RESUMO
Our previous studies have shown that overexpression of aromatase results in increased tissue estrogenic activity and induction of hyperplastic and dysplastic lesions in aromatase transgenic mammary glands. In this study, we have examined the effects of aromatase overexpression on biochemical changes in the aromatase transgenic mice. Our results show an increase in the expression of both estrogen and progesterone receptors, and their expression is maintained in the transgenic mammary tissue even without circulating ovarian estrogens. Our results also show an increase in the expression of several growth factors and cell cycle genes in the aromatase transgenic mammary glands, which is consistent with the observed increase in proliferating cell nuclear antigen levels and cellular proliferation. Interestingly, we have also observed a decrease in the expression of epidermal growth factor receptor and its ligands, epidermal growth factor and transforming growth factor alpha, as well as several tumor suppressor genes such as p53 and retinoblastoma. This study presents novel and interesting findings that are consistent with the current models of aromatase influence and the complex interactions of biochemical pathways leading to mammary tumorigenesis.
Assuntos
Apoptose/genética , Aromatase/biossíntese , Genes Supressores de Tumor/genética , Glândulas Mamárias Animais/enzimologia , Glândulas Mamárias Animais/patologia , Animais , Aromatase/genética , Ciclo Celular/genética , Divisão Celular/genética , Estradiol/sangue , Feminino , Regulação da Expressão Gênica , Substâncias de Crescimento/biossíntese , Substâncias de Crescimento/genética , Hiperplasia/enzimologia , Glândulas Mamárias Animais/fisiologia , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Lesões Pré-Cancerosas/enzimologia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genética , Receptores de Progesterona/biossíntese , Receptores de Progesterona/genética , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
Our recent studies have shown that the cellular gene at the mouse mammary tumor virus integration site in the int-5 locus is aromatase. To study the role of int-5/aromatase in normal mammary development and mammary neoplasia, we have generated transgenic mice that overexpress int-5/aromatase under the control of mouse mammary tumor virus enhancer/promoter. All the transgenic virgin (n = 10) and postlactational (n = 15) females that overexpress int-5/aromatase show various histological abnormalities. Overexpression of int-5/aromatase in mammary glands of virgin females leads to the enlargement of 40% of ducts, of which 65% had hyperplastic lesions, 20% had dysplastic lesions, and 15% had fibroadenoma lesions. Overexpression of int-5/aromatase in involuted mammary glands of transgenic females induces hyperplasia in 75-80% of ducts and glands that exhibit a range of morphological abnormalities, including formation of hyperplastic alveolar nodule (10%), ductal and glandular hyperplasia (70-80%), ductal and lobular dysplasia (15%), and nuclear abnormalities (2-5%) such as multinucleation and karyomegaly, which are all indicative of preneoplastic changes. Our results show that early exposure of mammary epithelium to in situ estrogen and continued exposure to in situ estrogen as a result of overexpression of int-5/aromatase appears to predispose mammary tissue to preneoplastic changes, which may, in turn, increase the risk of developing neoplasia and increase susceptibility to environmental carcinogens. These findings support clinical and experimental data that suggest that early estrogen exposure increases breast cancer risk.
Assuntos
Aromatase/genética , Glândulas Mamárias Animais/enzimologia , Lesões Pré-Cancerosas/enzimologia , Animais , Núcleo Celular/ultraestrutura , Feminino , Regulação Neoplásica da Expressão Gênica , Glândulas Mamárias Animais/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Lesões Pré-Cancerosas/ultraestrutura , RNA Neoplásico/genéticaRESUMO
Previous studies have reported the cloning and identification of the int-5, a novel mouse mammary tumor virus (MMTV) integration locus involved in mammary tumorigenesis. Here we report the characterization of the 5.5 kb region from this novel MMTV integration site. Our results show that the region after the MMTV integration site, a 258 bp sequence is homologous (100%) to the mouse early transposon (mETn) long-terminal repeat and other sequences of the this transposon are not present in the 5.5 kb region. Our results also show for the first time the tumor specific expression of mETn and expression of the region downstream of the MMTV integration site that is homologous to mETn-LTR in D2 mammary tumors.
