This project has two interrelated goals. The first goal is to answer the question of whether initiation in vivo is a common (e.g., 1 per 1000 cells or more frequent) or rare event at the cellular level. Initiation is defined here as a cellular change (lesion) that is directly and rapidly induced by carcinogen exposure and leads to the formation of a malignant neoplasm. This first goal will be addressed by quantitating the frequency of initiation in rat mammary epithelial cells following in situ exposure to each of the mammary carcinogens NMU and DMBA. This will be accomplished using a limiting dilution transplantation assay that we have developed for this purpose. The second related goal of this project is to examine the hypothesis that activation of the c-Ha-ras-1 oncogene is at least one possible primary cellular change responsible for cellular initiation of carcinogenesis in the rat mammary gland. Carcinomas developing from known numbers of exposed surviving target cells in the experiments relating to the first goal will be examined to determine if they possess an activated c-Ha-ras-1 gene, i.e. is this gene mutated at codon 12 (NMU-induced) or 61 (DMBA-induced). Restriction mapping will be the primary method used for this determination. This will be followed by oligonucleotide hybridization for determination of specific base substitutions in the altered c-Ha-ras-1. The data obtained from these measurements will allow us to answer the question of whether c-Ha-ras-1 activation is an early event. If it is an early event, its frequency per exposed mammary cell will also be calculated from these data. In addition, we will quantitate mutation frequency at other loci from similarly exposed cells in order to address the question of the relative """"""""hotness"""""""" (propensity to mutate) of c-Ha-ras-1. Finally, these experiments will be extended to a physical carcinogen, ionizing radiation. This carcinogen will be explored because, unlike DMBA and NMU, it is an efficient inducer of deletions. Thus, a different mutation spectrum would be expected. We feel that the experiments proposed here will aid our understanding of the etiology of mammary cancer. This understanding may lead to new approaches to cancer prevention.
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