Modulation of enzyme activity in azaguanine-resistant V79 cells selected by chronic drug exposure

Exposure of V79 cells to azaguanine (7-21 microM for 2-7 weeks) had little effect on growth or plating efficiency but resulted in gradual acquisition of resistance to 8-azaguanine (AZ) and 6-thioguanine (TG) and loss of ability to grow in HAT. The rate of evolution of the resistant phenotype was dependent on the concentration and duration of exposure to AZ. The increase in proportion of resistant cells was paralleled by a rise in phosphatase activity (pH optimum 7.0-7.5) expressed by intact cells and this preceded the fall in HGPRT activity. Elevated phosphatase activity and a resistant phenotype were stably expressed in clones isolated and cultured in the absence of AZ. Hypoxanthine guanine phosphoribosyl transferase (HGPRT) activity in cell extracts of three resistant clones ranged from 18 to 43% of wild-type levels but was unaltered with respect to substrate affinity and electrophoretic mobility. Mg2+-dependent activity dephosphorylated inosine 5'monophosphate (IMP), guanine 5'monophosphate (GMP), adenosine-5-monophosphate (AMP) and p-nitrophenylphosphate (PNPP) and was also elevated with respect to wild-type levels in resistant cell extracts. Purine nucleoside phosphorylase levels were similar in sensitive and resistant cell extracts. Cross-sensitivity studies with other purine analogues suggest that the elevated phosphatase activity does not contribute to the resistant phenotype. No karyotypic changes were observed in the resistant cell lines.