1. Eternal Life: Cell Immortalization
Chapter 10
Eternal Life: Cell Immortalization
and Tumorigenesis
10.1 ~
May 1, 8 & 15, 2007

2. 10.1 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.

3. Escape from senescence and become immortal or even further transformed to malignant cells.
Figure The Biology of Cancer (© Garland Science 2007)

4. “Hayflick limit” of fibroblasts in the culture

5. 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
Figure The Biology of Cancer (© Garland Science 2007)

6. senescence associated β-galactosidase (lysosomal β-D-galactosidase)
proliferating cells senescent cells
Figure The Biology of Cancer (© Garland Science 2007)

7. Young and old keratinocytes in the skin
Keratinocyte stem cells in the skin lose proliferative capacity with increasing age.
Figure 10.4b The Biology of Cancer (© Garland Science 2007)

8. 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

9. 10.2 & 3 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.

10. Cell populations in crisis show widespread apoptosis
Figure The Biology of Cancer (© Garland Science 2007)

11. senescence-associated
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
Figure 10.9c The Biology of Cancer (© Garland Science 2007)

12. 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

13. Telomeres detected by fluorescence in situ hybridization
(FISH)
telomeric DNA

14. 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)

15. 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)

16. truncation translocation aneuploidy
Figure 10.14b, c The Biology of Cancer (© Garland Science 2007)

17. Telomeric DNA shortens progressively
as cells divide
telomere shortening
chromosomes fuse
apoptotic death
Telomeres lose 50 to 100 bp of DNA during each cell generation.
Figure 10.13b The Biology of Cancer (© Garland Science 2007)

18. The length of telomeric DNA in cells (Southern blotting analysis)
Figure 10.13a The Biology of Cancer (© Garland Science 2007)

19. 10.5 Telomeres are complex molecular structures
that are not easily replicated
Telomeric DNA:
5’-TTAGGG-3’ hexanucleotide sequence, tandemly repeated thousands of times
Figure The Biology of Cancer (© Garland Science 2007)

20. Telomeric DNAs _____________________________________________________
Telomeric repeat
Organism sequence
Yeasts
Saccharomyces cerevisiae G1-3T
Schizosaccharomyces pombe G2-5TTAC
Protozoans
Tetrahymena GGGGTT
Dictyostelium G1-8A
Plant
Arabidopsis GGGTTTA
Mammal
Human GGGTTA ______________________________________________

21. Structure of the T-loop
Figure The Biology of Cancer (© Garland Science 2007)

22. Multiple telomere-specific proteins bound to telomeric DNA
Figure The Biology of Cancer (© Garland Science 2007)

23. Pot1 binds the single- and double-stranded telomeric DNA in order to stabilize its structure
s.s. telomeric DNA
Figure The Biology of Cancer (© Garland Science 2007)

24. Primers and the initiation of DNA synthesis

25. Primers and the initiation of DNA synthesis
this sequence
is not replicated

26. 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.)

27. human telomerase-associated RNA
(template for hTERT)
human telomerase reverse transcriptase
Figure 10.23a The Biology of Cancer (© Garland Science 2007)

28. The sequence of the catalytic subunit of telomerase is homologous to various reverse transcriptases
ciliates:
yeasts:
Figure 10.22a The Biology of Cancer (© Garland Science 2007)

29. The TRAP assay (telomeric repeat amplification protocol)
a synthetic primer
(in cell lysates)
+ dNTP
Figure 10.21a The Biology of Cancer (© Garland Science 2007)

30. The products of the TRAP reaction are analyzed by gel electrophoresis
heat treatment of cell lysates (inactivating telomerase activity)

31. Activation of telomerase activity following escape from crisis
Figure The Biology of Cancer (© Garland Science 2007)

32. Sidebar 10.5 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 (Section 8.9) and
Menin (the product of the MEN1 tumor suppressor
gene), deregulate the cell clock.

33. Prevention of crisis by expression of telomerase
HEK: human embryonic kidney cells
Figure The Biology of Cancer (© Garland Science 2007)

34. Sidebar 10.6 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?

35. 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

36. 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.
Figure The Biology of Cancer (© Garland Science 2007)

37. 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.

38. 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)
Figure The Biology of Cancer (© Garland Science 2007)

39. Telomerase activity and the prognosis of pediatric tumors
Figure 4.11
Figure The Biology of Cancer (© Garland Science 2007)

40. 10.8 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.

41. 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.

42. The ALT (alternative lengthening of telomerase ) mechanism (or copy-choice mechanism)
Figure The Biology of Cancer (© Garland Science 2007)

43. Exchange of sequence information between the telomeres of different chromosomes
neomycin-resistant gene was introduced into the midst of the telomeric DNA
Figure The Biology of Cancer (© Garland Science 2007)

44. 10.9 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.

45. Sidebar 10.8 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.

46. - 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).

47. SV40 and T antigens human fibroblasts, these cells will continue to
- 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.

48. SV40: the 40th simian virus in a series of isolates
papovavirus: papilloma, polyoma & vacuolating agent
Table 3.1 The Biology of Cancer (© Garland Science 2007)

49. SV40 launches a lytic cycle in monkey kidney cells (permissive host)
SV40 virus
formation of large cytoplasmic vacuoles prior to the death of the
cell and the release of progeny
virus particles
- vacuolating agent
SV40 launches a lytic cycle in monkey kidney cells (permissive host)
Figure 3.10 The Biology of Cancer (© Garland Science 2007)

50. double-stranded, circular DNA of SV40 (5 kb)
3.6 DNA Tumor virus genomes persist in virus-
transformed cells by becoming part of host cell DNA
double-stranded, circular DNA of SV40 (5 kb)
Cell transformation by SV40 depend on the integration of SV40 genome into host cell DNA and the continued presence of T (tumor-associated) antigens.
large T , middle T & small T Ags
Figure 3.11 & 18 The Biology of Cancer (© Garland Science 2007)

51. SV40 large T antigen can circumvent senescence
HEK: human embryonic kidney cells
Figure The Biology of Cancer (© Garland Science 2007)

52. HEK 293 HEK 293 cells were generated by transformation
of normal human embryonic kidney (HEK)
cells with adenovirus 5 DNA in the late 1977.
HEK 293T cell line contains, in addition, the SV40
large T antigen, that allows for episomal
replication of transfected plasmids containing the
SV40 origin of replication. This allows for
amplification of transfected plasmids and
expression of the desired gene products.

53. mTR -/- mice exhibit no telomerase activity in any
of their cells. They are indistinguishable phenotypically
from wild-type mice for at least 3 generations.
showing premature aging:
- wasting away of muscle
tissues & a hunched back
The progeny in the 6th generation show a diminished capacity to heal wounds, to respond properly to mitogenic signals, and suffer from substantially reduced fertility and tissue atrophy (loss of cells).
Figure The Biology of Cancer (© Garland Science 2007)