Donato Romagnolo

Interests

Research

Research focuses on; (i) mechanisms of epigenetic silencing of tumor suppressor genes by environmental and dietary xenobiotics; 2) role of dietary bioactive food components in the etiology and prevention of breast and colon cancer and inflammation.

Teaching

Teaching interests include Nutritional Biology - NSC408. Primary goals of this class are 1) to provide a comprehensive overview of how nutrients are utilized during various metabolic states, ex. in healthy subjects, diabetes, obesity, and cancer and 2) provide a foundation of nutrition concepts that will assist students in pursuing specialized/advanced courses in biochemistry, nutrition, physiology, and medicine.Another area of interest is metabolic integration (NSC 602). Objective of the course is to provide an analysis at the graduate level of current knowledge regarding the interactions between the intake, absorption, transport, processing, storage, catabolism and excretion of nutrients and the regulation of metabolic homeostasis in the intact organism. Emphasis areas include interrelationships between protein, carbohydrate and fat metabolism and their regulation by dietary, hormonal and genetic factors in humans. Syllabus includes topics relevant to nutritional regulation of gene expression and impact on obesity, diabetes, and cancer.A third area of teaching interest pertains to studies of Mediterranean dietary patterns and impact on health promotion. Epidemiologic studies revealed the protective role of adherence to this pattern on overall cancer incidence and mortality, prevention of obesity, type II diabetes, and cardiovascular diseases. On November 17, 2010, UNESCO recognized this diet pattern as an Intangible Cultural Heritage of Italy, Greece, Spain and Morocco, thus recognizing this Mediterranean component of life style as a contribution to the world. Objectives of this Summer Program include: 1) Provide students with information about the health benefits of foods associated with a Mediterranean diet and for the prevention of chronic diseases;2) Review and discuss the influence of bioactive compounds present in Mediterranean foods on metabolic pathways;3) Provide students with an opportunity to learn about the food industry in the Mediterranean region, and dietary patterns;4) Acquire hands-on experience with food preparation supervised by local food instructors;5) Experience the cultural diversity and influence of Mediterranean culture.

Courses

Scholarly Contributions

Books

• Romagnolo, D. -. (2014). Mediterranean Diet and Health.
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  Book on Mediterranean Diet and Health. Eds Romagnolo and Selmin. Springer Pub. (In press)

Chapters

• Donovan, M., Selmin, O., & Romagnolo, D. (2017). Prevention by food bioactives in relation to breast cancer subtype: Epigenetic mechanisms.. In Epigenetics of Chemoprevention(pp Chapter 16). Elsevier.
• Romagnolo, D. (2018). Mediterranean diet for the prevention of ER-negative breast cancers: Molecular interactions of olive oil and vegetable bioactives in HER2-enriched and triple negative breast cancers. Donovan MG, Selmin OI, Romagnolo DF.. In Molecular Nutrition: Cancer. Andrew Neilson and Eva Schmelz, editors.. Elsevier, New York, NY.
• Romagnolo, D. (2018). Prevention of breast cancer by food bioactives in relation to cancer subtypes: Epigenetic mechanisms. Donovan MG, Selmin OI, Romagnolo DF.. In Epigenetics of Chemoprevention: Bishayee A, editor.. New York, NY: Elsevier.
• Romagnolo, D. (2016). Building the Mediterranean Pyramid-PART A: Mediterranean Recipes.. In Mediterranean Diet: Impact on Health and Disease.. Humana Press/Springer.
• Romagnolo, D. (2016). Mediterranean Diet and Lifestyle in a Modern World Context. In Mediterranean Diet: Impact on Health and Disease.. Humana Press/Springer, 2016.
• Romagnolo, D. (2016). Mediterranean Diet and Neurodegenerative Diseases. In Mediterranean Diet: Impact on Health and Disease.. Humana Press/Springer.
• Romagnolo, D. (2016). Mediterranean Diet, Inflammatory Bowel Diseases and Colon Cancer.. In Mediterranean Diet: Impact on Health and Disease.. Humana Press/Springer Pubs,.
• Romagnolo, D., Jackson, K., Sparks, P., & Selmin, O. (2016). Building the Mediterranean Pyramid: Part B—Balancing the Plate. In Mediterranean Diet: Dietary Guidelines and Impact on Health and Disease(pp 275-288). Springer -Human Press. doi:DOI 10.1007/978-3-319-27969-5
• Romagnolo, D., & Selmin, O. (2015). Epigenetics of Endocrine Tumors in Women and Dietary Prevention. In Preventive Nutrition. Springer.
• Romagnolo, D. (2014). Epigenetics of BRCA-1-related breast tumorigenesis and dietary prevention. In The role of nutrition and metabolism on epigenetic regulation. Taylor&Francis.

