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Background Coordinated, multi-component school-based interventions can improve health behaviors in children, as well as parents, and impact the weight status of students. By leveraging a unique collaboration between Texas AgriLife Extension (a federal, state and county funded educational outreach organization) and the University of Texas School of Public Health, the Texas Grow! Eat! Go! Study (TGEG) modeled the effectiveness of utilizing existing programs and volunteer infrastructure to disseminate an enhanced Coordinated School Health program. The five-year TGEG study was developed to assess the independent and combined impact of gardening, nutrition and physical activity intervention(s) on the prevalence of healthy eating, physical activity and weight status among low-income elementary students. The purpose of this paper is to report on study design, baseline characteristics, intervention approaches, data collection and baseline data. Methods The study design for the TGEG study consisted of a factorial group randomized controlled trial (RCT) in which 28 schools were randomly assigned to one of 4 treatment groups: (1) Coordinated Approach to Child Health (CATCH) only (Comparison), (2) CATCH plus school garden intervention [Learn, Grow, Eat & Go! (LGEG)], (3) CATCH plus physical activity intervention [Walk Across Texas (WAT)], and (4) CATCH plus LGEG plus WAT (Combined). The outcome variables include student’s weight status, vegetable and sugar sweetened beverage consumption, physical activity, and sedentary behavior. Parents were assessed for home environmental variables including availability of certain foods, social support of student health behaviors, parent engagement and behavior modeling. Results Descriptive data are presented for students (n = 1369) and parents (n = 1206) at baseline. The sample consisted primarily of Hispanic and African American (53 % and 18 %, respectively) and low-income (i.e., 78 % eligible for Free and Reduced Price School Meals program and 43 % food insecure) students. On average, students did not meet national guidelines for vegetable consumption or physical activity. At baseline, no statistical differences for demographic or key outcome variables among the 4 treatment groups were observed. Conclusions The TGEG study targets a population of students and parents at high risk of obesity and related chronic conditions, utilizing a novel and collaborative approach to program formulation and delivery, and a rigorous, randomized study design.

Approximately 20% of early-stage breast cancers display amplification or overexpression of the ErbB2/HER2 oncogene, conferring poor prognosis and resistance to endocrine therapy. Targeting HER2+ tumors with trastuzumab or the receptor tyrosine kinase (RTK) inhibitor lapatinib significantly improves survival, yet tumor resistance and progression of metastatic disease still develop over time. Although the mechanisms of cytosolic HER2 signaling are well studied, nuclear signaling components and gene regulatory networks that bestow therapeutic resistance and limitless proliferative potential are incompletely understood. Here, we use biochemical and bioinformatic approaches to identify effectors and targets of HER2 transcriptional signaling in human breast cancer. Phosphorylation and activity of the Steroid Receptor Coactivator-3 (SRC-3) is reduced upon HER2 inhibition, and recruitment of SRC-3 to regulatory elements of endogenous genes is impaired. Transcripts regulated by HER2 signaling are highly enriched with E2F1 binding sites and define a gene signature associated with proliferative breast tumor subtypes, cell-cycle progression, and DNA replication. We show that HER2 signaling promotes breast cancer cell proliferation through regulation of E2F1-driven DNA metabolism and replication genes together with phosphorylation and activity of the transcriptional coactivator SRC-3. Furthermore, our analyses identified a cyclin-dependent kinase (CDK) signaling node that, when targeted using the CDK4/6 inhibitor palbociclib, defines overlap and divergence of adjuvant pharmacologic targeting. Importantly, lapatinib and palbociclib strictly block de novo synthesis of DNA, mostly through disruption of E2F1 and its target genes. These results have implications for rational discovery of pharmacologic combinations in preclinical models of adjuvant treatment and therapeutic resistance. Cancer Res; 76(6); 1463–75. ©2016 AACR.

Background Computer-based educational games present an opportunity for health education in school; however, their feasibility in school settings and effectiveness in changing behavior are poorly understood. Objective To evaluate the feasibility, acceptability, and effects of the Quest to Lava Mountain (QTLM) computer game on dietary behaviors, physical activity behaviors, and psychosocial factors among ethnically diverse children in Texas. Design Quasi-experimental group-randomized controlled trial conducted during the 2012-2013 school year. Participants/setting A total of 107 children in fourth and fifth grade consented. There was an attrition rate of 8.8% with a final sample size of 44 children in three intervention schools, and a sample of 50 children in three comparison schools. Dietary intake was measured using two random 24-hour recalls, whereas child self-report surveys measured diet, physical activity, and psychosocial factors before and after the intervention. Process data on QTLM usability and back-end server data on QTLM exposure and progress achieved were collected. Intervention QTLM was implemented as part of the in-school or afterschool program. Recommended game exposure duration was 90 min/wk for 6 weeks. Statistical analysis Analysis of covariance or logistic regression models evaluated effects of QTLM on diet, physical activity, and psychosocial factors. Post hoc exploratory analysis examined the changes before and after the intervention in outcome variables among children in the intervention group. Significance was set at P Results Children played an average of 274±110 minutes (approximately 4.6 hours) of QTLM during the 6 weeks (51% of recommended dosage). Compared with the comparison group, children in the intervention group reported decreased sugar consumption ( P =0.021) and higher nutrition/physical activity attitudes ( P =0.041) pre- to postintervention. There were no significant effects of QTLM on physical activity. However, post hoc analysis showed that higher QTLM exposure and gaming progress was associated with increased frequency of physical activity ( P Conclusions QTLM has some promising acceptability and initial effects on diet and physical activity behaviors among children in elementary school.

Chromatin assembly factor-1 contains a separable domain unrelated to histone deposition, which provides a previously unrecognized ability to maintain nucleolar protein and chromosome associations., Chromatin assembly factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element–containing loci. Of note, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishes nucleolar associations, whereas PCNA or HP1 interaction sites within p150 are not required for these interactions. In addition, acute depletion of SUMO-2 or the SUMO E2 ligase Ubc9 reduces α-satellite DNA association with nucleoli. The nucleolar functions of p150 are separable from its interactions with the other subunits of the CAF-1 complex because an N-terminal fragment of p150 (p150N) that cannot interact with other CAF-1 subunits is sufficient for maintaining nucleolar chromosome and protein associations. Therefore these data define novel functions for a separable domain of the p150 protein, regulating protein and DNA interactions at the nucleolus.

The SMRT coregulator functions as a dual coactivator and corepressor for estrogen receptor-α (ERα) in a gene-specific manner, and in several studies its elevated expression correlates with poor outcome for breast cancer patients. A specific role of SMRT in breast cancer progression has not been elucidated, but SMRT knock-down limits estradiol-dependent growth of MCF-7 breast cancer cells. In this study, small-interfering RNA (siRNA) and short-hairpin RNA (shRNA) approaches were used to determine the effects of SMRT depletion on growth of ERα-positive MCF-7 and ZR-75–1 breast cancer cells, as well as the ERα-negative MDA-MB-231 breast cancer line. Depletion of SMRT inhibited growth of ERα-positive cells grown in monolayer but had no effect on growth of the ERα-negative cells. Reduced SMRT levels also negatively impacted the anchorage-independent growth of MCF-7 cells as assessed by soft agar colony formation assays. The observed growth inhibitions were due to a loss of estradiol-induced progression through the G1/S transition of the cell cycle and increased apoptosis in SMRT-depleted compared with control cells. Gene expression analyses indicated that SMRT inhibits apoptosis by a coordinated regulation of genes involved in apoptosis. Functioning as a dual coactivator for anti-apoptotic genes and corepressor for pro-apoptotic genes, SMRT can limit apoptosis. Together these data indicate that SMRT promotes breast cancer progression through multiple pathways leading to increased proliferation and decreased apoptosis.

The silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) is an established histone deacetylase 3 (HDAC3)-dependent transcriptional corepressor. Microarray analyses of MCF-7 cells transfected with control or SMRT small interfering RNA revealed SMRT regulation of genes involved in DNA damage responses, and the levels of the DNA damage marker γH2AX as well as poly(ADP-ribose) polymerase cleavage were elevated in SMRT-depleted cells treated with doxorubicin. A number of these genes are established p53 targets. SMRT knockdown decreased the activity of two p53-dependent reporter genes as well as the expression of p53 target genes, such as CDKN1A (which encodes p21). SMRT bound directly to p53 and was recruited to p53 binding sites within the p21 promoter. Depletion of GPS2 and TBL1, components of the SMRT corepressor complex, but not histone deacetylase 3 (HDAC3) decreased p21-luciferase activity. p53 bound to the SMRT deacetylase activation domain (DAD), which mediates HDAC3 binding and activation, and HDAC3 could attenuate p53 binding to the DAD region of SMRT. Moreover, an HDAC3 binding-deficient SMRT DAD mutant coactivated p53 transcriptional activity. Collectively, these data highlight a biological role for SMRT in mediating DNA damage responses and suggest a model where p53 binding to the DAD limits HDAC3 interaction with this coregulator, thereby facilitating SMRT coactivation of p53-dependent gene expression.

Estrogen receptor (ER) antagonists are valuable in the treatment of ER-positive human breast cancer. In this study, we designed and synthesized nine new derivatives of 17β-estradiol (E2) with a bulky side chain attached to its C-7α position, and determined their ER antagonistic activity using in vitro bioassays. Four of the derivatives showed a strong inhibition of ERα transactivation activity in a luciferase reporter assay and blocked ERα interactions with coactivators. Similarly, these derivatives also strongly inhibited the growth of the ERα-positive human breast cancer cells. Computational docking analysis was conducted to model the interaction of these antagonists with the human ERα and showed that they could tightly bind to the ERα in a manner similar to that of ICI-182,780, a pure ER antagonist. These results provide an example that attachment of a bulky side chain to the C-7α position of E2 can produce ER antagonists with ER affinity comparable to that of ICI-182,780.

Estrogen receptor (ER) antagonists are generally thought to inhibit estrogen action through competitive inhibition, resulting in receptor binding to antagonist rather than agonist. However, microarray analyses reveal a group of genes for which ER agonist and antagonist cooperatively regulate expression, suggesting additional models of combined agonist/antagonist action must exist. In conjunction with a chimeric reporter gene and two modified ERs, one [ERα(GSCKV)] with a mutation in the DNA-binding domain and the other (ERα-G521R) with a ligand-binding specificity mutation, we herein demonstrate that ER agonist and antagonist cooperatively activate gene expression through an ER heteroligand dimer complex (ER-HLD) consisting of one subunit of the receptor dimer bound to agonist and another occupied by antagonist. Coimmunoprecipitation experiments confirmed interaction between the agonist-bound and antagonist-bound receptors. This cooperative activation of gene expression was enhanced by steroid receptor coactivator 3 coactivator, and required each ligand-bound subunit of the dimer to bind to DNA, as well as both activation function 1 domains for maximal transcriptional activity. Ligand combinations able to induce ER-HLD transcriptional activity include the agonists 17β-estradiol or conjugated estrogens with the antagonists tamoxifen, raloxifene, bazedoxifene, or fulvestrant. Moreover, ER-HLD can activate transcription in the context of a natural promoter. Taken together, these findings broaden our understanding of the complex relationship between ER agonist and antagonist, and suggest a novel model by which cell and tissue selective effects of antiestrogens may be achieved.

Silencing mediator of retinoic acid and thyroid hormone receptor (SMRT), also known as nuclear corepressor 2 (NCOR2) is a transcriptional corepressor for multiple members of the nuclear receptor superfamily of transcription factors, including estrogen receptor-α (ERα). In the classical model of corepressor action, SMRT binds to antiestrogen-bound ERα at target promoters and represses ERα transcriptional activity and gene expression. Herein SMRT mRNA and protein expression was examined in a panel of 30 breast cancer cell lines. Expression of both parameters was found to vary considerably amongst lines and the correlation between protein and mRNA expression was very poor (R (2) = 0.0775). Therefore, SMRT protein levels were examined by immunohistochemical staining of a tissue microarray of 866 patients with stage I-II breast cancer. Nuclear and cytoplasmic SMRT were scored separately according to the Allred score. The majority of tumors (67 %) were negative for cytoplasmic SMRT, which when detected was found at very low levels. In contrast, nuclear SMRT was broadly detected. There was no significant difference in time to recurrence (TTR) according to SMRT expression levels in the ERα-positive tamoxifen-treated patients (P = 0.297) but the difference was significant in the untreated patients (P = 0.01). In multivariate analysis, ERα-positive tamoxifen-untreated patients with high nuclear SMRT expression (SMRT 5-8, i.e., 2nd to 4th quartile) had a shorter TTR (HR = 1.94, 95 % CI, 1.24-3.04; P = 0.004) while there was no association with SMRT expression for ERα-positive tamoxifen-treated patients. There was no association between SMRT expression and overall survival for patients, regardless of whether they received tamoxifen. Thus while SMRT protein expression was not predictive of outcome after antiestrogen therapy, it may have value in predicting tumor recurrence in patients not receiving adjuvant tamoxifen therapy.

Bladder cancer is the fifth most common type of cancer in the USA, with over 70,000 new cases diagnosed each year. Treatment often involves invasive surgical therapies, as chemotherapy alone is often ineffective and associated with high recurrence rates. Identification of estrogen receptor-β (ERβ) in up to 75 % of urinary tumors raises the question of whether this receptor could be targeted to effectively treat bladder cancer. In this study, a panel of five bladder cancer cell lines representing a variety of disease stage and grades were treated with the antiestrogens 4-hydroxytamoxifen, raloxifene, or the pure antagonist ICI 182,780. All cell lines were ERβ positive while only a few expressed estrogen receptor-α (ERα). Notably, all but the TCCSUP cell line were growth inhibited 20-100 % by at least two antiestrogens. Using RT4 cells, we demonstrate that growth inhibition by raloxifene is ER dependent and either ERα or ERβ can mediate this response. Activation of caspase-3 and its effector poly-ADP ribose polymerase (PARP) demonstrate that raloxifene-induced growth inhibition is in part the result of increased apoptosis; this PARP cleavage was ER dependent. Moreover, changes in the expression of cell cycle genes indicate that cell proliferation is also affected. Specifically, raloxifene treatment results in the stabilization of p27 protein, likely via the downregulation of S-phase kinase-associated protein (SKP2). Expression of the negative cell cycle regulator B-cell translocation gene (BTG2) is also increased, while cyclin D1 transcription is reduced. These results indicate that antiestrogens may be useful therapeutics in the treatment of bladder cancer by targeting ER and inhibiting growth via multiple mechanisms.

Pure antiestrogens were clinically developed as alternative therapies for estrogen receptor (ER) positive breast cancers. Unlike the selective estrogen receptor modulators (SERMs), these antiestrogens are devoid of tissue-specific ER agonist activity. Many of these compounds are steroidal in nature, containing an estradiol (E2) structural core with long alkyl side chains at the C-7α position. Two novel 7α-substituted E2 derivatives were evaluated that retain high binding affinity for ER. Compared to known pure antiestrogens, these compounds, referred to as compound 13 (C13) and C14, contain shorter 7α alkyl side chains and differ only in their terminal substituent: a hydroxyl moiety versus a benzyloxy group, respectively. Herein we assessed the effects of these compounds on ER transcriptional activity and report that despite their similar overall structure, C13 and C14 produce distinct cell type-specific responses. Of note, C13 functions as a mixed agonist/antagonist in Hela cells, inducing only weak ER transcriptional activity while preventing coactivator recruitment and stabilizing ER expression. However, this compound effectively stimulates ER activity in MCF-7 cells, does not increase ER levels and promotes cell proliferation on par with E2. Conversely, C14 stimulates transcriptional activity in both cell types and enhances ER–coactivator interactions. The activities of both compounds were inhibited by the pure antiestrogen ICI 182,780. Taken together, these results reveal that C13 is a SERM while C14 is an ER agonist, and indicate that the terminal modification of the C-7α hexanyl side chain of these estradiol derivatives is an important determinant of the biocharacter of these ER ligands.

SUMMARY Small molecules stabilize specific protein conformations from a larger ensemble, enabling molecular switches that control diverse cellular functions. We show here that the converse also holds true, where the conformational state of the estrogen receptor can direct distinct orientations of the bound ligand. “Gain of allostery” mutations that mimic the effects of ligand in driving protein conformation allowed crystallization of the partial agonist ligand WAY-169916 with both the canonical active and inactive conformations of the estrogen receptor. The intermediate transcriptional activity induced by WAY169916 is associated with the ligand binding differently to the active and inactive conformations of the receptor. Analyses of a series of chemical derivatives demonstrated that altering the ensemble of ligand binding orientations changes signaling output. The coupling of different ligand binding orientations to distinct active and inactive protein conformations defines a novel mechanism for titrating allosteric signaling activity.

