N-Carbamoyloxyurea-resistant Chinese hamster ovary cells with elevated levels of ribonucleotide reductase activity

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Abstract
We describe the isolation and characterization of a Chinese hamster ovary cell line selected for resistance to N‐carbamoyloxyurea. Using the mammalian cell permeabilization assay developed in our laboratory, a detailed analysis of the target enzyme, ribonucleotide reductase (EC 1.17.4.1), was carried out. Both drug‐resistant and parental wild‐type cells required the same optimum conditions for enzyme activity. The Ki values for N‐carbamoyloxyurea inhibition of CDP reduction were 2.0 mM for NCR‐30A cells and 2.3 mM for wild‐type cells, while the Ki value for ADP reduction was 2.3 mM for both cell lines. Although the Ki values remained essentially unchanged, the Vmax values for NCR‐30A cells were 1.01 nmoles dCDP formed/5 × 106 cells/hour and 1.83 nmoles dADP/5 × 106 cells/hour, while those for the wild‐type cells were 0.49 nmoles dCDP produced/5 × 106 cells/hour and 1.00 nmoles dADP/5 × 106 cells/hour. This approximate twofold increase in reductase activity at least partially accounts for a 2.6‐fold increase in D10 value for cellular resistance to N‐carbamoyloxyurea exhibited by NCR‐30A cells. The NCR‐30A cell line was also cross‐resistant to the antitumor agents, hydroxyurea and guanazole. No differences in Ki values for inhibition of CDP and ADP reduction by these two drugs were detected and cellular resistance could be entirely accounted for by the elevation in activity of the reductase in the NCR‐30A cell line. The properties of N‐carbamoyloxyurea‐resistance cells indicate they should be useful for further investigations into the regulation of mammalian enzyme activity.