Early g1 induction of p21/waf1/cip1 in synchronized osteosarcoma cells is independent of p53

Enzymatic activities of cyclin-dependent protein kinases (cdks) are tightly regulated at the level of subunit composition, involving both positive (cyclins) and negative (p21Wafl/Cipl); p16Ink4; p27Kipl) effectors. In the present study, we examined the expression of p21/WAF1/CIP1 in highly synchronized human MG-63 osteosarcoma cells in which the sequential induction of specific cyclins was characterized previously (1). Two distinct peaks of p21/WAF1/CIP1 expression were detected by Northern analysis of serum-stimulated cells: one peak was detectable by 1 hour, reached a maximum at approximately 3 hours, and diminished markedly by 4-6 hours; and a second peak was observed during S phase. The early G1 induction of the 21 kDa gene product was further demonstrated by Western blotting. Both Northern analysis and Western blotting for the p53 tumor suppressor confirmed previous reports that its expression is not detectable in MG-63 cells at any time. The transient induction of p21/WAF1/CIP1 in early G1 was correlated with a transient decrease in p9Ckshs1-agarose precipitable histone H1 kinase activity, as determined by in vitro kinase assays. In contrast, the myelin basic protein kinase activity of anti-Cdk4 immune complexes was not attenuated to a significant extent. Taken together, these studies identify a novel biochemical pathway of p21/WAF1/CIP1 induction operating in p53-deficient osteosarcoma cells; a pathway whose independence from p53 may conceivably be exploited to therapeutic advantage in the treatment of proliferative disorders.