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.

The cell cycle-regulatory transcription factor E2F-1 is regulated by interactions with proteins such as the retinoblastoma gene product and by cell cycle-dependent alterations in E2F-1 mRNA abundance. To better understand this latter phenomenon, we have isolated the human E2F-1 promoter. The human E2F-1 promoter, fused to a luciferase cDNA, gave rise to cell cycle-dependent luciferase activity upon transfection into mammalian cells in a manner which paralleled previously reported changes in E2F-1 mRNA abundance. The E2F-1 promoter contains four potential E2F-binding sites organized as two imperfect palindromes. Gel shift and transactivation studies suggested that these sites can bind to E2F in vitro and in vivo. Mutation of the two E2F palindromes abolished the cell cycle dependence of the E2F-1 promoter. Thus, E2F-1 appears to be regulated at the level of transcription, and this regulation is due, at least in part, to binding of one or more E2F family members to the E2F-1 promoter.