TGF-BETA MEDIATED G(1) ARREST IN A HUMAN-MELANOMA CELL-LINE LACKING P15(INK4B) - EVIDENCE FOR COOPERATION BETWEEN P21(CIP1 WAF1) AND P27/

We have studied TGF-beta mediated G(1) arrest in WM35, an early stage human melanoma cell line. These cells have lost p15(INK4B) expression through loss of one chromosome 9 and rearrangement of the other. In as ynchronously growing WM35, TGF-beta caused reductions in cyclin D1, cy clin A and cdk4 proteins and their associated kinase activities and an increase in both p21(Cip1/WAF1) and p27(Kip1). These findings were co nfirmed in cells released from quiescence in the presence of TGF-beta, in which TGF-beta inhibited or delayed the reduction in the cdk inhib itors that normally occurs in late G(1). In contrast to observations i n other cell types, there was an increased association of both p21(Cip 1/WAF1) and p27(Kip1) with cyclin D1/cdk4 and with cyclin E/cdk2 durin g TGF-beta mediated arrest of asynchronously growing cells. Upregulati on of p21(Cip1/WAF1) preceded that of p27(Kip1). Furthermore, p21(Cip1 /WAF1) and p27(Kip1) were not present in the same cdk complexes but bo und distinct populations of target cdk molecules. Both p21(Cip1/WAF1) and p27(Kip1) immunoprecipitates from asynchronously growing cells con tained active kinase complexes. These KIP-associated kinase activities were reduced in TGF-beta arrested cells. It has been proposed that in TGF-beta arrested epithelial cells, up-regulation of p15(INK4B) and o f p15(INK4B) binding to cdk4 serves to destabilize the association of p27(Kip1) with cyclin D1/cdk4, promoting p27(Kip1) binding and inhibit ion of cyclin E/cdk2. Our findings demonstrate that this is not a univ ersal mechanism of G(1) arrest by TGF-beta, In TGF-beta arrested WM35, which lack p15(INK4B), the increased p21(Cip1/WAF1) may serve a simil ar function to that of p15(INK4B): initiating kinase inhibition and pr oviding an additional mechanism to supplement the effect of p27(Kip1) on G(1) cyclin/cdks.