RESUMO
Estrogen receptor beta (ERbeta) has potent antiproliferative and anti-inflammatory properties, suggesting that ERbeta-selective agonists might be a new class of therapeutic and chemopreventive agents. To understand how ERbeta regulates genes, we identified genes regulated by the unliganded and liganded forms of ERalpha and ERbeta in U2OS cells. Microarray data demonstrated that virtually no gene regulation occurred with unliganded ERalpha, whereas many genes were regulated by estradiol (E(2)). These results demonstrated that ERalpha requires a ligand to regulate a single class of genes. In contrast, ERbeta regulated three classes of genes. Class I genes were regulated primarily by unliganded ERbeta. Class II genes were regulated only with E(2), whereas class III genes were regulated by both unliganded ERbeta and E(2). There were 453 class I genes, 258 class II genes, and 83 class III genes. To explore the mechanism whereby ERbeta regulates different classes of genes, chromatin immunoprecipitation-sequencing was performed to identify ERbeta binding sites and adjacent transcription factor motifs in regulated genes. AP1 binding sites were more enriched in class I genes, whereas ERE, NFkappaB1, and SP1 sites were more enriched in class II genes. ERbeta bound to all three classes of genes, demonstrating that ERbeta binding is not responsible for differential regulation of genes by unliganded and liganded ERbeta. The coactivator NCOA2 was differentially recruited to several target genes. Our findings indicate that the unliganded and liganded forms of ERbeta regulate three classes of genes by interacting with different transcription factors and coactivators.
Assuntos
Receptor beta de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Pareamento de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Biologia Computacional , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Genoma Humano/genética , Humanos , Ligantes , Coativador 2 de Receptor Nuclear/metabolismo , Ligação Proteica/genética , Fator de Transcrição AP-1/metabolismoRESUMO
Hormonal, targeted and chemotherapeutic strategies largely depend on the expression of their cognate receptors and are often accompanied by intolerable toxicities. Effective and less toxic therapies for estrogen receptor negative (ER-) breast cancers are urgently needed. Here, we present the potential molecular mechanisms mediating the selective pro-apoptotic effect induced by BN107 and its principle terpene, oleanolic acid (OA), on ER- breast cancer cells. A panel of breast cancer cell lines was examined and the most significant cytotoxic effect was observed in ER- breast lines. Apoptosis was the major cellular pathway mediating the cytotoxicity of BN107. We demonstrated that sensitivity to BN107 was correlated to the status of ERalpha. Specifically, the presence of functional ERalpha protected cells from BN107-induced apoptosis and absence of ERalpha increased the sensitivity. BN107, an extract rich in OA derivatives, caused rapid alterations in cholesterol homeostasis, presumably by depleting cholesterol in lipid rafts (LRs), which subsequently interfered with signaling mediated by LRs. We showed that BN107 or OA treatment in ER- breast cancer cells resulted in rapid and specific inhibition of LR-mediated survival signaling, namely mTORC1 and mTORC2 activities, by decreasing the levels of the mTOR/FRAP1, RAPTOR and RICTOR. Cotreatment with cholesterol abolished the proapoptotic effect and restored the disrupted mTOR activities. This is the first report demonstrating possible concomitant inhibition of both mTORC1 and mTORC2 activities by modulating the levels of protein constituents present in these signaling complexes, and thus provides a basis for future development of OA-based mTOR inhibitors.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Gleditsia/química , Ácido Oleanólico/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Colesterol/metabolismo , Citocromos c/metabolismo , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/genética , Feminino , Imunofluorescência , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Microdomínios da Membrana/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Complexos Multiproteicos , Extratos Vegetais/farmacologia , Proteínas , Serina-Treonina Quinases TOR , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
Selective estrogen receptor modulators (SERMs), such as tamoxifen and raloxifene can act as estrogen receptor (ER) antagonists or agonists depending on the cell type. The antagonistic action of tamoxifen has been invaluable for treating breast cancer, whereas the agonist activity of SERMs also has important clinical applications as demonstrated by the use of raloxifene for osteoporosis. Whereas the mechanism whereby SERMs function as antagonists has been studied extensively very little is known about how SERMs produce agonist effects in different tissues with the two ER types; ERalpha and ERbeta. We examined the regulation of 32 SERM-responsive regions with ERalpha and ERbeta in transiently transfected MCF-7 breast, Ishikawa endometrial, HeLa cervical and WAR-5 prostate cancer cells. The regions were regulated by tamoxifen and raloxifene in some cell types, but not in all cell lines. Tamoxifen activated similar number of regions with ERalpha and ERbeta in the cell lines, whereas raloxifene activated over twice as many regions with ERbeta compared to ERalpha. In Ishikawa endometrial cancer cells, tamoxifen activated 17 regions with ERalpha, whereas raloxifene activated only 2 regions, which might explain their different effects on the endometrium. Microarray studies also found that raloxifene regulated fewer genes than tamoxifen in U2OS bone cancer cells expressing ERalpha, whereas tamoxifen was equally effective at regulating genes with ERalpha and ERbeta. Our studies indicate that tamoxifen is a non-selective agonist, whereas raloxifene is a relative ERbeta-selective agonist, and suggest that ERbeta-selective SERMs might be safer for treating clinical conditions that are dependent on the agonist property of SERMs.