archives@tulane.edu / BRCA1 faithfully repairs damaged DNA by promoting homology-directed repair (HDR). Loss of Brca1 and other HDR genes are incompatible with embryonic viability and cause severe genomic instability. Cells lacking BRCA1 are sensitive to cellular stresses such as DNA damage caused by ionizing radiation (IR). Homozygous loss of Brca1 is embryonic lethal in mice, and the few tissue-specific knockouts generated develop abnormally. Therefore, we created an inducible Cre mouse model to study Brca1 loss in all adult mouse tissues allowing for examination of viability, longevity, and stress response in the absence of HDR and the importance of HDR in different tissues of an adult mouse. After validating the inducible Cre system using a reporter allele in mice, we generated mice with alleles of the inducible Cre system and floxed Brca1 alleles. Cre was induced in adult mice at ten weeks of age, resulting in extensive, widespread deletion of Brca1. Contrary to the embryonic lethality observed in all previously tested germline Brca1 knockout mouse models, adult mice with Brca1 deletion displayed no overt phenotypes. Brca1Δ/Δ mice showed extensive, widespread deletion of Brca1 and survived up to 1 year after Brca1 recombination. Targeted, high-depth sequencing of recombined tissues indicated mutations accumulated in both the mammary gland and the intestine. However, only the mammary gland had an HDR deficiency signature. Next, we examined Brca1Δ/Δ mice survival after exposure to ionizing radiation and mitomycin C (MMC). Surprisingly, Brca1Δ/Δ mice responses are DNA damage specific. Brca1Δ/Δ mice deficient for HDR showed no increased sensitivity to IR but died four to eight days following MMC exposure. Our results show that BRCA1 is not required for long-term viability or DNA double-strand break repair, but BRCA1 is essential for DNA crosslink repair to maintain viability in an adult mouse. / 1 / JoyOlayiwola

PURPOSE:Selective Estrogen Receptor Modulators (SERMs) reduce the risk of breast cancer for women at increased risk by 38%. However, uptake is extremely low and the reasons for this are not completely understood. The aims of this study were to utilize time trade-off methods to determine the degree of risk reduction required to make taking SERMs worthwhile to women, and the factors associated with requiring greater risk reduction to take SERMs.METHODS:Women at increased risk of breast cancer (N=107) were recruited from two familial cancer clinics in Australia. Participants completed a questionnaire either online or in pen and paper format. Hierarchical multiple linear regression analysis was used to analyze the data.RESULTS:Overall, there was considerable heterogeneity in the degree of risk reduction required to make taking SERMs worthwhile. Women with higher perceived breast cancer risk and those with stronger intentions to undergo (or who had undergone) an oophorectomy required a smaller degree of risk reduction to consider taking SERMs worthwhile.CONCLUSION:Women at increased familial risk appear motivated to consider SERMs for prevention. A tailored approach to communicating about medical prevention is essential. Health professionals could usefully highlight the absolute (rather than relative) probability of side effects and take into account an individual's perceived (rather than objective) risk of breast cancer.

The tumor suppressor Breast Cancer Susceptibility Protein 1 (BRCA1) protects our cells from genomic instability in part by facilitating the efficient repair of DNA double-strand breaks. Other functions of BRCA1 include transcriptional regulation, apoptosis, DNA damage signaling, chromatin remodeling and protein ubiquitination. The major contribution of BRCA1 to maintaining genomic stability is thought to be through its role in DNA repair. BRCA1 promotes the error-free repair of double-strand breaks by homologous recombination, and is also implicated in the regulation of non-homologous end joining repair. Here we investigated the role of BRCA1 in maintaining the fidelity of non-homologous end joining repair following a double-strand break. We also examined the frequency of microhomology-mediated end joining (MMEJ) and the fidelity of double-strand break repair relative to BRCA1 protein levels in both control and tumorigenic breast epithelial cells. In addition to altered BRCA1 protein levels, we tested the effects of cellular exposure to mirin, an inhibitor of Meiotic recombination enzyme 11 (Mre11) 3' to 5' exonuclease activity. Knockdown or loss of BRCA1 protein resulted in an increased frequency of overall plasmid DNA repair mutagenesis and MMEJ following a double-strand break. Inhibition of Mre11 exonuclease activity with mirin significantly decreased the occurrence of MMEJ, but did not considerably affect the overall mutagenic frequency of plasmid double-strand break repair, although some of our data indicate that the size of sequence deletions may be reduced by mirin inhibition. The results suggest that BRCA1 protects DNA from mutagenesis during non-homologous double strand break repair in plasmid-based assays. The increased frequency of double-strand break mutagenesis and MMEJ repair in the absence of BRCA1 suggests a potential mechanism for carcinogenesis.

Camptothecin and its analogues (CPTs) represent one of the most potent classes of anticancer drugs used to treat several solid tumors. CPTs bind topoisomerase I during the replication process and cause DNA damage that results in cell death. However, its effectiveness is limited to 13-30 percent of patients. TopoI cuts and re-ligates DNA supercoiling but in the presence of CPT it fails to re-ligate DNA and collision of replication forks leads to DNA double strand break (DNA-DSB) and cell death. However, in resistant cells, due to deregulated kinase cascade, topoI is continually phosphorylated by DNA-PKcs and rapidly degraded by the ubiquitin proteasomal pathway (UPP). It has been found that BRCA1 plays a key role in imparting cellular resistance to topoI inhibitors. Importantly, BRCA1 ubiquitinates topoI in response to CPT. We hypothesize that disruption of BRCA1 binding to phosphorylated topoI would interrupt the resistance mechanism resulting in higher cellular sensitivity of CPT. Based on an in-silico drug screen, we identified a compound that inhibits topoI degradation by blocking BRCA1 binding. Imaging and survival assays findings are consistent with the hypothesis that BRCA1 plays a role in CPT resistance through its co-localization with topoI, and we speculate this role is through UPP degradation. CPTs are commonly used in combination with cytotoxic compounds, but this study focuses on discovering compounds that can overcome resistance without causing further cytotoxicity. / 2019-07-03T00:00:00Z

Thèse (M.Sc.)--Université Laval, 2005. / Titre de l'écran-titre (visionné le 14 mai 2007). Bibliogr.

