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Senescence and Immortalization

Senescence and Immortalization

Senescence and Immortalization

Lecture 18 November 5 2015 Assigned reading Chapter 10 Contact: [email protected] Normal Development

Differentiation functional maturation

Adult stem/ chronological aging progenitors Senescence PCD (apoptosis)

Death ESCs

Slide Credit: Dr. Dean G Tang Immortalization, Senescence, , and

1. Senescence: Characteristics 2. Telomerase, Senescence, and Cancer What is Senescence??

A state of cellular being characterized by: a) metabolic activity but b) irreversible loss of the capacity to enter active c) Growth factors help sustain viability but d) Are unable to elicit usual proliferative response

Leonard Hayflick and Paul Moorhead (1961) first showed the phenomenon that cells would stop growing after a certain number of divisions.

Phase I Cells divide actively to cover the culture dish Phase II Cells proliferate robustly Phase III Cells remain viable but do not proliferate Normal cells have predetermined population doublings Proliferative capacity decreases with age

The number of growth and division cycles is dictated by the and tissue of origin and age of the donor.

Regenerative capacity declines with age. Characteristics of senescent cells

• Permanent growth arrest– can not be reversed by physiological stimuli • Increased cell size– flat cells with huge cytoplasm (appearance of a fried egg) • Increased cytoplasmic granularity • Express senescence associated (SA) beta galactosidase • Metabolically active Influence of culture conditions

Physiological Laboratory incubator

Higher Levels Oxidative damage

Hypoxia Senescence Senescent cells exhibit inability to enter active cell cycle

Slide Credit: Dr. Dean G Tang Slide Credit: Dr. Dean G Tang Cyclin dependent kinase inhibitors are induced during senescence

Overexpression of (middle) can induce senescence phenotype as seen in cells that have entered senescence after extensive propagation in vitro. The INK4 locus

Slide Credit: Dr. Dean G Tang Two major tumor suppressors p53 and pRb regulate senescence

Inactivation of both p53 and pRb is needed to escape from senescence Both p53 and pRb are inactivated in majority of DNA damage response can activate cell senescence

Marker is gamma H2AX Normal Development

Differentiation functional maturation

Adult stem/ chronological aging progenitors Senescence PCD (apoptosis)

Death ESCs

Slide Credit: Dr. Dean G Tang Comparison between terminally differentiated and senescent cells in culture

Differentiated Senescent Morphology generally big big & flat SA-βgal positivity - + Motility little or low little Saturation density low low Differentiation markers + +/- Differentiation functions + -

Cell cycle “permanent” G0/G1 “permanent” G0/G1 Response to mitogens low low Karyotypic stability high low Resistance to apoptosis +/- + Essence “permanent” cell “permanent” cell cycle arrest to cycle arrest with evolve into func- continued growth tional cell types

Slide Credit: Dr. Dean G Tang Senescence associated heterochromatic foci (SAHFs)

Markers are: H3K9me HP1gamma Senescence & Senescent Cells 1) Big and flat, prominent cytoplasmic/nuclear vacuolization, less motile, decreased saturation density, and positive for SA-βgal. 2) Multiple alterations in gene expression, e.g., overexpression of collagenase and underexpression of TIMPs (tissue inhibitor of metalloproteinase). 3) Attenuated proliferative response to mitogens (EGF, PDGF, IGF-1) and unable to induce c-fos (but myc and ras induction ok).

4) “Irreversible” cell-cycle arrest at G1/S with 2N nuclear content (but increased nuclear size); <1 PD in 2 weeks. 5) Decrease in positive regulators (cyclin D/Cdk4, cyclin E/Cdk2, etc) and increase in negative regulators (p16, p21, p19ARF, hypo-phosphorylated RB, etc). 6) Resistance to apoptosis induction. 7) Senescent cells, to a degree, resemble terminally differentiated cells. 8) Presenescent cells often show dysfunction as revealed by markers ATM activation and formation of nuclear foci containing H2AX-γ, 53BP1, MDC1,NBS1, which disappear in fully senescent cells.* 9) Fully senescent cells often possess karyotypic instability: tetraploidy, endoreduplication, aneuploidy, and other abnormal karyotypes. 10) , like aging, is dominant. Therefore, results from recessive changes in negative regulators (tumor suppressive genes). 11) Senescent cells accumulate senescence-associated heterochromatin foci (SAHFs), in which HMG-A accumulate. 12) Senescent cells release pro-inflamatory cytokines (interleukins, IGFBPs, and TGF-beta) that act in an autocrine manner to promote senescence and in a paracrine fashion to recruit pro- inflamatory cells to promote tumorigenesis.

