CDK4/cyclin D1/PCNA complexes during staurosporine-induced G1 arrest and G0 arrest of human fibroblasts.
We have shown that staurosporine (STSP) arrests normal human diploid fibroblasts in the G1 phase of the cell cycle at a time approximately 3 h after release from low-serum-induced G0 arrest. This initial temporal mapping of the STSP-induced restriction point was based on flow cytometric analyses that measured the onset of DNA synthesis after release from STSP and low-serum treatment. Here we show that the STSP-mediated arrest point distinctly differs from low-serum G0 arrest. We have found that cyclin D1 is expressed in STSP-arrested G1 fibroblasts but not in 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 deprivation. Cdk4/cyclin D1/PCNA complexes are also formed in STSP-arrested G1 fibroblasts, but they are absent in serum-deprived G0 cells. The formation of cdk4/cyclin D1/PCNA complexes was found to coincide with the transcription 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 further supported by the observation that cyclin D1-GST fusion protein binds cdk4 and PCNA when added to G0 cell extracts. Circumstantial evidence obtained in our studies and by other investigators suggests that STSP-induced arrest may be due to the inhibition of cdk-activating kinase.
Gadbois
D M
DM
Life Sciences Division, Los Alamos National Laboratory, New Mexico 87545, USA.
Peterson
S
S
Bradbury
E M
EM
Lehnert
B E
BE
D000470:Alkaloids / Q000494:pharmacology*; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D019938:Cyclin D1; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000096:biosynthesis / Q000378:metabolism*; D004171:Diploidy; D005347:Fibroblasts / Q000166:cytology; D016193:G1 Phase / Q000502:physiology; D006801:Humans; D007399:Interphase / Q000502:physiology*; D015513:Oncogene Proteins / Q000096:biosynthesis / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D016192:Resting Phase, Cell Cycle / Q000502:physiology; D012867:Skin / Q000166:cytology; D019311:Staurosporine
D000470:Alkaloids; D016895:Culture Media, Serum-Free; D016213:Cyclins; D015513:Oncogene Proteins; D018809:Proliferating Cell Nuclear Antigen; D011518:Proto-Oncogene Proteins; D019938:Cyclin D1; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D019311:Staurosporine