Glutathione S-transferases (GSTs) are known as detoxification enzymes that catalyse the conjugation of glutathione to anticancer drugs. In addition to this activity, GSTM1, an isotype of the Mu class GSTs, has been suggested to act as a negative regulator of mitogen-activated protein kinase (MAPK) activation. About 40–60% of the population has a deficit in GSTM1 enzyme activity, and the patients possessing GSTM1 have been reported to have a greater risk of relapse in childhood acute lymphoblastic leukemia (ALL). However, the reasons for poor prognosis remain unclear. To establish our hypothesis that GSTM1-induced drug resistance was involved, GSTM1 was transfected into GSTM1-negative CCRF-CEM cell lines (hereafter, the transfectant is denoted as CEM/M1) and the drug sensitivity was compared with that of control cells (CEM/mock). CEM/M1 showed decreased sensitivity to melphalan and carmustine (relative resistance vs. CEM/mock: 1.6 and 2.1, respectively; p<0.05), but not to daunorubicin, vincristine or etoposide. Notably, sensitivity to dexamethasone (DEX) decreased to a degree of up to 8-fold (IC50:1.76 μM in CEM/M1 and 0.22 μM in CEM/mock; p<0.01). Glutathione depletion by buthionine sulfoximine abrogated the resistance of CEM/M1 to alkylating agents, but not to DEX. These results suggest that a different mechanism is involved in the resistance to DEX. To clarify the GSTM1-related DEX resistance, proapoptotic pathway via p38 MAPK was evaluated. In CEM/M1, DEX-induced apoptosis was significantly inhibited (annexin V-positive at 72hr: 27% in CEM/M1 and 57% in CEM/mock; p<0.01). Silencing of GSTM1 by siRNA restored the DEX resistance, indicating that the anti-apoptosis was caused by the expression of GSTM1. After the treatment with DEX, phosphorylation of p38 was upregulated in CEM/mock, but not in CEM/M1; and the level of Bim mRNA was higher in CEM/mock than it was in CEM/M1. These findings indicate that the overexpression of GSTM1 down-regulates the proapoptotic pathway via p38 MAPK-Bim. However, treatment with a p38 MAPK-inhibitor (SB202194) decreased the DEX-induced apoptosis in both CEM/mock and CEM/M1. The involvement in DEX-resistance seems to be partial. To investigate other mechanisms, NF-κB pathway was evaluated. The basal activity of NF-κB p50 was significantly higher in CEM/M1 than it was in CEM/mock. Inhibition of IκBα phosphorylation by BAY11-7082 increased DEX sensitivity in either cells. Of considerable importance, however, is that the increase by BAY11-7082 was prominent in CEM/M1 and the relative resistance decreased from 8 to 2-fold. It appears that the anti-apoptotic NF-κB pathway is also involved in a resistance to DEX. In conclusion, the expression of GSTM1 in CCRF-CEM caused the resistance to alkylating agents due to its catalytic activity and to DEX by its anti-apoptotic function. The latter is the first report demonstrating the relationship between the expression of GSTM1 and DEX resistance. The determination of GSTM1 expression could be a useful tool for the individualization of optimize therapy for childhood ALL.

(Supported by JSPS grant C19591102 and JRFCP grant)

Author notes

Disclosure: No relevant conflicts of interest to declare.