Received for publication September 6, 2006.
Revised December 28, 2006.
Accepted for publication December 29, 2006.

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

Abstract

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-1 (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 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 (MLEC) 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 MLEC 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 MLEC 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.

Key words: Bleomycin, DNA ligase, H2AX, Integrin, PARP-1, XRCC1

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