GENE AMPLIFICATION AND DUAL POINT MUTATIONS OF MOUSE IMP DEHYDROGENASE ASSOCIATED WITH CELLULAR-RESISTANCE TO MYCOPHENOLIC-ACID

Mouse neuroblastoma cells (NB) selected for 10000-fold increased resistance to mycophenolic acid (NB-Myco) showed a 200-500-fold increase in IMP dehydrogenase protein, and the enzyme (IMP:NAD(+) oxidoreductase, EC 1.1.1.205) also exhibited a 2400-fold increased ki for mycophenolic acid and reduced catalytic efficiency (Hedges, S.D., Fung, E., McKay, D.J., Renaux, B.S., and Snyder, F.F. (1989) J. Biol. Chem. 264, 18137-18141). The molecular basis of these changes is the subject of the present study. The nucleotide sequence of IMP dehydrogenase cDNA from NB-Myco cells revealed four nucleotide changes. One of these changes did not result in a codon change, and a second one corresponding to methionine-483 was present in the parental NB mouse line. The remaining two nucleotide substitutions and deduced residue changes are: the C to T transition at base 998 relative to initiation of translation, altering threonine-333 to isoleucine; and the C to A transversion at base 1052, altering serine-351 to tyrosine. Evidence was also obtained for IMP dehydrogenase having undergone gene amplification. IMP dehydrogenase mRNA levels were 500-fold increased in NB-Myco cells as compared to parental NB cells. DNA dot blot analysis showed a 25-fold increase in IMP dehydrogenase gene copy number and restriction enzyme analysis revealed similar gene structure for NB and NB-myco cells.