Journals/Publications

• Romagnolo, D. (2018). Special Issue on Breast Cancer and Nutrition. Romagnolo DF, Ross SA.. Frontiers in Clinical Nutrition.
• Romagnolo, D. (2019). Expression of an FXR transgene protects against colonic inflammation induced by n-6 linoleic acid from soybean oil. J. Nutr..
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  Wren S, Donovan MG, Doetschman TG, Selmin OI, Romagnolo DF. Expression of an FXR transgene protects against colonic inflammation induced by n-6 linoleic acid from soybean oil. J. Nutr.
• Romagnolo, D. (2020). n-6 High Fat Diet-Induced Gut Microbiome Dysbiosis Associates with Colonic Inflammation in a Mouse Model of Childhood to Adult Exposure. Journal of Nutrition.
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  Ornella I Selmin, Andreas Papoutsis, Sabine Hazan, Christopher Smith, Nick Greenfield, Micah G Donovan, Spencer N Wren, Thomas C Doetschman, Justin M Snider, Sherry H-H Chow, and Donato F Romagnolo
• Donovan, M. G., Selmin, O. I., Stillwater, B. J., Neumayer, L. A., & Romagnolo, D. F. (2020). Do Olive and Fish Oils of the Mediterranean Diet Have a Role in Triple Negative Breast Cancer Prevention and Therapy? An Exploration of Evidence in Cells and Animal Models. Frontiers in nutrition, 7, 571455.
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  Breast cancer is the most common malignancy and cause of cancer-related mortality among women worldwide. Triple negative breast cancers (TNBC) are the most aggressive and lethal of the breast cancer molecular subtypes, due in part to a poor understanding of TNBC etiology and lack of targeted therapeutics. Despite advances in the clinical management of TNBC, optimal treatment regimens remain elusive. Thus, identifying interventional approaches that suppress the initiation and progression of TNBC, while minimizing side effects, would be of great interest. Studies have documented an inverse relationship between the incidence of hormone receptor negative breast cancer and adherence to a Mediterranean Diet, particularly higher consumption of fish and olive oil. Here, we performed a review of studies over the last 5 years investigating the effects of fish oil, olive oil and their components in model systems of TNBC. We included studies that focused on the fish oil ω-3 essential fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in addition to olive oil polyphenolic compounds and oleic acid. Both beneficial and deleterious effects on TNBC model systems are reviewed and we highlight how multiple components of these Mediterranean Diet oils target signaling pathways known to be aberrant in TNBC including PI3K/Akt/mTOR, NF-κB/COX2 and Wnt/β-catenin.
• Donovan, M. G., Wren, S. N., Cenker, M., Selmin, O. I., & Romagnolo, D. F. (2020). Dietary fat and obesity as modulators of breast cancer risk: Focus on DNA methylation. British journal of pharmacology, 177(6), 1331-1350.
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  Breast cancer (BC) is the most common cancer and second leading cause of cancer mortality in women worldwide. Validated biomarkers enhance efforts for early detection and treatment, which reduce the risk of mortality. Epigenetic signatures have been suggested as good biomarkers for early detection, prognosis and targeted therapy of BC. Here, we highlight studies documenting the modifying effects of dietary fatty acids and obesity on BC biomarkers associated with DNA methylation. We focus our analysis on changes elicited in writers of DNA methylation (i.e., DNA methyltransferases), global DNA methylation and gene-specific DNA methylation. To provide context, we precede this discussion with a review of the available evidence for an association between BC incidence and both dietary fat consumption and obesity. We also include a review of well-vetted BC biomarkers related to cytosine-guanine dinucleotides methylation and how they influence BC risk, prognosis, tumour characteristics and response to treatment. LINKED ARTICLES: This article is part of a themed section on The Pharmacology of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.6/issuetoc.
• Selmin, O. I., Donovan, M. G., Stillwater, B. J., Neumayer, L., & Romagnolo, D. F. (2020). Epigenetic Regulation and Dietary Control of Triple Negative Breast Cancer. Frontiers in nutrition, 7, 159.
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  Triple negative breast cancer (TNBC) represents a highly heterogeneous group of breast cancers, lacking expression of the estrogen (ER) and progesterone (PR) receptors, and human epidermal growth factor receptor 2 (HER2). TNBC are characterized by a high level of mutation and metastasis, poor clinical outcomes and overall survival. Here, we review the epigenetic mechanisms of regulation involved in cell pathways disrupted in TNBC, with particular emphasis on dietary food components that may be exploited for the development of effective strategies for management of TNBC.
• Stillwater, B. J., Bull, A. C., Romagnolo, D. F., Neumayer, L. A., Donovan, M. G., & Selmin, O. I. (2020). Bisphenols and Risk of Breast Cancer: A Narrative Review of the Impact of Diet and Bioactive Food Components. Frontiers in nutrition, 7, 581388.
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  Data from preclinical studies suggest a link between increased risk of breast cancer and exposure to bisphenols at doses below what the United States Food and Drug Administration (FDA) considers as safe for consumption. Bisphenols exert estrogenic effects and are found in canned and plastic wrapped foods, food packaging, and plasticware. Mechanistically, bisphenols bind to the estrogen receptor (ER) and activate the expression of genes associated with cell proliferation and breast cancer. In this paper, we present a narrative literature review addressing bisphenol A and chemical analogs including bisphenol AF, bisphenol F, and bisphenol S selected as prototype xenoestrogens; then, we discuss biological mechanisms of action of these bisphenols in breast cells and potential impact of exposure at different stages of development (i.e., perinatal, peripubertal, and adult). Finally, we summarize studies detailing interactions, both preventative and promoting, of bisphenols with food components on breast cancer risk. We conclude the review with a discussion of current controversies in interpretation of the above research and future areas for investigation, including the impact of bisphenols and food components on breast tumor risk.
• Wren, S. N., Donovan, M. G., Selmin, O. I., Doetschman, T. C., & Romagnolo, D. F. (2020). A -Driven Transgene Modulates Enterohepatic Bile Acid Homeostasis and Response to an -6-Enriched High-Fat Diet. International journal of molecular sciences, 21(21).
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  A diet high in -6 polyunsaturated fatty acids (PUFAs) may contribute to inflammation and tissue damage associated with obesity and pathologies of the colon and liver. One contributing factor may be dysregulation by -6 fatty acids of enterohepatic bile acid (BA) metabolism. The farnesoid X receptor (FXR) is a nuclear receptor that regulates BA homeostasis in the liver and intestine. This study aims to compare the effects on FXR regulation and BA metabolism of a palm oil-based diet providing 28% energy (28%E) from fat and low -6 linoleic acid (LA, 2.5%E) (CNTL) with those of a soybean oil-based diet providing 50%E from fat and high (28%E) in LA (-6HFD). Wild-type (WT) littermates and a transgenic mouse line overexpressing the isoform under the control of the intestine-specific promoter () were fed the CNTL or -6HFD starting at weaning through 16 weeks of age. Compared to the CNTL diet, the -6HFD supports higher weight gain in both WT and littermates; increases the expression of α, and peroxisome proliferator-activated receptor-γ () in the small intestine, in the colon, and cytochrome P4507A1 () and small heterodimer protein () in the liver; and augments the levels of total BA in the liver, and primary chenodeoxycholic (CDCA), cholic (CA), and β-muricholic (βMCA) acid in the cecum. Intestinal overexpression of the augments expression of and ileal bile acid-binding protein ( in the small intestine and in the proximal colon. Conversely, it antagonizes -6HFD-dependent accumulation of intestinal and hepatic CDCA and CA; hepatic levels of ; and expression of in the small intestine. We conclude that intestinal overexpression represses hepatic de novo BA synthesis and protects against -6HFD-induced accumulation of human-specific primary bile acids in the cecum.
• Romagnolo, D. (2019). Arsenic‑induced BRCA1 CpG promoter methylation is associated with the downregulation of ERα and resistance to tamoxifen in MCF7 breast cancer cells and mouse mammary tumor xenografts.. Int J Oncol. 2019 Mar;54(3):869-878..
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  Selmin OI, Donovan MG, Skovan B, Paine-Murieta GD, Romagnolo DF.Arsenic‑induced BRCA1 CpG promoter methylation is associated with thedownregulation of ERα and resistance to tamoxifen in MCF7 breast cancer cells and mouse mammary tumor xenografts. Int J Oncol. 2019 Mar;54(3):869-878.
• Romagnolo, D. (2019). Dietary fat and obesity as modulators of BC risk: Focus on DNA methylation. Br J Pharmacol. 2019 Nov 5. doi: 10.1111/bph.14891..
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  Donovan MG, Wren SN, Cenker M, Selmin OI, Romagnolo DF. Dietary fat andobesity as modulators of BC risk: Focus on DNA methylation. Br J Pharmacol. 2019 Nov 5. doi: 10.1111/bph.14891.
• Romagnolo, D. (2019). Epigenetic Activation of BRCA1 by Genistein In Vivo and Triple Negative Breast Cancer Cells Linked to Antagonism toward Aryl Hydrocarbon Receptor.. Nutrients, 2019 Oct 23;11(11)..
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  Donovan MG, Selmin OI, Doetschman TC, Romagnolo DF. Epigenetic Activation ofBRCA1 by Genistein In Vivo and Triple Negative Breast Cancer Cells Linked toAntagonism toward Aryl Hydrocarbon Receptor. Nutrients. 2019 Oct 23;11(11).
• Romagnolo, D. (2019). n-6 Linoleic Acid Induces Epigenetics Alterations Associated with Colonic Inflammation and Cancer.. Nutrients. 2019 Jan 15;11(1)..
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  Romagnolo DF, Donovan MG, Doetschman TC, Selmin OI. n-6 Linoleic Acid Induces Epigenetics Alterations Associated with Colonic Inflammation and Cancer.Nutrients. 2019 Jan 15;11(1).
• Donovan, M. G., Selmin, O. I., & Romagnolo, D. F. (2018). Aryl Hydrocarbon Receptor Diet and Breast Cancer Risk. The Yale journal of biology and medicine, 91(2), 105-127.
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  Breast cancer is the most common type of cancer and leading cause of cancer mortality among women worldwide. However, the majority of breast malignancies are of sporadic etiology. Therefore, identifying risk-mitigating factors may significantly decrease the burden of breast cancer. Diet can have both a predisposing and protective role in breast tumorigenesis. However, establishing efficacy of dietary constituents for cancer prevention has been limited by suboptimal dietary assessment. There is a need to acquire new experimental evidence that can be used to discriminate beneficial from harmful dietary constituents. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is recognized as the mediator of halogenated and polycyclic aromatic hydrocarbon toxicities. Importantly, evidence points to a breast tumor-promoting role for the AhR. Preclinical and clinical studies suggest that the AhR is overexpressed in advanced and triple negative breast cancers. Several dietary constituents, namely flavonoid compounds, have demonstrated inhibitory effects on AhR activation. Given this background, in this paper we elaborate on the working hypothesis that a diet rich in AhR food agonists favors breast tumor development, whereas a diet rich in AhR food antagonists is protective. As an initial approach to developing an AhR diet hypothesis, we conducted a review of published studies reporting on the association between intake of AhR inhibitory foods and risk of breast cancer. To assist the reader with interpretation of the concepts leading to the AhR diet hypothesis, we have preceded this review with an overview of AhR biology and its role in breast cancer development.
• Romagnolo, D. (2018). Arsenic-induced BRCA1 CpG Promoter Methylation Associates with Downregulation of ERα and Resistance to Tamoxifen in MCF7 Breast Cancer Cells and Mouse Mammary Tumor Xenografts. International Journal of Oncology.
• Romagnolo, D. (2018). n-6 linoleic acid induces epigenetics alterations associated with colonic inflammation and cancer. Nutrients.
• Donovan, M. G., Selmin, O. I., Doetschman, T. C., & Romagnolo, D. F. (2017). Mediterranean Diet: Prevention of Colorectal Cancer. Frontiers in nutrition, 4, 59.
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  Colorectal cancer (CRC) is the third most common cancer diagnosis and the second and third leading cause of cancer mortality in men and women, respectively. However, the majority of CRC cases are the result of sporadic tumorigenesis via the adenoma-carcinoma sequence. This process can take up to 20 years, suggesting an important window of opportunity exists for prevention such as switching toward healthier dietary patterns. The Mediterranean diet (MD) is a dietary pattern associated with various health benefits including protection against cardiovascular disease, diabetes, obesity, and various cancers. In this article, we review publications available in the PubMed database within the last 10 years that report on the impact of a MD eating pattern on prevention of CRC. To assist the reader with interpretation of the results and discussion, we first introduce indexes and scoring systems commonly used to experimentally determine adherence to a MD, followed by a brief introduction of the influence of the MD pattern on inflammatory bowel disease, which predisposes to CRC. Finally, we discuss key biological mechanisms through which specific bioactive food components commonly present in the MD are proposed to prevent or delay the development of CRC. We close with a discussion of future research frontiers in CRC prevention with particular reference to the role of epigenetic mechanisms and microbiome related to the MD eating pattern.
• Romagnolo, D., & Selmin, O. (2017). Mediterranean Diet and Prevention of Chronic Diseases. Nutrition Today, Sep;52(5), 208-222.
• Romagnolo, D., Papoutsis, A., Donovan, M., & Selmin, O. (2017). Genistein reverses BRCA-1 CpG methylation in human breast cancer cells with activated AhR. Current Developments in Nutrition, 1(6), e000562. doi:https://doi.org/10.3945/cdn.117.000562
• Romagnolo, D. (2015). Epigenetics of endocrine tumors: modifying role of environmental and bioactive food compounds.. Mol Nutr Food Res.
• Papoutsis, A. J., Selmin, O. I., Borg, J. L., & Romagnolo, D. F. (2015). Gestational Exposure to the AhR Agonist 2,3,7,8-Tetrachlorodibenzo-p-dioxin Induces BRCA-1 Promoter Hypermethylation and Reduces BRCA-1 Expression in Mammary Tissue of Rat Offspring: Preventive Effects of Resveratrol. MOLECULAR CARCINOGENESIS, 54(4), 261-269.
• Romagnolo, D. (2015). Inactivation of Adenomatous Polyposis Coli Reduces Bile Acid/Farnesoid X Receptor Expression through Fxr gene CpG Methylation in Mouse Colon Tumors and Human Colon Cancer Cells.. J Nutr.
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  Authorship: Selmin OI, Fang C, Lyon AM, Doetschman TC, Thompson PA, Martinez JD, Smith JW,Lance PM, Romagnolo DF.
• Romagnolo, D. F., Papoutsis, A. J., Laukaitis, C., & Selmin, O. I. (2015). Constitutive expression of AhR and BRCA-1 promoter CpG hypermethylation as biomarkers of ERα-negative breast tumorigenesis. BMC cancer, 15(1), 1026.
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  Only 5-10 % of breast cancer cases is linked to germline mutations in the BRCA-1 gene and occurs early in life. Conversely, sporadic breast tumors, which represent 90-95 % of breast malignancies, have lower BRCA-1 expression, but not mutated BRCA-1 gene, and tend to occur later in life in combination with other genetic alterations and/or environmental exposures. The latter may include environmental and dietary factors that activate the aromatic hydrocarbon receptor (AhR). Therefore, understanding if changes in expression and/or activation of the AhR are associated with somatic inactivation of the BRCA-1 gene may provide clues for breast cancer therapy.
• Romagnolo, A. P., Romagnolo, D. F., & Selmin, O. I. (2015). BRCA1 as target for breast cancer prevention and therapy. Anti-cancer agents in medicinal chemistry, 15(1), 4-14.
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  The Breast Cancer 1 protein (BRCA1) is a tumor suppressor involved in basic cellular functions necessary for cell replication and DNA synthesis, but reduced expression of BRCA1, due to mutations or epigenetic inactivation, leads to impaired mammary gland differentiation and increased risk of breast cancer development. Although BRCA1 acts as a tumor suppressor and is present in all cells, where it is essential for the maintenance of the genome integrity, it is still not clear why mutations in the BRCA1 gene predispose to breast and ovarian, but not to other types of cancer. In the first part of this review, we briefly discuss the function and regulation of the BRCA1 protein, including its role associated with familial and sporadic breast cancer. The second part is an overview of the therapeutic compounds used for breast cancer treatment targeting BRCA1, and the natural food components that hold potential preventive effect against those types of breast cancer in which BRCA1 expression is either reduced or lacking. Further studies elucidating the interactions between dietary compounds and cellular pathways, involved in regulation of BRCA1expression, are necessary for the development of strategies that may successfully prevent or treat breast cancer.
• Papoutsis, A. J., Selmin, O. I., Borg, J. L., & Romagnolo, D. F. (2013). Gestational exposure to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin induces BRCA-1 promoter hypermethylation and reduces BRCA-1 expression in mammary tissue of rat offspring: Preventive effects of resveratrol. Molecular Carcinogenesis.
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  Abstract: Studies with murine models suggest that maternal exposure to aromatic hydrocarbon receptor (AhR) agonists may impair mammary gland differentiation and increase the susceptibility to mammary carcinogenesis in offspring. However, the molecular mechanisms responsible for these perturbations remain largely unknown. Previously, we reported that the AhR agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced CpG methylation of the breast cancer-1 (BRCA-1) gene and reduced BRCA-1 expression in breast cancer cell lines. Based on the information both the human and rat BRCA-1 genes harbor xenobiotic responsive elements (XRE=5'-GCGTG-3'), which are binding targets for the AhR, we extended our studies to the analysis of offspring of pregnant Sprague-Dawley rats treated during gestation with TCDD alone or in combination with the dietary AhR antagonist resveratrol (Res). We report that the in utero exposure to TCDD increased the number of terminal end buds (TEB) and reduced BRCA-1 expression in mammary tissue of offspring. The treatment with TCDD induced occupancy of the BRCA-1 promoter by DNA methyltransferase-1 (DNMT-1), CpG methylation of the BRCA-1 promoter, and expression of cyclin D1 and cyclin-dependent kinase-4 (CDK4). These changes were partially overridden by pre-exposure to Res, which stimulated the expression of the AhR repressor (AhRR) and its recruitment to the BRCA-1 gene. These findings point to maternal exposure to AhR agonists as a risk factor for breast cancer in offspring through epigenetic inhibition of BRCA-1 expression, whereas dietary antagonists of the AhR may exert protective effects © 2013 Wiley Periodicals, Inc.
