The long-term objectives of this study are l) to transform normal human breast epithelial cells (HBEC) in vitro stepwise to various stages of preneoplastic and malignant cells by chemical physical carcinogens and oncogenes; 2) to determine if one type of HBEC (Type I) with stem cell and luminal epithelial cell characteristics is the target cell for neoplastic transformation by various breast carcinogens; 3) to determine at which stage of neoplastic transformation, different known breast carcinogens are most effective in exerting their effects; and 4) to elucidate the genetic and molecular mechanisms of breast carcinogenesis. To these ends, using two best known agents that affect breast carcinogenesis (i.e. ionizing radiation and estrogenic compound), we propose 1) to determine if ionizing radiation is a potent initiator, promotor or progressor for neoplastic transformation of normal HBEC (with extended lifespan, immortality, weak tumorigenicity, and high tumorigenicity as end points); 2) to develop normal and preneoplastic HBEC that express estrogen receptor (ER) and respond to estrogens for cell proliferation; 3) to test if and how estrogenic compounds affect neoplastic transformation of ER-positive normal and preneoplastic HBEC; and 4) to characterize the biological and molecular nature of preneoplastic and neoplastic HBEC induced by ionizing radiation/estrogen to reveal the genetic and molecular mechanisms of breast carcinogenesis. To accomplish these specific aims, a new culture technique to grow two types of normal HBEC has been developed and molecular techniques, such as comparative genomic hybridization for molecular cytogenetic analysis and Northern, Western blot analysis, RT-PCR, DNA sequencing, immunostaining, laser cytometry will be used to determine the expression of various oncogene, tumor suppressor genes for preneoplastic and neoplastic cells at different stages. Besides the potential to elucidate the mechanism of breast carcinogenesis, the development of this system will be instrumental in identifying environmental carcinogens and in developing potential radiation protectors for mammography and chemopreventive/therapeutic strategies for breast cancer (e.g. differentiation of target normal or preneoplastic HBEC).
