Abstract
Abstract: :
Purpose: To investigate the mechanisms by which environmental (ultraviolet B, UVB) and chemical (hydrogen peroxide, H2O2; t–butyl hydroperoxide, TBHP) stress leads to cell death in a human lens epithelial (HLE) cell line. Methods: Cultures of human lens epithelial cells (SRA 01/04) were treated with UVB (0–1000 J/m2), H2O2 (0–800µM) and TBHP (0–600µM). Cell viability and morphology were determined. Apoptotic and necrotic mechanisms of cell death were differentiated by annexin V staining and DNA fragmentation. Activity of stress–activated proteins c–Jun NH2–terminal kinase (JNK), caspase–3 and DNA fragmentation factor 45 (DFF45) were determined by immunoblotting. Results: Treatment with UVB, H2O2 and TBHP significantly decreased HLE cell viability with LD50 values of 350 J/m2, 500µM and 300µM, respectively. Cellular morphology and DNA fragmentation consistent with apoptosis were observed only in UVB–treated cells. In contrast, cellular morphology and the absence of DNA fragmentation suggests necrosis as the primary mode of HLE cell death resulting from treatment with H2O2 or TBHP. Treatment of HLE cells with UVB (400 J/m2) significantly increased annexin V staining by 11% compared to untreated controls. Expression of stress–activated proteins, including JNK, caspase–3 and DFF45 were increased in UVB–treated cells. In contrast, chemical stressors did not alter the expression of stress–activated proteins and significantly increased propidium iodide (PI) staining in HLE cells. Conclusions: Environmental and chemical stress lead to distinct forms of death in HLE cells. Specifically, UVB induces morphological, biochemical and molecular changes consistent with apoptosis, while H2O2 and TBHP induce necrosis. UVB–induced apoptosis in the lens epithelium may represent a unique mechanism by which damaged and/or unwanted cells are removed from this tissue.
Keywords: apoptosis/cell death • stress response • radiation damage: light/UV