• Romagnolo, D., Papoutsis, A. J., Selmin, O. I., Borg, J. L., & Romagnolo, D. -. (2013). Gestational exposure to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin induces BRCA-1 promoter hypermethylation and reduces BRCA-1 expression in mammary tissue of rat offspring: Preventive effects of resveratrol. Molecular carcinogenesis.
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  Studies with murine models suggest that maternal exposure to aromatic hydrocarbon receptor (AhR) agonists may impair mammary gland differentiation and increase the susceptibility to mammary carcinogenesis in offspring. However, the molecular mechanisms responsible for these perturbations remain largely unknown. Previously, we reported that the AhR agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced CpG methylation of the breast cancer-1 (BRCA-1) gene and reduced BRCA-1 expression in breast cancer cell lines. Based on the information both the human and rat BRCA-1 genes harbor xenobiotic responsive elements (XRE = 5'-GCGTG-3'), which are binding targets for the AhR, we extended our studies to the analysis of offspring of pregnant Sprague-Dawley rats treated during gestation with TCDD alone or in combination with the dietary AhR antagonist resveratrol (Res). We report that the in utero exposure to TCDD increased the number of terminal end buds (TEB) and reduced BRCA-1 expression in mammary tissue of offspring. The treatment with TCDD induced occupancy of the BRCA-1 promoter by DNA methyltransferase-1 (DNMT-1), CpG methylation of the BRCA-1 promoter, and expression of cyclin D1 and cyclin-dependent kinase-4 (CDK4). These changes were partially overridden by pre-exposure to Res, which stimulated the expression of the AhR repressor (AhRR) and its recruitment to the BRCA-1 gene. These findings point to maternal exposure to AhR agonists as a risk factor for breast cancer in offspring through epigenetic inhibition of BRCA-1 expression, whereas dietary antagonists of the AhR may exert protective effects. © 2013 Wiley Periodicals, Inc.
• Papoutsis, A. J., Borg, J. L., Selmin, O. I., & Romagnolo, D. F. (2012). BRCA-1 promoter hypermethylation and silencing induced by the aromatic hydrocarbon receptor-ligand TCDD are prevented by resveratrol in MCF-7 Cells. Journal of Nutritional Biochemistry, 23(10), 1324-1332.
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  PMID: 22197621;Abstract: Epigenetic mechanisms may contribute to reduced expression of the tumor suppressor gene BRCA-1 in sporadic breast cancers. Through environmental exposure and diet, humans are exposed to xenobiotics and food compounds that bind the aromatic hydrocarbon receptor (AhR). AhR-ligands include the dioxin-like and tumor promoter 2,3,7,8 tetrachlorodibenzo- p-dioxin (TCDD). The activated AhR regulates transcription through binding to xenobiotic response elements (XREs=GCGTG) and interactions with transcription cofactors. Previously, we reported on the presence of several XREs in the proximal BRCA-1 promoter and that the expression of endogenous AhR was required for silencing of BRCA-1 expression by TCDD. Here, we document that in estrogen receptor-α-positive and BRCA-1 wild-type MCF-7 breast cancer cells, the treatment with TCDD attenuated 17β-estradiol-dependent stimulation of BRCA-1 protein and induced hypermethylation of a CpG island spanning the BRCA-1 transcriptional start site of exon-1a. Additionally, we found that TCDD enhanced the association of the AhR; DNA methyl transferase (DNMT)1, DNMT3a and DNMT3b; methyl binding protein (MBD)2; and trimethylated H3K9 (H3K9me3) with the BRCA-1 promoter. Conversely, the phytoalexin resveratrol, selected as a prototype dietary AhR antagonist, antagonized at physiologically relevant doses (1 μmol/L) the TCDD-induced repression of BRCA-1 protein, BRCA-1 promoter methylation and the recruitment of the AhR, MBD2, H3K9me3 and DNMTs (1, 3a and 3b). Taken together, these observations provide mechanistic evidence for AhR agonists in the establishment of BRCA-1 promoter hypermethylation and the basis for the development of prevention strategies based on AhR antagonists. © 2012 Elsevier Inc.
• Papoutsis, A., Borg, J., Selmin, O., & Romagnolo, D. (2012). BRCA-1 promoter hypermethylation and silencing induced by the aromatic hydrocarbon receptor-ligand TCDD are prevented by resveratrol in MCF-7 cells. J Nutr Biochem, 23(10), 1324-32.
• Romagnolo, D. -. (2012). Flavonoids and cancer prevention: a review of the evidence. J Nutr Gerontol Geriatr, 31(3), 206-238.
• Romagnolo, D. -. (2012). Proteomic approaches to predict bioavailability of fatty acids and their influence on cancer and chronic disease prevention. J Nutr, 142(7), 1.
• Romagnolo, D. F., & Milner, J. A. (2012). Opportunities and challenges for nutritional proteomics in cancer prevention. Journal of Nutrition, 142(7), 1360S-1369S.
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  PMID: 22649262;PMCID: PMC3374671;Abstract: Knowledge gaps persist about the efficacy of cancer prevention strategies based on dietary food components. Adaptations to nutrient supply are executed through tuning of multiple protein networks that include transcription factors, histones, modifying enzymes, translation factors, membrane and nuclear receptors, and secreted proteins. However, the simultaneous quantitative and qualitative measurement of all proteins that regulate cancer processes is not practical using traditional protein methodologies. Proteomics offers an attractive opportunity to fill this knowledge gap and unravel the effects of dietary components on protein networks that impinge on cancer. The articles presented in this supplement are from talks proffered in the "Nutrition Proteomics and Cancer Prevention" session at the American Institute for Cancer Research Annual Research Conference on Food, Nutrition, Physical Activity and Cancer held in Washington, DC on October 21 and 22, 2010. Recent advances in MS technologies suggest that studies in nutrition and cancer prevention may benefit from the adoption of proteomic tools to elucidate the impact on biological processes that govern the transition from normal to malignant phenotype; to identify protein changes that determine both positive and negative responses to food components; to assess how protein networks mediate dose-, time-, and tissue-dependent responses to food components; and, finally, for predicting responders and nonresponders. However, both the limited accessibility to proteomic technologies and research funding appear to be hampering the routine adoption of proteomic tools in nutrition and cancer prevention research. © 2012 American Society for Nutrition.
• Romagnolo, D. F., & Selmin, O. I. (2012). Flavonoids and Cancer Prevention: A Review of the Evidence. Journal of Nutrition in Gerontology and Geriatrics, 31(3), 206-238.
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  PMID: 22888839;Abstract: The objective of this work is to review data from epidemiological and preclinical studies addressing the potential benefits of diets based on flavonoids for cancer prevention. Flavonoids are subdivided into subclasses including flavonols, flavones, flavanones, flavan-3-ols, anthocyanidins, and isoflavones. Epidemiological studies suggest dietary intake of flavonoids may reduce the risk of tumors of the breast, colon, lung, prostate, and pancreas. However, some studies have reported inconclusive or even harmful associations. A major challenge in the interpretation of epidemiological studies is that most of the data originate from case-control studies and retrospective acquisition of flavonoid intake. Differences in agricultural, sociodemographics, and lifestyle factors contribute to the heterogeneity in the intake of flavonoids among populations residing in the United States, Europe, and Asia. Dose and timing of exposure may influence the anticancer response to flavonoid-rich diets. A limited number of intervention trials of flavonoids have documented cancer preventative effects. Proposed anticancer mechanisms for flavonoids are inhibition of proliferation, inflammation, invasion, metastasis, and activation of apoptosis. Prospective studies with larger sample sizes are needed to develop biomarkers of flavonoid intake and effect. Mechanistic studies are needed to ascertain how flavonoid-rich diets influence gene regulation for cancer prevention. © 2012 Copyright Taylor and Francis Group, LLC.
• Romagnolo, D. F., Dashwood, R., Stover, P. J., Waterland, R. A., & Ziegler, T. R. (2012). Nutritional regulation of epigenetic changes. Advances in Nutrition, 3(5), 749-750.
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  PMID: 22983864;PMCID: PMC3648767;
• Romagnolo, D. F., Davis, C. D., & Milner, J. A. (2012). Phytoalexins in cancer prevention. Frontiers in Bioscience, 17(6), 2035-2058.
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  PMID: 22652763;Abstract: Plant phytoalexins are a class of low molecular weight compounds that accumulate in response to biotic and abiotic elicitors such as pathogens, wounding, freezing, UV light, and exposure to agricultural chemicals. Phytoalexins have been identified in at least 75 plants including cruciferous vegetables, soybean, garlic, tomato, rice, beans, and potatoes suggesting plants may be a rich source of cancer-fighting compounds. Preclinical evidence suggests these compounds possess anticancer properties including an inhibition of microbial activity, cell proliferation, invasion and metastasis, hormonal stimulation, and stimulatory effects on expression of metabolizing enzymes. This review highlights the plausible molecular mechanisms through which phytoalexins regulate biological processes that can impinge cancer development. Targets of phytoalexins include signal transduction pathways, transcription factors, cell cycle checkpoints, intrinsic and extrinsic apoptotic pathways, cell invasion and matrix metalloproteinase, nuclear receptors, and the phase II detoxification pathway. Additional research should address physiological relevant dietary concentrations, combinations of phytoalexins and interactions with other dietary compounds, duration of exposure, and tissue specificity as variables that influence the effectiveness of phytoalexins on normal and cancerous processes.
• Romagnolo, D., Dashwood, R., Stover, P., Waterland, R., & Ziegler, T. (2012). Nutritional regulation of epigenetic changes. Adv Nutr, 3(5), 749-750.
• Romagnolo, D., Papoutsis, A. J., Borg, J. L., Selmin, O. I., & Romagnolo, D. -. (2012). BRCA-1 promoter hypermethylation and silencing induced by the aromatic hydrocarbon receptor-ligand TCDD are prevented by resveratrol in MCF-7 cells. The Journal of nutritional biochemistry, 23(10).
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  Epigenetic mechanisms may contribute to reduced expression of the tumor suppressor gene BRCA-1 in sporadic breast cancers. Through environmental exposure and diet, humans are exposed to xenobiotics and food compounds that bind the aromatic hydrocarbon receptor (AhR). AhR-ligands include the dioxin-like and tumor promoter 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). The activated AhR regulates transcription through binding to xenobiotic response elements (XREs=GCGTG) and interactions with transcription cofactors. Previously, we reported on the presence of several XREs in the proximal BRCA-1 promoter and that the expression of endogenous AhR was required for silencing of BRCA-1 expression by TCDD. Here, we document that in estrogen receptor-α-positive and BRCA-1 wild-type MCF-7 breast cancer cells, the treatment with TCDD attenuated 17β-estradiol-dependent stimulation of BRCA-1 protein and induced hypermethylation of a CpG island spanning the BRCA-1 transcriptional start site of exon-1a. Additionally, we found that TCDD enhanced the association of the AhR; DNA methyl transferase (DNMT)1, DNMT3a and DNMT3b; methyl binding protein (MBD)2; and trimethylated H3K9 (H3K9me3) with the BRCA-1 promoter. Conversely, the phytoalexin resveratrol, selected as a prototype dietary AhR antagonist, antagonized at physiologically relevant doses (1 μmol/L) the TCDD-induced repression of BRCA-1 protein, BRCA-1 promoter methylation and the recruitment of the AhR, MBD2, H3K9me3 and DNMTs (1, 3a and 3b). Taken together, these observations provide mechanistic evidence for AhR agonists in the establishment of BRCA-1 promoter hypermethylation and the basis for the development of prevention strategies based on AhR antagonists.
• Roos, B. d., & Romagnolo, D. F. (2012). Proteomic approaches to predict bioavailability of fatty acids and their influence on cancer and chronic disease prevention. Journal of Nutrition, 142(7), 1370S-1376S.
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  PMID: 22649259;PMCID: PMC3374672;Abstract: A low intake of fish and PUFA and high dietary trans- and SFA are considered to be among the main preventable causes of death. Unfortunately, epidemiological and preclinical studies have yet to identify biomarkers that accurately predict the influence of fatty acid intake on risk of chronic diseases, including cancer. Changes in protein profile and post-translational modifications in tissue and biofluids may offer important clues about the impact of fatty acids on the etiology of chronic diseases. However, conventional protein methodologies are not adequate for assessing the impact of fatty acids on protein expression patterns and modifications and the discovery of protein biomarkers that predict changes in disease risk and progression in response to fatty acid intake. Although fluctuations in protein structure and abundance and interindividual variability often mask subtle effects caused by dietary intervention, modern proteomic platforms offer tremendous opportunities to increase the sensitivity of protein analysis in tissues and biofluids (plasma, urine) and elucidate the effects of fatty acids on regulation of protein networks. Unfortunately, the number of studies that adopted proteomic tools to investigate the impact of fatty acids on disease risk and progression is quite small. The future success of proteomics in the discovery of biomarkers of fatty acid nutrition requires improved accessibility and standardization of proteomic methodologies, validation of quantitative and qualitative protein changes (e.g., expression levels, posttranslational modifications) induced by fatty acids, and application of bioinformatic tools that can inform about the causeeffect relationships between fatty acid intake and health response. © 2012 American Society for Nutrition.
• Palbykin, B., Borg, J., Caldwell, P. T., Rowles, J., Papoutsis, A. J., Romagnolo, D. F., & Selmin, O. I. (2011). Trichloroethylene induces methylation of the Serca2 promoter in H9c2 cells and embryonic heart. Cardiovascular Toxicology, 11(3), 204-214.
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  PMID: 21479763;Abstract: Trichloroethylene (TCE) is a halogenated hydrocarbon used as a solvent in industrial settings and in house-cleaning products. Exposure to TCE has been linked to increased risk for congenital heart malformations in both human and animal models. Previous studies showed TCE exposure reduced the expression and function of the ATPdependent calcium pump, Serca2a, which is important for regulating calcium flux in myocytes and maintaining physiological cardiac function. In this study, we investigated whether TCE reduced Serca2a expression by altering the methylation status of its proximal promoter region. Low doses of TCE exposure (10 ppb) induced DNA hyper methylation in the Serca2 promoter region in cardiac myoblast cells and rat embryonic cardiac tissue. TCE exposure induced DNA methylation in a region of the Serca2 promoter which is the target for SP1 binding site essential for regulation of Serca2a transcriptional activity. Chromatin immunoprecipitation data confirmed that TCE exposure reduced the binding of SP1 to the Serca2 promoter region adjacent to the methylated CpG dimer. Finally, low-dose TCE exposure reduced the concentration of S-adenosyl-methionine in exposed cells and embryos. These cumulative data indicate that epigenetic mechanisms, including DNA methylation, may be important in mediating the teratogenic effects of TCE in embryonic heart. © 2011 Springer Science+Business Media, LLC.
• Palbykin, B., Borg, J., Caldwell, P., Rowles, J., Papoutsis, A., Romagnolo, D., & Selmin, O. (2011). Tricholoroethylene induces methylation of the Serca2 promoter in H9c2 cells and embryonic heart. Cardiovasc Toxicol, 11(3), 204-214.
• Papoutsis, A. J., Lamore, S. D., Wondrak, G. T., Selmin, O. I., & Romagnolo, D. F. (2010). Resveratrol prevents epigenetic silencing of BRCA-1 by the aromatic hydrocarbon receptor in human breast cancer cells. Journal of Nutrition, 140(9), 1607-1614.
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  PMID: 20631324;PMCID: PMC3139234;Abstract: The BRCA-1 protein is a tumor suppressor involved in repair of DNA damage. Epigenetic mechanisms contribute to its reduced expression in sporadic breast tumors. Through diet, humans are exposed to a complex mixture of xenobiotics and natural ligands of the aromatic hydrocarbon receptor (AhR), which contributes to the etiology of various types of cancers. The AhR binds xenobiotics, endogenous ligands, and many natural dietary bioactive compounds, including the phytoalexin resveratrol (Res). In estrogen receptor-a (ERa)-positive and BRCA-1 wild-type MCF-7 breast cancer cells, we investigated the influence of AhR activation with the agonist 2,3,7,8 tetrachlorobenzo(p)dioxin (TCDD) on epigenetic regulation of the BRCA-1 gene and the preventative effects of Res. We report that activation and recruitment of the AhR to the BRCA-1 promoter hampers 17b-estradiol (E2)-dependent stimulation of BRCA-1 transcription and protein levels. These inhibitory effects are paralleled by reduced occupancy of ERa, acetylated histone (AcH)-4, and AcH3K9. Conversely, the treatment with TCDD increases the association of mono-methylated-H3K9, DNA-methyltransferase-1 (DNMT1), and methyl-binding domain protein-2 with the BRCA-1 promoter and stimulates the accumulation of DNA strand breaks. The AhR-dependent repression of BRCA-1 expression is reversed by small interference for the AhR and DNMT1 or pretreatment with Res, which reduces TCDD-induced DNA strand breaks. These results support the hypothesis that epigenetic silencing of the BRCA-1 gene by the AhR is preventable with Res and provide the molecular basis for the development of dietary strategies based on natural AhR antagonists. © 2010 American Society for Nutrition.
• Romagnolo, D. F., Papoutsis, A. J., & Selmin, O. (2010). Nutritional targeting of cyclooxygenase-2 for colon cancer prevention. Inflammation and Allergy - Drug Targets, 9(3), 181-191.
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  PMID: 20553228;Abstract: Factors related to diet and life style have been identified as primary determinants in about 80% of colorectal cancers. Non-steroidal anti-inflammatory drugs (NSAID) and selective cyclooxygenase-2 (COX-2) inhibitors (COXIB) reduce the relative risk of colon cancer. To overcome systemic COX inhibition associated with NSAID and COXIB, there is a growing interest in developing alternative colon cancer prevention strategies using diet-based approaches that target COX-2. The transition from aberrant crypt foci (ACF) to colon cancer is a multiyear process providing opportunities for nutritional targeting of genes influencing the course of this disease process at early stages of development. The activation of the proinflammatory gene COX-2 and PG production in the colonic mucosa are recognized risk factors in colon cancer. Many natural food components may impact colon cancer risk by interfering with ligand-activated receptors, signal transduction pathways, and transcription factors involved in stimulation of COX-2 expression. In this review, we highlight key upstream features of signaling pathways and transcriptional control of the COX-2 gene and discuss opportunities for dietary modulation of COX-2 expression in gastro-intestinal cancers with special emphasis on prevention of colorectal tumors. Review of the experimental evidence suggests that dietary strategies based on specific or cocktails of bioactive food components as well nutritional-pharmacological combinations targeted to regulation of COX-2 expression and activity may prove useful in the prevention of colon cancer. An integrated approach may offer the advantage of combined higher efficacies. Future studies should investigate the efficacy of combinations of bioactive food compounds on epigenetic regulation of the COX-2 gene and characterize potential synergies and amplification effects resulting from the concomitant use of bioactive food components and COX-2 inhibitors. © 2010 Bentham Science Publishers Ltd.
