Medema R H, Klompmaker R, Smits V A, Rijksen G
Department of Haematology, University Hospital Utrecht, The Netherlands.
Oncogene. 1998 Jan 29;16(4):431-41. doi: 10.1038/sj.onc.1201558.
p21waf1 has been shown to mediate the p53-dependent growth arrest induced by DNA-damaging agents. Several functions have been ascribed to p21waf1 that could be involved in this growth arrest. For one, p21waf1 is an efficient inhibitor of cyclin-dependent kinases (CDKs). Also, p21waf1 can interact with proliferating cell nuclear antigen (PCNA), and as such inhibit in vitro DNA-replication. Finally, p21waf1 has been reported to inhibit stress-activated protein kinases (SAPKs). In order to study these multiple functions of p21waf1 we have established U2OS-derived cell lines, in which the expression of p21waf1 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 p21waf1-expression. Both [3H]thymidine-incorporation and CDK2-activity were strongly inhibited by p21waf1. Upon induction of p21waf1 cells accumulated with a 2N or 4N DNA content suggesting events in G1 and G2 can be inhibited by p21waf1. Indeed, kinase activity associated with cyclin B was reduced dramatically upon induction of p21waf1, although cyclin B continues to be expressed. In contrast, p21waf1 does not seem to inhibit the function of PCNA in ongoing DNA replication, since cells expressing high levels of p21waf1 apparently progressed normally through S-phase. Also, the activity of SAPKs was not substantially affected by the high levels of p21waf1. We conclude that, at least in these U2OS-derived cells, p21waf1 functions as an inhibitor of CDK-activity in G1 and G2, but not as an inhibitor of PCNA or SAPKs.
p21waf1已被证明可介导DNA损伤剂诱导的p53依赖性生长停滞。p21waf1具有多种功能，可能参与这种生长停滞。其一，p21waf1是细胞周期蛋白依赖性激酶（CDK）的有效抑制剂。此外，p21waf1可与增殖细胞核抗原（PCNA）相互作用，从而在体外抑制DNA复制。最后，据报道p21waf1可抑制应激激活蛋白激酶（SAPK）。为了研究p21waf1的这些多种功能，我们建立了源自U2OS的细胞系，其中p21waf1的表达可由培养基中四环素的浓度调节。我们观察到诱导p21waf1表达后，细胞生长几乎完全但可逆地受到抑制。p21waf1强烈抑制[3H]胸苷掺入和CDK2活性。诱导p21waf1后，细胞积累了2N或4N的DNA含量，这表明G1期和G2期的事件可被p21waf1抑制。实际上，诱导p21waf1后，与细胞周期蛋白B相关的激酶活性显著降低，尽管细胞周期蛋白B仍在继续表达。相比之下，p21waf1似乎并不抑制正在进行的DNA复制中PCNA的功能，因为表达高水平p21waf1的细胞显然在S期正常进展。此外，SAPK的活性并未受到高水平p21waf1的实质性影响。我们得出结论，至少在这些源自U2OS的细胞中，p21waf1在G1期和G2期作为CDK活性的抑制剂发挥作用，但不是PCNA或SAPK的抑制剂。