Assuntos
Elementos de DNA Transponíveis/genética , Vírus do Tumor Mamário do Camundongo/genética , Sequências Repetitivas de Ácido Nucleico/genética , Animais , Sequência de Bases , Expressão Gênica , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência MolecularRESUMO
To test directly the role of breast-tissue estrogen in initiation of breast cancer, we have developed the aromatase-transgenic mouse model and demonstrated for the first time that increased mammary estrogens resulting from the overexpression of aromatase in mammary glands lead to the induction of various preneoplastic and neoplastic changes that are similar to early breast cancer. Continued overexpression of aromatase that leads to increased breast-tissue estrogen contributes to a number of epigenetic changes in mammary tissue such as alteration in the regulation of genes involved in apoptosis, activation of genes involved in cell cycle and cell proliferation, and activation of a number of growth factors. Our current studies show aromatase overexpression is sufficient to induce and maintain early preneoplastic and neoplastic changes in female mice without circulating ovarian estrogen. Preneoplastic and neoplastic changes induced in mammary glands as a result of aromatase overexpression can be completely abrogated with the administration of the aromatase inhibitor, letrozole. Consistent with complete reduction in hyperplasia, we have also seen downregulation of estrogen receptor and a decrease in cell proliferation markers, suggesting aromatase-induced hyperplasia can be treated with aromatase inhibitors. Our studies demonstrate that aromatase overexpression alone, without circulating estrogen, is responsible for the induction of breast hyperplasia and these changes can be abrogated using aromatase inhibitors.
Assuntos
Antineoplásicos/farmacologia , Inibidores da Aromatase , Aromatase/metabolismo , Inibidores Enzimáticos/farmacologia , Glândulas Mamárias Animais/enzimologia , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/enzimologia , Nitrilas/farmacologia , Triazóis/farmacologia , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Estrogênios/sangue , Feminino , Hiperplasia , Letrozol , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Transgênicos , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias Hormônio-Dependentes/metabolismo , Ovariectomia , Lesões Pré-Cancerosas/tratamento farmacológico , Lesões Pré-Cancerosas/metabolismo , Receptores de Estrogênio/metabolismoRESUMO
Estrogen exposure represents the major known risk factor for development of breast cancer in women and is implicated in the development of prostate cancer in men. Human breast tissue has been shown to be a site of oxidative metabolism of estrogen due to the presence of specific cytochrome P450 enzymes. The oxidative metabolism of 17beta-estradiol (E2) to E2-3,4-quinone metabolites by an E2-4-hydroxylase in breast tissue provides a rational hypothesis to explain the mammary carcinogenic effects of estrogen in women because this metabolite is directly genotoxic and can undergo redox cycling to form genotoxic reactive oxygen species. In this chapter, evidence in support of this hypothesis and of the role of P4501B1 as the 4-hydroxylase expressed in human breast tissue is reviewed. However, the plausibility of this hypothesis has been questioned on the grounds that insufficient E2 is present in breast tissue to be converted to biologically significant amounts of metabolite. This critique is based on the assumption that plasma and tissue E2 levels are concordant. However, breast cancer tissue E2 levels are 10-fold to 50-fold higher in postmenopausal women than predicted from plasma levels. Consequently, factors must be present to alter breast tissue E2 levels independently of plasma concentrations. One such factor may be the local production of E2 in breast tissue through the enzyme aromatase, and the evidence supporting the expression of aromatase in breast tissue is also reviewed in this chapter. If correct, mutations or environmental factors enhancing aromatase activity might result in high tissue concentrations of E2 that would likely be sufficient to serve as substrates for CYP1B1, given its high affinity for E2. This concept, if verified experimentally, would provide plausibility to the hypothesis that sufficient E2 may be present in tissue for formation of catechol metabolites that are estrogenic and which, upon further oxidative metabolism, form genotoxic species at levels that may contribute to estrogen carcinogenesis.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Neoplasias da Mama/etiologia , Neoplasias da Mama/metabolismo , Mama/metabolismo , Carcinoma/etiologia , Carcinoma/metabolismo , Estrogênios/metabolismo , Aromatase/metabolismo , Aromatase/fisiologia , Mama/enzimologia , Neoplasias da Mama/enzimologia , Carcinoma/enzimologia , Citocromo P-450 CYP1B1 , Sistema Enzimático do Citocromo P-450/metabolismo , Estrogênios/biossíntese , Feminino , Humanos , Hidroxilação , Modelos Químicos , OxirreduçãoRESUMO
