FUNCTIONAL AND PHYSICAL INTERACTIONS OF THE ARF TUMOR-SUPPRESSOR WITHP53 AND MDM2

The INK4a-ARF locus encodes two proteins, p16( INK4a) and p19(ARF), th at restrain cell growth by affecting the functions of the retinoblasto ma protein and p53, respectively. Disruption of this locus by deletion s or point mutations is a common event in human cancer, perhaps second only to the loss of p53, Using insect cells infected with baculovirus vectors and NIH 3T3 fibroblasts infected with ARF retrovirus, we dete rmined that mouse pl9(ARF) can interact directly with p53, as well as with the p53 regulator mdm2, ARF can bind p53 DNA complexes, and it de pends upon functional p53 to transcriptionally induce mdm2 and the cyc lin-dependent kinase inhibitor p21(Cip1), and to arrest cell prolifera tion, finding of p19(ARF) to p53 requires the ARF N-terminal domain (a mino acids 1-62) that is necessary and sufficient to induce cell cycle arrest. Overexpression of p19(ARF) in wild type or ARF-null mouse emb ryo fibroblasts increases the half-life of p53 from 15 to approximate to 75 min, correlating with an increased p53-dependent transcriptional response and growth arrest. Surprisingly, when overexpressed at supra -physiologic levels after introduction into ARF-null NIH 3T3 cells or mouse embryo fibro blasts, the p53 protein is handicapped in inducing this check point response. In this setting, reintroduction of p(19ARF) restores p53's ability to induce p21(Cip1) and mdm2, implying that, i n addition to stabilizing p53, ARF modulates p53-dependent function th rough an additional mechanism.