Alterations in differentiation and pyrimidine pathway enzymes in 5-azacytidine resistant variants of a myoblast line

5-azacytidine at concentrations higher than 5 muM inhibited the differentiation of a rat myoblast line in vitro. It was also somewhat cytotoxic at this level. Variants resistant to the cytotoxic effect of 5-azacytidine were obtained which were simultaneously unable to differentiate into myotubes and exhibited altered morphology. These characteristics were retained by the variants when subcultured in the absence of the drug for over 700 generations. Several of the azacytidine resistant cells were more susceptible than the parental line to the lethal action of 5-bromodeoxyuridine and adenosine, but not that of cytosine arabinoside, ouabain or 8-azaguanine. The variants were capable of transporting uridine, thymidine and 5-azacytidine. The uridine kinase activity was one-half to one-third of than in the parental cells but it was not missing completely in any of the variants. Two independently isolated variants selected for detailed study showed a 2- to 3-fold increase in the activity of orotidylic acid decarboxylase. This enzyme in the variants in contrast to that of the parental cells was completely insensitive to the inhibitory effect of a nucleotide generated from ATP and 5-azacytidine in cell extracts (probably 5-azacytidine monophosphate). These observations point to the possibility the 5-azacytidine resistance arises in myoblasts due to an alteration of the components of two target pathways of this drug, viz., the de novo pyrimidine pathway and an undefined sequence leading to the synthesis of membrane components.