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

5. 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
G1 Arrest
Apoptosis
(Bax)
(p21Waf1/Cip1)
DNA Damage
UV, Irradiation, Genotoxic Compounds
DNA Repair

6. Carcinogens can Damage the p53 Gene

7. p53 Mutations in Human Cancers

8. p53 mutations in human cancer

9. p53 mutations in colon cancer

10. p53 mutations in lung cancer

11. p53 mutations in liver cancer

12. Can we identify genes regulated by mutant p53?

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

14. Growth Rates of cells +/- mutant p53

15. 600 Gene Array

16. Gene Expression Array
p53 null
Mutant p53

17. Genes demonstrating altered expression in the presence of R248W p53
Induced Repressed
Multidrug resistance gene (MDR1) Tissue inhibitor of metalloproteinases-3 (TIMP-3)
Connexin 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

18. 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

19. TIMP-3 Reporter Construct
Promoter

20. Inhibition of TIMP promoter by mutant p53
Wt p53

21. 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

22. Deletions eliminate mutant but not wild type p53 repression

23. 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

24. Loss of p53-mediated repression

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

26. 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

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

28. A 25-bp element causes repression
by p53

29. 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.

30. RNAi inhibition of p53 CMV 248 Cl.2 Colo 320 SW 837 T98G 1 2 3 4
SW 837
T98G

31. p53 mRNA levels in Colo 320 siRNA expressing clones
Cl Cl Cl Cl Cl Cl Cl Cl.18

32. Inhibition of p53 leads to increase in TIMP-3 expression
untransfected siRNA expressing
Cl Cl Cl Cl.12
GAPDH

33. Cancer results from numerous genetic mutations

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

35. 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

36. ACKNOWLEDGEMENTS
Troy Loging
Sondra Spiegl
Tricia Solito
Shana Thomas