p53 mutations selected in vivo when mouse mammary epithelial cells form hyperplastic outgrowths are not necessary for establishment of mammary cell lines in vitro.

Abstract

Breast cancer is a consequence of multiple alterations occurring over a long period of time. Genetic changes in early stages of tumorigenesis have not been defined. A recently developed murine system permits the study of mammary preneoplastic cells in vivo and in vitro (F. S. Kittrell et al., Cancer Res., 52: 1924-1932, 1992). To assess the potential role of p53 mutations in early stages of breast cancer, the status of p53 was determined in a series of mouse mammary epithelial cell lines which give rise to preneoplastic outgrowths (hyperplastic alveolar nodules) when transplanted into cleared mammary fat pads of syngeneic mice. Protein stability and conformation were analyzed using immunoprecipitations and immunochemical assays; p53 transcripts were sequenced using a polymerase chain reaction approach. The parental cell lines (FSK lines) showed no evidence of p53 alterations at either the protein or the nucleic acid level, indicating that p53 mutations are not essential for the establishment of mammary epithelial cell lines in vitro. In contrast, cell lines (TM lines) derived from hyperplastic alveolar nodule outgrowths induced by FSK cells expressed only mutant p53 genes. The mutation in one outgrowth cell line (TM-2H) resulted in the loss of p53 protein synthesis, whereas two other outgrowth lines (TM-3, TM-4) overexpressed mutant p53 protein. Mutation of p53 appears to correlate with preneoplastic growth in vivo. Although it is not clear if the mutations occur before or after transplantation of cells in vivo, there appears to be a pronounced growth advantage in the mammary gland for cells expressing mutant p53.