How variable is a spontaneous mutation rate in cultured mammalian cells?

The Luria-Delbrück fluctuation analysis provides a method to estimate mutation rates and is commonly applied in somatic cell genetics and in cancer biology. We developed an assay for a Luria-Delbrück fluctuation analysis using the mouse lymphoma cell line, GRSL13. As these cells grow in suspension, one can handle hundreds of parallel cultures using multiwell dishes and dispensers. This assay thereby allows not only an accurate determination of the mutation rate per cell generation but also makes it possible to determine at which time after seeding mutations take place. Using approx. 8000 parallel cultures it has been possible to test whether the mutation rate is constant during the assay. It has been found that the spontaneous mutation rate of GRSL13 cells decreases in the course of a fluctuation test from 2 x 10(-6) to about 2 x 10(-7)/cell/generation. It was shown that this increased replication fidelity may partly be caused by cell density: maintenance of cells at high cell density resulted in a spontaneous mutation rate of 0.7 +/- 4.0 x 10(-7) compared to 4.0 +/- 3.1 x 10(-7) for the standard protocol. In contrast, growing the cells at extremely low cell density resulted in an enhanced mutation rate of 7.7 +/- 1.3 x 10(-7). Thus altogether the mutation rate can vary from 2 x 10(-6) to 0.7 x 10(-7) (approx. 30-fold). These results show that the spontaneous mutation rate is not constant, but highly dependent on experimental conditions. As incomplete expression and metabolic cooperation cannot explain the findings, the data suggest that the fidelity of DNA replication is not fixed but open to variation. Hence, determination of replication infidelity in cultured cells needs rigorous standardization or/and application of controlled variation in culture conditions.