B-cell Translocation Gene 2 (BTG2/TIS21/PC3) is an anti-proliferative tumor suppressor gene whose expression is significantly reduced in breast carcinomas, and in MCF-7 and T-47D breast cancer cell lines treated with estradiol (E2). In this study the mechanisms involved in E2 down regulation of BTG2 gene expression were examined. Depletion of ERalpha by siRNA indicated that the receptor is required for E2 down regulation of BTG2 mRNA levels, and cycloheximide experiments indicated that the effect of E2 on BTG2 expression was independent of intermediary protein synthesis. Chromatin immunoprecipitation analyses revealed that ERalpha interacts with the BTG2 promoter in a ligand-independent fashion whereas transfection experiments indicated that ERalpha's DNA and ligand binding domains are required for E2 repression of BTG promoter activity. Surprisingly, histone deacetylase (HDACs) activity is essential for basal expression as evidenced by trichostatin A inhibition of BTG2 mRNA levels. Estradiol treatment did not alter histone H3 acetylation although it did induce displacement of RNA polymerase II from the BTG2 gene. Depletion of the ER specific corepressor REA (Repressor of Estrogen Receptor Activity) significantly abrogated E2-mediated BTG2 repression. Taken together, our results reveal a requirement of HDAC activity for basal BTG2 expression and the ERalpha-REA interaction for estrogen repression of the BTG2 gene. The ability of E2-bound ERalpha and REA to suppress BTG2 expression indicates a positive role for this corepressor in regulation of breast cancer cell proliferation.

In 2001, the 77th Texas legislature passed Senate Bill 19 requiring students in publicly funded elementary schools to participate in physical activity and schools to implement a coordinated school health program by September 1, 2007. We report on awareness of and adherence to Senate Bill 19 in a statewide sample of elementary schools and a subsample in two public health regions located along the Texas-Mexico border. Statewide, structured interviews with principals indicated high awareness of Senate Bill 19's requirements, but lower awareness of the need for parental involvement. Only 43% of Texas schools had adopted their coordinated program 1 year or less before the implementation deadline. Principals reported an average of 179 min of physical education per week, higher than the 135-min mandate. Among the subsample border schools, principals' physical activity reports were consistent with teacher records and student reports. Further, direct observation of physical education indicated that 50% of class time was spent in moderate and vigorous physical activity, meeting the level of physical activity intensity recommended by Healthy People 2010. The differences observed by public health regions included: more physical activity minutes in Region 10 (231 min compared to 217 min in Region 11); higher adoption of coordinated school health programs (92% compared to 75%); more district-level school health advisory committees (58% compared to 38%) and school-level school health advisory committees (83% compared to 25%); and a lower prevalence of obesity in 4th grade students (21% compared to 32%). Differences by region suggest that Senate Bill 19 is not being adhered to equally across the state, and some regions may require further support to increase implementation. Results underscore the importance of continued monitoring of enacted legislation, and that legislation for child health that focuses on school programs and policies requires funding and refinement to produce the intended effect.

Multiple factors influence estrogen receptor alpha (ERalpha) transcriptional activity. Current models suggest that the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor functions within a histone deactylase-containing protein complex that binds to antiestrogen-bound ERalpha and contributes to negative regulation of gene expression. In this report, we demonstrate that SMRT is required for full agonist-dependent ERalpha activation. Chromatin immunoprecipitation assays demonstrate that SMRT, like ERalpha and the SRC-3 coactivator, is recruited to an estrogen-responsive promoter in estrogen-treated MCF-7 cells. Depletion of SMRT, but not histone deacetylases 1 or 3, negatively impacts estradiol-stimulated ERalpha transcriptional activity, while exogenous expression of SMRT's receptor interaction domains blocks ERalpha activity, indicating a functional interaction between this corepressor and agonist-bound ERalpha. Stimulation of estradiol-induced ERalpha activity by SMRT overexpression occurred in HeLa and MCF-7 cells, but not HepG2 cells, indicating that these positive effects are cell type specific. Similarly, the ability of SMRT depletion to promote the agonist activity of tamoxifen was observed for HeLa but not MCF-7 cells. Furthermore, impairment of agonist-stimulated activity by SMRT depletion is specific to ERalpha and not observed for receptors for vitamin D, androgen, or thyroid hormone. Nuclear receptor corepressor (N-CoR) depletion increased the transcriptional activity of all four tested receptors. SMRT is required for full expression of the ERalpha target genes cyclin D1, BCL-2, and progesterone receptor but not pS2, and its depletion significantly attenuated estrogen-dependent proliferation of MCF-7 cells. Taken together, these data indicate that SMRT, in conjunction with gene-specific and cell-dependent factors, is required for positively regulating agonist-dependent ERalpha transcriptional activity.

A number of available treatments provide relief of menopausal symptoms and prevention of postmenopausal osteoporosis. However, as breast safety is a major concern, new options are needed, particularly agents with an improved mammary safety profile. Results from several large randomized and observational studies have shown an association between hormone therapy, particularly combined estrogen-progestin therapy, and a small increased risk of breast cancer and breast pain or tenderness. In addition, progestin-containing hormone therapy has been shown to increase mammographic breast density, which is an important risk factor for breast cancer. Selective estrogen receptor modulators (SERMs) provide bone protection, are generally well tolerated, and have demonstrated reductions in breast cancer risk, but do not relieve menopausal symptoms (that is, vasomotor symptoms). Tissue-selective estrogen complexes (TSECs) pair a SERM with one or more estrogens and aim to blend the positive effects of the components to provide relief of menopausal symptoms and prevention of postmenopausal osteoporosis without stimulating the breast or endometrium. One TSEC combination pairing conjugated estrogens (CEs) with the SERM bazedoxifene (BZA) has completed clinical development and is now available as an alternative option for menopausal therapy. Preclinical evidence suggests that CE/BZA induces inhibitory effects on breast tissue, and phase 3 clinical studies suggest breast neutrality, with no increases seen in breast tenderness, breast density, or cancer. In non-hysterectomized postmenopausal women, CE/BZA was associated with increased bone mineral density and relief of menopausal symptoms, along with endometrial safety. Taken together, these results support the potential of CE/BZA for the relief of menopausal symptoms and prevention of postmenopausal osteoporosis combined with breast and endometrial safety.

Prostate cancer is an androgen-dependent disease; metastatic prostate cancer is typically treated by androgen receptor (AR) blockade. Recurrence after androgen ablation and evidence that AR continues to play a role in many prostate cancers has led to an examination of other factors that potentiate AR activity. AR is a ligand-activated transcription factor whose activity is regulated not only by hormone but also by the levels of coactivators recruited by AR to facilitate transcription. We sought to assess the consequences of reducing expression of the transcription intermediary factor 2 (TIF2) coactivator on prostate cancer cell growth and AR action in cell lines to examine TIF2 expression in prostate cancer and to correlate expression with clinical outcome. Depletion of TIF2 reduced expression of AR-induced target genes and slowed proliferation of AR-dependent and AR-independent prostate cancer cells. Remarkably, we found that TIF2 expression is directly repressed by high levels of androgens in multiple AR-expressing cell lines. Expression of a reporter containing 5′-flanking region of the TIF2 was repressed both by androgens and by the antagonist, Casodex. Expression of TIF2 correlates with biochemical (prostate-specific antigen) recurrence (P = 0.0136). In agreement with our in vitro findings, the highest expression of TIF2 was found in patients whose cancer relapsed after androgen ablation therapy, supporting the idea that AR blockade might activate pathways that lead to stimulation of AR-dependent and AR-independent proliferation of prostate epithelium. The elevated expression of TIF2 at low hormone levels likely aids in inducing AR activity under these conditions; treatment with Casodex has the potential to counteract this induction. (Cancer Res 2006; 66(21): 10594-602)

