p21^waf1 can block cells at two points in the cell cycle, but does not interfere with processive DNA-replication or stress-activated kinases

Abstract

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 [³H]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.