Telomere Stabilizing Mechanisms

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1 Telomere Stabilizing Mechanisms Cell Immortalization and Tumorigenesis Basic Seminar Malignant Diseases I N094 PhD Program Malignant Diseases N790 Dr Program Clinical Experimental Oncology N090 Dr Program Tumorbiology - Oncology Dr.Klaus Holzmann 1 Content Normal cell populations register the number of cell generations separating them from their ancestors in the early embryo Cancer cells need to become immortal in order to form tumors Cell-physiologic stresses impose a limitation on replication The proliferation of cultured cells is also limited by the telomeres of their chromosomes Telomeres are complex molecular structures that are not easily replicated Incipient cancer cells can escape crisis by expressing telomerase Telomerase plays a key role in the proliferation of human cancer cells Some immortalized cells can maintain telomeres without telomerase Telomeres play different roles in the cells of laboratory mice and in human cells The mechanisms underlying cancer pathogenesis in telomerasenegative mice may also operate during the development of human tumors Dr.Klaus Holzmann 2

2 Eternal Life: Cell Immortalization and Tumorigenesis Biologist August Weissmann (1881): Death takes place because a worn-out tissue cannot forever renew itself, and because a capacity for increase by means of cell division is not everlasting but infinite. -> Cancer cells must break the barrier that normally limits cell proliferative potential, so that they can successfully complete the multiple steps of tumor development. Literature: The Biology of Cancer: RA Weinberg (2007) Chapter 10 Dr.Klaus Holzmann 3 Serial passaging of living tissue cells in culture in vitro Dr.Klaus Holzmann 4

3 Hayflick Limit Nat Rev Mol Cell Biol Oct;1(1): Hayflick, his limit, and cellular ageing. Shay JW, Wright WE. STOP after population doublings (PDs) Lifespan calculated in PDs PD = log(number cells harvested)-log(number cells seeded) log 2 Dr.Klaus Holzmann 5 Loss of proliferative capacity with age same color = inter-individual variability -> Keratinocyte stem cells lose proliferative capacity with age Consequences observed in vivo: - thinning of the keratinocyte layer of the skin - loss of the ridge architecture Figure 10.4 The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 6

4 Senescent cells in vitro Phase II PD 20 Phase III - PD 55 Pre-sensecent human fibroblast cells Sensecent human fibroblast cells ->cease proliferation but remain viable ->express senescent-associated acidic ß-galactosidase enzyme (blue) ->change morphology fried egg Figure 10.3 The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 7 Dr.Klaus Holzmann 8

5 Cancer cells need to become immortal in order to form tumors Why normal cells lack immortalized growth properties? -> anti-cancer defense mechanism model based on facts: - human tumors are clonal (mean all the neoplastic cells in the tumor mass descend from a common ancestral cell that underwent transformation at one point in time) - required tumor cell population doublings to make a clinically detectable human tumor can be calculated Figure 10.5a The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 9 Some types of normal human cells are known to pass through 50 or 60 cycles of growth and division before they become senescent and stop growing Cell numbers are fare more than required to constitute a life-threatening tumor mass 2 40 = cells = 10 3 cm 3 = 1kg 2 60 = cells = 10 9 cm 3 = 10 6 kg! something is drastically wrong with these numbers Dr.Klaus Holzmann 10

6 ->cells do not only exponentially growth but also die off A number of defense mechanisms in the body s tissues exist to make life very difficult for cancer cells, e.g.: -withdrawal of growth factors -withdrawal of adequate oxygen -ability to eliminate metabolic waste via the vasculature In reality 2-3 times more PD may be required Dr.Klaus Holzmann 11 Generation-counting device? Properties: Cell-autonomous (intrinsic to the cell) Must be biochemically stable over extended period of time as it stores the past history of a cell lineage A counting molecule can not work over a concentration range of 2 50 or 2 60 two regulatory mechanism were defined: 1. One measures the cumulative physiologic stress that lineages of cells experience over extended period of time and halts further proliferation once that damage exceeds a certain threshold (phase termed senescence) 2. The second mechanism measures how many replicative generations a cell lineage has passed through and alarms if the allowed quota of replicative doublings has been used up (phase termed crisis) ->what is the device for 1 and 2? Dr.Klaus Holzmann 12

7 Induction of tumor suppressor proteins during in vitro culture Adult human endometrial fibroblasts PD 3 PD3+ectopic p16 PD 43 yellow: focal contacts orange: actin stress fibers Figure 10.7 The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 13 INK4a/ARF Locus =p16 Curr Opin Genet Dev Feb;12(1): Immortalisation and transformation revisited. Drayton S, Peters G. Dr.Klaus Holzmann 14