Mouse mammary tumorigenesis as a result of mouse mammary tumor virus (MMTV) integrations has helped to identify a wide variety of interesting genes that play a role in mammary development and tumorigenesis. Several such genes int1/wnt1, wnt3, wnt 10B, int2/fgf3, fgf4, int3/notch and int6 have been shown to be genetically altered in naturally formed mammary tumors as a consequence of MMTV integration. Some of these genes have been well characterised and examined in in vivo breast cancer transgenic models for their potential for tumorigenesis. Overexpression of one or more of these genes have resulted in a striking proliferation of mammary gland epithelium of both female and male transgenic mice. Our own studies have demonstrated overexpression of int5/aromatase in mammary glands of virgin and postlactational females leads to the induction of various preneoplastic and neoplastic changes that are similar to early breast cancer, that may, in turn, increase the risks for developing breast cancer. Therefore, further understanding of these genes should provide new insights to their involvement and mechanism of action in breast cancer.
Assuntos
Neoplasias da Mama/genética , Neoplasias Mamárias Animais/genética , Proteínas Wnt/fisiologia , Animais , Animais Selvagens , Neoplasias da Mama/fisiopatologia , Carcinógenos , Transformação Celular Neoplásica , Modelos Animais de Doenças , Fator de Iniciação 3 em Eucariotos/fisiologia , Feminino , Fator 3 de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica , Humanos , Masculino , Neoplasias Mamárias Animais/virologia , Vírus do Tumor Mamário do Camundongo/patogenicidade , Camundongos , Camundongos TransgênicosRESUMO
Our recent studies have shown that the cellular gene at the mouse mammary tumor virus (MMTV) integration site in the int-5 locus in BALB/c D2 precancerous hyperplastic alveolar nodules is identical to the gene encoding aromatase (CYP19), a member of the cytochrome P450 gene superfamily. MMTV integrated within the 3' untranslated region of the aromatase gene is responsible for the overexpression of this gene (int-5/aromatase) in mammary tumors. This paper describes the biological significance of overexpression of int-5/aromatase in D2 tumor cells. Using a cell line derived from the D2 tumor, we have demonstrated the effect of the aromatase substrate, androstenedione, on the proliferation of tumor cells. Proliferative effects of androstenedione were blocked by an aromatase inhibitor, providing evidence for the role of int-5/aromatase in this process. Furthermore, the androstenedione-mediated proliferation was inhibited by the addition of anti-estrogen ICI 164,384, suggesting that the estrogen formed from the conversion of androstenedione by int-5/aromatase acts like a mitogen to stimulate the growth of D2 tumor cells. This model with its known mechanism of aromatase activation should prove useful for studying the role of intra-tumoral estrogen in mammary cancer, for evaluating the effects of aromatase inhibitors, and for comparing breast cancer treatments.
Assuntos
Aromatase/genética , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/virologia , Vírus do Tumor Mamário do Camundongo/genética , Proteínas Virais/genética , Integração Viral/genética , Androstenodiona/metabolismo , Androstenodiona/farmacologia , Animais , Aromatase/metabolismo , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Estrogênios/biossíntese , Feminino , Expressão Gênica , Neoplasias Mamárias Experimentais/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Mitógenos/metabolismo , Dados de Sequência Molecular , Transplante de Neoplasias , Células Tumorais Cultivadas/efeitos dos fármacos , Proteínas Virais/metabolismoRESUMO
Our previous studies have resulted in the identification and cloning of int-5, a novel site of mouse mammary tumor virus (MMTV) integration, from BALB/c D2 precancerous hyperplastic alveolar nodules (HAN). This paper presents a detailed characterization of the int-5 locus from both D2 HAN and normal genome and expression of the unique gene from the MMTV integration site. Our results show that the cellular gene at the MMTV integration site in the int-5 locus is identical to the gene encoding aromatase (CYP19), a member of the cytochrome P-450 gene superfamily. MMTV is integrated within exon 10 in the 3' untranslated region of the aromatase gene. This gene (int-5/aromatase) is expressed in normal mammary gland and overexpressed in mammary tumors. These results suggest that the overexpression of this gene may be responsible for mammary tumorigenesis. This is the first demonstration of integration of MMTV in a cellular gene that plays a role in hormone-dependent breast cancers.
