Johnston P G, Drake J C, Trepel J, Allegra C J
NCI-Navy Medical Oncology Branch, Bethesda, Maryland 20892.
Cancer Res. 1992 Aug 15;52(16):4306-12.
Thymidylate synthase (TS) (EC 2.1.1.45) is an important cellular target for the fluoropyrimidine cytotoxic drugs that are widely used in the treatment of solid tumors. Using the TS 106 monoclonal antibody to human TS, we have compared the immunological quantitation of TS by Western immunoblot and immunofluorescent techniques to the conventional biochemical 5-fluorodeoxyuridine monophosphate binding assay in a panel of 5-fluorouracil (5-FU)-sensitive and -resistant human cancer cell lines. Densitometric quantitation of TS 106-labeled Western immunoblot analysis of cell lysates from two 5-FU-resistant colon carcinoma cell lines, NCI H630R1 and NCI H630R10, revealed 12.8- and 16-fold increases in TS, respectively, compared to the parent 5-FU-sensitive NCI H630 colon cell line. By biochemical analysis, the TS level was 15- and 23-fold higher, respectively, in these resistant cell lines. Similarly, immunoblot analysis of cell lysates from two 5-FU-resistant breast cancer cell lines, MCF-Ad5 and MCF-Ad10, detected a 2.3- and 6.3-fold increase in TS, respectively, compared to the parent MCF-7 cell line. By biochemical assay the TS activity was 1.8- and 7.0-fold higher in these resistant breast cell lines. Western immunoblotting analysis revealed a 35-fold range of TS protein concentrations among the 10 cell lines examined, compared to a 38-fold range demonstrated by the biochemical assay. Direct comparison of Western blotting and the biochemical assay revealed a highly significant correlation (r2 = 0.93) between the two assays. Moreover, using the monoclonal antibody TS 106, the Western blotting technique was capable of detecting TS protein levels as low as 0.3 fmol in cellular lysates. Quantitation of TS in intact cells by immunofluorescent TS labeling and flow cytometric analysis was performed using three of the cell lines, NCI H630, NCI H630R10, and MCF-Ad10. This revealed a 26-fold increase in TS in the 5-FU-resistant NCI H630R10 line compared to the parent NCI H630 line and a 3.5-fold increase in TS compared to the 5-FU-resistant MCF-Ad10 breast cell line. The 5-FU-resistant MCF-Ad10 breast cell line, in turn, displayed a 7.7-fold increase in TS, compared to the 5-FU-sensitive NCI H630 cell lines. TS immunofluorescent analysis was capable of measuring TS within individual cells. The development of these immunological assays using an anti-TS monoclonal antibody will facilitate the quantitation of TS in cell lines and tissue samples.
胸苷酸合成酶（TS）（EC 2.1.1.45）是氟嘧啶类细胞毒性药物的重要细胞靶点，这类药物广泛用于实体瘤治疗。我们使用针对人TS的TS 106单克隆抗体，在一组对5-氟尿嘧啶（5-FU）敏感和耐药的人癌细胞系中，将Western免疫印迹和免疫荧光技术对TS的免疫定量与传统的生化5-氟脱氧尿苷单磷酸结合试验进行了比较。对两种5-FU耐药结肠癌细胞系NCI H630R1和NCI H630R10的细胞裂解物进行TS 106标记的Western免疫印迹分析的光密度定量显示，与亲本5-FU敏感的NCI H630结肠癌细胞系相比，TS分别增加了12.8倍和16倍。通过生化分析，这些耐药细胞系中的TS水平分别高15倍和23倍。同样，对两种5-FU耐药乳腺癌细胞系MCF-Ad5和MCF-Ad10的细胞裂解物进行免疫印迹分析，与亲本MCF-7细胞系相比，检测到TS分别增加了2.3倍和6.3倍。通过生化测定，这些耐药乳腺癌细胞系中的TS活性分别高1.8倍和7.0倍。Western免疫印迹分析显示，在所检测的10个细胞系中，TS蛋白浓度范围为35倍，而生化测定显示为38倍。Western印迹法与生化测定的直接比较显示，两种测定之间具有高度显著的相关性（r2 = 0.93）。此外，使用单克隆抗体TS 106，Western印迹技术能够检测细胞裂解物中低至0.3 fmol的TS蛋白水平。使用三种细胞系NCI H630、NCI H630R10和MCF-Ad10，通过免疫荧光TS标记和流式细胞术分析对完整细胞中的TS进行定量。这显示，与亲本NCI H630细胞系相比，5-FU耐药的NCI H630R10细胞系中的TS增加了26倍，与5-FU耐药的MCF-Ad10乳腺癌细胞系相比增加了3.5倍。反过来，5-FU耐药的MCF-Ad10乳腺癌细胞系与5-FU敏感的NCI H630细胞系相比，TS增加了7.7倍。TS免疫荧光分析能够在单个细胞内测量TS。使用抗TS单克隆抗体开发这些免疫测定法将有助于对细胞系和组织样本中的TS进行定量。