BACKGROUND Estrogen receptors (ERs) are known to mediate important physiologic responses as well as the growth of some tumors in response to estradiol stimulation. In a previous study the selective ER modulator raloxifene was shown to induce apoptosis in an ERβ-positive bladder cancer cell line. However, the expression of ERβ in human bladder cancer has not been thoroughly investigated. METHODS ERα and ERβ expression in 224 bladder tumor samples was evaluated using tissue microarray and immunohistochemistry. Levels of ERα and ERβ protein and mRNA expression were determined in several bladder cancer cell lines using quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. The effect of estradiol and antiestrogen treatments on RT4 bladder cancer cell growth was determined by cell proliferation assays. RESULTS Analyses revealed that only 2 human bladder cancers weakly expressed ERα. In contrast, the expression of ERβ was detected in 141 tumors (63%). ERβ was expressed in 58% of WHO Grade 1 and 2 tumors, whereas 70% of Grade 3 tumors demonstrated expression (P=.085). Importantly, although only 53% and 55% of Ta and T1 tumors demonstrated ERβ expression, 80% of T2, 81% of T3, and 75% of T4 tumors showed ERβ expression. The differences in ERβ expression between Ta/T1 and T2/T3/T4 tumors were found to be highly significant (P

Prostate cancer is initially androgen dependent and there is evidence that androgen receptor continues to play a role in androgen-independent prostate cancer. Androgen receptor activity depends both on the level of androgens and on the level of coactivators that interact with androgen receptor. Our goal was to evaluate the role of the androgen receptor coactivator SRC-1 in prostate cancer progression. Using tissue arrays to measure SRC-1 protein levels, we found that increased SRC-1 expression in clinically localized, androgen-dependent cancer is associated with clinical and pathologic variables of increased tumor aggressiveness. Interestingly, there was variable expression of SRC-1 in normal prostate tissue which correlated with the staining intensity of the corresponding cancer tissue. To test the contribution of SRC-1, we examined its role in androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer cell lines. Using small interfering RNA to reduce expression of androgen receptor, we found that androgen receptor was required both for cell growth and for basal expression of prostate-specific antigen in the androgen-independent C4-2 cell line. Thus, although the cells can grow in an androgen-depleted medium, they remained androgen receptor dependent. Reduction of SRC-1 expression significantly reduced growth and altered androgen receptor target gene regulation in both LNCaP and C4-2 cell lines whereas it had no effect on the growth of the androgen receptor–negative PC-3 and DU145 prostate cancer cell lines. Although the requirement for androgens and androgen receptor in the development of prostate cancer is well established, our study implicates enhanced androgen receptor activity through elevated expression of SRC-1 in the development of more aggressive disease in men with prostate cancer.

SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1 is a primary transcriptional coregulator for estrogen receptor (ER). Six SRC-3 phosphorylation sites have been identified, and these can be induced by steroids, cytokines, and growth factors, involving multiple kinase signaling pathways. Using phosphospecific antibodies for six phosphorylation sites, we investigated the mechanisms involved in estradiol (E2)-induced SRC-3 phosphorylation and found that this occurs only when either activated estrogen receptor alpha (ERalpha) or activated ERbeta is present. Both the activation function 1 and the ligand binding domains of ERalpha are required for maximal induction. Mutations in the coactivator binding groove of the ERalpha ligand binding domain inhibit E2-stimulated SRC-3 phosphorylation, as do mutations in the nuclear receptor-interacting domain of SRC-3, suggesting that ERalpha must directly contact SRC-3 for this posttranslational modification to take place. A transcriptionally inactive ERalpha mutant which localizes to the cytoplasm supports E2-induced SRC-3 phosphorylation. Mutations of the ERalpha DNA binding domain did not block this rapid E2-dependent SRC-3 phosphorylation. Together these data demonstrate that E2-induced SRC-3 phosphorylation is dependent on a direct interaction between SRC-3 and ERalpha and can occur outside of the nucleus. Our results provide evidence for an early nongenomic action of ER on SRC-3 that supports the well-established downstream genomic roles of estrogen and coactivators.

Steroid receptor coactivator-3 (SRC-3/AIB1) is a coactivator for nuclear receptors and other transcription factors and an oncogene that contributes to growth regulation and development of mammary and other tumor types. Because of its biological functions, it is important to identify genes regulated by SRC-3. However, because coactivators do not bind DNA directly, extensive work is required to determine whether genes identified by RNA profiling approaches are direct or indirect targets. Here, we report the use of chromatin immunoprecipitation (ChIP)-based assays that involve genomic mapping and computational analyses of immunoprecipitated DNA to identify SRC-3-binding target genes in estradiol (E2)-treated MCF-7 breast cancer cells. We identified 18 SRC-3 genomic binding sites and demonstrated estrogen receptor-alpha (ERalpha) binding to all of them. Both E2-dependent and -independent SRC-3/ERalpha-binding sites were identified. RNA polymerase II ChIP assays were used to determine the correlation between SRC-3 and ERalpha binding and recruitment of the transcriptional machinery. These assays, in conjunction with analyses of RNA obtained from E2-treated cells, lead to the identification of SRC-3/ERalpha-associated genes. The ability of SRC family coactivators to regulate the expression of one of these genes, PARD6B/Par6, was confirmed by using cells individually depleted of SRC-1, SRC-2, or SRC-3 by small interfering RNA. The method described herein can be used to identify genes regulated by non-DNA-binding factors, such as other coactivators or corepressors, as well as DNA-binding transcription factors, and provides information on their binding location that can accelerate further gene characterization.

Regulation of gene expression requires dynamic changes in chromatin, but the nature of these changes is not well understood. Here, we show that progesterone treatment of cultured cells leads to recruitment of progesterone receptor (PR) and SWI/SNF-related complexes to Mouse Mammary Tumor Virus (MMTV) promoter, accompanied by displacement of histones H2A and H2B from the nucleosome containing the receptor binding sites, but not from adjacent nucleosomes. PR recruits SWI/SNF to MMTV nucleosomes in vitro and facilitates synergistic binding of receptors and nuclear factor 1 to the promoter. In nucleosomes assembled on MMTV or mouse rDNA promoter sequences, SWI/SNF catalyzes ATP-dependent sliding of the histone octamer followed only on the MMTV promoter by displacement of histones H2A and H2B. In MMTV nucleosome arrays, SWI/SNF displaces H2A and H2B from nucleosome B and not from the adjacent nucleosome. Thus, the outcome of nucleosome remodeling by SWI/SNF depends on DNA sequence.

The ability of steroid receptor RNA activator (SRA), an AF-1 coactivator, to contribute to differences in estrogen receptor (ER)-alpha and ERbeta transcriptional activity was tested. In transient transfections, SRA expression increased ERalpha- and ERbeta-dependent gene expression. However, when the receptors' amino-terminal A/B regions were examined as GAL4 DNA binding domain fusions, SRA enhanced the activity of GAL-ABalpha but not GAL-ABbeta. Exogenous SRA also enhanced AF-2 activity for both receptors, indicating that SRA effects are not limited to AF-1. Simultaneously mutating three phosphorylation sites within GAL-ABalpha domain only modestly reduced SRA coactivation of GAL-ABalpha, suggesting that phosphorylation does not play a major role in SRA function relative to this domain. SRA enhanced ERalpha activity stimulated by 4-hydroxytamoxifen, but was unable to convert this mixed antiestrogen to an ERbeta agonist. Thus, SRA is an ERalpha AF-1-specific coactivator that enhances the agonist activity of tamoxifen-bound ERalpha and may contribute to tamoxifen resistance.

Estrogen receptor (ER)alpha and ERbeta are transcription factors that can be activated by both ligand binding and growth factor signaling. Estradiol increases ER activity in part by enhancing interactions between its carboxy-terminal, ligand-binding domain (LBD) and the p160/SRC (steroid receptor coactivator) and p300/CBP (cAMP response element binding protein (CREB) binding protein) families of coactivators. In the absence of ligand and the LBD, these cofactors can also interact with the amino-terminal (A/B) domain of ERs in vitro. SRC-1 also enhances the ligand-independent activity of the full-length receptor. Both ligand-independent and estradiol-induced ER activity are increased by phosphorylation at specific serine (Ser) residues in the A/B domain (Ser104/106 and Ser118 in ERalpha). In the case of ERbeta, phosphorylation enhances the ligand-independent recruitment and action of SRC-1. We show here that unliganded ERalpha can activate endogenous gene expression in MCF-7 cells, and that this activation is mediated in part by a p160 coactivator. In transfected HeLa cells, we show that the full-length ERalpha can interact physically and functionally with p160/SRCs and CBP in the absence of ligand and that mutation of Ser104/106/118 affects these interactions. Accordingly, ERalpha dephosphorylation decreases its ligand-independent interaction with SRC-1 and CBP in vitro. In HeLa cells, both Ser104/106 and Ser118 impinge on SRC-1 action by two mechanisms: 1) a seemingly indirect effect on SRC-1 recruitment that requires other receptor domains in addition to the A/B, consistent with our finding that the ligand-independent interaction between the A/B and the LBD and its enhancement by SRC-1 depend in part on Ser104/106/118; and 2) an effect on SRC-1 coactivation that can be observed in the absence of the LBD. Ser104/106/118 can also affect coactivation by a subset of coactivators in the presence of hormone, albeit to a lesser extent than in its absence. Altogether, our observations suggest that the enhancement of ERalpha activity by p160/SRCs and CBP can be regulated by phosphorylation and stress the importance of using full-length receptors to assess the role of the activation function-1 in cofactor recruitment.