The human p63 gene discovered in 1997 encodes a series of protein isoforms that differ in their N- and/or C-terminal sequences. These isoforms have widely differing properties in promoting or repressing p53-related functions such as growth arrest and apoptosis. In addition, p63 appears to play important roles in the maintenance and differentiation of epithelial cell populations. Many studies have shown that p63, particularly Np63, is expressed in normal epithelium and also highly expressed in squamous cell carcinomas of surface epithelia. Methods. We have refined the analysis of the expression patterns of p63 isoforms by the use of a quantitative RT-PCR technique applied to micro-dissected normal oral mucosal samples. We have also studied p63 expression in squamous cell carcinoma of the head and neck (SCCHN) compared to normal oral mucosa taken from the same patient. Furthermore, tobacco-exposed buccal mucosa was compared to age and gender matched non exposed controls. RT-PCR for telomerase and immunohistochemical analysis for detection of p53 and Ki-67 proteins was further performed. We also explored the function of p63 in SCCHN cells by using small interfering RNA (siRNA) to silence the expression of different p63 isoforms in cell lines originating from SCCHN. The effect of p63 knockdown was studied using a Fluorescein Diacetate based cytotoxicity assay and immunohistochemistry looking at expression of differentiation markers. The response of the siRNA treated cells to radiation and cytostatic drug was also investigated. We have further studied normal oral wound healing using immunohistochemistry and quantitative PCR. By the use of a macro array comparing siRNA treated cells with non-treated cells a possible connection between the BRCA1 gene and p63 expression was shown and further studied with the use of cHIP and luciferase reporter assays. Results. The p63 isoforms are expressed in normal epithelium, with the highest levels consistently found in basal and parabasal layers. Extensive use of tobacco had no effect on p63. The quantitative PCR showed statistically increased levels of the ΔNp63 and p63isoforms. No correlation was found between p63-isoform expression patterns and proliferation. The exploration of the function of p63 in SCCHN cells by the use of small interfering RNA (siRNA) showed a statistically significant decreased survival for tumour cells when all p63 isoforms, the N-terminal truncated or the  isoforms were inhibited. No effect on cell proliferation or expression of epithelial differentiation markers was observed. We also demonstrated that inhibition of p63 expression sensitizes cells to the effects of ionizing radiation and cisplatin. The study of normal oral wound healing using immunohistochemistry and quantitative PCR showed significant changes in p63 isoform expression. The Np63 isoform was mainly expressed in the basal layer in the non proliferating and migrating cells while TAp63 was almost absent. The BRCA1 study showed p63 to bind to the BRCA1 promoter and activate the expression of BRCA1 protein. Summary. The p63 proteins have different functions and the balance between the isoforms and their localisation within the epithelium seems to be important for normal wound healing as well as cancer cell survival.

ABSTRACT Breast cancer is a complex disease, whose etiology is not well understood. A number of factors have been found to contribute to its development. Psychological stress has been recognized as such a factor in epidemiological studies, but few molecular mechanisms have been proposed to explain its association to breast cancer risk. This work addresses the lack of knowledge in the area of stress and breast cancer with the use of molecular and epidemiological techniques. Molecular experiments allowed the identification of a link between stress signaling and intracellular signaling pathways known to be affected in breast cancer development. Namely, the stress hormone hydrocortisone (cortisol) was found to down-regulate the Breast Cancer Susceptibility Gene 1 (BRCA1). Further study allowed identification of some of the mechanisms involved. Binding of the transcription factors GABPa/b and USF2 at specific sites of the BRCA1 promoter (the RIBS and UP sites) was shown to be negatively affected by hydrocortisone. In addition, a novel hormone-independent function of the receptor for hydrocortisone, the glucocorticoid receptor, was identified in the context of BRCA1 regulation. GR was determined to act as a positive regulator of BRCA1 in the absence of hydrocortisone through the RIBS and UP sites. Taken altogether these results represent a novel molecular mechanism linking stress signaling to breast cancer development. The second objective of this work was to design an epidemiological study which would determine whether stress-susceptible individuals are at higher risk of developing breast cancer. This study would be the first of its kind in the case of breast cancer and would allow the development of a genetic method of measuring stress exposure which can be used in future studies. The study was designed to look at glucocorticoid receptor iii polymorphisms known to produce phenotypic differences in GR activity in a population of women with incident breast cancer and population-based controls. In conclusion, the present work suggests an integrative model of the effect of stress on breast cancer development which incorporates genetic predisposition to the effects of stress and downstream changes in the expression and activity of the Breast Cancer Susceptibility Gene 1. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2008-01-02 23:42:20.89 / Canadian Breast Cancer Foundation-Ontario Chapter Canadian Institute of Health Research

Thèse (Ph. D.)--Université Laval, 2007. / Bibliogr.: f. [187]-210. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (Ph.D.). / Written for the Dept. of Human Genetics. Title from title page of PDF (viewed 2009/06/10). Includes bibliographical references.

Thèse (M.Sc.)--Université Laval, 2004. / Bibliogr. Publ. aussi en version électronique.