Related Experiment Videos
Nitric oxide induced poly(ADP-ribose) polymerase cleavage in RAW 264.7 macrophage apoptosis is blocked by Bcl-2
U K Messmer1, D M Reimer, J C Reed
1University of Erlangen-Nürnberg, Faculty of Medicine, Department of Medicine IV, Germany.
Abstract:
Endogenously generated or exogenously supplied nitric oxide causes cleavage of poly(ADP-ribose) polymerase (PARP) and apoptotic cell death in RAW 264.7 macrophages. With the use of NO donors such as S-nitrosoglutathione or spermine-NO we established that PARP digestion occurs in parallel with DNA fragmentation, and is preceded by accumulation of the tumor suppressor gene product p53. PARP cleavage in response to lipopolysaccharide and interferon-gamma treatment is prevented by NG-monomethyl-L-arginine, thus proving a NO requirement. Endogenous NO generation, p53 accumulation, and PARP degradation occurred prior to the detection of significant chromatin condensation. In contrast, in stable Bcl-2 transfected cells, NO-initiated PARP cleavage was almost completely blocked. Our data implicate PARP as a proteolytic substrate during NO-mediated apoptotic cell death in RAW 264.7 macrophages and establish Bcl-2 as an efficient signal terminator in this process.
Insights
Nitric oxide (NO) triggers poly(ADP-ribose) polymerase (PARP) cleavage and apoptosis in macrophages. Bcl-2 protein effectively blocks this NO-mediated cell death pathway.
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- Nitric oxide (NO) is a critical signaling molecule involved in various physiological and pathological processes.
- Apoptosis, or programmed cell death, is a tightly regulated process essential for development and tissue homeostasis.
- Poly(ADP-ribose) polymerase (PARP) is a DNA repair enzyme implicated in cell death pathways.
Purpose of the Study:
- To investigate the role of nitric oxide (NO) in inducing apoptotic cell death in RAW 264.7 macrophages.
- To elucidate the involvement of poly(ADP-ribose) polymerase (PARP) cleavage in NO-mediated apoptosis.
- To determine the effect of Bcl-2 on NO-induced apoptotic signaling.
Main Methods:
- Utilized NO donors (S-nitrosoglutathione, spermine-NO) to induce apoptosis in RAW 264.7 macrophages.
- Assessed PARP cleavage, DNA fragmentation, p53 accumulation, and chromatin condensation.
- Employed NG-monomethyl-L-arginine to inhibit endogenous NO production.
- Investigated the role of Bcl-2 by transfecting cells with a stable Bcl-2 construct.
Main Results:
- Exogenous and endogenous NO induced PARP cleavage and apoptotic cell death in macrophages.
- PARP digestion and DNA fragmentation occurred in parallel with p53 accumulation.
- NO-mediated PARP cleavage was dependent on NO generation, as inhibited by NG-monomethyl-L-arginine.
- Stable Bcl-2 expression significantly blocked NO-initiated PARP cleavage and subsequent apoptosis.
Conclusions:
- PARP acts as a proteolytic substrate during NO-mediated apoptosis in RAW 264.7 macrophages.
- Bcl-2 functions as an effective signal terminator, inhibiting NO-induced apoptotic cell death.