The p16INK4a cyclin-dependent kinase inhibitor is implicated in replicative senescence, the state of permanent growth arrest provoked by cumulative cell divisions or as a response to constitutive Ras-Raf-MEK signalling in somatic cells. Some contribution to senescence presumably underlies the importance of p16INK4a as a tumour suppressor but the mechanisms regulating its expression in these different contexts remain unknown. Here we demonstrate a role for the Ets1 and Ets2 transcription factors based on their ability to activate the p16INK4a promoter through an ETS-binding site and their patterns of expression during the lifespan of human diploid fibroblasts. The induction of p16INK4a by Ets2, which is abundant in young human diploid fibroblasts, is potentiated by signalling through the Ras-Raf-MEK kinase cascade and inhibited by a direct interaction with the helix-loop-helix protein Id1 (ref. 11). In senescent cells, where the Ets2 levels and MEK signalling decline, the marked increase in p16INK4a expression is consistent with the reciprocal reduction of Id1 and accumulation of Ets1.

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Deregulation of D-type cyclin-dependent kinases (CDK4 and 6) is widely observed in various human cancers, illustrating their importance in cell cycle control. Like other cyclin-dependent kinases (CDKs), assembly with cyclins is the most critical step for activation of CDK4/ 6. As previously reported elsewhere, we observed that the level of cyclinD1-CDK4 complex and its associated kinase activity were significantly low in asynchronously proliferating mouse embryo fibroblasts lacking both p21 Cip1 and p27 Kip1 (p21/p27-null MEFs). These evidences imply that p21 Cip1 and p27 Kip1 CDK inhibitors are 'essential activators' of cyclin D-kinases. We, however, discovered here that both the assembly and activation of cyclin D1-CDK4 complex occur when quiescent p21/p27-null MEFs were stimulated to re-enter the cell cycle. This mitogen-induced cyclin D1-kinase activity was blocked by overexpression of p16 INK4a and resulted in the inhibition of S phase entry in p21/p27-null MEFs. Furthermore, ectopic expression of p34 SEI-1 , a mitogen-induced CDK4 binding protein, increased the levels of active cyclinD1-CDK4 complex in asynchronously proliferating p21/p27-null MEFs. Together, our results suggest that there are several independent ways to stimulate the assembly of cyclin D1-CDK4 kinases. Although p21 Cip1 and p27 Kip1 play a role in this process, our results demonstrate that additional mechanisms must occur in G0 to S phase transition.

We have screened for CDKN2A germline mutations in 49 Jewish families with two or more cases of melanoma. The Val59Gly mutation, one of the three different alterations identified among these families, was also detected independently in two kindreds from France and one from Spain. The impact of the Val59Gly substitution on the function of the cyclin-dependent kinase inhibitor p16INK4a , a product of the CDKN2A gene, was assessed by protein -protein interaction and cell proliferation assays and related to potential structural alterations predicted by molecular modeling. Seven microsatellite markers in the vicinity of the CDKN2A gene were used to determine whether the mutation in these families is identical by descent, or represents a mutational hotspot in the CDKN2A gene. Our results show that the Val59Gly substitution impairs p16INK4a function, and this dysfunction is consistent with structural predictions. All melanoma-affected individuals tested in the families under study harbor this mutation. Interestingly, the Israeli pedigree includes an affected individual who is homozygous for the Val59Gly mutation. A common haplotype of microsatellite markers has been demonstrated for mutation carriers in all four pedigrees. The Israeli pedigree and one of the French melanoma families are of Moroccan and Tunisian Jewish descent, respectively, and the other families originate from regions of France and Spain close to the Pyrenees. We conclude that the Val59Gly mutation is a major contributor to melanoma risk in the families under study and that it may derive from a single ancestral founder of Mediterranean (possibly Jewish) origin.

