Multifaceted regulation of cell cycle progression by estrogen: regulation of Cdk inhibitors and Cdc25A independent of cyclin D1-Cdk4 function.
Estrogens induce proliferation of estrogen receptor (ER)-positive MCF-7 breast cancer cells by stimulating G(1)/S transition associated with increased cyclin D1 expression, activation of cyclin-dependent kinases (Cdks), and phosphorylation of the retinoblastoma protein (pRb). We have utilized blockade of cyclin D1-Cdk4 complex formation through adenovirus-mediated expression of p16(INK4a) to demonstrate that estrogen regulates Cdk inhibitor expression and expression of the Cdk-activating phosphatase Cdc25A independent of cyclin D1-Cdk4 function and cell cycle progression. Expression of p16(INK4a) inhibited G(1)/S transition induced in MCF-7 cells by 17-beta-estradiol (E(2)) with associated inhibition of both Cdk4- and Cdk2-associated kinase activities. Inhibition of Cdk2 activity was associated with delayed removal of Cdk-inhibitory activity in early G(1) and decreased cyclin A expression. Cdk-inhibitory activity and expression of both p21(Cip1) and p27(Kip1) was decreased, however, in both control and p16(INK4a)-expressing cells 20 h after estrogen treatment. Expression of Cdc25A mRNA and protein was induced by E(2) in control and p16(INK4a)-expressing MCF-7 cells; however, functional activity of Cdc25A was inhibited in cells expressing p16(INK4a). Inhibition of Cdc25A activity in p16(INK4a)-expressing cells was associated with depressed Cdk2 activity and was reversed in vivo and in vitro by active Cdk2. Transfection of MCF-7 cells with a dominant-negative Cdk2 construct inhibited the E(2)-dependent activation of ectopic Cdc25A. Supporting a role for Cdc25A in estrogen action, antisense CDC25A oligonucleotides inhibited estrogen-induced Cdk2 activation and DNA synthesis. In addition, inactive cyclin E-Cdk2 complexes from p16(INK4a)-expressing, estrogen-treated cells were activated in vitro by treatment with recombinant Cdc25A and in vivo in cells overexpressing Cdc25A. The results demonstrate that functional association of cyclin D1-Cdk4 complexes is required for Cdk2 activation in MCF-7 cells and that Cdk2 activity is, in turn, required for the in vivo activation of Cdc25A. These studies establish Cdc25A as a growth-promoting target of estrogen action and further indicate that estrogens independently regulate multiple components of the cell cycle machinery, including expression of p21(Cip1) and p27(Kip1).
Foster
J S
JS
Department of Obstetrics and Gynecology, Graduate School of Medicine, University of Tennessee Medical Center, Knoxville, Tennessee 37920, USA.
Henley
D C
DC
Bukovsky
A
A
Seth
P
P
Wimalasena
J
J
D000256:Adenoviridae / Q000235:genetics; D001483:Base Sequence; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins; D019938:Cyclin D1 / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016213:Cyclins / Q000235:genetics; D004967:Estrogens / Q000378:metabolism*; D005260:Female; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008954:Models, Biological; D009376:Neoplasms, Hormone-Dependent / Q000378:metabolism / Q000473:pathology; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D011960:Receptors, Estrogen / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D020687:cdc25 Phosphatases / Q000235:genetics / Q000378:metabolism*
C493956:CDKN1A protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004967:Estrogens; D008869:Microtubule-Associated Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D011518:Proto-Oncogene Proteins; D011960:Receptors, Estrogen; D016160:Retinoblastoma Protein; D025521:Tumor Suppressor Proteins; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495900:CDK4 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; C517004:CDC25A protein, human; D020687:cdc25 Phosphatases