Apoptosis and Cell Necrosis

Biochemistry
1st Year Section D1 Group 3

San Marcos, B. J. M.
Sandig, J. A. D.
Santos, E. M.
Santos, S. F. S.
Apoptosis

Programmed cell death

Active and controlled breakdown of a cell
due to an internal cellular program
Causes cell death by factors external to
the cell
involved in regulation of steady-state
levels of cells undergoing proliferation
Apoptosis vs. Necrosis
Apoptosis
Active and controlled program
Requires ATP
Individual cells appear involved
Cellular features:
•cell shrinkage
•Membrane remain intact
•condensation and fragmentation,
•phagocytosis of cell remnants
•Ladder-like DNA fragentation
Necrosis
Passive and inflammatory
ATP is depleted
Whole areas of tissue are affected
Cellular features:
•Cellular swelling
•membrane breaks down and
cellular contents leak out
•nucleus disintegrates
•cell ghosts
•DNA fragmentation is random
Two Pathways that Initiate
Apoptosis
Intrinsic/ Mitochondrial Apoptosis
◦ Regulated by Mitochondria
◦ Cytochrome c release

Extrinsic/ Death Receptor Apoptosis

◦ Activated by ligation of Death Receptors
◦ Fas, TNF alpha

These pathways intersect at the effector

caspases
Extrinsic Intrinsi
c
 Signals That Define The Apoptotic
Phenomenon

Internal Signals External Signals Pro- Anti-

apoptotic apoptotic
factors factors
•Glucocorticoids •Toxins •Bax •Bcl-2
•Radiation •Hormones •Bad •Bcl-xL
•Nutrient deprivation •Growth factors •Bak
•Viral infection •Nitric oxide •Bid
•Hypoxia •Cytokines
•Increased intracellular
calcium
Role of Apoptosis
Embryogenesis
A.I. D. S.
Cancer
Immune Defense
Role of Apoptosis
Embryogenesis
Role of Apoptosis
A. I. D. S.
◦ HIV enzymes
 deactivate anti-apoptotic Bcl-2
 primes the cell for apoptosis
 activate pro-apoptotic procaspase-8
◦ HIV
 increase the level of cellular proteins
 Fas-mediated apoptosis.
◦ decrease the amount of CD4 glycoprotein marker
◦ Released viral particles and proteins
 induce apoptosis in nearby "bystander" T helper cells.
◦ decreases the production of molecules involved in
marking the cell for apoptosis
◦ The infected CD4+ cell may also receive the death
signal from a cytotoxic T cell.
Role of Apoptosis
Cancer
◦ Human papilloma viruses (HPV)
 cervical cancer
 protein (E6)
 binds and inactivates the apoptosis promoter p53.

◦ Epstein-Barr Virus (EBV)

 mononucleosis and associated with some
lymphomas
 produces a protein similar to Bcl-2
 produces another protein that causes the cell to
increase its own production of Bcl-2
Role of Apoptosis
Cancer
◦ B-cell leukemias and lymphomas
 high levels of Bcl-2
 blocking apoptotic signals they may receive.
◦ Melanoma cells
 inhibits the expression of the gene encoding Apaf-1.
◦ Lung and colon cancer cells
 elevated levels of a soluble "decoy" molecule
 binds to FasL,
◦ Other cancer cells
 high levels of FasL
 kill any cytotoxic T cells (CTL)
Role of Apoptosis
Immune Defense
◦ B and T cell development
 eliminates cells
 nonfunctional B or T cell receptors
 autoreactive or self-destructive B or T cell receptors
 regulating activation of these cells with respect to the
duration and magnitude of an inflammatory response.
◦ Granulocyte development
 Increase life span at the site of infection
 Downregulates the inflammatory response
◦ prevents the release of enzymes and other
inflammatory mediators from senescent
granulocytes into surrounding healthy tissue,
preventing tissue destruction
Laboratory Indices
 Detection of apoptotic cells
◦ Microscopy
 Cells have classic features (eg. small darkly stained
nuclei)
 Detection of free 3’ ends of DNA by TUNEL
assay (terminal deoxytransferase-mediated dUTP-
biotin nick end labeling)
◦ Gel electrophoresis
 Detect DNA ladder of 180 bp intervals caused by
internucleosomal

 DNA cleavage
◦ Flow cytometry
 Measure externalization of phosphatidylserine (PS)
with fluorescently labeled Annexin-V
 Measure DNA fragmentation with propidium
iodide fluorescence
TUNEL Assay
TUNEL Assay
TUNEL Assay
Gel Electrophoresis
Flow Cytometry
Thank You for Listening!