Transcript Telomeric DNA

Eternal Life: Cell Immortalization
and Tumorigenesis
Molecular Biology of Cancer
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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.
Molecular Biology of Cancer
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“Hayflick limit” of Normal human cells
(Fibroblasts) in monolayer culture
They possess an
intrinsically
programmed limit
(now known as the
‘Hayflick limit’) to
their capacity for
proliferation
 even after a substantial
healthy period of cell
division, they undergo a
permanent growth arrest
(replicative senescence).
Molecular Biology of Cancer
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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.
 Accumulation 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.
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Molecular Biology of Cancer
Replicative senescence in vitro
Proliferating human
fibroblasts
Senescent cells in culture:
• “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
• Senescence associated β-galactosidase
(lysosomal β-D-galactosidase)
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Cell senescence does occur in vivo
Senescence-associated
β-galactosidase (SA-β-gal)
Treatment of lung cancer with
chemotherapeutic drugs
appear to induce senescence
in tumor cells
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Young and old keratinocytes in the skin
Keratinocyte stem cells in
the skin lose proliferative
capacity with increasing age.
Molecular Biology of Cancer
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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 linen established
in culture
 derived from the tissue of cervical adenocarcinoma
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cell cultures
derived from human
cancer tissues, once
successfully
established in vitro,
are often immortal
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Cell populations in crisis show
widespread apoptosis
Molecular Biology of Cancer
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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
Molecular Biology of Cancer
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Telomeres
detected by
fluorescence in
situ hybridization
(FISH)
telomeric DNA
Molecular Biology of Cancer
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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.
TRF2: Telomeric repeatbinding factor 2
Molecular Biology of Cancer
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Mechanisms of breakage-fusionbridge cycles
2 sister chromatids
during the G2 phase
of the cell cycle
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truncation
translocation
aneuploidy
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the end-replication problem:
Telomeric DNA shortens progressively as cells
divide
An inevitable consequence of semi-conservative
DNA replication in eukaryotic cells
 The free DNA ends of each chromosome are not duplicated
completely by DNA polymerase.
 Consequently, the ends of human chromosomes can lose up to 200
bp of DNA per cell division.
telomere shortening chromosomes
fuse apoptotic death
Molecular Biology of Cancer
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Primers and the initiation of DNA synthesis
this sequence
is not replicated
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Telomeres are complex molecular
structures that are not easily replicated
Telomeric DNA:
5’-TTAGGG-3’ hexanucleotide sequence, tandemly
repeated thousands of times
Molecular Biology of Cancer
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Structure of the T-loop
• The 3' DNA end at each telomere is always
longer than the 5’ end with which it is
paired, leaving a protruding single-stranded
• This protruding end has been shown to loop
back and tuck its single stranded terminus
into the duplex DNA of the telomeric repeat
sequence to form a t-loop
Molecular Biology of Cancer
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T-loops provide the normal ends of
chromosomes with a unique structure,
which protects them from degradative
enzymes and clearly distinguishes them
from the ends of the broken DNA molecules
that the cell rapidly repairs
Molecular Biology of Cancer
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Multiple telomere-specific proteins bound
to telomeric DNA
TRF: Telomeric repeatbinding factor
Molecular Biology of Cancer
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Cancer cells can escape crisis by
expressing telomerase
Telomerase activity (elongate telomeric
DNA)
Clearly detectable in 85 to 90% of human
tumor cell samples
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.)
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human telomeraseassociated RNA
(template for hTERT)
human telomerase
reverse transcriptase
Molecular Biology of Cancer
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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.
For example, the Myc protein and Menin (the product of
the MEN1 tumor suppressor gene), deregulate the cell
clock.
Molecular Biology of Cancer
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Prevention of crisis by expression of telomerase
HEK: human embryonic kidney cells
Molecular Biology of Cancer
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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?
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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
Molecular Biology of Cancer
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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.
Molecular Biology of Cancer
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Telomerase plays a key role in the
proliferation of human cancer cell
Expression of antisense RNA in the
telomerase (+) HeLa cells
They stop growing 23 to 26 days.
Expression of the dominant negative hTERT
subunit in telomerase (+) human tumor cell
lines:
They lose all detectable telomerase activity
with some delay, they enter crisis.
Molecular Biology of Cancer
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Suppression of telomerase results in the loss of
the neoplastic growth in 4 different human cancer
cell lines
(length of telomeric
DNA at the onset of
the experiment)
Molecular Biology of Cancer
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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 they 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.
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- the vast majority of the yeast Saccharomyces cervisiae
cells enter a state of crisis and die following inactivation
of genes encoding subunits of the telomerase holoenzyme.
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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.
Molecular Biology of Cancer
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The ALT (alternative lengthening of telomerase )
mechanism (or copy-choice mechanism)
Molecular Biology of Cancer
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Exchange of sequence information between the
telomeres of different chromosomes
neomycinresistant gene
was introduced
into the midst of
the telomeric
DNA
Molecular Biology of Cancer
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Telomeres play different roles in the cells
of laboratory mice and in human cells
Rodent cells, especially those of the laboratory
mouse strains, express significant levels of
telomerase throughout life.
The double-stranded region of mouse telomeric
DNA is as much as 30 to 40 kb long (~ 5 times
longer than corresponding human telomeric DNA).
Therefore, laboratory mice do not rely on telomere
length to limit the replicative capacity of their normal
cell lineages and that telomere erosion cannot serve as a
mechanism for constraining tumor development in these
rodents.
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Long telomeres (in mice) do not
suffice for tumor formation
Transgenic mice expressing mTERT (mouse
homolog of telomerase reverse transcriptase)
contributes to tumorigenesis even though the
mouse cells in which this enzyme acts already
possess very long (>30 kb) telomeres.
Thus, the mTERT enzyme aids tumorigenesis
through mechanisms other than simple telomere
extension.
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- Mouse cells can be immortalized relatively
easily following extended propagation in culture.
- Human cells require, instead, the introduction of
both the SV40 large T oncogene (to avoid
senescence) and the hTERT gene (to avoid
crisis).
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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 propotion of cells
(1 out of 106 cells) will proceed to proliferate
and continue to do indefinitely → becoming
immortalized.
Molecular Biology of Cancer
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SV40: the 40th simian virus in a series of isolates
papovavirus: papilloma, polyoma & vacuolating agent
Molecular Biology of Cancer
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SV40 large T antigen can circumvent senescence
HEK: human embryonic kidney cells
Molecular Biology of Cancer
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