDINE-5-MONOPHOSPHATE DECARBOXYLASE
INTERCONVERSION OF PYRIMIDINE NUCLEOTIDES

UTP can be converted to CTP by CTP synthetase (NH2 group provided by glutamine).
UMP can also be converted to TMP by first converting to UDP by UDP kinase, then to dUDP
by ribonucleotide reductase, to dUMP by nucleotidase (nucleotidase removes one phosphate
at a time from a nucleotide). (Examples: UTP to UDP to UMP to Uridine; dUTP to dUDP to
dUMP to deoxyuridine) dUMP is then converted to dTMP by the enzyme thymidylate
synthetase (Fig. 13).
14
79
FIGURE 13
O
HN
(UMP)
N
R- P
UDP
dUDP
dUMP
THYMIDYLATE
SYNTHETASE
(CH 3 group donated by
O Methylene Tetrahydrofolate )
UTP
GLUTAMINE
CTP SYNTHETASE
(SYNTHASE)
NH 2
CH3
HN
O
O
d R- P
TMP
(5 METHYL UMP)
OR THYMIDYLIC ACID
R- P
CTP
SALVAGE PATHWAYS FOR PYRIMIDINE SYNTHESIS

Unlike purine salvage pathway, PRPP is not involved in direct conversion of pyrimidine base to nucleotide.
Thymine is converted to thymidine by nucleoside phosphorylase, then to thymidylic acid (dTMP) by thymidine
kinase. Similarly, deoxyuridine is converted to dUMP (deoxyuridylic acid) by deoxyuridine kinase, and then to
dTMP by thymidylate synthetase (Fig. 14). Uracil and cytosine (like thymine) can be converted to uridine and
cytidine, respectively, by nucleoside phosphorylase. (This enzyme converts a base such as uracil to uridine. )
FIG URE 14
S AL V AGE PA T H W AYS FOR PYRIMID IN E BIO SYT HESIS

+
Pyrim id in e
phosp ho rylase
T HYMINE
Thymidine
kin ase
dT MP
TH YM IDINE
DEOXY URID INE
Deo xyuridine
kinase
dUMP
Thym idylate
synth etase
THE THY M IDINE KIN AS E AC TIV IT Y FL UC TU ATE S W IT H T HE CE L L C Y CLE ,

INC RE AS IN G T O M AX IM AL L E V E L D URIN G THE AC TIV E D NA S Y N TH ET IC PH AS E .
D IRE CT CO NV E RS IO N OF P Y RIM ID INE T O N UCL E O TIDE S B Y
P RP P IS RE L ATIVE L Y INS IG NIFIC ANT .
U RA CIL
U RID INE
CY TO S INE
CY T IDIN E
UMP
UDP
CM P
CD P
dU DP
dCD P
dUM P
d TMP
d CM P
+ C to U appear to use same nucleoside phosphorylase w hereas T uses its ow n enzyme. All three nucleosides
(Cytidine, Uridine, and Th ym idine) use their specific kinases to convert them to nucleotides.
15
80
Since it is a reversible enzyme, it can also convert uridine back to uracil.) Uridine and Cytidine are then converted
to UMP and CMP, respectively, by nucleoside kinase (nucleoside kinase participates in a reaction opposite to the
reaction catalyzed by the nucleotidase, i.e. adds a phosphate to nucleoside rather than removing a
phosphate from a nucleotide).
PYRIMIDINE CATABOLISM
In contrast to the purine catabolism where the ring structure is not cleaved (uric acid has a ring
structure), the pyrimidine ring can be opened and degraded to highly soluble structures such
as -alanine and -aminoisobutyrate (Fig. 15).
FIGURE 15
THYMINE
Dihydropyrimidine
CYTOSINE Dehydrogenase*
Dihydropyrimidine
Dehydrogenase*
DIHYDROTHYMINE
URACIL
-AMINO ISOBUTYRATE
DIHYDROURACIL
METHYL MALONYL
Co A
MALONLYL
CoA
- ALANINE
SUCCINYL CoA
ACETYL-CoA
TCA CYCLE
FATTY
ACID
SYNTHESIS
TCA
PYRIMIDINE CATABOLISM
*A defect in this enzyme will result in uracil and thymine accumulation.
5FU will be toxic at higher concentrations, specifically neurological toxicity.
REACTIONS OF PYRIMIDINE BIOSYNTHESIS As Anticancer Drug Targets

5-fluorouracil (a potent anticancer drug that is used against a variety of solid tumors) inhibits
the enzyme thymidylate synthetase.( Fig. 16A,16B) Methotrexate, another potent anticancer
drug, inhibits the enzyme dihydrofolate reductase that is involved in the synthesis of methylene
tetrahydrofolate, the CH3 donor for the enzyme thymidylate synthetase (Fig. 16A, 16C).
16
81
FIGURE 16A
INHIBITION OF REACTIONS IN THE PYRIMIDINE BIOSYNTHESIS PATHWAY

BY THREE ANTICANCER DRUGS
5FU AND METHOTREXATE

dTMP
dUMP
THYMIDYLATE
SYNTHETASE
DIHYDROFOLATE
(DHF)
N5 , N10
METHYLENE
TETRAHYDROFOLATE
DHF
REDUCTASE
METHOTREXATE
(METHYL DONOR)
TETRAHYDROFOLATE (THF)
FIGURE 16B
RIBONUCLEOTIDE
REDUCTASE
5FU
5FUMP 5FUDP 5FdUDP
URIDINE
KINASE
NUCLEOTIDASE
dUMP
5FdUMP
TMP
THYMIDYLATE
SYNTHASE
17
82
NH2
FIGURE 16C
C NH CH CH2 CH2 C OH
CH2 NH
N
N
C OH
O
OH
Folic Acid
N
NH2
CH3
C NH CH CH2CH2 C OH
CH2 N
N
N
C OH
O
NH2
Methotrexate
THF
DIHYDROFOLATE
FOLIC ACID
DHF
Reductase
METHYLENE THF
DHF
Reductase
(DHF)
Ara-C (where ribose in Cytidine is changed to Arabinose). Ara-C is converted to Ara-CTP

by the same enzymes that direct cytosine to CTP conversion. Ara-CTP acts as competitive
inhibitor
(with
respect
of
dCTP)
of
DNA
polymerase
(Fig
16
D).
FIGURE 16
NH 2
NH 2
ARA- C
O
N
HOH 2 C
H
HOH 2 C
H
OH
OH
H
H
ARA(CYTARABINE)
CYTOSINE - ARABINOSE
ARA-U
(ineffective ; not
incorporated
into DNA)
CYTIDINE
DEAMINASE
ARA-
ARA-CMP
H
H
OH
OH
CYTOSINE
CYTOSINE - RIBOSE (CYTIDINE)
ARAdCT
ARA-CTP
DN
DNA POLYMERASE
USED IN THE TREATMENT OF ACUTE MYELOGENOUS LEUKEMIA
(IN COMBINATION WITH OTHER ANTICANCER DRUGS) AND
OF NON-HODGKIN'S LYMPHOMAS
18
83
CLINICAL DISORDER OF PYRIMIDINE METABOLISM

Orotic aciduria
Low activities of orotate phosphoribosyl transferase and OMP decarboxylase lead to orotic
aciduria, causing abnormal growth, megaloblastic anemia, and the excretion of large amounts
of orotate in the urine. A diet rich in uridine improved the anemia and reduced orotate
excretion, because uridine is converted to UMP
19
84
MED 1 The Cell Block, Division 2

Lecture: Mechanisms of Cell Injury
12/13/11 10:30 11:30
Charles L. Hitchcock, M.D., Ph.D., Department of Pathology
Heart and Lung Research Institute, Room 081, 473 W. 12th Ave
Phone: Secretary 247-7485, charles.hitchcock@osumc.edu
1. Compare and contrast the factors that influence how a cell responds to injury.
2. Compare and contrast the mechanisms of cell injury from too little oxygen (i.e.
hypoxia/ischemia) and too much oxygen free radicals (i.e. reactive oxygen species)
a. Define the sequence of morphologic changes in cells subjected to hypoxic/ischemic
injury.
b. Define how oxygen free radicals are generated and eliminated by cells, and their impact
of cellular structures.
3. Compare and contrast mechanisms of cell death arising from altered plasma membrane
permeability and injury to mitochondria.
Learning Resource:
Pathologic Basis of Disease (PBD), 8th Ed, Chapter 1
I.
MECHANISMS OF CELL INJURY

A. Overview
Insulting
Agent
Cell
Injury
Cell
Response
Disease
State
Disease
Treatment
1. Pathology is the scientific study of disease process. This process involves the
sequential and cumulative changes in an organ that passes through sequential
stages from normal to abnormal.
2. Etiology of a disease (Insulting Agent) is the initiating event and its related risk
factors. Understanding the etiology leads to prevention.
3. Pathogenesis of disease is the mechanisms involved in the transition from normal to
abnormal (Cell Injury and Cell Response) as well as the actual structural and
functional changes occurring in the affected tissue. AS cell injury is common to all
pathologic processes, an understanding of the mechanisms of disease allows us to
develop means to interrupt the disease process.
4. Clinical expression of the disease (Disease State and Disease Treatment) includes
functional and morphologic abnormalities, as well as treatment. The clinical signs
and symptoms are several steps removed morphologic changes that are preceded
by the biochemical changes associated with cell injury.
85
B. Objective 1
1. Cellular response to injury is dependent upon the cells physiology, the nature of the
injurious stimulus, and the strength and duration of the injury.
2. The role of cell physiology is best demonstrated in the liver where the cells differ in
their physiology due to the changes in the content of the blood that flows past them.
a. Differences in drug metabolism in these hepatocytes will determine the extent of
cellular injury when exposed to a toxic substance (eg. acetaminophen overdose).
3. Numerous types of agents can cause cell injury.
a. Oxygen deprivation (hypoxia/ischemia) due to decreased or loss of blood flow,
anemia
b. Chemicals / drugs - tobacco, alcohol, poisons, Rx/OTC drugs
c. Physical injury - trauma, electricity, pollutants, burns, UV light, radiation
d. Infectious agents - bacteria, viruses, fungi, and parasites
e. Immune response - allergies and autoimmune disease
f. Nutritional imbalance - malnutrition, vitamin and essential nutrient deficiencies,
obesity
g. Genetic derangement chromosome abnormalities, gene mutations
4. Cell injury that is reversible is usually associated with an acute injury or low intensity.
5.
Chronic cell stress in the form of altered physiologic stimuli results in cellular
adaptations involving growth and differentiation.
6. Metabolic stress can lead to cellular accumulation of water, lipids, proteins or

carbohydrates. These changes are often reversible if the stress is relieved.
7. Cell injury that leads to cell death apoptosis or necrosis - is most often acute and of
high intensity.
C. Objective 2a Hypoxic/Ischemic Injury
1. Overview
a. Cell injury results from a disruption of one or more of the cellular components
that maintain cell viability including cell membranes, mitochondria, cellular
proteins, and DNA.
2. Various insulting agents can act at the same point on the cell to induce cell injury or
they can act at multiple cellular sites.
3. Hypoxia Ischemia Model Reduced ATP Production
a. Causes of reduced oxygen to cells include:
1) Ischemia, decreased blood flow, is the most common cause of decreased
oxygen reaching cells and is the prototypic mechanism of cell injury.
2) Hypoxia, reduced availability of O2 to cells, can be caused by:
a) Anemia - impaired absorption of O2
b) Leukemia malignancy of the disease of blood RBC mass
c) Vasculitis inflammation of blood vessels
d) Toxins/drugs e.g. CO
86
e) Shock - decreased blood flow O2 to cells

b. Selective permeability of the plasma membrane involves a variety of transporters
and channels.
1) Active transport of Na+/K+ ions against a concentration gradient utilizes ATPdependent ion pumps embedded within the membrane.
2) The ATP-dependent Na+/K+ Pump normally functions to transports 3 Na+/ions
out the cell and 2 K+ into the cell to provide the energy needed to capture and
the ions on the other side of the membrane.
3) This maintains differences in intracellular and extracellular ion concentration
and electrical charge.
4) ATP changes membrane permeability due to impaired Na+/K+ Pump
function.
1) a net in cytoplasmic concentrations of Na+
2) a net cytoplasmic K+ .
3) an iso-osmotic gain in water that results in cell and organelle swelling
5) Cell swelling, or hydropic change, is common seen in the renal tubules in
patients with shock. It is reversible if the cause of the shock is corrected. It
can also be seen in myocardial infarction where there is a zone of hydropic
myocytes at the periphery of the zone of cell death (necrosis).
c. ATP leads to anaerobic glycolysis resulting in lactic acid and production of
inorganic phosphates resulting in intracellular pH, also the release of H+ from
the mitochondria.
1) Acidic pH induces chromatin clumping and enzyme function.
d. ATP leads to decreased protein synthesis via swelling of the endoplasmic
reticum and detachment of ribosomes.
1) Protein misfolding due to various mechanisms followed by proteins
destruction within cytoplasmic and nuclear proteasomes.
D. Objective 2b
1. Normal cell respiration reduction of oxygen to water - generates a small amount of
partially reduced forms of oxygen in the form of superoxide, hydrogen peroxide and
hydroxyl radicals, or reactive oxygen species (ROS).
a. The reactive oxygen species are partially reduced intermediates produced during
normal cellular respiration where O2 is reduced by the transfer for four electrons
to H2 to form two H2O. Various oxidases located within the mitochondria,
endoplasmic reticulum, peroxisomes, and cytoplasm
b. ROS generation is common to all cells and is essential.
1) Thyroid follicular cells must make hydrogen peroxide in order to attach iodine
atoms to thyroglobulin in the synthesis of thyroxin.
87
2) Macrophages and neutrophils generate ROS in their lysosomes that merge

with phagosomes in order to kill ingested organisms alone or in the form of
hypochlorite ion (only in neutrophils).
c. Superoxide (O2) a one electron intermediate- is produced in the electron
transport chain in the mitochondria and in the cytosol, and exhibits little diffusion
and is quickly inactivated by superoxide dismutase (SOD).
d. Hydrogen peroxide (H2O2) two electron intermediate - is produced by autooxidation in mitochondria and by oxidases in peroxisomes from superoxide via
the activity of superoxide dismutase. It diffuses throughout the cell and is
converted to a free radical via the Fenton reaction.
e. Hydroxyl radicals (OH) three electron intermediate- are generated by
hydrolysis of water by ionizing radiation and from H2O2 by the Fenton reaction
that utilizes transitional metals such as Fe++ or Cu++.
2. ROS induced oxidative stress of cells is a major cause of the morbidity and
mortality associated with a wide number of pathologic processes that include:
a. Inflammation
b. Oxygen therapy
c. Reperfusion injury
d. Ionizing radiation
e. Chemical
f. Aging
3. ROS induced cellular damage is seen at the level of membrane fatty acids, proteins,
and DNA.
a. Peroxidation of membrane lipids, either free fatty acids or glycolipids, is an
autocatalytic process that involves several steps that alters membrane fluidity
and thus normal structure and function.
Initiation: R-HC=CH + OH R-HC=C + H2O
Propagation: HC=C + O2 R-HC=COO + RH HC=C + R-COOH
Termination: L + L L-L non-radical product
b. ROS induces fragmentation of cytoplasmic and membrane proteins by oxidation
of amino acids, formation of disulfide bonds (S-S), and oxidation of the protein
backbone.
1) Alters plasma membrane fluidity and function.
2) Alters the structure and function of the cytoskeletal proteins and of nonstructural proteins in the cytoplasm.
3) Increases the degradation of proteins by proteasomes
c. ROS cause oxidative damage of nuclear and mitochondrial DNA reaction,

primarily with thymidine and quanine residues, to induce single strand breaks
88
The resulting mutations can accumulate during life and/or result in a neoplastic
transformation of cells.
4. Antioxidants located in the in the serum and in cells inactivate ROS.
a. Extracellular compounds and elements have antioxidant properties.
1) Vitamins E, C, and A
2) Serum proteins that reduce the levels of free iron (transferrin, ferritin) and
copper (ceruloplasmin) needed to catalyze the formation of ROS
b. Superoxide dismutase (SOD) in the cytosol and mitochondria converts
superoxide to hydrogen peroxide.
c.
Hydrogen peroxide is converted to water by catalase in peroxisomes.
d. Glutathione peroxidase is the major intracellular ROS detoxification system, and

occurs primarily in the mitochondria. It converts both hydroxyl free radicals and
hydrogen peroxide to water.
i. Reduction in the cells ability to generate NADPH impairs the detoxification.
The best example of this is seen in G-6PD Deficiency an X-linked recessive
deficiency in the enzyme glucose-6 phosphate dehydrogenase in the
monophosphate shunt, especially in red blood cells.
ii. Glucose-6-phosphate dehydrogenase (G6DP) converts glucose-6-phosphate
into 6-phopshoglcuon--lactone which converts NADP to NADPH which is
then used in glutathione reduction.
iii. 6-phopshoglcuon-lactonase converts 6-phopshoglcuon--lactone which is
converted into 6-P-gluconate which is converted into Ribsoe-5-P and CO2 by
the action of phosphogluconate dehydrogenase. This reaction converts
NADP to NADPH which is then used in glutathione reduction which is then
converted into ribulose-5-phosphate.
iv. Glucose-6-dehydrogenase deficiency, is X-linked and one of the most
common enzyme deficiencies. Oxidative stress in these patients, often in the
form of infection treated with a sulfonamide containing antibiotic, induces a
hemolytic anemia in RBCs that are deficient in G6PD. The inability to
metabolize ROS leads to aggregation of hemoglobin (Heinz bodies) in RBCs.
E. Objective 3
1. Loss of calcium ion homeostasis is associated with ischemia and toxins that induced
alter membrane permeability and release of sequestered calcium ions in the cell,
mainly within the mitochondria.
2. The extracellular [Ca+2] is 104 times as great as the intracellular [Ca+2].
a. Ca+2 ions are sequestered in the cytoplasm, mitochondria and endoplasmic
reticulum.
b. Cell injury releases Ca+2 from storage depots and allows for increased diffusion
through the injured plasma membrane.
c. intracellular Ca+2 activates degradative enzymes causing auto-digestion.
89
1) Phospholipases leads to phospholipids synthesis that induces

mitochondrial permeability transition and digests phospholipids in all
membranes.
2) Proteases that breakdown cytoskeletal proteins that anchor the plasma
membrane to cytoplasmic constituents.
3) ATPase that breaks down ATP leading to decreased available energy.
4) Endonucleases that digest DNA and chromatin.
5) Induction of apoptosis.
3. Direct injury of the plasma membrane can occur during both an antibody-mediated
and a cell-mediated immune response.
a. Complement fixation leads to holes in the membrane of the target cell due to
formation of the membrane attack complex (MAC).
b. Cytotoxic T lymphocytes (CTL) release perforin that form a pore (hole) in the
membrane of the attached target cell. CTL release of granzyme induces target
cell apoptosis.
c. Cell membrane rupture is a common event with the release of viral particles from
an infected cell.
d. Membrane fluidity and thus membrane function can be altered by drugs.
4. Injury of the mitochondria results in a wide variety of events that include:
a. decrease in ATP production.
b. release of H+ ions contributes to acidosis.
c. release of stored Ca+2 ions that contribute to enzyme activation.
d. release of Cytochrome c and Apaf-1 pro-apoptosis factors.
90
Structure of Nucleic Acids

Dr. Lai-Chu Wu, D. Phil.
Davis Medical Center, Room S2077
Office Phone: 293-3042
e-mail: Laichu.wu@osumc.edu
Learning objectives:
Name the building blocks of nucleotides and nucleic acids.
Define seminal experiments showing DNA as the genetic materials.
Associate DNA double helix structure with faithful flow of genetic information.
Define DNA packaging of chromosomes, associate levels of DNA folding with specific
chromatin associated proteins, and how chromatin structure regulates gene expression.
Learning Resources:
Marks Basic Medical Biochemistry: A Clinical Approach, 2nd Edition, Chapter 12, pgs 207-215.
Slide 3
Slide 4
Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA)
Two types of nucleic acids:
o deoxyribonucleic acid (DNA), is the genetic materials for most organisms, including
human
o ribonucleic acids (RNA), some organisms use RNA as genetic materials
In eukaryotes DNA is located in the cell nucleus (prokaryotes do not have nuclei).
Human DNAs are linear molecules, and haploid genome contains 3 billion base pairs.
Human mitochondrion also has its own genome, circular molecule of 16,569 bp.
DNA and RNA are polymers of nucleotides.
Nucleoside = a base + a sugar.
Nucleotide = a base + a sugar + 1 to 3 phosphates = nucleoside + phosphate(s).
DNA and RNA are differed by:
o sugar (deoxyribose/ribose)
o base (thymine/uracil).
DNA contains phosphate, deoxyribose, pyrimdines and purines
RNA contains phosphate, ribose, pyrimdines and purines
Biochemistry of nucleotides
Sugar of DNA and RNA
Slide 5
Sugars of DNA and RNA
91
Pentose; 5 five-carbon ring

Carbon atoms in sugars are numbered with
primes () to differentiate the numbering
from the bases.
Ribose in RNA; C2, X = OH.
2-deoxy-ribose in DNA, C2, X = H.
The different of the sugar moiety makes
DNA more resistant than RNA to alkaline
hydrolysis.
Slide 6
Bases: Purine and Pyrimidine

Four principal bases in DNA or RNA.
Bases are heterocyclic rings of carbon and
nitrogen atoms.
There are two classes of bases:
Purines: 2 rings; a six-member ring and a
five-member ring; carbon or nitrogen atoms
are numbered anticlockwise in the sixmember ring and clockwise in the fivemember ring; and consist of adenine (A)
and guanine (G).
Pyrimidines: a six-member ring; atoms are
numbered clockwise; and consist of
cytosine (C), and thymine (T) in DNA and
uracil (U) in RNA.
Bases
Slide 7
Naming nucleosides and nucleotides

For purine bases, their nucleosides ended
with sine.
o Adenosine and guanosine
For pyrimidine bases, their nucleosides
ended with dine.
o Cytidine, thymidine, uridine
DNA has the prefix deoxyriboo Example, deoxyriboadenosine
RNA no prefix
o Example, adenosine but not
riboadenosine
For nucleotide, adds the number of
phosphate(s).
Example, adenosine diphosphate (ADP)
Nucleotide and Nucleoside
Slide 8
92
Slide 9
Polynucleotide chain of DNA

Nucleic acids are polymers of nucleotides.
Base is linked to sugar by glycosidic bonds.
Nucleotides are joined by a phosphodiester
linkage between 5' and 3' carbon atoms of the
sugar rings.
The 5' end of a polynucleotide chain, will have
a free ___________ group and the 3' end, a free
___________ group.
The linear sequence of nucleotides in a
polynucleotide chain is abbreviated by a oneletter code, and the sequence is always read
from the 5' end. In the example illustrated the
sequence is 5-A-C-G-T-3 or pApCpGpT.
Polynucleotide chain of DNA

glycosidic
bond
Slide 10
Chemotherapy using nucleotide analogues

Zidovudine (ZDV = AZT) is an analogue
of thymidine in which the 3-OH is
replaced by an azido group, and hence
blocks DNA synthesis.
Examples of chemotherapy using nucleotide analogues

A.
B.
antimetabolite
Block DNA synthesis
10
Fluorouracil (5-FU) is a drug that is

used to treat cancer, and belongs to a
family of drugs called anti-metabolites.
5-FU is converted into F-dUMP which
binds to thymine synthase, blocking DNA
synthesis by preventing the formation of
dTMP from dUMP.
Complementary base pairing

The shape and chemical structure of the
bases allow hydrogen bonds to form
efficiently only between A and T, and
between G and C, without distorting the
DNA double helix.
Two hydrogen bonds between A and T,
while three bonds between G and C.
The base pairing of the nucleotides between the two
strands of DNA, called ________________,
dictates the nucleotide sequence of RNA in
transcription and new DNA strand during
________________.
Slide 11
Complementary base pairing of
nucleotides
11
New nucleotides are added to a growing

DNA chain at the 3 OH position.
93
Slide 12
DNA structure: two anti-parallel strands

Nucleotides are covalently linked together
into polynucleotide chains with a sugar
phosphate backbone from which the bases
(A, C, G, and T) extend.
A DNA molecule is composed of two
polynucleotide chains (DNA strands) held
together by hydrogen bonds between the
paired bases.
The sugar phosphate backbone is located at
the outside, whereas the bases are located
of the DNA chains.
DNA has polarities - the two strands run
antiparallel to each other.
DNA is double helix made of two

anti-parallel strands.
12
Slide 13
DNA sequence and complementation
5-GATCTGTGCTAGTG-3
or pGpApTpCpTpGpTpGpCpTpApGpTpG
5-GATCTGTGCTAGTG-3
3-CTAGACACGATCAC-5
pGpApTpCpTpGpTpGpCpTpApGpTpG
pCpApCpTpApGpCpApCpApGpApTpC
13
Slide 14
DNA structure- double helix

Left, DNA is shown straightened out.
Right, in reality, DNA is wound into a
right-handed double helix.
The double helix DNA model was
proposed by Watson and Crick (1953),
which conforms to known properties of
DNA including X-ray diffraction data and
the fact that DNA samples from different
cells of the same species have the same
proportions of the four bases.
For examples: human DNA: 30% A & T;
20% G & C; E. coli: 24% A & T; 26% G &
C. The data suggest that the bases occur in
pairs.
DNA double helix

Watson and Crick (1953)
0.34 nm
14
94
Slide 15
Three forms of DNA: A, B, & Z

B form DNA: a right-handed helix, 3.4
between base pairs, 10.4 base pairs per
turn, and predominates in vivo.
A form: predominates in DNA-RNA
hybrids, and is more compact (2.3
between base pairs and 11 base pairs per
turn).
Z form: a left-handed helix, 3.8 between
base pairs and 12 base pairs per turn in Z
DNA. This form of the helix was
designated Z because, in each strand, a
line connecting the phosphates zigs and
zags.
Three forms of DNA : A, B, & Z

Left handed helix
right handed helices
Zig-zag
12 bases/turn
Found in regions
of active
transcription
10.4 bases/turn
predominant
11 bases/turn
DNA-RNA hybrids
15
Slide 16
DNA structure- faithful transmission of information

during DNA replication
The DNA model accounts for the storage
and faithful transfer of genetic information.
DNA replication or DNA synthesis is the
process of copying a double stranded DNA
strand in a cell, prior to cell division, or in
other words, the transfer of genetic
information from DNA into DNA.
Write the complementary sequence:
5-ATAGCTGATCGATGCTAG-3
The DNA model accounts for the faithful

transfer of genetic information during DNA
replication.
Replication
16
Slide 17
Complementary DNA sequence:
5-AATCCGATGCTGTGAT-3
3-TTAGGCTACGACACTA-5
17
95
Slide 18
DNA structure- faithful transmission of

information during transcription
Transcription is the process through
which a DNA sequence is enzymatically
copied by an RNA polymerase to produce a
complementary RNA, or in other words,
the transfer of genetic information from
DNA into RNA.
Write the RNA sequence for the DNA after
transcription:
The DNA model accounts for the faithful

transfer of genetic information during
transcription.
18
5-ATAGCTGATCGATGCTAG-3
Slide 19
Complementary RNA sequence
5-AATCCGATGCTGTGAT-3
3-UUAGGCUACGACACUA-5
19
Seminal experiments showing DNA as the genetic materials

Griffith identified transforming principal
Slide 20
Mouse lives when injected with heat killed
pathogenic S strain or non-lethal R strain
individually, but died when they are mixed.
Griffith concluded that a substance
(transforming principal) is present in the S
strain which transformed the nonlethal R strain
into deadly S strain.
Avery, MacLeod, and McCarty demonstrated DNA
is the genetic material
Test individual macromolecules (RNA,
protein, DMA, lipid and carbohydrate)
from the virulent S strain to transform the
R strain
Identified DNA as the transforming
principle that would permanently change
R- strain into the lethal S strain.
Providing the first evidence that DNA could serve
as the genetic material.
Erwin Griffith (1928) transformation
principal
Avery-Macleod-McCarty (1944) DNA = genetic

material
20
Figure 4-2 Molecular Biology of the Cell ( Garland Science 2008)
96
Slide 21
Hershey and Chase demonstrated DNA is the

genetic material.
Hershey & Chase (1952) label DNA with 32P and proteins with 35S to demonstrate DNA
was present in the progenies of viruses and hence was the genetic material.
21
Slide 22
Karyogram - Giemsa staining of human

chromosomes.
A human somatic cell contains pairs of
somatic chromosomes (22) plus two sex
chromosomes - 2 X chromosomes (female),
and X and Y chromosomes (male).
Somatic chromosomes are numbered in
approximate order of size.
These patterns are obtained by staining
chromosomes with Giemsa stain, which
produces dark bands in regions rich in A-T
nucleotide pairs.
Chromosomes contain telomeres,
centromeres, and are divided into short arm
(petit, p) and long arm (queue, q).
telomeres
Petit, p arm
Short arm
centromere
Quene
Q arm
Long arm
22
Sex chr
Slide 23
Bacteriophage contains only 2 macromolecules:

Proteins & DNA.
Virus injected its genetic material into a
bacterium.
32
Label DNA in one batch of viruses with P and
35
protein in a second batch with S.
These labeled viruses infected E. coli, and the
mixture disrupted by brief pulsing in a Waring
blender to separate the infected bacteria from
the empty viral heads.
32
P -labeled DNA entered the bacterial cells,
35
while most of the S-labeled proteins remained
in solution with the empty viral particles.
Demonstrated DNA as the genetic materials
DNA packaging
The DNA in each of our cells is ~1.8 meter long.

