Transfection with 4-hydroxynonenal-metabolizing glutathione S-transferase isozymes leads to phenotypic transformation and immortalization of adherent cells.
4-Hydroxy-2-trans-nonenal (4-HNE), one of the major end products of lipid peroxidation, has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. We show for the first time that incorporation of 4-HNE-metabolizing glutathione S-transferase (GST) isozyme, hGSTA4-4, into adherent cell lines HLE B-3 and CCL-75, by either cDNA transfection or microinjection of active enzyme, leads to their transformation. The dramatic phenotypic changes due to the incorporation of hGSTA4-4 include rounding of cells and anchorage-independent rapid proliferation of immortalized, rounded, and smaller cells. Incorporation of the inactive mutant of hGSTA4-4 (Y212F) in cells by either microinjection or transfection does not cause transformation, suggesting that the activity of hGSTA4-4 toward 4-HNE is required for transformation. This is further confirmed by the fact that mouse and Drosophila GST isozymes (mGSTA4-4 and DmGSTD1-1), which have high activity toward 4-HNE and subsequent depletion of 4-HNE, cause transformation whereas human GST isozymes hGSTP1-1 and hGSTA1-1, with minimal activity toward 4-HNE, do not cause transformation. In cells overexpressing active hGSTA4-4, expression of transforming growth factor beta1, cyclin-dependent kinase 2, protein kinase C betaII and extracellular signal regulated kinase is upregulated, whereas expression of p53 is downregulated. These studies suggest that alterations in 4-HNE homeostasis can profoundly affect cell-cycle signaling events.
Sharma
Rajendra
R
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77555-0647, USA.
Brown
David
D
Awasthi
Sanjay
S
Yang
Yusong
Y
Sharma
Abha
A
Patrick
Brad
B
Saini
Manjit K
MK
Singh
Sharda P
SP
Zimniak
Piotr
P
Singh
Shivendra V
SV
Awasthi
Yogesh C
YC
D000447:Aldehydes / Q000378:metabolism*; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000502:physiology*; D002453:Cell Cycle / Q000235:genetics; D002455:Cell Division; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000502:physiology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000502:physiology*; D048031:JNK Mitogen-Activated Protein Kinases; D008845:Microinjections; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D014162:Transfection
D000447:Aldehydes; D001426:Bacterial Proteins; D002352:Carrier Proteins; C105760:GstA protein, bacteria; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; C484555:GSTA1 protein, human; D005982:Glutathione Transferase; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D005978:Glutathione; C027576:4-hydroxy-2-nonenal