Molecular mechanisms responsible for the drug-induced posttranscriptional modulation of ribonucleotide reductase levels in a hydroxyurea-resistant mouse L cell line

Publisher Website
Google Scholar
Abstract
Ribonucleotide reductase, which catalyzes the formation of doxyribonucleotides from ribonucleoside diphosphate precursors, is the rate-limiting enzyme in DNA synthesis. The enzyme consists of two nonidentical subunits called M1 and M2, both of which are required for activity. Hydroxyurea, a specific inhibitor of DNA synthesis, acts by destroying the unique tyrosyl free radical of protein M2. Previously, we have described a mouse L cell line which exhibited a stable resistance to high concentrations of hydroxyourea. This mutant cell line contains elevated quantities of both proteins M1 and M2 as a result of corresponding increases in the levels of mRNAs for both subunits. Interestingly, both M1 and M2 protein levels were further elevated when mutant cells were cultured in the presence of hydroxyurea, and this elevation was not accompanied by increases in their corresponding mRNAs. These results indicated that hydroxyurea can modulate ribonucleotide reductase expression posttranscriptionally. In this report, we show that the level of both subunits of ribonucleotide reductase responds to hydroxyurea in a drug concentration dependent manner. Furthermore, results from kinetic studies indicate that protein M2 levels rise much more rapidly than protein M1. Pulse-chase experiments indicated that the half-lives of both the M1 and M2 polypeptides are increased by approximately 2-fold when the mutant cells are cultured in the presence of hydroxyurea. We also present evidence indicating that exposure of these cells to hydroxyurea leads to a relatively slow but specific increase in the rate of biosynthesis of both proteins M1 and M2, as assayed by pulse labeling. Therefore, we conclude that both components of ribonucleotide reductase are synthesized at an incr eased rate and turn over at a slower rate when these mutant cells are grown in the presence of hydroxyurea. In addition, experiments were performed to examine the effects of exogenously added iron on the biosynthesis of proteins M1 and M2. Interestingly, the results suggested a role for iron in regulating the level of M2 protein when cells are cultured in hydroxyurea-supplemented medium.