Microenvironmental influences on mutagenesis in mammary epithelial cells

Tumor progression may be viewed as an evolutionary process at the cellular level. Because blood supply to solid tumors is inadequate, the cancer cells face a hostile microenvironment characterized by hypoxia or anoxia, acidic extracellular pH and nutrient deficiencies. It has been proposed that these factors result in increased levels of spontaneous mutagenesis and thereby contribute to tumor progression. We have examined spontaneous mutagenesis in vitro and in vivo, using previously characterized cell lines (mammary epithelial cells [ME] and mammary fibroblast cells [MFib]) from the mammary gland of the BigBluetrade mark rat, carrying a transgene construct suitable for the detection of mutations. Cells were exposed in vitro to control conditions, low pH, or to glucose deprivation, under normoxic or hypoxic culture conditions, and were also grown as xenografted tumors in immune-deficient mice. We examined cell survival and mutant frequency/spectrum at the cII locus. Significant increases in mutant frequency were observed in ME cells exposed to hypoxia alone or in combination with no glucose; the latter condition also resulted in reduced clonogenic survival. Cells grown as xenografts and then recovered and expanded in culture also had elevated frequencies of spontaneous mutations. We observed a shift in the spontaneous mutation spectrum between the ME cells and the MET cells (cultured in vitro or isolated from mouse xenograft tumors). These results support the concept that the tumor microenvironment contributes to tumor progression by enhancing spontaneous mutagenesis, that different cell types from the same organ can respond differently to these stresses and that differences in microenvironment may influence the types of mutations that arise.