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ARF and p16 : One Locus, Two Tumor Suppressors

ARF and p16 : One Locus, Two Tumor Suppressors. Brandon Bunker Biology 445. CDK4. CDK4. CyclinD. p16. p16. Discovering ARF. ARF was discovered by a postdoc (Dawn Quelle) in the lab of Dr. Charles Sherr. Dawn was trying to clone the mouse p16 gene. Quick review…. ARF Discovery Cont.

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ARF and p16 : One Locus, Two Tumor Suppressors

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  1. ARF and p16: One Locus, Two Tumor Suppressors Brandon Bunker Biology 445

  2. CDK4 CDK4 CyclinD p16 p16 Discovering ARF • ARF was discovered by a postdoc (Dawn Quelle) in the lab of Dr. Charles Sherr. • Dawn was trying to clone the mouse p16 gene. • Quick review…

  3. ARF Discovery Cont. • Transfected mouse cDNA into cells. • Used known segment of p16 as probe. • Result: TWO genes! • One was p16. • One was unknown. • Overexpression of the unknown gene: • Cell cycle arrest: The new gene was TUMOR SUPPRESSOR! http://www.sciencewatch.com/march-april2001/sw_march-april2001_page3.htm Accessed on March 24, 2007

  4. The p16-ARF locus p16 reading frame: THE RED FOX HAD ONE HAT ARF reading frame: HUH ARE DFO XHA DON EHA T • ARF and p16 share NO sequence homology. • This economy of space is seen frequently in viruses- extremely rare in mammals. Sherr, C.J. Genes Dev.1998, 12, 2984-2991.

  5. ARF -/- Mouse Epidermal Fibroblasts (MEFs): Bypass senescence Become oncogenic with ras over-expression Less responsive to contact inhibition ARF Knockouts Kamijo, T; et al. Cell, 1997, 91, 649-659.

  6. ARF knockout mouse • Non-essential gene • Normal development, but… • Blindness • Spontaneous tumors (33%) • Rapidly inducible cancers • Mice died of cancer in 10-20 weeks. Kamijo, T; et al. Cell, 1997, 91, 649-659. McKeller, R.N.; et al. PNAS, 2002, 99(6), 3848-3853.

  7. Cancer Types in Arf null mice Kamijo, T; et al. Cell, 1997, 91, 649-659.

  8. Cancers in ARF -/- mice Sporadic lymphomas Fibrosarcomas Death occurred in 10-20 wks. Cancers in p53 -/- mice Sporadic lymphomas Osteosarcomas Death occurred in 10-20 wks. Analysis of ARF mutants • p53 -/- cells do not respond to ARF over-expression. • Tumors null for both p53 and ARF are rare. Donehower, L.A., et al. Nature, 1992, 356, 215-221. Kamijo, T; et al. Cell, 1997, 91, 649-659.

  9. Role and Importance of p53 • Tumor suppressor • Activated by: • DNA damage • Deregulated oncogene expression • Initiates cell cycle arrest • Gives cell time to repair its DNA • Initiates apoptotic response • Mutated in ~50% of all cancers Donehower, L.A., et al. Nature, 1992, 356, 215-221.

  10. How does ARF work? • ARFinteracts with Mdm2. • Remember Mdm2?? • E3 ubiquitin ligase that targets p53 for destruction. • ARF prevents Mdm2 shuttling ubiquitinated p53 into the cytoplasm. Pomerantz, J. et al. Cell, 1998, 92, 713-723. Llanos, S.; et al, Nat. Cell Bio,2001, 3, 445-451. Tao, W.; Levine, A.J. PNAS, 1999, 96, 6937-6941.

  11. ARF Pathway and the Players • ARF- Negatively regulates Mdm2 • Mdm2- E3 Ubiquitin Ligase of p53 • p53- Triggers cell cycle arrest or apoptosis Growth arrest/ apoptosis Eischen, C.M. Genes Dev. 1999, 13, 2658-2669.

  12. How is ARF regulated? • E2F protein/mRNA ↑  ARF ↑. • Kinetics of ARF transcription is slow. Bates, S. Nature, 1998, 395, 124-125.

  13. ARF Regulation: Normal Sherr, C.; Lowe, S.W. Curr Opin, 2003, 13, 77-83.

  14. ARF Regulation: Stressed Sherr, C.; Lowe, S.W. Curr Opin, 2003, 13, 77-83.

  15. What this means for us • The p16/ARF locus is mutated in ~40% of all cancers. • Mutation Types: • Point mutations- affects p16. • Deletions- affects both. • Hypermethylation- affects ARF, p16, or both. • ARF-only knockout mutations are rare. Kim, W.Y; Sharpless, N. E. Cell,2006, 127(2), 265-275.

  16. ARF mutations in Human Cancer • Germline Mutations • Melanoma predisposition • Cancers with ARF/p16 locus deletions: • Other Problems: • Chemotherapy resistant cancers • Highly aggressive tumors • Lung • Brain • Pancreatic • Skin • Kidney • Colon Laud, K.; et al, J. Med. Genet.2006, 43(6), 39-47. Kasahara, T. Anticancer Res. 2006, 6, 4299-4305. Kim, W.Y; Sharpless, N. E. Cell,2006, 127(2), 265-275.

  17. Summary • ARF and p16 are tumor suppressors that use different reading frames of the same locus. • ARF regulates p53 by inhibiting Mdm2. • ARF/p16 locus is deleted or mutated in many different cancer types.

  18. Questions? Comments? Concerns?

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