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Especificidade de Órgãos/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Linhagem Celular Tumoral , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Humanos , Cloridrato de Raloxifeno/farmacologia , Tamoxifeno/farmacologiaRESUMO
Microarrays have revolutionized many areas of biology due to our technical ability to quantify tens of thousands of transcripts within a single experiment. However, there are still many areas that cannot benefit from this technology due to the amount of biological material needed for microarray analysis. In response to this demand, chemistries have been developed that boast the capability of generating targets from nanogram amounts of total RnA, reflecting minimal amounts of biological material, on the order of several hundred or thousand cells. Herein, we describe the evaluation of four chemistries for RnA amplification in terms of reproducibility, sensitivity, accuracy, and comparability to results from a single round of T7 amplification. No evidence for false-positive measurements of differential expression was observed. In contrast, clear differences between chemistries in sensitivity and accuracy were detected. PCR validation showed an interaction of probe sequence on the array and target labeling chemistry, resulting in a chemistry-dependent probe set sensitivity varying over an order of magnitude.
Assuntos
Perfilação da Expressão Gênica/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Linhagem Celular Tumoral , Humanos , Tamanho da Amostra , Sensibilidade e EspecificidadeRESUMO
Glioblastoma, the most aggressive primary brain tumor in humans, exhibits a large degree of molecular heterogeneity. Understanding the molecular pathology of a tumor and its linkage to behavior is an important foundation for developing and evaluating approaches to clinical management. Here we integrate array-comparative genomic hybridization and array-based gene expression profiles to identify relationships between DNA copy number aberrations, gene expression alterations, and survival in 34 patients with glioblastoma. Unsupervised clustering on either profile resulted in similar groups of patients, and groups defined by either method were associated with survival. The high concordance between these separate molecular classifications suggested a strong association between alterations on the DNA and RNA levels. We therefore investigated relationships between DNA copy number and gene expression changes. Loss of chromosome 10, a predominant genetic change, was associated not only with changes in the expression of genes located on chromosome 10 but also with genome-wide differences in gene expression. We found that CHI3L1/YKL-40 was significantly associated with both chromosome 10 copy number loss and poorer survival. Immortalized human astrocytes stably transfected with CHI3L1/YKL-40 exhibited changes in gene expression similar to patterns observed in human tumors and conferred radioresistance and increased invasion in vitro. Taken together, the results indicate that integrating DNA and mRNA-based tumor profiles offers the potential for a clinically relevant classification more robust than either method alone and provides a basis for identifying genes important in glioma pathogenesis.
Assuntos
Neoplasias Encefálicas/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/genética , Hibridização de Ácido Nucleico , Adipocinas , Astrócitos/metabolismo , Neoplasias Encefálicas/patologia , Células Cultivadas/efeitos da radiação , Proteína 1 Semelhante à Quitinase-3 , Cromossomos Humanos Par 10/genética , DNA/genética , Glioblastoma/patologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Lectinas , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , RNA/genética , Tolerância a Radiação , Taxa de SobrevidaRESUMO
Over the past several years, microarray technology has evolved into a critical component of any discovery-based program. Since 1999, the Association of Biomolecular Resource Facilities (ABRF) Microarray Research Group (MARG) has conducted biennial surveys designed to generate a profile of microarray service laboratories and, more importantly, an overview of technology development and implementation. Survey questions addressed instrumentation, protocols, staffing, funding, and work flow in a microarray facility. Presented herein are the results of the MARG 2005 survey; where possible, trends in the field are discussed and compared to data collected from previous surveys.
