Selection of aphidicolin-resistant CHO cells with altered levels of ribonucleotide reductase.

Chinese hamster ovary cells were initially selected for resistance to aphidicolin at 0.3 microgram/ml. Serial cultivation with aphidicolin at concentrations up to 5.0 micrograms/ml yielded a series of mutants with increasing resistance. The most resistant mutant isolated was 44 times more resistant to aphidicolin than the parental CHO. The alpha-polymerases, assayed in the cytoplasmic extracts of the mutants, did not increase in specific activity or differ from the parental CHO in their sensitivity to aphidicolin. When cultured in the presence of deoxythymidine, deoxyadenosine, and 1-beta-D-arabinofuranosyl cytosine (araC) the mutants showed considerably more resistance to these inhibitors than did the parental CHO. The intracellular pools of all four deoxynucleoside triphosphates (dNTPs) in the mutants increased with increasing resistance to aphidicolin. The elevated dNTP pools in the mutant most resistant to aphidicolin appear to be the result of a 4- to 8-fold increase in the level of ribonucleotide reductase (2'-deoxyribonucleoside diphosphate:oxidized thioredoxin 2'-oxidoreductase, EC 1.17.4.1).