This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Johnston, P. G. Articles by Allegra, C. J. Search for Related Content PubMed PubMed Citation Articles by Johnston, P. G. Articles by Allegra, C. J.

Immunological Quantitation of Thymidylate Synthase Using the Monoclonal Antibody TS 106 in 5-Fluorouracil-sensitive and -resistant Human Cancer Cell Lines

NCI-Navy Medical Oncology Branch [P. G. J., J. C. D., C. J. A.] and Clinical Pharmacology Branch [J. T.], Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20892

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 H630_R1 and NCI H630_R10, 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 H630_R10, and MCF-Ad10. This revealed a 26-fold increase in TS in the 5-FU-resistant NCI H630_R10 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.

¹ To whom requests for reprints should be addressed, at National Cancer Institute, NMOB 8/5101, Bethesda, MD 20892.

Received 2/12/92. Accepted 6/ 4/92.

This article has been cited by other articles:

				T. Okabe, I. Okamoto, S. Tsukioka, J. Uchida, T. Iwasa, T. Yoshida, E. Hatashita, Y. Yamada, T. Satoh, K. Tamura, et al. Synergistic antitumor effect of S-1 and the epidermal growth factor receptor inhibitor gefitinib in non-small cell lung cancer cell lines: role of gefitinib-induced down-regulation of thymidylate synthase Mol. Cancer Ther., March 1, 2008; 7(3): 599 - 606. [Abstract] [Full Text] [PDF]

				G. Y. Locker, S. Hamilton, J. Harris, J. M. Jessup, N. Kemeny, J. S. Macdonald, M. R. Somerfield, D. F. Hayes, and R. C. Bast Jr ASCO 2006 Update of Recommendations for the Use of Tumor Markers in Gastrointestinal Cancer J. Clin. Oncol., November 20, 2006; 24(33): 5313 - 5327. [Abstract] [Full Text] [PDF]

				P. A. Wood, J. Du-Quiton, S. You, and W. J.M. Hrushesky Circadian clock coordinates cancer cell cycle progression, thymidylate synthase, and 5-fluorouracil therapeutic index. Mol. Cancer Ther., August 1, 2006; 5(8): 2023 - 2033. [Abstract] [Full Text] [PDF]

				P. Correale, M. T. Del Vecchio, G. Di Genova, G. G. Savellini, M. La Placa, C. Terrosi, M. Vestri, R. Urso, F. Lemonnier, A. Aquino, et al. 5-Fluorouracil-Based Chemotherapy Enhances the Antitumor Activity of a Thymidylate Synthase-Directed Polyepitopic Peptide Vaccine J Natl Cancer Inst, October 5, 2005; 97(19): 1437 - 1445. [Abstract] [Full Text] [PDF]

				W. L. Allen and P. G. Johnston Role of Genomic Markers in Colorectal Cancer Treatment J. Clin. Oncol., July 10, 2005; 23(20): 4545 - 4552. [Abstract] [Full Text] [PDF]

				S. Popat, R. Wort, and R. S. Houlston Relationship Between Thymidylate Synthase (TS) Genotype and TS Expression: A Tissue Microarray Analysis of Colorectal Cancers International Journal of Surgical Pathology, April 1, 2005; 13(2): 127 - 133. [Abstract] [PDF]

				T. Lecomte, J.-M. Ferraz, F. Zinzindohoue, M.-A. Loriot, D.-A. Tregouet, B. Landi, A. Berger, P.-H. Cugnenc, R. Jian, P. Beaune, et al. Thymidylate Synthase Gene Polymorphism Predicts Toxicity in Colorectal Cancer Patients Receiving 5-Fluorouracil-based Chemotherapy Clin. Cancer Res., September 1, 2004; 10(17): 5880 - 5888. [Abstract] [Full Text] [PDF]

				J. Boyer, E. G. McLean, S. Aroori, P. Wilson, A. McCulla, P. D. Carey, D. B. Longley, and P. G. Johnston Characterization of p53 Wild-Type and Null Isogenic Colorectal Cancer Cell Lines Resistant to 5-Fluorouracil, Oxaliplatin, and Irinotecan Clin. Cancer Res., March 15, 2004; 10(6): 2158 - 2167. [Abstract] [Full Text] [PDF]

