The sequential transcriptional activation of cyclins, the regulatory subunits of cell-cycle-specific kinases, is thought to regulate progress through the cell cycle. Cyclins are therefore potential oncogenes, and cyclin D1 overexpression and/or amplification at its genomic locus, 11q13, are common features of several human cancers. Induction of cyclin D1 is an early response to mitogenic stimulation in several cell types, but the consequences of altered expression of this gene in human cells of epithelial origin remain undefined. We assessd the effects of alterations of cyclin D1 expression in human breast cancer cells by generating T-47D cells expressing human cyclin D1 under the control of a zinc-responsive metallothionein promoter. In cycling cells induction of cyclin D1 after zinc treatment resulted in an increase in the number of cells progressing through G1 and in the rate of transition from G1 to S phase, indicating that cyclin D1 is rate-limiting for progress through G1 phase. In cells arrested in early G1 phase after growth factor deprivation, zinc induction of cyclin D1 was sufficient for completion of the cell cycle, a process requiring growth factor stimulation in control cells. These data demonstrate a critical role for cyclin D1 in human breast cancer cell-cycle control and suggest that deregulated expression of cyclin D1 is likely to reduce dependence on normal physiological growth stimuli, thereby providing a growth advantage to tumor cells and a potential mechanism of resistance to endocrine therapy.Cell-cycle progression in mammalian cells is coordinated at a series of control points, which ensure orderly progress through the complex and tightly regulated processes necessary for cell growth and division. The central mechanism for these control points is thought to be the sequential transcriptional activation of cycin genes and consequent transient accumulation of different cyclin/cycin-dependent kinase (CDK) complexes. In synchronized or growth factorstimulated cells, cyclins C, D1, D2, D3, and E are most abundant during G1 phase (1-3), suggesting that they function during G1. The D cyclins have closely related sequences but are differentially expressed: some cells express all three genes [for example, mammary epithelial cells (4)], but many cell types express only one or two (2,5,6). Furthermore, these cyclins are not coordinately regulated but, in general, appear sequentially during progress through G1 phase (2, 6-9), suggesting that they have complementary rather than redundant functions.Regulation of cyclin D1 gene expression is closely associated with changes in the proliferation rate of breast cancer cells. Increased expression of cyclin D1 occurs within 2 hr of stimulation of T-47D breast cancer cells by peptide mitogens-for example, insulin, insulin-like growth factor I, and basic fibroblast growth factor-and is followed by induction of other G1 cyclins, cyclins D3 and E, as cells progress through G1 phase (ref. 8, unpublished data). The proportion of cells that enter S phase is...

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. In breast cancer cells the predominant effect of synthetic progestins is long-term growth inhibition and arrest in G 1 phase. Progestin-mediated growth arrest of T-47D breast cancer cells was preceded by inhibition of cyclin D1-Cdk4, cyclin D3-Cdk4, and cyclin E-Cdk2 kinase activities in vitro and reduced phosphorylation of pRB and p107. This was accompanied by decreases in the expression of cyclins D1, D3, and E, decreased abundance of cyclin D1-and cyclin D3-Cdk4 complexes, increased association of the cyclin-dependent kinase (CDK) inhibitor p27 with the remaining Cdk4 complexes, and changes in the molecular masses and compositions of cyclin E complexes. In control cells cyclin E eluted from Superdex 200 as two peaks of ϳ120 and ϳ200 kDa, with the 120-kDa peak displaying greater cyclin E-associated kinase activity. Following progestin treatment, almost all of the cyclin E was in the 200-kDa, low-activity form, which was associated with the CDK inhibitors p21 and p27; this change preceded the inhibition of cell cycle progression. These data suggest preferential formation of this higher-molecular-weight, CDK inhibitorbound form and a reduced number of cyclin E-Cdk2 complexes as mechanisms for the decreased cyclin E-associated kinase activity following progestin treatment. Ectopic expression of cyclin D1 in progestininhibited cells led to the reappearance of the 120-kDa active form of cyclin E-Cdk2 preceding the resumption of cell cycle progression. Thus, decreased cyclin expression and consequent increased CDK inhibitor association are likely to mediate the decreases in CDK activity accompanying progestin-mediated growth inhibition.Steroid hormones regulate cellular proliferation and differentiation by cell cycle phase-specific actions (40). Estrogen, acting in concert with other hormones and growth factors, appears to be the main drive to proliferation in the female reproductive tract and mammary gland. In contrast with the proliferative effects of estrogen, progesterone acts as the differentiating female sex steroid. In this role it can either stimulate or inhibit proliferation in a cell type-and tissue-specific manner (5). For example, the primary function of progesterone in the uterus is to facilitate implantation, and in this organ progesterone acts synergistically with estrogen to stimulate proliferation of stromal cells but inhibits estrogen-induced mitosis in the epithelium. In the mammary gland progesterone stimulates proliferation and development of alveoli, a requirement for subsequent lactation. In breast cancer cells, a widely used model for studies of the effects of steroids on cell proliferation, treatment with synthetic progestins results in a biphasic change in the rate of cell cycle progression, consisting of an initial transient acceleration through G 1 phase and a subsequent increase in the S phase fraction, followed by cell cycle arrest and growth...

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