Studies of mammalian ribonucleotide reductase activity in intact permeabilized cells: A genetic approach

Abstract

We are using a genetic approach to study the complex properties of mammalian ribonucleotide reductase activity. Cytotoxic drugs (hydroxyurea, guanzole and N-carbamoyloxyurea) whose intracellular site of action is ribnonucleotide reductase have been used as selective agents in culture to isolate drug resistant cell lines with specific alterations in reductase activity. In general, cell lines resistant to “hydroxyurea-like” drugs exhibit genetic properties satisfying the majority of the criteria for classification as authentic somatic cell mutants. To study the mammalian enzyme properties, in a situation resembling physiological conditions, we have developed a convenient assay system for ribonucleotide reductase activity in whole cells. The procedure is relatively easy to perform, can accurately determine enzyme activity in as few as 10 6 cells grown conveniently on the surface of a tissue culture plate, and unlike cell-free enzyme preparations, activity is linear at low enzyme concentrations. The procedure can be adapted to investigate activity in a relatively small number of intact cells, it is very useful for studying cultures like human diploid fibroblasts, where large quantities of cells containing high enzyme activity may be difficult to obtain. Using this inact cell assay procedure, we have characterized the reductase activity in CHO cells with respect to pyrimidine (CDP) and purine (ADP) substrates, negative and positive effectors, and drug inhibition with the three antitumor agents employed as selective agents for isolating drug resistant cell lines. Some differences in the mode of action of N-carbamoyloxyurea when compared to hydroxyurea or guanazole were noticed and indicate the potential usefulness of the intact cell assay system for examining the action of specific drugs (e.g., 65) on intracellular enzyme activity. Intracellular ribonucleotide reductase activity was examined in the drug resistant cell lines and three different mutant classes were identified. 1. Cell lines containing an apparent structural alteration to the enzyme which results in reductase activity being less sensitive to drug inhibition. 2. Mutants containing elevated levels of reductase activity with a wild type sensitivity to the drug. 3. Cell lines containing a combination of the first two alterations described above; these mutants apparently contain elevated levels of a ribonucleotide reductase enzyme which has a structural alteration rendering it less sensitive to drug inhibition. Furthermore, some recent preliminary work suggests that additional mutant types with alterations in reductase activity can be isolated. The significance of these mutations in providing a better understanding of the regulation of mammalian ribonucleotide reductase was discussed.