Base excision repair proteins are required for integrin-mediated suppression of bleomycin-induced DNA breakage in murine lung endothelial cells.
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) alpha, 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-beta1 integrin antibody, and then BLM. 3'-OH in DNA and accumulation of phosphorylated histone H2AX (gammaH2AX), 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 Lig3alpha, PARP-1, or XRCC1, suppression of DNA breakage by integrin antibody was limited. BLM increased gammaH2AX levels, and integrin treatment inhibited this by 57 to 73% in MLECs transfected with control siRNA. Integrin engagement also inhibited increases in gammaH2AX in BLM-treated cells transfected with Lig1 siRNA. In contrast, Lig3alpha, PARP-1, and XRCC1 siRNAs prevented integrin-mediated inhibition of BLM-induced gammaH2AX levels. The results suggest that the BER proteins, Lig3alpha, PARP-1, and XRCC1, are required for integrin-mediated suppression of BLM-induced DNA breakage.
Rose
Jane L
JL
Division of Pharmacology, Ohio State University College of Pharmacy, 500 West Twelfth Avenue, Columbus, OH 43210, USA.
Reeves
Kevin C
KC
Likhotvorik
Rostislav I
RI
Hoyt
Dale G
DG
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000633:toxicity*; D001761:Bleomycin / Q000037:antagonists & inhibitors* / Q000633:toxicity*; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D004249:DNA Damage; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics; D004260:DNA Repair / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000235:genetics; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006657:Histones / Q000235:genetics; D020287:In Situ Nick-End Labeling; D016023:Integrins / Q000502:physiology*; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D000071137:Poly (ADP-Ribose) Polymerase-1; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000076105:X-ray Repair Cross Complementing Protein 1
D000964:Antimetabolites, Antineoplastic; D004268:DNA-Binding Proteins; D006657:Histones; D016023:Integrins; C000607894:Lig1 protein, mouse; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D034741:RNA, Small Interfering; D000076105:X-ray Repair Cross Complementing Protein 1; C000619352:Xrcc1 protein, mouse; C475961:gamma-H2AX protein, mouse; D001761:Bleomycin; D049452:Green Fluorescent Proteins; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D011088:DNA Ligases; D000072481:DNA Ligase ATP