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Cancer results from mutations in genes regulating cell growth

Cancer results from mutations in genes regulating cell growth. Two classes of genes are involved: 1) - positive regulators promote cancer by hyperactivity (one allele is enough)

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Cancer results from mutations in genes regulating cell growth

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  1. Cancer results from mutations in genes regulating cell growth • Two classes of genes are involved: • 1) - positive regulators promote cancer by hyperactivity (one allele is enough) • 2) - negative regulators, promote cancer by loss of activity (two allele must be mutated) Oncogenes Suppressor genes

  2. Rb • The first tumor suppressor- (retinoblastoma) • The most frequent mutated tumor suppressor - • p53 was first cloned at the weizmann institute and is mutated in > 50% of all cancer. • 25,000 papers published on p53 in 22 years p53 How p53 functions as a tumor suppressor?

  3. Induction of cell death by active p53 Parental M1 M1-p53Val135clones at 32oC

  4. Activation of temperature sensitive p53 prevents transformation Myc + Ras + no p53 temperature sensitive p53 ts p53 in mutant conformation ts p53 in wild type conformation

  5. Negative regulation of DNA binding Protein-protein interactions, Transcriptional repression apotosis

  6. (Ko LJ & Prives C, Genes & Dev. 10:1054-1072, 1996) Most of p53 mutations are found in the conserved regions of the central DNA binding domain

  7. (Cho Y et al., Science 265:346-55, 1994)

  8. Activated p53 Transactivation Other activities (C terminal = TFIIH binding?) (N terminal = SH3 binding?) p21/ Waf1 Bax, IGF-BP3, Fas, killer/DR5, Noxa, PIG3, p53AIP1, PIDD, Puma Other genes etc, etc, etc Apoptosis Growth arrest

  9. Growth arrest, apoptosis Functional p53 DNA damage, oncogene activation Loss of p53 function Cancer

  10. Three Experimental Systems 1. Primary and Secondary Targets of p53 2. Target genes related to apoptosis 3. Comparison of p53 and p73 A chip of 10,000 genes is more than ten thousand northern blots

  11. Filter hybridization MDM2 LIG1 PCNA p21

  12. Representation of 7000 genes on 1 cm2 chip

  13. DIRECT AND INDIRECT TARGETS CHX INHIBITS PROTEIN SYNTHESIS AND PREVENTS ACTIVATION OF SECONDARY TARGETS

  14. Effect of Cycloheximide on H1299 val135 Cells

  15. Only ˜10% of the genes changed expression in all 3 repeats(Coller et al. 2000) Primary p53 target genes in presence of CHX 38 24

  16. Clustering of 259 genes upregulated without CHX at least three times > 2.5 fold a. 9 genes (false?) b. All 38 primary c. 87% of primary

  17. GENE/ PROTEIN NAME p21waf1 MIC1 - member of TGF-b family MDM2 PCNA GADD45 Mitochondrial Stress 70 (Mortalin2) p57KIP2-CDK inhibitor 1C PIG3-p53 induced gene 3 FAS/APO1 BAX-Bcl2 associated X protein BAK1- Bcl2 antagonist/killer 1 38.0 10.0 8.3 3.9 3.0 1.5 11.0 3.8 3.7 1.5 p53 - Upregulated Genes in H1299-Val135 system RATIO FUNCTION CELL CYCLE APOPTOSIS

  18. GENE/ PROTEIN NAME DDB2-Nucleotide Excision Repair LIG1 - DNA ligase 1 ERCC5 - DNA Excision Repair related TDG - G/T mismatch DNA Glycosylase RPA1 - Replication Factor A Protein 1 MAPK14 MAP4K5 Activaes Jun N-term Kinase MAP2K1 - MEK1 MYD88 - Myeloid differentiation Retinoic Acid Receptor Beta FKBP4 HOXD3 - Homeobox protein CSPG2 - Chondroitin sulfate proteoglycan 2 6.5 2.3 1.9 1.8 1.6 3.8 1.6 1.5 5.4 4.6 3.1 1.8 2.0 P53 - Upregulated Genes in H1299-Val135 system RATIO FUNCTION DNA REPAIR KINASE RECEPTOR DEVELOP. IMMUNOPHIL. DEVELOP. ECM

