ARF and p16 : One Locus, Two Tumor Suppressors
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ARF and p16: One Locus, Two Tumor
Suppressors
Brandon Bunker
Biology 445
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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…
CyclinD
CDK4CDK4
p16
p16
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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
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• 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.
The p16-ARF locus
p16 reading frame: THE RED FOX HAD ONE HAT
ARF reading frame: HUH ARE DFO XHA DON EHA T
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ARF Knockouts
ARF -/- Mouse Epidermal Fibroblasts (MEFs): Bypass senescence Become oncogenic
with ras over-expression
Less responsive to contact inhibition
Kamijo, T; et al. Cell, 1997, 91, 649-659.
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ARF knockout mouse
Non-essential geneNormal 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.
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Cancer Types in Arf null mice
Kamijo, T; et al. Cell, 1997, 91, 649-659.
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Analysis of ARF mutants
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.
Kamijo, T; et al. Cell, 1997, 91, 649-659.
Donehower, L.A., et al. Nature, 1992, 356, 215-221.
• p53 -/- cells do not respond to ARF over-expression.
• Tumors null for both p53 and ARF are rare.
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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.
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How does ARF work?
• ARF interacts 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.
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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.
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How is ARF regulated?
• E2F protein/mRNA ↑ ARF ↑.
• Kinetics of ARF transcription is slow.
Bates, S. Nature, 1998, 395, 124-125.
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ARF Regulation: Normal
Sherr, C.; Lowe, S.W. Curr Opin, 2003, 13, 77-83.
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ARF Regulation: Stressed
Sherr, C.; Lowe, S.W. Curr Opin, 2003, 13, 77-83.
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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.
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ARF mutations in Human Cancer
Germline Mutations Melanoma predisposition
Cancers with ARF/p16 locus deletions:
Other Problems: Chemotherapy resistant cancers Highly aggressive tumors
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.
• Lung • Brain
• Pancreatic • Skin
• Kidney • Colon
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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.
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Questions?
Comments?
Concerns?