8 Cell-physiologic stresses impose a limitation on replication Human diploid fibroblasts Human epithelial cells (foreskin keratinocytes) Figure 10.6 The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 15 Role of large T antigen in circumventing senescence Inactivation of both prb and p53 is needed Figure 10.8 The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 16

9 Evidence of senescent cells in living tissues % of human fibroblast cells β -gal-positive passage number Acidic β-galactosidase (SAβ-gal) enzyme staining (blue) of lung carcinoma tissue from patient treated with the drugs carboplatin and taxol Figure 10.9c and suppl. Sidebar 14 The Biology of Cancer ( Garland Science 2007) 2 covalent alterations of histones exist in senescent cells but not in cells in the G0, quiescent state: 1. SAHF (senescence-associated heterochromatic foci) detected by antibodies against histone H3 methylated on lysine 9 residue 2. γ-h2ax foci (green spots) specific for dsdna breaks after X-irradiation (disappear after repair) and senescence cells (persist) Dr.Klaus Holzmann 17 The proliferation of cultured cells is also limited by the telomeres of their chromosomes Telomeres (green) detected by fluorescence in situ hybridisation (FISH) in human cells trapped in metaphase (using a microtubule antagonist), chromatids (red) Figure 10.11a The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 18

10 Telomere function Chromosomes that have lost functional telomeres at their ends soon fuse, end-to-end, with one another. One key protein in maintaining normal telomere structure, TRF2, was removed and resulted in formation of cells with virtually all the chromosomes fused into one giant one Figure 10.11b and 10.14a The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 19 Shortening of telomeric DNA in concert with cell proliferation each passage represented 3-6 PDs, loss of telomeric DNA can be calculated to be between 50 and 100 base pairs per cell generation Figure 10.13a The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 20

11 Shortening of telomeric DNA in concert with cell proliferation Figure 10.13b The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 21 Telomeres - Structure Figure The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 22

12 Lassos at the ends of chromosomal DNA Figure 10.17a The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 23 Structure of the T-Loop Figure 10.17b The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 24

13 Structure of the T-Loop Figure 10.17c The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 25 Multiple telomer-specific proteins bound to telomeric DNA Figure The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 26

14 How telomeres are replicated? End-replication problem = generation-counting device! (8-12 nt) All DNA synthesis occurs in 5 -to-3 direction RNA primers will be degraded -> leading strand is under-replicated (8-12 nt) Why lose of bp per cell generation? Exonucleases involved Figure The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 27 Incipient cancer cells can escape crisis by expressing the enzyme telomerase Nat Rev Cancer May;2(5): Modelling the molecular circuitry of cancer. Hahn WC, Weinberg RA. Dr.Klaus Holzmann 28

15 Signals from short telomeres J Clin Oncol May 15;21(10): Role of telomeres and telomerase in the pathogenesis of human cancer. Hahn WC. Dr.Klaus Holzmann 29 Telomerase Activity TRAP Assay Figure 10.21a+b, The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 30

16 The catalytic subunit of telomerase Enzyme first described 1985 by Carol W. Greider and Elizabeth H. Blackburn Identification of a specific telomere terminal transferase activity in tetrahymena extracts. In: Cell (1985) 43:405. Cloning History: 1996 p123 from the ciliate E.aediculatus (PNAS 93:10712) 1997 Est2 (ever-shorter telomeres) from S.cerevisiae (PNAS 94:9202) 1997 htert from H.sapiens (Science 277:955 and Cell 90:785) Figure 10.22a The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 31 Structure and function of the human telomerase holoenzyme htert 127kDa htr 451nt Figure 10.23a The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 32

17 Telomerase gene expression Nucleic Acids Res Feb 15;30(4): Natural and pharmacological regulation of telomerase. Mergny JL et al. Dr.Klaus Holzmann 33 Prevention of crisis by expression of telomerase Figure 10.25a The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 34

18 Suppression of telomerase activity -> Restoration of Crisis DNhTERT expression results in loss of the neoplastic growth program in 4 different human cancer cell lines DNhTERT expressing cells continue proliferation until the reach crisis Figure The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 35 Telomerase activity and the prognosis of pediatric tumors -> telomerase plays a key role in the proliferation of human cancer cells Figure The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 36