Summary Physical interaction between CDKN2A/p16 and CDK4 proteins regulates the cell cycle progression through the G1 phase and dysfunction of these proteins by gene mutation is implicated in genetic predisposition to melanoma. We analysed 15 Italian melanoma families for germ line mutations in the coding region of the CDKN2A gene and exon 2 of the CDK4 gene. One novel disease-associated mutation (P48T), 3 known pathological mutations (R24P, G101W and N71S) and 2 common polymorphisms (A148T and Nt500 G>C) were identified in the CDKN2A gene. In a family harbouring the R24P mutation, an intronic variant (IVS1, +37 G>C) of uncertain significance was detected in a noncarrier melanoma case. The overall incidence of CDKN2A mutations was 33.3%, but this percentage was higher in families with 3 or more melanoma cases (50%) than in those with only 2 affected relatives (25%). Noteworthy, functional analysis established that the novel mutated protein, while being impaired in cell growth and inhibition assays, retains some in vitro binding to CDK4/6. No variant in the p16-binding region of CDK4 was identified in our families. Our results, obtained in a heterogeneous group of families, support the view that inactivating mutations of CDKN2A contribute to melanoma susceptibility more than activating mutations of CDK4 and that other genetic factors must be responsible for melanoma clustering in a high proportion of families. In addition, they indicate the need for a combination of functional assays to determine the pathogenetic nature of new CDKN2A mutations.

The Id family (Idl-Id4) of helix-loop-helix proteins act as negative regulators of basic-helix-loop-helix (bHLH) transcription factors that drive cell lineage commitment and differentiation in diverse cell types. In general, tissue-specific bHLH proteins form heterodimers with the ubiquitously expressed bHLH proteins and activate the expression of specific genes carrying an E-box regulatory motif. Id proteins lack a basic region and act as dominant-negative antagonists of the bHLH transcription factors by forming heterodimers that are unable to bind DNA. We have previously shown that Id2 and Id3 undergo cell cycle dependent phosphorylation at a serine residue (Ser5) within a consensus target site for cyclin-dependent kinases (EMBO J. 1 6 332-342,1997). The timing of phosphorylation in the late G1 phase correlates with the activation of Cdk2 by cyclin E and/or cyclin A. We now show that Id4 can also be specifically phosphorylated by cyclin E-Cdk2 and cyclin A-Cdk2 in vitro. Analogous phosphorylation occurs in serum-stimulated F9 embryonal teratocarcinoma cells at a time in late G1 consistent with the appearance of active cyclin E-Cdk2. Phospho-mimicking (S5D) and phospho-ablating (S5A) mutants of Id4 have been constructed and are currently being evaluated in a model E-box band-shift assay and for functional properties in transfected cells. Our data suggest that modulation of Id function by Cdk-dependent phosphorylation could alter the patterns of bHLH-dependent gene expression during cell cycle progression. CLLThe transcription factors of the E2F (E2F-1 -E2F-6), cyklindependent-kinases (cdk) and cdk inhibitors play a key role in control of the cell cycle in eukaryotes. E2F proteins form heterodimers with the proteins, DP-I and DP-2. These complexes are regulated by the protein pRb, p107 and p130. E2F inhibitors are phosforylated during the cell cycle, releasing E2F/DP complexes. The phosphorylation is mediated by CyclinD/cdk4 or Cyklin D/cdk6 are mid G1 specific, Cyclin E/cdk2 late G1 specific and Cyklin A/cdk2 -S specific. The negative regulatory elements -p219(Cip), p27(Kip), p16(INK4A) block cdks activity. B-cell chronic lymphocytic leukaemia (B-CLL) is a disease with a low proliferative activity characterised by the progressive accumulation of clonal B lymphocytes in early phases (GO/Gl) of the cell cycle. Our goals were to study the mRNA expression of cell cycle proteins (E2F-1, E2F-4, DP1, DP2, cdks, p21, p27, p16), which primarily regulate the G1 phase of cell cycle, or Sphase entry, or both in B-CLL lymphocytes. We have examined mRNA expression in unstimulated and stimulated lymphocytes from 12 patients with B-CLL, used the ribonuclease protection assay. The leukaemic immunophenotype CD5/CD19 was demonstrate in more than 70% of lymphocytes in all studied cases. The constant mRNA expression E2F-4, E2F-1, DP2, cdk4 was shown in both unstimulated and stimulated lymphocytes. We did not find any mRNA expression p21, p27, p16 in patients whose stimulated PBL were in M phase of cell cycle.Objective We have previo...