Thymidylate synthase overproduction and gene amplification in fluorodeoxyuridine-resistant human cells.
Cytotoxicity to 5-fluoro-2'-deoxyuridine (FdUrd) derives from its conversion to 5-fluorodeoxyuridine-5'-monophosphate, which binds to and inhibits thymidylate synthase (TS) in the presence of the cofactor, 5,10-methylenetetrahydrofolate. We have selected FdUrd-resistant variants of the human cell line HEp-2 following adaptation to stepwise increases in drug concentration. In the initial selection, maximal drug resistance was associated with a 26-fold increase in the cellular level of TS. Greater TS overproduction (80-fold) was obtained by selection for FdUrd resistance in the presence of 10 microM folinic acid and 100 microM deoxyinosine. The latter agents were included to expand the folate pool to ensure adequate levels of cofactor during the selection process. Using cDNA plasmid pMTS-4, which is complementary to mouse TS mRNA, we show that TS overproduction in the HEp-2 variants is accompanied by a 100-fold increase in TS mRNA and a 100-fold amplification of the TS structural gene. Thus, TS overproduction and gene amplification is a mechanism of resistance to FdUrd in human cells.
Berger
S H
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Jenh
C H
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Johnson
L F
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Berger
F G
FG
D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D004351:Drug Resistance; D004586:Electrophoresis; D005467:Floxuridine / Q000494:pharmacology*; D005784:Gene Amplification; D006801:Humans; D007822:Laryngeal Neoplasms / Q000201:enzymology; D008970:Molecular Weight; D012333:RNA, Messenger / Q000096:biosynthesis; D013940:Thymidylate Synthase / Q000096:biosynthesis* / Q000235:genetics
D012333:RNA, Messenger; D005467:Floxuridine; D004247:DNA; D013940:Thymidylate Synthase