Although increases in intracellular cAMP can stimulate estrogen receptor-alpha (ER alpha) activity in the absence of exogenous hormone, no studies have addressed whether ER beta can be similarly regulated. In transient transfections, forskolin plus 3-isobutyl-1-methylxanthine (IBMX), which increases intracellular cAMP, stimulated the transcriptional activities of both ER alpha and ER beta. This effect was blocked by the protein kinase A inhibitor H89 (N-(2-(p-bromocinnamylamino)-ethyl)-5-isoquinolinesulfonamide) and was dependent on an estrogen response element. A 12-O-tetradecanoylphorbol-13-acetate response element (TRE) located 5' to the estrogen response element was necessary for cAMP-dependent activation of gene expression by ER beta but not ER alpha, indicating that the former subtype requires a functional interaction with TRE-interacting factor(s) to stimulate transcription. Both p160 and CREB-binding protein coactivators stimulated cAMP-induced ER alpha and ER beta transcriptional activity. However, mutation of the two cAMP-inducible SRC-1 phosphorylation sites important for cAMP activation of chicken progesterone receptor or all seven known SRC-1 phosphorylation sites did not specifically impair cAMP activation of ER alpha. The E/F domains of ER alpha are sufficient for activation by forskolin/IBMX, and this is accompanied by an increase in receptor phosphorylation. In contrast, cAMP signaling reduces the phosphorylation of the corresponding region of ER beta, and this correlates with the lack of forskolin/IBMX stimulated transcriptional activity. Our data suggest that cAMP activation of ER alpha transcriptional activity is associated with receptor instead of SRC-1 phosphorylation. Moreover, differences in the cofactor requirements, domains of ER alpha and ER beta sufficient for forskolin/IBMX activation, and the effect of cAMP on receptor phosphorylation indicate that this signaling pathway utilizes distinct mechanisms to stimulate ER alpha and ER beta transcriptional activity.

We find that Bax, a proapoptotic member of the Bcl-2 family, translocates to discrete foci on mitochondria during the initial stages of apoptosis, which subsequently become mitochondrial scission sites. A dominant negative mutant of Drp1, Drp1K38A, inhibits apoptotic scission of mitochondria, but does not inhibit Bax translocation or coalescence into foci. However, Drp1K38A causes the accumulation of mitochondrial fission intermediates that are associated with clusters of Bax. Surprisingly, Drp1 and Mfn2, but not other proteins implicated in the regulation of mitochondrial morphology, colocalize with Bax in these foci. We suggest that Bax participates in apoptotic fragmentation of mitochondria.

Skeletal unloading in an animal hindlimb suspension model and microgravity experienced by astronauts or as a result of prolonged bed rest causes site-specific losses in bone mineral density of 1%-2% per month. This is accompanied by reductions in circulating levels of 1,25-(OH)(2)D(3), the active metabolite of vitamin D. 1,25-(OH)(2)D(3), the ligand for the vitamin D receptor (VDR), is important for calcium absorption and plays a role in differentiation of osteoblasts and osteoclasts. To examine the responses of cells to activators of the VDR in a simulated microgravity environment, we used slow-turning lateral vessels (STLVs) in a rotating cell culture system. We found that, similar to cells grown in microgravity, MG-63 cells grown in the STLVs produce less osteocalcin, alkaline phosphatase, and collagen Ialpha1 mRNA and are less responsive to 1,25-(OH)(2)D(3). In addition, expression of VDR was reduced. Moreover, growth in the STLV caused activation of the stress-activated protein kinase pathway (SAPK), a kinase that inhibits VDR activity. In contrast, the 1,25-(OH)(2)D(3) analog, EB1089, was able to compensate for some of the STLV-associated responses by reducing SAPK activity, elevating VDR levels, and increasing expression of osteocalcin and alkaline phosphatase. These studies suggest that, not only does simulated microgravity reduce differentiation of MG-63 cells, but the activity of the VDR, an important regulator of bone metabolism, is reduced. Use of potent, less calcemic analogs of 1,25-(OH)(2)D(3) may aid in overcoming this defect.

Steroid receptor RNA activator (SRA) is a novel coactivator for steroid receptors that acts as an RNA molecule, whereas steroid receptor coactivator (SRC) family members, such as steroid receptor coactivator-1 (SRC-1) and transcriptional intermediary factor 2 (TIF2) exert their biological effects as proteins. Individual overexpression of each of these coactivators, which can form multimeric complexes in vivo, results in stimulated ERα transcriptional activity in transient transfection assays. However there is no information on the consequences of reducing SRC-1, TIF2, or SRA expression, singly or in combination, on ERα transcriptional activity. We therefore developed antisense oligodeoxynucleotides (asODNs) to SRA, SRC-1, and TIF2 mRNAs, which rapidly and specifically reduced the expression of each of these coactivators. ERα-dependent gene expression was reduced in a dose-dependent fashion by up to 80% in cells transfected with these oligonucleotides. Furthermore, treatment of cells with combinations of SRA, SRC-1, and TIF2 asODNs reduced ERα transcriptional activity to an extent greater than individual asODN treatment alone, suggesting that these coactivators cooperate, in at least an additive fashion, to activate ERα-dependent target gene expression. Finally, treatment of MCF-7 cells with asODN against SRC-1 and TIF2 revealed a requirement of these coactivators, but not SRA, for hormone-dependent DNA synthesis and induction of estrogen-dependent pS2 gene expression, indicating that SRA and SRC family coactivators can fulfill specific functional roles. Taken together, we have developed a rapid method to reduce endogenous coactivator expression that enables an assessment of the in vivo role of specific coactivators on ERα biological action and avoids potential artifacts arising from overexpression of coactivators in transient transfection assays.

It has previously been demonstrated that 19-nor contraceptive progestins undergo in vivo and in vitro enzyme-mediated A-ring double bond hydrogenation. Bioconversion of 19-nor progestins to their corresponding tetrahydro derivatives results in the loss of progestational activity and acquisition of estrogenic activities and binding to the ER. Herein, we report subtype-selective differences in ligand binding and transcriptional potency of nonphenolic synthetic 19-nor derivatives between ER alpha and ER beta. In this study, we have examined both ER- and PR-mediated transcriptional activity of a number of A-ring chemically reduced derivatives of norethisterone and Gestodene. Double bond hydrogenation decreased the transcriptional potency of norethisterone and Gestodene through both PR isoforms with a 100- to 1,000-fold difference, respectively. In terms of the effects of norethisterone and Gestodene and their corresponding 5 alpha-dihydro (5 alpha-norethisterone and 5 alpha-Gestodene), or 3 alpha,5 alpha-tetrahydro or 3 beta,5 alpha-tetrahydro derivatives (3 alpha,5 alpha-norethisterone/3 alpha,5 alpha-Gestodene and 3 beta,5 alpha-norethisterone/3beta,5 alpha-Gestodene, respectively) on estrogen-mediated transcriptional regulation, the 3 beta,5 alpha-tetrahydro derivatives of both norethisterone and Gestodene showed the highest induction when HeLa cells were transiently transfected with an expression vector for ER alpha. This activity could be inhibited with tamoxifen. These compounds did not activate gene transcription via ER beta, and none of them showed antagonistic activities through either ER subtype. The 3 beta,5 alpha-tetrahydro derivatives of both norethisterone and Gestodene were active in other cells in addition to HeLa cells and activated reporter expression through the oxytocin promoter. In summary, two ER alpha selective agonists have been identified. These compounds, with ER alpha vs. ER beta selective agonist activity, may be useful in evaluating the distinct role of these receptors as well as in providing useful insights into ER action.

