REGULATION OF P21(SDI1 CIP1/WAF1/MDA-6) AND EXPRESSION OF OTHER CYCLIN-DEPENDENT KINASE INHIBITORS IN SENESCENT HUMAN-CELLS/

Normal human diploid fibroblasts serially subcultured in vitro undergo senescence, a process whereby proliferation ceases after the cells ha ve undergone a limited number of population doublings. Many hypotheses have been proposed to account for this phenomenon, but the underlying cause(s) of cessation of growth is not known. We have found that mRNA and protein levels of the cyclin-dependent kinase inhibitors p21(Sdi1 ) and p16 are significantly higher in senescent than in actively proli ferating cells. We have also investigated the regulation of p21(Sdi1) in senescent cells and demonstrated that, unlike proliferation compete nt cells, senescent cells are unable to downregulate p21(Sdi1) protein in response to mitogen, and that the protein is always in the nucleus of these cells. p21(Sdi1) was associated with Cdk2, Cdk4, and Cdk6 co mplexes, with the largest amount present in Cdk2 complexes, suggesting that Cdk2 is an important target for the inhibitory action of p21 in senescence. Cdk4 complexes included a 16-kDa protein, and because p16 expression is upregulated in senescent cells it is a good candidate in hibitor of this complex. The expression of p18, p19, and p27 was the s ame in young and senescent cells, and p57 and p15 were not expressed. The results suggest that increased expression of both p21(Sdi1) and p1 6 in senescent cells and the inability to regulate these genes contrib utes to a block in G(1) and lack of entry into the cell cycle.