*Bakkenist CJ, Drissi R, Wu J, Kastan MB, Dome JS. Disappearance of the telomere dysfunction-induced stress response in fully senescent cells. Cancer Res. 2004 Jun 1;64(11):3748-52. Slide modified from Dr. Dean G Tang So how do we explain these effects seen in senescent cells? ** 1978: Telomere was first found as an unusual repeated sequence motif (GGGGTT) at chromosome termini in the ciliate Tetrahymena (Blackburn & Gall, J. Mol. Biol. 120, 33-53, 1978). **Tremendous variability: <50 bp in the hypotrichous ciliates but as long as 5100 kb in mice. **1985-1989: Telomerase activity and telomerase uncovered (Greider CW and Blackburn EH, Cell 43, 405-413, 1985; Cell 51, 887-898, 1987; 337, 331-337, 1989).

**In , telomeres are made up of an average of 5,000 -15,000 bp of G-rich (TTAGGG)n repeats and telomere-binding proteins. **Each loses 50-100 bp of telomeres **When a telomere loses a critical number of base pairs, it triggers a DNA damage signal to stop cell division and initiate senescence.

Telomere functions:

--- form specific complexes with telomere binding proteins --- protect chromosome ends from exonuclease digestion --- prevent aberrant recombination --- prevent the chromosome ends from activating cell-cycle and DNA damage checkpoints Slide Credit: Dr. Dean G Tang Telomere and the chromosome Telomere Synthesis

End replication problem: Watson JD. Nature New Biol. 239, 197-201, 1972. Olovnikov AM. J. Theoret. Biol. 41, 181-190, 1973. Slide Credit: Dr. Dean G Tang Telomere and Telomerase

Slide Credit: Dr. Dean G Tang

Telomere and Telomerase Assays

**Direct measurement of telomere length A. TRA (terminal repeat assay) 1. Digest DNA with Alu I or Hinf I. 2. Perform Southern with radiolabeled (TTAGGG)n

B. Q-FISH or flow cytometry using fluorescently-labeled probes 1. Metaphase spreads are hybridized with Cy3-labeled PNA

(CCCTAA)3 telomeric oligonucleotide. 2. Telomere fluorescence intensity analyzed by TFL-Telo. 3. 1 TFU = 1 kb telomere (PNAS 94, 7423-7428, 1997). 4. Flow cytometry is performed using similar procedures.

**TRAP (telomeric repeat amplification protocol) (Kim et al., Science 266, 2011-2015, 1994; Kim and Wu, NAR, 25, 2595-2597, 1997) 1. Prepare cell lysates (in CHAPS buffer). 2. Add an end-labeled telomere-specific oligonucelotide substrate (TS primer) to the lysates. 3. If telomerase is present, it adds TTAGGG repeats to the substrates.

4. PCR amplification of the extension products using TS primer and Slide Credit: Dr. Dean G Tang reverse primer (ACT or ACX). Loss of TRF2 leads to extensive end to end fusion

WT TRF2 deprived

TRF2 is a key in maintaining normal telomere structure Telomere, senescence and tumorigenesis

--- Telomeres of cultured somatic cells continuously erode until M1 --- Telomeres derived from elderly individuals tend to be shorter than those derived from young donors ---Telomeres derived from constant self-renewing tissues such as liver and GI systems tend to be shorter than most other tissues and organs. --- Telomere length is a predicator of proliferative potential ---If M1 is overcome by transformation with viral , telomeres continue to decrease in size until M2, a process that may be dictated by telomere length itself. --- Whereas telomere size continuously decreases during replicative senescence, immortalized cells reach an equilibrium, albeit at shorter-than-wild-type length

Slide Credit: Dr. Dean G Tang Telomere, Telomerase and the (Harley CB, 21: 494, 2002)

