Spatiotemporal control of estrogen-responsive transcription in ERα-positive breast cancer cells.

Recruitment of transcription machinery to target promoters for aberrant gene expression has been well studied, but underlying control directed by distant-acting enhancers remains unclear in cancer development. Our previous study demonstrated that distant estrogen response elements (DEREs) located on chromosome 20q13 are frequently amplified and translocated to other chromosomes in ERα-positive breast cancer cells. In this study, we used three-dimensional interphase fluorescence in situ hybridization to decipher spatiotemporal gathering of multiple DEREs in the nucleus. Upon estrogen stimulation, scattered 20q13 DEREs were mobilized to form regulatory depots for synchronized gene expression of target loci. A chromosome conformation capture assay coupled with chromatin immunoprecipitation further uncovered that ERα-bound regulatory depots are tethered to heterochromatin protein 1 (HP1) for coordinated chromatin movement and histone modifications of target loci, resulting in transcription repression. Neutralizing HP1 function dysregulated the formation of DERE-involved regulatory depots and transcription inactivation of candidate tumor-suppressor genes. Deletion of amplified DEREs using the CRISPR/Cas9 genomic-editing system profoundly altered transcriptional profiles of proliferation-associated signaling networks, resulting in reduction of cancer cell growth. These findings reveal a formerly uncharacterized feature wherein multiple copies of the amplicon congregate as transcriptional units in the nucleus for synchronous regulation of function-related loci in tumorigenesis. Disruption of their assembly can be a new strategy for treating breast cancers and other malignancies.

Activin A increases invasiveness of endometrial cells in an in vitro model of human peritoneum.

The aim of this study was to investigate whether activin A has an effect on the attachment and/or invasion of endometrial cells in a modeled peritoneum in vitro. Cultured endometrial stromal cells (ESCs) and endometrial epithelial cells (EECs) were treated with activin A (6.25-50 ng/ml) and with activin A (25 ng/ml) with and without inhibin A or follistatin. Fluorescent labeled cells were added to confluent peritoneal mesothelial cells (PMCs) and to a monolayer of confluent PMCs grown in a Matrigel invasion assay. The rate of endometrial cell attachment and invasion through PMCs was assessed. The expression of cell adhesion proteins N- and E-cadherin was evaluated with real-time RT-PCR. Activin A (25 ng/ml) promoted invasion of the endometrial cells through the modeled peritoneum (>2-fold versus control) and this effect was partially reversed by inhibin A and follistatin. Activin A had no effect on the rate of attachment of the endometrial cells to the PMCs or in the rate of proliferation. In addition, activin A induced a decreased mRNA expression of E-cadherin in cultured EECs. In conclusion, activin A increases invasion of EECs and ESCs into modeled peritoneum. In EECs, this effect may be related to down-regulation of E-cadherin expression. Further studies are warranted to evaluate the role of activin-A in the genesis of the endometriotic lesion.

Anti-aromatase chemicals in red wine.

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.

The effects of aromatase overexpression on mammary growth and gene expression in the aromatase x transforming growth factor alpha double transgenic mice.

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.

Regulation of colony stimulating factor-1 (CSF-1) in endometrial cells: glucocorticoids and oxidative stress regulate the expression of CSF-1 and its receptor c-fms in endometrial cells.

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.

Suppression of aromatase (estrogen synthetase) by red wine phytochemicals.

Estrogen promotes the proliferation of breast cancer cells. Aromatase is the enzyme that converts androgen to estrogen. In tumors, the expression of aromatase is upregulated compared to surrounding non-cancerous tissue. In this study, we found that wine contains phytochemicals that are capable of suppressing aromatase. Red wine was shown to be much more effective than white wine in the suppression of aromatase activity. Whole wine, lyophilized wine, and heat-treated extracts were examined for aromatase inhibition in a human placenta microsomal assay. C18 Sep-Pak cartridge (Waters Co.) separation of red wine extracts under an increasing acetonitrile (ACN) gradient found that the most active components were in the 20% ACN fraction, in that they inhibited the wild-type human placenta aromatase, wild-type porcine placenta and blastocyst aromatase in a dose-dependent fashion. The 20% ACN active fraction was heat stable and inhibited aromatase in a non-competitive manner. The aromatase-inhibitory action of red wine extracts was also examined with a transgenic mouse model in which aromatase is over-expressed in the mammary tissues. It was found that the intake of the 20% ACN fraction by gavage completely abrogated aromatase-induced hyperplasia and other changes in the mammary tissue. This is the first report demonstrating that wine, especially red wine, contains phytochemicals that can inhibit aromatase.

