Gene amplification and dual point mutations of mouse IMP dehydrogenase associated with cellular resistance to mycophenolic acid.
Mouse neuroblastoma cells (NB) selected for 10,000-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 (Hodges, 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.
Lightfoot
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Department of Paediatrics, Faculty of Medicine, University of Calgary, Alberta, Canada.
Snyder
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D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D004351:Drug Resistance; D005784:Gene Amplification; D007168:IMP Dehydrogenase / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009173:Mycophenolic Acid / Q000494:pharmacology*; D017354:Point Mutation; D012333:RNA, Messenger / Q000032:analysis; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology
D012333:RNA, Messenger; D007168:IMP Dehydrogenase; D009173:Mycophenolic Acid