Altered Ribonucleotide Reductase Activity in Drug-Resistant Mammalian Cells Detected by an Assay Procedure for Intact Cells

Abstract

Hydroxyurea, an antitumor drug which specifically inhibits ribonucleotide reductase, has been used as a selective agent to obtain single-step and double-step drug-resistant cell lines altered in reductase activity. Using an assay procedure developed for analyzing enzyme activity in intact cells a variety of novel changes in the properties of the enzyme activity was detected in the drug-resistant lines. Differences in cellular levels of enzyme activity, changes involving pyrimidine and purine substrate Km values, and altered enzyme sensitivity to hydroxyurea were found using the in vivo assay procedure. These changes could account for the drug-resistant phenotypes. The assay technique which is described in this report is easy to perform, can accurately determine enzyme activity in as few as 5 X 106 cells grown conveniently on the surface of a single tissue culture plate, and is a valuable quick screening method for identifying cells altered in different aspects of ribonucleotide reductase activity.

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

“…The approximate twofold increase in enzyme activity is sufficient to entirely account for the 1.5-to 1.6-fold increase in cellular resistance to hydroxyurea and guanazole, and at least partially explains t h e 2.6-fold increase in resistance to Ncarbamoyloxyurea. These cells resemble the hydroxyurea-resistant lines which contain elevated levels of drug-sensitive ribonucleotide reductase (Lewis and Wright, 1979;Koropatnick and Wright, 1980). Although the exact mechanism(s) leading to enhanced enzyme activity in these cells is not yet known, they promise to provide insights into the way in which cells regulate enzyme activity.…”

“…However, the V,,, values for NCH-30A and parental cells were significantly different; the drugresistant cells exhibited a V,,, Figure 1 shows that NCH-30A cells were cross-resistant to hydroxyurea and guanazole. Hydroxyurea-resistant cell lines, which contain an altered reductase activity, as judged by a decreased sensitivity to the inhibitory action of hydroxyurea and guanazole Wright, 1974, 1978b;Kuzik and Wright, 1980;Koropatnick and Wright, 1980) have been described. Therefore a series of kinetic studies were performed with the reductase activity in NC8-30A and WT cells in the presence of the antitumor agents to determine whether the drug-resistant cells contained an enzyme activity less sensitive to the three drugs.…”

“…reductase assay CHO cells were made permeable to nucleotides using the Tween 80 method developed in our laboratory (Lewis et al, 1978;Kuzik and Wright, 1980;Koropatnick and Wright, 1980;Dick and Wright, 1980). Exponentially growing cells were plated at a density of 2 x 10" cells/100 mm or 5 x 10W50 mm plastic tissue culture plate containing (r-MEM + 10% FCS and incubated at 37" C. After 40 hours for wildtype (WT) CHO cells and 32 to 36 hours for NCR-30A cells, the cells were removed from the surface of plates with trypsin solution, centri-fuged, washed once with a-MEM + 10% FCS, and counted by means of a Coulter particle counter (Coulter Electronics Co).…”

“…The drug-resistant phenotypes have been attributed to the production of structurally altered ribonucleotide reductase with reduced sensitivity to inhibition by hydroxyurea Wright, 1974, 1978a,b), the formation of elevated levels of drug-sensitive enzyme (Lewis and Wright, 1979), or the production of increased levels of ribonucleotide reductase with a structural alteration rendering it less sensitive to hydroxyurea inhibition (Kuzik and Wright, 1980;Koropatnick and Wright, 1980). It has been suggested that hydroxyurea owes its cytotoxicity to a n intracellular conversion to the antitumor agent N-carbamoyloxyurea (Jacobs and Rosenkranz, 1970;Cameron and Jeeter, 1973).…”

Ribonucleotide reduction in intact human diploid fibroblasts

“…We have chosen to study the enzyme activity in cultured normal human diploid fibroblasts. Since these cells have a limited lifetime in vitro, and it is difficult to obtain sflicient quantities of extract of high enzyme activity to attempt enzyme purification, we have modified a method developed in our labr oratory (Lewis et al, 1978;Kuzik and Wright, 1980;Koropatnick and Wright, 1980) for investigating ribonucleotide reductase activity in intact mammalian cells, and have used the method to examine some general properties of human ribonucleotide reductase in normal diploid fibroblasts.…”