A cell nucleus is ~6 mM in diameter.
DNA needs to be compacted ~10,000-fold to fit inside our cells.
Packaging of DNA needs to be orderly and regulated for the proper control of cellspecific gene expression.
DNA associates with proteins to form a complex known as chromatin.
Chromatin forms several hierarchical levels of folding to achieve necessary
compaction.
Chromatin has a profound influence on cellular processes that involve access to
chromosomal DNA (transcription, replication, recombination, and repair).
97
Slide 24
DNA Packaging
0.34 nm x 3 x 109 x 2
= 1.8 m
24
The first level of DNA packaging: Nucleosomes

DNA (146 base pairs) wraps around the histone
octamer (2 copies each of H2A, H2B, H3 and
H4), making 1 and 3/4 turns around the protein
complex. The octamer plus the DNA comprise
what is called the nucleosome core.
A small stretch of DNA (60 bp) runs between
adjacent nucleosome cores, and is known as the
linker, where H1/H5 binds.
A single nucleosome consists of one histone
core plus a linker. The total amount of DNA
involved in a single nucleosome is
approximately 206 bp.
Slide 25
25
Figure 4-23 (part 1 of 2) Molecular Biology of the Cell ( Garland Science 2008)
Slide 26

Slide 27
H1
Histone octamer =
2X (H2A, H2B, H3
and H4)
26
DNA (146 bp) wraps around

the histone octamer, making 1
3/4 turns around the protein
complex.
nucleosome core = octamer
plus DNA.
linker = a small stretch of
DNA (60 bp) runs between
adjacent nucleosome cores, and
binds by histone H1.
nucleosome = one
nucleosome core plus a linker.
The total amount of DNA
involved in a single
nucleosome is approximately
206 bp.
27
98
Slide 28
Slide 29
Histones
Regulation of chromatin structure by posttranslational modification of histones
Most abundant and most tightly associated

chromatin proteins.
Small and highly basic proteins
Divided into two classes:
histone tails
DNA
Core histones: H2A, H2B, H3 and H4

Linker histones: H1/5
Domain Structure
Globular core with histone fold
Unstructured N-terminal tails
histones
28
29
Histone acetylation and transcription
Slide 30
Histone acetylation
Fig. 16.9. Histone acetylation. HAC,

histone acetylase; HDAC, histone
deacetylase.
35
Sugar phosphate backbone of DNA is

negatively charged.
Histone acetylation (negative charge)
occurs on lysine residues, neutralizing their
positive charge, which changes electrostatic
interactions between histones and DNA.
Increased acetylation - decreased histone
DNA interaction - chromatin open structure
increased transcription
Decreased acetylation - chromatin close
configuration - decreased transcription
Slide 31
DNA packaging: 30 nm fibers
The second level of DNA packaging: 30 nM Filaments
DNA packaging involves many levels:

nucleosome, 30 nm fibers, and several
hierarchical levels of folding.
H1 molecules interact with each other,
causing the chromatin to form a spiral, with
6 to 8 nucleosomes per turn of the spiral.
This structure is known as a solenoid or 30
nm chromatin fiber
The 30 nm fiber is folded up further into
loops to make metaphase chromosomes.
Chromatin forms several hierarchical levels of
folding to achieve necessary compaction:
nucleosome, chromatin fibers, looped domains,
heterochromatins.
31
99
Slide 32
Slide 33
Formation of 30 nM Filaments
Formation of 30 nM Filaments
In vivo
Histone H1 phosphorylation is temporally
correlated with mitotic chromosome condensation.
Phosphorylation of H3 serine 10 is correlated with
chromosome condensation.
In vitro
Mutation of serine 10 in Tetrahymena leads to defects

in chromosome condensation and segregation.
Formation of 30 nM filaments is a reversible process that is modulated

by salt and Mg++ concentration.
Requires the core histone N-terminal tails.
Inhibited by tail acetylation.
32
30 nM filaments are stabilized by presence of H1.
Slide 34
33
Structural Maintenance of
Chromosomes (SMC) Proteins
Formation of higher order structures
Slide 35
ATPases that form the core of two distinct

complexes.
Condensin
Cohesin
Major protein components in the scaffold:
topoisomerase II, nuclease that prevent DNA tangling
Structural Maintenance of Chromosomes (SMC) proteins
34
35
Slide 36
Slide 37
condensin
Cohesines
Involved in chromosome condensation
Help hold sister chromatids together
36
37
100
10
Slide 38
Chromatin Proteins
Chromatin Is Heterogeneous and Dynamic
Slide 39
Chromatin structure varies both spatially and temporally.

Spatial
heterochromatin
Euchromatin
decondensed
transcriptionally competent
Heterochromatin
constitutively condensed
transcriptionally repressed
Temporal
euchromatin
Interphase: decondensed
Histones, class of proteins most tightly

associated with DNA.
Proteins such as high mobility group
proteins, topoisomerases, polymerases and
transcription factors are less tightly
associated with DNA.
SMC proteins are involved in higher-order
chromosome organization and dynamics
Metaphase: condensed
38
Slide 40
39
Summary
A nucleotide is composed of a base, a sugar and one to three phosphates.

Nucleotides are building blocks of DNA (RNA). DNA is a polymer of nucleotides.
DNA is the genetic materials of most organisms.
DNA has a double helix structure allowing the faithful transmission of genetic
information in transcription and DNA replication.
DNA in found in the nucleus and mitochondria. Nuclear chromosomes are linear
DNA; whereas mitochondrial DNAs are circular
DNA is packaged into chromatin by histone and chromatin proteins, and has many
levels of DNA folding, allowing condensation into the nucleus and gene expression
regulation.
Modifications of histones regulates gene expression and DNA metabolism
Sample Questions:
101
11
DNA Replication
Dr. Lai-Chu Wu, D. Phil.
Davis Medical Center, Room S2077
Office Phone: 293-3042
e-mail: Laichu.wu@osumc.edu
Define the mode of DNA replication.
Name the proteins involved in DNA replication.
Differentiate and compare the mechanisms by which the leading DNA strand and lagging DNA
strand are synthesized.
Solve the problem of replicating ends of linear DNAs.
DNA replication or DNA synthesis is the process
of copying a double stranded DNA strand.
Since DNA strands are antiparallel and
complementary, each strand serves as a
template for the reproduction of the
opposite strand.
The template strand is preserved as a whole
piece and the new strand is assembled from
nucleotide triphosphates.
This mode is called semiconservative replication.
Slide 43
DNA replication - overview
43
Slide 44
Three possible mode of DNA replication:

Conservative replication would leave the original
DNA molecule intact and generate a completely
new molecule.
Dispersive replication would produce two DNA
molecules with sections of both old and new DNA
interspersed along each strand.
Semi-conservative means that each DNA strand
(parent) serves as a template for the synthesis of a
new complementary strand (daughter), producing
two identical DNA molecules, each with one new
strand and one old strand.
Mode of DNA replication
44
102
12
Slide 45
Meselsen and Stahls Experiment showed that DNA

replication is semi-conservative - in the progeny
molecule one strand is "old" and another is "new".
Nitrogen is a component of nucleotide bases

and incorporated into DNA
Progeny of 1st
DNA replication
Progeny of 2nd
DNA replication
45
Slide 46
E. coli cells grown in medium containing

nutrients with an isotope of nitrogen, 15N,
produce macromolecules (including DNA)
that have this isotope incorporated into
them.
Because 15N is heavier (one additional
neutron) than 14N, the 15N molecules will
have a higher density that those containing
14N. Their strategy was to allow the
bacteria to grow in 15N or "heavy" medium
for several generations and then to shift
them to 14N or "light" medium.
Meselsen and Stahls Experiment:
Heavy DNA (N15) and light DNA (N14) can be

separated by gradient ultracentrifugation
46
Meselsen and Stahls Experiment:

15
E. coli grew in N medium has DNA heavier
14
that those grew in N medium.
15
After growing in N medium for several
14
generations, bacteria were shifted to N
medium.
DNA was extracted and subjected to
ultracentrifuge in a solution of CsCl, which
formed a density gradient.
The formation of an intermediate DNA species
in the first generation ruled out the conservative
model.
The formation of light and hybrid DNA strands
of the second progeny showed that DNA
replication is semiconservative.
Slide 47
At various times samples were taken, DNA

extracted and placed into an ultracentrifuge
in a solution of CsCl or sucrose.
This solution has the property that, under
the force of the centrifuge, a gradient forms
that has a density distribution.
DNA molecules will migrate in this
gradient until they reach a place where the
density of the solution equals the density of
the DNA.
103
13
Slide 48
Slide 49
Matthew Meselson and Franklin Stahl Experiment

showed that DNA replication is semi-conservative
No heavy
Hybrid to
light = 1: 1
What is the expected ratio of heavy, hybrid and

light DNA molecules after three generation ?
48
49
Slide 50
Steps involved in DNA replication:
Slide 51
1. Initiation
2. Extension
3. Unwinding
4. Processive synthesis to make long chains
5. Coordination of leading and lagging strand synthesis
6. Fidelity
7. Termination
51
Slide 52
Replication begins with the binding of

DnaA at the origin of replication,
designated oriC).
Origins contain region of high A/T content-facilitates DNA strand separation
Synthesis begins at the origin and
bidirectionally.
Replication ends on the other side of the
chromosome at a termination point.
Eukaryotes have multiple replication origins
Eukaryotic chromosomes are very long and
have multiple origins of replication to
duplicate large genomes in a shorter time
(within a few hours).
DNA replication starts at the origin and proceeds bidirectionally

Huberman JA & Tsai A J.Mol.Biol. 1973, 75:5-12
Demonstrate bidirectional replicaiton by pulse-labeling with
[3H]thymidine and DNA autoradiography
[3H]thymidine
thymidine
origin
[3H]thymidine
thymidine
52
104
14
Slide 53
Common Features of DNA replication origins

1. Recognized and bound by proteins (trans-acting factors)
2. Contain a short region (11 bp) of high A/T content--facilitates melting
3. Often contain palindromic sequences (inverted repeats)
4. Relatively small in bacteria, yeast, and viruses
5. Complex and large in metazoans--difficult to define sequence-specific
elements
DNA replication extension:
Slide 54
Extension
Nucleotides are added to the 3 OH group of

deoxyribose of a growing DNA chain using the
opposite (parental) strand as a template. The
pyrophosphate formed is quickly converted to Pi,
favoring the reaction to the right.
54
Slide 55
DNA unwinding:
55
Helicases (DnaB) separate the DNA strands

and unwind the parental duplex.
Single-strand binding proteins prevent the
strands from re-associating and protect
them from enzymes that cleave singlestranded DNA.
Topoisomerases, enzymes that can break
phosphodiester bonds and rejoin them,
relieve the supercoiling of the parental
duplex caused by unwinding.
DNA gyrase is a major topoisomerase in
bacterial cells.
105
15
Slide 56
Because DNA polymerase cannot synthesis

DNA de novo, a RNA primer is first
synthesized during replication.
Primase is the enzyme that synthesizes the
short RNA primers using DNA as a
template.
Unlike DNA polymerase, primase can start
a new polynucleotide chain by joining two
nucleoside triphosphates together.
The primase synthesizes a short
polynucleotide (~10 nucleotides) in the 5'to-3' direction and then stops, making the 3'
end of this primer available for the DNA
polymerase to continue DNA synthesis.
Elongation:
Priming
RNA primer
Because DNA polymerase cannot synthesis DNA de novo, a RNA

primer is first synthesized during replication.
Primase is the enzyme that synthesizes the short RNA primers using
DNA as a template.
Unlike DNA polymerase, primase can start a new polynucleotide
chain by joining two nucleoside triphosphates together.
The primase synthesizes a short polynucleotide (~10 nucleotides) in
the 5'-to-3' direction and then stops, making the 3' end of this primer
available for the DNA polymerase to continue DNA synthesis.
56
Slide 57
ELONGATION
Substrates: dNTP and a primer

(length n)/template
Products: PPi and primer (length
n+1)/template
Enzyme: DNA-dependent DNA

polymerase, requires Mg++
Catalytic step: Formation of
phosphodiester bond (high
stability bond requiring high
energy input)
Mechanism: Nucleophilic attack

by 3OH of sugar
57
Slide 58
Sliding Clamp
A sliding clamp is a protein fold that

serves as a processivity-promoting
factor in DNA replication.
binds DNA polymerase and prevents
the enzyme from dissociating from
the template DNA strand.
Because the rate-limiting step in the
DNA synthesis reaction is the
association of the polymerase with
the DNA template, the DNA clamp
protein increases the rate of DNA
synthesis up to 1,000-fold versus a
nonprocessive polymerase.
Example, proliferating cell nuclear
antigen (PCNA)
58
106
Substrates: dNTP and a primer (length

n)/template
Products: PPi and primer (length
n+1)/template
Enzyme: DNA-dependent DNA
polymerase, requires Mg++
Catalytic step: Formation of
phosphodiester bond (high
stability bond requiring high energy input)
Mechanism: Nucleophilic attack by 3OH
of sugar
A sliding clamp is a protein fold that serves
as a processivity-promoting factor in DNA
replication.
Binds DNA polymerase and prevents the
enzyme from dissociating from the
template DNA strand.
Because the rate-limiting step in the DNA
synthesis reaction is the association of the
polymerase with the DNA template, the
DNA clamp protein increases the rate of
DNA synthesis up to 1,000-fold versus a
nonprocessive polymerase.
Example, proliferating cell nuclear antigen
(PCNA)
16
Slide 59
DNA polymerases are responsible for duplication of DNA but have major
limitations
1. Require a template - no de novo synthesis
2.
Require a primer - extend from 3OH only. Primers are provided by

specialized DNA-dependent RNA polymerase (DNA primase)
3.
Polymerize in only one direction--5 to 3
4.
May have low inherent affinity for DNA
5.
Increased accuracy may slow extension
Slide 60
Pol III is involved in replication

All polymerases contain 3 to 5 exonuclease activity
60
There are 3 DNA polymerases in bacteria

o Pol I removes RNA primers and filling of gaps.
o Pol III is the major polymerase involves in DNA replication.
o Pol II is for DNA repair
Each DNA polymerase is a multi-protein complex. It catalysts DNA synthesis,
recruiting free deoxynucleotide 5-triphosphates to form hydrogen bonds with their
appropriate complementary dNTP on the template strand, and to form a covalent
phosphodiester bond with the 3' nucleotide of the growing DNA strand.
Preferential binding occurs between pairs of nucleotides (A with T, and G with C)
that can form base pairs. This enables each strand to act as a template for forming its
complementary strand.
An incoming nucleotide that forms correct hydrogen bonds with template in the active
site of a DNA polymerase is incorporated into the growing DNA chain. 3-OH of
primer attacks 5 phosphate of nucleotide, displacing a pyrophosphate, which is
rapidly metabolized. This results in the elongation of primer by one nucleotide. This
cycle is repeated as long as template is available.
107
17
Eukaryotic DNA polymerases :
Slide 61
Pol is the major DNA replication enzyme and Pol starts syntheis of the lagging strand
61
Pol : complex with primase (synthesizes a

RNA primer), and elongate DNA from RNA
primers until elongation is taken over by Pol
and .
Pol : involves in DNA repair.
Pol : replicates mitochondrial DNA.
Pol : is the main polymerase in replication, it
is highly processive and has 3' to 5' exonuclease
activity.
Pol : may substitute for Pol in lagging strand
synthesis in some tissues.
Pol , , , and are involved in the bypass of
DNA damage.
Only the polymerases that deal with the
elongation (, and ) have proofreading ability
DNA synthesis requires a RNA primer:

DNA polymerase cannot synthesize DNA
de novo.
DNA synthesis by DNA polymerase
requires a template and a 3 -OH end of a
growing strand.
The primase synthesizes a short
Slide 62
Movement of
Replication fork
polynucleotide (~30 nucleotides, RNA

62
primers) in the 5' to 3' direction using DNA

as a template and then stops, making the 3'
end of this primer available for the DNA
polymerase to continue DNA synthesis.
Slide 63
LEADING AND LAGGING DNA STRAND SYNTHESIS

Leading strand:
- Continuous elongation
- Direction of synthesis is 5 to 3
- Direction of chain growth same (5 to 3)
- Usually uses a processive DNA polymerase
Lagging strand:
- Replicated at same time as leading strand
- Template runs anti-parallel to that for leading strand
- Direction of synthesis is 5 to 3
- Direction of chain growth is 3 to 5
- Discontinuous synthesis allows for growth and synthesis to occur in different
directions
108
18
Slide 64
DNA replication in eukaryotes:

Primase associated with pol to produce
RNA primer (~10 nucleotides)
Pol adds about 20 dNTP to this RNA and
dissociates from the template.
On the leading strand, pol (the major
replicative enzyme) adds dNTP to this
RNA-DNA primer, continuously producing
this strand.
On the lagging strand, after pol
dissociates, pol adds nucleotides to the
primer, producing an Okazaki fragment,
and stops synthesizing when it reaches the
5 end of the previously synthesized
Okazaki fragment.
Eukaryotic DNA replication complex

Direction of DNA synthesis
64
DNA ligase:
DNA ligase joins two polynucleotide
chains together, in which the 3 OH at the
end of one fragment is ligated to the
phosphate group at the 5 end of the next
fragment, forming a phosphodiester bond.
DNA ligase joins two DNA chains after Pol
I (plus RNase H) removes RNA primers
and fills in the gaps.
Slide 65
DNA ligase sealing DNA gaps
65
Slide 66
66
109
19
Slide 67
Proofreading: remove errors during replication
Proofreading: remove errors during replication
DNA polymerases process polymerization and exonuclease activity that

removes the 3 nucleotide of the growing chain that is incorrectly base-paired
with the template strand.
Proofreading activity eliminates most base-pairing errors as they occur. The
error rate is reduced from 1/1000 to 1/1,000,000.
After replication, other mechanisms replace mismatched bases that escaped
proofreading so that the fidelity of DNA replication is very high.
The two processes of proofreading and postreplication mismatch repair result
in an overall error rate of about 1/10 billion.
67
68
Slide 68
Slide 69
Proofreading: Elimination of errors during replication

DNA polymerases process exonuclease activity, in additional to the polymerase
activity.
In bacteria, all three DNA polymerases (I, II, and III) have the ability to proofread,
using 3' to 5' exonuclease activity.
In eukaryotes only the polymerases that deal with the elongation (, and ) have
proofreading ability.
Nuclease activity removes the nucleotide at the end of the growing chain that is
incorrectly base paired with the template strand.
Proofreading activity eliminates most base-pairing errors as they occur. The error rate
is reduced from 1/thousand to 1/million.
After replication, other mechanisms replace mismatched bases that escaped
proofreading so that the fidelity of DNA replication is very high.
The two processes of proofreading and post replication mismatch repair result in an
overall error rate of about 1/10 billion.
End Replication problem of DNA ends:
Replication of the Ends of Chromosomes
Okazaki fragments
gap
gap
The problem to replicate DNA ends is that

there is no place to synthesize a primer that
would allow the daughter strand to be
completed, so the daughter strand will be
shorter than the parental strand.
In a linear DNA molecule, synthesis of the
lagging strand thus stops short just before
69
the end of the template.
110
20
Replication of DNA ends is solved by Telomeres

and telomerase:
Slide 70
Replication of DNA ends.
Telomeres:
Telomeres are found at the ends of linear
chromosomes.
Consist of many short tandem repeats.
Made up of heterochromatin and are highly
conserved in evolution
70
Slide 71
Telomerase:
Ribonucleoprotein enzyme complex with a
short RNA strand that catalyzes addition of new
telomere repeats to the 3 end of a DNA chain.
Telomerase RNA serves as the template for
nucleotide addition, while the protein
component functions as a reverse transcriptase
(synthesizing DNA using RNA template).
After a repeat is added, the enzyme dissociates,
binds DNA, and adds additional repeats.
Telomerase adds a series of repeats of a DNA
sequence to the template strand, which allows
the lagging strand to be completed by DNA
polymerase.
dNTPs
RNA
3
reversed
transcriptase
DNA
Reverse transcription
GTTAGGG
RNA template
71
Slide 72
Serve as a buffer to protect the end of

the chromosome from destruction.
dNTPs
GT TAGGG
GT TAGGGTTAGGG
G T T A G G G T T A G G G-3
3-U C C C A A U C C C-5
RNA primer
Pol
72
111
21
Slide 73
Summary
DNA replication is semi-conservative the mode of DNA replication which produces
two
DNA copies that each contained one of the original strands and one entirely new
strand.
DNA replication starts at the origin and proceeds bidirectionally.
Eukaryotes contain multiple origin of replication.
The two DNA strands are synthesized asymmetrically: continuously and
discontinuously.
Many proteins are involved in DNA synthesis.
DNA ends consist of telomere repeats.
Telomerase (RT and RNA) synthesize DNA ends.
Sample questions
Which of the following is true of pyrimidines?
1. Guanine is a pyrimidine.
2. Pyrimidines bind readily to deoxyribose, but not
to ribose.
3. Pyrimidines have a single-ringed structure.
4. Adenine's bonding to thymine is stronger than
that of guanine's to cytosine.
5. Pyrimidines are found in DNA but not in RNA.
In this diagram of the process of DNA replication at a

replication fork, the newly synthesized DNA strand
labeled B is the:
A.
B.
C.
D.
E.
coding strand
parental DNA
leading strand
lagging strand
RNA primer
75
76
If the genome of the bacterium E. coli requires about 20

minutes to replicate itself, how can the genome of the fruit
fly Drosophila be replicated in only three minutes?
Which histone protein is present as a monomer within the

nucleosome and is not a part of the core particle?
A. The Drosophila genome is smaller than the E. coli

genome.
B. Eukaryotic DNA polymerase synthesizes DNA at a much
faster rate than prokaryotic DNA polymerase.
C. The nuclear membrane keeps the Drosophila DNA
concentrated in one place in the cell, which increases the
rate of polymerization.
D. Drosophila DNA contains more origins of replication than
E. coli DNA.
E. Eukaryotes have more than one kind of DNA polymerase.
A. H1
B. H2A
C. H2B
D. H3
E. H4
77
78
112
22

Lecture: Cellular Adaptations
12/14/10 10:30 12:00
1. Define the cellular changes and the etiologies associated with atrophy.
2. Define the cellular changes and the etiologies associated with hypertrophy.
3. Define the cellular changes and etiologies associated with hyperplasia.
4. Define the cellular changes and etiologies associated with metaplasia.
Learning Resource:
Pathologic Basis of Disease (PBD), 8th Ed
II. CELLULAR ADAPATATIONS
A. Overview
1. Stress in the form of altered physiologic stimuli results in cellular adaptations
involving growth and differentiation.
a. Cell atrophy refers to a decrease in cell size.
b. Cell hypertrophy refers to an increase in cell size.
c. Hyperplasia refers to an increase in cell number (i.e., proliferation).
d. Metaplasia refers to a change in cell differentiation.
B. Objective 1
1. Atrophy is an active process leading to a reduction in cell size.
2. Clinically, atrophy is seen as shrinkage of an organ or tissue. It is accompanied by
decreased cell function and often by decreased tissue and organ function. The
etiology of atrophy is highly variable and includes:
a. Decreased functional demand
b. Ischemia
c. Inadequate nutrition
d. Decreased trophic stimulation denervation and reduced hormones
e. Persistent cell injury chronic inflammation, increased pressure, chronic disease
3. Decreased functional demand is commonly associated with atrophy of skeletal
muscle due to casting, prolonged bed rest, or general inactivity.
4. Decreased blood supply can lead to cell death via apoptosis as well as atrophy. This
is commonly encountered in peripheral vascular disease due to narrowing of arteries
due to atherosclerosis.
5. During starvation, energy is preferably derived from carbohydrates, then fat, then
muscle protein. The brain is the last source of energy.
a. The extent of skeletal muscle atrophy during starvation depends on the amount
of energy in the body at the beginning of starvation and on the length of
starvation.
113
b. Adequate nutrition reverses skeletal muscle atrophy due to starvation.

6. Trophic signals include nerve stimulation for skeletal muscle, growth factors, and
hormones. The loss of these stimuli, either physiologic or pathologic, is commonly
associated with atrophy of the target cells.
a. Denervation, of skeletal muscle cells will cause atrophy that is characterized by small
angulated skeletal muscle cells.
b. Withdraw of hormone/growth factor stimulation will cause both apoptosis (cell

death) and atrophy of the target cells. This is commonly seen in the breast and
ovaries of post-menopausal women due to loss of estrogen stimulation.
7. Chronic injury of cells can lead to their atrophy. Several diseases arise due to this.
a. Chronic gastritis refers to chronic inflammation of the gastric mucosa. It may
arise due to one of multiple etiologies that include infectious, autoimmune, or
idiopathic (no none cause).
b. Increased pressure over a long period of time is associated with hydrocephalus
and formation of Decubitus ulcers.
c. Cachexia refers to a loss of body mass due primarily to muscle atrophy - that can
not be reversed nutritionally, as is noted with starvation. It is mediated by TNF-
and other cytokines
8. At the cellular level, atrophy is an active process associated with a loss of
cytoplasmic constituents.
a. Decreased protein synthesis in skeletal muscle cells and decreased lipid
synthesis in adipocytes.
b. Increased degradation of proteins via proteasomes in skeletal muscles and
increased lipolysis in adipocytes.
c. There is upregulation and down regulation of various genes within cells
undergoing atrophy.
d. Up regulation of autophagy involves autophagosomes fusing with lysomes.
i. Autophagosomes are membrane bound vacuoles that contain one or more
cytoplasmic organelles and other cytoplasmic constituents that have been
autodigested.
ii. They fuse with lysosomes for degradation.
iii. Hard to digest lipid is seen as lipofuscin granules, the wear and tear
pigment of aging.
C. Objective 2
1. Hypertrophy refers to an increase in cell size due to increased synthesis of cellular
proteins and other cytoplasmic constituents, and is often associated with increased
functional capacity.
a. There is often a concurrent increase in tissue and/or organ size.
114
b. Cells capable of dividing may undergo both hypertrophy and hyperplasia.

c. Hypertrophy without hyperplasia occurs in cells that have limited proliferation
potential such as myocardial and skeletal muscle cells.
2. Clinical settings of hypertrophy include increased functional demand and hormone
stimulation. These can be physiologic or pathologic in nature.
a. Increased functional demand associated with increased skeletal muscle work
during weight lifting, or myocardial muscle hypertrophy due to systemic
hypertension or aortic valve stenosis.
b. Uterine smooth muscle hypertrophy due to estrogen stimulation during
pregnancy and the use of anabolic steroids to increase muscle mass are
examples of hormone stimulation.
3. At the molecular level, hypertrophy is an active process that involves:
a. Increased degradation of proteins that dont support hypertrophy and increased
synthesis of molecules needed for hypertrophy.
b. Skeletal muscle serves as a model for the factors that facilitate hypertrophy.
1) Mechanical stretch due to increased work load involves cell adhesion
molecule signaling.
2) Growth factors such as insulin-like growth factor-1 (IGF-1), TGF-, and FGF
help to initiate hypertrophy
3) Various agonists - endothelin-1, -adrenergic agonists, angiotensin II help to
maintain hypertrophy and required vascular changes
c. Signal transduction results in increased gene expression for transcription factors
(c-Jun and c-fos) and embryonic genes for contractile proteins (myosin and
actin).
d. Smooth endoplasmic reticulum enlargement in order to metabolize certain drugs
is an example of subcellular hypertrophy.
D. Objective 3
1. Hyperplasia is an increase in cell number, but usually not cell size. There is often a
concurrent increase in tissue/organ size and function.
a. Hyperplasia occurs in tissue where mature cells are capable of proliferation
and/or there is a population of tissue stem cells.
b. Hyperplasia is associated with an increased risk for neoplastic transformation.
2. Hyperplasia occurs in several different physiologic and pathologic settings including
hormone stimulation and stimulation by growth factors and various cytokines.
a. Physiologic hyperplasia results from either normal hormone stimulation or as a
compensation for loss of tissue.
1) Hormonal stimulation e.g., endometrial changes during the menstrual cycle.
2) Compensatory hyperplasia following damage - (e.g., wound healing) or
partial resection (e.g. partial hepatectomy).
115
a. Pathologic hyperplasia can result from excess hormone secretions. Examples

include:
1) Increased estrogen stimulation can result in epithelial hyperplasia in the
breast and the endometrium.
2) Nodular hyperplasia of the prostate due to increased dihydrotestosterone
(DHT) secretion by the fibromuscular stromal cells.
b. Pathologic hyperplasia can result from excess secretion of growth factors and
inflammatory cytokines.
1) Inflammation - autoimmune disease e.g., psoriasis.
2) Infections resulting in lymphoid hyperplasia.
3. Hyperplasia is mediated by trophic growth factors, hormones and cytokines whose
binding to target cells activates a wide variety of genes, including oncogenes that
lead to increased cell proliferation.
E. Objective 4
1. Metaplasia is an alteration in cell differentiation where one adult cell type is replaced
by another adult cell type. There is concurrent alteration in tissue/organ function.
2. Metaplasia most often arises from differentiation of reserve cells in the epithelium or
in the mesenchymal tissue.
a. Metaplasia leads to switching of the state of key developmental control genes.
b. Metaplasia may serve as fertile soil for neoplastic transformation of epithelial
cells.
c. The growth of cells is orderly as in their differentiation.
3. Clinical settings of metaplasia include:
a. Chronic irritation commonly associated with stones or catheters within the lumen
of a duct or tubular structure (e.g., calculi within salivary gland ducts, and
catheter with the lumen of ureter).
b. Chronic exposure to chemicals such as HCl, or toxins in cigarette smoke.
c. Lack of vitamin A in the diet.
d. Chronic inflammation associated with HPV viral infections of the cervix,
Schistosomias parasitic infection of the bladder, or H. pylori of the stomach.
4. Morphologic types of metaplasia include:
a. Squamous metaplasia involves the replacement of glandular, usually columnar,
epithelium with stratified squamous epithelium. Squamous metaplasia is
commonly seen in cases of:
1) smoking induced squamous metaplasia of respiratory epithelium.
2) vitamin A deficiency leads to squamous metaplasia of the goblet cells in the
conjunctiva. Blindness results if the Vitamin A deficiency is not corrected.
116
3) cervical epithelium commonly undergoes squamous metaplasia due to HPV

infection.
b. Intestinal metaplasia of squamous epithelium is associated with Barrett
esophagitis that occurs at the gastroesophageal (GE) junction. In chronic acid
reflux disease, the wear-and-tear squamous epithelium cannot buffer the HCl on
its surface. The resulting intestinal metaplasia involves differentiation of the basal
cells into the columnar cells and goblet cells (intestinal mucosal-type cells) that
secrete acid-buffering mucus onto the epithelial surface.
c. Osseous metaplasia in the lungs is associated with chronic inflammation.
Pulmonary stromal cells differentiate into osteoblasts and synthesize trabecular
bone which may form bone marrow.
117
Dr. Dale D. Vandre

Apoptosis
12/15/11, Thursday
8:30-10:00
MED 1 CELL BLOCK DIV 2

AUTUMN 2011
CELL BIOLOGY MODULE
Lecture: Apoptosis
Dr. Dale D. Vandre, Department of Physiology and Cell Biology
Hamilton Hall, Rm 002
Phone: office 292-4181, dale.vandre@osumc.edu
1. Define the role of apoptosis in regulating normal and abnormal cellular growth.
2. Describe the molecular components of an apoptotic pathway and differentiate between their
functional roles in programmed cell death.
3. Given an example of a pro-apoptotic or anti-apoptotic protein family member, be able to
define the proteins role in regulating cytochrome C release from mitochondria.
4. Describe the function of the apoptosome and the role of proximity induced cleavage of
procaspases in activation of an apoptotic pathway.
5. Describe the differences between the roles of initiator and executioner caspases in activation
of an apoptotic pathway.
6. Describe the molecular components of a death receptor mediated apoptotic pathway.
7. Differentiate between the molecular steps involved in intrinsic, extrinsic, and granzyme B
mediated apoptotic pathways.
8. Define the different roles that p53 plays in regulating apoptosis.
Learning Resources
Essential Cell Biology (ECB) Lectures will cover material in Chapters 18, pages 638-646.
Cancer Medicine 6th Edition web based book, see Chapter 4 Apoptosis and Cancer, by
Kufe et al. (see also appropriate topics in Molecular Biology of the Cell 4th Edition, and
Eurekah Bioscience Collection).
Open the following web addresses and scroll to the top of the page search for cancer medicine:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books
118
Dr. Dale D. Vandre