Slide Credit: Dr. Dean G Tang Apoptosis associated with cell populations in crisis BFB (Breakage-Fusion-Bridge) Cycle

Karyotypic chaos seen in human bladder cancer cell line Critical Differences in Human vs Rodent Cells

Mouse Human Population Doublings (PD) 10-30 60-80 Telomere length 60-100 kb 10-15 kb Involvement of ARF-mdm2-p53 Ye s Ye s Involvement of p16-cyclin D-pRb No Ye s Telomerase activity in somatic cells Yes No/low Rate of spontaneous immortalization Very high (10-4-10-5) Low (10-7) Conclusion Prematur e senescence Related to Induced by inappr o- telomere pr iate culture conditions shortening

Slide Credit: Dr. Dean G Tang

Is telomere shortening really important: The mTR-/- mouse model

1mTR-/- mice lacked detectable telomerase activity yet were viable for the 6 generations analyzed. Telomerase-deficient cells could be immortalized in culture, transformed by viral oncogenes, and generated tumors in nude mice following transformation. Cells from the 4th mTR-/- generation onward possessed chromosome ends lacking detectable telomere repeats, aneuploidy, and chromosomal abnormalities including end-to-end fusions.

2Late-generation mTR-/- mice show defects (decreased proliferation and increased apoptosis) in high-renewable organ systems such as spermatogenesis and hematopoietic cells in bone marrow and .

3mTR-/- ES cells slow down their proliferation after ~300 divisions and completely stop proliferation after 450 divisions.

4Late-generation mTR-/- mice demonstrate shortened telomere and genetic instability, shortened span and reduced capacity to respond to stresses such as wound healing and hematopoietic ablation. There was increased incidence of spontaneous malignancies.

1. Blasco et al., Cell 91, 25-34, 1997. 2. Lee et al., Nature 392, 569-574, 1998. 3. Niida et al., Nature , 19, 203-206, 1998. 4. Rudolph et al., Cell 96, 701-712, 1999.

Slide Credit: Dr. Dean G Tang Is telomere shortening really important: The mTR-/- mouse model

1mTR-/- significantly reduces tumor formation in p16INK4A/p19ARF null mice. Reintroduction mTR into cells restored the oncogenic potential, suggesting that telomerase activation is a cooperating event in the malignant transformation of cells containing critically short telomeres. Loss of telomere impairs, but does not prevent tumor formation.

2Late-generation mTR-/- cells show severe telomere shortening, genomic instability, and p53 activation, leading to cell-cycle arrest and/or apoptosis. The mTR-/-p53-/- mice showed significantly increased rate of epithelial cancer formation.

3mTR-/- mice show rapid liver cirrhosis when subjected to genetic, chemical, and surgical ablation. Telomerase gene delivery alleviated cirrhotic pathology and restored liver function.

4Telomere dysfunction in late-generation mTR-/- mice impairs DNA repair and enhances sensitivity to ionizing radiation.

5Telomere dysfunction, together with p53 deficiency, promotes non-reciprocal translocations and epithelial cancers in mice.

1. Greenberg et al., Cell 97, 515-525, 1999. 2. Chin et al., Cell 97, 527-538, 1999. 3. Rudoph et al., Science 287, 1253-1258, 2000. 4. Wong et al., Nature Genetics 26, 85-88, 2000. Slide Credit: Dr. Dean G Tang 5. Artandi et al., Nature 406, 641-644, 2000. Human vs Mouse Tumors

1. The majority of mouse tumors are sarcomas and leukemias whereas 80% of the human tumors are carcinomas - cancer of epithelia where rapid cell turnover occurs. 2. Most of the experimental therapeutics that work in mouse fail in human, why??? 3. The answer may partly lie in the behavior of telomeres, and the relation- ship between telomere shortening, replicative cell senescence, and genetic instability. 4. In human, telomerase is suppressed or shut down and telomere shortening leads to replicative cell senescence. In mice, cells have long telomeres and retain telomerase activity, thus no telomere-dependent replicative senescence. However, in the 5-6th generation of TERC-/- cells, the mice begin to show various abnormalities, including increased incidence of cancer, raising the possibility that natural telomere shortening helps to engender many human tumors. (TERC is telomerase RNA component).

