BIO + MED written by VIVEK ATHALYE
Discovering Cellular Stanford scientists unveil
The concept of immortality has
intrigued mankind since the very beginning.
From the first major literary work, the
Epic of Gilgamesh, to the Bible and other
religious texts to modern fantasies such
as Harry Potter, we find the theme of
immortality rooted in our culture and our
imagination. However, immortality is not
merely the subject of fiction. It exists on a
cellular level—when man is formed from
the division of embryonic stem cells, and
when cancer develops from malignant
cells’ uncontrollable growth. One important
player in cellular immortality is telomerase.
Telomerase is a massive enzyme complex
that prevents a cell from aging by preserving
the integrity of its DNA during cellular
division. Since its discovery in 1984, scientists
and pharmaceutical companies have been
“I call it the problem intensely interested in the enzyme because
of its implications in both cellular and
of telomerase systemic aging, stem cell proliferation, and
cancer.
dark matter. The
Yet, despite the enzyme’s profound
telomerase complex implications for science and medicine,
has a mass from 1 much of telomerase’s mechanisms and
composition remain a mystery. Dr. Steven
to 2 megadaltons, Artandi, Assistant Professor of Medicine at
Stanford University and telomerase and
but the portion of it cancer researcher, calls it “the problem of
telomerase dark matter.” He explains, “The
that we’ve identified telomerase complex has a mass from 1 to
2 megadaltons, but the portion of it that
weighs only 300 we’ve identified weighs only 300 kilodaltons.
kilodaltons. We’re We’re trying to discover the missing matter.”
trying to discover Now scientists in the Stanford University
School of Medicine are two steps closer.
the missing matter.” As published in the March 21, 2008 issue of
Cell, Dr. Artandi, graduate student Andrew
- Steven Artandi Venteicher and Dr. Timothy Veenstra, along
with NCI Frederick’s Dr. Zhaojing Meng and
University of Washington’s Dr. Philip Mason,
recently discovered two new proteins that
function in the telomerase complex.
stanford scientific
The two proteins are provocative as they
suggest a new level of complexity in
telomerase while providing a new direction
in which to search for other proteins.
Furthermore, their discovery presents
new approaches in cancer treatment that
could potentially inhibit cancer cells’ fatal
immortality.
In Pursuit of
Understanding
Immortality
According to emotion research, each of us
can regulate our emoTo begin to understand
cellular immortality, we must first consider
cellular division and the end replication
problem. Imagine for a moment that you
are a DNA polymerase enzyme, a protein
with the important job of copying the cell’s
genetic material so that when it divides,
each new cell can receive an exact copy
of the entire genome. You travel along
the chromosome, making just one error in
every 10,000 nucleotides copied, but as you
approach the end, you realize that it is out of
reach. Unable to copy these last few bases,
you leave the replica chromosome shorter
than the original. What is happening, and
what will be the consequences for the new
cell?
Because DNA polymerase can only extend
a growing chain, with each cellular division
the chromosomes become shorter and
shorter as the last bits of information
are snipped off. If the chromosome
were to end flush with its final gene,
this gene would eventually be lost after
successive replications, leading to cellular
abnormalities, cellular senescence—a state
of cellular aging marked by the cell’s inability
to divide—or apoptosis, programmed cell
death.
Evolution has solved this engineering
problem with the telomere, a region of
nucleotide repeats that do not code for
Immortality
two proteins functioning in telomerase
required proteins, but serve as buffers that
cap the ends of the chromosomes. Now
with each division the telomere shortens
instead of the essential genes. Not only
does this prolong the cell’s lifetime, but
it also suggests the specific length of the
telomere could function as a genetic clock,
regulating the number of times the cell can
divide before important genetic information
is lost and the cell senesces or dies.
Enter telomerase, the biological mechanism
of immortality. Telomerase works by
maintaining the length of the chromosome,
synthesizing the telomeres at the ends
where DNA polymerase could not
otherwise reach. Telomerase’s mechanism is
fascinating—the only reverse transcriptase
in human cells, it reads off an RNA template
to produce the DNA repeats of the telomere.
The expression of telomerase allows a
cell to divide indefinitely, hence the idea
of cellular immortality. While telomerase
is undetectable in normal adult cells, it is
naturally expressed in stem cells, progenitor
cells, and cells such as immune cells which
must divide prolifically. Cancer cells also
use telomerase to divide endlessly without
check, making cancer the deadly force we
know it to be today.
Path of Discovery:
Connecting Pontin and
Reptin to Telomerase
Scientists have been able to describe
how telomerase functions in maintaining
telomeres based on their knowledge of
its catalytic core, which has three primary
components. The first is TERT, the protein
catalytic subunit that synthesizes the
telomere. The second is TERC, the RNA
subunit that serves as the template for
telomere synthesis. The third is dyskerin, a
protein that assists in binding TERC to TERT.
While these are the main subunits, they
only add up to 15-30% of telomerase’s total
composition, leaving a biochemical mystery
for scientists to explore.
The first obstacle the Stanford group had
to overcome was the scant amounts of
telomerase available in an organism: normal
adult cells do not express telomerase. The
Stanford group solved the problem by
producing vats of HeLa cells (human cervical
cancer cells) that express and harness
telomerase in order to exist as an indefinitely
dividing malignancy.
Even with this vast quantity of cells, the
amount of available protein is very small.
However, “our research is backed by years of
technological advancement,” says Artandi.
“With technology such as Dual Affinity
Purification and the advances of the Human
Genome Project, we are actually able to
analyze the small quantities of protein.”
Dual Affinity Purification, which was adapted
from its normal use in yeast cells, infuses the
