CDK4 CYCLIN D1/PCNA COMPLEXES DURING STRAUROSPORINE-INDUCED G1 ARRESTAND G0 ARREST OF HUMAN FIBROBLASTS/

We have shown that staurosporine (STSP) arrests normal human diploid f ibroblasts in the G1 phase of the cell cycle at a time similar to 3 h after release from low-serum-induced G0 arrest. This initial temporal mapping of the STSP-induced restriction point was based on how cytomet ric analyses that measured the onset of DNA synthesis after release fr om STSP and low-serum treatment. Here we show that the STSP-mediated a rrest point distinctly differs from low-serum G0 arrest. We have found that cyclin D1 is expressed in STSP-arrested G1 fibroblasts but not i n low-serum-arrested G0 fibroblasts, whereas cyclin-dependent kinase 4 (cdk4) and proliferating cell nuclear antigen (PCNA) are equivalently expressed under conditions of both STSP treatment and serum deprivati on. Cdk4/cyclin D1/PCNA complexes are also formed in STSP-arrested G1 fibroblasts, but they are absent in serum-deprived G0 cells. The forma tion of cdk4/cyclin D1/PCNA complexes was found to coincide with the t ranscription and synthesis of cyclin D1, which indicates that the lack of available cyclin D1 is the limiting factor in cdk4/cyclin D1/PCNA complex formation in serum-deprived fibroblasts. This conclusion was f urther supported by the observation that cyclin D1-GST fusion protein binds cdk4 and PCNA when added to G0 cell extracts. Circumstantial evi dence obtained in our studies and by other investigators suggests that STSP-induced arrest may be due to the inhibition of cdk-activating ki nase. (C) 1995 Academic Press, Inc.