Overexpression of glutathione S-transferase A1-1 in ECV 304 cells protects against busulfan mediated G2-arrest and induces tissue factor expression.

Abstract

1. The antineoplastic drug busulfan is frequently used in preconditioning regimens for bone marrow transplantation. Pharmacokinetics vary tremendously between patients due to extensive metabolism in the liver via conjugation to glutathione catalysed by glutathione S-transferase (GST) A1-1. Since elevated busulfan plasma levels have been reported to be a risk factor for developing veno-occlusive disease (VOD), metabolism of busulfan may play a pivotal role in the induction of VOD. 2. Therefore, we developed a cell model to investigate the influence of busulfan metabolism on its biological effects. GSTA1-1 cDNA was transfected into the cell line ECV 304 and protein expression was demonstrated by Western blotting. Enzymatic activity could be detected by formation of tetrahydrothiophene. Additionally, effects of busulfan treatment on cell cycle and expression of tissue factor have been investigated. 3. A busulfan-induced G2-arrest was reduced in GSTA1-1-transfected cells, which consequently displayed a significantly higher activity of cdc2 kinase (24.1+/-1.5 AU mg(-1) protein) after busulfan treatment compared to controls (14.7+/-2.3 AU mg(-1) protein; P<0.01). Elevated basal expression of tissue factor in GSTA1-1-transfected ECV 304 cells could be 4 fold increased by busulfan treatment. 4. These data demonstrate that ECV 304 cells transfected with GSTA1-1 provide a valuable tool to assess busulfan metabolism in vitro. Furthermore, overexpression of GSTA1-1 leads to a partial protection against cell cycle effects of busulfan and affects tissue factor expression.