Amplification of a polymorphic dihydrofolate reductase gene expressing an enzyme with decreased binding to methotrexate in a human colon carcinoma cell line, HCT-8R4, resistant to this drug.
A methotrexate (MTX)-resistant human colon carcinoma cell line was obtained by growing HCT-8 cells in stepwise increasing concentrations of the drug. The resistant subline (HCT-8R4) was able to grow in the presence of 1 x 10(-4) M MTX and was found to have a 25-fold increase in the level of the target enzyme dihydrofolate reductase (DHFR), with a corresponding increase in DHFR gene copies as well as DHFR transcripts. Southern blot analysis of DNA from HCT-8R4 cells revealed the amplification of an altered gene. The amplified DHFR gene lacks an EcoRI restriction enzyme site in the coding region, normally present in other human cell lines. Sequence analysis of cDNA synthesized from transcripts in the MTX-resistant cell line revealed a base transition T----C at nucleotide position 91 resulting in a substitution of serine for phenylalanine. The dissociation constant for MTX binding to the HCT-8R4 enzyme was 1.25 nM, an 8-fold increase from the Kd 150 pM of purified wild type human DHFR. This decrease in binding of MTX to the HCT-8R4 DHFR is consistent with the predicted involvement of phenylalanine in the DHFR active site in hydrophobic interactions with MTX. This mutation plus the 25-fold increase in DHFR activity explains the high level of resistance of this subline to MTX.
Srimatkandada
S
S
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
Schweitzer
B I
BI
Moroson
B A
BA
Dube
S
S
Bertino
J R
JR
D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015139:Blotting, Southern; D003110:Colonic Neoplasms / Q000378:metabolism*; D015342:DNA Probes; D015246:Deoxyribonuclease EcoRI; D004351:Drug Resistance; D005784:Gene Amplification; D006801:Humans; D008727:Methotrexate / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D011110:Polymorphism, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D013762:Tetrahydrofolate Dehydrogenase; D015246:Deoxyribonuclease EcoRI; D008727:Methotrexate