Abstract
Abstract: :
Purpose: We have previously shown that human lens epithelial cells (HLE B–3) transfected with hGSTA4 undergo transformation and rapidly grow in an anchorage–independent manner with indefinite lifespan, as opposed to wild–type or empty vector–transfected cells which grow as adherent cells with a finite lifespan. hGSTA4–transfected cells overexpress hGSTA4–4 protein resulting in enhanced metabolism and depletion of 4–hydroxynonenal (HNE), which is known to be involved in various signaling mechanisms. In order to elucidate the mechanisms involved in the transformation of hGSTA4–4–overexpressing cells, we have now examined the effect of hGSTA4 transfection on genes involved in adhesion, apoptosis, cell cycle control, and proliferation. Methods: Wild–type and pTarget/hGSTA4–transformed HLE B–3 cells were harvested. Total RNA purified from these cells using RNAqueous–4PCR kit was subjected to microarray analysis using the HGUI133 Plus 2.0 chip. The results were analyzed by the UTMB Bioinformatics Program and fold–changes in expression of relevant genes were determined; results were verified by Western blot and real–time RT–PCR using 80 nanograms of RNA, normalized to 18s rRNA. Results: cDNA microarray studies demonstrated a profound decrease in the expression of the following genes: p16 (894 fold), laminin γ1 (598 fold), connexin 43 (185 fold), integrin α6 (70 fold), fibronectin 1 (65 fold), p53 (52 fold), and Fas (31 fold). A relatively moderate decrease in the expression of TGFα (8 fold), p21 (7 fold), c–jun (4 fold), and Bad (3 fold) was also observed in hGSTA4–transfected cells. In contrast, the expression of c–myc was upregulated (3 fold). The modulation of most of these genes in hGSTA4–overexpressing cells was verified by quantitative RT–PCR and Western blot analysis. These studies confirmed that the expression of p53, p21, p16, laminin γ1, fibronectin 1, integrin α6, connexin 43, Fas, c–jun, and TGFα was severely suppressed in hGSTA4–transfected cells, and c–myc, which was undetectable in wild–type or empty vector–transfected cells, was strongly expressed in hGSTA4–transfected cells. Conclusions: These studies demonstrate that hGSTA4 transfection, leading to lower constitutive HNE levels in HLE B–3 cells, causes profound changes in the expression of genes involved in cell survival/apoptosis, cell cycle control, proliferation, and adhesion. (ES 007254, EY 04396, ES 01217)
Keywords: gene microarray • gene/expression • cell adhesions/cell junctions