Assuntos
Biologia Computacional/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteômica/métodos , Proteômica/tendências , Animais , Interpretação Estatística de Dados , Genômica/métodos , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise Serial de Proteínas/instrumentação , Análise Serial de Proteínas/métodos , Proteômica/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SoftwareRESUMO
BACKGROUND: The most widely used amplification method for microarray analysis of gene expression uses T7 RNA polymerase-driven in vitro transcription (IVT) to produce complementary RNA (cRNA) that can be hybridized to arrays. However, multiple rounds of amplification are required when assaying very small amounts of starting RNA. Moreover, certain cRNA-DNA mismatches are more stable than the analogous cDNA-DNA mismatches and this might increase non-specific hybridization. We sought to determine whether a recently developed linear isothermal amplification method (ribo-SPIA) that produces single stranded cDNA would offer advantages over traditional IVT-based methods for microarray-based analyses of transcript expression. RESULTS: A single round of ribo-SPIA amplification produced sufficient sscDNA for hybridizations when as little as 5 ng of starting total RNA was used. Comparisons of probe set signal intensities obtained from replicate amplifications showed consistently high correlations (r = 0.99). We compared gene expression in two different human RNA samples using ribo-SPIA. Compared with one round IVT, ribo-SPIA had a larger dynamic range and correlated better with quantitative PCR results even though we used 1000-fold less starting RNA. The improved dynamic range was associated with decreases in hybridization to mismatch control probes. CONCLUSION: The use of amplified sscDNA may offer substantial advantages over IVT-based amplification methods, especially when very limited amounts of starting RNA are available. The use of sscDNA targets instead of cRNA targets appears to improve hybridization specificity.
Assuntos
DNA de Cadeia Simples/genética , Regulação da Expressão Gênica , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Primers do DNA/química , DNA Complementar/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Perfilação da Expressão Gênica/métodos , Técnicas Genéticas , Genômica/métodos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Reação em Cadeia da Polimerase , RNA/química , RNA/metabolismo , RNA Complementar/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Proteínas Virais/metabolismoRESUMO
Tamoxifen can stimulate the growth of some breast tumors and others can become resistant to tamoxifen. We previously showed that unliganded ERbeta inhibits ERalpha-mediated proliferation of MCF-7 cells. We investigated if tamoxifen might have a potential negative effect on some breast cancer cells by blocking the effects of unliganded ERbeta on gene regulation. Gene expression profiles demonstrated that unliganded ERbeta upregulated 196 genes in MCF-7 cells. Tamoxifen significantly inhibited 73 of these genes by greater than 30%, including several growth-inhibitory genes. To explore the mechanism whereby unliganded ERbeta activates genes and how tamoxifen blocks this effect, we used doxycycline-inducible U2OS-ERbeta cells to produce unliganded ERbeta. Doxycycline produced a dose-dependent activation of the NKG2E, MSMB and TUB3A genes, which was abolished by tamoxifen. Unliganded ERbeta recruitment of SRC-2 to the NKG2E gene was blocked by tamoxifen. Our findings suggest that tamoxifen might exert a negative effect on ERbeta expressing tumors due to its antagonistic action on unliganded ERbeta.
Assuntos
Proliferação de Células/efeitos dos fármacos , Antagonistas de Estrogênios/farmacologia , Receptor beta de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Tamoxifeno/farmacologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Antagonistas de Estrogênios/metabolismo , Receptor beta de Estrogênio/genética , Feminino , Humanos , Subfamília C de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Regiões Promotoras Genéticas , Tamoxifeno/metabolismoRESUMO
The aqueous extract of Anemarrhena asphodeloides (BN108) induces apoptosis in various cancer cell lines but is significantly less cytotoxic in non-transformed cells. Chemical fractionation of BN108 showed that its cytotoxicity is associated with timosaponins, steroidal saponins of coprostane type. Timosaponin BII (TBII) is a major saponin in BN108, but it shows little cytotoxicity. A much less abundant TAIII induces cell death in tumor cells but not in normal cells, reproducing the selectivity of the total extract BN108. Glycosidase treatment, by removing the extra sugar moiety in TBII, converts it to TAIII and confers cytotoxic activity. Analysis of the mechanisms of death induced by TAIII revealed activation of two distinct pro-apoptotic pathways: first, inhibition of mTORC1 manifested in much reduced phosphorylation of mTORC1 targets; second, induction of endoplasmic reticulum stress culminating in phosphorylation of eIF2alpha and activation of caspase 4. These pro-apoptotic pathways are activated by TAIII selectively in tumor cells but not in normal cells. Both pathways play a causative role in TAIII cytotoxicity, as restoration of either mTOR activity or relief of ER stress alone offer only partial protection from TAIII. Inhibition of mTORC1 and induction of ER stress apparently contribute to the induction of the previously reported autophagic response in TAIII-treated cells. TAIII induced autophagy plays a protective role in TAIII induced death signaling, and failure to mount autophagic response is associated with heightened sensitivity to TAIII induced apoptosis. The multiple death-promoting and apparently tumor-selective responses to TAIII, its ability to inhibit mTORC1, and the possibility of further enhancing its cytotoxicity by pharmacological inhibition of autophagy, make TAIII an attractive candidate for development as a cancer therapeutic agent.
