title: p21(Waf1/Cip1) inhibition of cyclin E/Cdk2 activity prevents endoreduplication after mitotic spindle disruption.
abstract: During a normal cell cycle, entry into S phase is dependent on completion of mitosis and subsequent activation of cyclin-dependent kinases (Cdks) in G1. These events are monitored by checkpoint pathways. Recent studies and data presented herein show that after treatment with microtubule inhibitors (MTIs), cells deficient in the Cdk inhibitor p21(Waf1/Cip1) enter S phase with a >/=4N DNA content, a process known as endoreduplication, which results in polyploidy. To determine how p21 prevents MTI-induced endoreduplication, the G1/S and G2/M checkpoint pathways were examined in two isogenic cell systems: HCT116 p21(+/+) and p21(-/-) cells and H1299 cells containing an inducible p21 expression vector (HIp21). Both HCT116 p21(-/-) cells and noninduced HIp21 cells endoreduplicated after MTI treatment. Analysis of G1-phase Cdk activities demonstrated that the induction of p21 inhibited endoreduplication through direct cyclin E/Cdk2 regulation. The kinetics of p21 inhibition of cyclin E/Cdk2 activity and binding to proliferating-cell nuclear antigen in HCT116 p21(+/+) cells paralleled the onset of endoreduplication in HCT116 p21(-/-) cells. In contrast, loss of p21 did not lead to deregulated cyclin D1-dependent kinase activities, nor did p21 directly regulate cyclin B1/Cdc2 activity. Furthermore, we show that MTI-induced endoreduplication in p53-deficient HIp21 cells was due to levels of p21 protein below a threshold required for negative regulation of cyclin E/Cdk2, since ectopic expression of p21 restored cyclin E/Cdk2 regulation and prevented endoreduplication. Based on these findings, we propose that p21 plays an integral role in the checkpoint pathways that restrain normal cells from entering S phase after aberrant mitotic exit due to defects in microtubule dynamics.
authors.affiliation: Departments of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Z A
authors.initials: ZA
authors.lastname: Stewart
authors.forename: S D
authors.initials: SD
authors.lastname: Leach
authors.forename: J A
authors.initials: JA
authors.lastname: Pietenpol
mesh_terms: D042846:CDC2-CDC28 Kinases; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D019927:Cyclin E; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000235:genetics / Q000502:physiology*; D004261:DNA Replication; D004791:Enzyme Inhibitors; D016193:G1 Phase; D006801:Humans; D016296:Mutagenesis; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D016196:S Phase; D008941:Spindle Apparatus; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000502:physiology
chemical_list: C534699:CCNB1 protein, human; C493956:CDKN1A protein, human; D019926:Cyclin B; D056744:Cyclin B1; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D018809:Proliferating Cell Nuclear Antigen; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495900:CDK4 protein, human; C495911:CDK6 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases