THYMIDYLATE SYNTHASE GENE AMPLIFICATION IN HUMAN COLON-CANCER CELL-LINES RESISTANT TO 5-FLUOROURACIL
THYMIDYLATE SYNTHASE GENE AMPLIFICATION IN HUMAN COLON-CANCER CELL-LINES RESISTANT TO 5-FLUOROURACIL
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DOI：
10.1016/0006-2952(95)00067-a
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发表时间：
1995-05-17
影响因子：
5.8
通讯作者：
CHU, E
中科院分区：
文献类型：
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作者：
COPUR, S;AIBA, K;CHU, E
通过将细胞持续暴露于 5-FU，建立了一系列 5-氟尿嘧啶 (5-FU) 耐药性人结肠 H630 癌细胞系。在亲代结肠系（H630）中抑制细胞增殖50%（IC50）所需的5-FU浓度为5.5μM。耐药H630-R1、H630-R10和H630-R细胞系的5-FU IC50值比亲代H630细胞系高11、29和27倍。使用放射性酶 5-氟-2'-脱氧尿苷-5'-单磷酸 (FdUMP) 结合和催化测定来测量胸苷酸合酶 (TS) 酶活性，耐药性 H630-R1（13 倍和 23 倍）、H630-R10（37 倍和 40 倍）和 H630-R（24 倍和 40 倍）中的 TS 活性显着增加。 34 倍）线，分别用于结合和催化测定，与亲本 H630 系相比。通过蛋白质免疫印迹分析确定，耐药 H630-R1（23 倍）、H630-R10（33 倍）和 H630-R（26 倍）细胞中 TS 蛋白水平显着增加。 Northern分析显示，相对于亲代H630细胞，H630-R1（18倍）、H630-R10（39倍）和H630-R（36倍）细胞中的TS mRNA水平升高。尽管 Southern 分析未发现 TS 基因发生重大重排，但 5-FU 抗性细胞中 TS 基因显着扩增，这通过 DNA 狭缝印迹分析得到证实。这些研究表明，人类结肠癌细胞持续暴露于 5-FU 会导致 TS 基因扩增和 TS 蛋白过度表达，从而导致氟嘧啶耐药性的发展。
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 mu M. 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.