Assuntos
Vírus do Tumor Mamário do Camundongo/genética , Proto-Oncogenes , Integração Viral , Animais , Aromatase/genética , Sequência de Bases , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Lesões Pré-Cancerosas , Mapeamento por RestriçãoRESUMO
We recently showed that the cellular gene int-5/aromatase in BALB/c mammary alveolar hyperplastic nodule (D2 HAN/D2 tumor cells) is activated as a result of mouse mammary tumor virus integration within the 3' untranslated region of the aromatase gene. In the present study, we evaluated the effect of various aromatase inhibitors on androstenedione-mediated tumor cell growth. Also, we compared the effect of the non-steroidal aromatase inhibitor (CGS 16949A) on the inhibition of tumor growth. Our results show that D2 tumor cells respond well to various aromatase inhibitors and antiestrogens. We examined the usefulness of this model by using D2 tumor cells to simulate postmenopausal breast cancer employing both in vitro cell culture and in vivo ovariectomized (OVX) nude mouse. Unlike DMBA-induced tumors or other models, D2 tumor cells form very rapid tumors within a few days in intact mice or OVX nude mice with androstenedione supplementation and respond well to an aromatase inhibitor. This model with its known mechanism of aromatase activation should be useful for studying the role of intra-tumoral estrogen in mammary cancer, for evaluating the effects of aromatase inhibitors and antiestrogens, and for comparing breast cancer treatments.
Assuntos
Androstenodiona/farmacologia , Inibidores da Aromatase , Neoplasias da Mama/patologia , Inibidores Enzimáticos/toxicidade , Fadrozol/toxicidade , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Experimentais/patologia , 9,10-Dimetil-1,2-benzantraceno , Aminoglutetimida/toxicidade , Análise de Variância , Anastrozol , Animais , Antineoplásicos Hormonais/uso terapêutico , Antineoplásicos Hormonais/toxicidade , Aromatase/biossíntese , Neoplasias da Mama/tratamento farmacológico , Divisão Celular/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Fadrozol/uso terapêutico , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nitrilas/toxicidade , Ovariectomia , Pós-Menopausa , Triazóis/toxicidade , Células Tumorais CultivadasRESUMO
Our studies using the aromatase transgenic mice model have shown that early exposure of mammary epithelium to in situ estrogen as a result of overexpression of aromatase predispose mammary tissue to preneoplastic changes. Here, we hypothesize that the preneoplastic changes induced by mammary estrogen in aromatase transgenic females may be susceptible to environmental carcinogens like 7,12-dimethylbenz[a]anthracene (DMBA), and may result in the acceleration and/or increase in the incidence of breast cancer. Results presented in this study show that tumors appeared in 25% of the mice that were treated with DMBA and all treated transgenic animals had microscopic evidence of neoplastic progression. Control non-transgenic females did not have significant changes even after treatment with DMBA. Consistent with increased neoplastic changes in DMBA-treated aromatase mice, we have seen an increase in the expression of genes involved in cell proliferation and cell cycle. We have also seen changes in the expression of oxidative stress markers and changes in estrogen-mediated growth factors. These studies indicate that more than one event is required for tumor formation, and that early estrogen exposure leading to preneoplastic changes in the mammary epithelial cells increases susceptibility to environmental carcinogens that may result in acceleration and/or an increase in the incidence of breast cancer.