protein ends with tags containing cleavage
sites. By using a specific protease, an
enzyme utilized to dissolve specific bonds
on a protein’s cleavage sites, the Stanford
group was able to cut the proteins into
sections only at the locations of the tags.
Dr. Artandi said, “These are gentle and very
specific steps that allowed us to get a huge
fold of enrichment.”
Having purified and cut the protein, the
group was able to analyze telomerase’s
composition using mass spectrometry, a
technique that measures the charge-to-
mass ratio of particles in order to generate
a spectrum of the telomerase components.
This information can be checked against a
database developed by the Human Genome
Project to identify the proteins included in
the complex.
According to the scientists’ publication
in Cell, analysis of the band in which “the
most prominent TERT-associated proteins”
exist allowed the Stanford group to identify
pontin and reptin as proteins within the
telomerase complex.
The Stanford group then went on to
study what physical associations the
proteins might have with each subunit of
telomerase’s catalytic core.
First, the group studied the relation of
pontin and reptin to TERT, the telomere-
synthesizer. Using purified pontin and
reptin the group was able to repeatedly
immunoprecipitate a polypeptide with the
same mass as TERT, demonstrating a strong
interaction between the three proteins.
After verifying this association, the group
sought to establish a connection between
the pontin, reptin, TERC and dyskerin.
By removing pontin and reptin from
the telomerase complex, subsequent
observation of TERC and dyskerin activity
clearly showed that pontin and reptin
are critical for accumulation of TERC and
dyskerin.
Finally, by depleting pontin and reptin with
shRNA, a short sequence that can be used
to silence gene expression through RNA
interference, the Stanford group observed
telomerase activity fall to a mere 10-20%
of its natural activity. These were exciting
results demonstrating pontin and reptin’s
crucial roles in each aspect of telomerase
functioning.
Biogenesis: Pontin,
Reptin, and the
Telomerase Creation
Story
There is more to the story than the strong
association of pontin and reptin with
telomerase. Indeed, the discovery of
pontin and reptin as essential proteins was
particularly provocative because they are
ATPases, enzymes that catalyze
volume VII
 BIO + MED
The Process of DNA
Replication
1. Helicase unwinds the complimentary
DNA strands.
2. RNA primase attaches RNA nucleotides
to the template. The first DNA
nucleotides will be added to this primer.
3. There are two separate elongation
processes for the two DNA strand
templates. The 5’ to 3’ template is
synthesized continuously. The daughter
strand that runs from 3’ to 5’ is called the
leading strand.
McMaster University History of Health Care in Hamilton Gallery
4. The 3’ to 5’ strand is copied in steps,
producing non-continuous pieces called
Okazaki fragments.
5. Finally, the RNA primers are removed
and replaced by DNA. Because of
the gap left by the RNA primer, DNA
polymerase cannot attach and seal the
last section of the lagging strand. This
is where the telomere comes into play
and explains the problem of telomere-
shortening.
stanford scientific
the breakdown of the cellular fuel ATP to
provide energy for other processes. The
question remains: for what process are they
required?
This question propelled the Stanford group
to look more specifically at the functions
of pontin and reptin, and the answer was
biogenesis. Pontin and reptin are central
to the assembly of the core telomerase
complex including TERT, TERC, and dyskerin.
The Cycle of Life: Cell
Cycle-Dependent
Telomerase
Construction
The Stanford group analyzed the patterns
of pontin and reptin expression and
telomerase production through each phase
of the cell cycle. Studies with yeast in 2000
had shown that telomerase lengthens
short telomeres in the S Phase of the cell,
the phase in which replication of genetic
material takes place. In accordance with that
study, the Stanford group found that the
amount of reptin and pontin bound to TERT
peaked during S Phase.
This proves pontin and reptin are involved
in cell cycle regulation of telomerase,
a discovery with significant scientific
implications. The study reveals that TERT
complexes are dynamic in nature as their
function in telomerase depends on the
phase of the cell cycle. Furthermore, the
results implied that pontin and reptin
are required for this cyclical telomerase
assembly.
Cancer and Disease:
Stopping the Dark Side
of Telomerase
Because cancer utilizes telomerase to
indefinitely expand its malignancy, scientists
and pharmaceutical companies alike have
been trying to find a treatment for cancer
that blocks telomerase’s activity. However,
attempts to identify telomerase inhibitors
have failed, “perhaps reflecting difficulty in
targeting the enzyme’s reverse transcriptase
function,” the Stanford study suggests. But
now, having halted telomerase activity
in vitro through depletion of pontin and
reptin, Dr. Artandi says, “The old approach
to treating cancer by inhibiting telomerase’s
active site was perhaps phrased incorrectly.
Our study suggests a new approach—the
blocking of pontin and reptin—for
inhibiting telomerase in cancer.”
Yet, while the discovery offers promise, there
is still a great void in compositional and
structural information available regarding
telomerase. Targeting telomerase as a cancer
treatment at this stage would still involve a
fair amount of guesswork. Dr. Artandi says, “I
would love to start working on a new cancer
treatment, but there is still so much we don’t
understand.”
The study of telomerase is a vastly
important topic as its implications reach
even beyond cancer. While one application
of understanding telomerase is to steal
cancer cells’ immortality, the complex is
also implicated with other diseases such
as dyskeratosis congenita, aplastic anemia,
and pulmonary fibrosis. Thus, the Stanford
group’s study suggests “important future
studies for these ATPases in human health
and disease.”
Tissue Engineering and
Stem Cells: The Power of
active, and the mice displayed vigorous
hair growth. Intriguingly, this result was
reproducible even without the expression
of the RNA subunit TERC. This function of
telomerase in stem cells independent of
telomere maintenance poses yet another
mystery for scientists to understand.

