During human pregnancy, the production of 17-beta-estradiol (E2) rises steadily to eighty fold at term, and placenta has been found to specifically bind estrogens. We have recently demonstrated the expression of estrogen receptor alpha (ER-alpha) protein in human placenta and its localization in villous cytotrophoblast (CT), vascular pericytes, and amniotic fibroblasts. In vitro, E2 stimulated development of large syncytiotrophoblast (ST) aggregates. In the present study we utilized ER-beta affinity purified polyclonal (N19:sc6820) and ER-alpha monoclonal (clone h-151) antibodies. Western blot analysis revealed a single ~52 kDa ER-beta band in chorionic villi (CV) protein extracts. In CV, strong cytoplasmic ER-beta immunoreactivity was confined to ST. Dual color immunohistochemistry revealed asymmetric segregation of ER-alpha in dividing villous CT cells. Prior to separation, the cell nuclei more distant from ST exhibited high ER-alpha, while cell nuclei associated with ST showed diminution of ER-alpha and appearance of ER-beta. In trophoblast cultures, development of ST aggregates was associated with diminution of ER-alpha and appearance of ER-beta immunoreactivity. ER-beta was also detected in endothelial cells, amniotic epithelial cells and fibroblasts, extravillous trophoblast (nuclear and cytoplasmic) and decidual cells (cytoplasmic only). In addition, CFK-E12 (E12) and CWK-F12 (F12) monoclonal antibodies, which recognize ~64 kDa ER-beta with hormone binding domain, showed nuclear-specific reactivity with villous ST, extravillous trophoblast, and amniotic epithelium and fibroblasts. Western blot analysis indicated abundant expression of a ~64 kDa ER-beta variant in trophoblast cultures, significantly higher when compared to the chorionic villi and freshly isolated trophoblast cell protein extracts. This is the first report on ER-beta expression in human placenta and cultured trophoblast. Our data indicate that during trophoblast differentiation, the ER-alpha is associated with a less, and ER-beta with the more differentiated state. Enhanced expression of ~64 kDa ER-beta variant in trophoblast cultures suggests a unique role of ER-beta hormone binding domain in the regulation of trophoblast Published: 15 April 2003 Reproductive Biology and Endocrinology 2003, 1:36 Received: 1 February 2003 Accepted: 15 April 2003 This article is available from: http://www.RBEj.com/content/1/1/36 © 2003 Bukovsky et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

We have previously demonstrated that the anti-apoptotic protein BAD is expressed in normal human breast tissue and shown that BAD inhibits expression of cyclin D1 to delay cell-cycle progression in breast cancer cells. Herein, expression of proteins in breast tissues was studied by immunohistochemistry and results were analyzed statistically to obtain semi-quantitative data. Biochemical and functional changes in BAD-overexpressing MCF7 breast cancer cells were evaluated using PCR, reporter assays, western blotting, ELISA and extracellular matrix invasion assays. Compared to normal tissues, Grade II breast cancers expressed low total/phosphorylated forms of BAD in both cytoplasmic and nuclear compartments. BAD overexpression decreased the expression of β-catenin, Sp1, and phosphorylation of STATs. BAD inhibited Ras/MEK/ERK and JNK signaling pathways, without affecting the p38 signaling pathway. Expression of the metastasis-related proteins, MMP10, VEGF, SNAIL, CXCR4, E-cadherin and TlMP2 was regulated by BAD with concomitant inhibition of extracellular matrix invasion. Inhibition of BAD by siRNA increased invasion and Akt/p-Akt levels. Clinical data and the results herein suggest that in addition to the effect on apoptosis, BAD conveys anti-metastatic effects and is a valuable prognostic marker in breast cancer.