19 Some immortalized cells can maintain telomeres without telomerase 85-90% of human tumors are telomerase positive, so 10-15% lack detectable telomerase activity but need to maintain their telomeres above some minimum length in order to proliferate indefinitely. ALT (alternative lengthening of telomeres) is telomerase independent; associated preferentially with ostesarcomas, soft-tissue sarcomas and glioblastomas FISH: in yellow telomere specific probe ALT -> heterogeneous telomere length crises TRF: ALT cell line Nat Rev Cancer Nov;2(11): Telomere maintenance and cancer -- look, no telomerase. Neumann AA, Reddel RR. Dr.Klaus Holzmann 37 The ALT mechanism Telomere sequence information exchanged between chromosomes in ALT cells -> interchromosomal recombination involved Figure The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 38

20 ALT model copy choice mechanism Der DNA Strang eines kurzen Telomers dient als Primer für die DNA Synthese bei einem anderen Telomer. ->Nettogewinn an Telomerlänge Gleiches Telomer, funktioniert über die T-Loop-Struktur Extrachromosomale DNA Elemente (circle, linear) existieren in ALT-Zellen, rolling circle wurde in der Hefe funktionell gezeigt, Möglichkeit zur fast unbegrenzte Verlängerung der Telomere Nat Rev Cancer Nov;2(11): Telomere maintenance and cancer -- look, no telomerase. Neumann AA, Reddel RR. Dr.Klaus Holzmann 39 Telomeres play different roles in the cells of laboratory mice and in human cells Erosion of telomeres over multiple generations in populations of mtr-/- mice The onset of human genetic disease with telomeres involved (e.g. X-linked form of dyskeratosis congenita ) is happen already in the first generation Figure The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 40

21 Telomerase-negative mice show both decreased and increased cancer susceptibility mtr-/- p16 INK4A /p19 ARF -/- mice: reduced rate of cancer mtr-/- p53-/- mice: increased rate of cancer Rate of tumor formation in cancer-prone mtr-/- p53-/- mice Figure The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 41 Mechanistic model of how breakage-fusion-bridge (BFB) cycles promote human carcinoma formation Figure The Biology of Cancer ( Garland Science 2007) Dr.Klaus Holzmann 42

22 Main concepts Take Home Message Two barriers prevent cultured cells from replicating indefinitely in culture (senescence and crisis) Senescence involves the long term residence of cells in a non growing but viable state Crisis involves the apoptotic death of cells Senescence is provoked by physiological stresses that cells experience in vitro (in vivo not yet clear) Crisis is provoked by the erosion of telomeres, which result in widespread endto-end chromosomal fusions, karyotypic chaos, and cell death Most pre-malignant cells escape from crisis by activating of telomerase (htert) and elongation of telomeric DNA Some cancer cells escape crisis by regenerating their telomeric DNA through the ALT mechanism Cells that have stabilized their telomeres through htert or ALT can than proliferate indefinitely and are therefore said to be immortalized Cell immortalization is a step that appears to govern the development of all human cancers The end-to-end chromosmal fusions that accompany crisis appear in turn to be responsible for much of the aneuploidy associated with the karytypes of many kinds of solid human tumors Dr.Klaus Holzmann 43 Telomerase and Immortality Hahn, W. C. J Clin Oncol; 21: Dr.Klaus Holzmann 44

23 Recent Findings TERRA/TelRNAs associate to telomeric chromatin and may be involved in regulation of telomere length. Model for a role of telomeric RNAs in the regulation of telomere length. TelRNAs are potent inhibitors of telomerase activity in vitro, possibly by forming of RNA:RNA hybrids with the template region of the telomerase RNA component (Terc). (from Schoeftner and Blasco 2009 Chromatin regulation and non-coding RNAs at mammalian telomeres. Seminars in Cell & Developmental Biology. Online) The TERT β-catenin connection. a, The telomerase complex functions in progenitor cells to repair chromosome ends, known as telomeres, during cell division. TERT provides reverse transcriptase activity to the complex, and uses TERC, the RNA component of telomerase, as a template. b, Park et al.1 find that TERT also increases the transcriptional activity of β-catenin/tcf complexes through interaction with BRG1, a factor that binds the Wnt signalling molecule β-catenin and alters the conformation of chromatin. These two separate functions of TERT may simultaneously prevent cellular senescence and increase proliferation of progenitor cells, permitting embryonic development and renewal of adult tissues. (from Millar 2009 The not-so-odd couple. Nature, 460:44) Dr.Klaus Holzmann 45