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J Biol Chem, Vol. 273, Issue 19, 11611-11618, May 8, 1998

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

Youzhi Tong, Xinyue Liu-Chen, Emine A. Ercikan-Abali^§, Gina M. Capiaux, Shi-Cheng Zhao^§, Debabrata Banerjee^§, and Joseph R. Bertino^§

From the ^§ Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center and the  Graduate School of Medical Sciences, Cornell University, New York, New York 10021

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.

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