Apoptosis
12/15/11, Thursday
8:30-10:00
Med1 IP, Cell Block, Division 2, Autumn 2011

Apoptosis lecture notes
Dr. Dale Vandr
Dept. of Physiology and Cell Biology
Rm 002, Hamilton Hall
292-4181, dale.vandre.@osumc.edu
Programmed Cell Death: Apoptosis

Survival of many cell types requires signals from other cells, and if deprived of these signals cells
will activate an intracellular suicide program and die. Programmed cell death is known as apoptosis.
During development of the nervous system more than half of the nerve cells normally die. Nerve sells are
produced in excess in the embryo, and those that do not find target cells die. In a normal adult, billions of
cells die in the bone marrow and intestines every hour. Thus, cell proliferation and cell death maintain a
balanced cell population. Apoptotic cells die without damaging their neighbors or inducing inflammation.
Apoptosis results from the activation of a cascade of proteases within the cytoplasm.
Apoptosis describes the built-in biochemical capacity of cells to undergo death, also known as
programmed cell death. In a sense, cells have an imprinted suicide pathway that can be activated in
response to a variety of signals or in response to cell damage. Apoptosis is as important to tumorigenesis
as is disruption of cell cycle regulation. Defects in apoptotic signaling pathways allow defective cells that
normally would die to survive and continue to proliferate. Thus malignant tumors often have defects
resulting in both the activation of growth promoting pathways and the inactivation of programmed death
pathways. Normally apoptotic pathways are involved in death of:
1.
2.
3.
4.
5.
6.
7.
Harmful cells such as thymocytes that will recognize self-antigens, cells with DNA damage,
viral infected cells
T and B lymphocytes with defective surface receptors
Excess cells, such as neurons that fail to make appropriate connections, which can be up to
80% of the neurons in some ganglia
Unnecessary cells such as the webbing between digits
Obsolete cells such as epithelial cells covering the midline of the palate, lactating vs, nonlactating breast
Virus infected cells
Chemotherapeutic killing of cells
119
Dr. Dale D. Vandre

Apoptosis
12/15/11, Thursday
8:30-10:00
Apoptosis Pathways
Key experiments leading to the identification of the molecular pathways regulating cell death
came from studies of the nematode Caenorhabditis elegans. During development, 1090 somatic cells are
generated in the worm, and of these 131 undergo programmed cell death. Mutations were identified that
regulate the death of these cells termed CED for C. elegans death genes. All 1090 cells survived in ced3 or ced-4 mutants and all 1090 cells died in ced-9 mutants. Additional studies have shown that these
genes work in sequence to regulate the apoptotic pathway. CED-9 inhibits activation of the apoptotic
pathway, so loss of function results in death of all 1090 cells. CED-9 binds directly to CED-4
suppressing its function. CED-4 binds directly to CED-3 and promoted its activation.
Studies of a chromosomal rearrangement in human follicular lymphoma identified an oncogene,
bcl-2, which promoted cell survival rather than proliferation. Bcl-2 was shown to be homologous to
CED-9, and could function in ced-9 mutant worms. These and other studies have led to the following
model:
C. elegans
Humans
Regulator
Adaptor
Ced-9 | Ced-4
Bcl-2 | Apaf-1
Effector
Ced-3 DEATH
Casp-9 Casp-3 DEATH
In humans, apoptosis regulator molecules include a family of 24 proteins related to bcl-2, six have
anti-apoptotic activity like bcl-2, while eighteen others have pro-apoptotic activity. Why do human cells
have so many different apoptosis regulator proteins? The different proteins allow the cell to respond to
different types of cellular stress.
Bcl-2 family proteins
Anti-apoptotic (protective)
Bcl-2, Bcl-XL, Bcl-w, Mcl-1,
Pro-apoptotic (killing)
Bax family Bax, Bak, Bok
BH3-only family Bid, Bim, Bad,
Noxa, Puma, Bmf, Bik
Adaptor proteins include the apoptotic protease activity factor Apaf-1, but also include proteins
known as death receptors such as the Fas receptor. Effector proteins are a class of 14 proteases known
as caspases. Caspases are present in the cell as proenzymes, an inactive form of the protease. Following
an apoptotic signal the procaspase binds to the adaptor protein and is cleaved to become an active
caspase effector molecule. Activation of caspase 9 or caspase 8 leads to subsequent activation of
additional caspases such as caspases 3, 6, and 7. Activated caspases target the destruction of critical
proteins required for cell survival, which leads directly to cell death.
120
Dr. Dale D. Vandre

Apoptosis
12/15/11, Thursday
8:30-10:00
Mechanisms of Apoptosis
The Intrinsic Apoptotic Pathway
Intrinsic apoptotic pathways by definition activate apoptosis through internal or stress mediated
signaling pathways that involve mitochondria. A key protein in understanding intrinsic apoptotic
pathways is the mitochondrial protein cytochrome C, which normally functions to transfer electrons
during oxidative phosphorylation. Cytochrome C resides between the inner and outer mitochondrial
membrane. However, during apoptosis, cytochrome C is released into the cytoplasm through channels in
the outer mitochondrial membrane. Bcl-2 and related anti-apoptotic proteins function to keep these outer
mitochondrial membrane channels closed by binding to pro-apoptotic proteins Bax and Bak, which are
also located in the outer mitochondrial membrane. As long as there is a balance between Bcl-2 family
proteins and the Bax family of proteins, the mitochondrial channels remain closed, cytochrome C is not
released, and the intrinsic apoptotic pathway cannot be activated. An excess amount of Bcl-2, as found in
some human follicular lymphomas and other human tumors, prevents the normal activation of intrinsic
apoptotic pathways, and thus promotes the survival of cells. When the levels of Bcl-2 family proteins
falls below that of the Bax family proteins, the outer mitochondrial membrane channels open, and
cytochrome C is released into the cytoplasm. Cytochrome C in the cytoplasm triggers the activation of
caspases (see below).
An imbalance between Bcl-2 and Bax that will trigger apoptosis is induced by the action of BH3only pro-apoptotic proteins. The different BH3-only family pro-apoptotic proteins are normally kept
inactive by binding to other cytoplasmic proteins, which sequesters them and keeps them inactive.
Different types of cellular stress or loss of survival cytokine signals can cause the release of a specific
BH3-only pro-apoptotic protein from its inactive storage location. The freed BH3-only pro-apoptotic
protein is now available to bind to the Bcl-2 family anti-apoptotic protein, effectively preventing it from
interacting with the Bax family pro-apoptotic protein. The balance between Bcl-2 and Bax family
proteins is disrupted, and the freed Bax proteins open the outer mitochondrial membrane channels. As a
result, cytochrome C is released into the cytoplasm.
Cytochrome C binds to Apaf-1 in the cytoplasm forming a multimeric complex known as the
apoptosome, which contains 7 molecules of each protein. The apoptosome can now bind procaspase 9,
and the close proximity of bound procaspase 9 allows for the enzymatic cleavage of the molecule forming
fully active caspase 9. Caspase 9 activates downstream procaspases 3, 6, and 7 into their active caspase.
These caspases cause the cleavage of >400 cellular proteins, eventually inducing cell death and the
characteristic morphology of apoptosis. Activation of caspases is similar to the amplification of a weak
signal by a kinase signal transduction pathway. In the caspase pathway, an initiator caspase, caspase 9,
activates the executioner caspases, caspases 3, 6, and 7. Additional regulators of apoptosis are also
released by the mitochondria. These proteins, known as Smac/DIABLO move into the cytoplasm to
inactivate regulatory proteins known as inhibitors of apoptosis (IAPs), which serve to inhibit any
inappropriate or low level caspase activity in the cytoplasm.
121
Dr. Dale D. Vandre

Apoptosis
12/15/11, Thursday
8:30-10:00
In addition to stress-mediated responses, the intrinsic apoptotic pathway can be triggered by

activation of p53. In response to cell damage, DNA damage, checkpoint activation, or anoxia, p53 levels
are increased. As a transcription factor, p53 can induce the synthesis of Bax or BH3-only pro-apoptotic
proteins Noxa and Puma that will trigger the intrinsic apoptotic pathway. In addition p53 can also
upregulate transcription of factors that are secreted by the cell and bind to survival cytokines. This
effectively blocks the survival cytokine signal pathway, and can trigger apoptosis. Lastly, p53 can also
increase the transcription of membrane receptors known as death receptors triggering an extrinsic
apoptotic pathways (see below).
The Extrinsic Apoptotic Pathway

A second apoptotic pathway can be triggered by signals outside of the cell. This apoptotic
pathway is known as the extrinsic apoptotic pathway and is plasma membrane receptor activated
involving the activation of pro-apoptotic cell surface death receptors. There are 30 death receptors in the
human genome, and they are members of the tumor necrosis factor (TNF) family of receptors, receptor
ligands include TNF-, TRAIL, and FasL. Ligand binding activates the cytoplasmic tail of the death
receptor to bind a cytoplasmic protein known as FADD (FAS-associated death domain). Association of
FADD with the receptor forms a complex known as the DISC (Death Iinducing Signaling Complex).
The DISC binds an initiator procaspase 8 (or initiator procaspase 10), and in a fashion similar to the
apoptosome complex, allows for cleavage and formation of active caspase 8. Caspase 8 will activate the
downstream executioner procaspases 3, 6, and 7. Activation of the death receptor pathway bypasses or
circumvents the Bcl-2 mitochondrial pathway of apoptosis.
Granzyme B Mediated Apoptotic Pathway

A third apoptotic pathway is triggered by the action of cytotoxic T lymphocytes and natural killer
cells of the immune system. These immune cells can activate death receptors or insert a pore forming
protein known as perforin into the plasma membrane of targeted cells. A protease within the T-cell,
granzyme B, is then released and is internalized by the target cell through the perforin pore. Once in the
cytoplasm granzyme B activates initiator procaspases 8 and 9, which subsequently activate downstream
executioner caspases. Granzyme B may also directly activate executioner caspase 3.
122

Lecture: Intracellular Accumulations and Cell Death
12/15/11 10:00 12:00
1. List the types of material that can be accumulated within cells and the four major
mechanisms of their accumulation.
2. Compare and contrast the etiologies, cellular mechanisms, and morphologic features of lipid
accumulation in hepatocytes and macrophages.
a. Look at steatosis in the liver.
b. Look at cholesterol accumulation in macrophages in the clinical setting of
hyperlipidemia, atherosclerosis and Niemann-Pick disease.
3. Compare and contrast the etiologies, cellular mechanisms, and morphologic features of
intracellular accumulation of proteins.
a. Look at alcoholic hepatitis and 1-antitrypsin deficiency.
4. Compare and contrast the etiologies, molecular and cellular mechanisms (biochemistry),
and morphologic features of intracellular accumulation of glycogen in glycogen storage
diseases.
5. Compare and contrast the etiologies, cellular mechanisms, and morphologic features of
intracellular accumulation of exogenous and endogenous pigment.
6. Correlate the etiologies with the gross and microscopic features of the four types of
necrosis.
a. What are the three possible nuclear changes that are commonly seen in
necrosis?
b. What are the cytoplasmic changes seen in necrosis?
c. What inflammatory responses are associated with necrosis?
7. Compare and contrast necrosis with apoptosis as to etiologies and cellular changes.
Learning Resource:
Pathologic Basis of Disease (PBD), 8th Ed, Various pages (for specific diseases noted in the
objectives)
III. INTRACELLULAR ACCUMULATIONS
A. Objective 1
1. Intracellular accumulations may not in themselves be harmful, but are indicative of
an underlying disease.
2. Injured cells can accumulate normal and abnormal substances including:
a. H2O, lipids, proteins and carbohydrates and their metabolites.
123
b. Exogenous and endogenous pigments.

3. The mechanisms of intracellular accumulation include:
a. Impaired metabolism of a normal substance is seen with water, triglycerides, and
protein in renal tubules.
b. Genetic or acquired defects result can result in abnormal metabolism due to a
lack of an enzyme, or alterations in packaging, transport, and/or secretion of
various products (e.g. lysosomal storage diseases, and protein folding
abnormalities)
c. Uptake of an abnormal exogenous substance (e.g. carbon pigment, suture) that
cannot be digested, or retention of an endogenous pigment.
B. Objective 2
1. Various lipids can be accumulated in cells depending on the cell type and the
underlying mechanism.
2. The accumulation of triglycerides is commonly seen in hepatocytes, e.g., steatosis
(fatty change), but can also occur in the heart, kidneys, and skeletal muscle.
a. There are multiple etiologies of steatosis.
1) Starvation, diabetes, steroid use, total parenteral nutrition (TPN), obesity, and
pregnancy give rise to increased serum levels of free fatty acids. These fatty
acids are taken up by various organs, including the liver, and converted into
triglycerides and stored.
2) Ethanol toxicity and hypoxia injury cause increased acetate formation that
increases the synthesis of fatty acids resulting in the accumulation of
triglycerides in hepatocytes.
3) Accumulation of triglycerides also arises due to a decrease in their secretion
resulting from impaired protein synthesis associated with toxins and
malnutrition.
b. The morphologic features of steatosis include:
1) On gross examination, the liver is small, greasy, and yellow as compared to
the normal brown-tan liver.
2) Microscopic examination demonstrates hepatocytes with small
(microvesicular) to large (macrovesicular), clear, lipid droplets that displace
the nucleus to one side (think of bubbles). The triglycerides in frozen tissue
sections stain the red with Oil-Red O.
3. Cholesterol Accumulation
a. Intracellular accumulation of cholesterol is common in macrophages.
1) The presence of cytoplasmic cholesterol yields a foamy appearance (i.e.,
foamy macrophages). This is commonly seen in xanthomas
(hyperlipidemia), atherosclerosis, and in inflammation.
2) Crystalline cholesterol accumulates extracellularly and is associated with a
giant cell reaction (multiple macrophages coming together to try to digest it).
124
This is common in disrupted atheromatous plaques.

4. Lipids can also accumulate in settings of lysosomal storage diseases where lipidcarbohydrate complexes accumulate due to enzyme deficiencies. Niemann-Pick
Disease represents an accumulation of cholesterol and glycolipids especially
sphingomyelin in macrophages in the lungs, liver, spleen, lymph nodes, bone
marrow, tonsils, and GI track. There are three molecular forms of the disease A, B,
and C that have form foamy macrophages.
D. Objective 3
1. Intracellular accumulation of proteins is characterized by pink (eosinophilic) staining
cytoplasmic droplets, vacuoles, or aggregates.
2. Mechanisms for intracellular accumulation of proteins are variable.
a. Alcohol damage of hepatocytes causes aggregation of cytoskeletal proteins (i.e.,
Mallory bodies).
b. Defects in protein folding can lead to intracellular accumulation of proteins.
1) Proper protein folding is required for proper functionality.
Chaperones facilitate protein folding, protein transport, and, along with
ubiquitin, facilitate degradation of damaged proteins in proteasomes and
autophagolysosomes.
2) The underlying mechanism of several well known diseases has been
attributed to misfolding of proteins.
3) The genetic mutation leading to alpha-1 anti-trypsin, a serine protease
inhibitor, deficiency leads to the accumulation of pink staining globules of
proteins within hepatocytes due to an inability to release them. The result is
a lack of protein needed in the alveoli of the lungs to degrade various
proteolytic enzymes released by alveolar macrophages. The end result is
emphysematous change (looks like a sponge).
E. Objective 4
1. Glycogen accumulation within cells appears as foamy to clear cytoplasmic vacuoles.
Compare the normal cardiac myocytes with those in a glycogen storage disease.
2. Glycogen storage diseases are characterized by an accumulation of glycogen due to
a mutation induced enzyme deficiency involved in glycogen synthesis or breakdown.
a. Intracellular accumulation of glycogen accumulation commonly occurs in
hepatocytes (hepatic forms) or in muscle cells (myopathic forms).
F. Objective 5
1. Intracellular accumulation of pigments is not usually harmful, but is often indicative of
an underlying disease.
2. The accumulation of exogenous carbon by alveolar macrophages (anthracosis) is
common in smokers and urban dwellers. There is no apparent cell injury.
a. Coal workers lung is a pneumoconiosis (fibrosis of the lungs) caused by an
inflammatory response to accumulation of carbon and silica dust that induces
lung injury and scarring.
125
b. Tattooing results from the accumulation of carbon and other dyes by dermal
macrophages.
3. Hemosiderin is a coarse golden, yellow-brown, intracellular pigment that can be
stained using Prussian Blue stain for iron.
a. Ferritin micelles that are derived from hemoglobin iron are normally taken up by
phagocytic cells within various organs.
b. Phagocytic cells and parenchymal cells will accumulate hemosiderin aggregates
of ferritin micelles when there is local (thick bruise) or systemic iron overload.
1) Systemic iron overload hemosiderosis is associated with a wide variety of
clinical conditions (e.g., hemolytic anemia, increased dietary iron,
transfusions, or impaired use of iron).
2) Hemochromatosis is an excessive accumulation of hemosiderin with
parenchymal cells and may be result from a mutation of the HFE gene or
secondary in its origin.
a) Hemochromatosis can result in cirrhosis of the liver, diabetes, abnormal
skin pigmentation, and heart failure. Prussian Blue staining clearly
demonstrates the intracellular accumulation of hemosiderin.
4. Bilirubin is a green-brown pigment normally found in the bile, and is the end product
of hemoglobin metabolism.
a. Intracellular accumulation of bilirubin a brown pigment within the hepatocytes occurs in hepatocytes in conjunction with:
1) excess production of bilirubin from hemoglobin e.g., hemolytic anemia,
ineffective erythropoiesis, internal hemorrhage.
2) reduced hepatic uptake.
3) impaired bilirubin conjugation.
4) impaired bile flow.
5) impaired excretion.
5. Lipofuscin (wear & tear pigment arrow)) is a coarse, golden-brown, pigment that
represents residual bodies in aging cells. It has no clinical significance.
6. Melanin is an endogenous brown-black pigment produced by melanocytes and
melanoma cells.
IV. CELL DEATH

A. Objective 6
1. Necrosis is a morphologic expression of cell death resulting from different patterns of
lysosomal enzyme degradation of cells, extracellular matrix, the type of necrotic
debris, and by bacterial products when present.
2. The etiologies of necrosis include:
a. Ischemic injury
b. Cancer
c. Chemical injury
126
1) Unaltered chemicals may directly injure cells (e.g. Hg, CN)

2) Metabolites of P-450 mixed function oxidases metabolism may result in toxic
intermediates or metabolites (e.g. CCl4 or acetaminophen) that damage cells
d. Infections such as tuberculosis.
3. Cellular changes common to the different morphologic types of necrosis include:
a. Cell swelling (hydropic change).
b. Nuclear changes may include:
1) Karyolysis fading of staining due to chromatin digestion.
2) Pyknosis nuclear shrinkage with increased staining.
3) Karyorrhexis nuclear fragmentation of a pyknotic nucleus.
4) Total loss.
c. Disrupted membranes leads to the release of lysosomal enzymes and increased
cytoplasmic free Ca+2 ions-induced activation of proteolytic enzymes. The end
result is denaturation of cytoplasmic proteins.
d. Inflammation may be acute or chronic depending on the etiology.
4. Sequela of necrosis include:
a. Release of cytoplasmic constituents. Many are clinically useful, including
proteolytic enzymes that can damage adjacent uninjured cells and digest stromal
elements (e.g., collagen that holds the tissue together resulting in the formation
of an abscess).
b. Subsequent healing may lead to scarring and reduced tissue function.
5. There are four basic morphologic types of necrosis. They differ in their etiology,
amount of enzymatic tissue destruction, and type of inflammation.
a. Coagulative necrosis, classically associated with ischemic/hypoxic injury (e.g.,
myocardial infarction) is can also be seen in certain chemical injuries. It is the
most common type of necrosis, and is characterized by:
1) Enlarged swollen cells.
2) Cytoplasmic eosinophilia of the dead cells is due to protein coagulation and
seen here as wavy lines.
3) The dead myocytes lack the central nucleus of the normal myocytes.
4) The dead myocytes retain the cell outline (tombstones) and overall
architecture of the tissue.
5) There is acute inflammation responsible for removing the dead cells and
cellular debris. The amount of acute inflammation will peak within the first 48
hours to be replaced by macrophages.
b. Liquefactive necrosis is associated with total digestion of the stroma by
lysosomal enzymes released from neutrophils.
127
1) The etiology of liquefactive necrosis includes purulent (pus producing)

bacterial and fungal infections, as well as hypoxic injury to the brain.
2) The released lysosomal enzymes digest (i.e. liquefy) the tissue beyond the
ability for repair leading to abscess formation (above).
3) The morphology is that of cellular debris and acute inflammatory cells.
c. Caseating necrosis combines features of coagulative and liquefactive necrosis.
1) Classically, caseating necrosis is associated with M. tuberculosis infections,
and rarely with other infections.
2) The gross appearance is that of white, "cottage cheese-like necrosis.
3) The microscopic appearance of caseating necrosis is that of destruction of
tissue architecture combined with an irregular focus of eosinophilic acellular
debris surrounded by granulomatous chronic inflammation.
d. Fat necrosis is often associated with direct trauma to the breast and with
pancreatitis.
1) The gross appearance is that of chalky white-yellow plaques in the adipose
tissue.
2) The ruptured fat cells (dead adipocytes) release fatty acids that are degraded
by lipases. A giant cell reaction is often seen as well. Deposition of calcium
yields a soap bubble appearance or saponification.
e. Autophagy occurs in response of cell stress, and is a means to save the cell
rather than a mechanisms of cell death.
B. Objective 7
1. Apoptosis differs from necrosis in several ways.
NECROSIS
APOTOSIS
Pathologic/Physiologic
Pathologic
Both
Gene Directed
No
Frequent
Cellular Changes
Size
Swelling
Shrinkage
Nucleus
Pyknosis, karyorrhexis, karyolysis Fragmentation
Cytoplasmic Contents
Digested
Intact
Plasma Membrane
Damaged
Altered but intact
Type of Cell Debris
Fragmented and/or coagulative
Apoptotic bodies
Removal of Cell Debris
Acute Inflammation
Macrophages
a. Morphologic features of apoptosis are characterized by cell shrinkage, nuclear
fragmentation, and formation of apoptotic bodies.
2. Clinical implications of apoptosis include:
a. Inappropriate activation of apoptosis is associated with AIDS, ischemic stroke,
neurodegenerative disease, and myelodysplasia.
b. Decreased apoptosis is associated with tumorigenesis and tumor progression,
autoimmune disease, and viral infections.
128

2011-2012
Review Questions of Facts on Cellular Injury, Adaptations, Accumulations and Death
Try answering these before you look at the answers.

1. The function of which plasma membrane pump is impaired by depletion of ATP production?
2. What are the general categories of enzymes to form reactive oxygen species?
3. What are the mechanisms behind cell damage due to reactive oxygen species?
4. What are the enzymes that function as intracellular anti-oxidants?
5. What are the general categories of enzymes activated by increased cytoplasmic calcium?
6. What are three consequences of mitochondria damage?
7. What cellular processes are increased in hypertrophy?
8. What cellular processes are increased in hyperplasia?
9. What cellular processes are increased in atrophy?
10. What cell type undergoes altered differentiation during squamous metaplasia of the lung?
11. What pigment is detectable using Prussian Blue staining?
12. What is the underlying mechanism of hydropic change?
13. Gene mutation resulting in damaged enzyme activity leads to increased cytoplasmic
accumulation of what general group(s) of metabolic substrates?
14. What are the mechanisms underlying hepatic steatosis?
15. What types of inflammation are associated with coagulative, caseating, and liquefactive
necrosis?
16. What are the possible causes of coagulative necrosis?
17. What are the possible causes of liquefactive necrosis?
18. How do apoptosis and necrosis differ relative to inflammation and cellular morphology?
129
Case Studies in Cellular Injury, Adaptations, Accumulations and Death

Case 1:
A 78-year-old obese male with a complaint of acute onset of shortness of breath and chest pain
that radiated to the left shoulder was transported to the Emergency Department. The patient
had a history of chest pain and cardiac arrhythmia. Laboratory studies demonstrated that
serum levels of the myocardial cytoplasmic proteins CK-MB (myocardial creatine kinase) and
cardiac muscle troponin I (cTnI) were elevated in each of three serum samples taken. The
patient subsequently died and an autopsy was performed. There is an infarction of the anterior
wall and IVS. (Image Case 1 - #1).
1. What were the mechanisms involved in injury to organelles and cellular systems in the
injured myocytes that led to their death?
2. What morphologic type of cell death would the injured myocytes most likely exhibit?
(Images Case 1 #2 and #3)
3. What type of inflammatory response would you expect to see adjacent to the injured
myocardial cells? (Images Case 1 #2 and #3)
4. How do you explain the increased serum levels of CK-MB and (cTnI) in the serum?
5. What type of adaptive cellular change would the uninjured myocardial cells most likely
exhibit over time if the patient had lived? (Arrows - Image Case 1 #4)
6. What accumulated pigment would most likely be seen in the uninjured myocytes? (Image
Case 1 #5)
130
Case 2
A 32-year-old woman was transferred to a hospital in acute liver failure due to an overdose of
acetaminophen. The patient was reported to have been despondent and took 50 500 mg
tablets of acetaminophen. Laboratory studies revealed markedly elevated levels of hepatic
enzymes in the serum, consistent with extensive hepatocytes injury. The patient was admitted
for a possible liver transplant. The patient died on the 5th hospital day and an autopsy was
performed.
Image Case 2 #1 represents a section taken of the patients liver. Note the portal triad on the
right (large portal vein (PV), small bile duct (BD), and small hepatic artery (HA). The central
vein is out of the field, but would be toward the left.
Oxidation of acetaminophen is complicated. It yields a toxic intermediate that binds to
microsomal proteins. Glutathione binds to this intermediate forming a harmless intermediate
that can be excreted, a process that led to oxidative stress due to exhaustion of glutathione.
Remember that glutathione is an antioxidant that inactivates reactive oxygen species that are
normally generated by a cell.
1. What substance has been accumulated by hepatocytes adjacent to the portal triad? What is
the mechanism of this process?
2. What is the morphologic process that is present in the hepatocytes to the left of the dotted
line?
3. How do free radicals injure cells? What other mechanisms of cell injury were subsequently
induced following free radical induced injury?
4. Why are the epithelial cells lining the bile ducts uninjured?
131
Case 3
A 67-year old woman was seen by a physician complaining of being tired and blood spotting on
her underwear. The physical examination was unremarkable. Laboratory studies demonstrated
normochromic normocytic anemia. No malignant cells were noted on the PAP smear.
Ultrasound and CT scans of the abdomen were normal. A biopsy of the endometrium was
obtained. Refer to Image Case 3 #1, and compare the normal endometrium on the left taken
from a 25 year-old woman of child bearing age with the image of the endometrium taken from
patient. The arrows denote the depth of the endometrium.
1. What type of adaptive cellular change would the glandular epithelial cells most likely exhibit
over time?
2. What cellular organelles would be directly involved in the morphologic changes seen in
glandular epithelial cells in this patient?
3. What is the likeliest pathway leading to apoptosis of the glandular epithelial cells?
4. What enzymes are the effectors of cell death in this case?
5. What proteins control the on and the off signals for apoptosis in the mitochondrial pathway
of apoptosis in this case?
6. How do these histologic changes differ from necrosis?
Case 4
A 73-year old male was seen by his physician for continued follow-up of his transfusiondependent hemolytic anemia. Laboratory studies demonstrated slightly elevated serum levels
of hepatic enzymes. A liver biopsy was obtained. (Image Case 4 #1)
1. What is the likeliest etiology of the of the cytoplasmic pigment accumulation within the
hepatocytes (B) and Kupffer cells (A)
2. What cell type (A or B) would most likely be first to have increased accumulations of this
pigment and why?
3. What stain would you use to clarify the nature of this pigment?
4. The ion within this pigment would impact the generation of free radicals in these
hepatocytes via which reaction?
132
Case 5
A 55-year-old male was seen by his physician for evaluation of a chronic non-productive cough
and shortness of breath after walking a few steps into his house. The patient reported that he
had a 50 pack-year history of smoking and was previously diagnosed with life-threatening
pulmonary emphysema. Physical examination demonstrates bitemporal wasting (atrophy) and
loss of subcutaneous fat. Pulmonary function tests demonstrate airflow restriction with
increased lung volume. A CT scan was interpreted as panacinar emphysema. A subsequent
liver biopsy demonstrated the accumulation of the eosinophilic (pink-staining) granules within
the hepatocytes. (Image Case 5 #1)
1. What is the mechanism behind this intracellular accumulation?
2. What is the type of substance being accumulated?
3. Why is the clinical manifestation of this process in the lungs rather than in the liver?
133
Answers to Review Questions

1.
The function of which plasma membrane pump is impaired by depletion of ATP

production?
Na+-K+ pump.
2.
What are the general categories of enzymes to form reactive oxygen species?
Oxidative enzymes Oxidases
3.
What are the mechanisms behind cell damage due to reactive oxygen species?
A-Lipid peroxidation in the cell membrane.
B-Oxidative modification of the proteins.
C-Single stranded DNA breaks.
4.
What are the enzymes that function as intracellular anti-oxidants?

A-Catalase
B-Superoxide dismutase
C-Glutathione peroxidase
5.
What are the general categories of enzymes activated by increased cytoplasmic

calcium?
A-Phospholipases.
B-Preoteases.
C-Endonucleases.
D-ATPases.
6.
What are three consequences of mitochondria damage?

A- Increase in the permeability of the mitochondrial membrane and decrease ATP
and finally necrosis.
B- Release of stored Ca2+ leads to activation of many enzymes.
C- Leakage of cytochrome c and other apoptotic proteins leads to apoptosis.
7.
What cellular processes are increased in hypertrophy?

Increased production of cellular proteins and other constituents
8.
What cellular processes are increased in hyperplasia?

Increased cell proliferation and synthesis of cytoplasmic components especially
proteins.
9.
What cellular processes are increased in atrophy?

Proteasome activity and autophagy
10.
What cell type undergoes altered differentiation during squamous metaplasia of the
lung?
Basal cell stem cell
11.
What pigment is detectable using Prussian Blue staining?

Hemosiderin (Iron).
12.
What is the underlying mechanism of hydropic change?
134
Decrease Na+-K+ pump function leads to alteration of membrane permeability and

inability to exchange Na and K in and outside the cell membrane, eventually
leakage of H2O inside following the Na concentration.
13.
Gene mutation resulting in damaged enzyme activity leads to increased cytoplasmic

accumulation of what general group(s) of metabolic substrates?
A-Carbohydrates
B-Lipids
C-Proteins
14.
What are the mechanisms underlying hepatic steatosis?

A- Excessive entry
B- Defective metabolism.
C- Defect in lipid exportation.
(Diabetes, starvation, TPN, hypoxia, toxins, obesity)
15.
What types of inflammation are associated with coagulative, caseating, and liquefactive
necrosis?
Acute inflammation.
16.
What are the possible causes of coagulative necrosis?

