Genetic complementation and resistance to 5-fluoro-2'-deoxyuridine in thymidine auxotrophs expressing a highly defective mutant of human thymidylate synthase.
A mutant human thymidylate synthase (TS) has been created in which a glutamine residue at position 214 has been replaced by glutamate. Glutamine at position 214 is postulated to be involved in maintaining the enzyme in a conformation that facilitates the binding of the substrate dUMP. Although the kcat/Km of the mutant protein for the substrate, dUMP, is 10(3) lower than that of wild-type TS, the mutant TS confers thymidine prototrophy on a TS-deficient bacterial strain when expressed at high levels. In the present investigation, a TS-deficient Chinese hamster lung cell line was transfected with DNA encoding the defective protein. Thymidine prototrophs were isolated that expressed the defective protein at levels that were physiologically relevant. The activities of the enzymes expressed endogenously in representative prototrophs were consistent with the activities observed for the purified proteins. At similar levels of TS expression, thymidine prototrophs expressing Glu214 TS were 8-fold more resistant to 5-fluoro-2'-deoxyuridine (FdUrd) cytotoxicity than are prototrophs expressing Gln214 TS. FdUrd is a prodrug of the tight-binding TS inhibitor, 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP). The resistance to FdUrd was associated with a significant decrease in the binding of FdUMP to the purified mutant enzyme. The data are consistent with the interpretation that TSs that are highly defective are capable of sufficient dTMP production for cell survival and optimal growth, yet may confer resistance to TS-directed inhibitors.
Zapf
J W
JW
Department of Basic Pharmaceutical Sciences, University of South Carolina, Columbia 29208, USA.
Zhao
P S
PS
Steadman
D J
DJ
Berger
S H
SH
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance; D005467:Floxuridine / Q000494:pharmacology*; D005468:Fluorodeoxyuridylate / Q000378:metabolism / Q000494:pharmacology; D005816:Genetic Complementation Test; D006801:Humans; D017354:Point Mutation; D013936:Thymidine / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D000964:Antimetabolites, Antineoplastic; D005467:Floxuridine; D005468:Fluorodeoxyuridylate; D013940:Thymidylate Synthase; D013936:Thymidine