  19. p53- DRIVEN APOPTOSIS A different cell line (M1) that undergoes apoptosis by p53 at 32

  20. APOPTOSIS : - Apoptosis is a genetically controlled program of cell death, also referred to as cell suicide or Programmed Cell Death (PCD). - It is an evolutionary conserved mechanism. • - It ultimately leads to elimination of undesired cells • either superfluous • or potentially harmful when damaged - It plays an essential role during developmental as well as adult stages by allowing tissue remodeling, tissue renewal and maintenance of tissue homeostasis

  21. Pro apop Anti apop

  22. APOPTOSIS IN LTR6 CELLS AT 32C. Sub G1

  23. A B 12h M1 2h LTR6 2h M1 2h M1 C D 9h LTR6 12h LTR6 2h M1 2h M1 SCATTER PLOTS OF 404 GENES THAT WERE REGULATED BY P53 IN LTR6 CELLS

  24. M1 2hr M1 12hr LTR6 2hr Control Cell type M1 LTR6 M1 LTR6 M1 LTR6 12 2 6 9 12 2 2 6 9 12 12 2 6 9 12 Time (hr) A B C D E F Clustering of 404 genes based on expression kinetics at 32C

  25. Kinetics of transcriptional activation Cluster E Cluster C Cluster D Relative ratio M1 2 6 9 12 M1 2 6 9 12 M1 2 6 9 12

  26. Apoptosis related genes upregulated by p53 H1299 Val Cells RATIO OF EXPRESSION (12h) ACCESSION NO. X63717 U82987 U00115 U16811 MOUSE ACCESSION NO. AB021961 M83649 U82532 J04953 Z16410 AW060710 X74504 AF064071 Fas/APO-1 cell surface antigen Bcl-2 binding component 3 (bbc3) PUMA Bcl-6 Bak p53 Fas antigen/TNFR6 TNFR18 Gelsolin Btg1 EST=PIG8 (Etoposide induced) T10 mRNA/human sentrin/SUMO-1 Apaf-1 9.8 30.5 6.8 7.4 57.8 54.5 11.3 2.9 9.0 6.2 4.3 7.2 LTR 6 Cells HUMAN ACCESSION NO. A1909620 X89101 A1923712 X04412 X61123 R11732 U83117 AL135220

  27. Northern Analysis of LTR6 Cells Apaf-1 mRNA induced at 32 C by ts-p53

  28. APAF-1 promoter contains p53 target at -604 bp 800 400 • RRRCWWGYYY N{0-13 bp} RRRCWWGYYY • APAF-1 AGACATGTCT GGAGACCCTAGGA cGACAAGCCC • BAX tcACAAGTTa G AGACAAGCCT • GADD45 GAACATGTCT AAGCATGCTg • MDM2 GGtCAAGTTg GGACAcGTCC

  29. p53 Binding to APAF-1 Target by Gel Shift Analysis Others Ab x’sX x’sA Oligo A A A A A A B C D p53 + + + + mut + + + Oligo A: AGACATGTCTGGAGACCCTAGGACGACAAGCCC Oligo B: AGACATGTCT CGACAAGCCC Oligo C: AGACATGTCTGGAGACCGACAAGCCC Oligo D: AGAaATGTCTGGAGACCCTAGGACGAaAAGCCC

  30. Apoptotic stimuli p53 Bax PUMA Bcl-2 Cyt c /dATP Smac p53 Apaf-1 Caspase-9 IAPs Caspase-3 Cellular targets Apoptosis

  31. p53 family members Comparison of p73 and p53 induced genes

  32. Scatter plot

  33. /Cip1

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