Slide Credit: Dr. Dean G Tang Telomerase-independent telomere maintenance

**A significant number of immortal or tumor cell lines have no detectable telomerase activity and also no defect in proliferation and growth. **These cells have unusually long telomeres (up to 50 kb; ~30 kb longer than that observed in the longest telomerase-positive cell lines). **The ALT (alternative lengthening of telomeres) pathway of telomere maintenance (EMBO J., 14, 4240-4248, 1995; Nature Genetics, 26, 447-450, 2000). ALT occurs by means of and copying switching (i.e., DNA sequences are copied from telomere to telomere).

Slide Credit: Dr. Dean G Tang Telomere-independent functions of telomerase

1. Telomerase is anti-apoptotic (Cao et al., Oncogene 21, 3130-3138, 2002). 2. Telomerase contributes to tumorigenesis by a telomere length-independent mechanism (Stewart et al., PNAS 99, 12606, 2002; Chang and DePinho, PNAS 99, 12520-12522, 2002). 3. Telomerase enhances DNA repair and genomic stability (Oncogene 22, 131-146, 2003). 4. TERT promotes cellular and organismal survival independently of telomerase activity. Lee J, Sung YH, Cheong C, Choi YS, Jeon HK, Sun W, Hahn WC, Ishikawa F, Lee HW. Oncogene. 2008 Jun 12;27(26):3754-60.

Slide Credit: Dr. Dean G Tang Dysregulation of Telomerase during Tumorigenesis

1) Telomerase activity and hTERT (telomerase reverse transcriptase) expression are low or absent in most somatic cells and primary tissues, due to a) transcriptional repression by WT1 and Mad, b) transcriptional repression by histone deacetylation.

2) In immortalized or cancer cells, hTERT activity is ‘reactivated’ due to a) transcriptional upregulation by myc, E2F1 etc, b) gene amplification, c) various signaling pathways such as c-Abl, bFGF, 14-3-3, Hsp90, Akt, PKC, etc, d) epigenetic chromatin remodeling.

3) Telomerase activity is normally associated with proliferation: cycling cells have high while differentiating cells have low telomerase activity. Due to this correlation, normal cells have relatively longer telomeres than tumor cells because the latter have undergone more cell divisions.

Slide Credit: Dr. Dean G Tang Replicative vs Premature Cell Senescence

Lloyd Ac. Nature Cell Biol. 4, E25-E27, 2002.

Inadequate culture conditions Tang, D.G., et al. Lack of replicative senescence in High O2 levels cultured rat oligodendrocyte precursor cells. Science 291: 868-871, 2001. p21-induced cell senescence appears to depend on ROS production (Macip et al., EMBO J. 21, 2180-2188, 2002).

Slide Credit: Dr. Dean G Tang Senescence, Aging, and Cancer “Because cancer incidence rises with age, it seems that cellular senescence is an imperfect tumor-suppressive mechanism that fails increasingly with age. In fact, late in life, accumulation of senescent cells probably even contributes to tumorigenesis because senescent cells overexpress, e.g., collagenases and underexpress their inhibitors”

--- Judy Campisi

1) Senescent promote premalignant and malignant cell proliferation in vitro and tumor development in vivo (Krtolica A et al., PNAS 98, 12072-12077, 2001) 2) Drug-induced senescent tumor cells secrete anti-apoptotic, mitogenic, and angiogenic factors that promote tumor development (Chang BD et al., PNAS 99, 389-394, 2002) 3) Ionization-induced, senescent-like fibroblasts promote breast cancer development (Tsai KK et al., Cancer Res. 65, 6734-6744, 2005) 4) Stromal fibroblasts from old (63-81 yr) prostates express and secrete higher levels of CXCL12 chemokine, which stimulates CXCR4-mediated epithelial cell proliferation (Begley L et al., 4, 291-298, 2005) 5) Senescent human prostate fibroblasts promote normal and cancerous prostate epithelial cell proliferation (Bavic C et al., Cancer Res. 66, 794-802, 2006) 6) Senescent human prostate epithelial cells promote tumor development (Bhatia et al Int. J. Cancer, 2008) Slide Credit: Dr. Dean G Tang Replicative cell senescence and cancer