Purpose The public has paid attention to green tea due to its health benefits. Epigallocatechin-3-gallate (EGCG), the major component of green tea, is well documented to induce apoptosis and cell cycle arrest in cancer cells by targeting multiple signal transduction pathways. However, the detailed mechanism(s) of action needs to be determined. Methods Cell growth was evaluated by MTT assay, cell cycle analysis, and caspase 3/7 activity. Protein expression was analyzed through Western blotting. Reverse transcription polymerase chain reaction was used for examining mRNA expression of p21 and cyclin D1. The promoter activity of p21 was assessed by the luciferase reporter system. Results We identified cyclin D1 and p21 as molecular targets of EGCG in human colorectal cancer cells. We observed that cyclin D1 was down-regulated, while p21 expression was up-regulated by EGCG in dose- and time-dependent manners. Furthermore, we found EGCG decreased cyclin D1 protein stability, therefore triggering ubiquitin-dependent proteasomal degradation. Meanwhile, EGCG increased p21 promoter activity, resulting in up-regulation of p21 mRNA and protein, which was likely dependent on extracellular-signal-regulated kinase (ERK), inhibitor of nuclear factor kappa-B kinase (IKK) and phosphoinositide 3-kinase (PI3 K). Conclusion The data presented here details a novel mechanism by which EGCG inhibits cell growth of colorectal cancer cells. Namely, EGCG-induced cyclin D1 degradation and p21 transcriptional activation partially contribute to growth suppression in these cells.

Obesity in post‐menopausal women doubles the breast cancer risk and metastasis. Concomitantly, nuclear factor kappa B (NF‐κB) is known to play a significant role in breast cancer cell metastasis. We used a novel three dimensional (3D) co‐culture system with breast cells (non‐cancerous, MCF10A, cancerous, MCF7 and invasive, MDA231) and adipocytes from pre‐ and post‐menopausal women in lean and obese groups. Comparison of cytokine profiles of co‐cultures revealed two‐way interaction of adipocytes and breast cells. Highly significant cytokine changes were reported for post‐menopausal adipocytes and MDA MB 231 co‐culture. Therefore, we assayed the NF‐κB activation effects of lean and obese post‐menopausal adipocytes on MDA MB 231 cells. Media transfer from lean/obese post‐menopausal adipocyte cultures in to growing MDA MB 231 breast cells resulted significant changes in NF‐κB activation pathway genes. This further verified our previous data that obese conditioned media increased the levels of NF‐κB family members (P65, P50, P52, Rel B and Rel C) compared to the lean counterpart. Therefore, our data indicates that adipocytes from post‐menopausal obese women can increase the NF‐κB activation in MDA MB 231 cells. In conclusion, this study suggests that increased breast cancer risk in obese post‐menopausal women may be at least in part due to obesity mediated NF‐κB activation. Support: UTORC, AgResearch and UTGSM.Grant Funding Source : Internal

Obesity and inflammation are associated with increased risk for breast cancer in postmenopausal women. Further, ù-6 arachidonic acid (AA) increases secretion of adipose pro-inflammatory cytokines compared to ù-3 eicosapentaenoic acid (EPA). We compared the effect of these fatty acids on breast cancer cells in relation to inflammation and obesity. We used a novel three dimensional (3D) co-culture system with breast cell lines (non-cancerous, MCF10A, cancerous, MCF7 and invasive, MDA231) and adipocytes from lean/obese women. Effects of AA and EPA on breast cell growth, morphology and inflammation were assessed. Compared to EPA, AA altered the non-cancerous regular spheroid morphology towards irregular cell structures and altered regular epithelial cell organization in adipocyte-MCF10A co-cultures. Alteration of mammary spheroid organization may leads to tumorigenesis. Minimal effects on cell growth were observed when breast cancer cells were exposed to conditioned media from EPA- compared to AA-treated adipocytes. Further, secretion of pro-inflammatory cytokines from adipocytes derived obese women significantly altered the mTOR/AMPK-related metabolic cell signaling in cancer cells. Thus, our data highlight the effects of adipocytes on breast cell growth, inflammation and metabolism as well as the role of fatty acids in modulating this interaction. Grant support: UT-ORC and TN AgResearch., UTGSM

