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 PTEN tumor suppressor gene encodes a phosphatidylinositol 3P P-phosphatase that is inactivated in a high percentage of human tumors, particularly glioblastoma, melanoma, and prostate and endometrial carcinoma. Previous studies showed that PTEN is a seryl phosphoprotein and a substrate of protein kinase CK2 (CK2). However, the sites in PTEN that are phosphorylated in vivo have not been identi¢ed directly, nor has the e¡ect of phosphorylation on PTEN catalytic activity been reported. We used mass spectrometric methods to identify Ser 370 and Ser 385 as in vivo phosphorylation sites of PTEN. These sites also are phosphorylated by CK2 in vitro, and phosphorylation inhibits PTEN activity towards its substrate, PIP3. We also identify a novel in vivo phosphorylation site, Thr 366 . Following transient over-expression, a fraction of CK2 and PTEN co-immunoprecipitate. Moreover, pharmacological inhibition of CK2 activity leads to decreased Akt activation in PTEN+/+ but not PTEN3 3/3 3 ¢broblasts. Our results contrast with previous assignments of PTEN phosphorylation sites based solely on mutagenesis approaches, suggest that CK2 is a physiologically relevant PTEN kinase, and raise the possibility that CK2-mediated inhibition of PTEN plays a role in oncogenesis. ß

Mutations of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a protein and lipid phosphatase, have been associated with gliomas, macrocephaly, and mental deficiencies. We have assessed PTENЈs role in the nervous system and find that PTEN is expressed in mouse brain late in development, starting at approximately postnatal day 0. In adult brain, PTEN is preferentially expressed in neurons and is especially evident in Purkinje neurons, olfactory mitral neurons, and large pyramidal neurons. To analyze the function of PTEN in neuronal differentiation, we used two well established model systemspheochromocytoma cells and cultured CNS stem cells. PTEN is expressed during neurotrophin-induced differentiation and is detected in both the nucleus and cytoplasm. Suppression of PTEN levels with antisense oligonucleotides does not block initiation of neuronal differentiation. Instead, PTEN antisense leads to death of the resulting, immature neurons, probably during neurite extension. In contrast, PTEN is not required for astrocytic differentiation. These observations indicate that PTEN acts at multiple sites in the cell, regulating the transition of differentiating neuroblasts to postmitotic neurons.

Because HER-2/neu overexpression is important in cancer development, we looked for a method of suppressing the cell transformation mediated by HER-2/neu overexpression. We have identified that the DNA-binding protein PEA3, which is encoded by a previously isolated gene of the ets family, specifically targeted a DNA sequence on the HER-2/neu promoter and downregulated the promoter activity. Expression of PEA3 resulted in preferential inhibition of cell growth and tumor development of HER-2/neu-overexpressing cancer cells. This is a new approach to targeting HER-2/neu overexpression and also provides a rationale to the design for repressors of diseases caused by overexpression of pathogenic genes.