chapter 10 eternal life: cell immortalization and tumorigenesis -10.1 ~ 10.9 - may 1, 8 & 15,...
TRANSCRIPT
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Chapter 10
Eternal Life: Cell Immortalization and Tumorigenesis
-10.1 ~ 10.9 -
May 1, 8 & 15, 2007
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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.
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Figure 10.2 The Biology of Cancer (© Garland Science 2007)
Escape from senescence and become immortal or even further transformed to malignant cells.
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“Hayflick limit” of fibroblasts in the culture
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Figure 10.3 The Biology of Cancer (© Garland Science 2007)
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
Replicative senescence in vitrosenescence-associated acidic β-galactosidase
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Figure 10.3 The Biology of Cancer (© Garland Science 2007)
proliferating cells senescent cells
senescence associated β-galactosidase
(lysosomal β-D-galactosidase)
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Figure 10.4b The Biology of Cancer (© Garland Science 2007)
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 line established in culture
- derived from the tissue of cervical adenocarcinoma
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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.
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Figure 10.10 The Biology of Cancer (© Garland Science 2007)
Cell populations in crisis show widespread apoptosis
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Cell senescence does occur in vivo
Figure 10.9c The Biology of Cancer (© Garland Science 2007)
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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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
10.4 The proliferation of cultured cells is limited by the telomeres of their chromosomes
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Telomeres detected by fluorescence in situ hybridization(FISH)
telomeric DNA
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Figure 10.11b The Biology of Cancer (© Garland Science 2007)
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.
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Figure 10.14a The Biology of Cancer (© Garland Science 2007)
Mechanisms of breakage-fusion-bridge cycles
2 sister chromatids during the G2 phase of the cell cycle
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Figure 10.14b, c The Biology of Cancer (© Garland Science 2007)
truncationtranslocationaneuploidy
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Figure 10.13b The Biology of Cancer (© Garland Science 2007)
Telomeric DNA shortens progressively as cells divide
telomere shorteningchromosomes fuse
apoptotic death
Telomeres lose 50 to 100 bp of DNA during each cell generation.
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Figure 10.13a The Biology of Cancer (© Garland Science 2007)
The length of telomeric DNA in cells(Southern blotting analysis)
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Figure 10.16 The Biology of Cancer (© Garland Science 2007)
10.5 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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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
______________________________________________
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Figure 10.17 The Biology of Cancer (© Garland Science 2007)
Structure of the T-loop
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Figure 10.19 The Biology of Cancer (© Garland Science 2007)
Multiple telomere-specific proteins bound to telomeric DNA
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Pot1 binds the single- and double-stranded telomeric DNA in order to stabilize its structure
s.s. telomeric DNA
Figure 10.18 The Biology of Cancer (© Garland Science 2007)
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Primers and the initiation of DNA synthesis
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Primers and the initiation of DNA synthesis
this sequence is not replicated
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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.)
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human telomerase reverse transcriptase
human telomerase-associated RNA
(template for hTERT)
Figure 10.23a The Biology of Cancer (© Garland Science 2007)
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Figure 10.22a The Biology of Cancer (© Garland Science 2007)
The sequence of the catalytic subunit of telomerase is homologous to various reverse transcriptases
ciliates:yeasts:
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Figure 10.21a The Biology of Cancer (© Garland Science 2007)
The TRAP assay (telomeric repeat amplification protocol)
(in cell lysates)
+ dNTP
a synthetic primer
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The products of the TRAP reaction are analyzed by gel electrophoresis
heat treatment of cell lysates (inactivating telomerase activity)
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Figure 10.24 The Biology of Cancer (© Garland Science 2007)
Activation of telomerase activity following escape from crisis
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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.
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Figure 10.25 The Biology of Cancer (© Garland Science 2007)
Prevention of crisis by expression of telomerase
HEK: human embryonic kidney cells
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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?
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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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Figure 10.26 The Biology of Cancer (© Garland Science 2007)
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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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.
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Suppression of telomerase results in the loss of the neoplastic growth in 4 different human cancer cell lines
Figure 10.27 The Biology of Cancer (© Garland Science 2007)
(length of telomeric DNA at the onset of the experiment)
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Figure 4.11
Telomerase activity and the prognosis of pediatric tumors
Figure 10.28 The Biology of Cancer (© Garland Science 2007)
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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.
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- 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.
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Figure 10.30 The Biology of Cancer (© Garland Science 2007)
The ALT (alternative lengthening of telomerase ) mechanism (or copy-choice mechanism)
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Figure 10.29 The Biology of Cancer (© Garland Science 2007)
neomycin-resistant gene was introduced into the midst of the telomeric DNA
Exchange of sequence information between the telomeres of different chromosomes
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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.
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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.
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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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Table 3.1 The Biology of Cancer (© Garland Science 2007)
SV40: the 40th simian virus in a series of isolates
papovavirus: papilloma, polyoma & vacuolating agent
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Figure 3.10 The Biology of Cancer (© Garland Science 2007)
SV40 virus
SV40 launches a lytic cycle in monkey kidney cells
(permissive host)
formation of large cytoplasmic vacuoles prior to the death of the cell and the release of progeny virus particles
- vacuolating agent
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3.6 DNA Tumor virus genomes persist in virus- transformed cells by becoming part of host cell DNA
Figure 3.11 & 18 The Biology of Cancer (© Garland Science 2007)
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
double-stranded, circular DNA of SV40 (5 kb)
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Figure 10.8 The Biology of Cancer (© Garland Science 2007)
SV40 large T antigen can circumvent senescence
HEK: human embryonic kidney cells
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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.
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mTR -/- mice exhibit no telomerase activity in any of their cells. They are indistinguishable phenotypically from wild-type mice for at least 3 generations.
Figure 10.31 The Biology of Cancer (© Garland Science 2007)
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).
showing premature aging: - wasting away of muscle tissues & a hunched back