Hydroxyurea-resistant mouse L cells with elevated levels of drug-resistant ribonucleotide reductase activity.
We describe the isolation and partial characterization of a mouse L-cell line which is resistant to normally highly cytotoxic concentrations of hydroxyurea. A detailed analysis of the target enzyme ribonucleotide reductase in both wild-type and hydroxyurea-resistant enzyme preparations suggests that the drug-resistant cells form a ribonucleotide reductase enzyme which contains a structural alteration, rendering it less sensitive to inhibition by hydroxyurea. K1 values for hydroxyurea inhibition of ribonucleotide reduction in enzyme preparations from hydroxyurea-resistant cells were significantly higher than corresponding values from preparations from wild-type cells. The Km for CDP reduction in enzyme preparations of drug-resistant cells was approximately threefold higher than the corresponding parental wild-type value. In addition, in vivo enzyme assays detected a major difference between the temperature profiles of ribonucleotide reduction in nucleotide-permeable drug-resistant and wild-type cells. When levels of ribonucleotide reductase activity were measured in vivo, it was found that the drug-resistant cells contained approximately 3 times the wild-type level of CDP reductase activity and twice wild-type level of GDP reductase activity. This combination of enhanced enzyme levels plus an altered sensitivity to drug inhibition can easily account for the drug-resistance phenotype. The properties of these hydroxyurea-resistant cells indicate that they will be useful for genetic and biochemical studies.
D000818:Animals; D002460:Cell Line; D002999:Clone Cells; D004351:Drug Resistance; D006918:Hydroxyurea / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D007739:L Cells / Q000201:enzymology*; D051379:Mice; D012262:Ribonucleoside Diphosphate Reductase / Q000235:genetics* / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000235:genetics*
D012264:Ribonucleotide Reductases; D012262:Ribonucleoside Diphosphate Reductase; D006918:Hydroxyurea