				S. Popat, A. Matakidou, and R. S. Houlston Thymidylate Synthase Expression and Prognosis in Colorectal Cancer: A Systematic Review and Meta-Analysis J. Clin. Oncol., February 1, 2004; 22(3): 529 - 536. [Abstract] [Full Text] [PDF]

				K.-J. Sohn, R. Croxford, Z. Yates, M. Lucock, and Y.-I. Kim Effect of the Methylenetetrahydrofolate Reductase C677T Polymorphism on Chemosensitivity of Colon and Breast Cancer Cells to 5-Fluorouracil and Methotrexate J Natl Cancer Inst, January 21, 2004; 96(2): 134 - 144. [Abstract] [Full Text] [PDF]

				J. M. Mariadason, D. Arango, Q. Shi, A. J. Wilson, G. A. Corner, C. Nicholas, M. J. Aranes, M. Lesser, E. L. Schwartz, and L. H. Augenlicht Gene Expression Profiling-Based Prediction of Response of Colon Carcinoma Cells to 5-Fluorouracil and Camptothecin Cancer Res., December 15, 2003; 63(24): 8791 - 8812. [Abstract] [Full Text] [PDF]

				P. J. Maxwell, D. B. Longley, T. Latif, J. Boyer, W. Allen, M. Lynch, U. McDermott, D. P. Harkin, C. J. Allegra, and P. G. Johnston Identification of 5-fluorouracil-inducible Target Genes Using cDNA Microarray Profiling Cancer Res., August 1, 2003; 63(15): 4602 - 4606. [Abstract] [Full Text] [PDF]

				Y. Mizutani, H. Wada, O. Yoshida, M. Fukushima, M. Nonomura, M. Nakao, and T. Miki Significance of Thymidylate Synthase Activity in Renal Cell Carcinoma Clin. Cancer Res., April 1, 2003; 9(4): 1453 - 1460. [Abstract] [Full Text] [PDF]

				P. G. Johnston, A. B. Benson III, P. Catalano, M. S. Rao, P. J. O'Dwyer, and C. J. Allegra Thymidylate Synthase Protein Expression in Primary Colorectal Cancer: Lack of Correlation With Outcome and Response to Fluorouracil in Metastatic Disease Sites J. Clin. Oncol., March 1, 2003; 21(5): 815 - 819. [Abstract] [Full Text] [PDF]

				M. Derenzini, L. Montanaro, A. Chilla, E. Tosti, C. Ceccarelli, F. Dall'Olio, D. Ofner, and D. Trere Evaluation of Thymidylate Synthase Protein Expression by Western Blotting and Immunohistochemistry on Human Colon Carcinoma Xenografts in Nude Mice J. Histochem. Cytochem., December 1, 2002; 50(12): 1633 - 1640. [Abstract] [Full Text] [PDF]

				M Derenzini, L Montanaro, D Trere, A Chilla, P L Tazzari, F Dall'Olio, and D Ofner Thymidylate synthase protein expression and activity are related to the cell proliferation rate in human cancer cell lines Mol. Pathol., October 1, 2002; 55(5): 310 - 314. [Abstract] [Full Text] [PDF]

				D. B. Longley, J. Boyer, W. L. Allen, T. Latif, P. R. Ferguson, P. J. Maxwell, U. McDermott, M. Lynch, D. P. Harkin, and P. G. Johnston The Role of Thymidylate Synthase Induction in Modulating p53-regulated Gene Expression in Response to 5-Fluorouracil and Antifolates Cancer Res., May 1, 2002; 62(9): 2644 - 2649. [Abstract] [Full Text] [PDF]

				D. B. Longley, P. R. Ferguson, J. Boyer, T. Latif, M. Lynch, P. Maxwell, D. P. Harkin, and P. G. Johnston Characterization of a Thymidylate Synthase (TS)-inducible Cell Line: A Model System for Studying Sensitivity to TS- and non-TS-targeted Chemotherapies Clin. Cancer Res., November 1, 2001; 7(11): 3533 - 3539. [Abstract] [Full Text] [PDF]

