. 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 .Estrogenic steroids, including 17--estradiol (E 2 ), regulate cellular function in a wide variety of tissues and influence proliferation in the female reproductive tract and mammary gland (31). A role for estrogens in breast cancer etiology is well established and clearly relates to their growth-stimulatory action (35). Estrogens elicit proliferative responses in breast cancer cells in vivo (85) and in vitro (43) and are essential for initiation and progression of breast cancer in animal models (35). Studies of estrogen receptor (ER)-positive breast cancer cell lines indicate that estrogens (41) and antiestrogens (86) act on sensitive populations of cells in early to mid-G 1 phase.G 1 /S transition is under the control of cyclin-dependent kinases (Cdks) activated by specific complex formation with regulatory cyclins. Cdk4 and Cdk6 are activated by binding to D-type cyclins and act early in G 1 phase, while Cdk2 kinase functions in conjunction with cyclins E and A and is necessary for progression through late G 1 and entry into S phase (81,83,92,98). A primary target of Cdk action in G 1 phase is the retinoblastoma susceptibility gene product (pRb), which mediates G 1 arrest through sequestration of transcriptional factors of the E2F-DP family. Phosphorylation of pRb and other members of the pocket protein family (p107 and p130) by active cyclin-Cdk complexes leads to release of E2F and DP transcription factors and transcription of requisite genes for S-phase entry (98). Recently a parallel, Cdk2-driven pathway promoting the G 1 /S transition independent of D cyclin-Cdk4 activation, pRb phosphorylation, and E2F release has been described in model systems utilizing cooperative Ras-Myc activation (40), and overexpression of cyclin E (45, 74).Cdk activation depends upon removal of inhibitory Thr/Tyr phosphorylation by members of the Cdc25 phosphatase family (17,21,25,77). Cdc25 phosphatases are candidate oncogenes and are overexpressed in a wide variety of tumors, including roughly 30% of breast carcinomas (20). Cdc25A expression is required for S-phase entry (17,27,33) and is induced in G 1 (3,27,33) by Myc (18,74) and E2F (7,19,30,93). Cdc25A is active from mid-G 1 through S phase and participates in activation of Cdk2 (3,27,33). Overexpression of Cdc25A is sufficient for transformation of Rb Ϫ/Ϫ fibroblasts and cooperates with Ras in causing tumors in mice (20).