Telomerase Undoubtedly there is still a whole world to

While telomerase may be an aiding factor
explore surrounding the tiny enzyme. Dr.
in cancer development, it is fundamentally
Artandi says, “We are barely scratching the
essential to our formation and our existence.
surface.” But given the potential to treat
Embryonic stem cells, the pluripotent cells
some of the worst diseases and health
that divide prolifically and contain the ability
problems today, it is worth spending
to differentiate into any type of cell, express
a lifetime in pursuit of understanding
telomerase to form adult humans. Stem cells
immortality.
and progenitor cells are able to regenerate
tissue because they both self-renew and
differentiate into the appropriate cell types.
Scientists have had great interest in taking
advantage of these features for treating a While telomerase may be an aiding factor
myriad of health conditions requiring tissue
regeneration such as spinal cord injuries, in cancer development, it is fundamentally
diabetes, heart disease, Parkinson’s disease,
Alzheimer’s disease, and numerous others. essential to our formation and our existence.
Despite knowing telomerase is expressed
in stem cells, its role in these cases is still
not understood. Dr. Artandi’s lab, which
conducts research on telomerase function in
stem cells as well, introduced a new enigma:
telomerase action independent of TERC and VIVEK ATHALYE is a sophomore majoring in Electrical Engineering with an interest in
telomere synthesis. Neuroscience. He takes great pleasure in creative writing, playing tennis, and violin.

Dr. Artandi’s lab expressed TERT in To Learn More

mouse skin, activating quiescent stem For more information, check out Dr. Artandi’s article entitled ‘Telomeres, Telomerase,
Credit: sxc.com

cell populations within the follicles. The and Human Disease’ in the September 2006 issue of New England Journal of Medicine,
Volume 355: 1195-1197, or his departmental website at http://www.stanford.edu/group/
results showed that the hair follicles of artandi/.
mice in which TERT was expressed became

volume VII