Elevated expression of M1 and M2 components and drug-induced posttranscriptional modulation of ribonucleotide reductase in a hydroxyurea-resistant mouse cell line

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Abstract
Ribonucleotide reductase, a rate-limiting enzyme in the synthesis of DNA, consists of two nonidentical subunits, proteins M1 and M2. Hydroxyurea, a specific inhibitor of DNA synthesis, acts by destroying the unique tyrosyl free radical of protein M2. In the past, we have described a mouse L cell line which exhibited a stable resistance to high concentrations of hydroxyurea [McClarty, G. A., Chan, A., and Wright, J. A. (1986) Somat. Cell Mol. Genet. 12, 121-131]. When this line was grown in the absence of hydroxyurea, the cells contained a modest but stable elevation in ribonucleotide reductase activity. However, the activity was further increased on the addition of drug to the culture medium. This was accompanied by an increase in protein M2 activity as shown by activity titration experiments. Likewise, removal of hydroxyurea resulted in a decrease in M2 activity. In the present study, we make use of recently isolated cDNAs and monoclonal antibodies for both the M1 and M2 proteins to further our understanding of the mechanism of hydroxyurea resistance at the molecular level in a subclone of this cell line. Our results indicated that protein M1 levels were elevated 2-3-fold and protein M2 levels were increased about 50-fold in the mutant cells when they were grown in the absence of hydroxyurea, compared to wild-type cells. These protein increases were accompanied by corresponding elevations in the levels of mRNAs for both subunits and increased rates of transcription of both genes. There was a 6-fold amplification in the gene copy number for protein M2. However, most interesting was the observation that 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 the corresponding mRNAs. These results indicate that hydroxyurea can modulate ribonucleotide reductase expression posttranscriptionally.