Assuntos
9,10-Dimetil-1,2-benzantraceno/toxicidade , Aromatase/metabolismo , Neoplasias Mamárias Experimentais/induzido quimicamente , 9,10-Dimetil-1,2-benzantraceno/administração & dosagem , Animais , Aromatase/genética , Testes de Carcinogenicidade , Modelos Animais de Doenças , Feminino , Incidência , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/epidemiologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Our recent studies have shown that overexpression of aromatase results in increased tissue estrogenic activity and induction of hyperplastic and dysplastic lesions in female mammary glands and gynecomastia and testicular cancer in male aromatase transgenic mice. Both aromatase mRNA and protein are overexpressed in transgenic mammary glands and its expression is not limited to epithelial cells. However, it is more in epithelial than in stromal cells. Our results also indicate aromatase overexpression-induced changes in mammary glands can be abrogated with very low concentrations of the aromatase inhibitor, letrozole. Low concentration of letrozole had no effect on normal physiology as indicated by no significant change in the circulating levels of estradiol and follicle stimulating hormone as well as no change in estrogen responsive genes such as the progesterone receptor and lactoferrin in the uterine tissue. These observations indicate that the expression of aromatase in both epithelial and stromal cells can influence the complex interactions of biochemical pathways leading to mammary carcinogenesis and that the aromatase inhibitor, letrozole can be used as chemopreventive agents without affecting normal physiology.
Assuntos
Aromatase/genética , Aromatase/fisiologia , Glândulas Mamárias Animais/enzimologia , Animais , Inibidores da Aromatase , Inibidores Enzimáticos/farmacologia , Células Epiteliais/enzimologia , Estradiol/sangue , Estrogênios/metabolismo , Feminino , Hormônio Foliculoestimulante/sangue , Expressão Gênica , Hiperplasia , Letrozol , Masculino , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/patologia , Glândulas Mamárias Animais/fisiologia , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/etiologia , Neoplasias Mamárias Experimentais/prevenção & controle , Camundongos , Camundongos Transgênicos , Nitrilas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Estromais/enzimologia , Distribuição Tecidual , Triazóis/farmacologiaRESUMO
Our previous studies have shown that overexpression of aromatase in mammary glands results in the induction of hyperplastic and dysplastic changes in female transgenic mice. In this study we show that overexpression of aromatase in male transgenic mice results in increased mammary growth and histopathological changes similar to gynecomastia. Increased estrogenic activity also results in an increase in estrogen and progesterone receptor expression in the mammary glands of transgenic males as compared to the nontransgenic males, as well as an increase in the expression of various genes involved in cell cycle and cell proliferation. We have also observed an increase in certain growth factors, such as bFGF and TGFbeta, as a result of aromatase overexpression in the male transgenic mammary glands. In order to obtain a better understanding of the biological significance of gynecomastia, a reliable model is necessary to explain the mechanisms and correlations associated with human cancers. This model, can potentially serve as a predictable and useful tool for studying gynecomastia, hormonal carcinogenesis and action of other carcinogens on hormone induced cancers.
Assuntos
Aromatase/genética , Ginecomastia/enzimologia , Glândulas Mamárias Animais/metabolismo , Animais , Aromatase/metabolismo , Sequência de Bases , Primers do DNA , Estrogênios/fisiologia , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Genes cdc , Masculino , Camundongos , Camundongos Transgênicos , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
The transforming growth factor alpha (TGFalpha) and its receptor (EGFR) are expressed in many breast cancers. Typically, the progression of estrogen dependent primary breast cancers into a hormone-independent state, due to the loss of the estrogen receptor, is associated with increased levels of TGFalpha and EGFR, leading to aggressive breast carcinomas. The relationship between breast tumorigenesis and TGFalpha is evident in the transgenic mice overexpressing TGFalpha in the mammary glands. In the aromatase transgenic mice, the mammary glands exhibit preneoplastic developments but do not form frank tumors. To test the interactions between growth factor overexpression with tissue estrogen, we have crossed the aromatase transgenic mice with the TGFalpha transgenic mice to produce a double transgenic strain. The histological data for the mammary glands of aromatase x TGFalpha double transgenic mice show that these mice develop hyperplastic changes similar to the aromatase parental strain but no tumors are formed. Consistently, the expression of cyclin D1 and PCNA is diminished in the double transgenic strain as compared to the parental strains. In addition, the expression of TGFalpha, EGF and EGFR are also decreased in the double transgenic strain, suggesting that continuous estrogen presence in the tissue due to aromatase overexpression downregulates the expression of EGFR and its ligands.
