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

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 X 10(6) cells/hour and 1.83 nmoles dADP/5 X 10(6) cells/hour, while those for the wild-type cells were 0.49 nmoles dCDP produced/5 X 10(6) cells/hour and 1.00 nmoles dADP/5 X 10(6) cells/hour. This approximate twofold increase in reductase activity as 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.

Ribonucleotide reductase R2 component is a novel malignancy determinant that cooperates with activated oncogenes to determine transformation and malignant potential

“…Infection of cells with SH/mR2 or control virus LXSH in the presence of polybrene was carried out (24), and stable infectants (Ն1 ϫ 10 4 clones) were obtained with hygromycin selection and pooled (19,24). Determinations of cell division times, plating efficiencies, and relative sensitivities to hydroxyurea cytotoxicity by estimating relative colony forming efficiencies, were carried out as described (1,20,25). Growth in soft agar was estimated in 10-cm tissue culture plates containing 15 ml of base agar (0.5% Bacto agar in ␣-MEM plus 10% calf serum) and 10 ml of growth agar (0.33% agar in ␣-MEM containing 10% calf serum).…”

“…*To whom reprint requests should be addressed. subconfluent cultures, and colonies were scored 10-15 days later (20,21,25). Transformation was also analyzed by determining focus formation after cells were infected with SH/mR2 or LXSH or transfected with T-24 Ras or V12 Rac-1 plasmids by calcium phosphate precipitation (22).…”

An Iron-Dependent 5'-Nucleotide Phosphoribohydrolase Activity in the Venom of Crotalus atrox and Its Effect upon the Determination of Mammalian Ribonucleotide Reductase

“…When CDP or ADP reductase activity is examined in permeabilized cells or cell-free extracts, a control experiment is performed consisting of the standard assay plus heatinactivated enzyme [3,10]. On completion of the assay, nucleotides are converted to nu cleosides by the action of snake venom en zymes, and labelled compounds are sepa rated on Dowex-1-borate columns as de scribed in Materials and Methods.…”

“…Ribonucleotide Reductase CHO cells were made permeable to nucleotides using the Tween 80 method as previously reported [3,10]. Exponentially growing cells were plated at a den sity of 5 X 106/150 mm plastic tissue culture plate with a-MEM + 10% FCS or 8% HS + 2% FCS and incubated at 37 °C.…”

“…Chinese hamster ovary (CHO) cells were cultured at 37 °C on the surface of glass bottles (Brockway Glass Co.) or on plastic tissue culture plates (Lux Scientific Ltd.) in a-minimal essential medium (a-MEM; Flow Laboratories, Ltd.) supplemented with antibiotics and 10% fetal calf serum (FCS; Gibco Ltd.) as previously described [3,10]. In some experiments 8% horse serum (HS; Gibco Ltd.) plus 2% FCS was substituted for FCS.…”