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 (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.
Footnotes
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This work was supported by the National Heart, Lung, and Blood Institute (Grant HL68054). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Heart, Lung, and Blood Institute or the National Institutes of Health.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.113498.
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ABBREVIATIONS: BLM, bleomycin; MLEC, mouse lung endothelial cell; PARP, poly(ADP-ribose) polymerase; BER, base excision repair; XRCC1, X-ray repair complementing defective repair in Chinese hamster cells 1; Lig3, ligase III; Lig1, ligase I; NHEJ, nonhomologous end joining; DNA-PKCS, DNA-dependent protein kinase catalytic subunit DNA; HRP, horseradish peroxidase; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; ET2, ethidium homodimer; siRNA, small inhibitory RNA; PCR, polymerase chain reaction; FBS, fetal bovine serum; PBS, phosphate-buffered saline; ISNT, in situ nick translation; H33342; 2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bis-1H-benzimidazole.
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↵1 Current affiliation: Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
- Received September 5, 2006.
- Accepted December 29, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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