A-Ischemia (most common)
B-Toxins
C-Thermal injury
D-Immune response
17.
What are the possible causes of liquefactive necrosis?

A- Bacterial or fungal infection leads to leukocytes accumulation and liquefaction
of the dead tissue and pus formation.
B- Cerebral ischemia
18.
How do apoptosis and necrosis differ relative to inflammation and cellular morphology?
NECROSIS
Pathologic/Physiologic
Gene Directed
Cellular Changes
Size
Nucleus
Cytoplasmic Contents
Plasma Membrane
Type of Cell Debris
Removal of Cell Debris
Pathologic
No
APOTOSIS
Both
Frequent
Swelling
Pyknosis, karyorrhexis, karyolysis
Digested
Damaged
Fragmented and/or coagulative
Acute Inflammation
Shrinkage
Fragmentation
Intact
Altered but intact
Apoptotic bodies
Macrophages
135
The Cell - Division 2

Lecture:
Neoplasia #1 Morphologic Features of Neoplastic Cells and Tumors

1/3/12 8:30 9:30
Heart and Lung Research Institute, Rm 081, 473 W 12th Ave
Phone: Secretary 247-7485,
E-mail: charles.hitchcock@osumc.edu
1. Define and be able to recognize the altered cellular features of a neoplastic cell. These
include: hypercellularity, pleomorphism, loss of polarity, and increased N/C ratio.
2. Define and be able to recognize the altered nuclear features of a neoplastic cell. These
include: hyperchromatic staining, multiple nuclei, uneven chromatin distribution, uneven
nuclear membrane, increased mitoses, abnormal mitotic figures, and abnormal nucleoli.
3. Define and recognize the cellular and nuclear morphologic features that distinguish a
dysplastic lesion from a metaplastic or hyperplastic lesion.
Learning Resources:
1. Pathologic Basis of Disease 8th Ed. Basics of Neoplasia Chapter 7
I.
MORPHOLOGY OF NEOPLASTIC CELLS

A. Objective 1
1. There is no single morphologic feature that defines a neoplastic cell. Many of the
cellular and nuclear features can be seen in cells that are proliferating in response to
wound repair, radiation and/or chemotherapeutic treatment, or infection.
2. Hypercellularity refers to an increased number of cells. There is usually a marked
increase in cell numbers as you go from normal cells to hyperplastic cells to
neoplastic cells.
3. Increased pleomorphism refers variability in the size and shape of cells and/or their
nuclei that is characteristic of malignant cells as seen in this lung carcinoma.
4. Anaplasia refers to large, bizarre looking tumor cells that lack morphologic evidence
of the lineage (histogenesis) of the tumor cells.
5. Loss of cellular polarity refers to the highly variable location of the nucleus in the
malignant cells (apical to basal) as compared to that of normal cells.
6. The nuclear to cytoplasm (N/C) ratio refers to the relative size of the nucleus as
compared to the volume of cytoplasm. Neoplastic cells tend to have an increased
N/C ratio as compared to normal cells. This is usually at the expense of the
cytoplasm, and is a fairly consistent feature of malignant cells. The increase N/C
ratio often yields the appearance of piling up of nuclei.
136
B. Objective 2
1. Hyperchromasia refers to dark blue staining on nuclei due to increased chromatin
condensation and/or increased DNA content. This is a fairly common feature of
neoplastic cells, but it is also a characteristic feature of lymphocytes. Compare the
nuclear staining in this cervical biopsy.
2. Multiple nuclei is not a common feature of most neoplasms, but is a characteristic
feature in some tumors such as this choriocarcinoma of the placenta (left image) and
giant cell tumor of bone (right image).
3. Piling up of nuclei can be seen in thicker tissue sections where there the tumor cells
have high N/C ratio, or in fine needle aspirations containing aggregates of cells.
4. Irregular chromatin distribution, often seen as nuclear clearing and irregular nuclear
membranes, is a common feature of neoplastic cells and reflects increased
chromatin material and abnormal chromosomes.
5. Increased mitoses reflect increased cell proliferation; this is especially true in
malignant tumors as compared to benign tumors. The finding of abnormal mitotic
figures is indicative of abnormal centrosomes within neoplastic cells.
6. Abnormal nucleoli refers to increased numbers of nuclei and/or irregularly shapes
nuclei, reflecting increased RNA synthesis. They are often seen as cherry red
macronucleoli. Note the enlarged nucleoli in neoplastic cells of this adenocarcinoma
of the colon.
A. Objective 3
1. Adaptive cellular changes can be confused with neoplastic changes.
a. Unwanted hyperplasia is associated with controlled cell proliferation and
increases the risk for developing a carcinoma. Excess estrogen stimulation can
lead to simple or complex hyperplasia of the endometrium. Estrogen can also
induce hyperplasia in the breast.
b. Metaplasia refers to the replacement of one mature cell type with another mature
cell type, increases the risk for subsequent progression to dysplasia that may
progress to a malignancy. These cells show little atypia and grow in an orderly
manner.
2. Dysplasia represents a disorderly cell growth and maturation that leads to
architectural disorganization.
a. Dysplasia is characterized by varying degrees of neoplastic cellular changes;
however, these changes are not definitive for the diagnosis as they can be seen
in reactive processes and adaptive cellular changes.
b. Abnormal basal appearing cells with hyperchromatic nuclei, prominent nucleoli,
high N/C ratio, and increased mitoses.
c. There is no invasion through the basement membrane.
137
d. Dysplasia is categorized as low to high grade (mild to severe) depending upon

how much of the thickness of the epithelium is replaced by abnormal basalappearing cells.
e. Dysplastic changes that do not involve the full thickness of the epithelium may be
reversible following removal of the inciting cause.
f.
Carcinoma in-situ (CIS) arises when dysplastic changes are marked and
encompass the full thickness of the epithelium, the lesion is considered to be preinvasive. This is commonly seen in the breast. The dysplastic cells exhibit
cytologic features of malignancy. However; the cells have not yet penetrated the
basement membrane. If left untreated, CIS may progress to an invasive
carcinoma.
g. Dysplasia does not inevitably progress to cancer, although dysplastic cell growth
is often found adjacent to foci of invasive carcinoma.
138
Dr. Dale D. Vandre

Cell Cycle Regulation
Tuesday 01/03/12
9:30-10:30

WINTER 2012
CELL BIOLOGY MODULE
Lecture: Cell Cycle Regulation
Hamilton Hall, Rm 002, 007
Learning objectives Be able to:
1. List the stages of the eukaryotic cell cycle, describe their function and duration, and define
the different types of checkpoints necessary to regulate progression through the different
stages of the cell cycle (Review).
2. Differentiate between the function of kinases and phosphatases in regulating entry into and
exit from mitosis.
3. Compare the protein amounts and the enzymatic activity of the components of maturation
promoting factor (MPF) during different stages of the cell cycle.
4. Describe the sequence of molecular events necessary to regulate activation and inactivation
of Cdk1/cyclin B complexes and their function in the transition from G2 phase to M phase.
5. Describe the functional role of the anaphase-promoting complex in regulating exit from
mitosis.
6. Differentiate between the molecular events associated with entry into mitosis versus those
associated with exit from mitosis
7. List the different Cdk/cyclin complexes, and associate each with the appropriate stage of the
cell cycle regulated by each complex.
8. Describe the molecular steps that control cdk4/cyclin D activity, and the effect of cdk4/cyclin
D phosphorylation of the Rb protein phosphorylation and progression through the cell cycle
9. Predict the consequences of mutations that cause gain of function, or loss of function, of
cdk1, cdk2, cdk4, cyclin B, cyclin D, cyclin E, cdc25 phosphatase, CAK, wee1, APC, Rb
protein, p21cip1, p27kip1, and p16ink4 on cell cycle progression and cellular growth
regulation.
Learning Resources
Essential Cell Biology (ECB) Lectures will cover material in Chapters 18 and 19.
Cancer Medicine 6th Edition web based book, see Chapter 3. Cell Proliferation and
Differentiation, and Chapter 4 Apoptosis and Cancer, by Kufe et al. See also the
Eurekah Bioscience Collection at the same address.
For a more in depth discussion of the material in Essential Cell Biology, see the
corresponding chapters in Molecular Biology of the Cell, which can also be accessed on
the web at the address below.
Open the following web address to access web based books:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books&itool=toolbar
139
Dr. Dale D. Vandre

Tuesday 01/03/12
9:30-10:30
Med1 IP, Cell Block, Division 2, Winter 2012

Regulation of the Cell Cycle Lecture notes
Dr. Dale Vandr
292-4181, dale.vandre@osumc.edu
Review Material
We have previously discussed the cell cycle and defined the stages of the cell cycle. As
a brief overview, the cell cycle refers to the orderly sequence of events in which a cell
duplicates its contents and divides into two. The eukaryotic cell cycle is divided into four
phases: 1) the period of time when cells are undergoing division (mitosis and cytokinesis) = M
phase; 2) the interval following completion of M phase, the G1 phase, where the cell is
transcribing genes, synthesizing proteins, growing in mass, and duplicating cell organelles; 3) S
phase, the period of time in which the cell is replicating DNA; and 4) the G2 phase, a
preparatory stage prior to mitosis. Movement through the cell cycle is progressive; however,
cells can delay progression through G1 phase for an extended period of time. These cells are
said to be in G0 phase, and effectively have been removed from the dividing population of cells
in the body. Cells can remain in G0 for days, weeks, or years, but can be stimulated to return to
the cell division cycle. All terminally differentiated cells such as neurons have lost the capacity
to divide and cannot return to the active cell cycle. The terminally differentiated state is
sometimes referred to as GD phase. The duration of the cell cycle varies greatly in normal cells
Intestinal epithelial cells may complete the cell cycle as often as once every 12 hours,
whereas a normal liver cell (a hepatocyte) may only complete the cell cycle once in every year.
Regulatory Conditions for Cell Cycle Control
Progression through the cell cycle is a highly regulated process. Why? Not only must cell cycle
processes occur in a sequential order, there are additional constraints known as the completion and
alternation problems. The following lists some of the conditions that must be regulated in order to
control cell cycle progression.
1.
2.
3.
4.
5.
The phases of the cell cycle must occur in sequential order.

Enzymes and proteins must be activated to carry out each process at the appropriate time.
These same proteins must be deactivated once the process is completed.
Completion problem. Need to ensure that each process is completed before the next process
begins. DNA synthesis must be complete before mitosis, mitosis must be complete before
cytokinesis, and cells must be of sufficient size before they divide.
Alternation problem. Processes like DNA synthesis must only be allowed to occur once per cell
cycle.
140
Dr. Dale D. Vandre

Tuesday 01/03/12
9:30-10:30
6.
7.
Cell cycle progression needs to be able to respond to extracellular signals, such as signals
stimulating cell division when more cells of a particular type are needed.
Damage to the cell or errors must be given time to be repaired or corrected before proceeding to
the next phase.
The Concept of Checkpoints

Control of cell cycle progression occurs by means of biochemical processes that sense completion
of critical events, and stop progression to the next phase of the cell cycle until completion of the previous
event. Such control mechanisms are termed cell cycle checkpoints. Most cells have a G1 checkpoint,
which monitors cell size, preventing progression until a critical size has been achieved. The G1
checkpoint can also be used to monitor external conditions and prevent committing cells to DNA
replication or S phase unless environmental conditions are favorable to support cell division. Once past
the G1 checkpoint a cell will normally proceed through the remainder of the cell cycle within12-24 hours.
Since this point in G1 is critical, and commits the cell to completing the cell cycle, it is often referred to as
Start or the Restriction point. Cells enter the G0 phase prior to reaching the restriction point. G2
checkpoints allow the cell cycle to halt prior to initiating mitosis. The DNA replication checkpoint
ensures that chromosome replication is completed prior to mitosis. The DNA damage checkpoint is
activated in response to DNA damage, and is capable of arresting the cell cycle in either G1 or G2 phase.
The spindle assembly checkpoint monitors attachment of all of the kinetochores to the mitotic spindle
prior to initiation of anaphase to ensure proper chromosome segregation. As a result, entry into mitosis
only occurs after all DNA is replicated and repaired, chromosome segregation only occurs after all
chromosomes (kinetochores) are attached to the mitotic spindle, and cells only complete cytokinesis after
mitosis (chromosome segregation) is completed.
Checkpoints therefore control key cell cycle transitions. Passage through a checkpoint commits
the cell to proceed through the next stage of the cell cycle. Activation of a checkpoint delays cell cycle
progression and can allow the cell time for repair of critical components. Checkpoint activation may also
lead to induction of programmed cell death or apoptosis if the damage is too severe and cannot be
repaired.
Cell-Cycle Control System.

Cell-cycle progression is governed by the phosphorylation of key regulatory proteins that initiate
DNA replication, mitosis, and cytokinesis. Protein phosphorylation and dephosphorylation are the
most common post-translational modification, and can lead to either the activation or inactivation of
cellular proteins. Enzymes that carry out phosphorylation reactions are designated as kinases. Enzymes
that carry out dephosphorylation reactions are designated as phosphatases. The most common amino
acids that are phosphorylated on proteins are serine, threonine, and tyrosine. There are two major classes
of protein kinases, the serine/threonine kinases and the tyrosine kinases. Protein kinases that regulate
the cell cycle are switched on at the appropriate time in the cell cycle and then switched off, whereas most
(but not all) phosphatase activity is less regulated. Phosphatases are generally less specific than kinases,
141
Dr. Dale D. Vandre

Tuesday 01/03/12
9:30-10:30
and many of these phosphatases are always active. Kinase activation will allow for phosphorylation of
substrates, and will overcome the basal phosphatase activity. The increased levels of phosphorylation will
be maintained until the kinase is inactivated and basal phosphatase activity removes the phosphate
residues. While the cell cycle regulatory kinases are always present in the cell, their activity rises and
falls in a cyclical fashion. This is accomplished in part through binding of regulatory proteins known as
cyclins, thus the regulatory kinases are known as cyclin-dependent kinases or Cdks. Cyclins have no
enzymatic activity themselves, but they must bind to Cdks before the kinases are active. Unlike the Cdks,
cyclin concentrations rise and fall during the cell cycle in a cyclical fashion. Thus the derivation of their
name cyclins. Cdk activity is further regulated by both phosphorylation and dephosphorylation
reactions (see below).
M-phase Promoting Factor (MPF): A Cyclin-CDK Regulatory Complex.
Discovery of MPF - Unfertilized eggs of the frog Xenopus are arrested in G2 phase of the first
meiotic division. If these cells are injected with a small amount of cytoplasm from a mitotic cell they are
rapidly driven into mitosis, whereas injection with cytoplasm from an interphase cell or cell undergoing
cytokinesis had no effect. The activity capable of inducing mitosis was called M-phase promoting
factor or MPF. By taking extracts from cells in different stages of the cell cycle it was shown that MPF
activity increased rapidly just before mitosis and fell rapidly to zero toward the end of mitosis.
When purified, MPF was shown to contain a kinase that phosphorylated key proteins responsible
for inducing chromosome condensation, nuclear envelope breakdown, and cause the microtubule
reorganization necessary to form the mitotic spindle. Additional biochemical studies identified proteins
that were gradually synthesized during interphase and were then destroyed at the end of mitosis, the
cyclins. Cyclin was also found to be required for MPF activity. Thus MPF contains two components a
regulatory subunit that is a mitotic cyclin, and a catalytic subunit that is a mitotic Cdk.
These same types of proteins were later found in other organisms including yeast and human cells,
indicating that this regulatory mechanism was highly conserved between organisms. In fact, yeast with a
defective mitotic cyclin or mitotic Cdk gene will fail to divide, but will divide normally if the appropriate
human gene is introduced into the defective cell. Therefore these proteins have functional
complementation across species. Many different cyclins and Cdks have subsequently been identified that
regulate progression through different stages of the cell cycle. The mitotic Cdk was the first discovered
and is known as Cdk1, and it will bind to three different cyclins, cyclin A, cyclin B, or cyclin B3.
Cdk1/cyclin A is involved in initiation of M phase, and Cdk1/cyclin B is involved in completion of
spindle assembly and progression to metaphase, and cyclin B3 is thought to play a role in anaphase.
Regulation of Cdk Activity by Phosphorylation/Dephosphorylation

While cyclin A or B binding to Cdk1 is required for activity of MPF, this binding is not sufficient to
activate MPF. Cyclins accumulate gradually during the cell cycle, but MPF switches on abruptly at the
end of interphase (G2 phase). Enzymatic activity of Cdk1 is tightly regulated by both phosphorylation
and dephosphorylation reactions. After binding its cyclin partner, Cdk1 is phosphorylated on two amino
acids, threonine 14 and tyrosine 15, which have inhibitory activity. Thus these phosphorylation sites are
142
Dr. Dale D. Vandre

Tuesday 01/03/12
9:30-10:30
considered to be negative regulatory sites since they inhibit or maintain the kinase in an inactive state.
Cdk1 is subsequently phosphorylated at a second regulatory amino acid, threonine 160. In contrast to the
Thr14 and Tyr15 sites, the Thr160 site has a positive regulatory effect on kinase activity, in other words it
stimulates enzymatic activity of the kinase. While phosphorylation of the activating site, Thr160, is
required for Cdk1 kinase activity, when Cdk1 is phosphorylated at both regulatory sites, Cdk1 is inactive.
Put another way, the negative regulatory phosphorylations at Thr14 and Tyr15 are dominant and keep the
CDK1 enzyme inactive even when the positive regulatory site at Thr160 is phosphorylated. Thus, the
final activation of Cdk1 requires the dephosphorylation of the negative regulatory or inhibitory
phosphorylation sites located on Thr14 and Tyr15. As a result of this complex regulatory control
mechanism, stockpiles of inactive Cdk1/cyclin A or Cdk1/cyclin B can be rapidly converted from the
inactive form to an active form by the action of an activating phosphatase that dephosphorylates Thr14
and Tyr15. Once a few molecules of Cdk1/cyclin complexes are activated, the kinase will phosphorylate
and activate more molecules of the activating phosphatase. This represents a positive feedback loop that
increases the activity of the activating phosphatase, and results in a sudden explosion of MPF
(Cdk1/cyclin) activity as the cell moves into M phase.
Important Cdk/cyclin regulatory proteins

wee1 nuclear kinase inhibits cdk1 phosphorylates tyrosine 15 (Tyr15).
myt1 cytoplasmic kinase inhibits cdk1 phosphorylates both threonine 14 and tyrosine 15 (Thr14,
Tyr15).
CAK (Cdk activating kinase) kinase promotes cdk activity phosphorylates threonine160
(Thr160).
cdc25b phosphatase promotes G2 to M phase dephosphorylates Thr14 and Tyr15.
cdc25a phosphatase promotes G1 to S phase.
Turning Off Cdk Activity by Cyclin Destruction

Degradation of proteins normally occurs in the lysosomes and is usually a continual process. A
second form of protein degradation has been identified that rapidly destroys regulatory proteins such as
cyclins. These regulatory proteins contain a series of 8-10 amino acids, termed the destruction box,
which labels them for degradation. For example, one protein substrate of activated Cdk1/cyclins are the
specific cyclin of the cdk/cyclin complex. Thus Cdk1/cyclin A will phosphorylate cyclin A, Cdk1/cyclin
B will phosphorylate cyclin B, and so on. Phosphorylation of cyclin A or B triggers a series of events that
lead to cyclin destruction. The phosphorylated cyclin protein (and only the phosphorylated version) is
recognized by ubiquitin ligase an enzyme that covalently attaches the small protein ubiquitin to the
cyclin. Once a protein has been polyubiquinated it is directed to a large RNA-protein complex called the
proteasome, where it is degraded by resident proteases. A large protein complex within the proteasome
is the anaphase promoting complex or APC. The APC is activated following phosphorylation of a
regulatory subunit of the APC by cdk1/cyclin complexes. Thus, activation of a Cdk/cyclin complex also
initiates the destruction of the regulatory cyclin, turning off the active Cdk complex. The Cdk/cyclin
complexes are activated sequentially, (Cdk1/cyclin A prior to Cdk1/cyclinB prior to Cdk1/cyclin B3), and
they are inactivated in the same order. For example, destruction of cyclin B is closely associated with the
143
Dr. Dale D. Vandre

Tuesday 01/03/12
9:30-10:30
triggering event for the initiation of anaphase, the so-called anaphase trigger. Once anaphase has been
initiated, the cell is committed to enter interphase.
Other Cell Cycle Stages and Cyclin/Cdk Regulation

Other cyclin/Cdk pairs function to initiate and control progression through various cell-cycle
stages. Cdk4/cyclin D and Cdk6/cyclin D pairs respond to extracellular signals and initiate the exit from
G0 phase and starts progression through the restriction point in G1 phase. Cdk2/cyclin E completes the
progression through the restriction point and regulates initiation of S phase, and Cdk2/cyclin A regulates
progression through S phase.
Kinase
Cdk1
Cdk1
Cdk1
Cdk2
Cdk2
Cdk3
Cdk4/Cdk6
Cdk5
Cdk7
Cdk8
Cdk9
Cyclin
A
B
B3
E
A
C
D1, D2, D3
G
H
C
T
Function
G2-early M phase
G2-late M phase
Anaphase
G1-S phase, completion of restriction point
S-G2 phase
G0-G1 phase
initiates passage of restriction point
neuronal differentiation
CAK
RNA polymerase II transcription
RNA polymerase II transcription
Cdk inhibitory proteins

One checkpoint method that blocks the assembly of or activity of certain Cdk/cyclin complexes
involves what are referred to as CDK Inhibitory proteins or CKIs. One example occurs in response to
damaged DNA. A G1 checkpoint arrests the cell to prevent replication of damaged DNA. Damaged
DNA causes the activation of kinases known as ATM and ATR. These kinases lead to the
phosphorylation of subsequent stabilization, and increase concentration of a transcription factor known as
p53. Activated p53 causes increased transcription of the gene for a Cdk inhibitory protein known as
Cip1/Waf1
Cip1/Waf1
p21
. The p21
protein inhibits Cdk2/cyclin E complexes arresting the cell in G1 phase.
The p53 protein is mutated in most human cancers. The p21
p27
Kip1
, and p57
Kip2
Cip1/Waf1
protein, and other family members
inhibit all Cdk kinases except Cdk4 and Cdk 6.
Another family of Cdk inhibitors are the four members of the INK4 family of proteins (Inhibitors
of Cdk4), which exclusively target Cdk4 and Cdk6 by disrupting association with D type cyclins.
Ink4a
Ink4b
Mutations in p15
and p16
proteins have been linked to human tumors including hereditary
melanoma.
144
Dr. Dale D. Vandre

Tuesday 01/03/12
9:30-10:30
Controlling Passage Through the Restriction Point: Rb the Gatekeeper of the Restriction Point
The Rb (retinoblastoma) gene codes for a large protein (~150,000 Daltons), which binds to and
regulates a family of transcription factors known as the E2F family. The Rb protein binds to and
inactivates E2F transcription factors preventing them from stimulating mRNA synthesis of specific
growth regulatory genes such as cyclin E. Thus Rb functions as a growth suppressor. To pass the
restriction point of the cell cycle, Rb must be inactivated each time through the cell cycle in order to
release E2F, allowing transcription of key cell cycle regulatory proteins including cyclin E and cyclin A.
Loss of the Rb gene function would therefore result in the constant activity of the E2F transcription
factors and continued synthesis of key growth regulatory proteins. Several DNA tumor viruses produce
proteins in infected cells that inactivate Rb, contributing to their ability to cause tumor formation.
The activity of normal Rb protein is regulated by Cdk/cyclin phosphorylation. The Rb protein is
unphosphorylated in G0 phase and it binds to and inhibits E2F transcription activity by sequestering the
E2F protein. Growth factor stimulation through receptor signaling pathways can lead to the transcription
of cyclin D and activation of Cdk4/cyclin D proteins. The D type cyclins are the only cyclins that are
regulated by extracellular signals; all other cyclins are controlled by programmed synthesis and
degradation mechanisms similar to those discussed above for Cdk1/cyclin B.
Rb protein is a substrate for phosphorylation by the Cdk4/cyclin D complex, and the Rb protein
becomes hypophosphorylated (a low level of phosphorylation) by Cdk4/cyclin D, Hypophosphorylated
Rb, however, still binds and inactivates E2F transcription factors. After hypophosphorylation by
Cdk4/cyclin D, Rb becomes a substrate for Cdk2/cyclin E. Additional phosphorylation by Cdk2/cyclin E
leads to the hyperphosphorylation (a high level of phosphorylation), of the Rb protein.
Hyperphosphorylation of Rb is required for the release of the E2F transcription factors necessary for
passage through the restriction point and the induction of S phase.
One of the genes upregulated by the E2F transcription factor is cyclin E. As a result, cyclin E
levels increase, which forms more Cdk2/cyclin E complexes to drive hyperphosphorylation of Rb protein.
Thus, a strong positive-feedback loop is triggered as cells pass through the restriction point. A second
positive-feedback further contributes to activation of Cdk2/cyclin E complexes. One substrate of
Kip1
Kip1
. Phosphorylation of p27
leads to its ubiquitination and destruction
Cdk2/cyclin E is the CKI p27
by the proteasome. Loss of this inhibitor of Cdk2/cyclin E triggers activation of more of the complex.
The combination of these positive-feedback mechanisms makes passage through the restriction point
essentially irreversible.
How is the passage through the restriction point initiated (or how is the kinase activity of cdk4
turned on)? Growth factor or mitogenic signal pathways must be activated to induce and maintain high
levels of cyclin D. Cyclin D is rapidly turned over or degraded, and when a growth factor/mitogenic
signal is withdrawn, the levels of cyclin D rapidly decrease. If growth factor/mitogenic signals are
withdrawn prior to passage through the restriction point, the Rb protein does not become
hypophosphorylated by Cdk4/cyclin D, or if the growth signal is not maintained long enough
hypophosphorylated Rb protein is dephosphorylated and is no longer a good substrate for Cdk2/cyclin E.
Once the cell has passed the restriction point, Rb protein is maintained in a hyperphosphorylated state by
145
Dr. Dale D. Vandre

Tuesday 01/03/12
9:30-10:30
the sequential activity of Cdk2/cyclin E, Cdk2/cyclin A, Cdk1/cyclin A, and Cdk1/cyclin B complexes.
Upon inactivation of the Cdk1/cyclin B at the end of mitosis, Rb phosphorylation ends, and phosphatases
convert the hyperphosphoryated Rb protein back to the unphosphorylated form. Rb once again binds E2F
transcription factors until the next round of the cell cycle is completed.
Cancer
Cancer, in part, develops as a result of the deregulation of normal cell growth control mechanisms.
Normal cell growth in tissues is a balance between signals that stimulate either proliferation or apoptosis
(programmed cell death). Therefore, loss of growth control in cancer cells may be the result of an
increased rate of cell proliferation or a decreased rate of cell death. Defects in the machinery that
regulates cell cycle progression can obviously contribute to an increase in cell proliferation. Almost all
human tumors have deregulated Rb control mechanisms. These mechanisms include mutation of the Rb
gene, increased levels of cyclin D, and inactivation of Rb by the viral proteins such as the E7 oncoprotein
of DNA tumor viruses (see next lecture on oncogenes). Similarly, defective cell cycle checkpoints can
allow for the proliferation of cells with damaged DNA or abnormal mitotic spindles, which will
contribute to the proliferation of defective cells. As we have also discussed, defects in the proteins
responsible for activating cell death pathways can allow for the continued proliferation of cells that should
otherwise be terminated.
146
Dr. Dale D. Vandre

Oncogenes and Tumor Suppressor Genes
01/03/12, Tuesday & 01/04/12, Wednesday
10:30-11:30
8:30-10:00

WINTER 2012
CELL BIOLOGY MODULE
Lecture: Oncogenes and Tumor Suppressor Genes
Hamilton Hall, Rm 002, 007
Learning objectives- Be able to:
1. Differentiate between the following terms: oncogene, proto-oncogene, and tumor suppressor
gene.
2. Based upon the life cycle of a tumor, relate the number of cells in the tumor mass with
number of tumor cell doublings, diagnostic capabilities, and potential therapeutic response.
3. Distinguish between the various molecular mechanisms underlying unregulated activation of
proto-oncogenes.
4. Identify the genes and their functions, which are part of the genome of an oncogenic
retrovirus.
5. Describe the mutations, as well as the functional consequences of these mutations, which
are responsible for the oncogenic properties of the src and ras genes.
6. Predict how different mutations in the Rb gene, or how alteration of Rb gene function, would
regulate cell growth and contribute to tumorigenesis.
7. Relate how mutations in p53, or how alteration in p53 gene function, could alter regulation of
apoptosis and tumorigenesis.
8. Classify the different families of oncogenes and relate them to signal transduction pathways.
Learning Resources
Essential Cell Biology (ECB) Lectures will cover material in Chapter 21, pages 726-738.
Cancer Medicine 6th edition, web based book, see address below (see also appropriate
topics in Molecular Biology of the Cell 4th Edition, and Eurekah Bioscience Collection.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books
Click on Cancer Medicine 6th edition logo, type in - Discovery and identification of oncogenes in
the search box, open the fourth item from the list: Discovery and identification of oncogenes
Click on Cancer Medicine 6th edition logo, type in mechanisms of oncogene activation, open
item 11 on the list Mechanism of oncogene activation
Click on Cancer Medicine 6th edition logo, type in retinoblastoma paradigm, open item 1 on the
list RetinoblastomaA Paradigm for Tumor-Suppressor Gene Function
Click on Cancer Medicine 6th edition logo, type in phosphoprotein p53, open item 3, The p53
gene
147
Dr. Dale D. Vandre

10:30-11:30
8:30-10:00
Med1 IP, Cell Block, Division 2, Winter 2012

Oncogenes and Tumor Suppressor Genes lecture notes
Dr. Dale Vandr
292-4181, dale.vandre@osumc.edu
Basic Definitions:
A gene, which upon mutation, produces a protein that is over expressed or hyperactive resulting in
excessive cell proliferation is defined as an oncogene. Oncogenes are typically components of signal
transduction pathways, and over 100 oncogenes have been identified. The normal functional gene
counterpart is known as a proto-oncogene. Normal diploid cells have two copies of most genes.
Generally, a mutation in only one copy of a proto-oncogene is sufficient to disrupt growth regulation.
Therefore oncogenic mutations are thought to be dominant, and tumorigenesis results from the gain of a
function in the mutated oncogene.
A mutation that inactivates a gene that has antiproliferative function can also lead to excessive
proliferation. In this case, the normal gene is referred to as a tumor-suppressor gene. In contrast to
proto-oncogenes, usually both copies of a tumor-suppressor gene must be mutated or inactivated for an
effect on growth regulation to become apparent. Tumorigenesis thus results from a loss of function of a
tumor-suppressor gene.
An oncogene can be generated from many of the genes encoding proteins involved in signaling
pathways that are involved in the response to growth factors or mitogens. The result being an overactive
gene product that over stimulates or maintains the growth regulation signaling pathway. Overactive gene
products can result from a number of different processes including the protein being produced: 1) in
excessive amounts (the activity of the protein is normal there is just too much of the protein which
generates a larger than normal proliferation signal or maintains the signal too long); 2) in a form whose
activity is not controlled (for example, the protein enzymatic activity cannot be shut off), or 3) by cells
that normally do not make it (for example, the inappropriate synthesis of an autocrine or selfstimulatory growth factor). In a similar fashion, mutations in proteins that regulate cell death (apoptosis)
can promote cell survival even in the absence of signals necessary to maintain cell survival.
Activation of Oncogenes
Three primary mechanisms underlie the conversion of a cellular proto-oncogene into an
oncogene: 1) Mutation, 2) Gene amplification, and 3) Chromosome rearrangement. Each of these
mechanisms may result in the unregulated activation of the proto-oncogene (in the case of tumor
suppressor genes the result is an inactivation of the gene). Mutations include DNA sequence base
substitutions, primarily single base changes or point mutations, which are one of the most common types
of mutations associated with human tumors. Deletion or insertion of bases in the gene sequence is
148
Dr. Dale D. Vandre

