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

2. 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
CDK4
p16
P16(INK4a) binds Cdk4 to inhibit the formation of CyclinD/Cdk4.
Dawn was an expert on tumor suppressors.

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!
Accessed on March 24, 2007

4. The p16-ARF locus ARF and p16 share NO sequence homology.
p16 reading frame: THE RED FOX HAD ONE HAT
ARF reading frame: HUH ARE DFO XHA DON EHA T
EXPLAIN CLEARLY!!
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,

5. 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,

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

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

8. Analysis of ARF mutants
Cancers in ARF -/- mice
Sporadic lymphomas
Fibrosarcomas
Death occurred in wks.
Cancers in p53 -/- mice
Sporadic lymphomas
Osteosarcomas
Death occurred in wks.
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,
Kamijo, T; et al. Cell, 1997, 91,

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,

10. 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,
Llanos, S.; et al, Nat. Cell Bio, 2001, 3,
Tao, W.; Levine, A.J. PNAS, 1999, 96,

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,

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

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

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

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),

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),
Kasahara, T. Anticancer Res. 2006, 6,
Kim, W.Y; Sharpless, N. E. Cell, 2006, 127(2),

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?