• Romagnolo, D., Papoutsis, A. J., Lamore, S. D., Wondrak, G. T., Selmin, O. I., & Romagnolo, D. -. (2010). Resveratrol prevents epigenetic silencing of BRCA-1 by the aromatic hydrocarbon receptor in human breast cancer cells. The Journal of nutrition, 140(9).
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  The BRCA-1 protein is a tumor suppressor involved in repair of DNA damage. Epigenetic mechanisms contribute to its reduced expression in sporadic breast tumors. Through diet, humans are exposed to a complex mixture of xenobiotics and natural ligands of the aromatic hydrocarbon receptor (AhR), which contributes to the etiology of various types of cancers. The AhR binds xenobiotics, endogenous ligands, and many natural dietary bioactive compounds, including the phytoalexin resveratrol (Res). In estrogen receptor- alpha (ER alpha )-positive and BRCA-1 wild-type MCF-7 breast cancer cells, we investigated the influence of AhR activation with the agonist 2,3,7,8 tetrachlorobenzo(p)dioxin (TCDD) on epigenetic regulation of the BRCA-1 gene and the preventative effects of Res. We report that activation and recruitment of the AhR to the BRCA-1 promoter hampers 17 beta -estradiol (E2)-dependent stimulation of BRCA-1 transcription and protein levels. These inhibitory effects are paralleled by reduced occupancy of ER alpha , acetylated histone (AcH)-4, and AcH3K9. Conversely, the treatment with TCDD increases the association of mono-methylated-H3K9, DNA-methyltransferase-1 (DNMT1), and methyl-binding domain protein-2 with the BRCA-1 promoter and stimulates the accumulation of DNA strand breaks. The AhR-dependent repression of BRCA-1 expression is reversed by small interference for the AhR and DNMT1 or pretreatment with Res, which reduces TCDD-induced DNA strand breaks. These results support the hypothesis that epigenetic silencing of the BRCA-1 gene by the AhR is preventable with Res and provide the molecular basis for the development of dietary strategies based on natural AhR antagonists.
• Degner, S. C., Papoutsis, A. J., & Romagnolo, D. F. (2009). Health Benefits of Traditional culinary and Medicinal Mediterranean Plants. Complementary and Alternative Therapies and the Aging Population, 541-562.
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  Abstract: This chapter focuses on the health benefits of traditional culinary and medicinal plants of the Mediterranean region. The Mediterranean diet is mentioned to be rich in fruits and vegetables, monounsaturated fatty acids and olive oil and the diet has a negative association with incidence of metabolic syndrome that contributes to an increased risk for cardiovascular disease. Mediterranean plants are believed to prevent or cure a wide range of ailments based on their bioactive components that can exert anti-oxidant, anti-inflammatory, anti-carcinogenic or anti-diabetic activities. A table is presented that gives a list of Mediterranean plants and their health benefits as well as brief accounts of the medicinal use of the plants and includes rosemary, licorice, chamomile and olive oil among others. The consumption of olive oil has a strong correlation with reduced hypertension, cancers of the prostate, breast and colon. It highlights the mechanism of action of the bioactive components and the molecular targets derived from these plants. © 2009 Elsevier Inc. All rights reserved.
• Degner, S. C., Papoutsis, A. J., Selmin, O., & Romagnolo, D. F. (2009). Targeting of aryl hydrocarbon receptor-mediated activation of cyclooxygenase-2 expression by the indole-3-carbinol metabolite 3,3 -diindolylmethane in breast cancer cells ^1,2. Journal of Nutrition, 139(1), 26-32.
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  PMID: 19056653;PMCID: PMC2646210;Abstract: Ligands of the aryl hydrocarbon receptor (AhR) include the environmental xenobiotic 2,3,7,8 tetrachlorodibenzo(p)dioxin (TCDD), polycyclic aryl hydrocarbons, and the dietary compounds 3, 3′-diindolylmethane (DIM), a condensation product of indol-3-carbinol found in Brassica vegetables, and the phytoalexin resveratrol (RES). The AhR and its cofactors regulate the expression of target genes at pentameric (GCGTG) xenobiotic responsive elements (XRE). Because the activation of cyclooxygenase-2 (COX-2) expression by AhR ligands may contribute to inflammation and tumorigenesis, we investigated the epigenetic regulation of the COX-2 gene by TCDD and the reversal effects of DIM in MCF-7 breast cancer cells. Results of DNA binding and chromatin immunoprecipitation (ChIP) studies documented that the treatment with TCDD induced the association of the AhR to XRE harbored in the COX-2 promoter and control CYP1A1 promoter oligonucleotides. The TCDD-induced binding of the AhR was reduced by small-interfering RNA for the AhR or the cotreatment with synthetic (3-methoxy-4-naphthoflavone) or dietary AhR antagonists (DIM, RES). In time course ChIP studies, TCDD induced the rapid (15 min) occupancy by the AhR, the histone acetyl transferase p300, and acetylated histone H4 (AcH4) at the COX-2 promoter. Conversely, the cotreatment of MCF-7 cells with DIM (10 μmol/L) abrogated the TCDD-induced recruitment of the AhR and AcH4 to the COX-2 promoter and the induction of COX-2 mRNA and protein levels. Taken together, these data suggest that naturally occurring modulators of the AhR such as DIM may be effective agents for dietary strategies against epigenetic activation of COX-2 expression by AhR agonists. © 2009 American Society for Nutrition.
• Romagnolo, D., Degner, S. C., Papoutsis, A. J., Selmin, O., & Romagnolo, D. -. (2009). Targeting of aryl hydrocarbon receptor-mediated activation of cyclooxygenase-2 expression by the indole-3-carbinol metabolite 3,3'-diindolylmethane in breast cancer cells. The Journal of nutrition, 139(1).
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  Ligands of the aryl hydrocarbon receptor (AhR) include the environmental xenobiotic 2,3,7,8 tetrachlorodibenzo(p)dioxin (TCDD), polycyclic aryl hydrocarbons, and the dietary compounds 3, 3'-diindolylmethane (DIM), a condensation product of indol-3-carbinol found in Brassica vegetables, and the phytoalexin resveratrol (RES). The AhR and its cofactors regulate the expression of target genes at pentameric (GCGTG) xenobiotic responsive elements (XRE). Because the activation of cyclooxygenase-2 (COX-2) expression by AhR ligands may contribute to inflammation and tumorigenesis, we investigated the epigenetic regulation of the COX-2 gene by TCDD and the reversal effects of DIM in MCF-7 breast cancer cells. Results of DNA binding and chromatin immunoprecipitation (ChIP) studies documented that the treatment with TCDD induced the association of the AhR to XRE harbored in the COX-2 promoter and control CYP1A1 promoter oligonucleotides. The TCDD-induced binding of the AhR was reduced by small-interfering RNA for the AhR or the cotreatment with synthetic (3-methoxy-4-naphthoflavone) or dietary AhR antagonists (DIM, RES). In time course ChIP studies, TCDD induced the rapid (15 min) occupancy by the AhR, the histone acetyl transferase p300, and acetylated histone H4 (AcH4) at the COX-2 promoter. Conversely, the cotreatment of MCF-7 cells with DIM (10 micromol/L) abrogated the TCDD-induced recruitment of the AhR and AcH4 to the COX-2 promoter and the induction of COX-2 mRNA and protein levels. Taken together, these data suggest that naturally occurring modulators of the AhR such as DIM may be effective agents for dietary strategies against epigenetic activation of COX-2 expression by AhR agonists.
• Hockings, J. K., Degner, S. C., Morgan, S. S., Kemp, M. Q., & Romagnolo, D. F. (2008). Involvement of a specificity proteins-binding element in regulation of basal and estrogen-induced transcription activity of the BRCA1 gene. Breast Cancer Research, 10(2).
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  PMID: 18377656;PMCID: PMC2397528;Abstract: Introduction: Increased estrogen level has been regarded to be a risk factor for breast cancer. However, estrogen has also been shown to induce the expression of the tumor suppressor gene, BRCA1. Upregulation of BRCA1 is thought to be a feedback mechanism for controlling DNA repair in proliferating cells. Estrogens enhance transcription of target genes by stimulating the association of the estrogen receptor (ER) and related coactivators to estrogen response elements or to transcription complexes formed at activator protein (AP)-1 or specificity protein (Sp)-binding sites. Interestingly, the BRCA1 gene lacks a consensus estrogen response element. We previously reported that estrogen stimulated BRCA1 transcription through the recruitment of a p300/ER-α complex to an AP-1 site harbored in the proximal BRCA1 promoter. The purpose of the study was to analyze the contribution of cis-acting sites flanking the AP-1 element to basal and estrogen-dependent regulation of BRCA1 transcription.Methods: Using transfection studies with wild-type and mutated BRCA1 promoter constructs, electromobility binding and shift assays, and DNA-protein interaction and chromatin immunoprecipitation assays, we investigated the role of Sp-binding sites and cAMP response element (CRE)-binding sites harbored in the proximal BRCA1 promoter.Results: We report that in the BRCA1 promoter the AP-1 site is flanked upstream by an element (5'-GGGGCGGAA-3') that recruits Sp1, Sp3, and Sp4 factors, and downstream by a half CRE-binding motif (5'-CGTAA-3') that binds CRE-binding protein. In ER-α-positive MCF-7 cells and ER-α-negative Hela cells expressing exogenous ER-α, mutation of the Sp-binding site interfered with basal and estrogen-induced BRCA1 transcription. Conversely, mutation of the CRE-binding element reduced basal BRCA1 promoter activity but did not prevent estrogen activation. In combination with the AP-1/CRE sites, the Sp-binding domain enhanced the recruitment of nuclear proteins to the BRCA1 promoter. Finally, we report that the MEK1 (mitogen-activated protein kinase kinase-1) inhibitor PD98059 attenuated the recruitment of Sp1 and phosphorylated ER-α, respectively, to the Sp and AP-1 binding element.Conclusion: These cumulative findings suggest that the proximal BRCA1 promoter segment comprises cis-acting elements that are targeted by Sp-binding and CRE-binding proteins that contribute to regulation of BRCA1 transcription. © 2008 Hockings et al.; licensee BioMed Central Ltd.
• Romagnolo, D., Hockings, J. K., Degner, S. C., Morgan, S. S., Kemp, M. Q., & Romagnolo, D. -. (2008). Involvement of a specificity proteins-binding element in regulation of basal and estrogen-induced transcription activity of the BRCA1 gene. Breast cancer research : BCR, 10(2).
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  Increased estrogen level has been regarded to be a risk factor for breast cancer. However, estrogen has also been shown to induce the expression of the tumor suppressor gene, BRCA1. Upregulation of BRCA1 is thought to be a feedback mechanism for controlling DNA repair in proliferating cells. Estrogens enhance transcription of target genes by stimulating the association of the estrogen receptor (ER) and related coactivators to estrogen response elements or to transcription complexes formed at activator protein (AP)-1 or specificity protein (Sp)-binding sites. Interestingly, the BRCA1 gene lacks a consensus estrogen response element. We previously reported that estrogen stimulated BRCA1 transcription through the recruitment of a p300/ER-alpha complex to an AP-1 site harbored in the proximal BRCA1 promoter. The purpose of the study was to analyze the contribution of cis-acting sites flanking the AP-1 element to basal and estrogen-dependent regulation of BRCA1 transcription.
• Romagnolo, D., Scheckel, K. A., Degner, S. C., & Romagnolo, D. -. (2008). Rosmarinic acid antagonizes activator protein-1-dependent activation of cyclooxygenase-2 expression in human cancer and nonmalignant cell lines. The Journal of nutrition, 138(11).
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  One mechanism through which bioactive food components may exert anticancer effects is by reducing the expression of the proinflammatory gene cyclooxygenase-2 (COX-2), which has been regarded as a risk factor in tumor development. Rosmarinic acid (RA) is a phenolic derivative of caffeic acid present in rosemary (Rosmarinus officinalis). Previous research documented that RA may exert antiinflammatory effects. However, the mechanisms of action of RA on COX-2 expression have not been investigated. Here, we report that in colon cancer HT-29 cells, RA (5, 10, and 20 micromol/L) reduced the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 promoter activity (P < 0.05) and protein levels (P < 0.05). In addition, the cotreatment with RA reduced (5 micromol/L, P < 0.05; 10 and 20 micromol/L, P < 0.01) TPA-induced transcription from a control activator protein-1 (AP-1) promoter-luciferase construct and repressed binding of the AP-1 factors c-Jun (10 micromol/L; P < 0.01) and c-Fos (10 micromol/L; P < 0.05) to COX-2 promoter oligonucleotides harboring a cAMP-response element (CRE). The anti-AP1 effects of RA were also examined in a nonmalignant breast epithelial cell line (MCF10A) in which RA antagonized the stimulatory effects of TPA on COX-2 protein expression (5 micromol/L, P < 0.05; 10 and 20 micromol/L, P < 0.01), the recruitment of c-Jun and c-Fos (10 micromol/L; P < 0.01) to the COX-2/CRE oligonucleotides, and activation of the extracellular signal-regulated protein kinase-1/2 (ERK1/2) (10 micromol/L; P < 0.01), a member of the mitogen-activated protein kinase pathway. Additionally, RA antagonized ERK1/2 activation in colon HT-29 and breast MCF-7 cancer cells (10 micromol/L; P < 0.01). Thus, we propose that RA may be an effective preventative agent against COX-2 activation by AP-1-inducing agents in both cancer and nonmalignant mammary epithelial cells.
• Scheckel, K. A., Degner, S. C., & Romagnolo, D. F. (2008). Rosmarinic acid antagonizes activator protein-1-dependent activation of cyclooxygenase-2 expression in human cancer and nonmalignant cell lines. Journal of Nutrition, 138(11), 2098-2105.
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  PMID: 18936204;PMCID: PMC3151436;Abstract: One mechanism through which bioactive food components may exert anticancer effects is by reducing the expression of the proinflammatory gene cyclooxygenase-2 (COX-2), which has been regarded as a risk factor in tumor development. Rosmarinic acid (RA) is a phenolic derivative of caffeic acid present in rosemary (Rosmarinus officinalis). Previous research documented that RA may exert antiinflammatory effects. However, the mechanisms of action of RA on COX-2 expression have not been investigated. Here, we report that in colon cancer HT-29 cells, RA (5, 10, and 20 μmol/L) reduced the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 promoter activity (P < 0.05) and protein levels (P < 0.05). In addition, the cotreatment with RA reduced (5 μmol/L, P < 0.05; 10 and 20 μmol/L, P < 0.01) TPA-induced transcription from a control activator protein-1 (AP-1) promoter-luciferase construct and repressed binding of the AP-1 factors c-Jun (10 μmol/L; P < 0.01) and c-Fos (10 μmol/L; P < 0.05) to COX-2 promoter oligonucleotides harboring a cAMP-response element (CRE). The anti-AP1 effects of RA were also examined in a nonmalignant breast epithelial cell line (MCF10A) in which RA antagonized the stimulatory effects of TPA on COX-2 protein expression (5 μmol/L, P < 0.05; 10 and 20 μmol/L, P < 0.01), the recruitment of c-Jun and c-Fos (10 μmol/L; P < 0.01) to the COX-2/CRE oligonucleotides, and activation of the extracellular signal-regulated protein kinase-1/2 (ERK1/2) (10 μmol/L; P < 0.01), a member of the mitogen-activated protein kinase pathway. Additionally, RA antagonized ERK1/2 activation in colon HT-29 and breast MCF-7 cancer cells (10 μmol/L; P < 0.01). Thus, we propose that RA may be an effective preventative agent against COX-2 activation by AP-1-inducing agents in both cancer and nonmalignant mammary epithelial cells. © 2008 American Society for Nutrition.
• Degner, S. C., Kemp, M. Q., Hockings, J. K., & Romagnolo, D. F. (2007). Cyclooxygenase-2 promoter activation by the aromatic hydrocarbon receptor in breast cancer MCF-7 cells: Repressive effects of conjugated linoleic acid. Nutrition and Cancer, 59(2), 248-257.
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  PMID: 18001219;Abstract: The role of the aromatic hydrocarbon receptor (AhR) in transcriptional regulation of the human cyclooxygenase-2 (COX-2) gene remains elusive. We report that the AhR-ligands benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced transcription activity of COX-2 in breast cancer MCF-7 cells. The TCDD-dependent activation of the COX-2 promoter was abrogated by mutation of 2 xenobiotic response elements (XREs) = CGTG). We found that TCDD stimulated the binding of the AhR to COX-2 and cytochrome P4501A1 (CYP1A1) oligonucleotides containing consensus XREs. Conversely, the cotreatment with TCDD plus a mixture of conjugated linoleic acid (CLA) or selected CLA isomers prevented (CLAmix = t10,c12-CLA>c9,t11-CLA) the induction of transcription from the COX-2 promoter. The TCDD-induced binding of the AhR to COX-2 and CYP1A1 oligonucleotides was repressed by cotreatment with CLA (t10,c12-CLA>c9,t11- CLA), and the AhR antagonists, 3-methoxy-4-naphthoflavone, and resveratrol. We conclude that the AhR may be a suitable target for prophylactic strategies that target COX-2 expression. Copyright © 2007, Lawrence Erlbaum Associates, Inc.
• Romagnolo, D., Degner, S. C., Kemp, M. Q., Hockings, J. K., & Romagnolo, D. -. (2007). Cyclooxygenase-2 promoter activation by the aromatic hydrocarbon receptor in breast cancer mcf-7 cells: repressive effects of conjugated linoleic acid. Nutrition and cancer, 59(2).
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  The role of the aromatic hydrocarbon receptor (AhR) in transcriptional regulation of the human cyclooxygenase-2 (COX-2) gene remains elusive. We report that the AhR-ligands benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced transcription activity of COX-2 in breast cancer MCF-7 cells. The TCDD-dependent activation of the COX-2 promoter was abrogated by mutation of 2 xenobiotic response elements (XREs) = CGTG). We found that TCDD stimulated the binding of the AhR to COX-2 and cytochrome P4501A1 (CYP1A1) oligonucleotides containing consensus XREs. Conversely, the cotreatment with TCDD plus a mixture of conjugated linoleic acid (CLA) or selected CLA isomers prevented (CLAmix = t10,c12-CLA > c9,t11-CLA) the induction of transcription from the COX-2 promoter. The TCDD-induced binding of the AhR to COX-2 and CYP1A1 oligonucleotides was repressed by cotreatment with CLA (t10,c12-CLA > c9,t11-CLA), and the AhR antagonists, 3-methoxy-4-naphthoflavone, and resveratrol. We conclude that the AhR may be a suitable target for prophylactic strategies that target COX-2 expression.
• Degner, S. C., Kemp, M. Q., Bowden, G. T., & Romagnolo, D. F. (2006). Conjugated linoleic acid attenuates cyclooxygenase-2 transcriptional activity via an anti-AP-1 mechanism in MCF-7 breast cancer cells. Journal of Nutrition, 136(2), 421-427.