Overexpression of aromatase in transgenic male mice results in the induction of gynecomastia and other biochemical changes in mammary glands.

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.

Acceleration of mammary neoplasia in aromatase transgenic mice by 7,12-dimethylbenz[a]anthracene.

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.

Overexpression of aromatase leads to hyperplasia and changes in the expression of genes involved in apoptosis, cell cycle, growth, and tumor suppressor functions in the mammary glands of transgenic mice.

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.

Aromatase overexpression transgenic mice model: cell type specific expression and use of letrozole to abrogate mammary hyperplasia without affecting normal physiology.

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.

Overexpression of aromatase leads to development of testicular leydig cell tumors : an in vivo model for hormone-mediated TesticularCancer.

Despite recent advances in diagnosis and treatment of testicular cancer, its causes remain unknown. The most common conditions known to be associated with testicular cancer are cryptorchidism, infertility, and overexposure to pesticides or radiation. Recent studies also indicate hormones may play a crucial role in testicular tumorigenesis. Our studies show that about half of the male transgenic mice overexpressing aromatase in testis were infertile and/or had larger than normal testicles. Gross pathology and histological analysis showed the mice to have Leydig cell tumors, unilaterally or bilaterally. Serum estradiol levels for transgenic mice were at least twice as high as those for nontransgenic mice. Expression of aromatase and estrogen receptor were also very high in testicular tissue of transgenic mice compared to nontransgenic mice. Consistent with increased estrogenic activity in the testicular tissue, we also saw an increase in the levels of genes involved in cell cycle that are regulated by the estrogen. To obtain a better understanding of the biological significance of testicular tumorigenesis, a reliable animal model is necessary to clarify the mechanisms and correlations associated with human cancers. Here we describe such a model, which shows that overexpression of aromatase results in increased estrogen production and a changed hormone milieu, leading to the induction of testicular cancer (Leydig cell tumors). This predictable and useful model is a potential tool for the study of testicular tumorigenesis, hormonal carcinogenesis, synergistic action of other carcinogens on hormone-induced tumors, and tumor dependency on endocrine factors.

Aromatase overexpression and breast hyperplasia, an in vivo model--continued overexpression of aromatase is sufficient to maintain hyperplasia without circulating estrogens, and aromatase inhibitors abrogate these preneoplastic changes in mammary glands.

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.

Detection of Clostridium botulinum type F using the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify a portion of the Clostridium botulinum type F toxin gene. An 1137-bp fragment was amplified from 11 different strains of type F C. botulinum with primers derived from the published sequence of type F strain no. 202. This fragment was not amplified from the DNA of C. botulinum types A, B and E, or from other clostridial organisms examined. When used as a hybridization probe, the 1137-bp PCR-generated fragment generated from one of the type F strains (the proteolytic strain type F Langeland) hybridized to the PCR products from all other type F toxin-producing strains tested. Portions of fragments amplified from the type F Langeland strain were sequenced. The sequence of this strain was found to exhibit approximately 3% variation from the published sequence of the non-proteolytic type F strain no. 202. Primers designed to pair with the regions of maximum sequence variation between strain 202 and the Langeland strain gave amplification products only with DNA from type F strains that exhibited the same proteolytic properties as the strain from which the primer sequences were derived. These findings underscore the need to consider variations in sequence when designing oligonucleotide probes and PCR primers in order to avoid false negative results.