Both cyclin D1 and estrogens have an essential role in regulating proliferation of breast epithelial cells. We show here a novel role for cyclin D1 in growth regulation of estrogen-responsive tissues by potentiating transcription of estrogen receptor-regulated genes. Cyclin D1 mediates this activation independent of complex formation to a CDK partner. Cyclin D1 activates estrogen receptor-mediated transcription in the absence of estrogen and enhances transcription in its presence. The activation of estrogen receptor by cyclin D1 is not inhibited by anti-estrogens. A direct physical binding of cyclin D1 to the hormone binding domain of the estrogen receptor results in an increased binding of the receptor to estrogen response element sequences, and upregulates estrogen receptor-mediated transcription. These results highlight a novel role for cyclin D1 as a CDK-independent activator of the estrogen receptor.

The estrogen receptor (ER) is an important regulator of growth and differentiation of breast epithelium. Transactivation by ER depends on a leucine-rich motif, which constitutes a ligand-regulated binding site for steroid receptor coactivators (SRCs). Cyclin D1 is frequently amplified in breast cancer and can activate ER through direct binding. We show here that cyclin D1 also interacts in a ligand-independent fashion with coactivators of the SRC-1 family through a motif that resembles the leucine-rich coactivator binding motif of nuclear receptors. By acting as a bridging factor between ER and SRCs, cyclin D1 can recruit SRC-family coactivators to ER in the absence of ligand. A cyclin D1 mutant that binds to ER but fails to recruit coactivators preferentially interferes with ER activation in breast cancer cells that have high levels of cyclin D1. These data support that cyclin D1 contributes significantly to ER activation in breast cancers in which the protein is overexpressed. Our present results reveal a novel route of coactivator recruitment to ER and establish a direct role for cyclin D1 in regulation of transcription.

. This study demonstrates a novel aspect of cyclin D1 as a regulator of the transition between G 1 and G 0 . Overexpression of cyclin D1 in MCF7 breast tumor cells resulted in a continued proliferation under low-serum conditions, whereas nonoverexpressing cells ceased to grow. This difference in growth was due to a reduced exit from G 1 to G 0 in cyclin D1-overexpressing cells. Our data therefore suggest a model in which cyclin D1 overexpression in tumor cells is responsible for hyperproliferation under growth factor-deprived conditions.The proliferation of mammalian cells is primarily regulated by a decision that occurs at a unique point in the first gap phase (G 1 ) of the cell cycle: to remain in the cell cycle and proliferate or to leave the cell cycle to become quiescent. Mitogenic signals regulate cell division by triggering a cascade of events and ultimately induce the expression of G 1 cyclins, including the D-type cyclins and cyclin E (33). These cyclins are presumed to control progression through the cell cycle by governing the activity of cyclin-dependent kinases (CDKs). Passage through the unique control point at late G 1 defines the stage at which cells no longer require growth factors. Once a cell passes this checkpoint, also called the restriction point, it will complete the cycle, even in the absence of mitogens (23). Because the G 1 cyclins are critical to the decision to pass the restriction point, it is not surprising that a deregulated expression of G 1 cyclins and/or their CDK partners is often found in malignant cells. For instance, many aberrations in G 1 -associated processes are observed in cancer cells, in which cyclin D1 abnormalities are the most prominent. Deregulation of cyclin D1 was found in many different tumor types, such as breast and squamous cell carcinomas (2,15,20,30), parathyroid adenomas (21), and in esophageal carcinomas (11).Several lines of evidence support the hypothesis that cyclin D1 is rate limiting for the transition of G 0 to S phase: (i) cyclin D1 expression levels peak in the G 1 phase and are associated with either CDK4 or CDK6, which are active during G 1 phase (1,2,3,19,32); (ii) microinjection of either antisense oligonucleotides or antibodies against cyclin D1 into various cell types arrests the cells in the G 1 phase (1, 2, 25); and (iii) in synchronized (M-phase or G 0 -phase) cells, cyclin D1 overexpression causes an increase in the percentage of S-phase cells (12,22,25,27). To further explore the role of cyclin D1 overexpression in G 1 -associated processes, we investigated the role of cyclin D1 overexpression in cell cycle exit. For that purpose, we have generated clones from the human epithelial breast cancer cell line MCF7, which can be induced to overexpress cyclin D1. This cell line is normally composed of a high percentage of noncycling cells and is therefore very useful for this study. MCF7 cells do not contain an amplification of the cyclin D1 gene (although cyclin D1 amplification is quite common in this cell type), they are well differentiate...

There is growing evidence that not only the total amount of fat, but also the distribution of body fat determines risks for metabolic and cardiovascular disease. Developmental studies on factors influencing body fat distribution have been hampered by a lack of appropriate techniques for measuring intraabdominal fat in early life. Sonography, which is an established method for assessing abdominal fat distribution in adults, has not yet been evaluated in infants. To adapt the sonographic measurement of abdominal fat distribution to infants and study its reliability. The Generation R study, a population-based prospective cohort study. We included 212 one-and 227 two-year old Dutch infants in the present analysis. Sixty-two infants underwent replicate measurements to assess reproducibility. We developed a standardized protocol to measure the thickness of (1) subcutaneous and (2) preperitoneal fat in the upper abdomen of infants. To this end we defined infancy specific measurement areas to quantify fat thickness. Reproducibility of fat measurements was good to excellent with intraclass correlation coefficients of 0.93-0.97 for intra-observer agreement and of 0.89-0.95 for inter-observer agreement. We observed a pronounced increase in preperitoneal fat thickness in the second year of life while subcutaneous fat thickness increased only slightly, resulting in an altered body fat distribution. Gender did not significantly influence fat distribution in the first two years of life. Our age specific protocol for the sonographic measurement of central subcutaneous and preperitoneal fat is a reproducible method that can be instrumental for investigating fat distribution in early life.