Mutations resistant to bromodeoxyuridine in mouse lymphoma cells selected by repeated exposure to EMS. Characteristics of phenotypic instability and reversion to HAT resistance by 5-azacytidine

Abstract

Mutations induced by repeated EMS treatments were investigated by using mouse L5178Y cells. The frequency of TGr mutations increased linearly with the number of EMS treatments whereas the yield of BrdUrdr mutations showed a curvilinear dose-response curve. The BrdUrdr frequency was roughly proportional to the square of the TGr frequency and the results were compatible with the hypothesis that BrdUrdr colonies isolated after 6 EMS treatments, however, were unstable. When BrdUrdr colonies that had arisen in BrdUrd medium after 2 weeks' incubation were isolated in normal medium, the descendant cells showed a nearly normal level of thymidine incorporation and low plating efficiencies of about 1% in BrdUrd medium. In contrast, after isolation of the same colonies in BrdUrd medium, a low level of thymidine incorporation and high plating efficiencies in BrdUrd medium were observed in the descendant cells. Reverse selection from BrdUrdr to HATr was accomplished with frequencies of 10(-6)-10(-3) for the descendants grown in BrdUrd medium, and AzaCyd treatment drastically increased the reversion frequency to nearly 10(-1). Further re-revertants from HATr to BrdUrdr were also found with frequencies of 10(-3)-10(-2) without treatment. These results indicate that the initial BrdUrdr cells did not result from inactivation of the thymidine-kinase gene but that the mode of gene expression was altered in some way.