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  PMID: 16424122;Abstract: Overexpression of cyclooxygenase-2 (COX-2) is regarded as a causative factor in the onset of tumorigenesis of the breast. In this study, we investigated the effects of conjugated linoleic acid (CLA) on COX-2 transcription in MCF-7 breast cancer cells. Results of transient transfection studies revealed that treatment with a CLA mix or selected isomers (c9, t11-CLA; t10, c12-CLA) at concentrations ranging from 20 to 80 μmol/L, attenuated COX-2 transcription induced by the proinflammatory agent 12-O- tetradecanoylphorbol-13-acetate (TPA). In addition, the CLA mix inhibited TPA-induced activity of the collagenase-1 promoter. Using electrophoretic mobility shift assays, we found that the CLA mix reduced TPA-induced recruitment of nuclear proteins to a cAMP response element (CRE) in the COX-2 promoter and a consensus TPA-responsive element (TRE) in the collagenase-1 promoter. Both CRE and TRE are binding sites for activator protein-1 (AP-1). Binding studies revealed that the t10, c12-CLA isomer was more effective than the CLA mix or c9, t11-CLA in reducing binding of cJun to either the COX-2 CRE or collagenase-1 TRE, whereas linoleic acid increased binding to both elements. Overexpression of the AP-1 member, c-Jun, reversed the inhibitory effects of the CLA mix on COX-2 transcription, and restored binding of nuclear proteins to the CRE and TRE. Collectively, these results suggest that CLA represses AP-1-mediated activation of COX-2 transcription. © 2006 American Society for Nutrition.
• Hockings, J. K., Thorne, P. A., Kemp, M. Q., Morgan, S. S., Selmin, O., & Romagnolo, D. F. (2006). The ligand status of the aromatic hydrocarbon receptor modulates transcriptional activation of BRCA-1 promoter by estrogen. Cancer Research, 66(4), 2224-2232.
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  PMID: 16489025;Abstract: In sporadic breast cancers, BRCA-1 expression is down-regulated in the absence of mutations in the BRCA-1 gene. This suggests that disruption of BRCA-1 expression may contribute to the onset of mammary tumors. Environmental contaminants found in industrial pollution, tobacco smoke, and cooked foods include benzo(a)pyrene [B(a)P] and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which have been shown to act as endocrine disruptors and tumor promoters. In previous studies, we documented that estrogen (E2) induced BRCA-1 transcription through the recruitment of an activator protein-1/estrogen receptor-α (ERα) complex to the proximal BRCA-1 promoter. Here, we report that activation of BRCA-1 transcription by E2 requires occupancy of the BRCA-1 promoter by the unliganded aromatic hydrocarbon receptor (AhR). The stimulatory effects of E2 on BRCA-1 transcription are counteracted by (a) cotreatment with the AhR antagonist 3′-methoxy-4′-nitroflavone; (b) transient expression in ERα-negative HeLa cells of ERα lacking the protein-binding domain for the AhR; and (c) mutation of two consensus xenobiotic-responsive elements (XRE, 5′-GCGTG-3′) located upstream of the ERα-binding region. These results suggest that the physical interaction between the unliganded AhR and the liganded ERα plays a positive role in E2-dependent activation of BRCA-1 transcription. Conversely, we show that the AhR ligands B(a)P and TCDD abrogate E2-induced BRCA-1 promoter activity. The repressive effects of TCDD are paralleled by increased recruitment of the liganded AhR and HDAC1, reduced occupancy by p300, SRC-1, and diminished acetylation of H4 at the BRCA-1 promoter region flanking the XREs. We propose that the ligand status of the AhR modulates activation of the BRCA-1 promoter by estrogen. ©2006 American Association for Cancer Research.
• Kemp, M. Q., Liu, W., Thorne, P. A., Kane, M. D., Selmin, O., & Romagnolo, D. F. (2006). Induction of the transferrin receptor gene by benzo[a]pyrene in breast cancer MCF-7 cells: Potential as a biomarker of PAH exposure. Environmental and Molecular Mutagenesis, 47(7), 518-526.
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  PMID: 16721748;Abstract: Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental DNA-damaging agents regarded as risk factors for human disease, including lung and breast cancer. The biotransformation of PAHs to carcinogenic metabolites is mediated by the aromatic hydrocarbon receptor (AhR), which activates transcription at xenobiotic responsive elements (XREs = 5′-GCGTG-3′) found in the promoter regions of genes encoding for detoxifying enzymes, including CYP1A1 and CYP1B1. In this study, we wished to identify novel biomarkers that may be useful in monitoring critical carcinogenic events of the breast induced by PAHs. Using a GeneMAP™ CancerArray, we analyzed in breast cancer MCF-7 cells the temporal effects of the AhR agonist benzo[a]pyrene (B[a]P), which is a prototype PAH and known environmental carcinogen. Genes upregulated at least threefold by B[a]P and containing potential XREs within their promoter regions included CYP1A1, CYP1B1, paired box gene 3 (PAX3), cortactin (CTTN/EMS1), β-2-microglobulin [B2M], and transferrin receptor (TfR]. The stimulatory effects of B[a]P on expression of these genes were abrogated by cotreatment with the AhR antagonist flavonoid, α- napthoflavone (ANF). The TfR gene was selected for further analysis as its promoter region contains two potential XREs and its expression has been shown to be increased in breast cancer cells. Accumulation of TfR mRNA in B[a]P-treated cells was confirmed by quantitative real time PCR. Transient transfection studies indicated that the transcriptional activity of the TfR promoter was stimulated by B[a]P, whereas ANF counteracted this induction. These results indicate that the TfR gene may be a potential biomarker of PAH exposure. © 2006 Wiley-Liss, Inc.
• Liu, W., Capuco, A. V., & Romagnolo, D. F. (2006). Expression of cytosolic NADP⁺-dependent isocitrate dehydrogenase in bovine mammary epithelium: Modulation by regulators of differentiation and metabolic effectors. Experimental Biology and Medicine, 231(5), 599-610.
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  PMID: 16636309;Abstract: The cytosolic NADP+-dependent isocitrate dehydrogenase (IDH1) catalyzes the conversion of isocitrate to a-ketoglutarate in the cytosol, and generates NADPH as a primary source of reducing equivalents for de novo fatty acid synthesis in bovine mammary gland. The enzymatic activity of IDH1 increases dramatically in early lactation in bovine mammary tissue. We hypothesized that the expression of IDH1 in bovine is modulated by regulators of mammary epithelial differentiation. To test this hypothesis, we first examined the changes in IDH1 expression in late pregnancy (-20 days) and at various stages (14, 90, 120, and 240 days) of lactation in bovine mammary tissue. IDH1 mRNA levels increased by 2.3-fold after parturition compared to late pregnancy and remained elevated thereafter. Next, we examined the effects of extracellular matrix and lactogenic hormones on the expression of IDH1 in cultured BME-UV bovine mammary epithelial cells. We found that expression of IDH1 mRNA increased in parallel with β-casein expression induced by extracellular matrix. Fetal calf serum and insulin repressed, whereas prolactin stimulated the expression of IDH1 mRNA in a dose-dependent fashion. The inhibitory effects of insulin on IDH1 mRNA levels were antagonized by cotreatment with prolactin. In contrast, treatment with prolactin in the presence of extracellular matrix further increased IDH1 mRNA and protein accumulation. Prolactin-induced IDH1 expression was inhibited by the mitogen-activated protein kinase (MAPK) inhibitors PD98059 and U0126, and Janus tyrosine kinase 2 (Jak2) inhibitor AG490, suggesting that both MAPK and Jak2 contribute to regulation of IDH1 expression by prolactin. Finally, we report that treatment of BME-UV cells with a-ketoglutarate and palmitic acid reduced IDH1 transcript levels. Taken together, our data suggest that the expression of IDH1 in bovine mammary epithelium is modulated by regulators of differentiation including extracellular matrix and lactogenic hormones as well as metabolic effectors. Copyright © 2006 by the Society for Experimental Biology and Medicine.
• Liu, W., Degner, S. C., & Romagnolo, D. F. (2006). Trans-10, cis-12 conjugated linoleic acid inhibits prolactin-induced cytosolic NADP⁺-dependent isocitrate dehydrogenase expression in bovine mammary epithelial cells. Journal of Nutrition, 136(11), 2743-2747.
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  PMID: 17056794;Abstract: Conjugated linoleic acid (CLA) has been found to exert beneficial effects on lipid profile and repress de novo fatty acid synthesis in mammary gland during lactation. However, the underlying mechanisms responsible for the antilipogenic effects of CLA have not been established. The cytosolic NADP +-dependent isocitrate dehydrogenase (IDH1) plays a critical role in cholesterol and fatty acid biosynthesis by providing reducing equivalents as NADPH. In previous studies, we documented that the expression of IDH1 in bovine mammary epithelium was modulated by regulators of mammary differentiation and metabolic effectors. In this study, we investigated the short-term effects of prolactin (PRL) and CLA on IDH1 expression in BME-UV bovine mammary epithelial cells. In time-course experiments, we found that the treatment with PRL for 60 and 90 min elicited a significant increase in IDH1 transcript levels. Conversely, the cotreatment of BME-UV cells with PRL plus a CLA mixture for 90 min prevented the accumulation of IDH1 mRNA induced by PRL. In addition, we found that the trans-10, cis-12 CLA, but not the cis-9, trans-11 CLA isomer, inhibited basal- and PRL-induced IDH1 mRNA expression. The inhibitory effects of the trans-10, cis-12 CLA isomer on PRL-induced IDH1 expression accumulation were confirmed by quantitative real time PCR and western-blotting analysis. We propose that the inhibitory effects of CLA on milk fat synthesis in mammary epithelial cells may derive, at least in part, from repression of IDH1 expression. © 2006 American Society for Nutrition.
• Romagnolo, D. F., Degner, S. C., Kemp, M. Q., Hockings, J. K., & Selmin, O. (2006). Role of dietary xenobiotics-gene interactions in carcinogenesis: Protective effects of nutritional factors. Current Nutrition and Food Science, 2(3), 205-214.
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  Abstract: In this paper, we discuss how interactions between dietary xenobiotics and nutrients influence cancer risk by modulating overlapping biochemical pathways leading to repression of tumor suppressor genes, activation of tumor promoters, or both. Epidemiologic studies in humans suggested that diet is an important vehicle of exposure to various xenobiotics, including polycyclic aromatic hydrocarbons (PAHs), dioxins, and chlorinated hydrocarbons (CHs). The activation of the aromatic hydrocarbon receptor (AhR) pathway by PAHs and dioxins stimulates the expression of several genes including cytochrome P450s, which metabolize PAHs to highly mutagenic compounds that cause fixation of mutations in the p53 gene and repress the expression of the tumor suppressor gene, BRCA-1. Conversely, PAHs and dioxins have been shown to activate the expression of cyclooxygenase-2 (COX-2), whose protein product participates in the production of reactive PAH-metabolites and synthesis of proinflammatory prostaglandins (PGs). The carcinogenicity of CHs has been attributed to their ability to activate the expression of oncogenes including c-myc, and fatty acid activation of the peroxisome proliferator-activated receptor-α (PPARα). Dietary components that may protect against the activation of the AhR pathway include flavonoids, which comprise a large family of dietary phenolic phytochemicals found in fruits and vegetables. Nutrients such as dietary fatty acids may differentially influence the risk of cancer by inducing (linoleic and arachidonic acid) or repressing (conjugated linoleic acid (CLA); n-3PUFA) the expression of COX-2. Finally, we present original findings produced in our laboratory documenting the protective effects of natural (genestein) and synthetic (α-naphthoflavone) flavonoids against PAH-induced changes in gene expression. © 2006 Bentham Science Publishers Ltd.
• Romagnolo, D., Degner, S. C., Kemp, M. Q., Bowden, G. T., & Romagnolo, D. -. (2006). Conjugated linoleic acid attenuates cyclooxygenase-2 transcriptional activity via an anti-AP-1 mechanism in MCF-7 breast cancer cells. The Journal of nutrition, 136(2).
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  Overexpression of cyclooxygenase-2 (COX-2) is regarded as a causative factor in the onset of tumorigenesis of the breast. In this study, we investigated the effects of conjugated linoleic acid (CLA) on COX-2 transcription in MCF-7 breast cancer cells. Results of transient transfection studies revealed that treatment with a CLA mix or selected isomers (c9, t11-CLA; t10, c12-CLA) at concentrations ranging from 20 to 80 micromol/L, attenuated COX-2 transcription induced by the proinflammatory agent 12-O-tetradecanoylphorbol-13-acetate (TPA). In addition, the CLA mix inhibited TPA-induced activity of the collagenase-1 promoter. Using electrophoretic mobility shift assays, we found that the CLA mix reduced TPA-induced recruitment of nuclear proteins to a cAMP response element (CRE) in the COX-2 promoter and a consensus TPA-responsive element (TRE) in the collagenase-1 promoter. Both CRE and TRE are binding sites for activator protein-1 (AP-1). Binding studies revealed that the t10, c12-CLA isomer was more effective than the CLA mix or c9, t11-CLA in reducing binding of cJun to either the COX-2 CRE or collagenase-1 TRE, whereas linoleic acid increased binding to both elements. Overexpression of the AP-1 member, c-Jun, reversed the inhibitory effects of the CLA mix on COX-2 transcription, and restored binding of nuclear proteins to the CRE and TRE. Collectively, these results suggest that CLA represses AP-1-mediated activation of COX-2 transcription.
• Romagnolo, D., Hockings, J. K., Thorne, P. A., Kemp, M. Q., Morgan, S. S., Selmin, O., & Romagnolo, D. -. (2006). The ligand status of the aromatic hydrocarbon receptor modulates transcriptional activation of BRCA-1 promoter by estrogen. Cancer research, 66(4).
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  In sporadic breast cancers, BRCA-1 expression is down-regulated in the absence of mutations in the BRCA-1 gene. This suggests that disruption of BRCA-1 expression may contribute to the onset of mammary tumors. Environmental contaminants found in industrial pollution, tobacco smoke, and cooked foods include benzo(a)pyrene [B(a)P] and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which have been shown to act as endocrine disruptors and tumor promoters. In previous studies, we documented that estrogen (E2) induced BRCA-1 transcription through the recruitment of an activator protein-1/estrogen receptor-alpha (ER alpha) complex to the proximal BRCA-1 promoter. Here, we report that activation of BRCA-1 transcription by E2 requires occupancy of the BRCA-1 promoter by the unliganded aromatic hydrocarbon receptor (AhR). The stimulatory effects of E2 on BRCA-1 transcription are counteracted by (a) cotreatment with the AhR antagonist 3'-methoxy-4'-nitroflavone; (b) transient expression in ER alpha-negative HeLa cells of ER alpha lacking the protein-binding domain for the AhR; and (c) mutation of two consensus xenobiotic-responsive elements (XRE, 5'-GCGTG-3') located upstream of the ER alpha-binding region. These results suggest that the physical interaction between the unliganded AhR and the liganded ER alpha plays a positive role in E2-dependent activation of BRCA-1 transcription. Conversely, we show that the AhR ligands B(a)P and TCDD abrogate E2-induced BRCA-1 promoter activity. The repressive effects of TCDD are paralleled by increased recruitment of the liganded AhR and HDAC1, reduced occupancy by p300, SRC-1, and diminished acetylation of H4 at the BRCA-1 promoter region flanking the XREs. We propose that the ligand status of the AhR modulates activation of the BRCA-1 promoter by estrogen.
• Romagnolo, D., Kemp, M. Q., Liu, W., Thorne, P. A., Kane, M. D., Selmin, O., & Romagnolo, D. -. (2006). Induction of the transferrin receptor gene by benzo[a]pyrene in breast cancer MCF-7 cells: potential as a biomarker of PAH exposure. Environmental and molecular mutagenesis, 47(7).
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  Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental DNA-damaging agents regarded as risk factors for human disease, including lung and breast cancer. The biotransformation of PAHs to carcinogenic metabolites is mediated by the aromatic hydrocarbon receptor (AhR), which activates transcription at xenobiotic responsive elements (XREs = 5'-GCGTG-3') found in the promoter regions of genes encoding for detoxifying enzymes, including CYP1A1 and CYP1B1. In this study, we wished to identify novel biomarkers that may be useful in monitoring critical carcinogenic events of the breast induced by PAHs. Using a GeneMAP CancerArray, we analyzed in breast cancer MCF-7 cells the temporal effects of the AhR agonist benzo[a]pyrene (B[a]P), which is a prototype PAH and known environmental carcinogen. Genes upregulated at least threefold by B[a]P and containing potential XREs within their promoter regions included CYP1A1, CYP1B1, paired box gene 3 (PAX3), cortactin (CTTN/EMS1), beta-2-microglobulin (B2M), and transferrin receptor (TfR). The stimulatory effects of B[a]P on expression of these genes were abrogated by cotreatment with the AhR antagonist flavonoid, alpha-napthoflavone (ANF). The TfR gene was selected for further analysis as its promoter region contains two potential XREs and its expression has been shown to be increased in breast cancer cells. Accumulation of TfR mRNA in B[a]P-treated cells was confirmed by quantitative real time PCR. Transient transfection studies indicated that the transcriptional activity of the TfR promoter was stimulated by B[a]P, whereas ANF counteracted this induction. These results indicate that the TfR gene may be a potential biomarker of PAH exposure.
• Romagnolo, D., Liu, W., Capuco, A. V., & Romagnolo, D. -. (2006). Expression of cytosolic NADP+-dependent isocitrate dehydrogenase in bovine mammary epithelium: Modulation by regulators of differentiation and metabolic effectors. Experimental biology and medicine (Maywood, N.J.), 231(5).
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  The cytosolic NADP+-dependent isocitrate dehydrogenase (IDH1) catalyzes the conversion of isocitrate to alpha-ketoglutarate in the cytosol, and generates NADPH as a primary source of reducing equivalents for de novo fatty acid synthesis in bovine mammary gland. The enzymatic activity of IDH1 increases dramatically in early lactation in bovine mammary tissue. We hypothesized that the expression of IDH1 in bovine is modulated by regulators of mammary epithelial differentiation. To test this hypothesis, we first examined the changes in IDH1 expression in late pregnancy (-20 days) and at various stages (14, 90, 120, and 240 days) of lactation in bovine mammary tissue. IDH1 mRNA levels increased by 2.3-fold after parturition compared to late pregnancy and remained elevated thereafter. Next, we examined the effects of extracellular matrix and lactogenic hormones on the expression of IDH1 in cultured BME-UV bovine mammary epithelial cells. We found that expression of IDH1 mRNA increased in parallel with beta-casein expression induced by extracellular matrix. Fetal calf serum and insulin repressed, whereas prolactin stimulated the expression of IDH1 mRNA in a dose-dependent fashion. The inhibitory effects of insulin on IDH1 mRNA levels were antagonized by cotreatment with prolactin. In contrast, treatment with prolactin in the presence of extracellular matrix further increased IDH1 mRNA and protein accumulation. Prolactin-induced IDH1 expression was inhibited by the mitogen-activated protein kinase (MAPK) inhibitors PD98059 and U0126, and Janus tyrosine kinase 2 (Jak2) inhibitor AG490, suggesting that both MAPK and Jak2 contribute to regulation of IDH1 expression by prolactin. Finally, we report that treatment of BME-UV cells with alpha-ketoglutarate and palmitic acid reduced IDH1 transcript levels. Taken together, our data suggest that the expression of IDH1 in bovine mammary epithelium is modulated by regulators of differentiation including extracellular matrix and lactogenic hormones as well as metabolic effectors.
