Ink4a-Arf 9q21

1. Ink4a-Arf9q21 Samuel A. Hayes Biology 169: Cancer 03/28/06

2. INK4a/Arf locus yields two transcripts derived from alternative first exons, E1 E1 through alternative promoters…. Both splice into a common Exon 2 and 3. • Transcripts  +  have distinct translation start sites, producing alternative reading frames and therefore different ORFs. • Do not share amino acid homology • That is great! But does this ARF code a protein? • Many presumed  transcripts would not. 7,9,12

3. Quelle et al surveyed some mouse tissues and cell lines. • Northern Blotting detected both  +  mRNAs through 1 + 1 exon probes. (not shown) • This was supported by RT-PCR using the Exon 1 and 1 primers. Lead to specific amplification of  transcripts and hybridization of the products with exon 1 and exon 2 probes. • Amplified  transcripts did not generate a signal when hybridized with exon 1 probe, and vice versa. • Also tested expression in cell lines- absent only in NIH3T3 fibroblasts and BAC1.2F5 macrophages- both of which include INK4a locus deletions. (not shown) 7

4.  and  cDNAs were transcribed and translated, then blotted. • Use of C-terminal amino acid specific antiserum to  and  mRNA produced specific precipitation with no cross-reaction. 7

5. NIH3T3 cells injected with viruses encoding: p19 {N-terminus tagging also with hemagglutinin (HA)} • Cells cytospined + immunofluorescence through antibodies to p19 C-terminus or the N-terminal HA epitope. • Revealed Nucleus Localization! 7

6. Locus • Ink4a-Arf locus encodes two proteins • p16Ink4a: acts as tumor suppressor | inhibiting cdk4/6 and Rb phosphorylation • p19ARF (humans: p14ARF): ? • Proteins are encoded in part by the same nucleotide sequences • Read in alternative reading frames during translation. 1,7,12

7. p16INK4a 11

8. Does unitary inheritance of p16INK4a and p19ARF hint towards p19ARF functionality?

9. p19ARFInduce cell arrest p19ARF induces cell cycle arrest in NIH3T3 cells @ G1/S and G2/M- functionality independent from p16INK4a But how? Structure very different from cdk-inhibitors; lysis of MEL cells: CDC2, CDK2, CDK4-6, Cyclins- D, E and A immunoprecipitates blotted with anti-p19ARF - no coprecipitation 7

10. p19ARF Role 1

11. INK4a/ARF locus may be sensitive/negatively regulated to extracellular signals- growth factors 6

12. Mice Knockouts • Arf/Mdm2/p53 knockouts- produce broader spectrum of tumors than those with justloss of p53 or Arf. • Triple knockouts- rapid and spontaneously independent sites: neural, mesenchymal, epithelial… • ARF-null mice- blind soon after birth • ARF- expressed in the vitreous of the eye, responsible for vascular regression | w/o- blood vessels over proliferate and destroy retina and lens 1

13. Role in Cancer • INK4a/ARF common target for deletion and point mutation in human cancers, possibly 2nd only to p53. • Both familial and sporadic • Mutations in Exon 2 frequent for INK4a- displace ARF from Nucleus • Homozygous deletions, micro-deletions, insertions and single nucleotide substitutions • Reduce Arf ability to block p53 export to cytoplasm and stabilization. • Deletion in exon 1 resulted in mutated ARF unable to arrest the cell cycle. 2

14. Burkitt-typeLymphoma • Myc transgene under immunoglobulin heavy chain enhancer-promoter. • Early- increased number of B-cells in lymph nodes sent to apoptosis… response eventually fails • 1/2 of lymphomas exhibit mutations in ARF or p53 • Lymphoma with w+ p53 cured by cyclophosphamide (DNA-damage agent) • Lymphomas lacking ARF, responsive • Lymphomas lacking INK4a, resistant • ARF contributes to acceleration of disease, however, INK4a results in poor response to drug treatment. 1,5

15. Burkitt-typeLymphoma a: w+b: Rb+/-c:Ink4a-Arf+/-d: p53+/-e: Ink4a-Arf+/-/p53+/- 5

16. Unitary inheritance purposeful? • Ink4a-Arf locus encodes two proteins • p16Ink4a: acts as tumor suppressor | inhibiting cdk4/6 and Rb phosphorylation • p19ARF (humans: p14ARF): tumor suppressor | acts on Mdm2 | upstream from p53 • Proteins are encoded in part by the same nucleotide sequences • Read in alternative reading frames during translation. • Dual requirement for cell-cycle control! 1,7,12

17. Bibliography 1. Lowe, Scott W and Charles J Sherr. Tumor suppression by InK4a-Arf: progress and puzzles. Current Opinion in Genetics & Development 2003, 13:77-83. 2. Zhang, Yanping and Yue xiong. Mutations in Human Arf Exon 2 Disrupt Its Nuclear Localization and Impair Its Ability to block Nuclear Export of MDM2 and p53. Molecular Cell 1999,Vol. 3, 579-591. 3. Sherr, Charles J and Jason D Weber. The ARF/p53 pathway. Current Opinion in Genetics and Development 2000, 10: 94-9. 4. Sherr, Charles J. Tumor Surveillance via the ARF-p53 pathway. Genes & Development 1998,12:2984-2991. 5. Schmitt, Clemens A. et al. INK4a/ARF mutations accelerate lymphomagenesis and promote chemoresistance by disturbing p53. Genes & Dev. 1999, 13:2670-7. 6. Zindy, Frederique et al. Myc signaling via the ARF tumor suppressor regulates p53-dependant apoptosis and immortalization. Genes & Dev. 1998, 12:2424-33. 7. Quelle, Dawn E. et al. Alternative Reading Frames of the INK4a Tumor Suppressor Gene Encode Two Unrelated Proteins capable of Inducing Cell Cycle Arrest. Cell 1995, Vol. 83:993-1000. 8. Lodish, et al. Molecular Cell Biology, 5th Edition. New York, W.H. Freeman and Company, 2004. 9. Landry, Josette-Renee et al. Complex Controls: the role of alternative promoters in mammalian genomes. Trends in Genetics 2003, Vol. 19: 11. 10. Zhang, Yanping et al. ARF Promotes MDM2 Degradation and Stabilizes p53:Arf-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell 1998, Vol. 92, 725-34. 11. Duronio, Bob and Mark Peifer. Class Notes-E2F PowerPoint. 02/23/06. 12. Serrano, Manuel. The Ink4a/Arf locus in murine tumorigenesis. Carcinogensis 2000, Vol 21 no.5, 865-869.