Pig kidney cells exhibit resistance to puromycin when cultured at high cell densities, growing progressively in otherwise lethal concentrations of drug. Comparison of population growth of cultures inoculated at different densities indicates that survival in puromycin is a function of cell density. Uptake of puromycin from culture media is less at higher cell densities resulting in decreased inhibition of protein synthesis by puromycin. Enhanced survival in puromycin at high population densities is attributed to reduced accumulation of puromycin from the culture media.
In a study of resistance to puromycin in mouse L cells, Lieberman and Ove ('59a,b) calculated that one-step mutations from sensitivity to resistance occur with a frequency of 3.6 X 10-6 per cell per generation. In contrast, Harris ('67) found in studies of puromycin resistance in pig kidney cells that apparent rates of mutation vary considerably with experimental conditions. Mutation rates were greater in samples tested at higher population densities, indicating that resistance in pig kidney cells is influenced by cellular interactions.
Data from numerous studies of heritable drug resistance in cultured mammalian cells support the view that stable resistance arises by discrete random changes rather than by a process of physiological adaptation (Harris, '64). However, these studies of drug resistance were based on the assumption that sensitive and resistant cells could be sharply differentiated in mixed populations by exposure to inhibitor. Although stable, heritable variants can be obtained by selection with puromycin (Harris, '67), occurrence of non-genetic resistance obscures estimates of absolute mutation rates obtained through simple one-step selection procedures. Unless non-genetic resistance is suitably controlled in experimental assays, selection data are invalid as proof for a genetic basis of resistance to metabolic inhibitors.
These investigations are concerned with factors responsible for non-genetic resistance to puromycin in pig kidney J. CELL. PHYSIOL., 79: 13S146.