• Romagnolo, D., Liu, W., Degner, S. C., & Romagnolo, D. -. (2006). Trans-10, cis-12 conjugated linoleic acid inhibits prolactin-induced cytosolic NADP+ -dependent isocitrate dehydrogenase expression in bovine mammary epithelial cells. The Journal of nutrition, 136(11).
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  Conjugated linoleic acid (CLA) has been found to exert beneficial effects on lipid profile and repress de novo fatty acid synthesis in mammary gland during lactation. However, the underlying mechanisms responsible for the antilipogenic effects of CLA have not been established. The cytosolic NADP+ -dependent isocitrate dehydrogenase (IDH1) plays a critical role in cholesterol and fatty acid biosynthesis by providing reducing equivalents as NADPH. In previous studies, we documented that the expression of IDH1 in bovine mammary epithelium was modulated by regulators of mammary differentiation and metabolic effectors. In this study, we investigated the short-term effects of prolactin (PRL) and CLA on IDH1 expression in BME-UV bovine mammary epithelial cells. In time-course experiments, we found that the treatment with PRL for 60 and 90 min elicited a significant increase in IDH1 transcript levels. Conversely, the cotreatment of BME-UV cells with PRL plus a CLA mixture for 90 min prevented the accumulation of IDH1 mRNA induced by PRL. In addition, we found that the trans-10, cis-12 CLA, but not the cis-9, trans-11 CLA isomer, inhibited basal- and PRL-induced IDH1 mRNA expression. The inhibitory effects of the trans-10, cis-12 CLA isomer on PRL-induced IDH1 expression accumulation were confirmed by quantitative real time PCR and western-blotting analysis. We propose that the inhibitory effects of CLA on milk fat synthesis in mammary epithelial cells may derive, at least in part, from repression of IDH1 expression.
• Jeffy, B. D., Hockings, J. K., Kemp, M. Q., Morgan, S. S., Hager, J. A., Beliakoff, J., Whitesell, L. J., Bowden, G. T., & Romagnolo, D. F. (2005). An estrogen receptor-α/p300 complex activates the BRCA-1 promoter at an AP-1 site that binds Jun/Fos transcription factors: Repressive effects of p53 on BRCA-1 transcription. Neoplasia, 7(9), 873-882.
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  PMID: 16229810;PMCID: PMC1501940;Abstract: One of the puzzles in cancer predisposition is that women carrying BRCA-1 mutations preferentially develop tumors in epithelial tissues of the breast and ovary. Moreover, sporadic breast tumors contain lower levels of BRCA-1 in the absence of mutations in the BRCA-1 gene. The problem of tissue specificity requires analysis of factors that are unique to tissues of the breast. For example, the expression of estrogen receptor-α (ERα) is inversely correlated with breast cancer risk, and 90% of BRCA-1 tumors are negative for ERα. Here, we show that estrogen stimulates BRCA-1 promoter activity in transfected cells and the recruitment of ERα and its cofactor p300 to an AP-1 site that binds Jun/Fos transcription factors. The recruitment of ERα/p300 coincides with accumulation in the S-phase of the cell cycle and is antagonized by the antiestrogen tamoxifen. Conversely, we document that overexpression of wild-type p53 prevents the recruitment of ERα to the AP-1 site and represses BRCA-1 promoter activity. Taken together, our findings support a model in which an ERα/AP-1 complex modulates BRCA-1 transcription under conditions of estrogen stimulation. Conversely, the formation of this transcription complex is abrogated in cells overexpressing p53. Copyright © 2005 Neoplasia Press, Inc. All rights reserved.
• Romagnolo, D., Selmin, O., Thorne, P. A., Blachere, F. M., Johnson, P. D., & Romagnolo, D. -. (2005). Transcriptional activation of the membrane-bound progesterone receptor (mPR) by dioxin, in endocrine-responsive tissues. Molecular reproduction and development, 70(2).
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  We originally identified the membrane-bound progesterone receptor (mPR) using a screening for genes differentially expressed in liver of rats exposed to dioxin. Recent findings have suggested a role for the mPR in sperm cells, ovary, and brain; however, its mechanisms of action are largely unknown. In this study, we examined the expression pattern of the mPR in liver of rats exposed to dioxin and identified possible mechanisms of its regulation. We observed that mPR expression was induced by dioxin, but was also dependent on the hormonal responsiveness of the tissue. In particular, in male, but not female liver, dioxin induced the expression of the mPR. However, in control, untreated female liver the level of mPR transcript was higher than in control males. Moreover, in breast cancer cells MCF-7 dioxin induced mPR expression. Promoter studies using the luciferase assay indicated that a fragment of approximately 350 bp of the mPR promoter was able to induce luciferase activity in the presence of dioxin, suggesting that the presumptive XREs sites contained in this mPR promoter region are responsive to dioxin. Analysis of mPR protein level confirmed the results observed at the RNA level, both in rat liver and MCF-7 cells. Taken together, these observations suggest the existence of a novel cross-talk between steroid and aromatic hydrocarbon receptors (AhR), and underline the importance of the mPR as a mediator of physiologic effects of the sex hormones.
• Selmin, O., Thorne, P. A., Blachere, F. M., Johnson, P. D., & Romagnolo, D. F. (2005). Transcriptional activation of the membrane-bound progesterone receptor (mPR) by dioxin, in endocrine-responsive tissues. Molecular Reproduction and Development, 70(2), 166-174.
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  PMID: 15570619;Abstract: We originally identified the membrane-bound progesterone receptor (mPR) using a screening for genes differentially expressed in liver of rats exposed to dioxin. Recent findings have suggested a role for the mPR in sperm cells, ovary, and brain; however, its mechanisms of action are largely unknown. In this study, we examined the expression pattern of the mPR in liver of rats exposed to dioxin and identified possible mechanisms of its regulation. We observed that mPR expression was induced by dioxin, but was also dependent on the hormonal responsiveness of the tissue. In particular, in male, but not female liver, dioxin induced the expression of the mPR. However, in control, untreated female liver the level of mPR transcript was higher than in control males. Moreover, in breast cancer cells MCF-7 dioxin induced mPR expression. Promoter studies using the luciferase assay indicated that a fragment of ∼350 bp of the mPR promoter was able to induce luciferase activity in the presence of dioxin, suggesting that the presumptive XREs sites contained in this mPR promoter region are responsive to dioxin. Analysis of mPR protein level confirmed the results observed at the RNA level, both in rat liver and MCF-7 cells. Taken together, these observations suggest the existence of a novel cross-talk between steroid and aromatic hydrocarbon receptors (AhR), and underline the importance of the mPR as a mediator of physiologic effects of the sex hormones. © 2005 Wiley-Liss, Inc.
• Kemp, M. Q., Jeffy, B. D., & Romagnolo, D. F. (2003). Conjugated Linoleic Acid Inhibits Cell Proliferation through a p53-Dependent Mechanism: Effects on the Expression of G1-Restriction Points in Breast and Colon Cancer Cells. Journal of Nutrition, 133(11), 3670-3677.
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  PMID: 14608092;Abstract: Previous reports have documented the antiproliferative properties of a mixture of conjugated isomers (CLA) of linoleic acid [LA (18: 2)]. In this study, we investigated the mechanisms of CLA action on cell cycle progression in breast and colon cancer cells. Treatment with CLA inhibited cell proliferation in breast cancer MCF-7 cells containing wild-type p53 (p53 +/+). At cytostatic concentrations, CLA elicited cell cycle arrest in G1 and induced the accumulation of the tumor suppressors p53, p27 and p21 protein. Conversely, CLA reduced the expression of factors required for G1 to S-phase transition including cyclins D1 and E, and hyperphoshorylated retinoblastoma Rb protein. In contrast, the overexpression of mutant p53 (175Arg to His) in MFC-7 cells prevented the CLA-dependent accumulation of p21 and the reduction of cyclin E levels suggesting that the expression of wild-type p53 is required for CLA-mediated activation of the G1 restriction point. To futher elucidate the role of p53, the effects of CLA in colon cancer HCT116 cells (p53+/+) and p53-deficient (p53-/-) HCT116 cells (HCTKO) were examined. The treatment of HCT116 cells with CLA increased the levels of p53, p21, p27 and hypophosphorylated (pRb) protein and reduced the expression of cyclin E, whereas these effects were not seen in p53-deficient HCTKO cells. The t10,c12-CLA isomer was more effective than c9,t11-CLA in inhibiting cell proliferation of MCF-7 breast cancer cells and enhancing the accumulation of p53 and pRb. We conclude that the antiproliferative properties of CLA appear to be a function, at least in part, of the relative content of specific isomers and their ability to elicit a p53 response that leads to the accumulation of pRb and cell growth arrest.
• Romagnolo, D. F., Chirnomas, R. B., Jennifer, K. u., Jeffy, B. D., Payne, C. M., Holubec, H., Ramsey, L., Bernstein, H., Bernstein, C., Kunke, K., Bhattacharyya, A., Warneke, J., & Garewal, H. (2003). Deoxycholate, an endogenous tumor promoter and DNA damaging agent, modulates BRCA-1 expression in apoptosis-sensitive epithelial cells: Loss of BRCA-1 expression in colonic adenocarcinomas. Nutrition and Cancer, 46(1), 82-92.
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  PMID: 12925308;Abstract: Deoxycholate, a bile salt present at high levels in the colonic lumen of individuals on a high-fat diet, is a promoter of colon cancer. Deoxycholate also causes DNA damage. BRCA-1 functions in repair of DNA and in induction of apoptosis. We show that, when cultured cells of colonic origin are exposed to deoxycholate at different concentrations, BRCA-1 expression is induced at a low noncytotoxic concentration (10 μM) but is strongly inhibited at higher cytotoxic concentrations (≥ 100 μM). Indication of phosphorylation of BRCA-1 by deoxycholate (100 μM) at a lower dose was seen by Western blot analysis, whereas, at a higher dose, deoxycholate (200 and 300 μM) caused a complete loss of BRCA-1 expression. We show that BRCA-1 is substantially lower in colon adenocarcinomas from five patients compared with associated non-neoplastic colon tissue from the same patients, suggesting that the loss of BRCA-1 expression contributes to the malignant phenotype. In the non-neoplastic colon tissue, BRCA-1 was localized to the nongoblet cells. Our results imply that reduced expression of BRCA-1 may be associated with carcinoma of the colon.
• Romagnolo, D., Kemp, M. Q., Jeffy, B. D., & Romagnolo, D. -. (2003). Conjugated linoleic acid inhibits cell proliferation through a p53-dependent mechanism: effects on the expression of G1-restriction points in breast and colon cancer cells. The Journal of nutrition, 133(11).
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  Previous reports have documented the antiproliferative properties of a mixture of conjugated isomers (CLA) of linoleic acid [LA (18:2)]. In this study, we investigated the mechanisms of CLA action on cell cycle progression in breast and colon cancer cells. Treatment with CLA inhibited cell proliferation in breast cancer MCF-7 cells containing wild-type p53 (p53(+/+)). At cytostatic concentrations, CLA elicited cell cycle arrest in G1 and induced the accumulation of the tumor suppressors p53, p27 and p21 protein. Conversely, CLA reduced the expression of factors required for G1 to S-phase transition including cyclins D1 and E, and hyperphoshorylated retinoblastoma Rb protein. In contrast, the overexpression of mutant p53 (175Arg to His) in MFC-7 cells prevented the CLA-dependent accumulation of p21 and the reduction of cyclin E levels suggesting that the expression of wild-type p53 is required for CLA-mediated activation of the G1 restriction point. To further elucidate the role of p53, the effects of CLA in colon cancer HCT116 cells (p53(+/+)) and p53-deficient (p53(-/-)) HCT116 cells (HCTKO) were examined. The treatment of HCT116 cells with CLA increased the levels of p53, p21, p27 and hypophosphorylated (pRb) protein and reduced the expression of cyclin E, whereas these effects were not seen in p53-deficient HCTKO cells. The t10,c12-CLA isomer was more effective than c9,t11-CLA in inhibiting cell proliferation of MCF-7 breast cancer cells and enhancing the accumulation of p53 and pRb. We conclude that the antiproliferative properties of CLA appear to be a function, at least in part, of the relative content of specific isomers and their ability to elicit a p53 response that leads to the accumulation of pRb and cell growth arrest.
• Jeffy, B. D., Chirnomas, R. B., & Romagnolo, D. F. (2002). Epigenetics of breast cancer: Polycyclic aromatic hydrocarbons as risk factors. Environmental and Molecular Mutagenesis, 39(2-3), 235-244.
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  PMID: 11921194;Abstract: In the absence of a causal relationship between the incidence of sporadic breast cancer and occurrence of mutations in breast cancer susceptibility genes, efforts directed to investigating the contribution of environmental xenobiotIcs in the etiology of sporadic mammary neoplasia are warranted. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants, which have been shown to induce DNA damage and disrupt cell cycle progression. In this report we discuss published data pointing to PAHs as a risk factor in carcinogenesis, and present findings generated in our laboratory suggesting that the mammary tumorigenicity of PAHs may be attributable, at least in part, to disruption of BRCA-1 expression by reactive PAH-metabolites. We report that benzo[a]pyrene (B[a]P), selected as a prototype PAH, disrupts BRCA-1 transcription in estrogen receptor (ER)-positive but not ER-negative breast cancer cells. The reduced potential for BRCA-1 expression in B[a]P-treated cells coincides with disruption of cell cycle kinetics and accumulation of p53. These effects are counteracted by the AhR-antagonist α-naphthoflavone (ANF), and in breast cancer cells expressing mutant p53 or the E6 human papilloma virus protein. We suggest that exposure to PAHs may be a predisposing factor in the etiology of sporadic breast cancer by disrupting the expression of BRCA-1. © 2002 Wiley-Liss, Inc.
• Jeffy, B. D., Chirnomas, R. B., Chen, E. J., Gudas, J. M., & Romagnolo, D. F. (2002). Activation of the aromatic hydrocarbon receptor pathway is not sufficient for transcriptional repression of BRCA-1: Requirements for metabolism of benzo[a]pyrene to 7r,8t-dihydroxy-9t,10-epoxy-7,8,9,10 tetrahydrobenzo[a]pyrene. Cancer Research, 62(1), 113-121.
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  PMID: 11782367;Abstract: Reduction of BRCA-1 expression through nonmutational events may be a predisposing event in the onset of sporadic breast cancer. In this study, we investigated the mechanisms through which the environmental carcinogen benzo[a]pyrene (B[a]P) lowered BRCA-1 mRNA levels in breast cancer MCF-7 cells. We report that B[a]P does not compromise the stability of BRCA-1 mRNA, but represses transcriptional activity of a 1.69-kb BRCA-1 (pGL3-BRCA-1) promoter fragment that contains both exon-1A and exon-1B transcription start sites. The loss of BRCA-1 promoter activity was accompanied by accumulation of CYP1A1 and BAX-α mRNA and p53 and p21 protein, whereas levels of Bcl-2 mRNA were reduced. The aromatic hydrocarbon receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is not metabolized, did not affect BRCA-1 promoter activity or the cellular levels of BRCA-1 and p53 protein, but it did induce a CYP1A1-like promoter. Conversely, treatment with the B[a]P metabolite 7r,8t-dihydroxy-9t,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) repressed BRCA-1 promoter activity and protein, while increasing p53 and p21 protein levels. Transient expression of dominant negative p53 (175 Arg→His) counteracted the detrimental effects of BPDE on BRCA-1 promoter activity and protein levels. Similarly, treatment with B[a]P, TCDD, or BPDE failed to repress transcription from the pGL3-BRCA-1 construct transfected into ZR75.1 breast cancer cells containing mutated p53 (152Pro→Leu). We conclude that activation of the aromatic hydrocarbon receptor is not sufficient for down-regulation of BRCA-1 transcription, which is, however, inhibited by the B[a]P metabolite BPDE through a p53-dependent pathway.
• Romagnolo, D., Jeffy, B. D., Chirnomas, R. B., & Romagnolo, D. -. (2002). Epigenetics of breast cancer: polycyclic aromatic hydrocarbons as risk factors. Environmental and molecular mutagenesis, 39(2-3).
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  In the absence of a causal relationship between the incidence of sporadic breast cancer and occurrence of mutations in breast cancer susceptibility genes, efforts directed to investigating the contribution of environmental xenobiotics in the etiology of sporadic mammary neoplasia are warranted. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants, which have been shown to induce DNA damage and disrupt cell cycle progression. In this report we discuss published data pointing to PAHs as a risk factor in carcinogenesis, and present findings generated in our laboratory suggesting that the mammary tumorigenicity of PAHs may be attributable, at least in part, to disruption of BRCA-1 expression by reactive PAH-metabolites. We report that benzo[a]pyrene (B[a]P), selected as a prototype PAH, disrupts BRCA-1 transcription in estrogen receptor (ER)-positive but not ER-negative breast cancer cells. The reduced potential for BRCA-1 expression in B[a]P-treated cells coincides with disruption of cell cycle kinetics and accumulation of p53. These effects are counteracted by the AhR-antagonist alpha-naphthoflavone (ANF), and in breast cancer cells expressing mutant p53 or the E6 human papilloma virus protein. We suggest that exposure to PAHs may be a predisposing factor in the etiology of sporadic breast cancer by disrupting the expression of BRCA-1.
• Romagnolo, D., Jeffy, B. D., Chirnomas, R. B., Chen, E. J., Gudas, J. M., & Romagnolo, D. -. (2002). Activation of the aromatic hydrocarbon receptor pathway is not sufficient for transcriptional repression of BRCA-1: requirements for metabolism of benzo[a]pyrene to 7r,8t-dihydroxy-9t,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Cancer research, 62(1).
