Thymidylate synthase gene amplification in human colon cancer cell lines resistant to 5-fluorouracil

Sitki Copur, Keisuke Aiba, James C. Drake, Carmen J. Allegra, Edward Chu

Research output: Contribution to journalArticlepeer-review

161 Scopus citations

Abstract

A series of 5-fluorouracil (5-FU)-resistant human colon H630 cancer cell lines were established by continuous exposure of cells to 5-FU. The concentration of 5-FU required to inhibit cell proliferation by 50% (ic50) in the parent colon line (H630) was 5.5 μM. The 5-FU ic50 values for the resistant H630-R1, H630-R10, and H630-R cell lines were 11-, 29-, and 27-fold higher than that for the parent H630 cell line. Using both the radioenzymatic 5-fluoro-2′-deoxyuridine-5′-monophosphate (FdUMP) binding and catalytic assays for measurement of thymidylate synthase (TS) enzyme activity, there was significantly increased TS activity in resistant H630-R1 (13- and 23-fold), H630-R10 (37- and 40-fold), and H630-R (24- and 34-fold) lines, for binding and catalytic assays, respectively, compared with the parent H630 line. The level of TS protein, as determined by western immunoblot analysis, was increased markedly in resistant H630-R1 (23-fold), H630-R10 (33-fold), and H630-R (26-fold) cells. Northern analysis revealed elevations in TS mRNA levels in H630-R1 (18-fold), H630-R10 (39-fold), and H630-R (36-fold) cells relative to parent H630 cells. Although no major rearrangements of the TS gene were noted by Southern analysis, there was significant amplification of the TS gene in 5-FU-resistant cells, which was confirmed by DNA slot blot analysis. These studies demonstrate that continuous exposure of human colon cancer cells to 5-FU leads to TS gene amplification and overexpression of TS protein with resultant development of fluoropyrimidine resistance.

Original languageEnglish (US)
Pages (from-to)1419-1426
Number of pages8
JournalBiochemical Pharmacology
Volume49
Issue number10
DOIs
StatePublished - May 17 1995
Externally publishedYes

Keywords

  • 5-FU resistance
  • gene amplification
  • thymidylate synthase

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

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