The two distinct proteins encoded by the CDKN2A locus are specified by translating the common second exon in alternative reading frames. The product of the α transcript, p16 INK4a , is a recognized tumour suppressor that induces a G 1 cell cycle arrest by inhibiting the phosphorylation of the retinoblastoma protein by the cyclin-dependent kinases, CDK4 and CDK6. In contrast, the product of the human CDKN2A β transcript, p14 ARF , activates a p53 response manifest in elevated levels of MDM2 and p21 CIP1 and cell cycle arrest in both G 1 and G 2 /M. As a consequence, p14 ARFinduced cell cycle arrest is p53 dependent and can be abrogated by the co-expression of human papilloma virus E6 protein. p14 ARF acts by binding directly to MDM2, resulting in the stabilization of both p53 and MDM2. Conversely, p53 negatively regulates p14 ARF expression and there is an inverse correlation between p14 ARF expression and p53 function in human tumour cell lines. However, p14 ARF expression is not involved in the response to DNA damage. These results place p14 ARF in an independent pathway upstream of p53 and imply that CDKN2A encodes two proteins that are involved in tumour suppression.

The CDKN2A tumour suppressor locus encodes two distinct proteins, p16 INK4a and p14 ARF , both of which have been implicated in replicative senescence, the state of permanent growth arrest provoked in somatic cells by aberrant proliferative signals or by cumulative population doublings in culture. Here we describe primary ®broblasts from a member of a melanomaprone family who is homozygous for an intragenic deletion in CDKN2A. Analyses of the resultant gene products imply that the cells are p16 INK4a de®cient but express physiologically relevant levels of a frameshift protein that retains the known functions of p14 ARF . Although they have a ®nite lifespan, the cells are resistant to arrest by oncogenic RAS. Indeed, ectopic expression of RAS and telomerase (hTERT) results in outgrowth of anchorage-independent colonies that have essentially diploid karyotypes and functional p53. We ®nd that in human ®broblasts, ARF is not induced demonstrably by RAS, pointing to signi®cant differences between the proliferative barriers implemented by the CDKN2A locus in different cell types or species.

After a limited number of population doublings (PDs), cultures of normal mammalian diploid cells undergo an irreversible growth arrest known as replicative senescence [1]. As well as contributing to cellular ageing, senescence is viewed as an important mechanism of tumour suppression by preventing the emergence of immortal cell clones [2-4]. Senescent cells have a number of characteristics that distinguish them from cycling or quiescent cells including elevated levels of two cyclin-dependent kinase (Cdk) inhibitors, p16INK4a and p21CIP1 [5-11]. Here, we demonstrate that both of these Cdk inhibitors, as well as other members of their protein families (the INK4 and CIP/KIP families, respectively [12]), induce several facets of the senescent phenotype when ectopically expressed in young human diploid fibroblasts. These include a reduced proliferative capacity, an altered size and shape, the presence of underphosphorylated retinoblastoma protein (pRb), increased expression of plasminogen activator inhibitor (PAI-1) and the appearance of senescence-associated beta-galactosidase (SA-beta-gal) activity [2,3,13-15]. A 20 amino acid peptide from p16INK4a that inhibits Cdks active in the G1 phase of the cell cycle [16] produces similar effects in a dose-dependent manner suggesting that, in primary fibroblasts, inhibition of G1-specific Cdk activity is sufficient to induce phenotypic changes that normally occur at the end of their finite lifespan.

No abstract