Effect of GSTM1 and GSTM4 inhibition on their mRNA levels and on sensitivity towards MTX.

<p>Effect on mRNA levels: MCF7 cells (6×10⁴) were plated in 1 ml of medium and transfected 18 h later with specific hairpins against GSTM1 (hpGSTM1) (A) or GSTM4 (hpGSTM4) (B). mRNA levels were determined 30 h after transfection. Results are expressed as changes in expression compared to the control non-transfected cells and are the mean ± SE of at least 3 different experiments. A scrambled hairpin was used as a negative control (hpSC). Effect on MTX cytotoxicity: MCF7 cells (6×10⁴) were plated in 1 ml of medium and transfected 18 h later with either hpGSTM1 (C) or hpGSTM4 (D). MTX was added 24 h after transfection and viability assayed by MTT 3 days later. Results are expressed as the percentage of cell survival compared to the control non-transfected cells and are the mean ± SE of at least 3 different experiments. *<i>p</i><0.05, **<i>p</i><0.01. E) <i>In vitro</i> MTX glutathionylation reaction. It was determined by calculating the differences between the fluorescence of the MCB-GSH adduct in the control reaction minus that in the presence of MTX. GST activity is expressed as nmols of GSH transferred by GST per minute. F) MTX glutathionylation reaction in cell free extracts. The formation of MCB-GSH adduct was followed by its fluorescence using cell free extracts from sensitive or resistant MCF7 cells in the absence or in the presence of MTX. Results are expressed as relative fluorescence units (RFU) per mg of total protein.