Abstract Alteration of organized breast epithelial ductal architecture accompanies neoplastic transformation. Obesity and chronic inflammation are associated with increased risk of postmenopausal breast cancer (BC). Further, omega 6 arachodonic acid (AA) significantly increases the secretion of pro-inflammatory cytokines by adipocytes compared to omega 3 eicosapentaenoic acid (EPA), therefore, we compared the effect of adipocytes and these two fatty acids on primary breast epithelial cell growth and formation of acini-like spheroid architecture. We used three dimensional (3D) MatrigelTM cultures of both basal and luminal primary breast cells and a novel 3D co-culture system with primary breast epithelial cells (basal/luminal) and adipocytes from lean or obese women. Effects of adipocytes as well as fatty acids on cellular architecture were assessed. Culture of basal or luminal epithelial cells with obese adipocytes/AA showed a significant increase in the irregularity of acini-like spheroid formation compared to normal spheroid formation in the absence of adipocytes or AA. Conditioned media transfer from lean/obese adipocytes into growing breast cell cultures showed remarkably different effects on LKB1/AMPK/mTOR metabolic signaling systems specifically in invasive MDA MB 321 cells. Obese adipocyte media decreased the activation of LKB1, AMPK, Acetyl-CoA carboxylase (ACC) and mTOR levels compared to lean adipocyte media. Further the expression of LKB1, pLKB1, pACC and mTOR were significantly higher in MDA MB 231 cells compared to less invasive MCF7 or epithelial MCF10A cells. Our data highlights the regulatory influence of adipocytes and inflammatory fatty acids on breast cellular architecture as well as metabolism and such alterations may underlie the effects of obesity on transformation of normal breast cells to BC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3111. doi:10.1158/1538-7445.AM2011-3111

Among the various pathologies obesity is associated with is an increase in incidence of some cancers. There is a substantial body of evidence that post menopausal breast-cancer (BC) incidence as well as mortality is increased in obese (BMI above 30) women. The patho-physiological basis for this association is obscure, even though increased estrogen levels in obese women could contribute to BC. Given the fact that the vast majority of stromal cells in the breast is made of adipocytes and the emerging importance of stroma in malignancy, we investigated the cellular interactions between adipocytes and BC cells (MCF 7, MDAMB23l) and breast epithelial cells (MCF10A) in a novel 3 dimensional (3D) culture system as well as by media transfer experiments. Our data show that obese and lean adipocytes had differential effects on cell growth of the 3 breast cell lines with obese adipocytes having the largest growth promoting effect. We measured 26 cytokines using the Luminex platform and found significant changes in eleven (including G-CSF, GM-CSF, IL8, IL6, MCP1). The changes in cytokines were cell type specific for obese and lean adipocytes and the breast cell type. In media transfer experiments we found that lean and obese adipocyte conditioned media regulated breast cell TNF alpha content, Furthermore these media had distinct effects on breast cell NFkB family members activity. Remarkably, the greatest increases were observed in MDAMB231 cells exposed to obese adipocyte conditioned media. Our novel results with co-culture/media transfer experiments strongly suggests that adipocytes and BC cells have a two way communication which is likely to regulate BC growth/function.

