Characterization of a mouse cell line selected for hydroxyurea resistance by a stepwise procedure: Drug-dependent overproduction of ribonucleotide reductase activity

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Abstract
Hydroxyurea was used as a selective agent in culture, to isolate by a stepwise procedure, a unique mouse L cell line called LHF which exhibited a stable resistance to high concentrations of drug (5 mM). LHF cells contained an elevation in ribonucleotide reductase activity which depended upon whether cells were previously cultured in the presence or absence of hydroxyurea. M1 immunoprecipitation and M2 titration experiments indicated that both ribonucleotide reductase subunits were elevated in drug-resistant cells. Interestingly, a very large drug-dependent change in the M2 activity (about a 100-fold) was observed. Studies on enzyme activity with cycloheximide and actinomycin D indicated that the hydroxyurea-dependent increase in activity required de novo protein synthesis and transcriptional activity. These results are different from other ribonucleotide reductase overproducing cell lines previously described, and indicate that hydroxyurea modulates enzyme activity by an interesting mechanism.