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CELLULAR AND MOLECULAR

Base Excision Repair Proteins Are Required for Integrin-Mediated Suppression of Bleomycin-Induced DNA Breakage in Murine Lung Endothelial Cells

Division of Pharmacology, Ohio State University College of Pharmacy (J.L.R., K.C.R., R.I.L., D.G.H.), Columbus, Ohio; and the Dorothy M. Davis Heart and Lung Research Institute (D.G.H.), Columbus, Ohio

Engagement of integrin cell adhesion receptors suppresses bleomycin (BLM)-induced DNA strand breakage in endothelial cells. Previous investigation of cells from poly(ADP-ribose) polymerase (PARP)-1 knockout mice and with an inhibitor of the enzyme indicated that this facilitator of base excision repair (BER) is required for integrin-mediated suppression of DNA strand breakage. Here, small inhibitory RNA (siRNA) was used to assess the requirement for the BER proteins, DNA ligase III (Lig3) , PARP-1, and X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1), and for the long-patch BER ligase, DNA ligase I (Lig1), in integrin-mediated protection from BLM-induced DNA breakage. Murine lung endothelial cells (MLECs) were transfected with siRNA, treated with anti-1 integrin antibody, and then BLM. 3'-OH in DNA and accumulation of phosphorylated histone H2AX (H2AX), which reflects double-strand breakage, were measured. Integrin antibody inhibited the increases in 3'-OH caused by BLM in MLECs transfected with either control or Lig1 siRNA. However, after knockdown of Lig3, PARP-1, or XRCC1, suppression of DNA breakage by integrin antibody was limited. BLM increased H2AX levels, and integrin treatment inhibited this by 57 to 73% in MLECs transfected with control siRNA. Integrin engagement also inhibited increases in H2AX in BLM-treated cells transfected with Lig1 siRNA. In contrast, Lig3, PARP-1, and XRCC1 siRNAs prevented integrin-mediated inhibition of BLM-induced H2AX levels. The results suggest that the BER proteins, Lig3, PARP-1, and XRCC1, are required for integrin-mediated suppression of BLM-induced DNA breakage.

Address correspondence to: Dr. Dale G. Hoyt, Division of Pharmacology, Ohio State University College of Pharmacy, 500 West Twelfth Avenue, Columbus, OH 43210. E-mail: hoyt.27{at}osu.edu

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