10:30-11:30
8:30-10:00
another mutational mechanism. Gene amplification results in the expansion of the copy number of the
gene, thus increased amounts of the protein product are produced. Amplified genomic DNA can contain
upwards of several hundred copies of the gene. Oncogenes commonly amplified include the myc
oncogene, which is found amplified in 20-30% of breast cancer, erbB in 50% of glioblastomas, and ras in
15-30% of breast cancer. Chromosome rearrangements are most frequently found in hematological
tumors and can result in transcriptional activation or production of a chimeric or fusion gene product.
Transcriptional activation involves the movement of a proto-oncogene to a region under regulatory
control of another gene like an immunoglobulin or T-cell receptor gene. Formation of an abnormal
chimeric protein with oncogenic activity also usually results from rearrangements of immunoglobulin or
T-cell receptors genes with the coding portions of oncogenes. For example the fusion of the tyrosine
kinase domain of abl with bcr results in the formation of a new protein product the bcr/abl oncogene.
Oncogenic Retroviruses
Retroviruses are a class of RNA viruses that have the capacity to induce neoplastic
transformation. They replicate within cells via a DNA intermediate, known as a provirus, which
becomes integrated into the host chromosomal DNA or genome. It is the ability to integrate into the host
genome that is key to the oncogenic potential of the retrovirus. Upon infection the retroviral RNA is
copied into DNA by viral reverse transcriptase, which is carried by the viral particle. The proviral DNA
is replicated along with the host DNA during S phase, and is also transcribed into progeny viral RNA and
viral mRNA for viral proteins. A typical retrovirus genome consists of transcription enhancer regions in
the long terminal repeat (LTR) sequence followed by coding sequence for three genes. The gag gene
codes for internal structural proteins, the pol gene codes for the viral reverse transcriptase, and the env
gene codes for the viral particle surface glycoprotein.
Most commonly, retroviruses induce cellular transformation as a result of the location within the
host genome of the proviral DNA. Proviral insertion may result in: 1) placement of LTR enhancer
sequences in proximity of a cellular proto-oncogene resulting in disruption of normal regulation of the
proto-oncogene. 2) insertion within the coding sequence of a cellular proto-oncogene resulting in the
production of an altered protein product that may have lost critical regulatory regions. 3) insertion into
regions of the gene that regulate proto-oncogene mRNA stability or protein stability, resulting in longer
lived and/or greater amounts of protein.
Some retrovirus genomes may also contain a fourth region that codes for an oncogenic protein.
This tumor inducing region of the viral genome is known as the onc gene. These are cellular protooncogene sequences that were captured by the retrovirus genome after insertion of the provirus in close
proximity to the cellular proto-oncogene. When the viral genome was transcribed the coding sequence
for cellular protein was picked up and included in the packages retrovirus. This is usually accompanied
by mutation of the proto-oncogene sequence that results in production of a mutated gene product with
higher, or unregulated, activity. The first oncogenes were identified in retroviruses. When normal
cellular homologues were found they were designated as the c-onc or proto-oncogene, and the mutated
version carried by the retrovirus was designated v-onc for the viral oncogene.
149
Dr. Dale D. Vandre

10:30-11:30
8:30-10:00
Examples of Oncogenes
The Src gene
In 1911 Francis Rous isolated a virus that causes cancer in chickens, later called Rous Sarcoma
Virus. This was the first isolation of a tumor causing virus. Other related viruses did not cause cancer,
and it was hypothesized that the Rous Sarcoma Virus contained an extra gene that was oncogenic. With
the development of molecular techniques, it was shown that the transforming virus contained an extra
gene named v-src (viral-sarcoma). A similar gene product was identified in normal cells, and was
designated c-src (cellular-src). The viral oncogene is therefore v-src, and the normal cellular protooncogene c-src. Analysis of the two genes indicated that they both code for membrane associated
proteins of 60,000 Daltons. The only differences in the coding sequences between c-src and v-src
includes the substitution of the C-terminal 19 amino acids in c-src with 12 unrelated amino acids in v-src.
Thus when the ancestral Rous sarcoma virus picked up the src gene from the chicken genome, a small
portion of the normal gene was altered. This small change is enough to alter the regulation of the protein,
and upon infection and expression of the v-src gene the infected cells lose growth control and proliferate
uncontrollably.
Src proteins are tyrosine kinases, and were the first tyrosine kinases identified. Myristic acid, a
14-carbon fatty acid, is covalently attached to the N-terminus of Src. Myristilation enables Src to attach
to the cytoplasmic face of the plasma membrane. Binding to the plasma membrane is required for the
transforming activity of v-src. The major difference between v-src and c-src is their kinase activities. Vsrc activity is ~20 times greater than that of c-src. In v-src the deregulation of kinase activity is primarily
due to the loss of a phosphorylated tyrosine at residue 527, which is present in the C-terminus of c-src,
and the phosphorylation of tyrosine residue 416 that is not normally phosphorylated in c-src. While
some substrates for Src have been identified, the phosphorylated substrate proteins have not been
identified that are specifically responsible for cellular transformation by v-src.
Src kinase is normally activated through growth factor dependent receptor tyrosine kinase
pathways. As you will recall, a consequence of growth factor binding is the phosphorylation of the
cytoplasmic tail of a receptor tyrosine kinase. This modification allows for the binding of cellular
proteins to the phosphorylated cytoplasmic tail of the receptor molecule. One of the first proteins
identified that binds to activated receptor tyrosine kinases was c-src. This binding is mediated through a
binding region on Src spanning ~100 amino acids known as the SH2 domain (Src-homology 2). Similar
SH2 and the related sequences known as SH3 domains have been identified in many proteins that bind to
receptor tyrosine kinases. A family of other kinases related to Src have been identified, and include Src,
Fyn, Yes, Fgr, Lek, Yrk, Hek, Lyn, and Blk, making up a group of signaling kinases known as the
membrane-associated non-receptor tyrosine kinases.
The Ras gene

The oncogenic potential of this protein was first discovered in the study of rat sarcomas (Ras). In
fact, the protein was first identified as an oncogenic protein prior to the discovery of its normal cellular
150
Dr. Dale D. Vandre

10:30-11:30
8:30-10:00
counterpart. Two murine sarcoma viruses, Harvey murine sarcoma and Kirsten murine sarcoma, have
been identified that carry the v-ras gene. Ras oncogenes encode for mutated forms of a cell signaling Gprotein known as c-ras. There are three Ras genes in humans designated as H-ras, K-ras, and N-ras. A
single point mutation at amino acid residue 12 or 61 can convert a cellular form of Ras into an oncogenic
form. Mutations present in the Ras oncogene lead to disruption of the GTPase activity of the subunit of
the G-protein. Thus increased Ras activity is related to its transforming activity. In fact, overexpression
of c-ras by increasing the mRNA as little as ~10 times is capable of transforming some cells. Nearly 30%
of all human tumors have mutations in their Ras genes, making it one of the most common mutations
found in human cancers.
Proto/
Oncogene
Amino acid at
12 59 61
Source of tumor
c-ras(H,K,N)
H-ras
Gly Ala
Gly Ala
Val Ala
Cys Ala
Arg Ala
Val Ala
Gly Ala
Gly Ala
wild type protooncogene

lung carcinoma Hs242
bladder carcinoma T24
lung carcinoma Calu-1
lung carcinoma LC-10
colon carcinoma SW480
neuroblastoma SK-N-SH
lung carcinoma SW1271
K-ras
N-ras
Gln
Leu
Gln
Gln
Gln
Gln
Lys
Arg
Retroviruses and Human Cancer

While we have discussed two animal retroviruses and the oncogenes that they carry in their
genome, the search for oncogenic human retroviruses has not identified retroviruses with oncogenic
potential similar to that of the animal retroviruses. Although about 1/5 of human cancers can be linked to
viruses, in fact very few human cancers appear to be caused directly by retroviruses. Unlike retroviruses,
human oncogenic viruses are usually DNA containing viruses (see below). For example, the human
papilloma virus HPV16 responsible for some types of cervical cancer, Epstein-Barr virus associated with
some types of nasopharyngeal carcinoma, and human herpesvirus-8 associated with Kaposis sarcoma are
DNA viruses that introduce viral oncogenes that contribute to tumorigenesis. Hepatitis B and hepatitis C
viruses are associated with liver cancer, but primarily as a result of chronic inflammation associated with
viral infection.
The Rb gene as an Example of a Tumor-suppressor Gene
Retinoblastoma is a childhood disease that takes the form of tumors on the retina. It occurs as a
heritable trait and sporadically; however, the sporadic form is extremely rare. Retinoblastoma occurs
only when both copies of the Rb gene are inactivated. In the inherited form of the disease, one parental
copy of the gene is defective. A somatic mutation in the remaining functional copy of the gene in retinal
151
Dr. Dale D. Vandre

10:30-11:30
8:30-10:00
cells results in tumor formation. This supports the two-hit hypothesis, which states that two mutations
are needed to induce tumors.
Genotype
+
Rb Rb
Rb Rb
Rb Rb
Phenotype
+
Normal individual, 2 Rb alleles

Loss of one allele, somatic cells no effect, germ cells creates
carrier
Loss of second allele induces tumor
formation
As we have already discussed, the Rb gene codes for a large protein (~150,000 daltons) that
functions as a growth suppressor by binding to and sequestering a family of transcription factors known
as E2F. Phosphorylation of Rb by Cdk4/cyclin D and Cdk2/cyclin E results in hyperphosphorylation and
release of the E2F transcription factors. Release and function of the E2F transcription factors allows cells
to progress through the restriction point of the cell cycle.
In addition to mutation and inactivation of the Rb gene, and disregulation of its phosphorylation
during the cell cycle, a third mechanism exists to disrupt Rb regulation of the restriction point. Upon
infection by certain DNA tumor viruses, viral genome encoded proteins are expressed such as the E7
oncoprotein of certain human papillomavirus associated with cervical cancer or the E1A oncoprotein of
human adenovirus type 5 that bind to unphosphorylated or hypophosphorylated forms of the Rb protein.
The binding of E7 or E1A oncoproteins to Rb sequesters and inactivates the Rb protein, and can induce
Rb protein degradation. Thus, while functional Rb protein is in the infected cell, the DNA tumor virus
oncoproteins inactivate the protein, which results in the release and activity of E2F transcription factors
and progression through the restriction point.
The p53 tumor Suppressor Gene: Regulator of Apoptosis and Tumorigenesis

The p53 protein is the most extensively studied of the tumor suppressor genes, and it is the most
commonly mutated tumor suppressor gene in human cancer (>50% of all human tumors have mutations in
the p53 gene). The protein has antiproliferative activity that promotes checkpoint activation when it is
activated in response to cellular stresses such as DNA damage, oncogene expression, and hypoxia.
Disruption of the gene promotes checkpoint defects, immortalization of cells, genomic instability, and
inappropriate cell survival. The tumor suppressor activity of p53 is closely linked to its ability to trigger
apoptosis (see next lecture on apoptosis). p53 is a transcription factor that binds to DNA in a sequencespecific fashion, and regulates the expression of proapoptotic genes including bax and bid, the apoptotic
adaptor gene Apaf-1, and effector gene caspase 6. The p53 gene also appears to regulate extrinsic
pathways of apoptosis by sensitizing cells to death receptor ligands. The function of p53 is highly
regulated and context dependent. In other words, p53 regulates apoptosis and cell cycle arrest in different
152
Dr. Dale D. Vandre

10:30-11:30
8:30-10:00
ways in different cell types depending upon the regulatory pathways that function in the specific cell type.
Thus there is no universal response to loss of p53 activity. In some tumors with wild type p53, the
downstream effectors of p53 activity are defective. In other tumors the cells are defective in apoptosis,
but not cell cycle arrest. In different tumors, the p53 network may be disabled at distinct points or times
in progression of tumorigenesis, which may further contribute to tumor heterogeneity. Like the DNA
tumor virus oncoproteins that bind to and inactivate Rb protein, the human papilloma virus produces the
oncoprotein E6 that inactivates functional p53, allowing cells to replicate despite having mitotic spindle
abnormalities.
Multistep Process of Neoplastic Transformation

The mouse skin model of carcinogenesis indicates that neoplastic transformation is a multistep
process that requires the mutation of several genes. A single treatment of mouse skin with a chemical
mutagen such as 7,12 dimethyl-benzanthracene (DMBA) has been shown to cause a mutation in H-ras,
but tumors do not develop. This is known as tumor initiation, and is an irreversible step. Treatment of
the DMBA treated skin with a proliferation-promoting compound such as the phorbol ester TPA rapidly
induces the formation of benign papillomas from the initiated skin cells. This second step is known as
promotion. After extended exposure to TPA some of the benign papillomas will develop into malignant
carcinomas indicating an additional step in tumor development known as progression. Analysis of other
benign skin papillomas showed that H-ras was commonly mutated and activated. Thus H-ras activation is
an early step in carcinogenesis that occurs in premalignant lesions.
Statistical evidence supports the hypothesis that 5-6 independent mutations are required for the
development of solid human tumors, whereas 3-4 mutations are required in human leukemia. Cells from
a single tumor are likely to have mutations in both proto-oncogenes as well as tumor suppressor proteins.
They are also likely to have mutations in proteins that regulate programmed cell death or apoptosis.
Families of Oncogenes
Oncogenic proteins have been identified at each major step in a typical signal transduction
pathway. The major classes of oncogenic proteins are listed below:
1)
Growth factors
2)
Tyrosine kinase growth factor receptors
3)
Membrane-associated non-receptor tyrosine kinases
4)
Non-tyrosine kinase growth factor receptors
5)
G-proteins
6)
Cytoplasmic regulators
7)
Cytoplasmic protein kinases
8)
Nuclear transcription regulators
9)
Cell cycle regulatory proteins
153

Lecture:
Neoplasia #2 Benign vs. Malignant Tumors and Nomenclature #1

1/4/12 10:00 11:30
1. Distinguish between a benign and a malignant tumor based its gross and/or microscopic
features image, and be able to predict its biologic and clinical behavior.
a. Categorize a tumor as being well differentiated, poorly differentiated and anaplastic
based on its morphologic features.
b. Define appearance of metastases on peritoneal surfaces, in lymph nodes and solid
organs, and how carcinomas differ from sarcomas in route of primary metastasis.
2. Distinguish between benign and malignant epithelial based on their gross and microscopic
features. Focus on these.
a. Squamous cell carcinoma vs. squamous papilloma
b. Squamous cell carcinoma vs. adenocarcinoma
c. Adenocarcinoma vs. adenoma
d. Transitional vs. squamous and gland epithelial neoplasms
Learning Resources:
II. CHARACTERISTICS OF BENIGN AND MALIGNANT NEOPLASMS

A. Objective 1
1. There are several criteria that differentiate malignant from benign tumors. Tumors of
intermediate behavior exhibit some of these features.
2. Benign and malignant tumors exhibit several features that help to differentiate them
from each other.
Feature
Benign
Malignant
Invasiveness
Seldom
Common
Capsule
Often
Seldom
Margin
Pushing or expansile
Infiltrative
Growth Rate
Slow
Rapid
Differentiation
Well
Well to anaplastic
Metastases
No
Yes
Fatal
Rare
Common if untreated
3. Invasiveness
a. Invasiveness is a reliable feature that helps to define the malignant behavior of a
tumor. Fingers of invading tumor cells help to define a tumor as being malignant.
154
b. Benign tumors often have a pushing or expansile margin that does not infiltrate
into the adjoining tissue. In contrast, malignant tumors have a poorly defined
infiltrative margin. Which one would be easier to remove?
c. Fibrous tumor capsules are commonly associated with benign tumors. Invasion
through the capsule is indicative of a malignant behavior. Many benign tumors
are encapsulated, i.e. surrounded by a connective tissue capsule.
d. Malignant tumors are usually unencapsulated and invade into the adjacent
normal tissue.
4. Tumor growth is influenced by the rates of cell death (apoptosis) and cell proliferation.
The latter being associated with increased telomerase activity, mutations of tumor
suppressor genes, and activation of oncogenes. The rate of growth of benign tumors
is less than that of malignant tumors.
9
a. Classically, the smallest clinically detectable mass is 10 cells (1 gm) which
represents about 30 population doublings.
1) It is generally accepted that the proliferation rate as measured by the number
of mitotic figures is higher in a malignant tumor as compared to the benign
tumor. However; as tumors progress, the fraction of proliferating cells in a
tumor decreases, which impacts on the utility of chemotherapy directed at
inhibiting cell proliferation.
2) Malignant tumors tend to be more cellular than their benign counterparts.
However; malignant tumors also tend to have more apoptotic neoplastic cells.
3) The rate tumor growth, both proliferation and apoptosis, is dependent upon
trophic factors and blood supply.
5. Tumor differentiation refers to how close tumor cells resemble comparable normal
cells, both morphologically and functionally.
a. Think of differentiation as how much a painting differs from a photograph of the
same subject.
6. Metastases are defined as neoplastic implants that are discontinuous with the
primary site. Benign tumors lack the ability to metastasize, whereas malignant
tumors are defined by this ability.
a. The likelihood of metastasis increases with large, growing tumors, and anaplasia.
There three routes of metastasis.
b. Hematogenous metastasis is the primary route of metastasis for sarcomas, and
tends to occur after lymphatic spread.
c. Lymphatic spread to regional and distant lymph nodes is the typical route of
metastasis for carcinomas.
1) The sentinel lymph node (SLN) concept is based on the premise that these
are the first node (s) encountered by metastatic tumor cells. Tumor is
injected with radioactive dye SLN is detected by vision and hearing (Geiger
counter) SLN removed if negative surgery is stopped.
155
a. Seeding body cavities occurs when the tumor invades a body cavity (e.g.
carcinomas of the ovary and colon and mesothelioma).
III. NOMENCLATURE OF NEOPLASIA
A. Objective 2
1. Tumors are classified on the basis of their cell of origin, histogenesis.
a. The following are general rules in naming a tumor:
b. Suffix of oma tend to equate with a benign behavior there are exceptions.
c. The term carcinoma means a malignant epithelial tumor.
d. The term sarcoma means a malignant mesenchymal tumor.
e. The term blastoma refers to a malignant tumor of embryonic cell of origin.
2. Epithelial cells give rise to approximately 85% of all neoplastic lesions in the body.
3. Epithelial tumors are classified according to their growth patterns and histogenesis,
i.e. epithelial cells of origin.
4. Varying growth patterns of neoplastic epithelial cells are used to describe the benign
and malignant epithelial tumors (see below).
a. Polyp refers to a projection above a mucosal surface. It is a non-specific term
that doesnt describe behavior.
b. Papillary refers to finger-like or warty projections with fibrovascular core lined by
neoplastic cells. They have been described in multiple organs.
1) Papillomas are usually exophytic superficial benign tumors with a broad base.
2) Papillary carcinomas have the same growth pattern, but the lined epithelial
cells are malignant.
c. Cyst or cystic refers to fluid filled spaces lined by benign or malignant epithelium.
5. In most cases, epithelial tumors are classified on the basis of the histologic cell type
(histogenesis) of that underwent neoplastic transformation.
TISSUE
Squamous
Gland / ducts
Transitional epithelium
Renal epithelium
Neuroendocrine
BENIGN BEHAVIOR
Papilloma
Adenoma
Papilloma
Cystadenoma
Papilloma
Adenoma
Carcinoid
MALIGNANT BEHAVIOR
Squamous cell carcinoma
Adenocarcinoma
Papillary carcinoma
Cystadenocarcinoma
Transitional cell carcinoma
Renal cell carcinoma
Small cell carcinoma
Neuroendocrine carcinoma
6. Squamous cell tumors arise from squamous epithelium found in multiple sites skin,
mouth, cervix and vagina, larynx and true vocal cords, esophagus, and anus - or
from squamous metaplasia glandular epithelium from various sites respiratory tract,
urinary bladder, or endocervix.
156
a. Benign squamous papillomas are cover with multiple layers of neoplastic cells,
but lack stromal invasion. In contrast, squamous cell carcinoma contains
invasive nests and islands of malignant neoplastic cells.
b. Malignant squamous cells may be large, with a low N/C ratio, with a pavementstone-like appearance or in whorls, or they can be small with a high N/C ratio.
c. A neoplastic lesions arising from squamous cell are characterized by the
presence of intercellular bridges that are normally seen in stratified squamous
epithelium such as the epidermis. However, these structures may be hard to
identify in poorly differentiated squamous cell carcinomas.
d. Classic squamous cell carcinomas are keratinized due to the aggregation of
cytoplasmic keratin filaments. This gives the tumor cells a hard look. Keratin
pearls represent concentric layers of keratinized malignant cells. The malignant
cells may also form concentric whorls that are not as dense as the pearls.
e. Malignant squamous cells can be small (high N/C ratio), often with individual
keratinized cells (dyskeratotic cells), or no keratinization, but still with intercellular
bridges.
7. Glandular epithelium is found in endocrine and exocrine glands scattered throughout
the body including the GI tract, respiratory tract, liver, pancreas, gall bladder, kidneys,
uterus, ovaries, prostate, and breast.
a. Adenomas are benign tumors arising from glandular epithelium (e.g. thyroid
adenoma), mucosal epithelium (colonic polyp with a villous adenoma), or from
parenchymal epithelial cells (hepatic adenoma).
b. Adenocarcinomas are malignant tumors arising from a mucosal surface (e.g.
adenocarcinoma of the colon) or from an endocrine or exocrine (breast
carcinoma) gland.
c. Tumor cells may be arranged in a wide variety of histologic patterns
a. gland-like structures often back-to-back - with a central lumen formed by
tumor cells (think donuts).
b. Cords
c. Nests isolated or scattered in a sea of mucin
d. Solid sheets
e. Trabeculae
f. Papillary projections
g. Signet ring - individual tumor cells with a central mucinous vacuole that
pushes the nucleus to the side yielding a appearance.
8. Transitional epithelium lines the renal pelvis, the ureters, and the urinary bladder.
a. Tumors arising from them are either a benign papilloma or a transitional cell
carcinoma (TCC). The latter are further categorized as papillary or flat (nonpapillary)
157

Lecture:
Neoplasia #3 Nomenclature #2, Epidemiology, and Carcinogenesis

1/4/12 8:30 10:00- 8:30
1. Distinguish between benign and malignant mesenchymal tumors based on their gross and
microscopic features. Focus on the following neoplasms.
a. Osteosarcoma vs. chondrosarcoma
b. Leiomyoma vs. leiomyosarcoma
c. Leukemia vs. lymphoma
d. Hamartoma
2. Recognize the basic morphologic features of the following tumors.
a. Brain tumors
b. Germ cell tumors
c. Hamartoma
3. Recognize the roles of geographic location, genetics, age, gender, and environmental
factors in increasing the risk for cancer.
a. Distinguish an inherited cancer syndrome from a sporadic cancer based on clinical and
pathologic features.
4. Define the critical features of and initiator and a promoter and be able to accurately evaluate
a potential chemical carcinogen as one of them (Look at Figures 7-41 and 7-42)
5. Define the role of UV and ionizing radiation as carcinogenesis.
6. Define the molecular consequences of HPV, EBV and hepatitis B infection on infected cells,
and the role of chronic inflammation that lead to neoplastic transformation.
Learning Resources:
2. Cancer Statistics 2011 ( CA 61(4): 205-282, 2011 http://caonline.amcancersoc.org )
III. NOMENCLATURE OF NEOPLASIA
A. Objective 1
1. Mesenchymal tumors comprise approximately 15% of all neoplastic tumors arise
from mesenchymal cells and their nomenclature of mesenchymal tumors is very
straight forward based on the cells of origin.
158
TISSUE
Adipose
Cartilage
Bone
Smooth Muscle
Skeletal Muscle
Blood vessel
Leukocytes
Lymphocytes
BENIGN BEHAVIOR
Lipoma
Chondroma
Osteoma
Leiomyoma
Rhabdomyoma
Hemangioma
-
MALIGNANT BEHAVIOR
Liposarcoma
Chondrosarcoma
Osteosarcoma
Leiomyosarcoma
Rhabdomyosarcoma
Angiosarcoma
Leukemia
Lymphoma
2. Soft tissue tumors arising from adipose cells are either benign lipomas or
liposarcomas.
3. Bone tumors arising from cartilage are either benign chondromas or malignant
chondrosarcomas. In general, cartilaginous tumors arise from the medullary cavity
or cortex of a bone and produce cartilaginous matrix material.
4. Soft tissue and bone tumors arising from osteocytes are either benign osteomas or
malignant osteosarcomas. Osseous tumors synthesize osteoid eosinophilic bone
matrix material that is not calcified.
5. Fibroblast tumors are characterized by spindle shaped cells with spindle shaped
nuclei; fibromas and fibrosarcomas. Many mesenchymal tumors are characterized
as spindle cell tumors.
6. Leiomyomas and leiomyosarcomas arise from smooth muscle cells anywhere in the
body. They commonly arise from smooth muscle in the uterine myometrium and in
the intestinal wall.
7. Leukemias arise from bone marrow derived hematopoietic cells. Lymphomas are
malignancies that arise from lymphoid tissue located anywhere in the body.
B. Objective 2
1. Metastases to the brain out number the primary brain tumors.
2. The most common primary brain tumors arise from various cells types including
a. Glial cells astrocytomas, brain stem gliomas, ependymoma, oligodendroglioma
1) Glioblastoma multiforme is a high grade, highly aggressive, astrocytoma
b. Neuroectodermal cells medulloblastoma
c. Meninges meningioma
d. Schwann cells schwannoma
e. Germ cells - germinoma
3. Germ cell tumors arise from totipotential ells that are capable of differentiating into
other cell types.
a. Germ cell tumors arise in the gonads - testes and ovaries as well as in
extragonadal locations (e.g. intracranial, oral cavity, mediastinum, and pelvis).
159
b. Germ cell tumors may be benign or malignant and are classified according to
their differentiation.
c. Teratomas of the ovary (dermoid cysts) are benign tumors that contain mature
tissue elements derived from the three germ layers.
2. Hamartoma benign, non-neoplastic tumors that are made up of disorganized normal
tissue that occurs in its normal location. Their clinical significance comes from their
confusion with a neoplastic lesion due to location.
IV. EPIDEMIOLOGY
A. Objective 3
1. Cancer is commonly encountered throughout the world. The incidence is highest in
developing countries, but it is increasing in the developing world.
a. In the U.S, close to 1.5 million individuals will be diagnosed with a cancer other
than the common tumors of the skin.
b. Among men and women in the U.S., lung cancer is the number one cancer.
However, carcinoma of the breast is the most common cancer in women and
prostatic carcinoma is the most common cancer in men.
2. There are geographic differences in specific cancer incidence as shown by ed by
when a population relocates around the world. These differences may be due
environmental factors as well as habits.
3. Cancer is a genetic disease. (Refer to Dr. Vandres and Dr. Westmans lectures)
a. Inherited cancer syndromes exhibit common clinical and pathologic features.
1) Clinically apparent at a younger age.
2) Involve specific sites and tissues.
3) Often bilateral organ involvement.
4) Multifocality, i.e. more than one tumor in the same organ.
5) Often associated with a specific phenotype.
6) There is usually incomplete penetrance and variable expression.
b. von Hippel-Lindau (VHL) Syndrome is a prototypic inherited cancer syndrome.
Its common features include:
1) Kidney cancer renal cell carcinoma
a) Usually develops in young patients - 20-30s as compared to the 6th and
7th decades for most renal cell carcinomas.
b) VHL syndrome is autosomal dominant as compared to most renal
carcinomas which are sporadic.
c) Tumors are often bilateral nature as compared to most sporadic renal
carcinomas which only involve only one kidney.
d) Each kidney may have multiple tumors as compared to most sporadic
renal carcinomas that involve one tumor mass in one kidney.
2) VHL syndrome is characterized by concurrent tumors in other organs. These
include:
a) Cancer of the adrenal medulla - pheochromocytoma
b) Vascular tumors in the brain - hemangioblastomas 40% of cases.
c) Vascular tumors in the retina - angiomas.
160
d) Multiple cysts (think water balloon) in the kidneys and pancreas.

