Thymidylate synthase gene amplification in human colon cancer cell lines resistant to 5-fluorouracil.
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 microM. 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.
Copur
S
S
NCI-Navy Medical Oncology Branch, National Cancer Institute, Bethesda, MD 20889-5105, USA.
Aiba
K
K
Drake
J C
JC
Allegra
C J
CJ
Chu
E
E
D002455:Cell Division / Q000187:drug effects; D002460:Cell Line / Q000187:drug effects; D003110:Colonic Neoplasms / Q000201:enzymology; D004273:DNA, Neoplasm / Q000096:biosynthesis; D004351:Drug Resistance; D005472:Fluorouracil / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D005784:Gene Amplification; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis; D013936:Thymidine / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000235:genetics*; D015854:Up-Regulation
D004273:DNA, Neoplasm; D012333:RNA, Messenger; D013940:Thymidylate Synthase; D005472:Fluorouracil; D013936:Thymidine