Abstract The role of ER alpha as the mediator of estrogen action in breast cancer (BC) cells is well known, however, the role of ER beta is controversial. The majority of BC expressing ER alpha also express ER beta. Some clinical studies suggest that ER beta expression is a good prognosticator; however, in others, especially where expression levels of ER beta > ER alpha, ER beta may be associated with more advanced BC. Previously, we showed that ER beta may regulate a number of signaling pathways (AACR, 2008). Our recent work demonstrates that ER beta has even wider actions in MCF7 and T47D cells stably overexpressing ER beta. Thus, ER beta decreased phospho (p)/ total Stat1, p Stat3, p Stat5/total Stat5. Expression of the oncogenic miR cluster 17-92 was highly reduced (p<0.01) when ER beta overexpressing vs. control cells were compared. ER beta also decreased PPAR gamma, superoxide dismutase, as well as the concentration of the mRNA processing factor, RBM5, and the activated form of AMPK. All these changes were significant (p<0.05) in triplicate measurements. While ER beta overexpression increased migration through Matrigel, it significantly decreased CXCR4 expression (p<0.05, n=4). All of these effects of ER beta were estrogen-independent. On the other hand, estrogen increased CXCR4 expression in MCF7 cells significantly. In our study, we analyzed the expression of ER beta and BAD using tissue microarrays with normal and neoplastic breast tissue. Our immunohistochemical (IHC) data revealed a significant increase of ER beta cytoplasmic staining in neoplastic cells compared to normal cells (p<0.001). In addition, the nuclear expression of ER beta was significantly decreased in neoplastic cells compared to normal cells (p<0.01). From our correlation analysis of IHC data obtained from tissue microarray, we identified that cytoplasmic expression of BAD was negatively correlated with cytoplasmic expression of ER beta. In addition, the nuclear expression of BAD was significantly positively correlated with nuclear ER beta. These results suggest that ER beta has a wide variety of actions in BC cells, as predicted by the regulation of 62 miRs (p<0.01) as determined by Exiqon Inc. Taken together our results suggest that ER beta may have a multitude of actions in breast cancer and that the ratio of ER alpha/beta may determine the prognostic outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4591.

Abstract BAD is a multifunctional protein in breast cancer cells R. Fernando1, M. Cekanova2, J. Woraratphoka2, M. Sukhthankar2, J. Bahn2, S. Dharmawardhana3, N. Siriwardana2, N. Moustaid-Moussa2, A. Strom4, S. Baek2, Q. Wong5, M. Zou5, R. Donnell2, J. Wimalasena2 1NCI, Bethesda, MD; 2University of Tennessee, Knoxville, TN; 3University of Puerto Rico Medical Sciences, San Juan, PR; 4University of Houston, Houston, TX; 5University of Oklahoma Medical Center, Oklahoma City, OK. BAD is a typical BH3 protein and, like many other BCL-2 family proteins, regulates apoptosis. However, several recent studies suggest that BAD has non-apoptotic roles. Previously, we had shown that BAD inhibits the MCF7 cell cycle via abrogation of cyclin D1 synthesis (JBC 282, 28864, 2007). Using tetracycline-regulated stable BAD overexpression in MCF7 cells, we now report that BAD regulates a wide variety of functions: since BAD binds to c-Jun (JBC, 2007), we investigated the ability of BAD to regulate the AP1 reporter activity. BAD inhibited MEK and activated RAS (V12, S35) regulated AP1 activity, but not the increase due to AKT. BAD overexpression blocked ERK activation by MEK1, but not by active AKT. Wildtype BAD inhibited estrogen- or serum-induced activation of c-Jun, JNK, and ERK, but not p38 MAPK. However, the S112/S136 double mutant BAD had no inhibitory effects, suggesting a requirement for phosphorylation. We then explored if BAD can regulate signaling pathways and found that it can inhibit beta catenin mRNA and protein expression. Several signaling proteins, including total AKT, active GSK 3 beta, and apoptosis-related soluble FasL, TNF alpha, TRAIL, EGFR, and RBM5, which may regulate FasL were significantly (p<0.01) regulated by BAD. BAD decreased invasion of MCF7 cells through Matrigel (p<0.05) and decreased cellular VEGF (p50% (p<0.01). Surprisingly, BAD decreased protein expression of PPAR gamma, active AMPK, LKB1, and active acetylCoA carboxylase (all at p<0.01). We analyzed the expression of several markers in breast cancer specimens: phospho (p)-BAD, BAD and cyclin D1 using tissue microarrays containing normal and neoplastic tissue. In addition, we checked the expression levels of ERK-1, phospho (p)-ERK1, AKT, and phospho-AKT in surgical pathology blocks with normal and neoplastic breast epithelial cells. As expected, cytoplasmic p-BAD expression was significantly and positively correlated with nuclear BAD, nuclear p-BAD, as well as nuclear cyclin D1. Our data showed that cytoplasmic cyclin D1 was positively correlated with cytoplasmic ERK-1, and cytoplasmic cyclin D1 was negatively correlated with nuclear AKT expression. Nuclear BAD and AKT were negatively correlated with nuclear cyclin D1 and nuclear ER-beta. Cytoplasmic expression of p-ERK1 was negatively correlated with nuclear AKT and positively correlated with nuclear cyclin D1, and nuclear ER-beta. Taken together, these results suggest that BAD may be a multifunctional protein. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1046.