				J. L. Grem, K. D. Danenberg, K. Behan, A. Parr, L. Young, P. V. Danenberg, D. Nguyen, J. Drake, A. Monks, and C. J. Allegra Thymidine Kinase, Thymidylate Synthase, and Dihydropyrimidine Dehydrogenase Profiles of Cell Lines of the National Cancer Institute's Anticancer Drug Screen Clin. Cancer Res., April 1, 2001; 7(4): 999 - 1009. [Abstract] [Full Text]

				B. Van Triest, B. M. Loftus, H. M. Pinedo, H. H.J. Backus, P. Schoenmakers, F. Telleman, T. Tadema, G. W. Aherne, C. J. Van Groeningen, F. A.N. Zoetmulder, et al. Thymidylate Synthase Expression in Patients with Colorectal Carcinoma Using a Polyclonal Thymidylate Synthase Antibody in Comparison to the TS 106 Monoclonal Antibody J. Histochem. Cytochem., June 1, 2000; 48(6): 755 - 760. [Abstract] [Full Text]

				G. Gribaudo, L. Riera, D. Lembo, M. De Andrea, M. Gariglio, T. L. Rudge, L. F. Johnson, and S. Landolfo Murine Cytomegalovirus Stimulates Cellular Thymidylate Synthase Gene Expression in Quiescent Cells and Requires the Enzyme for Replication J. Virol., June 1, 2000; 74(11): 4979 - 4987. [Abstract] [Full Text]

				S. J. Welsh, J. Titley, L. Brunton, M. Valenti, P. Monaghan, A. L. Jackman, and G. W. Aherne Comparison of Thymidylate Synthase (TS) Protein Up-Regulation after Exposure to TS Inhibitors in Normal and Tumor Cell Lines and Tissues Clin. Cancer Res., June 1, 2000; 6(6): 2538 - 2546. [Abstract] [Full Text]

				A. S. Haqqani, R. T. Cowling, J. A. Maroun, and H. C. Birnboim Characterization of a Polyclonal Antibody to Human Thymidylate Synthase Suitable for the Study of Colorectal Cancer Specimens J. Histochem. Cytochem., December 1, 1999; 47(12): 1563 - 1574. [Abstract] [Full Text]

				B. van Triest, H. M. Pinedo, Y. van Hensbergen, K. Smid, F. Telleman, P. S. Schoenmakers, C. L. van der Wilt, J. A. M. van Laar, P. Noordhuis, G. Jansen, et al. Thymidylate Synthase Level as the Main Predictive Parameter for Sensitivity to 5-Fluorouracil, but not for Folate-based Thymidylate Synthase Inhibitors, in 13 Nonselected Colon Cancer Cell Lines Clin. Cancer Res., March 1, 1999; 5(3): 643 - 654. [Abstract] [Full Text] [PDF]

				A.-S.A. Ismail, C. J. Van Groeningen, A. Hardcastle, Q.-F. Ren, G. W. Aherne, F. Geoffroy, C. J. Allegra, and J. L. Grem. Modulation of Fluorouracil Cytotoxicity by Interferon-alpha and -gamma Mol. Pharmacol., February 1, 1998; 53(2): 252 - 261. [Abstract] [Full Text]

				E. Chu, J. L. Grem, P. G. Johnston, and C. J. Allegra New Concepts for the Development and Use of Antifolates Oncologist, August 1, 1996; 1(4): 255 - 259. [Abstract] [Full Text] [PDF]

				E Chu, J. Grem, P. Johnston, and C. Allegra New concepts for the development and use of antifolates Stem Cells, January 1, 1996; 14(1): 41 - 46. [Abstract]

				E. L. Schwartz, N. Baptiste, S. Wadler, and D. Makower Thymidine Phosphorylase Mediates the Sensitivity of Human Colon Carcinoma Cells to 5-Fluorouracil J. Biol. Chem., August 11, 1995; 270(32): 19073 - 19077. [Abstract] [Full Text] [PDF]