Reduction of Cdc25A contributes to cyclin E1-Cdk2 inhibition at senescence in human mammary epithelial cells.
Replicative senescence may be an important tumor suppressive mechanism for human cells. We investigated the mechanism of cell cycle arrest at senescence in human mammary epithelial cells (HMECs) that have undergone a period of 'self-selection', and as a consequence exhibit diminished p16INK4A levels. As HMECs approached senescence, the proportion of cells with a 2N DNA content increased and that in S phase decreased progressively. Cyclin D1-cdk4, cyclin E-cdk2 and cyclin A-cdk2 activities were not abruptly inhibited, but rather diminished steadily with increasing population age. In contrast to observations in fibroblast, p21Cip1 was not increased at senescence in HMECs. There was no increase in p27Kip1 levels nor in KIP association with targets cdks. While p15INK4B and its binding to both cdk4 and cdk6 increased with increasing passage, some cyclin D1-bound cdk4 and cdk6 persisted in senescent cells, whose inhibition could not be attributed to p15INK4B. The inhibition of cyclin E-cdk2 in senescent HMECs was accompanied by increased inhibitory phosphorylation of cdk2, in association with a progressive loss of Cdc25A. Recombinant Cdc25A strongly reactivated cyclin E-cdk2 from senescent HMECs suggesting that reduction of Cdc25A contributes to cyclin E-cdk2 inhibition and G1 arrest at senescence. Although ectopic expression of Cdc25A failed to extend the lifespan of HMECs, the exogenous Cdc25A appeared to lack activity in these cells, since it neither shortened the G1-to-S phase interval nor activated cyclin E-cdk2. In contrast, in the breast cancer-derived MCF-7 line, Cdc25A overexpression increased both cyclin E-cdk2 activity and the S phase fraction. Thus, mechanisms leading to HMEC immortalization may involve not only the re-induction of Cdc25A expression, but also activation of this phosphatase.
Sandhu
C
C
Cancer Research, Sunnybrook and Women's College Health Sciences Centre, Toronto, Ontario, Canada.
Donovan
J
J
Bhattacharya
N
N
Stampfer
M
M
Worland
P
P
Slingerland
J
J
D000328:Adult; D000595:Amino Acid Sequence; D001940:Breast / Q000166:cytology; D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins; D016922:Cellular Senescence / Q000187:drug effects / Q000502:physiology; D019927:Cyclin E / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D051361:Cyclin-Dependent Kinase 6; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050763:Cyclin-Dependent Kinase Inhibitor p18; D050764:Cyclin-Dependent Kinase Inhibitor p19; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D004791:Enzyme Inhibitors; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects; D005260:Female; D005656:Fungal Proteins / Q000378:metabolism; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D020409:Molecular Motor Proteins; D008969:Molecular Sequence Data; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D029701:Saccharomyces cerevisiae Proteins; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D020687:cdc25 Phosphatases / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
C493956:CDKN1A protein, human; C493966:CDKN2B protein, human; C493971:CDKN2C protein, human; C493974:CDKN2D protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D019927:Cyclin E; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050763:Cyclin-Dependent Kinase Inhibitor p18; D050764:Cyclin-Dependent Kinase Inhibitor p19; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; C075341:KIP2 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; D020409:Molecular Motor Proteins; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495911:CDK6 protein, human; D051357:Cyclin-Dependent Kinase 2; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases; C517004:CDC25A protein, human; D020687:cdc25 Phosphatases