Senescence and Telomeres

Normal cell populations register the number

of cell generations separating them from
their ancestors in the early embryo

- Normal cells have a limited proliferative potential.

- Cancer cells need to gain the ability to proliferate
indefinitely – immortal.
- The immortality is a critical component of the
neoplastic growth program.
Escape from
senescence and
become
immortal or
even further
transformed to
malignant cells.
“Hayflick limit” of fibroblasts in the culture
 Replicative senescence in vitro
senescence-
associated acidic
β-galactosidase

proliferating human senescent cells in culture

fibroblasts
- “fried egg” morphology
- remain metabolically active, but
lost the ability to re-enter into the
active cell cycle
- the downstream signaling pathways
seem to be inactivated
Cells need to become immortal in order
to form cancers
Two regulatory mechanisms to govern the
replicative capacity of cells:
1. senescence - Cumulative physiologic stress over extended
periods of time halts further proliferation.
These cells enter into a state of senescence.
cumulation of oxidative damage contributes to senescence,
e.g., reactive oxygen species (ROS), DNA damage

2. crisis - Cells have used up the allowed “quota” of

replicative doublings. These cells enter into a state
of crisis, which leads to apoptosis.
Cancer cells and embryonic stem cells share
some replicative properties
- Embryonic stem (ES) cells show unlimited replicative
potential in culture and are thus immortal.
- The replicative behavior of cancer cells resembles
that of ES cells.
- Many types of cancer cells seem able to proliferate
forever when provided with proper in vitro culture
conditions.
- HeLa cells (Henrietta Lacks, 1951):
- the 1st human cell line and 1st human cancer cell line
established in culture
- derived from the tissue of cervical adenocarcinoma
10.4 The proliferation of cultured cells is
limited by the telomeres of their
chromosomes
Barbara McClintoch discovered (1941) specialized
structures at the ends of chromosomes, the telomeres, that
protected chromosomes from
end-to-end fusions.

(She also demonstrated movable genetic elements

in the corn genome, later called transposons.)

- Nobel prize in Physiology & Medicine in 1983

 Telomeres are complex DNA repeats found at the chromosome ends that ensure genomic
stability. In mammalian cells, they are constituted by TTAGGG repeats, which could vary from few
to tens of kb pairs between species.

 Telomeres are protected by a complex of six proteins, named shelterin. Shelterin includes TRF1,
TRF2, Pot1-TPP1 heterodimer, Rap1, and Tin2.

 During each cell replication cycle, telomeres shorten because of the end-replication problem, a
situation aggravated by the shortage of telomerase in the majority of the cells constituting adult
tissues.

 Telomerase is constituted by a catalytic unit with reverse-transcriptase activity (Tert) and RNA
component (Terc) that serves as template for telomere extension.

 Telomerase compensates the telomere attrition through a concerted action of its two subunits.
Telomerase is the main cellular protein responsible for telomere maintenance and elongation.
Telomerase activity is, however, silenced in most cells of adult tissues and is almost only present in
adult stem cell compartments.

 From studies in vitro, the shortening rate was described to be between 30 bp and 120 bp per cycle.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824275/
Telomeres
detected by
fluorescence in
situ
hybridization
(FISH)
telomeric
DNA
 The telomeres lose
their protective
function in cells that
have been deprived
of TRF2, a key
protein in
maintaining normal
telomere structure.

In an extreme form, all the chromosomes of the cell fused into one giant
chromosome.
Figure 10.11b The Biology of Cancer (© Garland Science 2007)
 Mechanisms of breakage-fusion-bridge cycles

2 sister chromatids during the G2 phase of the

cell cycle

Figure 10.14a The Biology of Cancer (© Garland Science 2007)

truncation
translocation
aneuploidy
10.5 Telomeres are complex molecular structures
that are not easily replicated

Telomeric DNA:
5’-TTAGGG-3’ hexanucleotide sequence, tandemly
repeated thousands of times
 Structure of the T-loop

This overhanging strand is often

found in a most unusual
molecular configuration
termed the t-loop. It was
discovered in the late 1990s, when
electron microscopy showed the
ends of telomeric DNA to be
configured in a loop, in effect a
lariat.

Both the relatively long double-stranded telomeric DNA and the short overhang-
ing end are bound by specific proteins.
 Multiple telomere-specific proteins bound to
telomeric DNA

As is indicated here, by binding

to the D-loop, POT1 is able to stabilize
this structure and thus the structure of
the t-loop as a whole.

Two of the latter’s proteins—TRF1 and TRF2—bind to the double-stranded DNA portion of telomeres while a third
protein—POT1—binds to both the single-stranded 3ʹ overhang created by the G-rich strand and the ssDNA in the
displacement (D-) loop.
 10.6 Incipient cancer cells can escape crisis by
expressing telomerase

- Telomerase activity (elongate telomeric DNA) is

clearly detectable in 85 to 90% of human tumor
cell samples, while being present at very low
levels in most types of normal human cells.

telomerase holoenzyme:
1. hTERT catalytic subunit
2. hTR RNA subunit
(At least 8 other subunits may exist in the
holoenzyme but have not been characterized.)

The available evidence indicates that strong expression of the telomerase enzyme is
human telomerase-associated RNA
(template for hTERT)

human telomerase reverse

transcriptase

 The telomerase enzyme was initially characterized in

baker’s yeast using genetic analyses
 and in ciliates through biochemical purification.

