p21waf1 can block cells at two points in the cell cycle, but does not interfere with processive DNA-replication or stress-activated kinases

p21(waf1) has been shown to mediate the p53-dependent growth arrest induced by DNA-damaging agents, Several functions have been ascribed to p21(waf1) that could be involved in this growth arrest, For one, p21(waf1) is an efficient inhibitor of cyclin-dependent kinases (CDKs). Also, p21(waf1) can interact with proliferating cell nuclear antigen (PCNA), and as such inhibit in vitro DNA-replication. Finally, p21(waf1) has been reported to inhibit stress-activated protein kinases (SAPKs). In order to study these multiple functions of p21(waf1) we have established U2OS-derived cell lines, in which the expression of p21(waf1) can be regulated by the concentration of tetracycline in the culture medium, We observed a virtually complete, but reversible inhibition of cell growth upon induction of p21(waf1)-expression. Both [H-3]thymidine-incorporation and CDK2-activity were strongly inhibited by p21(waf1). Upon induction of p21(waf1) cells accumulated with a 2N or 4N DNA content suggesting events in G1 and G2 can be inhibited by p21(waf1). Indeed, kinase activity associated with cyclin B was reduced dramatically upon induction of p21(waf1), although cyclin B continues to be expressed, In contrast, p21(waf1) does not seem to inhibit the function of PCNA in ongoing DNA replication, since cells expressing high levels of p21(waf1) apparently progressed normally through S-phase. Also, the activity of SAPKs was not substantially affected by the high levels of p21(waf1). We conclude that, at least in these U2OS-derived cells, p21(waf1) functions as an inhibitor of CDK-activity in G1 and G2, but not as an inhibitor of PCNA or SAPKs.