4. In general, the incidence of cancer increases with age in both men and women.
a. Some tumors exhibit a bimodal age distribution (e.g. Hodgkins lymphoma), an
initial increase in infants and another later in life (e.g. leukemia, brain cancers), or
occur in a younger age group (e.g. testicular germ cell cancers).
5. Various environmental factors have been associated with cancer.
a. An environment of economic poverty limits access to medical care.
b. The types and amount of food intake may increase a patients risk for cancer.
1) Morbid obesity has been linked to an increased risk for developing several
different types of cancer including carcinoma of the breast.
2) Some foods can directly cause cancers (e.g. Aflatoxin B1 a potent
hepatocarcinogen produced by Aspergillus flavus growing on improperly
stored grain and peanuts), while other ingredients are metabolized into
cancer inducing agents (e.g. nitrates and nitrosable amines ingredients are
metabolized into nitrosamines and amides gastric bacteria).
c. Exposure to various chemicals, encountered in the workplace or by habit, has
been associated with an increased risk of cancer. These include:
1) Asbestos exposure increases the risk of mesothelioma.
2) Exposure to aniline dyes in rubber workers increases their risk of transitional
cell carcinoma of the urinary bladder.
3) Carcinogens in tobacco smoke increase a persons risk for cancer of several
organs including those of the upper aerodigestive tract and the lungs.
4) Ethanol combined with smoking work together to increase a persons risk of
squamous cell carcinoma of the mouth and pharynx.
d. There is a marked increase in the risk of skin cancers and melanoma in those
locations with increased sun light.
e. Certain viral, bacterial, and parasitic infectious agents increase the risk specific
types of cancer.
V. CARCINOGENESIS
A. Objective 4
1. Carcinogens cause non-lethal genetic mutations of oncogenes, tumor suppressor
genes, DNA repair genes, and/or genes of apoptosis. These mutations lead to
neoplastic transformation of the stem cell population.
a. Carcinogenic agents work alone or in combination and include: chemicals,
radiation, and oncogenic viruses, as well as certain bacterial and parasitic
infections.
161
b. Carcinogenesis is a two step process:

1)
Initiation leads to permanent non-lethal DNA damage making a cell
susceptible to transformation. It alone is not sufficient for tumor development,
but may induce hyperplasia or dysplasia.
2)
Promotion leads to cell proliferation which are reversible, but do not alter
DNA.
2. Initiators are electrophiles that react with nucleophilic sites to induce DNA- adducts,
i.e. the carcinogenic chemical is covalently bound to DNA nucleosides.
a. Carcinogen-altered cells must undergo at least one cycle of proliferation so that
the DNA change is permanent. No single or unique alteration of DNA is
associated with chemical carcinogenesis.
b. Direct acting initiators do not require chemical transformation for carcinogenicity
(e.g. alkylating & acylating agents).
c. Indirect acting initiators require metabolic conversion for carcinogenicity.
d. Carcinogenic potency of a chemical is determined by its inherent reactivity plus
the balance between metabolic activation and inactivation (detoxifying) reactions.
Cytochrome p450 (CYP1A1) metabolism activates many procarcinogens.
Glutathione-S-transferase (GST) detoxifies polycyclic aromatic hydrocarbons.
e. No mutation is unique to chemical carcinogens, however, patterns of mutations
may implicate certain chemicals (e.g. aflatoxin B1 is associated with p53 gene
mutations in hepatocellular carcinoma.)
3. Promotion
a. Initiated cells respond differently to promoters than do normal cells, and tumors
only develop when exposed first to an initiator and then to an appropriate
promoter.
b. Promoters induce cell proliferation but are not mutagenic.
c. Initiated cells may have reduced requirements for growth factors, or cells may be
less responsive to growth inhibitors.
d. Sustained proliferation enhances opportunities for further mutagenesis
e. There is ample evidence to show that promotion must follow initiation for
tumorigenesis to occur.
4. There are several types of chemical carcinogens.

a. Direct-acting alkylating agents are weak carcinogens that directly damage DNA
(e.g. chemo - cyclophosphamide, chlorambucil, busulfan, melphalan).
b. Polycyclic aromatic hydrocarbons are potent carcinogens in tobacco smoke,
broiling meat fats, and smoked meat.
162
c. Hepatic metabolism of aromatic amines and azo dyes generates carcinogenic

metabolites (e.g. -naphthylamine is associated with bladder cancer in aniline
dye and rubber workers).
d. Aflatoxin B1 is a potent hepatocarcinogen produced by the fungus Aspergillus
flavus growing on improperly stored grain and peanuts.
e. Other chemical carcinogens include:
1) asbestos - bronchogenic carcinoma, mesothelioma, GI cancer
2) vinyl chloride - hemangiosarcoma of liver
3) chromium/nickel - lung cancer
4) arsenic - skin cancer
f.
Promoters of chemical carcinogenesis include: cigarette smoke, hormones (e.g.

estrogen) and bile salts.
INITIATION
Detoxification
Carcinogen Metabolic Electrophilic DNA Adduct Repaired

Normal cell
Activation
Intermediate
Formation
Cell Death
Preneoplastic
Cell
Initiated Cell
Clone
Proliferation
PROMOTION
Cell Proliferation
Additional mutations
Malignant Neoplasm
B. Objective 5
1. UV, ionizing, and particulate radiation induce human cancer, and have both a latency
and cumulative effects.
a. UV effects vary with the wavelength, length of exposure, and the quantity of
melanin in the skin.
UVA 320-410 nm
UVB 280-320 nm
UVC 200-280 nm, which is filtered by the ozone layer
b. UVB induces formation of pyrimidine dimers in DNA, repaired by nucleotide
excision repair (NER) mechanisms that are overwhelmed by excess sun
exposure.
c. Xeroderma pigmentosum is an autosomal recessive disease associated with
mutations of NER genes that increase the risk of skin cancers by 2000x.
2. Inonizing radiation includes both electromagnetic radiation (X- rays and -rays) and
particulate radiation (-particles, -particles, protons, and neutrons.
a. Their clinical significance is associated with :
1) X-rays increase the risk of developing skin cancer.
163
2) Therapeutic radiation of the thymus is associated with increased risk of

thyroid cancer following childhood exposure.
3) Atomic bomb survivors had an increased risk of developing leukemia and
carcinomas of the breast, colon, thyroid, and lung.
C. Objective 6
1. DNA Oncogenic viruses Human Papilloma Viruses (HPV)
a. There are >70 different types of oncogenic viruses
b. Types 1, 2, 4, and 7 - associated with benign papillomas (warts).
c. Types 16 and 18 - in 85% of cervical squamous cell carcinomas and its
precursors.
d. Types 6 and 11 - associated with low grade dysplasia and are low risk for cancer.
e. Infection with HPV acts as an initiating event.
f. Mechanism is associated with two early viral gene products E6 and E7
1) E6 binds to p53 and hastens its degradation
2) E7 binds to Rb protein to displace the E2F transcription factor
2. Epstein-Barr Virus (EBV)
a. EBV infection is associated with various lymphomas
1) Burkitt lymphoma, African form via c-myc activation (co-factor)
2) B-cell lymphomas in immunocompromised hosts
3) T cell lymphomas and Hodgkins disease unknown mechanism
b. EBV predisposes the infected B cell to rearrange immunoglobulin genes, acquire
the t(8;14), and releases cell from normal growth control.
c. Several viral gene products immortalize the cells.
1) LMP-1 prevents apoptosis by up regulating bcl-2 and activating c-myc
growth- promoting pathways (i.e. prevents death and stimulates proliferation).
3. Hepatitis B Virus
a. Hepatitis B viral infection is endemic in the Asia and Africa and is associated with
hepatic carcinoma.
b. Viral infection causes chronic liver cell injury and regenerative hyperplasia.
c. Hbx protein binds to p53 and interferes with its growth suppressing activities.
Kaposi sarcoma herpes virus (KSHV)
3. KSHV is a herpes virus (HHV8) associated with Kaposi sarcoma (KS) and B-cell
lymphoma in HIV-infected patients.
b. KS can occur in a wide variety of locations. It represents a neoplasm of
lymphatic endothelial cells that form blood filled vessels.
5. Human T-cell Leukemia Virus Type I (HTLV-1) RNA oncogenic virus
a. Viral infection is endemic in Japan and Caribbean.
b. Tropic for CD4+ (helper) T cells and T cell leukemia/lymphoma develops in 1% of
infected patients after long latent period.
6. Helicobacter pylori is a bacteria in the stomach that is linked to lymphoma and
carcinoma of the stomach.
164
7. Schistosoma haematobium infection leads to inflammation resulting in squamous

metaplasia of the transitional epithelium that can progress to dysplasia and finally to
squamous cell carcinoma of the urinary bladder.
165

Lecture:
Neoplasia #4 Tumorigenesis and Tumor Biology

1/6/12 8:30 10:00
1. Define the ten proposed hallmarks/characteristics of a malignant tumor cancer (Cell 144,
March 4, 2011) and their roles in tumorigenesis. (Refer to lectures by Drs. Vandre and
Westman)
2. Define role of clonality, morphologic and genetic changes in tumorigenesis.
3. Define the behavior of tumor stem cells and their potential clinical impact.
4. Describe the role of the extracellular matrix and its components in tumor growth and
metastasis.
a. What is the impact on tumorigenesis of the constant two-way interaction between the
neoplastic cells and the stromal cellular and acellular components?
b. What is the role of cell adhesion molecules and protease and TIMPS activity on tumor
mobility through the extracellular matrix invasiveness?
5. Define the steps that a tumor cell must accomplish to for a successful metastasis.
Learning Resources:
2. Cell 144, March 4, 2011
3. Cancer Stem Cells, Jordan et al. New England Journal of Medicine, 355;1253-1261, Sept.
21, 2006.
4. Cancer Stem Cells in Solid Organs: Accumulating Evidence and Unresolved Questions.
Visvader and Lindeman. Nature Review Cancer, 8(10); 755-768, 2008.
VI. TURMORGENESIS AND TUMOR BIOLOGY

A. Objective 1
1. Carcinogenesis results in a series of gene mutations that lead to malignant
transformation of cells. The end result of these gene mutations is a series of
changes in the phenotype (traits) of the transformed cells that alters the normal
control mechanisms of cells within a population, including cell proliferation,
apoptosis, differentiation, senesce.
166
2. It has been proposed that malignant cells have 10 hallmarks or characteristic s that
include:
a. Sustaining proliferative signalling (Liston, Vandre, Westman)
b. Evading growth suppressors (Vandre, Westman)
c. Avoiding immune destruction
d. Enabling replicative immortality (Westman)
e. Tumor-promoting inflammation
f. Activating invasion and metastasis
g. Inducing angiogenesis (Vandre)
h. Genome instability and mutation (Westman)
i. Resisting cell death (Vandre)
j. Deregulating cellular energetics
3. Oncogene activation due to mutation, translocation, or gene amplification
results sustains proliferative signalling and reduces the dependence on growth
signals.
a. The resulting oncoproteins promote cell proliferation. They are devoid of
regulatory elements and their production is independent of growth factors or
external signals. (Review lectures from Drs. Liston, Vandre & Westman)
4. Loss of sensitivity to growth Inhibitory signals results from two mutations of a tumor
suppressor gene (i.e. loss of heterozygosity). ((Review lectures from Drs. Vandre
& Westman)
5. Alterations in both the innate and the adaptive immune responses are involved in the
he mechanisms of tumor cell avoidance of the immune response, but the specifics
are still unknown.
6. Immortalization or limitless replicative potential of tumor cells results from the
activation of telomerase in the tumor cells. (Review lecture from Dr. Westman)
7. Tumor infiltration by subsets of inflammatory cells (eg. lymphocytes, macrophages,
and plasma cells) is commonly seen. Instead of destroying the tumor, inflammatory
cells elaborate growth factors, chemokines, and cytokines that serve as promoters.
In addition, the reactive oxygen species they elaborate serve as initiators.
8. Activating invasion and metastasis. (See below)
9. Angiogenesis is required before a tumor can enlarge beyond 1-2 mm and before it
can metastasize. (Review lecture from Dr. Vandre) The abnormal blood vessels of
tumors are characterized by:
a. Irregular shape with dilatation and often closed ended.
b. Lacking definitive arterioles, venules and capillaries.
c. Increased permeability that improves the exchange of nutrients and waste
products.
d. Endothelial cells with up-regulated receptors for various factors, including VEGF
and TGF.
167
10. Genome instability and mutation results in a multistep process that involves
sequential genetic changes. (Refer to Dr. Vandres and Dr. Westmans lectures)
a. These changes result in unregulated growth is due to serial acquisition of genetic
events leading to the expression of genes that promote cell proliferation with
concomitant silencing of growth inhibitory genes and blunting of cell death.
b. Tumors contain a heterogeneous population of neoplastic cells differing in their
genetic mutations and their phenotype.
11. Reduced cell death (apoptosis) can arise from a mutation of anyone of several
genes, e.g. Bcl-2 gene family and p53. (Review lecture from Dr. Vandre)
12. Deregulation of cellular energetic results in tumor cells being dependent on
glycolysis for generation of ATP, i.e. aerobic glycolysis. This is termed the Warburg
effect.
a. This effect permits tumor to take up 18F-fluorodeoxyglucose (FDG) that can then
be measured using a PET (positron emission tomography) scan that can
visualize metabolically active tumor cells.
b. This switch from oxidative phosphorylation to glycolysis is thought to generate
additional carbon molecules for synthesis of amino acids, lipids and other
molecules needed by the proliferating tumor cells.
B. Objective 2
1. Carcinogenesis transforms a single normal cell into a malignant cell. The
progression from transformed cells to metastatic tumor cells requires a sequential
multiple genetic mutations.
2. The best example of this is the production of monoclonal immunoglobulin (Ig) with
restricted kappa or lambda light chains by neoplastic plasma cells in multiple
myeloma or B cells in a non-Hodgkin lymphoma or leukemia.
3. The hypothesis that all tumor cells are clonal is best demonstrated by study of G6PD
isoenzymes (X-linked) in women who are heterozygous at this locus.
a. Allelic differences can be detected by protein electrophoresis or at the DNA level.
b. One X chromosome is randomly inactivated early in embryogenesis (Lyon
hypothesis) resulting in only one G6PD allele will be expressed in any given cell.
4. In tissues, composed of a mosaic of many cells, expression of both alleles is
normally detected. However, examination of benign and malignant neoplasms
demonstrates that all cells in an individual tumor express either one or the other
allele, but not both, indicating that the neoplasm is derived from a single progenitor
cell.
5. Note that clonality does not invariably predict malignant behavior, since benign
neoplasms are also clonal proliferations.
C. Objective 3
1. Tumors arise from cancer stem cells that have properties of normal stem cells,
particularly self-renewal and multipotentiality (a minority) of tumor cells.
168
2. Unregulated cell growth is due to a disruption in the regulatory mechanism in stem

cell renewal.
3. Although this is not a new model, there is growing experimental and clinical data to
support is.
4. In the Stem Cell Model, there is a small population of stem cells that is responsible
for the growth and progression of cancer.
a. Key to stem cell survival is the ability for self-renewal; at the time of cell division,
one or both daughter cells retain the same biologic properties as the parent cell.
b. Stem cells are quiescent (in G0). They are long-lasting cells that have the
potential to proliferate extensively
c. The capability to develop into multiple lineages, i.e. making all of the cells in the
tumor.
5. Cancer stem cells have been identified in acute myelogenous leukemia (AML),
chronic lymphocytic leukemia (CLL), and in carcinomas of the breast, colon, head
and neck, and astrocytomas of the brain.
6. Oncogenic mutations confer self-renewal potential by activating pathways used by
normal stem cells, irrespective of whether the mutations occur in stem cells or other
cells. Key genetic pathways include the Wnt pathway and the Notch pathway, as
well as mutations of Bmi-1 and PTEN
7. It is thought that cancer stem cells are relatively resistant to conventional
chemotherapies and radiation therapies, and that that they give rise to progressive
disease and to metastases.
D. Objective 4
1. As an organ, a tumor contains both cellular and acellular components.
a. Neoplastic cells comprise the parenchymal cells of a tumor.
b. Fibroblasts, macrophages, and endothelial cells are the major stromal cells of a
tumor.
c. The extracellular matrix (ECM) plays a critical role in structural supports and
tumorigenesis.
2. The microenvironment, with its supporting stromal cells and extracellular matrix
(ECM), is needed to foster tumor cell proliferation, angiogenesis, invasion and
metastasis.
3. The composition of the microenvironment differs between tumors and within the
same tumor.
1. The structural components of the extracellular matrix include structural proteins
(collagens and elastin), specialized proteins (fibrillin, fibronectin, and laminin),
and proteoglycans.
2. Many of the factors stored in the ECM and released (think sponge-like) play
pivotal a role in induction, selection, and proliferation of neoplastic cells and
169
angiogenesis.
3. Differences in microenvironments extend to organs as well. This accounts for
why certain tumors have a predilection for metastasis to certain sites; specific
microenvironments support organ-specific metastases i.e. the seed and soil
concept.
4. There is extensive cross-talk between the tumor cells and the microenvironment.
1. The tumor microenvironment contributes positive and negative signals to tumor
cells that are mediated by bioactive factors and cell-cell and cell-matrix contacts
2. Tumor cells modify the microenvironment to produce bioactive factors such as:
growth factors, chemokines, matrix-degrading enzymes that enhance the
proliferation, survival, invasion, and metastasis of tumor cells. These may have
autocrine, paracrine and/or endocrine functions.
3. The microenvironment is constantly changing as the tumor progresses. This is
especially true of angiogenesis that is required for tumor growth beyond 2mm
and subsequent metastasis.
4. It has been postulated that there is a niche of tumor stem cells within the
microenvironment that places them close to the blood vessels. Within this niche
there is extensive cross-talk.
E. Objective 5
1. Metastasis is the major cause of the morbidity and mortality associated with cancer.
Invasion and metastasis help to define a tumor as being malignant. This inefficient
process follows a complicated multistep sequence of events that include:
a. Detachment from the primary mass
b. Invasion through the extracellular matrix
c. Penetration of blood and/or lymphatic vessels
d. Survival within the circulation
e. Arrest in the distant organs microcirculation (capillaries, sinuses or venules)
f. Penetration of vessels at the distant site
g. Invasion through the distant sites parenchyma
h. Adapt to a new microenvironment
2. Detachment from the primary mass begins with an altered expression of cell
adhesion molecule that leads to discohesiveness.
1. Epithelial-mesenchymal transition, normal to embryogenesis and critical to tumor
cell invasion and metastasis, involves an alteration of cell adhesion molecule
expression by the epithelial cells and increased motility, i.e. a mesenchymal
phenotype (little cell-cell adhesion, and expression of mesenchymal proteins)
2. Cell adhesion molecules are transmembrane cell surface proteins whose
extracellular domain is responsible for a cells ability to bind to other cells and/or
to the extracellular matrix and intracellular domain interacts with cytoskeletal
proteins such as actin. They play a pivotal role in both tumor cell metastasis,
inflammation and wound healing. There are four protein families of cell adhesion
molecules.
1) Cadherins
170
2) Integrins
3) Immunogobulin superfamily
4) Selectins
3. Cell adhesion molecules may be homophilic (binding to like molecules on
another cell) or heterophilic (binding to a different cell adhesion molecules on a
cell or to various extracellular matrix components).
4. Cadherins are calcium dependent, homophilic, cell adhesion molecules that bind
to cytoplasmic actin via -catenin and -catenin. These molecules play a critical
role in epithelial cell adhesion. E-cadherin is commonly expressed by epithelial
cells, and inhibition of its expression and replacement with N-cadherin has been
associated with the ability of neoplastic epithelial cells to invade.
5. Integrins are hetrodimeric, 18 chains and 8 chains, proteins. These
heterophilic transmembrane proteins function in both cell-cell and cellextracellular matrix interaction.
1) Integrins mediate epithelial cell adhesion to the laminin and fibronectin in the
basal lamina. Altered expression of integrins during tumorigenesis regulates
the cell-cell interactions and tumor cell migration through the ECM (i.e.
invasion and metastasis).
2) The intracellular domain interacts with various cytoskeletal proteins, e.g.
actin, tropomyosin, talin and vinculin.
3) Integrins are also signaling molecules linked to various tyrosine kinases that
promote tumor cell proliferation and survival. Thus, binding to ECM induces
them to cluster leading to signal transduction via the MAP kinase pathway.
6. Immunoglobulin superfamily (aka CAMS) proteins bind to other cell CAMS
(homophilic) or integrins (hetrophilic) involved in cell-to cell interaction. Their
altered expression in certain cancers is reported to enhance tumor cell motility
through the extracellular matrix.
7. Selectins are transmembrane, single chain, proteins whose extracellular domain
binds to sialylated carbohydrate attached to mucin-like molecules on cells and in
the extracellular matrix. They play a role in extravasation tumor cells during
metastasis.
3. Invasion of the neoplastic cells requires proteolytic digestion of the basement
membrane and components of the extracellular matrix. This involves a wide variety
of extracellular proteases secreted by both tumor cells and stromal cells.
a. Proteases such as: serine proteases (plasmin, urokinase, plasminogen activator)
and cathepsins.
b. Matrix metalloproteinases (MMP) are a diverse family of Zn+2 and Ca+2
dependent endopeptidases (collagenases, matrilysin, gelatinase, stromelysin).
In addition to all stages of tumor progress, MMPs play a pivotal role in embryonic
development, tissue repair, remodeling, and in a variety of non-neoplastic
diseases (e.g. arthritis, atherosclerosis).
171
c. There are numerous substrates upon which these proteolytic enzymes work.
1) Inactive MMPs
2) Matrix proteins
3) Receptors for cell-matrix adhesion
4) Matrix proteins yielding fragments with new biologic activity
5) Matrix proteins releasing embedded growth factors
6) Receptors in cell-cell adhesion
7) Cell surface bound ligands releasing biologically active growth factors
8) Cell surface receptors leading to their inactivation
d. Tissue Inhibitors of Metalloproteinases (TIMPS) are a group of endogenous
inhibitors of MMP activity by forming a complex with them. A disturbed balance
between TIMPS and MMPs characterizes a variety of diseases.
4. Multiple factors are required for tumor cell motility, i.e. invasion through the
extracellular matrix. These include: appropriate gene expression, correct
microenvironment, and release of MMPs and other proteolytic enzymes.
a. Invasion is mediated by tumor cell derived motility factors (e.g. the autocrine
hepatic growth factor), the MMP cleavage the ECM, and by ECM-integrin
interactions.
b. ECM regulates tumor cell motility through it. Not only does the ECM provide the
scaffolding of the tumor, integrin-mediated tumor cell binding ECM components
trigger numerous cellular responses including release of proteolytic enzymes and
motility.
c. Integrins binding to ECM components transmit the force from the ECM to the
cytoskeleton resulting in cell infiltration of the ECM. Integrin molecules are
recycled along the cell surface. Movement is directional toward higher
concentrations of chemokines and chemotactic factors.
5. Vascular Dissemination and Homing of Tumor Cells
a. Tumor cells are vulnerable to destruction while in the circulation, primarily by NK
cells.
b. Cell adhesion molecules mediate formation of tumor cell clumps with each other
and with platelets in the circulation.
1) Aggregates have a higher likelihood of survival and imprintability.
2) Tumor aggregates may embolize a small vessel.
3) Cell adhesion molecules (e.g. CD44) mediate adhesion of tumor cells to
endothelial cells.
c. Not all tumors have the same route of metastasis. Some tumors commonly
metastasize to certain sites (organ tropism) due to differences in cell adhesion
molecule capability with endothelial cells, presence of chemotactic factors, or an
unfavorable microenvironment.
172

Lecture:
Neoplasia #5 Clinical Aspects of Cancer

1/9/12 8:30 9:30 and 11/10/11 10-30-11:30 (if needed)
1. What are the features of an ideal screening test and how are they met or not met in
screening for cancer of the cervix, breast, and colon in the general population.
2. Define the basic signs and symptoms of cancer and the significance of paraneoplastic
syndromes.
a. What are the mechanisms and clinical features associated with an endocrinopathy
arising in cancer of the lungs, pancreas, GI tract, kidney, liver, and brain; Eaton-Lambert
syndrome, acanthosis nigricans, and coagulopathy associated with cancer?
3. Define grade tumors and to stage tumors as to their variables and clinical utility.
Learning Resources:
2. Cancer screening in the United States, 2011 : A Review of Current American Cancer
Society Guidelines and Issues in Cancer Screening (pages 830) Smith et al. CA 61(1) 830, 2011.
VII. CLINICAL ASPECTS OF CANCER
A. Objective 1
1. Screening is defined as systematic testing of individuals who are asymptomatic with
respect to a target disease.
2. The purpose of screening is to prevent, interrupt, or delay the development of
advanced disease in the subset with a pre-clinical form of the target disease through
early detection and treatment. During the progression of disease, there is a critical
interval of time between detection and the onset of signs and symptoms where
screening is the most effective. (Review Epidemiology module)
a. The ideal screening test is highly sensitive, highly specific, non-invasive,
available, and inexpensive.
b. Critical to screening is to identify patient population at risk for a disease. This
translates in the identification of the patient population with the highest disease
prevalence.
3. Diagnosis differs from screening in several aspects.
Diagnosis
Screening
Patient is symptomatic
Patient is asymptomatic
Test is diagnostic
Test is rarely diagnostic
There is high disease prevalence
There is low disease prevalence
173
4. There are several types of screening:

a. Physical examination for masses in the testes, breasts, prostate, mouth, or eyes
are easily performed screening tests.
b. The PAP smear for screening women for squamous cell cancer has significantly
reduced the incidence of the disease in those countries screening fro the
disease.
1) The test in an easy to perform collection of cells from the exo- and the
endocervix.
2) The collected cells are transferred into a liquid media and a cellular
monolayer is produced for staining and microscopic analysis.
3) The slides are assessed for the presence of HPV infected virus and for the
presence of dysplastic or malignant cells. (Classic PAP Smear)
4) The brush is placed into the liquid media and is available for analysis for viral
DNA using hybrid capture technology.
c. Clinical breast examination and mammography are used as a routine screening
tools for breast cancer. The presence of a mass and/or microcalcifications ona
mammogram often prompts a biopsy.
d. Screening of the general population for possible colorectal carcinoma involves
the Hemoccult assay for possible blood. Colonoscopy for possible colorectal
carcinoma is used for patients over 50 or at increased risk of developing cancer.
B. Objective 2
1. The site of the tumor, benign or malignant, often determines the nature of signs and
symptoms.
2. American Cancer Society's Seven Warning Signs
a. nagging cough or hoarseness
b. sore that does not heal
c. unusual bleeding or discharge
d. thickening or lump in breast or other area
e. indigestion or difficulty swallowing
f. obvious change in wart or mole
g. change in bowel or bladder habits
3. Unexplained weight loss cachexia vs. anorexia
4. Anemia bleeding, bone marrow suppression
5. Infections secondary to ulcerations or obstructions
6. Fever of unknown origin (FUO)
7. Paraneoplastic syndromes
a. Symptom complexes not readily be explained by local or distant tumor spread or
by the elaboration of hormones indigenous to the tissue from which the tumor
arose (i.e. ectopic hormone production)
174
1)
2)
3)
4)
Occur in up to 10% of cases

3)
Often the 1st sign of tumor
Clinically significant and even fatal
May mimic metastatic disease
b. Endocrinopathies (ectopic production of hormone)

1) Hypercalcemia (most common) due to PTH-related peptide, TGF, TNF,
and IL-1 associated with:
a) Squamous cell carcinoma of the lung
b) Breast carcinoma
c) Multiple myeloma
d) Ovarian carcinoma
e) Adult T cell leukemia/lymphoma
2) Cushing syndrome due to ACTH or ACTH-like substance associated with:
e. Small cell lung cancer
f. Pancreatic carcinomas
g. Neural tumors
h. Thymoma
3) IADH due to ADH commonly associated with small cell carcinoma of the
lung and to lesser extent CNS tumors.
4) Hypoglycemia due to insulin or insulin-like substance that has been
associated with ovarian carcinomas and fibrosarcoma sarcomas
5) Carcinoid syndrome due serotonin secretion by carcinoids and pancreatic
and gastric carcinomas)
6) Polycythemia ( RBCs) due to due erythropoietin (renal carcinoma)
c. Eaton-Lambert Syndrome is a good example of neuromyopathic syndromes
associated with thymomas and small cell lung carcinoma. This autoimmune
disease involves autoantibodies that inhibit voltage-gated calcium channels. The
clinical picture is similar to that of myasthenia gravis.
d. Acanthosis nigricans refers to velvety hyperpigmentation of the skin of the axilla,
groin, and skin folds. It is usually associated with an endocrinopathy, but in
about 40% of cases it is associated with an adenocarcinoma, with the GI tract
being the most common.
e. Hypercoagulability
1) Venous thrombosis
a) Trousseau syndrome - migratory thrombophlebitis and pulmonary
embolus
b) Nonbacterial thrombotic endocarditis
2) Associated with mucin secreting carcinomas arising in the pancreas, GI tract,
breast and lungs.
175
3) DIC associated with promyelocytic leukemia (M3 AML)

C. Objective 3
1. General Concepts
a. Cancer grading and staging are used to determine therapy, assess prognosis,
and compare results of therapeutic trials among institutions.
b. Specific guidelines are used for grading and staging each type of neoplasm.
2. Tumor Grading
1. Grade is a morphologic assessment of the degree of differentiation of a
neoplasm.
2. In general, grading is based on the degree of pleomorphism - morphologic
assessment of how closely the neoplasm resembles the normal cells from which
it arose - and number of mitoses
3. Grading is presumed correlates of the neoplasm's aggressiveness.
4. Example of a grading system for prostate adenocarcinoma is based on
architectural patterns.
a. It goes from Grade I most well-differentiated to Grade 5 which is the least
differentiated.
5. Problems with grading
a. Subjective: three level systems more reproducible than four level systems.
b. There is not uniform system for every neoplasm.
c. Sampling errors: reflect morphologic heterogeneity within a tumor
d. Grading is not as predictive of survival as staging for some tumors.
3. Tumor Stage
a. Concepts
1) The Stage of a tumor assesses the extent of tumor growth and metastasis.
2) Based on tumor extent locally, as well as regional and distant metastases.
3) Both clinical and pathologic staging criteria exist for each tumor.
4) Pathologic staging is based on gross and microscopic assessment of tumor
invasion and metastasis, however The pathologist can only evaluate what
he/she is given.
5) Staging provides uniform nomenclature for discussion and for basing
treatment decisions, and is usually of greater prognostic significance than
grading.
b. TNM System
1) T - size of primary lesion, ranked on a scale of 0 (in situ) to 4
2) N - presence or absence of lymph node metastasis, ranked on a scale of 0-3
3) M - presence or absence of distant metastasis, ranked on a scale of 0-2
4) The stage is assessed according to the combination of T, N, and M findings,
which may be different for different tumor types.
176
Practice Pathology Questions

The Cell Division 2
General Review Questions on Neoplasia
1. What are the nuclear features that may define a cell as being neoplastic?
2. What are the cellular features that may define a process as neoplastic?
3. What are the characteristic features of dysplastic epithelium?
4. How do low grade dysplasia and carcinoma in-situ differ?
5. How does carcinoma in situ differ from carcinoma?
6. What are the key differences between benign and malignant tumors?
7. What are the morphologic features that distinguish squamous papilloma from
squamous cell carcinoma?
8. What are the morphologic features that distinguish adenocarcinoma from
9. What are the features that differentiate a leiomyoma from a leiomyosarcoma?
10. What are the features that differentiate an osteosarcoma and classic
chondrosarcoma?
11. What are the features that differentiate leukemia from a lymphoma?
12. What are the features that differentiate a hamartoma from a choristoma?
13. What is the most common tumor in men?
14. What is the most common tumor in women?
15. What is the most common tumor in men and women?
16. At what age does the incidence of cancer increase?
17. What features, patient and tumor, that help to define a patient a having an inherited
cancer syndrome?
18. What does the term carcinogenesis is a multistep process mean?
19. What are the hallmark features of a cancer?
20. What are the essential steps in chemical carcinogenesis?
21. What is the relationship between radiation and carcinogenesis?
22. What are examples of tumors that are associated with the proceeding or
concurrent infectious disease?
23. What is the molecular mechanism of HPV induced cervical cancer?
24. What are the steps in tumor cell invasion and metastasis?
25. What are the roles of cell adhesion molecules in tumor cell invasion?
26. Why is angiogenesis important in tumor cell behavior?
27. What are the common paraneoplastic syndromes?
28. What are the components used in staging of a tumor?
177
Images of Cancer
Well differentiated adenocarcinoma of the colon
Poorly differentiated adenocarcinoma of the colon
178
Well differentiated squamous cell carcinoma
Intercellular
Bridges
Keratin
Pearl
Invasive breast carcinoma
179
A Squamous Cell Carcinoma

B Adenocarcinoma
C Anaplastic carcinoma
D. Leiomyosraoma
180
Answers for General Review Questions on Neoplasia

1.
What are the nuclear features that may define a cell as being neoplastic?
Loss of polarity (nucleus not located at base), increased nucleus to cytoplasm
ratio i, hyperchromasia, multiple nuclei, piling up of nuclei, irregular nuclear
membrane, irregular chromatin distribution, increased mitosis, abnormal nucleoli,
and abnormal mitotic figures.
2.
What are the cellular features that may define a process as neoplastic?
Loss of polarity. hypercellularity, increased pleomorphism, and anaplasia.
3.
What are the characteristic features of dysplastic epithelium?