Assuntos
Aromatase/genética , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Fator de Crescimento Transformador alfa/genética , Animais , Neoplasias da Mama/genética , Receptores ErbB/genética , Feminino , Expressão Gênica , Humanos , Neoplasias Mamárias Experimentais/etiologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/fisiopatologia , Camundongos , Camundongos Transgênicos , Neoplasias Hormônio-Dependentes/genéticaRESUMO
Estrogen synthesized in situ plays a more important role in breast cancer cell proliferation than does circulating estrogen. Aromatase is the enzyme that converts androgen to estrogen and is expressed at a higher level in breast cancer tissue than in surrounding noncancer tissue. A promising route of chemoprevention against breast cancer may be through the suppression of in situ estrogen formation using aromatase inhibitors. A diet high in fruits and vegetables may reduce the incidence of breast cancer, because they contain phytochemicals that can act as aromatase inhibitors. In our previous studies, we found that grapes and wine contain potent phytochemicals that can inhibit aromatase. We show that red wine was more effective than white wine in suppressing aromatase activity. Interestingly, our results from white wine studies suggest a weak inductive effect of alcohol on aromatase activity. On the other hand, the potent effect of anti-aromatase chemicals in red wine overcomes the weak inductive effect of alcohol in wine. Several purification procedures were performed on whole red wine to separate active aromatase inhibitors from non-active compounds. These techniques included liquid-liquid extraction, silica gel chromatography, various solid phase extraction (SPE) columns, and high performance liquid chromatography. An active Pinot Noir red wine SPE C18 column fraction (20% acetonitrile:water) was more effective than complete Pinot Noir wine in suppressing aromatase assay. This red wine extract was further analyzed in a transgenic mouse model in which aromatase was over-expressed in mammary tissue. Our gavaged red wine extract completely abrogated aromatase-induced hyperplasia and other neoplastic changes in mammary tissue. These results suggest that red wine or red wine extract may be a chemopreventive diet supplement for postmenopausal women who have a high risk of breast cancer. Further research is underway to purify and characterize the active compounds in red wine that are responsible for the inhibition of aromatase.
Assuntos
Inibidores da Aromatase , Inibidores Enzimáticos/farmacologia , Vinho/análise , Quercetina/farmacologia , Resveratrol , Rutina/farmacologia , Estilbenos/farmacologiaRESUMO
OBJECTIVE: This study was designed (1) to characterize the resident leukocyte population in ectopic endometrium (EE), (2) to assess proliferative activity of cellular components in EE, (3) to assess whether resident leukocytes in EE express IFN gamma mRNA and (4) to demonstrate endometrial epithelial cell IFN gamma receptors in EE. STUDY DESIGN: Biopsies of EE and normal eutopic endometrium (UE) were studied immunocytochemically using monoclonal antibodies specific for CD45 leukocyte common antigen, CD3 (a T cell marker), CD11c (a macrophage marker), and Ki67 (proliferation marker). Leukocyte types were identified immunocytochemically, followed by in situ hybridization to assess expression of IFN gamma mRNA. IFN gamma receptor expression was assessed by immunocytochemistry. RESULTS: The percentage of scattered stromal cells staining for each CD marker was greater in EE than in UE. The proliferative activity of endometrial stromal cells and epithelial cells was significantly less in EE than in UE. The overall concentration of T cells and macrophages expressing IFN gamma mRNA was significantly greater in EE than in UE. The percentage of each leukocyte type expressing IFN gamma mRNA was also greater in EE than in UE, and IFN gamma receptors were present in glandular epithelium of EE. CONCLUSIONS: These findings support a possible paracrine role for resident leukocytes and IFN gamma in regulating cell proliferation in endometriosis.
