The evolution of breast cancer is a complex, multistep process. Using the mouse model of mammary carcinogenesis, our research has three long-term objectives: 1) defining the steps involved in neoplastic progression of mouse mammary epithelial cells (MMEC) to malignancy, 2) identifying the factors and underlying events responsible for advancement from one stage to the next, and 3) developing markers to detect cells in different stages of progression. Most of these steps probably involve alterations in gene structure and/or expression. We have recently shown that many preneoplastic and malignant MMEC have marked elevation in expression of the endogenous retrotransposons, intracisternal A particles (IAPs), compared to normal cells. In contrast, expression of other endogenous retrotransposons is unchanged, substantially decreased, or only occassionally increased. IAPs are mobile elements in mouse DNA with the potential for contributing to neoplastic transformation by their ability to transpose and cause insertional mutagenesis, thereby increasing genetic instability of the host genome. The hypothesis of the proposed research is that amplified expression with subsequent transposition of IAP sequences are key events in at least one pathway of mouse mammary carcinogenesis, i.e. induction by high doses of 7,12-dimethylbenzanthracene (DMBA). To investigate the possible role of IAPs in development of mammary cancer, the proposed research will use three approaches: 1) mouse mammary carcinogenesis induced by DMBA will be analyzed to define when altered expression of IAP sequences begins during neoplastic progression, the identity of the cells affected, and which class(es) of IAP elements is involved; 2) an oligonucleotide probe specific for the main class of IAP element expressed in DMBA tumors will be used to determine if any IAP copies are located in new sites of the host genome; 3) the effects of IAP transposition on MMEC will be investigated by transfecting them with an IAP element linked to a selectable marker that is expressed only after transposition occurs. After selection, cells in which the marked element has transposed will be isolated, characterized, and tested for altered growth properties, including tumorigenicity in vivo. The major methodologies for these studies will include the use of cDNA probes and restriction nucleases in Northern and Southern blotting, in situ hybridization, cell culture procedures, transfection techniques, and nude mice for testing tumorigenicity. As ten percent of the human genome consists of transposable elements, several of which have been implicated in mutagenizing human DNA, these elements might be operative in human, as well as mouse, breast cancer. In fact, oncogene activation due to transposition and insertional mutagenesis by one such element has already been documented in a human breast ductal carcinoma.
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