Construction and Characterization of Human Mammary Epithelial Cell Lines Containing Mutations in the p53 or BRCAl Genes

Abstract : To study the effects of inactivating mutations in the p53 and BRCA1 genes early in the breast cancer pathway, we will develop genetically defined human mammary epithelial cell (HMEC) lines by introducing heterozygous and homozygous mutations of each gene using homologous recombination. Additionally, we will construct HMEC lines deficient in p53 protein by expressing the E6 gene of human papillomavirus type 16 (HPV16), which increases the rate of degradation of the p53 protein. The consequences of these genetic changes for cell metabolism will be discovered through controlled in vitro comparisons between genetically altered derivatives and their isogenic parent cells. One level of comparison will involve observations of their growth properties, expression of certain cell lineage markers (e.g. keratins, integrins), morphology and behavior. At another level, we will take a global approach to the discovery of metabolic changes associated with genetic alterations in early tumorigenesis by constructing substraction cDNA libraries and by differential display to reveal changes in mRNA transcription that are associated with loss of activity of each of these genes. Clones showing differential expression will be sequenced in our Sequence Core Facility at the University of Utah to reveal genes potentially critical in growth control, by reference to databanks.