Assuntos
Divisão Celular , Citocinas/fisiologia , Endometriose/patologia , Adulto , Endométrio/imunologia , Endométrio/patologia , Feminino , Humanos , Imunofenotipagem , Hibridização In Situ , Interferon gama/genética , Macrófagos/imunologia , Macrófagos/metabolismo , RNA Mensageiro/análise , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
OBJECTIVE: To evaluate the regulation and expression of CSF-1 and its receptor c-fms in endometrial cells. DESIGN: In vitro study. SETTING: Research and teaching institution. PATIENT(S): None. INTERVENTION(S): In vitro experimental study. MAIN OUTCOME MEASURE(S): The effect of glucocorticoid and oxidative stress on the expression of CSF-1 and c-fms in endometrial cells. RESULT(S): Cultured nonmalignant EM42 cells not only express CSF-1 and c-fms but are also capable of responding to exogenous CSF-1. We have also seen that glucocorticoids can regulate the expression of CSF-1/c-fms in endometrial cells. Furthermore, this study shows that oxidative stress plays a significant role in the induction of CSF-1 and its receptor c-fms. CONCLUSION(S): The results suggest that CSF-1 may promote the growth of nonmalignant endometrial cells in both an autocrine and paracrine manner and that endometrial cells under oxidative stress induce CSF-1 and c-fms.
Assuntos
Dexametasona/farmacologia , Endométrio/metabolismo , Glucocorticoides/farmacologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Estresse Oxidativo/fisiologia , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Linhagem Celular , Endométrio/citologia , Feminino , Humanos , Fator Estimulador de Colônias de Macrófagos/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologiaRESUMO
OBJECTIVES: To quantitate and compare granulosa cell alpha-inhibin messenger RNA (mRNA) levels in IVF-ET poor and good responders and thereby learn how alpha-inhibin mRNA levels change in states of diminished ovarian responsiveness. DESIGN: Ribonucleic acid analysis of stored luteinized granulosa cell samples. SETTING: Academic tertiary care institution. PATIENTS: Fifty-three women undergoing follicle aspiration for IVF-ET were studied. Patients were classified as poor responders (n = 16) or good responders (n = 37) according to their E2 concentration on the day of hCG; the E2 of poor responders was < 1,000 pg/mL (3,671 pmol/L) and that of good responders was > or = 1,000 pg/mL (3,671 pmol/L). MAIN OUTCOME MEASURES: Messenger RNA levels were measured using dot blot RNA analysis. The following parameters were determined or derived: total mRNA levels, total alpha-inhibin mRNA levels, alpha-inhibin mRNA per follicle, and proportional alpha-inhibin mRNA as the ratio of alpha-inhibin mRNA:total mRNA. RESULTS: Proportional alpha-inhibin mRNA and alpha-inhibin mRNA per follicle were not significantly different between poor responders and good responders. Total mRNA and total alpha-inhibin mRNA levels, however, were diminished significantly in poor responders. CONCLUSIONS: The observations that proportional alpha-inhibin mRNA and alpha-inhibin mRNA per follicle do not significantly change in poor responders, whereas total alpha-inhibin mRNA does, indicate that the decrease in total alpha-inhibin mRNA in poor responders reflects a decreased pool of total mRNA, likely because of a reduction in follicle number. These findings are in contrast to other recent reports that describe a change in granulosa cell function accompanying states of decreased ovarian responsiveness.
Assuntos
Transferência Embrionária , Fertilização in vitro , Inibinas/genética , Peptídeos/genética , RNA Mensageiro/biossíntese , Adolescente , Adulto , Feminino , Células da Granulosa/química , Humanos , RNA Mensageiro/análiseRESUMO
OBJECTIVE: To investigate the interdependent role of macrophage colony-stimulating factor (CSF-1) and its receptor (c-fms) on their induction and their role in granulosa cell tumorigenesis. METHODS: Normal ovarian granulosa cells were used to develop stable transfectants that overexpress CSF-1 or CSF-1/c-fms. CSF-1 was expressed under the control of tissue/cell specific alpha-inhibin promoter, and c-fms was expressed constitutively using a viral promoter. Stable transfectants were used to examine the effect of overexpression of these molecules on the proliferation, induction of autocrine loop, and tumorigenesis. RESULTS: Expression vectors were developed for CSF-1 and its receptor, c-fms, and used to generate stable transfects overexpressing these genes in granulosa cells. Data show that overexpression of CSF-1 leads to the induction of its receptor. Stable transfectants that overexpress CSF-1 show about a 2.5-fold increase in cell proliferation compared with normal granulosa cells, and these cells are also converted to anchorage-independent and tumorigenic phenotype. Using an antisense RNA approach, we also demonstrated that the increased cell proliferation is CSF-1 specific. Concomitant overexpression of CSF-1 and c-fms further results in increased cell proliferation (sixfold), rapid anchorage-independent growth, and aggressive tumor formation. CONCLUSION: CSF-1 is capable of inducing its own receptor, and, similarly, the CSF-1 receptor, c-fms, can also induce its growth factor ligand. These studies also demonstrate the interdependent role of these genes in transformation of normal ovarian granulosa cells to a tumorigenic phenotype and suggest the possibility of a similar role for these genes in progression of ovarian cancer.