Bax is a member of the Bcl-2 family of proteins known to regulate mitochondria-dependent programmed cell death. Early in apoptosis, Bax translocates from the cytosol to the mitochondrial membrane. We have identified by confocal and electron microscopy a novel step in the Bax proapoptotic mechanism immediately subsequent to mitochondrial translocation. Bax leaves the mitochondrial membranes and coalesces into large clusters containing thousands of Bax molecules that remain adjacent to mitochondria. Bak, a close homologue of Bax, colocalizes in these apoptotic clusters in contrast to other family members, Bid and Bad, which circumscribe the outer mitochondrial membrane throughout cell death progression. We found the formation of Bax and Bak apoptotic clusters to be caspase independent and inhibited completely and specifically by Bcl-XL, correlating cluster formation with cytotoxic activity. Our results reveal the importance of a novel structure formed by certain Bcl-2 family members during the process of cell death.

Estrogen receptor-alpha (ER alpha) is downregulated in the presence of its cognate ligand, estradiol (E2), through the ubiquitin proteasome pathway. Here, we show that ubiquitin proteasome function is required for ER alpha to serve as a transcriptional activator. Deletion of the last 61 amino acids of ER alpha, including residues that form helix 12, abolishes ligand-mediated downregulation of the receptor as do point mutations in the ligand binding domain that impair coactivator binding. In addition, coactivators also are subject to degradation by the 26S proteasome, but their intrinsic transcriptional activity is not affected. These data provide evidence that protein interactions with ER alpha coactivator binding surfaces are important for ligand-mediated receptor down-regulation and suggest that receptor and coactivator turnover contributes to ER alpha transcriptional activity.

Bax, a pro-apoptotic member of the Bcl-2 family, translocates from the cytosol to the mitochondria during programmed cell death. We report here that both gain-of-function and loss-of-function mutations can be achieved by altering a single amino acid in the Bax hydrophobic C-terminus. The properly mutated C-terminus of Bax can target a non-relevant protein to the mitochondria, showing that specific conformations of this domain alone allow mitochondrial docking. These data along with N-terminus epitope exposure experiments suggest that the C- and the N-termini interact and that upon triggering of apoptosis, Bax changes conformation, exposing these two domains to insert into the mitochondria and regulate the cell death machinery.

In this study, we found that the E6-associated protein (E6-AP/UBE3A) directly interacts with and coactivates the transcriptional activity of the human progesterone receptor (PR) in a hormone-dependent manner. E6-AP also coactivates the hormone-dependent transcriptional activities of the other members of the nuclear hormone receptor superfamily. Previously, it was shown that E6-AP serves the role of a ubiquitin-protein ligase (E3) in the presence of the E6 protein from human papillomavirus types 16 and 18. Our data show that the ubiquitin-protein ligase function of E6-AP is dispensable for its ability to coactivate nuclear hormone receptors, showing that E6-AP possesses two separable independent functions, as both a coactivator and a ubiquitin-protein ligase. Disruption of the maternal copy of E6-AP is correlated with Angelman syndrome (AS), a genetic neurological disorder characterized by severe mental retardation, seizures, speech impairment, and other symptoms. However, the exact mechanism by which the defective E6-AP gene causes AS remains unknown. To correlate the E6-AP coactivator function and ubiquitin-protein ligase functions with the AS phenotype, we expressed mutant forms of E6-AP isolated from AS patients and assessed the ability of each of these mutant proteins to coactivate PR or provide ubiquitin-protein ligase activity. This analysis revealed that in the majority of the AS patients examined, the ubiquitin-protein ligase function of E6-AP was defective whereas the coactivator function was intact. This finding suggests that the AS phenotype results from a defect in the ubiquitin-proteosome protein degradation pathway.

The classical receptor for estradiol is a member of a super-family of nuclear receptors that function as hormone-regulated transcription factors. The ability of the estrogen receptor (ER)-alpha to activate target gene transcription is mediated by two transcriptional activation functions (AF): AF-1 located in the amino-terminal domain and AF-2 found in the carboxyl-terminal portion of the molecule. The ligand binding domain overlaps AF-2, and upon estrogen binding this region undergoes a conformational change that enables it to contribute to the receptor's transcriptional activity. ER activation is accompanied by increased phosphorylation, and in the absence of ligand, activators of protein kinase A or inhibitors of protein phosphatases are able to stimulate ER-dependent gene expression. More importantly, polypeptide growth factors, such as epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I), also stimulate the ER's transcriptional activity in an estrogen-independent manner. The AF-1 domain appears to be required for activation by EGF and IGF-I, and point mutation of a single phosphorylation site located within this domain inhibits the ability of growth factor to activate the ER. Thus, steroid receptor function may be regulated by estrogenic ligands as well as by pathway "cross-talk" from membrane receptors for growth factors.

The Golgi complex is a dynamic organelle engaged in both secretory and retrograde membrane traffic. Here, we use green fluorescent protein–Golgi protein chimeras to study Golgi morphology in vivo. In untreated cells, membrane tubules were a ubiquitous, prominent feature of the Golgi complex, serving both to interconnect adjacent Golgi elements and to carry membrane outward along microtubules after detaching from stable Golgi structures. Brefeldin A treatment, which reversibly disassembles the Golgi complex, accentuated tubule formation without tubule detachment. A tubule network extending throughout the cytoplasm was quickly generated and persisted for 5–10 min until rapidly emptying Golgi contents into the ER within 15–30 s. Both lipid and protein emptied from the Golgi at similar rapid rates, leaving no Golgi structure behind, indicating that Golgi membranes do not simply mix but are absorbed into the ER in BFA-treated cells. The directionality of redistribution implied Golgi membranes are at a higher free energy state than ER membranes. Analysis of its kinetics suggested a mechanism that is analogous to wetting or adsorptive phenomena in which a tension-driven membrane flow supplements diffusive transfer of Golgi membrane into the ER. Such nonselective, flow-assisted transport of Golgi membranes into ER suggests that mechanisms that regulate retrograde tubule formation and detachment from the Golgi complex are integral to the existence and maintenance of this organelle.

Urothelial carcinoma of the bladder is a common malignancy that causes approximately 150,000 deaths per year worldwide. So far, no molecularly targeted agents have been approved for treatment of the disease. As part of The Cancer Genome Atlas project, we report here an integrated analysis of 131 urothelial carcinomas to provide a comprehensive landscape of molecular alterations. There were statistically significant recurrent mutations in 32 genes, including multiple genes involved in cell-cycle regulation, chromatin regulation, and kinase signalling pathways, as well as 9 genes not previously reported as significantly mutated in any cancer. RNA sequencing revealed four expression subtypes, two of which (papillary-like and basal/squamous-like) were also evident in microRNA sequencing and protein data. Whole-genome and RNA sequencing identified recurrent in-frame activating FGFR3-TACC3 fusions and expression or integration of several viruses (including HPV16) that are associated with gene inactivation. Our analyses identified potential therapeutic targets in 69% of the tumours, including 42% with targets in the phosphatidylinositol-3-OH kinase/AKT/mTOR pathway and 45% with targets (including ERBB2) in the RTK/MAPK pathway. Chromatin regulatory genes were more frequently mutated in urothelial carcinoma than in any other common cancer studied so far, indicating the future possibility of targeted therapy for chromatin abnormalities.

For several decades, a barrier has existed between research and clinical medicine, making it difficult for aspiring scientists to gain exposure to human pathophysiology and access to clinical/translational research mentors during their graduate training. In 2005, the Howard Hughes Medical Institute announced the Med Into Grad initiative to support graduate programs that integrate clinical knowledge into PhD biomedical training, with the goal of preparing a new cadre of translational researchers to work at the interface of the basic sciences and clinical medicine. Two institutions, Baylor College of Medicine and the Cleveland Clinic/Case Western Reserve University, developed new PhD programs in translational biology and/or molecular medicine. These programs teach the topics and skills that today’s translational researchers must learn as well as expose students to clinical medicine. In this article, the authors compare and contrast the history, implementation, and evaluation of the Translational Biology and Molecular Medicine program at Baylor College of Medicine and the Molecular Medicine program at the Cleveland Clinic/Case Western Reserve University. The authors also demonstrate the feasibility of creating a multidisciplinary graduate program in molecular medicine that integrates pathophysiology and clinical medicine without extending training time. They conclude with a discussion of the similarities in training approaches that exist despite the fact that each program was independently developed and offer observations that emerged during their collaboration that may benefit others who are considering developing similar programs.

