Cellular levels of downstream products of membrane lipid oxidation appear to regulate differentiation in K562 human erythroleukemia cells. 4-Hydroxynonenal (4-HNE) is a diffusible and relatively stable product of peroxidation of arachidonic and linoleic acids, cellular levels of which are regulated through metabolism to glutathione (GSH) conjugate by glutathione S-transferases (GSTs). A group of immunologically related alpha-class mammalian GSTs expressed in mice (mGST A4-4), rat (rGST A4-4), human (hGST A5.8), and other species, as well as the more distantly related human hGST A4-4, preferentially utilize 4-HNE as a substrate and are suggested to be major determinants of intracellular levels of 4-HNE. Present studies were designed to examine the effects of 4-HNE on K562 cells and to study the effect of transfection of mGSTA4-4 in these cells. Exposure of K562 cells to 20 microM 4-HNE for 2 h resulted in a rapid erythroid differentiation of K562 cells, as well as apoptosis evidenced by characteristic DNA laddering. Stable transfection of cells with mGST A4-4 resulted in a fivefold increase in GST-specific activity toward 4-HNE compared with wild-type or vector-only transfected cells. The mGST A4-4-transfected cells were resistant to the cytotoxic, apoptotic, and differentiating effects of 4-HNE. The mGST A4 transfection also conferred resistance to direct oxidative stress (IC(50) of H(2)O(2) 22, 23, and 35 microM for wild-type, vector-transfected, and mGST A4-transfected cells, respectively). mGST A4-4-transfected cells also showed a higher rate of proliferation compared with wild-type or vector-transfected K562 cells (doubling time 22.1 +/- 0.7, 31 +/- 1.2, and 29 +/- 0.6 h, respectively). Cellular 4-HNE levels determined by mass spectrometry were lower in mGST A4-4-transfected cells compared to cells transfected with vector alone (5.9 pmol/5 x 10(7) cells and 62.9 pmol/5 x 10(7) cells, respectively). Our studies show that 4-HNE can induce erythroid differentiation in K562 cells and that overexpression of mGST A4 suppresses 4-HNE levels and inhibits erythroid differentiation and apoptosis.