Date of Award

2006

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Environmental Science

First Advisor

Dr. Sherif M. Karam

Second Advisor

Prof. Gerald Buzzell

Third Advisor

Dr. Chatherine Tomasetto

Abstract

Breast cancer is the most common type of cancer and the leading cause of cancer deaths among women in the United Arab Emirates and all over the world. Although many factors contribute to the high incidence of breast cancer, a considerable number of cases are related to environmental factors. In this study, breast cancer was induced in female rats using a single dose, 80 mg/kg body wt, of the environmental carcinogen 7,12- dimethylbenz[a]anthracene (DMBA). The aim was to characterize some of the early cellular and molecular changes that occur during breast cancer development in the DMBA-treated rat model. Mammary gland tissues of control and DMBA-treated rats were processed for: i) routine histological examination, to define the initial histopathological changes that occur in mammary gland tissues, ii) immunohistochemical probing using anti-PCNA and anti-BRCA1 antibodies to characterize and correlate the localization of these cell cycle proteins during progression to cancer, iii) Western blotting, to analyze the alteration of p53 protein expression in pre-neoplastic and neoplastic lesions of the mammary glands, iv) polymerase chain reactions using primers specific for PCNA, BRCA1, and p53 genes followed by single stranded conformational polymorphism (SSCP) or restriction fragment length polymorphism (RFLP) assays to detect possible mutations in these genes during development of breast cancer.

Microscopic examination revealed a wide range of pre-neoplastic and neoplastic lesions in the mammary glands of DMBA-treated rats. They provided a sequence representing the multistep process of breast cancer formation. The earliest morphological change observed was moderate dilatation of the terminal ducts and accumulation of extruded dead cells in their lumens. These changes were referred to as the stage of "cell death". It was followed by stages of hyperplasia, dysplasia, and then in situ cribriform carcinoma. Finally, the invasive cribriform and papillary carcinomas developed. In addition, some DMBA-treated rats developed benign tumors: lactating adenoma and squamous cell papilloma.

Immunohistochemical localization of PCNA revealed an initial down regulation during the stage of cell death followed by a gradual increase in the number of PCNA-labeled cells during the following stages of breast cancer development. Probing for BRCA1 protein suggested a gradual defect in its translocation from the cytoplasm to the nucleus during breast cancer progression. In control rats, BRCA1 was present in the nuclei of terminal duct epithelial cells. However, in the pre-neoplastic lesions, BRCA1 was localized in both the cytoplasm and nuclei of the epithelial duct cells. In all malignant lesions, BRCAI was mainly found in the cytoplasm.

Western blotting revealed that the expression of p53 protein during breast cancer development was initially down regulated. However, with progression toward malignancy, upregulation of the mutant form of p53 was observed. These changes were associated with polymorphism in p53 gene, which was detected in exon 5 using SSCP assay. However, no polymorphisms were detected in the PCNA and BRCA1 genes.

Because some recent studies suggested that the use of multiple alkaloids may have some synergistic inhibitory influence on cancer cell growth, the effect of crude extract of Vinca alkaloids on the progression of breast cancer was tested in DMBA-treated rats. A daily treatment of these rats with 200 mg/kg body wt of Vinca alkaloids for 21 days starting 1 week after DMBA administration induced about 50% decrease in the incidence of mammary gland neoplasia. When these tumors were compared with those of DMBA-treated rats, the former were smaller in size, exhibited a smaller number of PCNA-labeled cells, and showed down regulation in the expression of both PCNA and mutant p53 proteins.

In conclusion, characterization of the early changes that occur during breast cancer development may provide some clues for better understanding of its pathogenesis and would hopefully improve modalities for its prevention and/or early detection.

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