Squirrel monkey (Saimiri sciureus) corticosteroid-binding globulin (CBG) is the product of a 1.6-kilobase mRNA in the liver. Analyses of two overlapping cDNAs revealed that the squirrel monkey CBG precursor comprises 406 amino acids, the first 22 residues of which exhibit 91% identity with the human CBG leader sequence. The mature form of squirrel monkey CBG, therefore, very likely comprises 384 amino acids and has a polypeptide mol wt of 42,854. Compared to human CBG, the squirrel monkey protein contains an additional residue (threonine) at position 144, and the two proteins exhibit 86% sequence identity if this is taken into account. Squirrel monkey CBG contains five consensus sites for N-glycosylation, four of which are located in analogous positions in human CBG, and has two cysteine residues in the same relative positions as the cysteines in human CBG. Unlike CBG in most other species, squirrel monkey CBG appears to circulate as a dimer, and its affinity for glucocorticoids is remarkably low. We, therefore, expressed cDNAs for human and squirrel monkey CBGs in Chinese hamster ovary (CHO) cells and compared the physico-chemical properties of the products with those of the corresponding serum proteins. Squirrel monkey CBG is produced by CHO cells as a dimer, and its subunit size heterogeneity is similar to that associated with CBG in serum. In addition, the cortisol-binding affinity of squirrel monkey CBG produced by CHO cells is similar to that of the natural protein and is 5- to 8-fold lower than that of natural or recombinant human CBG. Mutants in which a threonine at position 144 was either added to human CBG or subtracted from squirrel monkey CBG were also expressed in CHO cells. This demonstrated that this additional amino acid in the squirrel monkey CBG sequence may actively contribute to its propensity for spontaneous dimerization, but does not account for its relatively low steroid-binding affinity.

H IGH INTAKE OF SUGAR-SWEETENED BEVERAGES and energy-dense foods, as well as low intake of healthy foods such as fruits, vegetables, and whole grains, has been linked to childhood obesity in the United States. School and after-school settings are promising locations to promote healthy nutrition to children, but barriers such as the need to expend resources for staff trainings and frequent staff turnover hamper successful implementation. Innovative approaches are needed to mitigate these barriers while providing educational fidelity, seamless implementation within the school day, and minimal teacher oversight. Educational video games may contribute to a solution. During the last decade, increasing research and application of serious games has demonstrated a positive impact on children’s cognitive development, dietary habits, andphysical activity behavior. However, few evidence-based educational games are currently available for elementary school children. The purpose of this article is to describe the theoretical foundation, function, andpreliminary feasibility results of a new three-dimensional (3D) computer-based video game for nutrition education in children (8 to 12 years of age): The Quest to Lava Mountain (QTLM).

Confocal microscopy produces sharp images of structures within relatively thick specimens (up to several hundred microns). It is particularly useful for examining fluorescent specimens, such as fluorescent-stained cells or intact organisms (e.g., Drosophila or zebrafish embryos), and can localize intracellular antigens in dissociated cells. Its sensitivity even allows the monitoring of fluorescence in living organisms, making it possible to follow the movements of fluorescent probes such as GFP or dyes for specific organelles. This unit presents the theory of optical sectioning, describes the instrumentation of the technique, and offers practical guidelines for sample preparation and analysis.

It has been previously demonstrated that several members of the steroid receptor superfamily may be activated by the neurotransmitter dopamine in the apparent absence of cognate ligand. We have examined wild-type and mutant human estrogen receptors (ERs, [Gly400]ER and [Val400]ER, respectively) for their abilities to activate ER-dependent transcription of a transgene in a ligand-independent manner. In cells expressing the wild-type ER, dopamine was nearly as effective as 17 beta-estradiol at inducing the chloramphenicol acetyltransferase activity of the reporter gene in a dose-dependent manner; simultaneous addition of suboptimal concentrations of 17 beta-estradiol and dopamine stimulated transcription more than either compound alone. Dopamine alone was unable to induce gene expression in cells expressing [Val400]ER mutant receptors, but concomitant treatment with 17 beta-estradiol produced a synergistic increase in transcription, suggesting that the ligand may alter the mutant receptor's conformation such that it can be activated subsequently by a dopaminergic signaling mechanism. In the presence of the antiestrogen ICI 164,384, dopamine-stimulated gene expression was undetectable in cells expressing either form of ER. However, simultaneous treatment of cells expressing wild-type ER with trans-4-hydroxytamoxifen and dopamine resulted in transgene expression that was additive in nature compared to either compound alone; similar treatment of cells expressing [Val400]ER produced a synergistic increase. Our results suggest that ligand and ligand-independent activation of the ER initiate from distinct pathways and that the latter may occur in a variety of target tissues subject to modulation by receptor ligands.

Elevated expression of steroid receptor coactivator-3 (SRC-3), a member of the p160 family of nuclear receptor coactivators, has been implicated in tamoxifen resistance of breast tumors while the involvement of the two other members of this family, SRC-1 and SRC-2, is less well characterized. In this study, using small interfering RNA-based silencing, the role of each SRC coactivator in the growth of the LCC2 estrogen-independent and tamoxifen-resistant breast cancer cell line was evaluated. The loss of SRC-1, SRC-2, or SRC-3 did not significantly alter LCC2 proliferation or cell cycle distribution of 4-hydroxytamoxifen- versus vehicle-treated cells. However, depletion of SRC-2 and SRC-3, but not SRC-1, decreased basal cell proliferation and increased apoptosis. Cell cycle analyses further illustrated the divergent contributions of SRC-2 and SRC-3 with depletion of the former increasing the percentage of cells in the G0G1 and sub-G0G1 phases of cell cycle yet maintaining sensitivity to estradiol and ICI 182 780 antiestrogen, while SRC-3 depletion increased cells in the sub-G0G1 phase and ablated response to estrogen receptor a (ERa) ligands. Surprisingly, the effects of SRC coactivator depletion on ERa transcriptional activity, as measured by luciferase reporter gene, did not correspond to the observed effects on proliferation (e.g. SRC-1 knockdown increases ERa activity). Collectively, these data indicate that SRC control of basal and hormone-regulated proliferations is not solely mediated by ERa, and suggest that targeting growth inhibition by disrupting SRC-2 and SRC-3 function may be an effective approach to inhibit the growth of tamoxifen-resistant breast cancer. Endocrine-Related Cancer (2011) 18 113‐127

Although the ability of coactivators to enhance the expression of estrogen receptor-alpha (ERalpha) target genes is well established, the role of corepressors in regulating 17beta-estradiol (E2)-induced gene expression is poorly understood. Previous studies revealed that the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor is required for full ERalpha transcriptional activity in MCF-7 breast cancer cells, and we report herein the E2-dependent recruitment of SMRT to the regulatory regions of the progesterone receptor (PR) and cyclin D1 genes. Individual depletion of SMRT or steroid receptor coactivator (SRC)-3 modestly decreased E2-induced PR and cyclin D1 expression; however, simultaneous depletion revealed a cooperative effect of this coactivator and corepressor on the expression of these genes. SMRT and SRC-3 bind directly in an ERalpha-independent manner, and this interaction promotes E2-dependent SRC-3 binding to ERalpha measured by co-IP and SRC-3 recruitment to the cyclin D1 gene as measured by chromatin IP assays. Moreover, SMRT stimulates the intrinsic transcriptional activity of all of the SRC family (p160) coactivators. Our data link the SMRT corepressor directly with SRC family coactivators in positive regulation of ERalpha-dependent gene expression and, taken with the positive correlation found for SMRT and SRC-3 in human breast tumors, suggest that SMRT can promote ERalpha- and SRC-3-dependent gene expression in breast cancer.

