p53: To Be or Not to Be…

1. p53: To Be or Not to Be… Ashim Malhotra Bio 722 Instructor: Dr. Lockshin March, 24, 2003

2. What is p53? • Tumor suppressor phosphoprotein with 4 domains; Conserved in vertebrates • Gene name: Human: TP53, 17p13.1; Mouse : Trp53 • Genestructure:19.21 kb; 11 exons • Subcellular Location: Nuclear; in a punctate granular/vesicular patter • Evolution: Xenopus (68% homology) • Sequence comparison: shows 5 highly conserved regions, coinciding with mutation clusters found in p53 in human cancers

3. Why talk of p53? • 27, 458 • Molecule of the Year, Science, 1993 • Guardian of the Genome; Cellular Gatekeeper for Growth & Division (Levine, 1997 review) • Emergency brake in the cell cycle • Bio 722! Involved in PCD

4. What does it do? • Suppresses progression through the cell cycle in response to DNA damage • Initiates apoptosis if the damage to the cell is severe • Often as a tumor suppressor • A potent transcription factor

5. Involvement in diseases; putative applications • p53 gene mutationsare the most frequently observed genetic lesions in human cancers • p53 and radiation sensitivity: Problems in radiotherapy • Mutant p53: problems in tumor hypoxia • Gene therapy with p53; Nature 1996 • Clinical Phase trials: (I) Adenovirus vectors expressing p53 in tumors casues regression of mouse lymphomas (tumor cells with mP53 do not die even though O2 starved)

6. Come into the mad world of p53…

7. Discovery of p53 The viral approach The serological approach

8. Courtesy:

9. Courtesy:

10. The IARC Human p53 Mutation Database Current release has nearly 7000 entries detailing: • Location of point mutations • Description of mutation • Resultant tumor type • Exposure information • Pointer to original reference article http://www.iarc.fr/p53

11. Protein structure & DNA binding • Domain: Specific interactions with bcl-2 require ankyrin & SH3 domains • p53 binds as a tetramer to PBS & activates expression of downstream genes inhibiting growth &/or invasion

12. Mechanisms of p53 inactivation • p53 binding to PBS. Deletion of one or both p53 alleles= expression of tetramers & expression of the growth inhibitory genes. • Nonsense or splice site mutations result in protein-truncation preventing oligomerization reducing tetramers: lung & esophagus. • Missense mutations resulting in dominant-negative effects & even greater reduction of functionally active tetramers: colon, brain, lung, breast, skin & bladder. • Expression of the HPV - E6 gene causes functional inactivation of p53 through binding and degradation. • The p53 pathway may also be disrupted by alteration of a cellular gene, MDM2.

13. p53 Polymorphisms • 2 p53 variants, 1 containing arginine & the other containing proline. • Arginine containing p53 kills cancer cell more effeciently than the one containing proline. • The proline form has an enhanced frequency in African Americans & darker skinned people living closer to the equator!! • Arginine form travels out of the nucleus and into the Mt better than the proline form. Dumont, Leu & Pietra, Nature Genetics, March 2003

14. What does it do? • Suppresses progression through the cell cycle in response to DNA damage • Initiates apoptosis if the damage to the cell is severe • Often as a tumor suppressor • A potent transcription factor

15. Convergence of distinct stimuli at p53 de Stanchina et al, Genes and Development, 1998

16. Pathways! Pathways! P on S-15 UV DNA- damage P on S-20 Mdm2 Cytostasis p53 p53

17. P53 induced cell cycle arrest I N H I B I T I O N p53 p21 Cyclin-Cdk2 complex Hyp-Rb/E2F complex Cytostasis

18. S-20 phosphorylation in response to DNA damage Cheha, Malikzay, Stavridi & Halazonetis. Cell Biology, 1999

19. Summary of p53 stabilization pathways after DNA damage Giaccia, and Kastan, Genes and Development, 1998

20. p53 / Rb connection Sherr, Science, 1996

21. What does it do? • Suppresses progression through the cell cycle in response to DNA damage • Initiates apoptosis if the damage to the cell is severe • Often as a tumor suppressor • A potent transcription factor

22. p53 associated apoptosis • Of 7,202 transcripts identified, only 14 (0.19%) are markedly increased in p53-expressing cells. • Many of these genes encode proteins that generate or respond to oxidative stress. • These observations suggest that p53 causes apoptosis through a three-step process: 1. Transcriptional induction of redox-related genes. 2. Formation of reactive oxygen species and. 3. Oxidative degradation. Polyak, Xia, Zweier, Kinzler & Vogelstein, Nature, 1997.

23. Possible steps in p53 mediated apoptosis: step I UV p53 DNA PK DNA damage

24. DNA PKcs mediates apoptosis but not cytostasis via p53 Wang, Guo,Ouyang , Li,Cardo ,Kurimasa,Chen,Fuks, Ling , & Li, Cell Biology, 2000

25. P53! Anoint thee wench! Peg3/PW1 Transcriptional Activation Bax p53 PUMA

26. Bax sublocalization with Peg3 Yibin & Wu, Cell Biology, 2000

27. Peg3/PW1 causes translocation of Bax to Mitochondria Yibin & Wu, Cell Biology, 2000

28. Scheme for PUMA induced Mt translocation of Bax PUMA Mt Translocation Bax Bcl-Xl p53

29. What does it do? • Suppresses progression through the cell cycle in response to DNA damage • Initiates apoptosis if the damage to the cell is severe • Often as a tumor suppressor • A potent transcription factor

30. To P or not to P:c-Myc & p53 p53 C-Myc p53 Inhibits p21/WAF1 Miz

31. Summary • p53, a tumor suppressor, exists as a polymorph & this may be responsible for cancer susceptibility across differing ethnicities. • At a molecular level, it switches cells from cytostasis to apoptosis. • p53 mediated apoptosis occurs via a number of molecular processes: transcriptional upregulation of Bax, hence a change in Bcl2/Bax ratio; of PUMA & Peg3/PW1 & an increase in translocation of Bax to Mt. • p53 cytostasis occurs through the p21/WAF1, Rb pathway.

32. Summary • p53 is being employed for ablation of tumors using gene therapy. • p53 interacts with a number of other proteins, such as JNKs, etc and is involved in a variety of pathways. • P53 offers a profound insight into the working of cancer cells & an opportunity for future therapeutic treatments.

33. Me! Running against time to finish the presentation!