The long term objective of the proposed research is to understand processes which lead to tumorigenesis of breast epithelial cells. This goal will be addressed by studying virus-induced mammary cancer in the mouse to identify a host gene whose abnormal expression can cause a cell to lose control of its proliferation. This gene may be transcriptionally activated by control sequences of the retrovirus mouse mammary tumor virus (MMTV), which may be integrated adjacent to the affected gene in mammary tumors. Several genes which are affected in this way have been isolated and characterized as mammary proto-oncogenes, but these genes do not serve as insertion sites for MMTV in all mammary tumors induced by the virus. The central hypothesis for the proposed research is that there exist genes other than those previously identified which may be transcriptionally activated by MMTV to induce tumorigenesis. A fragment of cellular DNA adjacent to an integration site for MMTV in a virus-induced mammary tumor has been cloned. The tumor had acquired a single new provirus which was not integrated near any of three genes known to be frequently affected by MMTV in mammary tumors. The working hypothesis for the proposed studies is that the cloned cellular DNA fragment represents a portion of a genetic locus which may function as another """"""""mammary proto-oncogene"""""""".
The Specific Aims are 1) To obtain a molecular clone of the new integration region from normal tissue 2) To determine if other virus-induced mammary tumors utilize the new integration region as an insertion site for MMTV 3) To map the structure of the new integration region by restriction enzyme analysis and to determine where on this map integrations of MMTV in mammary tumors occur 4) To characterize the transcriptional activity of the gene within the integration region in normal and neoplastic tissues 5) To determine the nucleotide sequence of the gene and compare its sequence with that of known genes and 6) To examine phenotypic properties of cultured mammary epithelial cells expressing RNA from an introduced copy of the gene. The experimental strategy is to identify DNA clones representing normal copies of the locus by hybridization to the fragment which was adjacent to MMTV in the original tumor. The locus will be mapped by restriction analysis and Southern blotting to identify rearrangements of the locus in other tumors. RNA transcribed from the region will be identified by Northern analysis. The DNA sequence of the transcribed region will be determined by dideoxy sequencing reactions and compared to known genes by computer assisted homology searches. A vector will be constructed which forces expression of the gene; this will be introduced into cultured mouse mammary epithelial cells to determine the effect of expression of the gene on growth and differentiation potential of the cells. Similarities in the characteristics of breast cancer in humans and mice predict that genes identified in this way may be involved in human cancer, and that the insight gained through work in this system will be of value in understanding the human disease.