Effects of mGST A4 transfection on 4-hydroxynonenal-mediated apoptosis and differentiation of K562 human erythroleukemia cells.
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.
Cheng
J Z
JZ
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas, 77555-1067, USA.
Singhal
S S
SS
Saini
M
M
Singhal
J
J
Piper
J T
JT
Van Kuijk
F J
FJ
Zimniak
P
P
Awasthi
Y C
YC
Awasthi
S
S
D000447:Aldehydes / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division; D004920:Erythropoiesis / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D051379:Mice; D051381:Rats; D014162:Transfection
D000447:Aldehydes; D005982:Glutathione Transferase; D005978:Glutathione; C027576:4-hydroxy-2-nonenal