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  1. TYPES OF GENETIC INSTABILITY IN CANCER Aneuploidy Chromosome breakage Deletions and translocations Gene Amplification HSRs - homogeneously staining regions DMs - double minutes Elevation of mutation rates Epigenetic changes "CIMP" (CpG island methylator) phenotype

  2. Chromosomal Instability in Cancer

  3. Microsatellite Instability in Cancer

  4. GENETIC BASIS OF INSTABILITY Mismatch-repair defects Base-excision repair defect Cell-cycle checkpoint defects p53 mutations (Li-Fraumeni syndrome) Spindle-checkpoint defects (BUB1, MAD2) Other defects in repair or recombination Chromosome breakage syndromes

  5. Pathways of DNA repair O6-alkylguanine DNA alkyltransferase suicide protein transfers methyl group Base excision repair glycosylase, AP endonuclease, DNA polymerase, ligase Nucleotide excision repair transcription-coupled and global Cross-link repair Fanconi’s anemia genes and BRCA2 Double-strand break repair Homologous recombination and non-homologous end- joining Heteroduplex and mismatch repair Post-replication repair; translesion synthesis by pol eta, zeta, iota or kappa

  6. Familial cancer syndromes with defects in DNA damage response ataxia telangiectasia (AT), Nijmegen breakage syndrome, AT-like disorder - cell cycle checkpoints and DNA repair (dsb repair). ATM, NBS1, MRE-11 Fanconi’s anemia (FA) - DNA repair (cross-link repair). BRCA2 and FANC-A, B, C, D1, and E Hereditary non-polyposis colorectal cancer (HNPCC) – DNA repair (mismatch repair) and cell cycle checkpoints. hMSH2, hMLH1, PMS2, hMSH6 xeroderma pigmentosum (XP) – DNA repair (nucleotide excision repair, translesion synthesis) and transcriptional regulation. XPA, B, C, D, E, F, G and V Familial breast cancer I – cell cycle checkpoints. BRCA1 Li-Fraumeni syndrome (LFS) - cell cycle checkpoints, DNA repair and transcriptional regulation. p53, Chk2 Bloom’s syndrome, Werner syndrome, Rothmund-Thompson syndrome – DNA repair and cell cycle checkpoints. Blm, Wrn, RecQL

  7. HUMAN MICROSATELLITES Repeat units of 1 - 5 base pairs Tracts of ~ 6 - 30 repeat units Highly interspersed >100,000 tracts/genome Many are polymorphic

  8. Lynch & de la Chapelle, New Eng. J. Med. 348:919 (2003)

  9. MISMATCH-REPAIR PROTEINS Bacteria: MutH, MutS, MutL Human: hMSH2 hMSH3 = MutS homologues hMSH6 hMLH1 hPMS2 = MutL homologues

  10. mplex MSH2-MSH6 complex MSH2-MSH3 complex MLH1-PMS2 complex

  11. Familial cancer syndromes Ataxia telangiectasia - cell cycle checkpoint function, DNA repair. ATM, NBS1, MRE-11 Fanconi’s anemia - DNA repair. BRCA2 and 5 other genes HNPCC - mismatch repair, hMSH2, hMLH1, PMS2, hMSH6 Xeroderma pigmentosum - nucleotide excision repair and post-replication repair. 8 XP genes Familial breast cancer I - S and G2 checkpoint responses. BRCA1 Li-Fraumeni syndrome – cell cycle checkpoint function and DNA repair. P53, Chk2 Bloom’s syndrome, Werner syndrome, Rothmund-Thompson syndrome – chromosomal instability. Blm, Wrn, RecQ

  12. Checkpoint genes and cancer ATM – mutated in ataxia telangiectasia, a familial cancer syndrome; related to DNA-PK and ATR; phosphorylates NBS1, Chk2, p53, Abl, BRCA1 P53 – mutated in Li-Fraumeni syndrome with early onset breast cancer and fibrosarcoma; transactivates p21Waf1, p48/XPE, 14-3-3 BRCA1 – familial breast cancer; interacts with BASC, ATM, ATR, BRCA2, Rad51 and Rad52

  13. Checkpoints and carcinogenesis Cancer is characterized by enhanced growth and genetic instability Cell cycle checkpoints slow growth and preserve genetic stability Defects in cell cycle checkpoint function enhance growth and genetic instability, thereby fueling malignant progression

  14. Inactivation of p53-dependent G1 checkpoint function in diploid human fibroblasts Use replication-defective retrovirus to transduce HPV16E6 or dominant-negative p53 alleles HPV16E6 ablates p53 function by ubiquitin-mediated proteolysis p53 dominant-negative alleles (V143A, H179Q) compete for substrates

  15. Flow cytometric analysis of cell cycle checkpoint response to DNA damage: normal human fibroblasts FITC-anti-BrdU Phospho-histone H3 DNA/PI G1 checkpoint G2 checkpoint

  16. P53-dependent G1 checkpoint function

  17. Inactivation of p53 extends cellular lifespan but leads to crisis; immortal lines may emerge from crisis

  18. Chromosomal aberrations in aging E6-expressing fibroblasts

  19. Genetic instability in human fibroblasts Finite lifespan Extended lifespan Immortal G1 + G1 - G1 - G2 + G2 +/- G2 +/- diploid aneuploid aneuploid,polyploid

  20. What is the source of polyploidy? E6-expressing cells start with diploid stable genomes Attenuation of DNA damage G2 checkpoint function in ataxia telangiectasia is not associated with polyploidy P53-defective cells are prone to polyploidy when they experience stress on the mitotic spindle

  21. Polyploidization in aging E6-expressing cells

  22. DNA inputs to the G2 checkpoint

  23. Decatenation checkpoint monitors chromatid separation by topoII

  24. Cyclin B1/Cdk1 Activity Model of ATR/ATM-dependent G2 checkpoints Catenated Chromatids DNA dsb ATR ATM/ATR BRCA1 Plk1 Chk1 Cdc25C Cyclin B1/Cdk1 Localization G2 M

  25. Attenuation of decatenation checkpoint in aging E6-expressing cells

  26. Expression of telomerase suppresses chromosomal destabilization in aging E6-expressing cells Aberrations per 50 cells • Cell Strain PDL Dicentrics/ Breaks/ %Polyploidy %Evading G2 Rings Exchanges Delay • F7-neo 10 0 2 8 7  5 30 0 2 4 0, 2 • F7-E6 10 0 1 2 17  5 30 19 6 4 22, 48 58 25 38 33 133  66 • F7-E6-TRT(-) 58 40 18 36 88  11 • F7-E6-TRT(+) 58 0 2 3 9  7

  27. Defective G2 checkpoint function in cancer lines

  28. Figure 5. DNA damage G2 checkpoint function in normal human melanocytes and melanoma cell lines. Upper left panels: flow cytometric quantification of mitotic cells labeled with Anti-phospho-histone H3 antibody. Upper right panel: percent of cells in mitosis 2 h after 1.5 Gy IR, equivalent to the percent of cells evading IR-induced G2 delay. Bottom left panel: G2 checkpoint function in melanoma lines with wildtypeN-Ras and B-Raf alleles (wt, n=4), with mutant N-Ras (N, n=8) or mutant B-Raf (B, n=6).