Effects of bleomycin and integrin engagement on physical DNA breakage in endothelial cells

Abstract

We previously showed that pretreatment of endothelial cells (EC) with anti-beta1 integrin antibody (B1) inhibited bleomycin (BLM)-induced DNA damage, as indicated by DNA 3′OH end labeling or by histone 2A.X phosphorylation. The mechanism of this integrin-mediated suppression remains unclear. Here we used single cell gel electrophoresis (Comet Assay) to measure the effect of integrin engagement on physical DNA breaks induced by BLM. EC were treated with either 1 ug B1/ml, or the corresponding non-immune IgG for 1 h, followed by the addition of 2 ug goat anti-rat secondary antibody/ml for 4 h. The cells were then treated with 100 ug BLM/ml for 45 min, scraped, resuspended in 0.5% low melt agarose, and placed on slides between two layers of 0.5% normal agarose. DNA was allowed to unwind in alkaline buffer for 20 minutes before alkaline electrophoresis at 25V for 20 min and staining with 20 ug Ethidium Bromide/ml. The resulting fluorescence microscopic images were analyzed to determine the degree of migration of DNA (% of DNA in the tails of “comets”), which reflects breakage. BLM alone caused significant breakage that was reversible after the drug was removed. Surprisingly, B1 did not suppress this evidence DNA breakage. The results suggest that B1 integrin engagement does not limit the physical DNA breakage caused by 100 ug BLM/ml. Integrin engagement may operate on the cellular reactions to DNA breakage rather than on its production.