Overproduction of the free radical of ribonucleotide reductase in hydroxyurea-resistant mouse fibroblast 3T6 cells

Abstract

Hydroxyurea inhibits the activity of ribonucleotide reductase (ribonucleoside-diphosphate reductase; 2'-deoxy-ribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) in bacteria and mammalian cells. The reductase from Escherichia coli consists of two nonidentical subunits (B1 and B2) and hydroxyurea acts by specifically destroying a tyrosine free radical of B2 required for enzyme activity. The mammalian enzyme also consists of two nonidentical subunits (M1 and M2), only one of which (M1) has been obtained in pure form. By continuous culture at stepwise increasing drug concentrations, we have now obtained a 3T6 mouse fibroblast cell line with a 100-fold increased resistance to hydroxyurea. Extracts from resistant cells showed a 3- to 15-fold increase in reductase activity. The amount of M1 protein was not increased. The amount of M2 protein could not be measured directly, but the M2 activity in extracts from resistant cells (but not normal cells) showed an EPR spectrum very similar to that of the tyrosine radical of the bacterial B2 subunit. We propose that resistance to hydroxyurea is caused either by overproduction of the complete M2 subunit or by increased generation of the tyrosine radical within the M2 protein. It seems that either alternative mirrors a possible normal regulatory mechanism for the activity of the reductase.