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  Reduction of BRCA-1 expression through nonmutational events may be a predisposing event in the onset of sporadic breast cancer. In this study, we investigated the mechanisms through which the environmental carcinogen benzo[a]pyrene (B[a]P) lowered BRCA-1 mRNA levels in breast cancer MCF-7 cells. We report that B[a]P does not compromise the stability of BRCA-1 mRNA, but represses transcriptional activity of a 1.69-kb BRCA-1 (pGL3-BRCA-1) promoter fragment that contains both exon-1A and exon-1B transcription start sites. The loss of BRCA-1 promoter activity was accompanied by accumulation of CYP1A1 and BAX-alpha mRNA and p53 and p21 protein, whereas levels of Bcl-2 mRNA were reduced. The aromatic hydrocarbon receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is not metabolized, did not affect BRCA-1 promoter activity or the cellular levels of BRCA-1 and p53 protein, but it did induce a CYP1A1-like promoter. Conversely, treatment with the B[a]P metabolite 7r,8t-dihydroxy-9t,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) repressed BRCA-1 promoter activity and protein, while increasing p53 and p21 protein levels. Transient expression of dominant-negative p53 ((175)Arg-->His) counteracted the detrimental effects of BPDE on BRCA-1 promoter activity and protein levels. Similarly, treatment with B[a]P, TCDD, or BPDE failed to repress transcription from the pGL3-BRCA-1 construct transfected into ZR75.1 breast cancer cells containing mutated p53 ((152)Pro-->Leu). We conclude that activation of the aromatic hydrocarbon receptor is not sufficient for down-regulation of BRCA-1 transcription, which is, however, inhibited by the B[a]P metabolite BPDE through a p53-dependent pathway.
• Samuelson, D. J., Denise, S. K., Roffler, R., Ax, R. L., Armstrong, D. V., & Romagnolo, D. F. (2001). Response of Holstein and Brown Swiss cows fed alfalfa hay-based diets to supplemental methionine at two stages of lactation. Journal of Dairy Science, 84(4), 917-928.
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  PMID: 11352168;Abstract: In this study, we evaluated the effects of dietary supplementation at two stages of lactation with various levels of Mepron85 (M85) and M85 plus DL-methionine (DL-Met) on milk production and composition of Holstein and Brown Swiss cows fed an alfalfa-hay and corn grain-based diet. In experiment 1, control diets were formulated to supplement, in early lactation [days in milk (DIM) = 73.2], concentrations of metabolizable methionine at 104% of the estimated requirements based on the Cornell Net Carbohydrate and Protein System. Treatment groups were fed the control diet plus 10, 20, or 30 g/d of M85 at 116, 128, or 139% of the estimated requirements for metabolizable methionine. The supplementation with 10 g/d in Brown Swiss and 30 g/d of M85 in Holstein cows increased milk yields and fat percentage, but had no effects on protein percentage. These data suggested that the estimated postruminal supply of metabolizable methionine in the control ration was limiting for maximum milk fat synthesis. Conversely, in experiment 2, the cosupplementation with M85 (15 g/d) plus DL-Met (15 g/d) to cows in midlactation (DIM = 140.5) did not influence fat percentage, but increased protein yield and percentage (+0.1%) in both Holstein and Brown Swiss, and lactose percentage (+0.18%) in Holstein cows. The supplementation with 15 g/d of M85 reduced milk and protein yields, whereas 15 g/d of DL-Met reduced protein percentage in four of the five experimental weeks for Holstein cows. We conclude that supplementation with M85, alone or in combination with DL-Met, may be used to influence milk composition, but these effects are influenced by dosage and type of supplemental methionine, breed, and stage of lactation.
• Samuelson, D. J., Powell, M., Lluria-Prevatt, M., & Romagnolo, D. F. (2001). Transcriptional activation of the gp91phox NADPH oxidase subunit by TPA in HL-60 cells. Journal of Leukocyte Biology, 69(1), 161-168.
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  PMID: 11200061;Abstract: The exposure to epigenetic effectors capable of inducing copious production of reactive oxygen species (ROS) has been associated with chronic inflammation, tumor initiation, and promotion. The objective of this study was to examine the regulation of gp91phox, the catalytic subunit of the NADPH oxidase, and the kinetics of ROS production in promyelocytic leukemia HL-60 cells induced with 12-O-tetradeconylphorbol-13-acetate (TPA). The treatment of HL-60 cells with TPA (0.1 μM) induced cellular differentiation, which was followed after 48 h by a tenfold increase in chemiluminescence from lucigenin and a 2.5-fold increase in the intracellular oxidation of 2′,7′-dicholorofluorescin (DCFH). Whereas higher concentrations (1.0 μM) of TPA did not stimulate further ROS production, repeated stimulation with 0.1 μM TPA of differentiated cells induced a modest (1.2-fold) but rapid (15 min) increase in chemiluminescence. In cells treated with TPA, the burst in ROS at 48 h was preceded by accumulation at 12 h of gp91phox (8.8-fold) and p47phox mRNA (threefold), whereas untreated cells contained steady-state levels of both transcripts. Time-course experiments with actinomycin D to inhibit transcription revealed that TPA did not improve the stability of gp91phox. In transient transfections, luciferase reporter activity directed from a 1.5-kb gp91phox promoter fragment was enhanced threefold upon treatment with TPA for 24 h. We conclude that TPA can commit HL-60 cells to differentiation and elicit transcription from the proximal gp91phox promoter.
• Jeffy, B. D., Chen, E. J., Gudas, J. M., & Romagnolo, D. F. (2000). Disruption of cell cycle kinetics by benzo[a]pyrene: Inverse expression patterns of BRCA-1 and p53 in MCF-7 cells arrested in S and G₂. Neoplasia, 2(5), 460-470.
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  PMID: 11191113;PMCID: PMC1507983;Abstract: The effects of a ligand of the aromatic hydrocarbon receptor (AhR), benzo[a]pyrene (B[a]P), and its metabolite, BPDE (7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene), on BRCA-1 levels and cell cycle kinetics were determined in MCF-7 breast cancer cells. Exposure of asynchronous MCF-7 cells for 72 hours to a non-cytotoxic dose of 0.5 μM B[a]P triggered a three-fold reduction in BRCA-1 protein. In MCF-7 cells resistant (20% to 30%) to genotoxic concentrations of B[a]P (1 to 5 μM), the loss of BRCA-1 protein was coupled with pausing in S-phase and G2/M, and accumulation of p53, mdm2 and p21. Treatment of MCF-7 cells synchronized in S-phase (72%) with B[a]P prolonged the arrest in S-phase, although this checkpoint was transient since cells resumed to G2/M after 12 hours with reduced levels of BRCA-1. In these cells, levels of p53 were increased, whereas the cellular content of p21 remained unaltered. In contrast, the co-treatment with the AhR antagonist, α-naphthoflavone (ANF), abrogated the deleterious effects of B[a]P on BRCA-1 expression, while preventing the accumulation of p53 and disruption of cell cycle profile. These findings suggest that the AhR mediated the inverse expression patterns of BRCA-1 and p53 upon exposure to B[a]P. The treatment with BPDE induced S-phase arrest and reduced BRCA-1 mRNA levels. The negative effects of BPDE on BRCA-1 expression were not transient since removal of BPDE did not allow complete reversal of the repression. These cumulative data suggest that the B[a]P metabolite, BPDF, may play a key role in disruption of BRCA-1 expression and cell cycle kinetics in breast epithelial cells. Neoplasia (2000) 2, 460-470.
• Jeffy, B. D., Schultz, E. U., Selmin, O., Gudas, J. M., Bowden, G. T., & Romagnolo, D. (1999). Inhibition of BRCA-1 expression by benzo[a]pyrene and its diol epoxide. Molecular Carcinogenesis, 26(2), 100-118.
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  PMID: 10506754;Abstract: The objective of this study was to investigate whether polycyclic aromatic hydrocarbons (PAHs) contribute to the etiology of sporadic breast cancer by altering the expression of BRCA-1. Acute exposure to the PAH benzo[a]pyrene (B[a]P) inhibited in a time- and dose-dependent fashion cell proliferation and levels of BRCA-1 mRNA and protein in estrogen receptor (ER)-positive breast MCF-7 and ovarian BG-1 cancer cells. Moreover, the acute exposure to B[a]P abrogated estrogen induction of BRCA-1 in MCF-7 cells. The loss of BRCA-1 expression was prevented by the aromatic hydrocarbon receptor (AhR) antagonist α-naphthoflavone, suggesting participation of the AhR pathway. BRCA, 1 exon 1 a transcripts were downregulated by B[a]P faster than exon 1 b mRNA was. Long-term exposure to B[a]P (40 nM for 15 mo) lowered BRCA-1 mRNA levels in subclones of MCF-7 and BG-1 cells, whereas expression of BRCA-1 in these clones was reverted to normal levels by washing out of B[a]P. The mechanisms of BRCA-1 repression by B[a]P were further investigated by examining the effects of the halogenated aryi hydrocarbon 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) and the B[a]P metabolite 7r,8t-dihydroxy- 9t, 10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). While TCDD did not influence basal BRCA- 1 mRNA and protein levels at any of the doses (from 10 nM to 1 μM) tested in this study, treatment with 50 nM BPDE drastically reduced BRCA-1 mRNA levels, indicating that metabolism of B[a]P to BPDE may contribute to downregulation of BRCA-1. Conversely, ER-negative breast MDA- MB-231 and HBL-100 cancer cells were refractory to treatment with B[a]P or TCDD and expressed constant levels of BRCA-1 mRNA and protein. We conclude that B[a]P may be a risk factor in the etiology of sporadic breast cancer.
• Romagnolo, D., Annab, L. A., Thompson, T. E., Risinger, J. I., Terry, L. A., Barrett, J. C., & Afshari, C. A. (1998). Estrogen upregulation of BRCA1 expression with no effect on localization. Molecular Carcinogenesis, 22(2), 102-109.
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  PMID: 9655254;Abstract: Alterations in the expression of the breast and ovarian cancer susceptibility gene BRCA1 may contribute to the development of mammary and ovarian neoplasia. The sex-steroid estrogen modulates cell proliferation of normal and neoplastic breast and ovarian epithelial cells, but the role of estrogen regulation on the expression of BRCA1 remains to be defined. In this study, estrogen-regulated BRCA1 expression was examined in breast and ovarian cancer cells. Estrogen stimulated the proliferation of estrogen receptor (ER)-positive breast MCF-7, C7-MCF-7, and ovarian BG-1 cells as well as the expression of the estrogen-inducible pS2 gene. This was concomitant with upregulation of BRCA1 mRNA (2.5- to 5.0-fold) and a 3- to 10-fold induction of BRCA1 protein (230 kDa). Cell fractionation studies localized the BRCA1 protein to the nucleus in both unstimulated and estrogen-stimulated cells. The antiestrogen ICI-182780 inhibited estrogen-induced cell proliferation, BRCA1 mRNA induction, and BRCA1 protein expression in ER-positive cells. Conversely, estrogen did not influence expression of BRCA1 in HBL-100 cells that lacked the estrogen receptor, although the constitutive levels of BRCA1 mRNA (but not protein) in these cells were 5- to 30-fold higher than in other breast and ovarian cancer cells. Secretion of the BRCA1 protein into the cell medium did not account for the discrepancy between the mRNA and protein levels in HBL-100 cells. Proliferation of HBL-100 cells was not affected by either estrogen or ICI-182780. Taken together, these data support a role for the steroid estrogen and the involvement of the estrogen receptor pathway in the modulation of expression of BRCA1. We therefore propose that stimulation of cell proliferation may be a prerequisite for upregulation of BRCA1 in breast and ovarian cancer cells.
• Romagnolo, D., & DiAugustine, R. P. (1994). The mammary gland: Protein factory of the future. Environmental Health Perspectives, 102(8), 644-646.
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  PMID: 7895703;PMCID: PMC1567316;
• Romagnolo, D., & DiAugustine, R. P. (1994). Transgenic approaches for modifying the mammary gland to produce therapeutic proteins. Environmental Health Perspectives, 102(10), 846-851.
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  PMID: 9644191;PMCID: PMC1567344;Abstract: Bioengineering of the mammary gland to produce proteins of therapeutic and industrial value is the result of extensive investigation of thy physiology of the mammary gland and the ability to generate transgenic animals. Targeting the expression of heterologous proteins to mammary tissue requires a thorough understanding of the biochemical events that coordinate growth and differentiation of the mammary gland and of the hormonal and developmental regulation of expression of milk protein genes. The characterization of mammary-specific promoter regions in milk protein genes and knowledge of the mechanisms that confer integration site-independent expression of transgenes have significantly contributed to modifying the mammary gland to produce heterologous proteins of therapeutic interest. The generation of large transgenic farm animals provides the opportunity for large-scale production of proteins in milk that have a therapeutic value are naturally present at low concentrations in biological fluids. Transgenic mammary epithelial cells offer a versatile research model in biomedical, environmental health, and neonatal toxicology research.
• Romagnolo, D., Akers, R. M., Byatt, J. C., Wong, E. A., & Turner, J. D. (1994). IGF-I-Induced IGFBP-3 potentiates the mitogenic actions of IGF-I in mammary epithelial MD-IGF-I cells. Molecular and Cellular Endocrinology, 102(1-2), 131-139.
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  PMID: 7523204;Abstract: Limited information is available concerning the molecular and cellular mechanisms that regulate expression of insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs) in bovine mammary epithelial cells. Here, we report on the autocrine mechanisms of action of IGF-I and hormonal regulation of expression of IGFBPs in bovine mammary epithelial MD-IGF-I cells which express recombinant IGF-I under the control of the glucocorticoid-inducible mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Levels of IGFBP-3 mRNA and secretion of IGFBP-3 by MD-IGF-I cells were stimulated by IGF-I, insulin (INS), and IGF-I analogs but not prolactin (PRL). Conversely, parental MAC-T cells expressed little IGF-I and secreted primarily IGFBP-2 (29-32 kDa) in response to stimulation with INS, dexamethasone (DEX), or IGF-I analogs. Secretion of recombinant IGF-I caused a 26.5-fold increase in secretion of IGFBP-3, as measured by densitometric analysis of ligand blots, which was associated with a 1.7-fold increase in total DNA. Conditioned media (CM) from MD-IGF-I cells induced with DEX stimulated a 2.8-fold increase in [3H]thymidine incorporation into DNA of parental MAC-T cells, compared with uninduced cells. Moreover, inclusion of exogenous IGF-I with CM from MD-IGF-I cells triggered an additional 3.0-fold increase in label incorporation, but only a 1.6-fold increase in the presence of IGFBP-2-containing media conditioned by MAC-T cells. Des(l-3)IGF-I added to CM from both MAC-T and MD-IGF-I cells respectively, stimulated a 10.2 and 6.9-fold increase in [3H]thymidine incorporation into DNA of MAC-T cells. We suggest that expression of IGF-I-induced IGFBP-3 is an important component of an autocrine loop which potentiates the local mitogenic actions of IGF-I in bovine mammary epithelial cells. © 1994.
• Romagnolo, D., Akers, R. M., Byatt, J. C., Wong, E. A., & Turner, J. D. (1994). Regulation of expression of IGF-I-induced IGFBP-3 and IGF-I-receptor by constitutive vs regulated expression of recombinant IGF-I in transfected mammary epithelial cells. Endocrine, 2(5), 375-384.
• Romagnolo, D., Polan, C. E., & Barbeau, W. E. (1994). Electrophoretic analysis of ruminal degradability of corn proteins.. Journal of dairy science, 77(4), 1093-1099.
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  PMID: 8201044;Abstract: Albumin, globulin, and prolamin fractions were extracted from corn meal in water, .5 M NaCl, and 50% (vol/vol) 1-propanol and examined by SDS-PAGE and densitometric scanning to investigate fractional degradation rates of corn proteins. Several protein fractions were identified for globulins, zein prolamins, and glutelins. Ruminal degradability of individual subfractions was evaluated by suspension of corn and corn gluten meal samples in the rumen of lactating dairy cows from 0 to 72 h. Electrophoretic and densitometric analysis of protein residues revealed that the prolamin fraction zein for corn and corn gluten meal was more resistant to ruminal degradation than albumins, globulins, and glutelins. Relative rates of degradation of zein and the fraction containing albumins, globulins, and glutelins were .060, .026, and .018, .015/h for corn and corn gluten meal, respectively. Total degradabilities of corn and corn gluten meal, measured by summation of degradability of subfractional components, were 52.2 and 18.6%. Quantitative measurement of ruminally degradable subfractions and estimation of their degradation rates by electrophoretic and densitometric scanning are useful in understanding ruminal degradability of corn proteins.
• Romagnolo, D., Selmin, O., Romagnolo, D., & Selmin, O. (1994). Renovating Italian science [1]. Science, 263(5144), 1669-.
• Romagnolo, D., & Nebel, R. L. (1993). The accuracy of enzyme-linked immunosorbent assay and latex agglutination progesterone test for the validation of estrus and early pregnancy diagnosis in dairy cattle. Theriogenology, 39(5), 1121-1128.
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  Abstract: The accuracy of the enzyme-linked immunosorbent assay (ELISA) and the latex agglutination (LA) on-farm progesterone kit for detecting estrus and diagnosing early pregnancy was investigated in this study. Italian Friesian dairy cows (n=82) from 6 dairy herds were used for the collection of foremilk samples at the time of breeding and at 19, 21, and 23 days post insemination. Pregnancy status was ascertained by uterine palpation per rectum 40 to 60 days post insemination. Progesterone levels were affected by herd, percentage of milk fat, and the day of testing × diagnosis interaction. Validation of estrus by qualitative on-farm tests was 74.6% (LA) and 100.0% (ELISA) accurate using 0.5 ng/ml of progesterone as the RIA estimate for estrus. The accuracy rate for early pregnancy diagnosis by RIA was 68.4 to 83.8% for day 19 and day 21, respectively, while the detection rate for nonpregnancy was 84.6 to 100% on day 19 and day 21, respectively, as compared with uterine palpation per rectum. The average accuracy rate for early pregnancy diagnosis ranged from 84.7 to 92.3% for the LA and ELISA tests, respectively; the nonpregnancy rate was correctly predicted 93.9% to 68.2% for the LA and ELISA tests, respectively. © 1993.
• Romagnolo, D., Akers, R. M., Wong, E. A., Boyle, P. L., McFadden, T. B., & Turner, J. D. (1992). Overexpression of ovine insulin-like growth factor-1 stimulates autonomous autocrine or paracrine growth in bovine mammary-derived epithelial cells. Molecular Endocrinology, 6(11), 1774-1780.
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  PMID: 1480169;Abstract: To test the hypothesis that insulin-like growth factor-I (IGF-I) affects the growth of bovine mammary epithelial cells through an autocrine and/or paracrine pathway, a cell line (MD-IGF-I) was originated from MAC-T cells by cotransfection with a construct containing the cDNA for an ovine exon 2-encoded prepro-IGF-l under control of the mouse mammary tumor virus-long terminal repeat promoter. Clone MD-IGF-I contained multiple copies of the plasmid integrated into the genome, expressed the highest level of IGF-I mRNA, and secreted radioimmunoactive IGF-I into the medium. The mitogenic activity of MD-IGF-I cells was stimulated 80% by dexamethasone (DEX). The total DNA in MD-IGF-I cells was 2.5-fold higher than that in parental MAC-T cells in the presence of DEX. Conditioned medium from MD-IGF-I cells, induced with DEX, stimulated [3H]thymidine incorporation into DNA of MAC-T cells and uninduced MD-IGF-I cells. These data provide evidence that IGF-I was secreted into medium by MD-IGF-I cells. It is suggested that IGF-I can stimulate the growth of mammary epithelial cells by an autocrine and/or paracrine mode of action. The MD-IGF-I cell line may be a suitable system to study translational and posttranslational modifications of IGF-I pep-tides. Copyright © 1992 by The Endocrine Society.