Objective. Endometrial cancer (EC) is the most frequent cancer of the female genital tract. It has been hypothesized that those ECs that occur in the postmenopausal period, might be sensitive to elevated levels of luteinizing hormone/human chorionic gonadotropin (LH/hCG). Based on previous indications, we analyzed the functional expression of LH/hCG receptors (LH/hCG-R) in primary ECs.Methods. We studied a cohort of primary ECs, in which both the LH/hCG-R mRNA and the LH/hCG-R protein were analyzed. Results were correlated with both clinical-pathological data and the effects of LH addition on cell invasion in vitro.Results. The LH/hCG-R mRNA levels ranged from 4.67 e(-02) to 2.36 e(+03). The transcript was properly translated into a functional LH/hCG-R protein. The analysis of cell invasion in vitro in response to LH/hCG allowed us to divide the EC samples into two groups, one with a null or very low response (non-responders=NR) and the other with a significant response to LH (responders=R). The two groups had significantly different levels of LH/hCG-R mRNA expression: the NR roup had a median value of 1.40 e(+00), while the R group of 7.42 e(+01) (p=0.043).Conclusion. In primary ECs a statistically significant correlation emerged between the levels of LH/hCG-R mRNA and the LH-induced cell invasion in vitro. These results suggest that therapies aimed at decreasing LH levels, through Gonadotropin Releasing Hormone (Gn-RH) analogues, could produce benefits in the treatment of recurrent or metastatic EC, especially in patients displaying high LH/hCG-R levels. (C) 2008 Elsevier Inc. All rights reserved.

