Differential catalytic efficiency of allelic variants of human glutathioneS-transferase Pi in catalyzing the glutathione conjugation of thiotepa

Alkylating agents are extensively used in the treatment of cancer. The clin ical usefulness of this class of anticancer drugs, however, is often limite d by the emergence of drug-resistant tumor cells. Increased glutathione (GS H) conjugation through catalysis by GSH S-transferases (GSTs) is believed t o be an important mechanism in tumor cell resistance to alkylating agents. In the present study, we report that the allelic variants of human Pi class GST (hGSTP1-1), which differ: in their primary structures at amino acids i n positions 104 and/or 113, exhibit significant differences in their activi ty in the GSH conjugation of alkylating anticancer drug thiotepa. Mass spec trometry revealed that the major product of the reaction between thiotepa a nd GSH was the monoglutathionyl-thiotepa conjugate. While nonenzymatic form ation of monoglutathionyl-thiotepa was negligible, the formation of this co njugate was increased significantly in the presence of hGSTP1-1 protein. Th e hGSTP1-1-catalyzed GSH conjugation of thiotepa was time and protein depen dent and followed Michaelis-Menten kinetics. The catalytic efficiency of hG STP1-1(I104,A113) variant was approximately 1.9- and 2.6-fold higher compar ed with hGSTP1-1(V104,A113) and hGSTP1-1(V104,V113) isoforms, respectively. The results of the present study indicate that the hGSTP1-1 polymorphism m ay be an important factor in GST-mediated tumor cell resistance to thiotepa , and that subjects homozygous for the hGSTP1-1(I104,A113) allele, which is most frequent in human populations, are likely to be at a greater risk for developing GST-mediated resistance to thiotepa than heterozygotes or homoz ygotes with valine 104 background. (C) 1999 Academic Press.