The regulation of the D-type cyclin-dependent kinase (CDK4 and CDK6) activity appears to be the key step in the progression of eukaryotic cells through the G 1 cell cycle phase. One of the mechanisms involved in this process is the binding of some small proteic inhibitors, with a molecular mass ranging between 14 and 20 kDa, to these CDKs. We have evaluated the amount of two such inhibitors, namely p16INK4 and p18, in normal and transformed cells, as well as the biochemical features of the macromolecular complexes containing these proteins. The results obtained indicated that (i) p18 gene expression, unlike p16 INK4 gene, is not regulated by pRb status, (ii) no evident relationship exists between the expression of p16 INK4 and p18 genes, (iii) significant amounts of the two proteins are not bound to CDKs but occur as free molecules, (iv) each inhibitor forms a complex with the CDK protein with a 1:1 stoichiometry, and (v) a competition exists between cyclin D and the inhibitor protein toward the CDK protein resulting in the absence of detectable cellular free kinase. Moreover, employing the human native partially purified p16 INK4or the pure recombinant protein, we have been able to demonstrate in vitro the dissociation of CDK4-cyclin D1 complex and the formation of CDK4-p16 INK4 bimolecular complex. Our findings suggest that during the cell division cycle the members of the p16 INK4 protein family and cyclin Ds compete for binding to CDK4/CDK6 and that their quantitative ratio is essential for G 1 3 S transition.In eukaryotes the progression through the cell cycle is due to a biochemical cycle in which distinct cyclin-dependent serinethreonine kinases (CDKs) 1 are sequentially activated by different cyclins (1, 2). Then activated CDKs regulate their target molecules by phosphorylation. Finally, these downstream molecules carry out the steps that ultimately allow the ordered development of the cell division cycle (3-7). Thus, the regulation of CDK activity is the key event in the cell cycle progression.The level of the various CDK proteins generally does not vary remarkably during the cycle, whereas the amount of each cyclin undergoes dramatic changes. During the early G 1 phase, in particular, there occurs the accumulation of three D-type cyclins (D1, D2, and D3) (8 -11), which assemble into holoenzymes with either CDK4 (5,7,12) or, in a minor number of cell types, CDK6 (13,14). Interaction between cyclin Ds and these CDKs is not sufficient to activate the kinase activity of the holoenzyme, since functioning CDKs also require phosphorylation at a single threonyl residue (15). Additional proteins are normal constituents of complexes formed between D-type cyclins and CDK4 (or CDK6), including the proliferating cell nuclear antigen and a small protein named p21. Proliferating cell nuclear antigen, the processivity factor required by eukaryotic DNA polymerases ␦ and ⑀ (16 -21), is involved in the control of the rate of chromosome replication as well as in the repair of damaged DNA (22,23). p21 is a negative regul...

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The status of the CDKN2A gene family, including CDKN2A, CDKN2B, and CDKN2C, was investigated in 24 cases of neuroblastoma. These genes were selected on the basis of 1) high incidence of their inactivation in several human cancers and 2) their localization on chromosomal regions (9p and 1p) frequently rearranged in neuroblastomas. Detailed molecular analyses indicated the absence of homozygous deletions and point mutations involving these genes in all investigated tumor samples. However, when loss of heterozygostity for chromosome 9p21 (the region where CDKN2A and CDKN2B are localized) was investigated, 16% of cases showed abnormalities in an area telomeric to the CDKN2A locus. To study transcriptional silencing of the CDKN2A gene, the methylation status of exon 1 was examined. In about 35% of cases, a partial methylation was evidenced. Analysis of the CDKN2A mRNA expression, however, did not show any relationship between methylation status and gene transcription. Finally, expression of the CDKN2B gene was demonstrated in all stage IV neuroblastomas, whereas none of stage I tumors expressed this gene. This finding suggests the occurrence of a correlation between CDKN2B transcription and tumor phenotype.

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