Study of dihydrofolate reductase in methotrexate resistant Chinese hamster lung cells
Abstract
A methotrexate (MTX)-resistant Chinese hamster lung (CHL) cell line, DC-3F8/A55, was studied in order to understand the molecular basis of its drug-resistant phenotype. To identify the basis for the disproportional relationship between drug resistance and dihydrofolate reductase (DHFR) expression levels the DHFR coding region in DC-3F8/A55 cells was examined by cloning the appropriate cDNAs into an expression vector and transforming them into bacteria. Results indicated that the majority of DHFR overexpressed in DC-3F8/A55 cells is a Leu22 {dollar}\to{dollar} Phe mutant form of the Asn95 (20k allele) enzyme encoded by one of two alleles present in DC-3F cells. Characterization of purified DHFR from DC-3F8/A55 cells displayed a 6.7-fold higher dissociation constant ({dollar}K\sb{lcub}d{rcub}{dollar}) for MTX than the wild type 20k enzyme while the catalytic properties of the DC-3F8/A55 enzyme were indistinguishable from the wild type. This explained the lowered sensitivity of the DC-3F8/A55 DHFR to MTX. It was also found that DC-3F8/A55 cells do not accumulate MTX. Subsequently, biochemical study of purified DHFRs from antifolate-resistant CHL sublines illustrated that the DC-3F8/A55 enzyme (20k mutant form) displayed a 4-fold lower {dollar}K\sb{lcub}d{rcub}{dollar} value for MTX than did the DC-3F/A3 enzyme, which is the same mutant (Leu22 {dollar}\to{dollar} Phe) form of the 21k (Asp95) allele. Indeed, the 21k and 20k wild type enzymes, both containing Leu at position 22, differed by 2.7-fold (1.58 {dollar}\pm{dollar} 0.08 p scM and 0.58 {dollar}\pm{dollar} 0.10 p scM respectively) in their {dollar}K\sb{lcub}d{rcub}{dollar}'s for MTX. In order to address whether the amino acid residue at this position affects enzyme function, the amino acid at position 95 was changed from Asp and/or Asn to a polar (Glu or Ser) or a nonpolar (Ala, Gly, or Leu) residue by oligonucleotide-directed in vitro mutagenesis. The recombinant wild type CHL DHFRs (Asp95 and Asn95 enzymes) were indistinguishable from each other in their steady-state parameters. Mutant enzymes displayed Michaelis constant ({dollar}K\sb{lcub}m{rcub}{dollar}) and {dollar}K\sb{lcub}d{rcub}{dollar} values for H{dollar}\sb2{dollar}folate comparable to those of the recombinant wild type DHFRs, whereas the Glu95 and Ala95 enzymes showed increased K{dollar}\sb{lcub}m{rcub}{dollar} and {dollar}K\sb{lcub}d{rcub}{dollar} values for NADPH (between 2.2 and 2.9-fold compared to the Asp95 enzyme). Determination of the inhibition constants ({dollar}K\sb{lcub}i{rcub}{dollar}) for MTX and trimetrexate (TMTX) showed that, in comparison with the recombinant wild type Asp95 enzyme, the Leu95 enzyme has a 2.2-fold higher {dollar}K\sb{lcub}i{rcub}{dollar} value for MTX, while the Ser95 enzyme displayed a 2.4-fold higher {dollar}K\sb{lcub}i{rcub}{dollar} for TMTX. These results suggest that position 95 is involved in NADPH binding and that it also influences the interaction between inhibitor and enzyme. (Abstract shortened by UMI.)Description
University of Maryland, Baltimore. Ph.D. 1993Keyword
Biology, MolecularBiology, Genetics
Chemistry, Biochemistry
Chinese hamster
methotrexate resistant
Cricetulus
Lung
Tetrahydrofolate Dehydrogenase