Assuntos
Anemarrhena/metabolismo , Retículo Endoplasmático/metabolismo , Regulação Neoplásica da Expressão Gênica , Extratos Vegetais/farmacologia , Proteínas Quinases/metabolismo , Saponinas/farmacologia , Esteroides/farmacologia , Apoptose , Linhagem Celular Transformada , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Citometria de Fluxo , Glicosilação , Humanos , Relação Estrutura-Atividade , Serina-Treonina Quinases TORRESUMO
Estrogens produce biological effects by interacting with two estrogen receptors, ERalpha and ERbeta. Drugs that selectively target ERalpha or ERbeta might be safer for conditions that have been traditionally treated with non-selective estrogens. Several synthetic and natural ERbeta-selective compounds have been identified. One class of ERbeta-selective agonists is represented by ERB-041 (WAY-202041) which binds to ERbeta much greater than ERalpha. A second class of ERbeta-selective agonists derived from plants include MF101, nyasol and liquiritigenin that bind similarly to both ERs, but only activate transcription with ERbeta. Diarylpropionitrile represents a third class of ERbeta-selective compounds because its selectivity is due to a combination of greater binding to ERbeta and transcriptional activity. However, it is unclear if these three classes of ERbeta-selective compounds produce similar biological activities. The goals of these studies were to determine the relative ERbeta selectivity and pattern of gene expression of these three classes of ERbeta-selective compounds compared to estradiol (E(2)), which is a non-selective ER agonist. U2OS cells stably transfected with ERalpha or ERbeta were treated with E(2) or the ERbeta-selective compounds for 6 h. Microarray data demonstrated that ERB-041, MF101 and liquiritigenin were the most ERbeta-selective agonists compared to estradiol, followed by nyasol and then diarylpropionitrile. FRET analysis showed that all compounds induced a similar conformation of ERbeta, which is consistent with the finding that most genes regulated by the ERbeta-selective compounds were similar to each other and E(2). However, there were some classes of genes differentially regulated by the ERbeta agonists and E(2). Two ERbeta-selective compounds, MF101 and liquiritigenin had cell type-specific effects as they regulated different genes in HeLa, Caco-2 and Ishikawa cell lines expressing ERbeta. Our gene profiling studies demonstrate that while most of the genes were commonly regulated by ERbeta-selective agonists and E(2), there were some genes regulated that were distinct from each other and E(2), suggesting that different ERbeta-selective agonists might produce distinct biological and clinical effects.
Assuntos
Receptor beta de Estrogênio/agonistas , Regulação da Expressão Gênica/efeitos dos fármacos , Western Blotting , Linhagem Celular , Estradiol/farmacologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Lignanas , Nitrilas/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenóis/farmacologia , Propionatos/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
We used microarray analysis with Affymetrix rat chips to determine gene expression profiles of freshly isolated rat type I (TI) and TII cells and cultured TII cells. Our goals were 1) to describe molecular phenotypic "fingerprints" of TI and TII cells, 2) to gain insight into possible functional differences between the two cell types through differentially expressed genes, 3) to identify genes that might indicate potential functions of TI cells, since so little is known about this cell type, and 4) to ascertain the similarities and differences in gene expression between cultured TII cells and freshly isolated TI cells. For these experiments, we used preparations of isolated TI and TII cells that contained <2% cross-contamination. With a false discovery rate of 1%, 601 genes demonstrated over twofold different expression between TI and TII cells. Those genes with very high levels of differential expression may be useful as markers of cell phenotype and in generating novel hypotheses about functions of TI and TII cells. We found similar numbers of differentially expressed genes between freshly isolated TI or TII cells and cultured TII cells (698, 637 genes) and freshly isolated TI and TII cells (601 genes). Tests of sameness/difference including cluster dendrograms and log/log identity plots indicated major differences between the phenotypes of freshly isolated TI cell and cultured type II cell populations. The latter results suggest that experiments with TII cells cultured under these conditions should be interpreted with caution with respect to biological relevance to TI or TII cells.