Hypothesis 1: Replicative senescence is anti-cancer mechanism and an added barrier that cancer cells have to break through. Hypothesis 2: Cells in most tissues never actually reach replicative senescence in the course of a human lifetime, and the “immortality” of cancer cells is merely a side-effect of selection for some other property they need to have. Hypothesis 3: Many cells in normal tissue undergo replicative senescence and slow down or halt their proliferation as a person ages, and this creates circumstances in which cancer cells can thrive all the better by continuing to divide at full throttle. Thus, replicative senescence, far from protecting us from the growth of tumors, creates a breeding ground for mutant cells that evade the normal controls and overrun the tissue because they face no competition from their senescent normal neighbors. The mutations that allow continued proliferation may confer at the same time other cancerous traits, such as genetic instability or a general disregard for cell-cycle controls, leading on progressively more disordered behavior. In this way, replicative cell senescence in self-renewing tissues might be expected to favor the genesis of cancer; it could be a part of the reason why cancer is predominantly a of .

Martin Raff: in “Molecular Biology of the Cell”, the 4th edition, pp1324, 2002.

Slide Credit: Dr. Dean G Tang Replicative cell senescence and cancer

“The Hayflick limit presents a block to normal cell growth in culture, but because cancer cells invariably inactivate Rb and p53 pathways, it has been difficult to document a direct role for shortened telomere-induced senescence in tumor suppression in vivo. We favor the hypothesis that crisis plays a more prominent role than senescence in tumorigenesis. Although crisis is a potent barrier to immortal growth in culture, the massive genetic instability associated with this state may well be the mechanism by which the rare cells surviving crisis acquire the constellation of genetic alterations needed for malignant transformation. These rare cells emerge by activating telomere maintenance mechanisms - most commonly by activating telomerase.”

Maser & DePinho: Science, 297, 565, 2002.

Slide Credit: Dr. Dean G Tang Evidence of senescence as a tumor suppression mechanism Kang TW et al., Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature. 2011 Nov 9;479(7374):547-51.

Upon the aberrant activation of oncogenes, normal cells can enter the cellular senescence program, a state of stable cell-cycle arrest, which represents an important barrier against tumour development in vivo. Senescent cells communicate with their environment by secreting various cytokines and growth factors, and it was reported that this 'secretory phenotype' can have pro- as well as anti-tumorigenic effects. Here we show that oncogene- induced senescence occurs in otherwise normal murine hepatocytes in vivo. Pre-malignant senescent hepatocytes secrete chemo- and cytokines and are subject to immune-mediated clearance (designated as 'senescence surveillance'), which depends on an intact CD4(+) T-cell- mediated adaptive immune response. Impaired immune surveillance of pre-malignant senescent hepatocytes results in the development of murine hepatocellular carcinomas (HCCs), thus showing that senescence surveillance is important for tumour suppression in vivo. In accordance with these observations, ras-specific Th1 lymphocytes could be detected in mice, in which oncogene-induced senescence had been triggered by hepatic expression of Nras(G12V). We also found that CD4(+) T cells require monocytes/macrophages to execute the clearance of senescent hepatocytes. Our study indicates that senescence surveillance represents an important extrinsic component of the senescence anti-tumour barrier, and illustrates how the cellular senescence program is involved in tumour immune surveillance by mounting specific immune responses against antigens expressed in pre-malignant senescent cells.

Slide Credit: Dr. Dean G Tang Tumor Development

oncogenic malignant Normal cells immortalization transformation transformation (stem/progenitor Immortal Tumor cells or their cells cells progeny) progression Cell Metastases Cancer senescence cells

Slide Credit: Dr. Dean G Tang Senescence as a Therapeutic Alternative

1. DNA damage is able to induce senescence in p53-wt tumor cells in vitro and in vivo. p53 and p21 appear to play a critical role in the onset of senescence while p16 is involved in maintenance of senescence (te Poele et al., Cancer Res. 62, 1876-1883, 2002). 2. Senescence induction appears to contribute significantly to the efficacy of anti- neoplastic drugs (Schmitt et al., Cell 109, 335-346, 2002; Cancer Cell 1, 289-296, 2002; JCI, 113, 169-174, 2004).

Slide Credit: Dr. Dean G Tang

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