Eternal Life: Cell Immortalization and Tumorigenesis

1. Eternal Life: Cell Immortalization and Tumorigenesis

2. 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. “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).

4. 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.

5. 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 (lysosomalβ-D-galactosidase)

6. 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

7. Young and old keratinocytes in the skin Keratinocyte stem cells in the skin lose proliferative capacity with increasing age.

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 linen established in culture • derived from the tissue of cervical adenocarcinoma

9. cell cultures derived from human cancer tissues, once successfully established in vitro, are often immortal

10. Cell populations in crisis show widespread apoptosis

11. 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

12. Telomeres detected by fluorescence in situhybridization (FISH) telomeric DNA

13. 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

14. Mechanisms of breakage-fusion-bridge cycles 2 sister chromatids during the G2 phase of the cell cycle

15. truncation translocation aneuploidy

16. 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

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

18. Telomeres are complex molecular structures that are not easily replicated Telomeric DNA: 5’-TTAGGG-3’ hexanucleotide sequence, tandemly repeated thousands of times

19. 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

20. 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

21. Multiple telomere-specific proteins bound to telomeric DNA TRF: Telomeric repeat-binding factor

22. 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.)

23. human telomerase-associated RNA (template for hTERT) human telomerase reversetranscriptase

24. Oncoproteins and tumor suppressor proteins play critical roles in governinghTERT 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.

25. Prevention of crisis by expression of telomerase HEK: human embryonic kidney cells

26. 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?

27. 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

28. 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.

29. 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.

30. 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)

31. 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.

32. 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.

33. The ALT (alternative lengthening of telomerase ) mechanism (or copy-choice mechanism)

34. Exchange of sequence information between the telomeres of different chromosomes neomycin-resistant gene was introduced into the midst of the telomeric DNA

35. 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.

36. 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.

37. - 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).

38. 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.

39. SV40: the40thsimianvirus in a series of isolates papovavirus: papilloma, polyoma & vacuolating agent

40. SV40 large T antigen can circumvent senescence HEK:human embryonic kidney cells