Isolation and characterization of Thymitaq (AG337) and 5-fluoro-2- deoxyuridylate-resistant mutants of human thymidylate synthase from ethyl methanesulfonate-exposed human sarcoma HT1080 cells Journal Article
| Authors: | Tong, Y.; Liu-Chen, X.; Ercikan-Abali, E. A.; Capiaux, G. M.; Zhao, S. C.; Banerjee, D.; Bertino, J. R. |
| Article Title: | Isolation and characterization of Thymitaq (AG337) and 5-fluoro-2- deoxyuridylate-resistant mutants of human thymidylate synthase from ethyl methanesulfonate-exposed human sarcoma HT1080 cells |
| Abstract: | Thymidylate synthase plays an essential role in the synthesis of DNA. Recently, several new and specific thymidylate synthase inhibitors that occupy the folate binding site, including Tomudex®, BW1843U89, and Thymitaq, have demonstrated therapeutic activity in patients with advanced cancer. In order to find drug-resistant forms of human thymidylate synthase for gene therapy applications, human sarcoma HT1080 cells were exposed to ethyl methanesulfonate and Thymitaq selection. Thymitaq-resistant clonal derived sublines were established, and analysis indicated that both gene amplification and point mutations contributed to drug resistance. Eight mutant cDNAs that were identified from Thymitaq-resistant sublines were generated by site-directed mutagenesis and transfected into thymidylate synthase-negative cells. Only K47E, D49G, or G52S mutants retain enzyme activity. Moreover, cytotoxicity studies demonstrated that D49G and G52S transfected cells, besides displaying resistance to Thymitaq with IC50 values 40- and 12-fold greater than wild-type enzyme transfected cells, respectively, also lead to fluorodeoxyuridine resistance (26- and 97-fold in IC50 values, respectively) but not to Tomudex or BW1843U89. Characterization of the purified altered enzymes obtained from expression in Escherichia coli is consistent with the cell growth inhibition results. We postulate that the D49G or G52S mutation leads to the structural perturbation of the highly conserved Arg50 loop, decreasing the binding of thymidylate synthase to the inhibitors, Thymitaq and fluorodeoxyuridylate. |
| Keywords: | gene mutation; human cell; sequence analysis; dna synthesis; protein domain; amino acid substitution; alkylating agent; cancer cell culture; enzyme activation; drug resistance; tumor cells, cultured; sarcoma; cloning, molecular; genetic transfection; kinetics; enzyme inhibitors; escherichia coli; recombinant proteins; gene therapy; mutagenesis, site-directed; folic acid; drug cytotoxicity; point mutation; thymidylate synthase inhibitor; thymidylate synthase; structure analysis; folic acid antagonists; quinazolines; site directed mutagenesis; ligand binding; mutagenesis; gene expression regulation, enzymologic; sarcoma cell; raltitrexed; nolatrexed; polymorphism, single-stranded conformational; humans; human; priority journal; article; fluorodeoxyuridylate; 2 [5 [[(1,2 dihydro 3 methyl 1 oxobenzo[f]quinazolin 9 yl)methyl]amino] 1 oxo 2 isoindolinyl]glutaric acid; flucytosine deoxyriboside; mesylic acid ethyl ester; ethyl methanesulfonate |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 273 |
| Issue: | 19 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 1998-05-08 |
| Start Page: | 11611 |
| End Page: | 11618 |
| Language: | English |
| DOI: | 10.1074/jbc.273.19.11611 |
| PUBMED: | 9565579 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Article -- Export Date: 12 December 2016 -- Source: Scopus |
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