Ectopic overexpression of theta class glutathione transferases in a human keratinocyte cell line.

Jackson, K. M. and Allinson, Sarah L. and McMillan, Trevor J. (2007) Ectopic overexpression of theta class glutathione transferases in a human keratinocyte cell line. Mutagenesis, 22 (6). pp. 444-445. ISSN 1464-3804

Abstract

UVA induces oxidative stress in cells via photosensitiser mediated processes, one consequence of which is elevated levels of lipid peroxidation. Lipid peroxidation products include lipid hydroperoxide and unsaturated aldehyde species and these can result in a number of downstream consequences, including the production of potentially genotoxic DNA damage. It is therefore important that cells are able to process lipid peroxidation products. There are several classes of glutathione transferases (GSTs), all of which are dimeric, phase II detoxification enzymes that catalyse the conjugation of reduced glutathione to various electrophilic compounds. There are two theta class GSTs in humans: hGSTT1-1 and hGSTT2-2. Both show activity towards organic hydroperoxide species, however only hGSTT2-2 seems to be active against unsaturated aldehydes. The overall aim of the present study is to see the effect of overexpression of hGSTT1-1 and hGSTT2-2 in the HaCaT keratinocyte cell line on a range of biological endpoints following treatment with UVA. To achieve the overexpression of hGSTT1-1 and hGSTT2-2 in the HaCaT cell line, the cDNA was firstly cloned into the pET28 bacterial expression vector to introduce a His tag at the N-terminus of the protein and to allow expression and purification of recombinant enzyme. The inserts including the His tag were then sub-cloned into the pZeoSV2þ mammalian expression vector and transfected into the HaCaT cell line. Overexpression of hGSTT1-1 and hGSTT2-2 in isolated clones was confirmed by the use of RT-PCR and western blotting. Preliminary results suggest that overexpression of hGSTT2- 2 increases the survival of the HaCaT cell line compared to that of the empty vector control cell line on treatment with UVA. More data is required to confirm these results and to see the effects of various specific lipid peroxidation products on the overexpressing cell lines but initial results are promising.