title: p21waf1 can block cells at two points in the cell cycle, but does not interfere with processive DNA-replication or stress-activated kinases.
abstract: 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.
authors.affiliation: Department of Haematology, University Hospital Utrecht, The Netherlands.
authors.forename: R H
authors.initials: RH
authors.lastname: Medema
authors.forename: R
authors.initials: R
authors.lastname: Klompmaker
authors.forename: V A
authors.initials: VA
authors.lastname: Smits
authors.forename: G
authors.initials: G
authors.lastname: Rijksen
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D042846:CDC2-CDC28 Kinases; D002166:Camptothecin / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D019926:Cyclin B / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000502:physiology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000032:analysis / Q000378:metabolism; D016213:Cyclins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004249:DNA Damage; D004261:DNA Replication; D004273:DNA, Neoplasm / Q000187:drug effects / Q000528:radiation effects; D005434:Flow Cytometry; D016193:G1 Phase / Q000235:genetics / Q000502:physiology*; D016195:G2 Phase / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008938:Mitosis / Q000235:genetics / Q000502:physiology; D011003:Ploidies; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D016196:S Phase / Q000502:physiology; D013752:Tetracycline / Q000494:pharmacology; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects
chemical_list: D000972:Antineoplastic Agents, Phytogenic; C493956:CDKN1A protein, human; D019926:Cyclin B; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004273:DNA, Neoplasm; D018809:Proliferating Cell Nuclear Antigen; D011500:Protein Synthesis Inhibitors; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D013752:Tetracycline; D002166:Camptothecin