Project 1: Repair of formaldehyde-induced DNA-protein crosslinks in Drosophila
PI: Jeff Sekelsky, PhD, Associate Professor of Biology and Program in Molecular Biology & Biotechnology, Biology Dept.
Project 2: Heterotypic interactions in breast responses to ionizing radiation
Project 3: Dietary protein mediates bacterial regulation of host energy balance
Project 4: Oxidative stress in the progression of experimentally induced osteoarthritis
Project 5: Preclinical evaluation of genistein and soy extract in combination with conventional cytotoxic and hormonal treatment regimens for endometrial cancer
Project 6: Gastrointestinal stem cells and risk factors for colorectal adenoma
Project: Repair of formaldehyde-induced DNA-protein crosslinks in Drosophila
DNA-protein crosslinks (DPCs) arise from both exogenous and endogenous sources. One important source the environmental carcinogen formaldehyde. Cells can repair DPCs, but the repair mechanisms are poorly understood. Previous studies in E. coli and mammalian cells suggests that repair involves components of two well-studied repair pathways: nucleotide excision repair (NER) and homologous recombination (HR). We propose to study repair of DPCs in vivo in the model organism Drosophila melanogaster. We will first determine whether the NER endonucleases XPG and XPF-ERCC1 (the excinucleases) are required to excise DPCs caused by exogenous formaldehyde. We will then investigate the role of HR in the response to DPCs induced by exogenous formaldehyde. To explore the contribution of endogenous formaldehyde to DNA damage, we will attempt to block spontaneous mitotic crossing, which may arise during repair of DPCs, with a formaldehyde quencher. These experiments will further our understanding of in vivo repair of DPCs in animals.
Project: Heterotypic interactions in breast responses to ionizing radiation.
Heterotypic interactions between initiated epithelium and surrounding stroma play a critical role in development and progression of cancer. There is strong evidence that the stroma does not just passively respond to initiated cells, but plays a more active role in carcinogenesis. This project will test the hypothesis that stromal-epithelial interactions determine the ionizing radiation (IR) response phenotype of the breast. In Aim 1, the gene expression changes induced by stromal-epithelial interactions will be assessed in coculture. This aim will develop a model culture system for characterizing cell-cell interactions. In Aim 2, we will use this culture system to study IR responses. Using cells in monoculture and coculture, we will identify gene expression changes that occur in coculture and are correlated with phenotypic endpoints such as growth arrest and apoptosis. Responses will also be compared with what is observed in cultures of tissue explants. This project will develop and validate a model coculture system for testing hypotheses about the role of heterotypic interaction in environmental carcinogenesis and will provide important biological insights about breast responses to ionizing radiation.
Project: Dietary protein mediates bacterial regulation of host energy balance
The obesity epidemic is presenting a major public health challenge. Identifying new strategies for regulating energy storage is therefore an important goal. The proposed research will investigate the interaction between two environmental factors that influence energy storage: the diet and intestinal bacteria. The presence of bacteria in the gut leads to an increase in body fat due to microbial suppression of intestinal expression of Fasting-induced adipose factor (Fiaf). The mechanisms underlying bacterial suppression of Fiaf are undefined, and represent potential therapeutic targets for manipulating human energy storage. We have used the zebrafish model to show that the host-bacteria interactions controlling Fiaf expression are sensitive to dietary protein levels. Moreover, we have identified a signaling mechanism used by bacteria to control Fiaf expression. The objective of this proposal is to determine if dietary protein levels influence the composition and activity of the intestinal bacterial community and thereby regulate host metabolism. The proposed research will address the central hypothesis that dietary protein potentiates the bacteria-mediated suppression of Fiaf production by the intestinal epithelium.
Project: Oxidative stress in the progression of experimentally induced osteoarthritis.
Evidence is accumulating to implicate environmental toxicants and oxidative stress in a complimentary role in osteoarthritis (OA). Epidemiological studies show positive associations between OA severity and blood lead levels as well as selenium insufficiency. The problem with our understanding of the influence of the environment is that the precise link between oxidative stress and OA remains elusive. This compels us to examine the effect of oxidative stress on joint pathology using a murine model of surgically-induced knee instability, to determine if the time-course of disease is altered in animals maintained on a selenium-deficient diet. We will also examine the role of the transcription factor Nrf2, critically important for expression of antioxidant enzymes, by following OA in animals that lack Nrf2, and normal animals fed a potent activator of Nrf2. We hypothesize that oxidative stress will accelerate tissue damage, and conversely that enhancement of antioxidant gene expression will slow progression as observed by MRI and histolopathogy. The natural extension of this pilot data is to explore the mechanisms of heavy metal exposures as oxidant stressors in the progression of OA.
Project: Preclinical evaluation of genistein and soy extract in combination with conventional cytotoxic and hormonal treatment regimens for endometrial cancer.
Treatment of women with recurrent or advanced endometrial cancer has been met with limited success. This has prompted a search for an additional agent which could be used in combination with more traditional therapies to dramatically increase efficacy while not increasing toxicities. Genistein, the bioactive isoflavone of soybeans, acts as a potential radiosensitizer for prostate cancer and has been shown to enhance the cytotoxicity of chemotherapeutic drugs in a variety of tumor types. Thus, our overall goal is to evaluate cell proliferation, telomerase activity and apoptosis in a novel endometrial cancer co-culture model system after exposure to genistein or soy extract in combination with cytotoxic and hormonal chemotherapeutic agents commonly used in the treatment of this disease. The effects of genistein or soy extract on estrogen and progesterone receptor signaling will also be explored. We hope that this will provide valuable evidence that phytoestrogens may potentiate the effects of other cytotoxic and hormonal agents in endometrial cancer, and that combination therapy may be a more effective treatment option for women with recurrent or advanced stage disease.
Project: Gastrointestinal stem cells and risk factors for colorectal adenoma.
Colorectal cancer is thought to be initiated by inappropriate survival or expansion of crypt stem cells. Low apoptosis of crypt cells strongly predicts precancerous adenomas in multiple patient populations supporting this model. Elevated insulin, obesity, and high fat diet increase adenoma risk, and correlate with low apoptosis. However, definitive evidence for a role of stem cells in adenoma, or an effect of environmental factors on stem cells is lacking because no valid stem cell markers were available. Recent findings by others, and here at UNC, define GPR49/Lgr5 and SOX9 as markers of multipotent intestinal stem cells, and SOX4 as a potential biomarker of a subset of stem cells. Our studies in mice demonstrate that insulin signaling promotes survival of SOX9-positive stem cells and colon tumorigenesis. This pilot aims to translate these exciting findings to humans and directly test the hypothesis that adenoma, elevated plasma insulin, obesity, or high fat diet promote expansion of GPR49/Lgr5, SOX9 or SOX4 positive stem cells in normal colon, leading to selective expansion of the same stem-cell sub-type in adenomas.
|Funded by NIEHS Grant # P30 ES010126|