Regulation of Bleomycin-Induced DNA Breakage and Chromatin Structure in Lung Endothelial Cells by Integrins and Poly(ADP-Ribose) Polymerase
Abstract
Activation of endothelial cell integrins inhibits DNA breakage by diverse agents, including the DNA-damaging agent bleomycin. DNA breaks activate nuclear poly(ADP-ribose) polymerase (PARP), which regulates chromatin structure and DNA repair. We determined the role of PARP in suppression of bleomycin genotoxicity by integrins using wild-type and PARP knockout mouse lung endothelial cells (MLEC), and the PARP inhibitor, 3-aminobenzamide (3AB). Activation of β1 integrins by antibody clustering enhanced the sensitivity of wild-type nuclei to digestion with micrococcal nuclease and deoxyribonuclease I, indicating that chromatin structure was altered. 3AB blocked this effect. Knockout and 3AB-treated wild-type MLEC were hypersensitive to deoxyribonuclease I compared with wild-type cells, demonstrating that PARP regulates chromatin structure. Integrin clustering reduced the hypersensitivity of knockout cells, suggesting additional, PARP-independent mechanisms that inhibit nuclease interaction with chromatin. Bleomycin caused DNA breakage in wild-type and knockout MLEC. Breaks were eliminated after 60 min incubation of wild-type cells in drug-free medium, whereas 3AB or PARP knockout inhibited DNA repair. Integrin clustering protected wild-type cells from DNA breakage, and 3AB and PARP knockout inhibited this protection. Bleomycin caused large increases in PARP activity in wild-type but not knockout MLEC, and integrin clustering inhibited the activation of PARP. The results indicate that the antigenotoxic effects of integrin activation require PARP and that integrins alter chromatin structure by PARP-dependent and -independent mechanisms.
Footnotes
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Send reprint requests to: Dale G. Hoyt, Ph.D., The Ohio State University, Division of Pharmacology, College of Pharmacy, 500 West Twelfth Avenue, Columbus, Ohio. E-mail: hoyt-27{at}osu.edu
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This work was supported by an award from the American Heart Association, Ohio Valley Affiliate (C.B.J.), American Lung Association Career Investigator Award CI-009N (D.G.H.), and by National Institutes of Health Grants HL56018 and P30-CA16058.
- Abbreviations:
- BLM
- bleomycin
- PARP
- poly(ADP-ribose) polymerase
- ADPR
- ADP-ribose
- 3AB
- 3-aminobenzamide
- DNase
- deoxyribonuclease
- VCAM-1
- vascular cell adhesion molecule-1
- PECAM
- platelet endothelial cell adhesion molecule
- MLEC
- mouse lung endothelial cells
- ISNT
- in situ nick translation
- MNase
- micrococcal nuclease
- PARSIS
- poly(ADPR) synthesis in situ
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- Received March 6, 2000.
- Accepted October 11, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
