Genetic complementation and resistance to 5-fluoro-2 '-deoxyuridine in thymidine auxotrophs expressing a highly defective mutant of human thymidylatesynthase

Citation
Jw. Zapf et al., Genetic complementation and resistance to 5-fluoro-2 '-deoxyuridine in thymidine auxotrophs expressing a highly defective mutant of human thymidylatesynthase, BIOCH PHARM, 58(6), 1999, pp. 973-981
Citations number
38
Categorie Soggetti
Pharmacology & Toxicology
Journal title
BIOCHEMICAL PHARMACOLOGY
ISSN journal
00062952 → ACNP
Volume
58
Issue
6
Year of publication
1999
Pages
973 - 981
Database
ISI
SICI code
0006-2952(19990915)58:6<973:GCART5>2.0.ZU;2-T
Abstract
A mutant human thymidylate synthase (TS) has been created in which a glutam ine residue at position 214 has been replaced by glutamate. Glutamine at po sition 214 is postulated to be involved in maintaining the enzyme in a conf ormation that facilitates the binding of the substrate dUMP. Although the k (cat)/K-m of the mutant protein for the substrate, dUMP, is 10(3) lower tha n 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 i nvestigation, a TS-deficient Chinese hamster lung cell line was transfected with DNA encoding the defective protein. Thymidine prototrophs were isolat ed that expressed the defective protein at levels that were physiologically relevant. The activities of the enzymes expressed endogenously in represen tative prototrophs were consistent with the activities observed for the pur ified proteins. At similar levels of TS expression, thymidine prototrophs e xpressing 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'-mono phosphate (FdUMP). The resistance to FdUrd was associated with a significan t decrease in the binding of FdUMP to the purified mutant enzyme. The data are consistent with the interpretation that TSs that are highly defective a re capable of sufficient dTMP production for cell survival and optimal grow th, yet may confer resistance to TS-directed inhibitors. BIOCHEM PHARMACOL 58;6:973-981, 1999. (C) 1999 Elsevier Science Inc.