Cyclin D1 plays an important role in the development of breast cancer and is required for normal breast cell proliferation and differentiation associated with pregnancy. We show that ectopic expression of cyclin D1 can stimulate the transcriptional activity of the estrogen receptor in the absence of estradiol and that this activity can be inhibited by 4-hydroxytamoxifen and ICI 182,780. Cyclin D1 can form a specific complex with the estrogen receptor. Stimulation of the estrogen receptor by cyclin D1 is independent of cyclin-dependent kinase 4 activation. Cyclin D1 may manifest its oncogenic potential in breast cancer in part through binding to the estrogen receptor and activation of the transcriptional activity of the receptor.The three D cyclins are differentially expressed in a cell lineage-specific manner as part of a delayed early response to mitogens. D-type cyclins are rate limiting and essential for progression through the G 1 phase of the cell cycle (48, 49). One of the known biochemical functions of D cyclins is to bind to and activate cyclin-dependent kinase 4 (cdk4) and cdk6. In addition, cyclins D1, D2, and D3 can bind to the retinoblastoma protein Rb, and related proteins, in the absence of kinase in vitro. This binding is thought to direct cdk4 and cdk6 to Rb, allowing for efficient phosphorylation of the substrate. In support of the notion that Rb is a critical downstream target of D cyclins, cells lacking functional Rb do not require cyclin Ddependent kinases for passage from G 1 into S phase (50). Emerging evidence suggests that D-type cyclins are not redundant. The three D cyclins have different affinities for Rb (15,17,31). Ectopic expression of cyclins D2 and D3, but not cyclin D1, can inhibit granulocyte differentiation (32). Cyclin D1-and D2-deficient mice show different, specific developmental defects (19, 51, 52). Cyclin D1, and not cyclins D2 and D3, is overexpressed in a high percentage of certain tumors (24).Cyclin D1 is amplified or overexpressed in a high percentage (Ͼ50%) of human breast adenocarcinomas (3,8,12,21,41,57) and is oncogenic in vivo, in breast epithelial cells, and in vitro (26,38,56). While cyclin D1 is not essential for the development of most murine tissues and organs, female cyclin D1 Ϫ/Ϫ mice are markedly deficient in breast epithelial cell proliferation associated with pregnancy (19, 52). Specifically, ductal side branching and lobuloalveolar development are severely impaired in these mice despite normal levels of circulating ovarian hormones. It has been suggested that steroid hormone-induced breast epithelial cell proliferation and/or differentiation during pregnancy requires the action of cyclin D1.Here, we describe the functional interaction of cyclin D1 with the estrogen receptor. MATERIALS AND METHODS Plasmids.The following plasmids have been described previously: Ϫ1745CD1Luc (human cyclin D1 promoter-luciferase reporter) (2); p(ERE) 2 -tk-luc (estrogen response element [ERE]-luciferase reporter) (34), a gift from P. Chambon; pCMV-hER (60), a gift from D. J...

The Ras proto-oncogene is a central component of mitogenic signal-transduction pathways, and is essential for cells both to leave a quiescent state (G0) and to pass through the G1/S transition of the cell cycle. The mechanism by which Ras signalling regulates cell-cycle progression is unclear, however. Here we report that the retinoblastoma tumour-suppressor protein (Rb), a regulator of G1 exit, functionally links Ras to passage through the G1 phase. Inactivation of Ras in cycling cells caused a decline in cyclin D1 protein levels, accumulation of the hypophosphorylated, growth-suppressive form of Rb, and G1 arrest. When Rb was disrupted either genetically or biochemically, cells failed to arrest in G1 following Ras inactivation. In contrast, inactivation of Ras in quiescent cells prevented growth-factor induction of both immediate-early gene transcription and exit from G0 in an Rb-independent manner. These data suggest that Rb is an essential G1-specific mediator that links Ras-dependent mitogenic signalling to cell-cycle regulation.

Independent of its antiapoptotic function, Bcl-2 can, through an undetermined mechanism, retard entry into the cell cycle. Cell cycle progression requires the phosphorylation by cyclin-dependent kinases (Cdks) of retinoblastoma protein (pRB) family members to free E2F transcription factors. We have explored whether retarded cycle entry is mediated by the Cdk inhibitor p27 or the pRB family. In quiescent fibroblasts, enforced Bcl-2 expression elevated levels of both p27 and the pRB relative p130. Bcl-2 still slowed G 1 progression in cells deficient in pRB but not in those lacking p27 or p130. Hence, pRB is not required, but both p27 and p130 are essential mediators. The ability of p130 to form repressive complexes with E2F4 is implicated, because the retardation by Bcl-2 was accentuated by coexpressed E2F4. A plausible relevant target of p130/E2F4 is the E2F1 gene, because Bcl-2 expression delayed E2F1 accumulation during G 1 progression and overexpression of E2F1 overrode the Bcl-2 inhibition. Hence, Bcl-2 appears to retard cell cycle entry by increasing p27 and p130 levels and maintaining repressive complexes of p130 with E2F4, perhaps to delay E2F1 expression.In addition to its well-established function in controlling cell survival, the Bcl-2 family (1) has been found to influence the cell cycle. Although Bcl-2 and its prosurvival relatives do not affect the growth rate in proliferating cultures, they can both accelerate withdrawal from the cycle (46) and retard reentry (4,29,30,35). Conversely, a shortened G 1 is found in lymphocytes that lack Bcl-2 (29) or express the Bcl-2 antagonist Bax (4). Thus, Bcl-2 appears to have a physiological role in influencing the transition between the quiescent and cycling states. That this ability is separate from its role in cell survival (46) is most clearly shown by mutations of Bcl-2 that eliminate its cell cycle activity but spare its antiapoptotic function (17,45).The cell cycle control function of Bcl-2 has ramifications for cellular homeostasis. Cycling cells are often more vulnerable to apoptosis, perhaps because, under conditions unfavorable for proliferation, certain cell cycle effectors promote apoptosis (11). Hence, promoting quiescence under conditions of stress may provide Bcl-2 with an additional, albeit indirect, means to enhance cell survival (30,46). Interference with Bcl-2's cell cycle effect may also augment its oncogenic role (see Discussion).Progression through the cell cycle requires the action of cyclin-dependent kinases (Cdks) (38, 39). As cells enter the cycle, newly synthesized D-type cyclins associate with and activate their Cdk-4/6 catalytic partners in mid-to late G 1 phase, while cyclin E appears later in G 1 and activates its Cdk-2 kinase subunit near the G 1 /S boundary. Opposing their activity are Cdk inhibitors (Cki) of two classes: INK4 proteins, such as p16, specifically inhibit D-cyclin kinases, whereas Cip/Kip proteins, such as p21 and p27, also inhibit Cdk-2 (38, 39). Other key negative regulators include the best-known Cdk substrates...