Abstract

TER286 [γ-glutamyl-α-amino-β(2-ethyl-N,N,N′,N′-tetrakis(2-chloroethyl)phosphorodiamidate)-sulfonyl-propionyl-(R)-(−) phenylglycine] is a novel nitrogen mustard prodrug that is preferentially activated by glutathione S-transferase P1-1 (GSTP1-1). A human promyelocytic leukemia /TER286-resistant cell line was selected by chronic, long-term exposure to the prodrug. Although resistance was not readily achieved, eventually a 5-fold resistant clone was isolated. Cross-resistance to melphalan occurred, but not to doxorubicin (Adriamycin), taxol, and γ-glutamyl-S-(benzyl)cysteinyl-R(−)-phenyl glycine diethyl ester, a GSTP1-1 inhibitor. The protein and transcript levels and enzymatic activity of GSTP1-1 were reduced significantly in the selected resistant line. GSTα levels were unchanged, and GSTμ was undetectable. Although glutathione levels were elevated in human promyelocytic leukemia/TER286 cells, no changes in the expression of thiol-related genes including γ-glutamylcysteine synthetase, γ-glutamyl transpeptidase, or multidrug resistance protein were found. A 7-fold increase in catalase expression in the resistant cell line indicated an adaptive response to oxidative and electrophilic stress, and this was also reflected in the lower prevalence of drug-induced DNA single-strand breaks in the resistant cells. Mouse embryo fibroblast GSTP1-1^−/− cells exhibited 2-fold resistance to TER286 compared with GSTP1-1^+/+ cells. NIH3T3 cells transfected with combinations of γ-GCS and multidrug resistance protein exhibited enhanced resistance to TER286, although the degree of resistance was impaired by cotransfection of GSTP1-1. These results are consistent with responses in the TER286-resistant cells indicative of GSTP1-1-mediated mechanism of activation. In consequence, these data support the rationale that tumors expressing high levels of GSTP1-1 will be more sensitive to the cytotoxic effects of the drug.