Breast cancer is the most common malignancy among Swedish women. Although the mechanism behind the tumorigenesis remains unclear, estrogen receptor α (ERα) plays an important role in the progression of breast cancer and is regarded as a target for endocrine therapy. In this thesis, focus is on the second estrogen receptor, ERβ and its function in breast cancer. In addition, the significance of the transcription factors Hes-1 and Hes-6 in breast cancer and their relation to ERα has been studied. By using T47D breast cancer cells with inducible ERβ expression, the role of ERβ has been characterised with respect to proliferation and cell-cycle regulation. In contrast to ERα, expression of ERβ inhibited the proliferation of 17β-estradiol (E2) treated breast cancer cells and caused significantly changed levels of cell-cycle regulators. In response to ERβ expression, the levels of the Cdk2-activating phosphatase Cdc25A as well as cyclin E and E2F1 were reduced with a subsequent decrease of the Cdk2activity. Moreover, expression of ERβ reduced the number of tumor associated blood vessels as well as tumor volume in a mouse xenograft model. In addition to the anti-tumorigenic effects, expression of ERβ reduced the levels of secreted growth factors in vitro as well as in vivo. The transcriptional repressor Hes-1 has been described as an essential factor during embryonic development. However, Hes-1 is also important in breast cancer cells where it inhibits proliferation. Downregulation of Hes-1 by ERα is a crucial step in E2 stimulated proliferation of breast cancer cells. We expressed Hes-1 in breast cancer cells to study the mechanism behind its antiproliferative properties upon E2 treatment. Hes-1 expression induced a G1 cell-cycle phase arrest and a concomitant reduction of the E2F1-level. By real-time quantitative PCR and electrophilic mobility shift assay, we can conclude that Hes-1 inhibits E2F1 at the promoter level. Hes-6 is an inhibitor of Hes-1 and has been associated with tumorigenesis and metastasis. When Hes-6 was expressed in T47D breast cancer cells, proliferation was increased as well as tumor growth in immunodeficient mice. Furthermore, E2F1 was identified as an important target gene, induced by Hes-6 in breast cancer cells. In conclusion, these studies have significantly contributed to the knowledge of estrogen receptor function in breast cancer as well as to elucidate important roles of Hes-1 and Hes-6 in estrogen signalling. LIST OF PUBLICATIONS I. Anders Ström, Johan Hartman, James S Foster, Jay Wimalasena and JanÅke Gustafsson. Estrogen receptor β inhibits 17β-estradiol-stimulated proliferation of the breast cancer cell line T47D. Proc Natl Acad Sci U S A. 2004 Feb 10;101(6):1566-71 II. Johan Hartman, Karolina Lindberg, Andrea Morani, José Inzunza, Anders Ström and Jan-Åke Gustafsson. Estrogen receptor β inhibits angiogenesis and growth of T47D breast cancer xenografts. Cancer Res. 2006 Dec 1;66(23):11207-13. III. Johan Hartman, Patrick Müller, James S Foster, Jay Wimalasena, JanÅke Gustafsson and Anders Ström. HES-1 inhibits 17β-estradiol and heregulin-β1-mediated upregulation of E2F-1. Oncogene. 2004 Nov 18;23(54):8826-33 IV. Johan Hartman, Eric W.-F. Lam, Jan-Åke Gustafsson and Anders Ström. Hes-6, an inhibitor of Hes-1, is regulated by 17β-estradiol and increases the malignant behaviour of breast cancer cells. Submitted manuscript.

Recent investigations suggest that functions of the proapoptotic BCL2 family members, including BAD, are not limited to regulation of apoptosis. Here we demonstrate that BAD inhibits G(1) to S phase transition in MCF7 breast cancer cells independent of apoptosis. BAD overexpression inhibited G(1) transit and cell growth as well as cyclin D1 expression. Inhibition of cyclin D1 expression was mediated through inhibition of transcription activated by AP1. Chromatin immunoprecipitation assays indicated that BAD is localized at the 12-O-tetradecanoylphorbol-13-acetate-response element (TRE) and cAMP-response element (CRE) in the cyclin D1 promoter. This was shown to reflect direct binding interactions of BAD with c-Jun, and this interaction inhibited the activity of AP1 complexes at TRE. BAD did not interact with phosphorylated forms of c-Jun. Our data suggest that inhibitory TRE/CRE-c-Jun-BAD complexes are present at the cyclin D1 promoter in quiescent cells. Estrogen stimulation displaced BAD from TRE/CRE elements in MCF7 cells, whereas BAD overexpression inhibited estrogen-induced cyclin D1 synthesis and cell proliferation. Inhibition of endogenous BAD in MCF7 cells markedly increased the proliferative fraction and DNA synthesis, activated Cdks, and increased cyclin D1 protein levels. This action of BAD required serine residues Ser(75) and Ser(99). Both phosphorylated and unphosphorylated forms of BAD localized to the nuclei of human breast epithelial cells. Thus, we demonstrate a novel role for BAD in cell cycle regulation dependent upon its phosphorylation state and independent of the BAD/BCL2 interaction and apoptosis.

