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J. Xi, F. Bai, H.C. Patel, U.P. Andley; Identification of Genes Responsive to UVA Radiation in Human Lens Epithelial Cells using cDNA Microarrays . Invest. Ophthalmol. Vis. Sci. 2003;44(13):302.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Lens epithelial cells exposed to UVA radiation undergo apoptosis after four hours (Andley et al., 1998, J. Biol. Chem.). The present study was designed to identify sets of differentially expressed genes in UVA-irradiated lens epithelial cells using cDNA microarrays. Methods: Human lens epithelial cells with extended lifespan, HLE B-3, were exposed to UVA radiation at 365 nm, at a fluence rate of 0.007 watts cm-2 and a total fluence of 37.8 J cm-2. Cells were incubated for 1-24 hours in normal culture medium. Flow cytometric analysis showed that cells undergo maximum apoptosis 4 hours after the stress. RNA isolated from six replicates of control or UVA irradiated cells was pooled in order to minimize non-specific effects. The data represent the average of 18 replicate cultures under each condition. Biotinylated cRNA was purified using the Qiagen RNAeasy mini cleanup protocol, cRNA was fragmented and allowed to hybridize to Human Genome U95A chips containing about 12,000 genes (Affymetrix). The GeneSpring program was used to analyze the data from three different sets of chips for control and UVA irradiated samples. GeneSpring data analysis was performed by T. Harter. Semi-quantitative and real-time PCR was performed using gene-specific primers. Western immunoblotting with specific antibodies was used to detect protein levels. Results: We identified approximately 80 genes abnormally expressed in UVA irradiated cells (showing >2.5 fold change at p<0.05). These genes are implicated in various biological processes, including signal transduction, nucleic acid binding and enzymes. A majority of the genes were downregulated. Among the genes that were upregulated, most were involved in signal transduction. This analysis revealed that the expression of genes for the transcription factors ATF-3 and Pilot, and the calpain large polypeptide 3 (CANP3) increased four to eight-fold. In addition, peroxisomal biogenesis factor 7, NAPOR-1 and tumor associated calcium signal transducer genes decreased six to eight-fold. PCR analysis and immunoblotting confirmed the increase in ATF-3 in the irradiated cells. Surprisingly, none of these genes had previously been shown modulated by UVA radiation. Conclusions: Our results show that human lens epithelial cells respond to a single dose of UVA radiation by differentially enhancing or suppressing functionally distinct sets of genes around the time at which maximum apoptosis occurs.
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