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 synthesis of cyclin D1 and its assembly with cyclin-dependent kinase 4 (CDK4) to form an active complex is a rate-limiting step in progression through the G 1 phase of the cell cycle. Using an activated allele of mitogen-activated protein kinase kinase 1 (MEK1), we show that this kinase plays a significant role in positively regulating the expression of cyclin D1. This was found both in quiescent serum-starved cells and in cells expressing dominant-negative Ras. Despite the observation that cyclin D1 is a target of MEK1, in cycling cells, activated MEK1, but not cyclin D1, is capable of overcoming a G 1 arrest induced by Ras inactivation. Either wild-type or catalytically inactive CDK4 cooperates with cyclin D1 in reversing the G 1 arrest induced by inhibition of Ras activity. In quiescent NIH 3T3 cells expressing either ectopic cyclin D1 or activated MEK1, cyclin D1 is able to efficiently associate with CDK4; however, the complex is inactive. A significant percentage of the cyclin D1-CDK4 complexes are associated with p27 in serum-starved activated MEK1 or cyclin D1 cell lines. Reduction of p27 levels by expression of antisense p27 allows for S-phase entry from quiescence in NIH 3T3 cells expressing ectopic cyclin D1, but not in parental cells.Both positive and negative extracellular growth factors exert their influence upon proliferation during the G 1 phase of the cell cycle. At a point in late G 1 , termed the restriction point (R), cells become largely refractory to these factors and once past R are committed to completing the mitotic cycle (62,87,97). The action of serum growth factors is mediated, in part, through their activation of receptor tyrosine kinases. Ras, an inner plasma membrane-bound GTPase, plays a significant role in receiving and transducing extracellular signals by functioning as a downstream mediator of several membrane-bound receptor and nonreceptor tyrosine kinases (5,45,46,69). Ras influences, and is required for, both the G 0 /G 1 transition and passage through G 1 to a point temporally coincident with the restriction point. In certain situations, Ras has been shown to be sufficient for progression from G 0 to S phase, as demonstrated by the ability of microinjected activated or wild-type Ras to induce DNA synthesis from quiescence in the absence of serum factors (19,84). Furthermore, microinjection of murine fibroblasts with anti-Ras neutralizing antibodies prevents efficient serum-induced S-phase entry from a G 0 state (16,52), and expression of a dominant-negative Ras protein (Ras N17 ) inhibits the proliferation of NIH 3T3 and 32D myeloid cells, resulting in a G 1 arrest (8,18,59).In terms of nuclear events influencing progression through the G 1 phase of the cell cycle, a key role is played by G 1 cyclins and their catalytic subunits, the cyclin-dependent kinases (CDKs). The three D-type cyclins associate with and activate CDK4 and CDK6. Cyclin E is required for the activation of CDK2. The synthesis of D-type cyclins, whose expression is rate limiting for G 1 progression, is ...
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