Assuntos
Divisão Celular , Tumor de Células da Granulosa/genética , Células da Granulosa/metabolismo , Fator Estimulador de Colônias de Macrófagos/genética , Neoplasias Ovarianas/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Animais , Linhagem Celular Transformada , Feminino , Expressão Gênica , Vetores Genéticos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Ratos , Ratos Endogâmicos , TransfecçãoRESUMO
OBJECTIVE: Using an analogy with renin gene overexpression, low-renin hypertension animal models, we wished to test the hypothesis that renin gene expression is increased in decidua basalis in human gestation with preeclampsia. METHODS: Human placentas were obtained immediately after delivery from 11 control (C) and 11 preeclamptics (PE). Tissue samples were microdissected and renin gene expression in decidua basalis (DB), chorionic villi (CV), and decidua vera (DV) was measured using dot-blot hybridization. RESULTS: Overall renin gene expression is highest in decidua basalis (mean +/- SEM, 2.66 +/- 0.69 densitometry area units) compared to chorionic villi (mean +/- SEM, 1.85 +/- 0.5) or compared to decidua vera (mean +/- SEM, 1.63 +/- 0.9) (both t-tests p = 0.001 two-tailed and analysis of variance p = 0.0001). Renin gene expression in DB and in CV was similar in both preeclamptic and normal pregnancies (DB mean +/- SEM C 2.79 +/- 0.96 versus PE 2.54 +/- 1.04, and CV mean +/- SEM C 2.11 +/- 0.91 versus PE, 1.59 +/- 0. 44). Renin gene expression in DV was approximately threefold higher in tissues from preeclamptics compared to control (mean +/- SEM PE 2. 44 +/- 1.76 versus C 0.82 +/- 0.42). Using the median value of 0.5 units for DV as cutoff, the preeclamptics displayed higher renin gene expression (chi square p = 0.033, two tailed). CONCLUSION: Our data suggest that renin gene expression is increased in preeclampsia in decidua vera. This may explain previously reported increased renin secretion in uterine circulation in preeclampsia.
Assuntos
Vilosidades Coriônicas/química , Decídua/química , Expressão Gênica/genética , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/patologia , Renina/análise , Renina/genética , Adulto , Análise de Variância , Animais , Estudos de Casos e Controles , Modelos Animais de Doenças , Feminino , Humanos , Immunoblotting , Circulação Placentária , Pré-Eclâmpsia/fisiopatologia , Gravidez , Renina/fisiologiaRESUMO
Proline-, glutamic acid- and leucine-rich protein-1 (PELP1) is a scaffolding oncogenic protein that functions as a coregulator for a number of nuclear receptors. p53 is an important transcription factor and tumor suppressor that has a critical role in DNA damage response (DDR) including cell cycle arrest, repair or apoptosis. In this study, we found an unexpected role for PELP1 in modulating p53-mediated DDR. PELP1 is phosphorylated at Serine1033 by various DDR kinases like ataxia-telangiectasia mutated, ataxia telangiectasia and Rad3-related or DNAPKc and this phosphorylation of PELP1 is important for p53 coactivation functions. PELP1-depleted p53 (wild-type) breast cancer cells were less sensitive to various genotoxic agents including etoposide, camptothecin or γ-radiation. PELP1 interacts with p53, functions as p53-coactivator and is required for optimal activation of p53 target genes under genomic stress. Overall, these studies established a new role of PELP1 in DDRs and these findings will have future implications in our understanding of PELP1's role in cancer progression.