One subunit
 is a DNA polymerase, more specifically a reverse
transcriptase, which functions, like
 the enzymes made by retroviruses

 RNA template—the second essential subunit. A short

segment of this

 451-nucleotide-long RNA molecule serves as the template

that instructs the reverse
transcriptase activity of the holoenzyme.
 Oncoproteins and tumor suppressor
proteins play critical roles in
governing hTERT expression

- The mechanisms that lead to the de-repression of

hTERT transcription during tumor progression in
humans are complex and still quite obscure.
- Multiple transcription factors appear to collaborate
to activate the hTERT promoter.
The myc gene can activate the hTERT
Gene transcription. The promoter of hTERT geneb
Has atleast two binding sites for bHLH.
 SV40 and T antigens
- If the SV40 large T oncoprotein is expressed in
human fibroblasts, these cells will continue to
replicate another 10 to 20 cell generations and
then enter crisis.

- On rare occasion, a small proportion of cells

(1 out of 106 cells) will proceed to proliferate
and continue to do indefinitely → becoming
immortalized.
The role of telomeres in
replicative senescence

- In cultured human fibroblasts, senescence can be

postponed by expressing hTERT prior to the
expected time for entering replicative senescence.
- However, senescence is also observed in cells that
still possess quite long telomeres.

Why?
Possible explanations:

- When cells encounter cell-physiologic stress or

the stress of tissue culture, telomeric DNA loses
many of the single-stranded overhangs at the
ends.
The resulting degraded telomeric ends
may release a DNA damage signal, thereby
provoking a p53-mediated halt in cell
proliferation that is manifested as the senescent
growth state
Replicative senescence and the actions of telomerase

This is a still-speculative
mechanistic model of how
and why telomerase
expression can prevent
human cells from entering
into replicative senescence.
 While telomerase expression is high in embryonic and adult stem cells, telomerase activity is absent in
differentiated somatic cells due to transcriptional silencing of the telomerase reverse transcriptase (TERT)
gene, which encodes its catalytic subunit that synthesizes TTAGGG nucleotide repeats at chromosome
ends.

 Multiple transcription factors, including Myc, β-catenin and NF-κB have been documented to control
human TERT promoter activity and several studies have suggested that the chromatin environment plays a
critical role in the regulation of TERT transcription.

 Recently, two highly recurrent point mutations in the TERT promoter were identified in a multitude of
cancer types including melanoma (where these mutations were initially described), glioblastoma
multiforme (GBM), urothelial cancer, thyroid carcinoma and hepatocellular carcinoma (HCC).

 Occurring at nucleotide residues -124 and -146 upstream from the ATG start site (commonly referred to as
C228T and C250T, respectively) in a mutually exclusive manner, the two somatic mutations resulted in
the de novo generation of consensus binding motifs for E-twenty-six (ETS) transcription factors.

 Indeed, recent cumulative evidence from a number of laboratories supports the notion
that TERT promoter mutations are gain of function, driver events in cancer progression.

Telomerase reactivation in cancers: Mechanisms that govern transcriptional activation of the wild-
type vs. mutant TERT promoters
Yinghui Lia and Vinay Tergaonka Transcription. 2016 Mar-Apr; 7(2): 44–49.
10.7 Telomerase plays a key role in the proli-
feration of human cancer cells

- Expression of antisense RNA in the telomerase (+)

HeLa cells causes them stop growing 23 to 26 days.
- Expression of the dn (dominant negative) hTERT
subunit in telomerase (+) human tumor cell lines
also causes them to lose all detectable telomerase
activity and, with some delay, to enter crisis.
Suppression of telomerase results in the loss of the neoplastic growth in 4 different human cancer cell
lines
Some immortalized cells can maintain
telomeres without telomerase

- 85 to 90% of human tumors have been found to be

telomerase-positive.
- The remaining 10 to 15% lack detectable telomerase
activity, yet these cells need to maintain their
telomeres above some minimum length in order to
proliferate indefinitely.
- These cells obtain the ability to maintain their
telomeric DNA using a mechanism that does not
depend on the actions of telomerase.
- In the yeast Saccharomyces cervisiae, following
inactivation of genes encoding subunits of the
telomerase holoenzyme, the vast majority of the
cells enter a state of crisis and die.
- Rare variants emerged from these populations of
dying cells that used the alternative lengthening of
telomerase (ALT) mechanism to construct and
maintain their telomeres.
- This ALT mechanism is also used by the minority of
human tumor cells that lack significant telomerase
activity, e.g., 50% osteosarcomas and soft-tissue
sarcomas and many glioblastomas.
 The ALT (alternative lengthening of telomerase )
mechanism (or copy-choice mechanism)

Figure 10.30 The Biology of Cancer (© Garland Science 2007)

Human cells require, instead, the introduction of
both the SV40 large T oncogene (to avoid
senescence) and the hTERT gene (to avoid crisis).

If the SV40 large T oncoprotein is expressed in

human fibroblasts, these cells will continue to
replicate another 10 to 20 cell generations and
then enter crisis.

- On rare occasion, a small propotion of cells

(1 out of 106 cells) will proceed to proliferate
and continue to do indefinitely → becoming
immortalized.