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 10(6) 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.
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