TY - JOUR
T1 - Thymidylate synthase gene amplification in human colon cancer cell lines resistant to 5-fluorouracil
AU - Copur, Sitki
AU - Aiba, Keisuke
AU - Drake, James C.
AU - Allegra, Carmen J.
AU - Chu, Edward
N1 - Funding Information:
SITKI COPUR,* KEISUKE AIBA,t JAMES C. DRAKE,* CARMEN J. ALLEGRA* and EDWARD CHU*$ *NCI-Navy Medical Oncology Branch, National Cancer Institute, Bethesda, MD 20889-5105, U.S.A.; and tJapanese Foundation for Cancer Research, Cancer Chemo Center, Tokyo, Japan
PY - 1995/5/17
Y1 - 1995/5/17
N2 - 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.
AB - 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.
KW - 5-FU resistance
KW - gene amplification
KW - thymidylate synthase
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U2 - 10.1016/0006-2952(95)00067-A
DO - 10.1016/0006-2952(95)00067-A
M3 - Article
C2 - 7763285
AN - SCOPUS:0029052988
SN - 0006-2952
VL - 49
SP - 1419
EP - 1426
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 10
ER -