In order to elucidate the mechanisms by which estrogens and antiestrogens modulate the growth of breast cancer cells, we have characterized the changes induced by estradiol that occur during the G 1 phase of the cell cycle of MCF-7 human mammary carcinoma cells. Addition of estradiol relieves the cell cycle block created by tamoxifen treatment, leading to marked activation of cyclin E-cdk2 complexes and phosphorylation of the retinoblastoma protein within 6 h. Cyclin D1 levels increase significantly while the levels of cyclin E, cdk2, and the p21 and p27 cdk inhibitors are relatively constant. However, the p21 cdk inhibitor shifts from its association with cyclin E-cdk2 to cyclin D1-cdk4, providing an explanation for the observed activation of the cyclin E-cdk2 complexes. These results support the notion that cyclin D1 has an important role in steroiddependent cell proliferation and that estrogen, by regulating the activities of G 1 cyclin-dependent kinases, can control the proliferation of breast cancer cells.A variety of models have been proposed to explain how estrogen drives the proliferation of normal mammary epithelial cells and breast cancer cells (8). By acting through the estrogen receptor (ER), estrogen can regulate the transcription of a cohort of responsive genes and in this way appears to regulate cell cycle progression. Even in the face of still-incomplete mechanistic insight into how estrogen regulates growth, effective antitumor therapies directed against the ER have been developed around the use of antiestrogens such as tamoxifen (20). Many studies of the effects of tamoxifen have indicated that tamoxifen acts in a cytostatic fashion on breast cancer cells, causing them to arrest in the G 0 /G 1 phases of their growth cycle (36,49). For these reasons, it is important to understand how estrogens and antiestrogens control G 1 progression.The central regulator of this process is the cell cycle clock apparatus, which operates in the cell nucleus and is assembled from an array of cyclins and cyclin-dependent kinases (cdks) (45). The activities of the cdks are positively controlled by their association with cyclins and restrained by cdk inhibitors. Included among the latter are p21, p27, and p57, which can inhibit a wide range of cyclin-cdk complexes, and the INK4 family (p15, p16, p18, and p19), which specifically inhibits cdk4 and cdk6 (46). Extracellular signals such as those conveyed by growth factors affect the activity of cyclins and cdks largely during the G 1 phase of the cell cycle. The most important components of the cell cycle clock apparatus during this period are (i) the D-type cyclins together with their catalytic partners cdk4 and cdk6 and (ii) cyclin E, which interacts with cdk2. Both classes of G 1 cyclin-cdk complexes are known to drive the phosphorylation of the retinoblastoma protein (pRb) (18,19,21,42). This phosphorylation represents a key event in G 1 progression (53). Hypophosphorylated pRb is active in mediating G 1 arrest while hyperphosphorylated pRb appears to be inactive ...