Disorderly cell growth and maturation with immature cells (high N/C ratio)
extending upward leading architectural disorganization. The epithelial cells
exhibit varying degrees of neoplastic cellular/nuclear changes (answers to
questions 1 and 2), and cells DO NOT invade the basement membrane.
4.
How do low grade dysplasia and carcinoma in-situ (CIS) differ?

CIS refers to dysplasia that involves the full thickness of the epithelium from
basement membrane to the lumen, there is no maturation of the epithelium. Low
grade dysplasia classically involves the cells in the lower 1/3 of the epithelium,
while there is concurrent normal maturation in the upper portion.
5.
How does carcinoma in situ differ from carcinoma?

CIS does not invade the basement membrane, but carcinoma does. The cells
many have the same appearance.
6.
What are the key differences between benign and malignant tumors?
Feature
Invasiveness
Capsule
Margin
Growth Rate
Differentiation
Metastases
Fatal
7.
Benign
Seldom
Often
Pushing or expansile
Slow
Well
No
Rare
Malignant
Common
Seldom
Infiltrative
Rapid
Well to anaplastic
Yes
Common if untreated
What are the morphologic features that distinguish squamous papilloma from
Feature
Squamous
Squamous Cell
Papilloma
Carcinoma
Behavior
Papillary growth pattern
Fibrovascular core
Viral etiology
Increased mitotic figure
Pleomorphism
Benign
Malignant
Yes
Sometimes
Yes (HPV)
No
No
Sometimes
Yes
Yes
181
8.
What are the morphologic features that distinguish adenocarcinoma from

Feature
9.
Adenocarcinoma
Yes
No
Derived from squamous

epithelium
Derived from squamous
metaplastic epithelium
Keratinized
Yes
No
Some
No
Keratin pearls
Some
No
Dyskeratotic cells
Intercellular bridges
Dense cytoplasm
Staining for squamous
specific cytokeratins
Derived from glandular
epithelium
Derived from glandular
metaplastic epithelium
Malignant glands
Mucin production
Staining for glandular
epithelial specific
cytokeratins
Yes
Yes
Some
No
No
No
Yes
No
No
Yes
No
Yes
No
No
Some
Some
No
Yes
What are the features that differentiate a leiomyoma from a leiomyosarcoma?

Feature
Behavior
Cellularity
Pleomorphism
Bland oval nuclei
Well circumscribed
10.
Squamous Cell
Carcinoma
Leiomyosarcoma
Leiomyoma
Malignant
Yes
Yes
No
No
Benign
No
No
Yes
Yes
What are the features that differentiate an osteosarcoma and classic

chondrosarcoma?
Feature
Osteosarcoma
Chondrosarcoma
Behavior
Age at onset
Cells in lacuna
Pleomorphism
Matrix
Malignant
75% < 20
No
Yes
Yes
Malignant
osteoid
Malignant
40
Yes
Yes
Yes
Malignant hyaline
Osteosarcomas often arise in the metaphysis form an intramedullary mass

that invades through the cortical bone.
182
11.
What are the features that differentiate leukemia from a lymphoma?

Lymphoid neoplasms have T cells, B cells or B cell origins.
Myeloid neoplasms are derive from early hematopoietic progenitors.
Leukemias are malignant neoplasms have widespread involvement of the bone
and usually the peripheral blood. These can either lymphoid or myeloid.
Lymphomas are lymphoid malignant neoplasms that arise as discrete tissue
masses. There can be overlap between them.
12.
What are the features that differentiate a hamartoma from a choristoma?

Choristomas and hamartomas are tumors, but contain normal cells rather than
neoplastic cells. Hamartoma disorganized mass of cells that are indigenous to
the site but are benign. Choristomas aggregates of normal cells that are NOT
indigenous to the site, i.e. a heterotopic rest.
13.
What is the most common tumor in men? Prostate adenocarcinoma
14.
What is the most common tumor in women? Breast adenocarcinoma
15.
What is the most common tumor in men and women? Lung carcinoma
16.
At what age does the incidence of cancer increase?

75% of cases are in patients 55 years of age
17.
What features, patient and tumor, that help to define a patient a having an
inherited cancer syndrome?
Clinically apparent at a younger age, dominant gene with incomplete penetrance
and variable expression, involve specific sites and tissues (Retinoblastoma, LiFraumeni, colon, neurofibromatosis, breast and ovarian, Cowen epithelial, renal,
von Hippel-Lindau syndrome), often bilateral organ involvement, multifocality, i.e.
more than one tumor in the same organ and often associated with a specific
phenotype.
18.
What does the term carcinogenesis is a multistep process mean?

Not one event makes a cell neoplastic. There are multiple events that lead to the
cell becoming cancerous. There are many factors that have to get out of
whack (such as cell cycle, apoptosis, growth signals, DNA repair/mismatch,
tumor suppressor gene function, oncogene function, etc)
19.
What are the hallmark features of a cancer?

Self-sufficiency in growth signals; insensitivity to growth-inhibitory signals
evasion of apoptosis; limitless replicative potential; sustained angiogenesis
ability to invade and metastasize; defects in DNA repair; and shift from
mitochondria to glycolysis and consume more glucose by the tumor cells.
183
20.
What are the essential steps in chemical carcinogenesis?

An initiator has to introduce some sort of cell damage, but alone is not
carcinogenic. Then promotor events have to occur AFTER the initiator event,
without the initiator these promotor events arent carcinogenic either. But with
repeated promotor events after an initiator event in close time proximity, cancer
arises.
21.
What is the relationship between radiation and carcinogenesis?

UV rays (UVB at 280-320 nm) cause pyrimindine dimmers in DNA, so sun
exposure, esp burns can lead to melanoma. Ionizing radiation included
electromagnetic neutrons (x-rays, gamma rays) or particulate (alpha or beta
particles, protons) and usually health workers are at risk for this type of exposure
(x rays) or those exposed to the atom bomb in Japan
22.
What are examples of tumors that are associated with the proceeding or
concurrent infectious disease?
HPV - squamous cell carcinoma of the cervix and oral cavcity
HTLV-1- T cell leukemia/lymphoma
Hepatitis B Virus Hepatocellular carcinoma
Kaposi sarcoma herpes virus (KSHV) - Kaposi sarcoma (KS) and B-cell
lymphoma in HIV-infected patients.
Helicobacter pylori - a gastric bacteria linked to lymphoma and carcinoma
Schistosoma haematobium - squamous cell carcinoma of the urinary
bladder
23.
What is the molecular mechanism of HPV induced cervical cancer?

Two early viral gene products E6 and E7 in the infected cells - E6 binds to p53
and hastens its degradation thus inhibiting apoptosis and E7 binds to Rb protein
to displace the E2F transcription factor.
24.
What are the steps in tumor cell invasion and metastasis?

a. Detachment from the primary mass.
1) Begins with an altered expression of cell adhesion molecule that leads to
discohesiveness Loss of lateral surface E-cadherin expression.
2) Involves the Epithelial-mesenchymal transition, which is an alteration of cell
adhesion molecule expression by the epithelial cells and increased motility, i.e. a
mesenchymal phenotype
b. Invasion through the extracellular matrix involve matrix metalloproteinases and
cell interaction with the extracellular matrix via integrin binding, stimulated by various
chemokines
c. Penetration of blood and/or lymphatic vessels involves proteolytic enzymes
(metalloproteinases)
d. Survival within the circulation
184
e. Arrest in the distant organs microcirculation (capillaries, sinuses or venules)

involves cell adhesion molecules on endothelial cell and tumor cells as well as the
sites microenvironment.
f. Penetration of vessels at the distant site
g. Invasion through the distant sites parenchyma
h. Adapt to a new microenvironment
25.
What are the roles of cell adhesion molecules in tumor cell invasion?
Tumor cell interaction with the ECM at the original site, on endothelial cells at the
distant site, and the ECM at the distant site
26.
Why is angiogenesis important in tumor cell behavior?

Angiogenesis is need for growth beyond 2mm, metastasis and establishment of a
successful metastatic site
27.
What are the common paraneoplastic syndromes?

Paraneoplastic
Syndrome
Carcinoid syndrome
Polycythemia
Hypercalcemia
Cushing Syndrome
Hyponatremia - IADH
Eaton-Lambert
myastenia gravis
Acanthosis nigricans
Hypercoagulation
28.
Factor
Serotonin
Tumor
Erythropoietin
PTH-like peptide
ACTH
ADH
Auto antibodies
GI - neuroendocrine tumors
(carcinoids)
Renal cell carcinoma
Lung Squamous cell carcinoma
Lung small cell carcinoma
Lung small cell carcinoma
Thymoma
Mucin
GI adenocarcinomas
Mucinous adenocarcinomas
What are the components used in staging of a tumor?

T tumor extent size, invasion of local structures usually varies from T1 to T4
N regional lymph node metastasis Usually includes N0 (no mets), N1 and N2
mets to nodes at varying differences
M distant metastasis M0(-) M1 (+)
185
Case Studies in Neoplasia

Case 1
A 49 year-old woman was seen by a physician for evaluation of menometrorrhagia
(bleeding from the uterus) and significant anemia. Physical examination revealed an
enlarged uterus, and a hysterectomy was recommended. Gross pathology
demonstrated a single 10 x 5 x 8 cm soft, fleshy, well-circumscribed mass arising from
the myometrium. This is an image taken from the uterus.
Normal
Tumor
1.
2.
3.
What is the most likely diagnosis?

What is the likeliest clinical behavior of this tumor?
What is the normal cell type that gives rise to this tumor?
186
Case 2:
A 17 year-old male presents to the Emergency Department for treatment of a fractured
left tibia, that occurred as a result of falling off of his bicycle. An x-ray demonstrates a
pathologic fracture due to an intramedullary mass arising in the metaphysis of the
proximal tibia. A biopsy shows the following features.
1. What is the most likely diagnosis?

2. What is the likeliest clinical behavior of this tumor?
3. What is the normal cell type that gives rise to this tumor?
187
Case 3:
A 48 year-old man is seen by a physician complaining of being tired. His father died of
metastatic colon cancer at age 48, and a 34 year-old paternal cousin had a colectomy
for multiple colonic polyps one year ago. Laboratory studies demonstrate anemia, but
no other abnormalities. Physical examination demonstrates blood in his stool. A
colonoscopy demonstrates a mass in the right colon that on resection has the following
microscopic appearance.
1.
2.
3.
4.
What type of tumor is this?

What is the normal function of the genes that have undergone a germline
mutation associated with this tumor?
Which stage would be representative of a hepatic metastasis in this patient?
188
Case 4:
A 53 year-old female presented to her family physician complaining of fatigue and
menometrorrhagia. Lab studies demonstrated significant anemia. Physical
examination revealed a cervical lesion that was biopsied. This image is characteristic of
the lesion.
1.
2.
3.

What is the likeliest etiology of this lesion (be specific)?
The function of what cell cycle protein is altered by the etiologic agent in this
case?
189
Case 5:
A 71 year-old male presented to a physician with a complaint of a persistent cough,
confusion, polyuria/polydipsia. A chest X-ray demonstrated a 2.5 cm mass in the hilum
of the left lung. Lab tests revealed marked elevated serum calcium (hypercalcemia). A
biopsy was obtained, a microscopic image of which is seen below.
1.
2.
What is the likeliest etiology of this lesion?
3.
What is the likeliest cause of the patients hypercalcemia?
190
Case 6:
A six year-old boy is brought to a missionary hospital in the Republic of the Congo with
a mass on his left jaw. A biopsy and a peripheral blood stain (Image VII-Case 6-1and
inset) were obtained.
1. What is the most likely diagnosis?

2. What is the likeliest etiology of this lesion (be specific)?
3. Increased expression of which oncogene is associated with this disease
process?
191
Answers to Case Study Questions

Case 1
1.
leiomyoma
2.
benign
3.
Smooth muscle cells
Case 2
1.
Osteosarcoma
2.

Malignant
3.
Osteoblasts
Case 3
1.
What type of tumor is this?
Adenocarcinoma
2.
What is the normal function of the genes that have undergone a germline
mutation associated with this tumor?
The patient is too old for APP this is HNPCC Hereditary nonpolyposis
colorectal cancer or Lynch Syndrome. Involves methylation or mutation of
mismatch repair genes that include; MSH2, MLH1, MSH6, pms2
3.

Malignant
4.
Which stage would be representative of a hepatic metastasis in this patient?

M1 means you have you have a Stage IV cancer
192
Case 4
1.
2.
What is the likeliest etiology of this lesion (be specific)?

HPV 16 and 18 (high risk)
3.
The function of what cell cycle protein is altered by the etiologic agent in this
case?
HPV E6 enhances p63 degradation which inhibits apoptosis and inhibits p21 that
would inhibit cell proliferation thus leading to increased proliferation
HPV E7 inhibits p21 as well. E7 binds to RB protein displacing the E2F
transcription factor thus allowing for progression through the cell cycle.
Case 5
1.
2.
What is the likeliest etiology of this lesion?

Paraneoplastic syndrome due to PTH-like peptide elaboration leading
hyperparathyroidism
3.
What is the likeliest cause of the patients hypocalcemia?

Squamous cell carcinoma of the lung
193
Dr. Dale D. Vandre

Angiogenesis
01/09/12 Monday
10:30-11:30

WINTER 2012
CELL BIOLOGY MODULE
Lecture: Angiogenesis
Hamilton Hall, Rm 002, Lab 007
1.
2.
3.
4.
5.
6.
7.
8.
Describe what is meant by heterotypic interactions, and define the role that these types of
interactions play in tumorigenesis.
Define angiogenesis, and describe important processes that depend upon angiogenesis.
Describe how tumor cells can induce angiogenesis.
Describe examples of angiogenic factors and define their functional role in angiogenesis.
Compare and contrast the properties of tumor capillaries in relation to normal capillaries,
and describe the impact on tumor growth properties.
Describe what is meant by the angiogenic switch.
Describe examples of anti-angiogenic factors and define their functional role.
Describe the importance of targeting chemotherapeutic agents to the process of
angiogenesis.
Learning Resources
This handout and corresponding power point slides.
194
Dr. Dale D. Vandre

Angiogenesis
01/09/12 Monday
10:30-11:30
Tissues and Cancer
Cancer is a disease of tissues not just cells. As you recall from tissue histology, the
epithelium is avascular, and epithelial cells must receive nutrients by diffusion from blood
vessels that lie under the basement membrane. In most carcinomas (carcinomas are derived
from epithelial tissue and comprise over 80% of human cancers), 90% or more of the cells are
normal and serve as stroma for the tumor cells (in Hodgkins disease 99% of the cells in the
tumor are normal). Cancer cells communicate with and recruit normal stromal cells to the tumor
site including fibroblasts, endothelial cells, pericytes, smooth muscle, adipocytes, macrophages,
lymphocytes, mast cells, and other cells derived from mesenchyme. Communication between
various different cell types is defined as heterotypic interactions. The most important stromal
support required by the tumor is access to the circulation. The threshold for O2 diffusion through
tissue is 0.2 mm. Therefore tumor cells cluster around blood vessels, and if they are more than
0.2 mm from the vessel will die as a result of hypoxia.
Angiogenesis
New blood vessel growth is a process termed angiogenesis. The development of new
vasculature plays an important functional role in: 1) embryonic development; 2) implantation of
the placenta; 3) wound healing; 4) disease states such as diabetic retinopathy, psoriasis, and
rheumatoid arthritis; and 5) tumorigenesis. Tumors do not invade normal tissue and capture
existing blood supply, rather they recruit endothelial cells to form new capillaries.
How Do Tumors Assemble Microvasculature?

Cells monitor the O2 tension in tissue environment through proteins known as VHL proteins.
Under hypoxic conditions (low O2 tension), the VHL proteins allow for functional HIF-1
transcription factor, which subsequently regulates the production of a protein known as
vascular endothelial growth factor (VEGF). The cells, in this case tumor cells, release VEGF
into the interstitial fluid. VEGF binds to receptors on the surface of endothelial cells and
stimulate the proliferation of these cells. In addition, endothelial cells migrate towards areas of
higher VEGF concentration. The hypoxic tumor cells not only stimulate the replication of nearby
endothelial cells, but also attract them to the tumor mass along a gradient of VEGF in the
extracellular matrix. The tumor cells are not alone however. Myofibroblasts in the tumor stroma
release the chemotactic factor SDF-1 (stromal derived factor-1), which attract endothelial cells,
as well as VEGF, which stimulates endothelial cell proliferation and maturation. VEGF and
SDF-1 would be defined as angiogenic factors. Many other angiogenic factors have been
identified using assays in animal systems that detect blood vessel growth towards a source
implanted in tissues such as the cornea or the chick chorioallantoic membrane. Angiogenic
factors identified to date include TGF-, FGF, interleukin-8, angiopoietin, angiogenin, and
PDGF.
195
Dr. Dale D. Vandre

Angiogenesis
01/09/12 Monday
10:30-11:30
Initially the tumor recruits local endothelial cells, but it has been shown that as the tumor
matures it can recruit precursor cells from the bone marrow to the tumor site even prior to
metastasis of the tumor.
As you will learn in other lectures, tumors consist of a heterogeneous population of cells having
different morphological and tumorigenic properties. One of these heterogeneous characteristics
is angiogenic capacity. Different clonal populations of cells derived from a tumor have been
shown to vary in angiogenic capacity. Some clones have high angiogenic capacity while others
have little or no angiogenic capacity. Therefore it appears that highly angiogenic cells within the
tumor population support the growth of those tumor cells without angiogenic capacity.
Properties of Tumor Capillaries

The capillaries supporting tumor growth are not normal. Tumor capillaries are typically up to 3
times the diameter of normal capillaries. They have a much more chaotic layout, pericytes are
not as tightly associated, and smooth muscle organization is disrupted. Gaps of several
microns between the endothelial cells allow for greater blood plasma leakage, and the walls of
the capillaries are up to 10 times more permeable than normal. Greater interstitial fluid volume
contributes to an increased pressure. As a result the lymphatic drainage is poor due to collapse
of lymphatic vessels. This higher tissue fluid pressure in the tumor also lowers the effective
penetration of anti-tumor drugs. Increased capillary density within the tumor has a poor
prognosis for survival. It is not known whether increased vascularization leads to more
aggressive tumors or whether more aggressive tumors attract greater amounts of
vascularization.
The Angiogenic Switch

As a tumor progresses it gains the capacity for angiogenesis. In animal models of pancreatic
cancer, many small tumors will develop clustered around normal capillaries, but only a few of
these will grow out as they gain angiogenic capabilities. Therefore, normal condition in body is
to prevent cells from triggering angiogenesis, by overcoming this block the tumor progresses
releasing factors that stimulate angiogenesis. As such the tumor undergoes what is termed an
angiogenic switch. The recruitment of new blood vessels to underlying connective tissue near
a tumor can be observed prior to the tumor penetrating the basement membrane, thus benign
tumors demonstrate angiogenesis. Angiogenesis is also a necessary and required step in the
progression of a tumor towards malignancy. As the tumor breaks through the basement
membrane, blood vessels invade and gain access to the tumor.
196
Dr. Dale D. Vandre

Angiogenesis
01/09/12 Monday
10:30-11:30
Inhibitors of Angigenesis
A protein known as thrombospondin-1 is released by many different cell types into the
extracellular matrix that binds to receptors on endothelial cells and inhibits their proliferation.
This protein also induces apoptosis (programmed cell death) in endothelial cells. Thus in
normal tissues there exists a balance between angiogenic factors and inhibitors of
angiogenesis such as thrombospondin-1. Paradoxically, it appears that tumors also produce
anti-angiogeneic factors. Often after the surgical removal of a large primary tumor, cryptic
secondary tumors start growing. Thus it appears that the primary tumor is preventing or
inhibiting the growth of distant secondary tumors through the release of anti-angiogenic factors.
Once the source of the anti-angiogenic factors is removed, in this case the primary tumor, the
secondary tumors begin to grow as a result of new angiogenesis by these tumors.
There have been many anti-angiogenic factors identified including thrombospondin-1, arrestin,
tumstatin, angiostatin, and endostatin. Many of these inhibitors are derived from the
proteolytic cleavage of normal extracellular matrix proteins. Plasminogen a protein involved in
coagulation gives rise to angiostatin, collagen XVIII gives rise to endostatin, and collagen IV
gives rise to both tumstatin and arrestin.
Therapeutic Opportunity?
As you recall, tumors cannot grow above a diameter of 0.2 mm without angiogenesis to support
further expansion of the tumor mass. Thus without angiogenesis tumors are growth restricted.
If the angiogenic capacity of large tumors is, the tumor will collapse. This provides a
chemotherapeutic advantage because anti-angiogenic compounds target normal cells the tumor
is dependent upon, and eliminates the problem of tumor resistance to chemotherapy due to
heterogeneity and unstable genomes of the cells within the tumor. Also since normal
endothelial cells divide once over 100s of days up to several years, therefore in the adult blood
vessels are stable and chemotherapeutic drugs that target proliferating cells do not affect them.
In contrast, endothelial cells in tumors divide rapidly (every 50-60 hours), and standard anticancer drugs that target proliferating cells may kill the proliferating normal endothelial cells
present in tumors.
Are there other heterotypic pathways that can be taken advantage of to prevent tumor growth?
197

Lecture:
Biomarkers
1/11/12 8:30 9:20
Phone: office 247-7469, charles.hitchcock@osumc.edu
1. Define the basic types of biomarkers, their potential clinical utility, and the variables that
impact clinical value.
2. In a patient with a solid tumor, ascertain the best role of a serum protein biomarker of in
patient management.
a. What are clinical roles of ER/PR and HER2/NEU assessment of breast cancer?
3. Define the role molecular markers in patient diagnosis and management.
a. What is the clinical significance of assessing both EGFR and KRAS mutations prior to
treatment of a patient with adenocarcinoma of the lung or colon.
4. Define the steps needed to best validate tumor biomarkers.
Learning Resources:
1. Ludwig JA and Weinstein JN. Biomarkers in Cancer Staging, Prognosis and Treatment
Selection. Nature Rev Cancer 5:844-856 2005.
2. Ciardiello F and Tortora G. EGFR Antagonists in Cancer Treatment. NEJM 358:1160-1174,
2008
TUMOR MARKERS
A. Objective 1
1. Cancer biomarkers are substances produced by tumor cells or by other cells of the
body in response to cancer or to a benign or noncancerous condition that are found
in tissue, serum, urine or a body fluid. These substances include:
a. Proteins altered expression, mislocation, altered enzymatic activity, altered
structure
b. Chromosomes polyploidy, translocations, loss of heterozygosity (LOH)
c. DNA point mutations, microsatellite alterations, hypermethylation of promoter
regions, viral sequences
d. RNA altered expression, point mutations
2. Tumor biomarkers can be useful in multiple steps from ascertaining risk to monitoring
treatment response. These include:
a. Assessing risk for developing cancer
b. Screening to detect premalignant lesions or early cancer.
c. Diagnosis of a specific disease
1) Differentiating between benign and malignant disease
2) Determine type of malignancy
3) Improve staging
198
d. Predict prognosis: relapse or recurrence of a primary malignancy, progression of

metastatic disease, survival
e. Predict response to therapy
1) Chemotherapy
2) Hormone therapy
3) Novel therapy
f.
Monitoring disease
1) Predict relapse of primary disease
2) Follow detectable metastatic disease
3. There are several goals for using biomarkers.

a. The ultimate goal of using biomarkers is to accurately subclassify a patients
cancer in order to individualize therapy. Understand that tumor grading and
staging predict probable clinical course and outcome and that prognosis varies
with the treatment being used.
b. Their use needs to have a significant impact on clinical decision making. In the
end the, the benefits outweigh the toxicity risks.
c. Their routine use requires a standardized reproducible assay. Well designed
clinical trials whose results are validated in subsequent studies.
4. The FDA has approved several cancer biomarkers for clinical use; however, the
majority of more recent biomarkers are classified as analyte-specific reagents
(ASRs) that are used for research purposes only. They are not used routinely
because test is not reimbursed by Medicare/Medicaid or by private insurers.
5. Caveats:
a. The clinical impact of a biomarker varies with treatment toxicity and the patients
perspective.
b. Sensitivity and specificity of a biomarker varies with disease prevalence. For
example, a marker to be used in screening the general population must have an
extremely high specificity to minimize false positives that necessitate costly or
invasive follow-up studies and scare patients and their families needlessly. The
same marker need not be so specific if used for high-risk populations and can be
even less so once a cancer has been detected.
6. Research into the development and use of biomarkers brings together numerous
technologies (omics) to design potential panels that will be specific for an
individuals tumor, i.e. promote individualized medicine.
B. Objective 2
1. Classically, cancer biomarkers in the serum or urine have been quantitated using
monoclonal antibodies. These proteins range from small peptides to large
glycoproteins.
199
2. Numerous serum tumor markers have routine clinical utility.

Protein
Marker
Primary Tumor
Other Tumors
CEA
Colorectal carcinoma
CA19-9
CA125
Pancreatic, biliary tract

carcinomas
Hepatocellular,
non-seminomatous
(GCT)
non-seminomatous
GCT, gestational
trophoblastic disease
Ovarian carcinoma
PSA
Prostate
None
Ig
monoclonal
B Cell Lymphoma
B cell Leukemia
Myeloma
Plasmacytoma
AFP
-hCG
Benign Conditions
GI, breast, lung,

pancreatic etc
Colon,
esophageal, liver
Gastric, biliary,
pancreatic
Smoking, PUD,IBD,
pancreatitis, cirrhosis
Pancreatitis, biliary disease,
cirrhosis
Cirrhosis, viral hepatitis,
pregnancy
Rare GI
Hypogonadal states,
marijuana
Endometrial,
breast, lung, GI,
liver, pancreas
Menstruation, pregnancy,
fibroids, ovarian cysts, pelvic
inflammation,
endometriosis, cirrhosis,
ascites, pleural effusions
Prostatitis, BPH, trauma,
ejaculation
MUGUS
a. Serum CEA (carcinoembryonic antigen) is increased in colon, breast and lung

cancer, but also in many benign conditions. Its lack of specificity precludes it use
as a screening tool, but it is useful in detecting recurrent disease.
b. Although AFP (-fetoprotein) and -HCG (human chorionic gonadotropin-),
increased in several non-malignant setting, are reliability used in assessing
testicular germ cell tumors (think Lance Armstrong). The AJCC (American Joint
Committee on Cancer) has incorporated these two biomarkers, along with lactate
dehydrogenase (LDH), into the TNM system staging of testicular nonseminomatous germ cell tumors.
c. Serum protein biomarkers for cancer are not sensitive enough or specific enough
to be used as screening tools. To date, no randomized controlled study has
demonstrated their use as a screening tool provides any survival advantage.
d. Serum protein biomarkers are useful in the setting of monitoring patient for
recurrent disease. They should only be used when there is meaningful treatment.
1) Although the routine use of prostate specific antigen (PSA), in several
different forms, for screening males over the age of 50 for prostate cancer is
controversial, there is not disagreement about its value in monitoring
diagnosed prostate cancer or its treatment.
3. More recent studies have are using various techniques of proteomics to identify
protein-based fingerprints that will outperform individual protein biomarkers of
cancer.
200
4. Several cellular proteins are new biomarkers that are being used to predict
response to targeted therapy. They are the first generation of biomarkers for
individualized treatment of cancer.
a. Estrogen and progesterone receptors (ER/PR), identified by immunohistochemical staining, in the nucleus of breast cancer cells are routinely used as
a predictor of response to treatment with tamoxifen or aromatase inhibitors.
b. HER2/NEU, a member of the epidermal growth factor family of growth factor cell
membrane receptors, was original used as a negative prognostic biomarker
independent of TNM stage in patients with breast carcinoma.
1) Trastuzumab (Herceptin) is a monoclonal antibody used to target HER2/NEU
positive tumor cells. Thus, it is now a biomarker for predicting response to
treatment.
2) Both immunohistochemical staining and fluorescence in-situ hybridization
(FISH) techniques are being used to identify its presence in tumor cells.
c. CD20 is a transmembrane phosphoprotein that is expressed on all stages of B
lymphocyte development. It is used to help classify hematologic malignancies as
B cell a non-Hodgkin lymphoma or lymphocytic leukemias.
1) Rituximab is a monoclonal antibody that targets CD20 in the treatment of B
cell malignancies.
d. The presence of the CD117, a platelet-derived growth factor receptor-
(PDGFRA), in tissue is characteristic of a mutation of the c-KIT oncogene. The
tyrosine kinase activity of the receptor in gastrointestinal stromal tumors is
blocked by the drug Imatinib.
C. Objective 3
1. Molecular biomarkers are characterized by high specificity and high sensitivity due to
PCR technology.
2. The presence of absence of specific molecular markers have been used to assess
risk of cancer development (e.g. BRCA1 and BRCA2), improved classification of
diseases (e.g. acute myeloid leukemias), staging (e.g. molecular panels to
distinguish primary from recurrent tumor), predict response to treatment (e.g. BCRABL translocation), and monitoring of recurrent disease (eg. leukemias).
3. Chromosome deletions and gains as well as translocations are common in
neoplastic lesions. However, few of these changes are consistent enough to be
useful as a biomarker. However; they are being used with greater frequency to
subclassify patients with leukemia.
4. The classic chromosome biomarker is t(9;22)(q34;q11) giving rise to the Philadelphia
chromosome BCR-ABL fusion protein with tyrosine kinase activity in chromic myeloid
leukemia (CML). It is common demonstrated using FISH techniques.
a. The fusion proteins tyrosine kinase activity is the target of the drug Imatinib.
201
b. The presence of the Philadelphia chromosome in a subset of patients with acute

lymphoblastic leukemia, as demonstrated by PCR, predicts a poor prognosis,
and can be used to monitor the disease for recurrence.
5. DNA biomarkers of cancer include mutations in oncogenes, tumor-suppressor
genes, and mismatch-DNA repair genes.
6. Single-nucleotide polymorphisms (SNPs) are responsible for a vast majority of
genetic polymorphisms among individuals. The advent of high-throughput
technologies has allowed researchers to detect allelic variants of SNPs in breast
cancer. However; their use as a biomarker for predicting increased risk of sporadic
breast cancer is not as accurate as more conventional tools.
7. Cancer susceptibility genes are used as biomarkers that predict an increased risk for
cancer. The classic examples include:
a. BRCA1 and BRCA2 gene mutations predict increase risk for breast and ovarian
cancer.
b. MLH1, MLH2, MSH6, and PMS2 gene mutations predict an increased risk for
Lynch syndrome.
c. TP53 germline mutations (LiFraumeni syndrome) increase patient risk for
developing cancers.
8. Human papilloma virus (HPV) DNA typing in a cervical scraping is used as a
screening tool in the diagnosis of cervical dysplasia in women.
a. Viral types 6 and 11 impart a low risk for cervical squamous cell carcinoma
b. Viral types 16 and 18 impart a high risk for cervical squamous cell carcinoma
9. Mutational analysis of multiple genes (e.g. KRAS, B-RAF) and allelic imbalance
using microsatellite markers (e.g. 1p, 3p, 5q, 9p, 10q, 17p, 18q, 21q, 22q) are being
commercially used to generate cumulative mutational data to better distinguish
between low and high grade dysplasia. This same approach is used to improve
staging in cases where a new lesion must be defined as metastasis or a new primary
tumor.
10. Gene expression profiling, mutational analysis of multiple genes is being used
commercially as a prognostic biomarker, predicting recurrence, in those patients with
node-negative breast cancer.
11. Pretreatment analysis of specific gene mutations is being used to predict response to
targeted chemotherapy.
a. Mutation of EGFR (epidermal growth factor receptor) gene in adenocarcinoma,
but no other non-small cell carcinoma, of the lung predicts positive response to
tyrosine kinase inhibitors (Erlotinib and Gefitinib). The response to these drugs is
eliminated by the presence of a KRAS gene mutation which is downstream from
the EGF-receptors tyrosine kinase activity.
b. FISH analysis of EGFR copy number is predictive of response in patients with
colorectal carcinomas. This positive response is abrogated by the
presence of a KRAS gene mutation. Thus, KRAS mutation analysis is a
biomarker that predicts a poor response to antibody (Cetuximab) treatment to
EGFR alone or in combination with chemotherapy.
202
12. High through-put technologies are being used to evaluate multi-gene RNA patterns
or fingerprints as potential biomarkers in cancer. This approach depends heavily on
the development of new applications in biostatistics, bioinformatics and data
visualization.
13. As with DNA gene analysis, assessment of multiple miRNAs has the potential of
having a greater clinical impact as compared to assessment of a single miRNA.
D. Objective 4
1. The genetic paradigm of cancer does not account for the molecular complexity of
cancers. This complexity ranges from epigenetic modulation of mRNA expression or
altered protein expression, post-translational modification, and/or function. The
introduction of high-throughput technologies is providing an ever growing list of
potential biomarkers for cancer. Few of the current and emerging biomarkers
available have been integrated into clinical practice.
2. Potential cancer biomarkers undergo a series tests before they can be considered
for clinical practice. Five Levels of Evidence) of biomarker development have been
proposed.
Levels of Evidence (LOE) (Hayes et al., JNCI 88:1456, 1996)
Level
Type of Evidence
I
Evidence from a single high-powered prospective controlled study
specifically designed to test the marker, or evidence from meta-analysis,
pooled analysis, or overview of level II and III studies.
II
Evidence from a study in which marker data are determined in
relationship to a prospective therapeutic trial that is performed to test a
therapeutic hypothesis but not specifically designed to test marker utility.
III
Evidence from large retrospective studies
IV
Evidence from small retrospective studies
V
Evidence from small retrospective pilot studies
3. The problem lies in the fact that no biomarkers have gone beyond phase III testing.
Most studies are small and are generally use retrospective material, and prospective
studies, often hampered by a small patients set, have often yielded inconsistent
results.
4. Standardization of technologies and statistical validations is critical to the routine
clinical application of biomarkers.
5. Ideally, biomarkers should be validated analogously in prospective, well-controlled
clinical studies of diverse patients across multiple institutions, with well-established
standards for all steps in the process. Those steps include, for example, tissue
collection, purification, amplification (if necessary), hybridization or ligand-binding,
data capture, normalization, statistical analysis and scoring. Furthermore, there
should be reliable reproducibility within and among laboratories. However, those
ideal conditions rarely apply.
6. Economics will play a large role in driving this process.
203
Dr. Dale Vandre

Proteomics
01/12/12, Thursday
8:30-9:30
WINTER 2012
CELL BIOLOGY MODULE
Lecture: Proteomics (Cancer Biomarkers)
Hamilton Hall, Rm 002, Lab 007
Learning objectives: Be able to 1.
2.
3.
4.
5.
6.
7.
Describe how biomarkers could be applied to clinical situations.

