ESTROGEN-INDUCED ACTIVATION OF CDK4 AND CDK2 DURING G(1)-S-PHASE PROGRESSION IS ACCOMPANIED BY INCREASED CYCLIN D1 EXPRESSION AND DECREASEDCYCLIN-DEPENDENT KINASE INHIBITOR ASSOCIATION WITH CYCLIN-E-CDK2

Estrogens induce cell proliferation in target tissues by stimulating p rogression through G(1) phase of the cell cycle, but the underlying mo lecular targets remain undefined, To determine the role of the cyclin/ cyclin-dependent kinase (CDK)/retinoblastoma protein (pRB) pathway in this response we treated MCF-7 breast cancer cells with the pure estro gen antagonist ICI 182780 to inhibit estrogen-induced gene expression and induce G(1) phase arrest, Subsequent treatment with 17 beta-estrad iol resulted in the synchronous entry of cells into S phase commencing at 12 h, The proportion of cells in S phase reached a maximum of 60% at 21-24 h, Cells subsequently completed mitosis and entered a second semi-synchronous round of replication. Entry into S phase was preceded by increased activity of both Cdk4 and cyclin E-Cdk2 and hyperphospho rylation of pRB, all within the first 3-6 h of estradiol treatment, Th e increase in Cdk4 activity was accompanied by increases in cyclin D1 mRNA and protein, indicating that an initiating event in the activatio n of Cdk4 was increased cyclin D1 gene expression, In contrast, the le vels of Cdk2 and the CDK inhibitors p21 (WAF1/CIP1/SDI1) and p27 (KIP1 ) in total cell lysates and in cyclin E immunoprecipitates were unalte red at these early time points. However, an inhibitory activity was pr esent in antiestrogen-pretreated cell lysates toward recombinant cycli n E-Cdk2 and was relieved by estradiol treatment, This activity was at tributable predominantly to p21, These apparently conflicting data wer e resolved by performing gel filtration chromatography, which revealed that only a minority of cyclin E-Cdk2 complexes were active following estradiol treatment, Active complexes eluted at a higher molecular we ight than inactive complexes, were relatively deficient in both p21 an d p27, and contained Cdk2 with increased threonine 160 phosphorylation , consistent with a mechanism of activation of cyclin E-Cdk2 involving both reduced CDK inhibitor association and CDK-activating kinase-medi ated phosphorylation of Cdk2, These results provide an explanation for the early activation of both cyclin D1-Cdk4 and cyclin E-Cdk2 complex es that accompany G(1)-S phase progression in response to estradiol.