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

• Published in: The Journal of pharmacology and experimental therapeutics Vol. 321; no. 1; pp. 318 - 326
• Main Authors: Rose, Jane L, Reeves, Kevin C, Likhotvorik, Rostislav I, Hoyt, Dale G
• Format: Journal Article
• Language: English
• Published: United States American Society for Pharmacology and Experimental Therapeutics 01.04.2007
• Subjects: Animals; Antimetabolites, Antineoplastic - toxicity; Bleomycin - antagonists & inhibitors; Bleomycin - toxicity; Blotting, Western; Cell Survival - drug effects; DNA Damage; DNA Ligase ATP; DNA Ligases - genetics; DNA Repair - drug effects; DNA-Binding Proteins - genetics; Endothelial Cells - cytology; Endothelial Cells - drug effects; Endothelial Cells - metabolism; Fluorescent Antibody Technique; Green Fluorescent Proteins - biosynthesis; Green Fluorescent Proteins - genetics; Histones - genetics; In Situ Nick-End Labeling; Integrins - physiology; Lung - cytology; Lung - drug effects; Lung - metabolism; Mice; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases - genetics; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering - pharmacology; X-ray Repair Cross Complementing Protein 1
• ISSN: 0022-3565; 1521-0103
• DOI: 10.1124/jpet.106.113498

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) α, 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-β1 integrin antibody, and then BLM. 3′-OH in DNA and accumulation of phosphorylated histone H2AX (γH2AX), 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 Lig3α, PARP-1, or XRCC1, suppression of DNA breakage by integrin antibody was limited. BLM increased γH2AX levels, and integrin treatment inhibited this by 57 to 73% in MLECs transfected with control siRNA. Integrin engagement also inhibited increases in γH2AX in BLM-treated cells transfected with Lig1 siRNA. In contrast, Lig3α, PARP-1, and XRCC1 siRNAs prevented integrin-mediated inhibition of BLM-induced γH2AX levels. The results suggest that the BER proteins, Lig3α, PARP-1, and XRCC1, are required for integrin-mediated suppression of BLM-induced DNA breakage.