Ink4a-Arf9q21

Samuel A. HayesBiology 169: Cancer

03/28/06

• 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.

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• 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)

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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.

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• 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!

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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.

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p16INK4a

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Does unitary inheritance of p16INK4a and p19ARF hint

towards p19ARF functionality?

p19ARF Induce 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

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p19ARF Role

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• INK4a/ARF locus may be sensitive/negatively regulated to extracellular signals- growth factors

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Mice Knockouts•Arf/Mdm2/p53 knockouts- produce broader spectrum of tumors than those with just loss 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

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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.

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Burkitt-type Lymphoma• 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.

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Burkitt-type Lymphoma

a: w+

b: Rb+/-

c:Ink4a-Arf+/-

d: p53+/- e: Ink4a-Arf+/-/p53+/-

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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!

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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.