Abstract
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.
Highlights
From the $Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510and the §MemorialSloan-Kettering Cancer Center, New York, New York 10021
Genomic Organization of dihydrofolate reductase (DHFR) Genes in HCT-8 and HCT8R Cells-Fig. 1shows the Southernblot pattern of restricted DNAs from HCT-8 and HCT-8R4 cells hybridized with the 32P-labeledhuman DHFR cDNA probe pHD80
The relative intensities of fragments hybridizing with the probe revealed an amplification of theDHFR gene in HCT-8R cells as compared to HCT-8cells
Summary
Cell Lines and Culture-Human colon adenocarcinoma cells HCT8 [13] were grown as monolayers in RPMI-1640 medium supplemented with 10% horse serum (GIBCO). RNAs were electrophoresed on 1%agarose and 7% formaldehyde gels and transferred to nitrocellulose filters as described by Maniatis et al [15].Prehybridization, hybridization,and washing of nonhybridized label were essentially as mentioned for Southern transfers. Cloning and Sequencing of DHFR cDNAs-In order toobtain DHFR-specific cDNA clones, HCT-8R4 Poly(A+) RNA was enriched for DHFRtranscripts by hybridizationselection[22] to pHD80 immobilized on nitrocellulose filters. This was followed by synthesis and cloning of double-stranded cDNA in the PstI site of pBR322 by dG/dCtailingmethod as described by Maniatis et al [15]. Linear regression of a [L]/[EL] uersus [L] plotresults in a line with aslope equal to 1/ [E] and anx-intercept of -Kd
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