Background Estrogen receptor α (ERα) phosphorylation is important for estrogen-dependent transcription of ER-dependent genes, ligand-independent receptor activation and endocrine therapy response in breast cancer. However ERα phosphorylation at the previously identified sites does not fully account for these receptor functions. To determine if additional ERα phosphorylation sites exist, COS-1 cells expressing human ERα were labeled with [32P]H3PO4 in vivo and ERα tryptic phosphopeptides were isolated to identify phosphorylation sites. Results Previously uncharacterized phosphorylation sites at serines 46/47, 282, 294, and 559 were identified by manual Edman degradation and phosphoamino acid analysis and confirmed by mutagenesis and phospho-specific antibodies. Antibodies detected phosphorylation of endogenous ERα in MCF-7, MCF-7-LCC2, and Ishikawa cancer cell lines by immunoblot. Mutation of Ser-282 and Ser-559 to alanine (S282A, S559A) resulted in ligand independent activation of ERα as determined by both ERE-driven reporter gene assays and endogenous pS2 gene expression in transiently transfected HeLa cells. Mutation of Ser-46/47 or Ser-294 to alanine markedly reduced estradiol dependent reporter activation. Additionally protein kinase CK2 was identified as a kinase that phosphorylated ERα at S282 and S559 using motif analysis, in vitro kinase assays, and incubation of cells with CK2 kinase inhibitor. Conclusion These novel ERα phosphorylation sites represent new means for modulation of ERα activity. S559 represents the first phosphorylation site identified in the extreme C-terminus (F domain) of a steroid receptor.

Oncogenesis in breast cancer often requires the overexpression of the nuclear receptor coactivator AIB1/SRC-3 acting in conjunction with estrogen receptor-alpha (ERalpha). Phosphorylation of both ERalpha and AIB1 has been shown to have profound effects on their functions. In addition, proteasome-mediated degradation plays a major role by regulating their stability and activity. CK1delta, a member of the ubiquitous casein kinase-1 family, is implicated in the progression of breast cancer. In this study, we show that both ERalpha and AIB1 are substrates for CK1delta in vitro, and identify a novel AIB1 phosphorylation site (S601) targeted by CK1delta, significant for the co-activator function of AIB1. CK1delta is able to interact with ERalpha and AIB1 in vivo, while overexpression of CK1delta in breast cancer cells results in an increased association of ERalpha with AIB1 as confirmed by co-immunoprecipitation assays from cell lysates. Using an siRNA-based approach, luciferase reporter assays and qRT-PCR, we observe that silencing of CK1delta leads to reduced ERalpha transcriptional activity, despite increased ERalpha levels, similarly to proteasome inhibition. We provide evidence that AIB1 protein levels are reduced by CK1delta silencing, in an estradiol-dependent manner; such destabilization can be inhibited by pre-treatment with the proteasome inhibitor MG132. We propose that differing activities adopted by ERalpha and AIB1 as a consequence of their interactions with and phosphorylation by CK1delta, particularly AIB1 stabilization, influence the transcriptional activity of ERalpha, and therefore have a role in breast cancer development.

In 2005, the Texas State Legislature passed Senate Bill 42 (SB42), which required public middle school students (grades 6-8) to participate in 30 minutes of daily structured physical activity. The purpose of this study was to assess awareness of and adherence to SB42 in Texas middle schools, and to assess the impact of SB42 on the frequency and quality of structured physical activity.Key informant (school principals, physical education [PE] instructors, nurses, or designated personnel) telephone interviews on the implementation of SB42 were conducted from a statewide representative sample of public middle schools (n=112). Direct observation, key informant, and student report of physical activity in PE classes at 17 Texas-Mexico border middle schools assessed the frequency and quality of structured physical activity.State level (94% +/- 4.5%) and border district (94% +/- 13.5%) key informants reported a high level of overall awareness of SB42. Postimplementation of SB42 border districts reported a minimum of 4 days per week of PE instruction and more than 58 minutes per PE class, exceeding the 30-minute minimum of structured physical activity per day or 135 minutes per week as required by SB42 (range: 58.2-61.4 minutes). A significant increase in the number of days of PE class was observed in the border sample between 2004 and 2005 and 2006 and 2008, with eighth grade students reporting an average of 2.0 days and 3.7 days of PE per week, respectively (p.001). Additionally, border districts met the Healthy People 2010 objective of 50% time in moderate-to-vigorous physical activity (mean 54.9% +/- 5.1%) during PE class.Implementation of SB42 appears to have impacted the frequency of school PE in Texas and the prevalence of child self-reported physical activity behaviors along the Texas-Mexico border. General awareness of and adherence to SB42 was high in both statewide and among the border districts. Our mixed findings on adherence to specific components of the legislation suggest the need for further investigation of the factors that both facilitate and inhibit local leadership around school policy and the mechanisms to ensure the school policy is being implemented.

The interaction between human corticosteroid binding globulin and activated leukocytes is restricted to the granulocyte population, and is characterized by specific proteolytic cleavage of corticosteroid binding globulin which markedly reduces its steroid binding activity. A direct interaction between corticosteroid binding globulin and the activated cells appears to enhance this event, and does not involve cellular internalization of corticosteroid binding globulin or its proteolytic degradation products, which resemble those obtained after incubation of corticosteroid binding globulin with neutrophil elastase. These data suggest that corticosteroid binding globulin interacts with elastase on the surface of activated neutrophils, and may promote glucocorticoid delivery to these cells during inflammation.

In human blood, cortisol is transported by a plasma protein known as corticosteroid-binding globulin (CBG). As anticipated from primary structure comparisons of CBG and alpha 1-proteinase inhibitor (A1-PI), CBG acts as a substrate for neutrophil elastase. However, unlike A1-PI, CBG does not alter the activity of this enzyme, but is cleaved by it at a single location close to its carboxy-terminus, and this reduces its molecular size by 5 kDa with the concomitant release of more than 80% of CBG-bound cortisol. Three small molecular size fragments are detected after elastase cleavage, and carbohydrate analysis of these fragments suggests that they represent the same polypeptide fragment which has been differentially glycosylated. To assess the biological significance of these observations, CBG was incubated with either mononuclear cells or granulocytes obtained from patients with acute inflammation (sepsis) and from a normal volunteer. Only granulocytes from septic patients reduced the mol wt of CBG by about 5 kDa and destroyed its steroid-binding activity. Preincubation with A1-PI prevented this, which demonstrates that neutrophil elastase plays a key role in this event. These results suggest a physiological role for CBG in the delivery of cortisol to sites of inflammation.

Lysosomes are the stomachs of the cell-terminal organelles on the endocytic pathway where internalized macromolecules are degraded. Containing a wide range of hydrolytic enzymes, lysosomes depend on maintaining acidic luminal pH values for efficient function. Although acidification is mediated by a V-type proton ATPase, a parallel anion pathway is essential to allow bulk proton transport. The molecular identity of this anion transporter remains unknown. Recent results of knockout experiments raise the possibility that ClC-7, a member of the CLC family of anion channels and transporters, is a contributor to this pathway in an osteoclast lysosome-like compartment, with loss of ClC-7 function causing osteopetrosis. Several mammalian members of the CLC family have been characterized in detail; some (including ClC-0, ClC-1 and ClC-2) function as Cl--conducting ion channels, whereas others act as Cl-/H+antiporters (ClC-4 and ClC-5). However, previous attempts at heterologous expression of ClC-7 have failed to yield evidence of functional protein, so it is unclear whether ClC-7 has an important function in lysosomal biology, and also whether this protein functions as a Cl- channel, a Cl-/H+ antiporter, or as something else entirely. Here we directly demonstrate an anion transport pathway in lysosomes that has the defining characteristics of a CLC Cl-/H+ antiporter and show that this transporter is the predominant route for Cl- through the lysosomal membrane. Furthermore, knockdown of ClC-7 expression by short interfering RNA can essentially ablate this lysosomal Cl-/H+ antiport activity and can strongly diminish the ability of lysosomes to acidify in vivo, demonstrating that ClC-7 is a Cl-/H+ antiporter, that it constitutes the major Cl- permeability of lysosomes, and that it is important in lysosomal acidification.

Objectives To evaluate estrogen receptors as a therapeutic target for human bladder cancer. Methods The ability of the selective estrogen receptor modulators (SERMs) tamoxifen and raloxifene to inhibit 5637 human transitional cell carcinoma cell proliferation was determined in vitro and in xenograft studies using 5637 cells in female athymic BALB/c nu/nu mice. Results Treatment with tamoxifen, raloxifene, or the pure antiestrogen ICI 182,780 inhibited proliferation of 5637 cells in vitro. In the first xenograft study, raloxifene (10, 100, or 1000 μg/day) administered by oral gavage inhibited the growth of tumors compared with placebo or untreated controls (P