The functional consequences of luteinizing hormone/human chorionic gonadotropin signaling via neuronal luteinizing hormone/human chorionic gonadotropin receptors expressed throughout the brain remain unclear. A primary function of luteinizing hormone (LH) in the gonads is the stimulation of sex steroid production. As LH can cross the blood-brain barrier, present in cerebrospinal fluid and is expressed by neuronal cells, we tested whether LH might also modulate steroid synthesis in the brain. Treatment of differentiated rat primary hippocampal neurons and human M17 neuroblastoma cells with LH (100 mIU/mL) resulted in a twofold increase in pregnenolone secretion in both cell types, suggesting an increase in P450scc-mediated cleavage of cholesterol to pregnenolone and its secretion from neurons. To explore how LH might regulate the synthesis of pregnenolone, the precursor for steroid synthesis, we treated rat primary hippocampal neurons with LH (0, 10 and 100 mIU/mL) and measured changes in the expression of LH receptor and steroidogenic acute regulatory protein (StAR). LH induced a rapid (within 30 min) increase in the expression of StAR, but induced a dose-dependent decrease in LH receptor expression. Consistent with these results, the suppression of serum LH in young rats treated with leuprolide acetate for 4 months down-regulated StAR expression, but increased LH receptor expression in the brain. Taken together, these results indicate that LH induces neuronal pregnenolone production by modulating the expression of the LH receptor, increasing mitochondrial cholesterol transport and increasing P450scc-mediated cleavage of cholesterol for pregnenolone synthesis and secretion.

5417 Previously we had shown that BAD plays a critical role in apoptosis of MCF-7 breast CA cells (MBC, 15:3266,2004). Recently we have observed that BAD is a regulator of cell proliferation in the MCF-7 cell line (AACR 2004 Abs# 4824). App 20% of endogenous BAD is localized in the nucleus. Phosphorylated (p) BAD (S136) can also be observed in the nucleus where as both S136 and S112 pBAD is localized in the nuclear fraction upon over expression of wild type (wt) BAD. Over expressed wtBAD inhibited E2 induced proliferation fraction (PF) but dual phosphorylation site mutant (S136A/S112A (dmBAD)) did not. Removal of endogenous BAD by specific siRNA appeared to increase PF following E2 treatment. Endogenous cyclin D1 (CD1) was also increased by siRNA for BAD. WtBAD but not dmBAD inhibited serum or E2 stimulated CD1 synthesis. Using CRE and AP-1 mutants of the CD1 reporter we found that BAD inhibits expression of CD1 mainly through the CRE element, whereas dmBAD was ineffective. This inhibition appears to be mediated by the ability of wtBAD but not dmBAD to interact with c-jun. BAD appears to inhibit phospho c-jun formation by inhibiting Erk as well as JNK. Thus we demonstrate a novel role for BAD in cell cycle regulation which is dependent on phosphorylation of S112/136 residues but not on BAD/BCL2 interaction. (supported by NIH)

The origin of oocytes and primary follicles in ovaries of adult mammalian females has been a matter of dispute for over 100 yr. The prevailing belief that all oocytes in adult mammalian females must persist from the fetal period of life seems to be a uniquely retrogressive reproductive mechanism requiring humans to preserve their gametes from the fetal period for several decades. The utilization of modern techniques during last 10 yr clearly demonstrates that mammalian primordial germ cells originate from somatic cell precursors. This indicates that if somatic cells are precursors of germ cells, then somatic mutations can be passed on to progeny. Mitotically active germline stem cells have been described earlier in ovaries of adult prosimian primates and recently have been reported to also be present in the ovaries of adult mice. We have earlier shown that in adult human females, mesenchymal cells in the ovarian tunica albuginea undergo a mesenchymal-epithelial transition into ovarian surface epithelium cells, which differentiate sequentially into primitive granulosa and germ cells. Recently, we have reported that these structures assemble in the deeper ovarian cortex and form new follicles to replace earlier primary follicles undergoing atresia (follicular renewal). Our current observations also indicate that follicular renewal exists in rat ovaries, and human oocytes can differentiate from ovarian surface epithelium in fetal ovaries in vivo and from adult ovaries in vitro. These reports challenge the established dogma regarding the fetal origin of eggs and primary follicles in adult mammalian ovaries. Our data indicate that the pool of primary follicles in adult human ovaries does not represent a static but a dynamic population of differentiating and regressing structures. Yet, the follicular renewal may cease at a certain age, and this may predetermine the onset of the natural menopause or premature ovarian failure. A lack of follicular renewal in aging ovaries may cause an accumulation of spontaneously arising or environmentally induced genetic alterations of oocytes, and that may be why aging females have a much higher chance of having oocytes with more mutations in persisting primary follicles.

