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Regulation of gene expression by mutant and oncogenic forms of the p53 tumor suppressor

Regulation of gene expression by mutant and oncogenic forms of the p53 tumor suppressor. “The Cell Cycle”. DNA Damage Leads to p53-Mediated Arrest of Cell Cycle. p53 Regulatory Pathways. DNA Repair. Phosphorylation Acetylation Methylation. Oncogene Expression c-Myc and Ras. G 2 Arrest

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Regulation of gene expression by mutant and oncogenic forms of the p53 tumor suppressor

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  1. Regulation of gene expression by mutant and oncogenic forms of the p53 tumor suppressor

  2. “The Cell Cycle”

  3. DNA Damage Leads to p53-Mediated Arrest of Cell Cycle

  4. p53 Regulatory Pathways DNA Repair Phosphorylation Acetylation Methylation Oncogene Expression c-Myc and Ras G2 Arrest (14-3-3s, Gadd45) (-) p19ARF DNA Damage UV, Irradiation, Genotoxic Compounds p53 MDM2 ATM Apoptosis (Bax) G1 Arrest DNA Damage UV, Irradiation, Genotoxic Compounds (p21Waf1/Cip1) DNA Repair

  5. Carcinogens can Damage the p53 Gene

  6. p53 Mutations in Human Cancers

  7. p53 mutations in human cancer

  8. p53 mutations in colon cancer

  9. p53 mutations in lung cancer

  10. p53 mutations in liver cancer

  11. Can we identify genes regulated by mutant p53?

  12. Expression of mutant p53 • p53-null cells 10(3) expressing p53 mutant(248) from an expression vector

  13. Growth Rates of cells +/- mutant p53

  14. 600 Gene Array

  15. Gene Expression Array p53 null Mutant p53

  16. Genes demonstrating altered expression in the presence of R248W p53 • Induced Repressed • Multidrug resistance gene (MDR1) Tissue inhibitor of metalloproteinases-3 (TIMP-3) • Connexin-32 Monocyte chemotactic protein-3 (MCP-3) • Ribosomal protein gene L37 • Ribosomal protein gene RPP-1 • Ribosomal protein gene S1 • Transcription/Termination Factor I (TTF-1) • PW29, Calcium-binding protein • Heatshock 86Kd protein

  17. Some Properties of TIMP-3 • Inhibitor of matrix metalloproteinases • Required for proper tissue remodeling, vascularization and matrix turnover • Loss of expression in a mouse tumor progression model - potential tumor suppressor • Reduced expression observed in metastatic human colon cancer • Reconstituted/elevated expression of TIMP-3 leads to reduced metastasis and reduced angiogenesis

  18. TIMP-3 Reporter Construct TIMP-3 Promoter

  19. Inhibition of TIMP promoter by mutant p53

  20. Deletions in the TIMP-3 promoter -2500 -2000 -1000 -600 -2900 AP-1 2xNFkB 4xAP-1 p53 AP-1 2xSp1 TATA -500 -400 -300 -200 -100

  21. Deletions eliminate mutant but not wild type p53 repression

  22. Deletions in the TIMP-3 promoter -2500 -2000 -1000 -600 -2900 AP-1 2xNFkB 4xAP-1 p53 AP-1 2xSp1 TATA -500 -400 -300 -200 -100

  23. Loss of p53-mediated repression

  24. Inhibition of TIMP-3 in cells expressing mutant p53

  25. Deletions in the TIMP-3 promoter -2500 -2000 -1000 -600 -2900 AP-1 2xNFkB 4xAP-1 p53 AP-1 2xSp1 TATA -500 -400 -300 -200 -100

  26. TIMP3(1) TIMP3(4) mutant p53 - + + + - + + + -2800 * * -2800 Two Nuclear Factors Bind to TIMP-3 in the Presence of Mutant p53 -2900

  27. A 25-bp element causes repression by p53

  28. RNAi-mediated inhibition of mutant p53 expression • RNA-guided regulation of gene expression • conserved in most eukaryotic organisms. • involves dsRNA inhibiting the expression of genes with complementary nucleotide sequences. • thought to have evolved as a form of innate immunity in defense against viruses, • plays major regulatory roles in development and genome maintenance.

  29. 1 2 3 4 1 2 3 4 5 6 7 SW 837 T98G 1 2 3 4 5 6 7 8 1 2 3 4 5 6 RNAi inhibition of p53 CMV 248 Cl.2 Colo 320

  30. 1 2 3 4 5 6 7 8 9 p53 mRNA levels in Colo 320 siRNA expressing clones Cl.4 Cl.5 Cl.7 Cl.12 Cl.10 Cl.11 Cl.17 Cl.18

  31. 1 2 3 4 5 6 7 8 9 GAPDH Inhibition of p53 leads to increase in TIMP-3 expression untransfected siRNA expressing Cl. 4 Cl.5 Cl.7 Cl.12

  32. Cancer results from numerous genetic mutations

  33. Model for role of mutant p53 expression in progression to metastasis

  34. Future Goals Characterize factors that bind to the TIMP promoter Determine role of mutant p53 in regulating these factors Determine whether inhibition of mutant p53 can result in elevated TIMP and inhibition of tumor progression 4. Assess contribution of the pathway to cancer progression

  35. ACKNOWLEDGEMENTS Troy Loging Sondra Spiegl Tricia Solito Shana Thomas

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