A series of N-carbamoyloxyurea resistant cell lines with alterations in ribonucleotide reductase: lack of coordination in pyrimidine and purine reductase activity.

Abstract

N-Carbamoyloxyurea is cytotoxic for cells in culture and, like hydroxyurea and guanazole, the drug is an effective inhibitor of mammalian ribonucleotide reductase and thus DNA synthesis. In addition to ribonucleotide reductase, N-carbamoyloxyurea has a second site of action which also appears to be in the pathway of DNA synthesis. A series of drug-resistant cell lines, which contain alterations in ribonucleotide reduction, have been sequentially selected in the presence of increasing concentrations of N-carbamoyloxyurea. CDP and ADP reductase activities in these drug-resistant lines have been investigated and two types of alterations have been identified: elevated levels of enzyme activity with wild-type sensitivity to drug and altered levels of reductase with reduced drug sensitivity, probably owing to structural modification of the enzyme. Furthermore, N-carbamoyloxyurea resistant lines contain another alteration as well, presumably at a second site of drug action. They are also cross-resistant to hydroxyurea and guanazole, and studies on enzyme activity levels support our previous findings with cells selected for resistance to hydroxyurea, which showed changes in CDP reductase activity are not always coordinated with changes in ADP reductase. Although several possibilities exist, these observations are most easily explained by the existence of independent enzyme substrate binding subunits which are regulated by different mechanisms. Moreover, increases in cellular resistance were accompanied by significant increases in CDP but not ADP reductase, suggesting that an ability to maintain an adequate level of CDP reductase activity is especially important to achieve resistance to DNA synthesis inhibitors like N-carbamoyloxyurea, hydroxyurea, and guanazole.