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Y.C. Awasthi, D. Brown, R. Sharma, Y. Yang, A. Sharma, N.K. Tewari, S.K. Manjit, B. Patrick; Alternation in 4–Hydroxynonenal Homeostasis in Human Lens Epithelial Cell (HLE B–3)Leads to their Phenotypic Transformation . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1714.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:4–hydroxy–2–trans–nonenal (4–HNE), the major end–product of lipid peroxidation has been shown to induce apoptosis in a variety of cell lines. In present studies, we have investigated the effect of 4–HNE depletion in human lens epithelial cells (HLE B–3). Methods:HLE B–3 cells were either transfected with 4–HNE metabolizing glutathione S–transferase (GST) isozyme, hGSTA4–4 or microinjected with hGSTA4–4 protein, or its expression vector and the effect of hGSTA4–4 overexpression was studied on intracellular levels of 4–HNE, morphology of cells, and the key genes involved in cell cycle regulation. Results: our results demonstrate for the first time that depletion of intracellular 4–HNE by incorporating hGSTA4–4 in adherent cell line HLE–B3, either by cDNA transfection or by microinjection of active enzyme leads to phenotypic transformation of cells. The dramatic phenotypic changes due to the incorporation of hGSTA4–4 and reduction of 4–HNE levels include, rounding of cells and anchorage–independent rapid proliferation of immortalized rounded, smaller cells. When an inactive mutant of hGSTA4–4, (Y212F) is incorporated in cells either by microinjection or by transfection, neither lowering of 4–HNE levels is observed nor the cells undergo transformation suggesting that lowering the intracellular 4–HNE level is an essential prerequisite for transformation. Depletion of 4–HNE in cells overexpressing active hGSTA4–4 is accompanied with the upregulation of the expression of transforming growth factor beta, (TGFß1), cyclin–dependent kinase (cdk2), protein kinase C beta (PKCßII), and extracellular signal regulated kinase (ERK) and downregulation of p–53. In cells overexpressing inactive mutant hGSTA4–4, the expression of none of these genes is affected suggesting that lowering of 4–HNE levels brings about these changes. Similar results are obtained when hGSTA4–4 is overexpressed in another adherent cell line from human lung fibroblasts (CCL–75). Conclusions:These studies provide first–direct evidence that alterations in 4–HNE homeostatis can profoundly affect cell cycle signaling events. (supported by EY grant 04396)
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