List the characteristics of an ideal biomarker, and differentiate biomarkers based
upon data presented in receiver operating characteristic (ROC) curves.
List the relevant sources of clinical material for biomarker discovery.
Define proteomics and describe how proteins are identified by mass spectroscopy
in a typical proteomics analysis.
Describe the advantages and disadvantages of human serum as a source for
proteomics based discovery of biomarkers.
Define and differentiate the following proteomics methodologies: 2D-DIGE,
MALDI-TOF, SELDI-TOF, ESI-LC-MS.
List the validated cancer biomarkers discovered by proteomics currently used in
clinical applications.
Learning Resources
This handout and the accompanying powerpoint presentation.
For a more complete description of ROC analysis visit:
http://www.anaesthetist.com/mnm/stats/roc/Findex.htm
What is a Biomarker?
A biomarker is any biological compound or process that provides a molecular fingerprint
of disease. A cancer biomarker would be a molecule or process that can be measured,
which differentiates the patient with a tumor from a patient without the tumor. Thus a
cancer biomarker could be DNA, mRNA, microRNA, metabolites, proteins, or processes
such as increased proliferation/mitosis, decreased apoptosis, or angiogenesis, as long as
the molecule or process differentiates patients with tumors from those without. To
substantiate the use of a biomarker clinically, it must ultimately be associated with
proven improvements in patient survival and/or quality of life. We will focus on proteins
as cancer biomarkers, and the utilization of proteomics technologies as they have been
applied to the discovery of new biomarkers.
204
Dr. Dale Vandre

Proteomics
01/12/12, Thursday
8:30-9:30
What types of clinical situations could biomarkers be applied?

Biomarkers could be used in a variety of clinical situations, and could be utilized to
individualize patient care. If sufficient types of biomarkers are available they could be
utilized to design a personalized treatment plan for each individual patient, one type of
personalized medicine based upon molecular fingerprints. Some typical clinical
applications of biomarkers are listed below:
1.
2.
3.
4.
5.
Screening of the general population to detect occurrence of disease. Ideally such

a screen would use a non-invasive test to indicate cancer risk or development.
Clinical staging of disease. This type of biomarker would either allow for early
detection and/or differentiate between benign and malignant disease, or be able to
differentiate between early and advanced stages of malignant disease such as
metastasis. Such markers might also be used to estimate tumor mass.
Differential diagnosis of disease. This type of biomarker would allow for the
classification of subtypes of individual cancers, for example distinguish between
aggressive versus less aggressive forms of a particular cancer.
Predictive biomarker. These biomarkers would be utilized to determine the
probability of response to treatment. They would be used to stratify the cancer to
determine the most appropriate course of therapy, or be used to monitor response
of the tumor to therapy.
Prognostic biomarker. These biomarkers would be used to monitor disease
progression, regression, or recurrence. They would also be markers for likelihood
of cure.
What are the ideal characteristics of a cancer biomarker?

The ideal biomarker for practical clinical use must:
1.
2.
3.
4.
4.
5.
Be measured easily using non-invasive methods.

Be quantifiable.
Be measured using cost effective methods.
Be present at early or preclinical stages of disease.
Have high sensitivity.
Have high specificity.
Specificity with relation to a clinical biomarker test refers to the proportion of the normal
population that test negative. Therefore a highly specific biomarker would have few if
any false positives. Sensitivity with relation to a clinical biomarker test refers to the
proportion of the diseased population that test positive. Therefore a highly specific
biomarker would have few if any false negatives.
205
Dr. Dale Vandre

Proteomics
01/12/12, Thursday
8:30-9:30
If we consider some test for a disease (in this case the presence of biomarker X, see Table
below), the following results could be obtained for a group of diseased and normal
patients examined for the presence of the biomarker. If the test is positive for a diseased
individual this is the true positive fraction (TPF), if the test is negative for a diseased
individual this represents the false negative fraction (FNF). The TNF + FNF must = 1,
the total of the diseased individuals. Similarly, if the test is positive for a normal
individual this represents the false positive fraction (FPF), and if the test is negative for a
normal individual this represents the true negative fraction (TNF). As with the diseased
population, the normal population also shows FPF + TNF = 1. The fraction of TPF, FPF,
FNF, and TNF will change depending upon where the cutoff levels for the level of X are
set (see graphical representation in corresponding lecture powerpoint presentation, or for
more in depth information go to the following web site:
http://www.anaesthetist.com/mnm/stats/roc/Findex.htm).
Test for disease = biomarker X
Diseased
Normal
Postive test*
TPF
FPF
Negative test*
FNF
TNF
*cutoff levels for values of X are based upon relative/arbitrary levels
As the specificity of the test increases, the false positive fraction (FPF) decreases. As the
sensitivity of the test increases, the false negative fraction (FNF) would decrease.
Typically as the sensitivity of an assay increases the specificity decreases, and vice versa.
A graphical representation between sensitivity and specificity is the receiver operating
characteristic (ROC) curve, and the ROC can be used to evaluate the efficacy of a tumor
marker at various cut-off points. The ideal ROC graph would have the maximum area
under the curve (AUC), being characterized by a 100%TPF at the same time as a 0%
FPF. Using ROC analysis, and comparing the AUC between different tests (biomarkers)
allows for the determination of whether a new test (biomarker) has greater clinical
efficacy (see corresponding powerpoint slides for this lecture).
A possible biomarker for ovarian cancer having a specificity of 95% has been described
in the literature. Currently there is no diagnostic test for ovarian cancer, and it is
usually found as a widespread metastatic disease at initial diagnosis. There is also no
current method of early detection of the disease. The frequency of ovarian cancer is
1/2500. Based upon this information, would you recommend the use of this new
biomarker test to screen the general population for ovarian cancer? Why or why not?
206
Dr. Dale Vandre

Proteomics
01/12/12, Thursday
8:30-9:30
Sources of Biomarkers
Human biological fluids provide the must relevant source of material for biomarker
discovery. The biological sample of choice is blood plasma or serum, since this can be
collected by standard non-invasive procedures, and it is thought that compounds
produced by the tumor may enter the circulation. An example of this type of marker, and
one that is used for clinical purposes is prostate specific antigen (PSA). This protein is
normally shed into the glandular lumen of the prostate, but low levels of the protein can
enter the circulation by diffusion through normal epithelial junctions and the basement
membrane (serum levels of 0.5-2 ng/ml in normal individuals). As the tissue architecture
is destroyed, increasing levels of PSA can enter the blood. In advanced prostate cancer,
PSA levels between in range of 10-1000 ng/ml can be detected. In addition to blood
serum or plasma, other fluids sources for biomarkers would include, cerebrospinal fluid,
nipple aspirate fluid, cervicovaginal fluid, stool, pleural effusion, bronchoalveolar lavage,
saliva, ascites fluid, pancreatic juice, seminal fluid, and urine.
Proteomics
Proteomics is the large-scale study of proteins, and the proteome refers to the complete
protein complement of a specific organism or system including all of the modifications
associated with that set of proteins. Proteomics has only become possible as the result of
several technological advances, which include: 1) standardized and reproducible methods
for the two-dimensional separation of proteins based upon charge (isoelectric focusing)
and size (SDS polyacrylamide gel electrophoresis); 2) sequencing of the human genome
and the availability of the corresponding bioinformatics resources; and 3) development of
mass spectrometers suitable for the analysis of peptides.
A standard proteomics analysis would involve the following steps. Some form of protein
sample preparation is required. This could include isolation of a cell type or organelle, or
the collection of a biological fluid such as serum under standardized conditions. The
prepared sample normally will contain a complex mixture of proteins, and the proteins
must be separated and/or fractionated to reduce the complexity of the mixture. If proteins
are separated by two-dimensional electrophoresis, individual protein spots can be
isolated. After staining of the gels to detect the individual proteins, these protein spots
are then cut from the gel matrix, and the proteins are digested using specific proteases
such as trypsin or chymotrypsin. The peptides derived from the digested proteins are
analyzed by mass spectrometry and the accurate mass of the peptide is determined.
Based upon the peptide mass information, genome databases can be searched to identify
possible proteins that contain peptides of the appropriate mass. If the mass of a sufficient
number of peptides is determined, the identity of the specific protein from which these
peptides were derived can be determined. If the peptides are derived from a mixture of
two or more proteins, it becomes impossible to determine the protein identification from
the accurate mass of the peptides alone. Other types of mass spectrometers that are
capable of determining the sequence of individual peptides can be used to identify
proteins present in mixtures.
207
Dr. Dale Vandre

Proteomics
01/12/12, Thursday
8:30-9:30
Serum as a source for proteomics based biomarker analysis.
Serum is thought to be an ideal source of cancer biomarkers, since it is hypothesized that
tumors will shed small amounts of protein into the blood. Thus blood may contain a
molecular protein fingerprint of the cancer. Since blood is also easily obtained, this
makes it an ideal candidate source for identifying new biomarkers. The first report of a
biomarker pattern for cancer using proteomics technologies was reported for ovarian
cancer in 2002. Despite the excitement generated by this report, other laboratories could
not reproduce the ovarian cancer markers that were detected, and these markers were
never validated. Similar early promising reports of other cancer biomarkers detected in
serum were also never proven. As a result, subsequent studies have brought into question
this approach, which was based upon protein peak patterns present in serum as
determined by SELDI-TOF (see below for mass spectroscopy methods).
It is thought that serum may contain >10,000 proteins, and that the dynamic range of
protein concentration in serum may vary over 10 orders of magnitude. The analytical
tools used to examine the serum proteome suffer from being limited to a sensitivity of
only 3 orders of magnitude. Thus, there is no method that can detect the range of protein
concentrations found in serum. To complicate matters further, only 14 different serum
proteins account for 95% of the total protein present in serum (albumin alone accounts
for ~60% of the total protein concentration in serum). If you include these proteins and
the next 8 most abundant serum proteins (the 22 most abundant serum proteins) they
account for 99% of the concentration of all serum proteins. In general, biomarker
proteins derived from tumors are thought to be minor serum protein components, and
these abundant proteins likely mask the presence of lower abundance biomarkers. In
part, the initial biomarker studies using SELDI-TOF mass spectroscopy were looking at
possible non-specific proteins or fragments generated from abundant serum proteins that
were not associated with the tumor. Practical searches for biomarkers in serum using
proteomics approaches require prefractionation of the serum to remove or deplete the
serum of these abundant proteins. Antibody depletion reagents are now commonly used
to deplete the serum prior to analysis. Additional methods have also been shown to be
necessary to further prefractionate the serum to get meaningful proteomics results.
Proteomics and mass spectrometry terminology and basic principles or operation
New proteomics methodologies are being developed at a rapid pace, and advanced mass
spectrometers can range in price from $200,000 to over 1 million dollars. Several
common types of proteomics analysis and mass spectroscopy are briefly listed here.
2D-DIGE (two dimensional-difference gel electrophoresis) is a two-dimensional gel
electrophoresis method where two different samples (for example normal and diseased)
are mixed together and separated on the same gel. Proteins in one sample would be
tagged with one fluorescent dye (red) and proteins in the other sample would be tagged
with a second fluorescent dye (green). When the mixed proteins from the two samples
are separated, differences in protein composition can be readily detected on the single gel
by observing the color of the separated spots. The red fluorescent staining pattern is
208
Dr. Dale Vandre

Proteomics
01/12/12, Thursday
8:30-9:30
overlayed on that of the green pattern, and where proteins overlap a yellow color is
produced (green + red = yellow). Differences in green and red spots intensity indicate
distinct differences in relative protein amount between the two samples.
SELDI-TOF (surface enhanced laser desorption ionization-time of flight) is a type of
mass spectroscopy where proteins attach to the surface of a sample plate depending upon
the properties of the plate surface (hyodrophobic, positive charge, negative charge,
hydrophilic surfaces). A mixture of proteins will adhere to the surface and these proteins
are then ionized and separated in a time-of-flight mass analyzer. The resulting data is a
pattern of protein peaks at various molecular weights. The pattern can be distinct for a
disease when compared to the patterns obtained from control samples. The major
disadvantage of SELDI-TOF is that the protein peaks cannot be identified, and thus
verification of the potential biomarker peaks is very difficult if not impossible.
MALDI-TOF (matrix assisted laser desorption ionization-time of flight) is a type of mass
spectroscopy suited for the determination of the accurate mass of peptides. If the
peptides are derived from a single protein, the peptide masses that are determined can
define the identity of the protein. MALDI-TOF is not suited for the analysis of mixtures
of peptides derived from different proteins.
ESI-LC-MS (electrospray ionization-liquid chromatography-mass spectroscopy) is a
mass spectroscopic technique suited for the analysis of peptide mixtures that are derived
from multiple proteins. The peptides enter the mass spectrometer following liquid
chromatographic separation where the peptides are ionized in the liquid stream and
separated by mass based upon magnetic deflection in the mass spectrometer. Peptides of
individual mass can be collected and further fragmented within the machine and
subjected to another round of mass separation, which yields a peptide sequence. From
the peptide sequence information the protein from which the peptide was derived can be
determined.
Validated cancer biomarkers obtained from proteomics studies
To date there are no validated cancer biomarkers that have been identified using
proteomics approaches. Early optimism, based upon SELDI-TOF studies, was proven to
be premature. Despite these early setbacks, there remains a significant interest in and
potential for development of new biomarkers using proteomics approaches. With
improved approaches for the prefractionation of serum and advances in mass
spectrometer technology it is likely only a matter of time before useful cancer biomarkers
are identified, validated and appear in clinical settings.
A current example of the state-of-the-art in proteomics biomarker analysis is a report in
the Journal of Proteome Research entitled 2D-DIGE as a strategy to identify serum
markers for the progression of prostate cancer by J.C Byrne et al. They describe the
analysis of serum from prostate cancer patients using a 2D-DIGE approach coupled with
ESI-LC-MS. Two proteins differentially expressed in prostate cancer were identified,
209
Dr. Dale Vandre

Proteomics
01/12/12, Thursday
8:30-9:30
pigment epithelium derived factor (PEDF), and zinc-alpha2-glycoprotein (ZAG).
Differences in protein expression were also shown when comparing low grade (Gleason
score5) from aggressive (Gleason score 7) prostate cancer. Interestingly, PEDF is a
protein having anti-angiogenic activity. And the levels of protein expression go down in
prostate cancer patients. Using ROC analysis, they demonstrate that these two marker
proteins can be used to distinguish prostatic cancer from benign prostatic hyperplasia
better than using prostatic specific antigen (PSA), which is the standard clinical
biomarker currently used (see powerpoint slides that accompany this lecture).
210

Lecture:
microRNA : From Bench to Clinic -1/13/12
Melissa Piper Ph.D., Division of Pulmonary, Allergy, Critical Care and Sleep Medicine
Heart and Lung Research Institute, Rm 201
Phone: 247-7642, Melissa.piper@osumc.edu
1. Define microRNA processing and roles of key mediators
2. Understand the regulatory role that microRNAs play in biological processes
3. Understand concepts of microRNA/mRNA targeting and gene/protein regulation
4. Define the homeostatic role of miRNAs in tissue and peripheral blood
5. Understand applications of microRNA to pathogenesis of lung disease
6. Define the utility of miRNAs as diagnostic and prognostic biomarkers of disease
7. Introduction into viral miRNAs hat hijack the host immune system
Learning Resources:
Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 116(2), 281-297
(2004)
Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for
small expressed RNAs. Science. 294(5543), 853-858 (2001).
Calin GA , Croce CM. MicroRNA signatures in human cancers. Nat.Rev.Cancer. 6(11), 857-866
(2006).
Sonkoly E, Sthle M, Pivarcsi A. MicroRNAs and immunity: novel players in the regulation of
normal immune function and inflammation. Semin Cancer Biol. 2008 Apr;18(2):131-40. Epub
2008 Jan 15. Review.
Barbarotto E, Schmittgen TD, Calin GA.MicroRNAs and cancer: profile, profile, profile.
Int J Cancer. 2008 Mar 1;122(5):969-77. Review.
Cullen BR. Viruses and microRNAs: RISCy interactions with serious consequences. Genes
Dev. 2011 Sep 15;25(18):1881-94.
Nana-Sinkam SP, Karsies T, Riscili B, Ezzie M, Piper M. Lung microRNA: from development to
disease. Expert Rev Respir Med. 2009 Aug;3(4):373-85. PubMed PMID: 20477329.
Hunter MP, Ismail N, Zhang X, Aguda BD, Lee EJ, Yu L, Xiao T, Schafer J, Lee ML, Schmittgen
TD, Nana-Sinkam SP, Jarjoura D, Marsh CB. Detection of microRNA expression in human
peripheral blood microvesicles. PLoS One. 2008;3(11):e3694. Epub 2008 Nov 11.
A. microRNAs: General Considerations
1. MicroRNAs (miRNAs) are small non-coding (19-22 nucleotide) RNAs that were first
identified in C. elegans larval development
2. To date, miRNAs have been identified in plants, animals and fungi. The generation of
miRNAs are evolutionarily conserved
211
3. MicroRNAs may located in introns of mRNAs or in independent transcription untis

4. The biogenesis of miRNAs is a multi-step process beginning in the nucleus and
culminating in the cytoplasm and involves numerous enzymes and accessory
proteins
5. MiRNAS are also encoded in clusters with their own promoter and open reading
frame. They are then subjected to mechanisms that regulate gene expression such
as transcriptional regulation.
6. MiRNAs are involved in numerous biological processes including metabolism, cell
cycle, apoptosis, and disease regulation
7. By regulating hundreds of genes simultaneously, miRNAs have the capacity for
regulation of biological networks
8. MiRNAs may be responsible for the regulation of up to one third of the genome.
9. To date, over 1000 miRNAs have been identified in mammals, yet their biological
relevance and functional targets remain largely unknown.
10. Viruses have miRNAs that target the immune response
B. microRNA Processing: Generation of mature miRNAs requires multiple steps:
1. Within the nucleus, a long primary (pri)- miRNA transcript ranging from hundreds to
thousands of nucleotides in length is transcribed by RNA polymerase II
2. The pri-miRNAs are then bound to a double-stranded RNA-binding domain (dsRBD)
protein known as Pasha or Partner of Drosha for invertebrates or DiGeorge
syndrome critical region gene 8 (DGCR8) for vertebrates
3. The processing of the pri-miRNA to smaller stem-loop ~70 nucleotide (nt) precursor
miRNA (pre-miRNA) molecules is facilitated by an universal RNase III endonuclease,
RNASEN, better known as Drosha in invertebrates.
4. The pre-miRNAs are then transported to the cytoplasm by the double-stranded
(ds)RNA binding protein, Exportin 5
5. Once in the cytoplasm, the pre-miRNA is further cleaved to double stranded ~22 nt
miRNA molecules by the RNase III endonuclease, Dicer. The miRNA is separated
into two single stranded molecules; the anti-sense strand is incorporated in the RNAinduced silencing complex (RISC) through its interaction with Argonaute (AGO)
proteins, while the sense strand is targeted for degradation
6. In the mRNA processing bodies (P-bodies), AGO proteins bind to the 3 UTR of the
mRNA and facilitate the alignment and stabilization of the miRNA:mRNA duplex. In
addition to the AGO proteins, other proteins important in the degradation and/or
silencing of mRNA targets are found in the RISC complex including a variety of
helicases, mRNA decapping proteins, deadenylases, and methyltransferases
212
C. microRNA/mRNA Targeting
1. Conserved base-pairing between the 3 UTR (untranslated region) of the mRNA and
the seed sequence located in the 5 end of the miRNA plays a role in determining the
fate of the mRNA
2. A high degree of complementary between the seed sequence of the miRNA and the
mRNA is sufficient for the miRNA:mRNA duplex to form.
3. If a perfect match exists, then generally the target is degraded through ubiquitination. In
some cases, the perfect match of one miRNA is not sufficient to degrade its target but
rather multiple miRNAs are required to bind the 3 UTR of the mRNA molecule. However,
imperfect matches mainly predict inhibition of protein translation.
4. There have been a few reports indicating that miRNAs can also bind the 5 UTR of the
mRNA, however the end result of this interaction is not as efficient as 3 UTR silencing
5. Using target prediction modeling, there are four 5-dominant interaction sites for the
formation of the miRNA:mRNA duplex and are designated; 6mer, 7mer-m8, 7mer-A1, and
8mer.
6. The 6mer site matches have perfect complementary to the seed sequence, while the
7mer-m8 matches the seed sequence perfectly as well as an additional nucleotide in the
8th position. The 7mer-A1 also matches the seed sequence but has an adenine in the first
position of the miRNA which is believed to augment the binding of the miRNA to the
mRNA. Lastly, the 8mer interaction site is a combination of the two 7mer sites. It has the
match seed sequence with the adenine in the first position and the matched nt at the 8th
position of the miRNA.
213
7. The presence of polymorphisms in the 3 UTR of the target gene may also compromise
the miRNA binding and regulation
D. Target Prediction Analysis

1. Several databases have been developed to predict the targets of the miRNAs. Many of
these evaluate the free-energy generated by the formation of the miRNA:mRNA,
phylogentic conservation, and/or the interaction sites present. These include: TargetScan,
Microcosm and MirTarget2
E. MicroRNA Detection
1. Northern blotting
2. Microarrays
3. Bead-based flow cytometry
4. Quantitative PCR
5. Deep sequencing
6. Nanostring Technology
F. MicroRNA Regulation: There multiple manners in which miRNAs may be regulated:

1. Chromosomal location
A. Fragile chromosomal regions that are susceptible to translocations, microdeletions and amplifications
214
B. Allelic loss is a frequent genetic alteration in human lung cancers.

Cytogenetic and molecular analyses have revealed chromosomal deletion on 3p, 8p, 9p,
11p, 13q, 17p, 18q and 22q
2. Epigenetic Regulation
3. miRNA/miRNA interaction
4. Environmental factors: diet, smoking, infection
G. MicroRNAs in Human Lung Disease

1. Lung Development
A. Animal studies demonstrate that miRNA are aberrantly expressed during lung
development
B. The changes in the miRNA expression patterns between the fetal and adult
lung may reflect the transition of immature cell types that are found in the
developing lung to those present in the adult tissue. In contrast, miRNA
expression that is unchanged throughout development may be functionally
important in regulating homeostasis within the lung or maintenance of cell
phenotypes.
C. MiRNAs appear to be quiescent after development to maintain lung
homeostasis, but may become perturbed in disease states of the lung.
2. Inflammatory Response
215
A. Research to date suggests a vital role for miRNA in inflammatory cell

differentiation, regulation of the innate immune response, and regulation of the
adaptive immune response, both from T and B lymphocytes
B. Cigarette smoke causes changes in miRNA expression in both animal and
human study
3. Lung Diseases
A. Lung Cancer
1. The number one cause of cancer related deaths in the United States
among both men and women
2. Aberrant expression of miRNAs in several malignancies compared to
normal tissue as well as their frequent location in fragile chromosomal
regions suggests that they may be important to pathogenesis of disease
3. MiRNAs have the capacity to function as either oncogenes or tumor
suppressors
4. miRNAs can also mediate angiogenesis.
5. Alterations in miRNA processing appear to contribute to lung
tumorigenesis. Silencing of Drosha (RNASEN), DGCR8 and Dicer and
decrease global miRNA production caused both in vivo and in vitro
promotion of lung tumor growth
6. miRNAs as Biomarkers of Disease: expression patterns have been
linked to survival
B. Idiopathic pulmonary fibrosis (IPF)
1. Has a 2-5 year mortality rate after diagnosis with no know treatment.
2. Disease is associated with increase collagen and fibrotic gene
expression in the lung.
3. Loss of the miRNA cluster miR-17~92 is associated with the disease.
The miRNAs in this cluster target many fibrotic genes and regulators of
epigenetic gene expression.
C. Idiopathic Pulmonary Arterial Hypertension
1. Abnormal highly arterial pressure alter cardiac and lung function.
2. Previously thought to be associated with EBV infection
3. Detected miRNAs from HSV, EBV and KS in peripheral blood.
D. Sepsis
1. A common and costly disease often overlooked as a complication of
other diseases (underappreciated).
2. Early and effective antibiotics are the only proven therapy
3. No effective mechanisms to optimize organ function or to alter the
immune response.
4. Identification of miRNA profile early in disease compared to late when
multiple organ dysfucntion
216
217

Cell, Division 2 Course Packet 2011-12

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Cell, Division 2 Course Packet 2011-12

Uploaded by

Copyright:

Available Formats

The Ohio State University College of Medicine

Med 1 Integrated Pathway

The Cell, Division 2

Med 1, Cell Block (Division 2)

Dr. Hai, Handout cover page

MED 1. CELL BLOCK

Date and Time

The Cell, Division 2 2011-12

Med 1, Cell Block (Division 2)

Dr. Hai, Handout cover page

Practice Exam Questions for Dr. Hais Lectures

Med 1, Cell Block (Division 2)

Dr. Hai lecture 1, RNA Property

The Cell, Division 2 2011-12

Med 1, Cell Block (Division 2)

Dr. Hai lecture 1, RNA Property

(1) Different types of RNA

The Cell, Division 2 2011-12

Med 1, Cell Block (Division 2)

Dr. Hai lecture 1, RNA Property

Some modified bases:

The Cell, Division 2 2011-12

Med 1, Cell Block (Division 2)

Dr. Hai lecture 1, RNA Property

Med 1, Cell Block (Division 2)

Dr. Hai lecture 1, RNA Property

Some unusual base pairing (non-Watson Crick base pairing):

Wobble base pair

The Cell, Division 2 2011-12

Med 1, Cell Block (Division 2)

Dr. Hai lecture 1, RNA Property

L-shape tertiary structure

The Cell, Division 2 2011-12

Med 1, Cell Block (Division 2)

Dr. Hai lecture 1, RNA Property

The Cell, Division 2 2011-12

Med 1, Cell Block (Division 2)

Dr. Hai lecture 1, RNA Property

All from exons

(4) tRNA: Adapter molecule

The Cell, Division 2 2011-12

Med 1, Cell Block (Division 2)

Dr. Hai lecture 1, RNA Property

the stem to accept the amino acids

The Cell, Division 2 2011-12

Med 1, Cell Block (Division 2)

Dr. Hai lecture 1, RNA Property

(5) rRNA: Component of ribosomes (most abundant RNA)

Structure provides functions.

The Cell, Division 2 2011-12

Med 1, Cell Block (Division 2)

Dr. Hai lecture 1, RNA Property

(6) microRNAs (miRNAs, miRs)

The Cell, Division 2 2011-12

Med 1, Cell Block (Division 2)

Dr. Hai lecture 1, RNA Property

(7) Other types of RNAs

(8) Clinical discussions

Med 1, Cell Block (Division 2)

Dr. Hai lecture 1, RNA Property

ribosomes. Because mitochondrial ribosomes are similar to the bacterial ribosomes,

Summary of RNA properties

The Cell, Division 2 2011-12

Med 1, Cell Block (Division 2)