2606 Estrogen receptor β has been found to counteract the activity of estrogen receptor α in many systems. In agreement with this we find that induced expression of estrogen receptor β in the breast cancer cell line T47D causes inhibition of proliferation at less than equal level of expression of estrogen receptor β compared to estrogen receptor α mRNA. Reduced levels of both mRNA and protein for the G1 to S phase regulating factor cyclin E was found in cells expressing estrogen receptor β, which could explain the inhibition of proliferation. The latter described factor is a critical component of the active cdk2 complex important for cell cycle progression through the G1/S-phase transition. Surprisingly, cyclin D1 mRNA and protein were induced earlier in the G1 phase and also reached a higher level when estrogen receptor β was expressed in the cells. Estrogen receptor β mediated down regulation of the cdc25A phosphatase was found but ruled out as the limiting component important for cell cycle progression in these cells. These findings establish a role of estrogen receptor β in breast cancer and imply that estrogen receptor β specific ligands might reduce the incidence of breast cancer. In order to explore the function of ERβ, stable transformants of ERα-positive breast cancer MCF-7 cells with an ERβ inducible expression vector were established, the expression of ERβ was confirmed by western blot and real-time PCR, respectively. By using the gene chip technology we found that when ERβ was expressed in syncronized MCF-7 cells treated with 17β-estradiol for 24 hours, expression of a number of genes normaly increased by ERα were down regulated. Some examples are thymidine kinase, thymidylate synthetase, trefoil factor 1, and c-myc. The regulated genes strongly indicates that ERβ have an anti-proliferative effect on these cells which is in agreement with the cell cycle analysis of ERβ expressing T47D cells.

We have previously shown that expression of the transcription factor HES-1 is required for the growth-inhibitory effect of all-trans retinoic acid on MCF-7 cells. In this study, we have used T47D cells with tetracyclin-regulated expression of wild-type or a dominant-negative form of HES-1. Expression of HES-1 in T47D cells inhibited G1/S-phase transition and activation of Cdk2 elicited by estrogen. Estrogen treatment of T47D cells caused increased expression of E2F-1, and this expression was inhibited by cotreatment with all-trans retinoic acid. We show that the effect is mediated through HES-1, which directly downregulates E2F-1 expression through a CACGAG-site within the E2F-1 promoter. Furthermore, proliferation caused by heregulin-beta1 treatment of T47D cells was inhibited by all-trans retinoic acid and this effect was mediated by HES-1. Interestingly, heregulin-beta1-mediated upregulation of E2F-1 expression was directly inhibited by HES-1 through the same CACGAG-site as seen with estrogen-stimulated induction. In addition, we found that two important downstream target genes of estrogen and heregulin-beta1 that are regulated through E2F-1, cyclin E and NPAT, were both regulated in a similar fashion by all-trans retinoic acid, and these effects were antagonized by dominant-negative HES-1. These findings establish that HES-1 inhibits both estrogen- and heregulin-beta1-stimulated growth of breast cancer cells, and further suggest that growth inhibition induced in these cells by all-trans retinoic acid occurs via HES-1-mediated downregulation of E2F-1 expression.