Proceedings Publications

• Turbyville, T., Blonder, J., Das, S., Ye, X., Prieto, D., Veenstra, T., Milner, J., & Romagnolo, D. (2010, Fall). Comparative Proteomic Profiling of Human Breast Cell Lines. In T2012 FASEB Meetings.

Presentations

• Romagnolo, D. (2019, 01/25). AhR and breast cancer metastasis. Seminar series of the Metastatic Breast Cancer Program. Arizona Cancer Center, Rm 3968.
• Romagnolo, D. (2019, 02/13). AhR in breast cancer development and dietary prevention. Research Day - College of Medicine Data Blitz. Arizona Research Building - The University of Arizona.
• Romagnolo, D. (2019, 03/04). Mediterranean Diet and Disease Prevention. CALS Alumni Event. La Jolla, CA.
• Romagnolo, D. (2019, 04/25). Mediterranean Diet&Health: Disease Landscape and Prevention Through Diet. CALS-Alumni Ag 100 Club.. Yuma, AZ.
• Romagnolo, D. (2019, 09/10 - 09/12). Dietary Prevention of Breast Cancer: A case for the Mediterranean Diet. CALS Alumni. Salinas and San Jose', CA.
• Romagnolo, D. -. (2011, August). BRCA-1 promoter methylation induced by the activated AhR and prevention by the dietary antagonist resveratrol. 2011 Era of Hope US DOD Breast Cancer Program. Orlando, FL.
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  Broadcast
• Romagnolo, D. -. (2011, December). Epigenetics of Breast Cancer. AZCC Townhall MeetingAZCC.
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  Broadcast
• Romagnolo, D. -. (2011, November). Epigenetics of BRCA-1 and Sporadic Breast Cancer. 2011 American Institute for Cancer Research Conference. Washington DC: American Institute for Cancer Research.

Poster Presentations

• Romagnolo, D. (2019, 04/01). Modulating effects of genistein in a mouse model of conditional BRCA1 heterozygous deletion and triple negative breast cancer cells.. 2019 American Association for Cancer Research Conference. Session category: Nutrition, Screening, and Early Detection for Cancer Prevention. Abstract #1604.. Georgia World Congress Center, Atlanta, GA..
• Romagnolo, D. (2019, 05/15/). n-6 linoleic acid induces epigenetic alterations associated with colonic inflammation and cancer.. 2019 Meetings of the American Institute for Cancer Research. Chapel Hill, NC. May 15th, 2019..
• Romagnolo, D. (2019, 11/20). BRCA1: targeting triple negative breast cancer with dietary isoflavones. Reimagine Health: Is my fate in my genes? -. 2nd Annual University of Arizona Research Symposium. Biomedical Sciences Partnership Building.. University of Arizona College of Medicine-Phoenix. Phoenix, AZ, November 20th, 2019..
• Romagnolo, D. (2018, April 14-18th). Galangin is an epigenetic modulator of BRCA1 and induces estrogen receptor alpha in triple negative breast cancer cells.. 2018 American Association for Cancer Research Conference. Session category: Molecular and Cellular Biology/Genetics: Epigenetic Therapy. Abstract #7597.. Chicago, IL,: AACR.
• Romagnolo, D. (2018, March 23). Flavones counteract epigenetic silencing of BRCA1 in breast cancer cells with activated AhR.. CALS Poster Forum. ENR2 Bldg -University of Arizona: CALS.
• Romagnolo, D. (2018, September 28th). • Arsenic-induced BRCA1 CpG promoter methylation associates with downregulation of ERα and resistance to tamoxifen in MCF7 breast cancer cells and mouse mammary tumor xenografts.. 3rd Annual University of Arizona Cancer Center Scientific Symposium and Retreat. Westward Look Resort, Tucson, AZ.: University of Arizona Cancer Center.

Reviews

• Selmin, O., & Romagnolo, D. (2017. Epigenetics of Nuclear Receptors in Breast Cancer - The Handbook of Nutrition, Diet and Epigenetics.

Creative Productions

• Romagnolo, D. (2021. Mediterranean Diet and Health promotionUA Life and Work Connections.
• Romagnolo, D., & Selmin, O. (2017. UA Cancer Center Research Team Explores Anti-Breast Cancer Properties of SoyArizona Cancer Center.
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  http://deptmedicine.arizona.edu/news/2017/ua-cancer-center-research-team-explores-anti-breast-cancer-properties-soy

Other Teaching Materials

• Romagnolo, D., & Selmin, O. (2016. Appendix: 2015 Dietary Guidelines for Americans. Spinger - Humana Press.

Others

• Lyon, A., Selmin, O., Fang, C., Smith, J., Doetschman, T., Thompson, P., Martinez, J., Lance, P., & Romagnolo, D. (2015, APR). Epigenetic Regulation of the FXR by High Fat Diet and APC in Colon Cells. FASEB JOURNAL.
• Romagnolo, D. -. (2012, December). Flavonoids for Cancer Prevention: A Closer Look. http://www.aicr.org/cancer-research-update/december_5_2012/cru-flavonoids-prevention.html
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  Exact Date: 12/6/2012