ISOLATION AND CHARACTERIZATION OF THYMITAQ (AG337) AND 5-FLUORO-2-DEOXYURIDYLATE-RESISTANT MUTANTS OF HUMAN THYMIDYLATE SYNTHASE FROM ETHYLMETHANESULFONATE-EXPOSED HUMAN SARCOMA HT1080 CELLS

Thymidylate synthase plays an essential role in the synthesis of DNA, Recently, several new and specific thymidylate synthase inhibitors tha t occupy the folate binding site, including Tomudex(R), BW1843U89, and Thymitaq, have demonstrated therapeutic activity in patients with adv anced cancer. In order to find drug-resistant forms of human thymidyla te synthase for gene therapy applications, human sarcoma HT1080 cells were exposed to ethyl methanesulfonate and Thymitaq selection. Thymita q-resistant clonal derived sublines were established, and analysis ind icated that both gene amplification and point mutations contributed to drug resistance. Eight mutant cDNAs that were identified from Thymita q-resistant sublines were generated by site-directed mutagenesis and t ransfected 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 fluorod eoxyuridine resistance (26- and 97-fold in IC50 values, respectively) but not to Tomudex or BW1843U89. Characterization of the purified alte red enzymes obtained from expression in Escherichia coli is consistent with the cell growth inhibition results, We postulate that the D49G o r G52S mutation leads to the structural perturbation of the highly con served Arg(50) loop, decreasing the binding of thymidylate synthase to the inhibitors, Thymitaq and fluorodeoxyuridylate.