Eternal Life: Cell Immortalization and Tumorigenesis

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Eternal Life Cell Immortalization and
Tumorigenesis
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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.

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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).

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Cells need to become immortal in order to form
cancers

• Two regulatory mechanisms to govern the
  replicative capacity of cells
• 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
• 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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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 ß-galactosidase (lysosomal
  ß-D-galactosidase)

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Cell senescence does occur in vivo
Senescence-associated ß-galactosidase (SA-ß-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.
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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
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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

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Telomeres detected by fluorescence in situ
hybridization (FISH)
telomeric DNA
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• Telomeric repeat-binding factor

• Telomeric repeat-binding factor

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 repeat-binding factor 2
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Mechanisms of breakage-fusion-bridge 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 problemTelomeric 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
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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
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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

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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

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Multiple telomere-specific proteins bound to
telomeric DNA
TRF Telomeric repeat-binding factor
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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
• hTERT catalytic subunit
• hTR RNA subunit
• (At least 8 other subunits may exist in the
  holoenzyme but have not been characterized.)

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human telomerase-associated RNA (template for
hTERT)
human telomerase reverse transcriptase
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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.

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Prevention of crisis by expression of telomerase
HEK human embryonic kidney cells
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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

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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.
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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.

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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)
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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.
• 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.

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The ALT (alternative lengthening of telomerase )
mechanism (or copy-choice mechanism)
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Exchange of sequence information between the
telomeres of different chromosomes
neomycin-resistant gene was introduced into the
midst of the telomeric DNA
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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
  (gt30 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.

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SV40 the 40th simian virus in a series of
isolates papovavirus papilloma, polyoma
vacuolating agent
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SV40 large T antigen can circumvent senescence
HEK human embryonic kidney cells