Numerous somatic mutations have been identified in human and experimental mammary neoplasia. In this project we propose to make use of a well defined chemical carcinogenesis model to facilitate the identification of relevant chromosomal and genetic events in mammary carcinogenesis. It is the main hypothesis of this proposal that specific chromosomal abnormalities and allelic losses play critical roles in mouse mammary tumor development and progression. In human breast cancer allelic losses and specific chromosome abnormalities were reported affecting various chromosome regions. Several of these areas appear to be the locale of putative tumor suppressor genes. However, the relevance and role in breast cancer of several of this abnormalities is still unclear. It is very important to determine whether some of these anomalies are cause or effect of tumor progression. Similar karyotypic and allelotypic studies have not been performed in the widely used model of mouse mammary carcinogenesis. Based on previous observations from our laboratory, we can speculate a likely involvement of areas of the mouse genome homologous to those observed affected in humans, thus mimicking genetic mechanisms of human carcinogenesis. The main goal of this investigation will be not only to identify such abnormalities but also to determine their temporal relation from preneoplasia to the development of aggressive ovary independent malignant phenotypes. We propose to monitor the cytogenetic progression of chemically induced mouse mammary tumors coupled with studies to identify areas of the mouse genome with putative tumor suppressor activity. These studies will hopefully provide information of relevance for understanding mechanisms of mammary tumor development and also of chemical carcinogenesis in general.
The specific aims of this proposal therefore are: 1) To analyze the cytogenetic profile of chemically induced mouse mammary tumors at different stages of development. Two different chemical carcinogenesis protocols will be used a) a high DMBA dose complete carcinogenesis protocol and b) a single NMU treatment in conjunction with hormone stimulation using medroxyprogesterone acetate. These studies will be performed using F1 hybrid mice for the purpose described in the following aims. 2) To analyze the relevance, timing and sequence for loss of heterozygosity in specific mouse chromosome areas. Primary target for study will be the homologous areas to those that were shown to be nonrandomly affected in human breast cancer. These studies will be performed using F1 hybrid mice polymorphic at multiple loci (B6D2Fl and CD2Fl mice). The search for the specific LOHs will be performed by means of conventional RFLP analysis from Southern blots and by PCR analysis of microsatellites. 3) To identify specific allelic losses at premalignant stages by analyzing mammary dysplasias by means of the PCR of microsatellites strategy, on material obtained from mammary wholemounts. 4) To assess the role of specific allelic losses and karyotypic abnormalities in the comparison of ovary dependent vs independent tumors. 5) To suppress tumorigenicity of mammary tumor lines by means of microcell fusion techniques that allow the introduction of specific human donor chromosomes.
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