The int-1 gene is activated by insertions of mouse mammary tumor virus (MMTV) proviral DNA in MMTV-induced tumors and its protein product is strongly implicated in tumorigenesis. The gene has transforming activity in certain mammary epithelial cell lines but not in fibroblasts. int-1 is also implicated in the development of the central nervous system in mid-gestational mouse embryos. Recent data suggest that int-1 protein products may enter the secretory pathway, and that the gene may act via secretion of a diffusible factor. The purpose of this research is to investigate the mechanism of int-1 mediated transformation, and the role of this gene in the pathway of tumorigenesis. Specifically, we shall test the hypothesis that int-1 encodes a novel growth factor. Polyclonal antibodies will be raised against int-1 fusion proteins expressed in E. coli, and used for detection of secreted forms of int-1 protein in cell culture medium. We shall also seek evidence of an int-1 dependent secreted factor by means of its predicted mitogenic activity on approriate mammary cells. In order to begin a functional analysis of int-1 proteins, specific deletions and site-directed mutations will be generated in int-1; their biological activities will then be tested in int-1 responsive cells in conjunction with characterization of their protein products. In addition, the target cell specificity of int-1 action will be investigated by infecting a variety of cell types with retroviral vectors carrying the gene. As a step towards investigating distal stages in the mechanism of int-1 action, attempts will be made to isolate mutant cell lines resistant to int- 1 transformation. We shall also perform direct tests of functional cooperation between int-1 and other oncogenes implicated in mammary tumorigenesis, and determine whether an assay based on DNA transfer into partially transformed mammary epithelial cells can be used to identify novel oncogenes that contribute to tumor formation. Mouse mammary carcinomas provide a model system for studying oncogene action in epithelial cells: studies of the role of int-1 in these tumors may reveal novel mechanisms of carcinogenesis with relevance to other epithelial tumors including human cancers.
