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.)