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Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method
Published on: September 16, 2017
Induction of oxidative DNA damage in u937 cells by TNF or anti-Fas stimulation
I Nathan1, M Dizdaroglu, L Bernstein
1Laboratory of Molecular Immunology, National Cancer Institute, Frederick, MD 21702-1201, USA.
Abstract:
TNF and Fas signaling pathways are reported to induce mitochondrial damage associated with production of oxygen radicals. We examined whether such radical production elicited detectable nuclear DNA damage in U937 cells following treatment with TNF or with anti-Fas antibodies. Using GC-mass spectroscopy for analysing base oxidation, several oxidized species increased significantly following TNF treatment, whereas anti-Fas resulted in less detectable oxidative damage using this assay. Cytogenetic analysis showed that, in the presence of aphidicolin, which blocks several types of DNA repair, TNF induced extensive chromosomal damage. Aphidicolin also synergized with TNF and anti-Fas in inducing cell death which was prevented by reducing atmospheric oxygen or addition of n -acetyl cysteine, a scavenger of oxygen radicals. Thus, several lines of evidence point to the TNF and Fas pathways inducing extensive oxidative DNA damage and repair, and suggest potential roles for these pathways in mutagenesis and aging.
Insights
Tumor necrosis factor (TNF) and Fas pathways cause oxidative DNA damage and cell death, particularly when DNA repair is blocked. These findings suggest roles in aging and mutagenesis.
Area of Science:
- Molecular Biology
- Cellular Biology
- Genetics
Background:
- Tumor necrosis factor (TNF) and Fas signaling pathways are known to induce mitochondrial damage and reactive oxygen species (ROS) production.
- The direct impact of these pathways on nuclear DNA integrity and damage has been less characterized.
Purpose of the Study:
- To investigate whether TNF and Fas signaling induce detectable nuclear DNA damage in U937 cells.
- To explore the role of oxygen radical production in mediating this DNA damage.
- To assess the involvement of these pathways in cell death and their potential contribution to mutagenesis and aging.
Main Methods:
- Treatment of U937 cells with TNF or anti-Fas antibodies.
- Gas chromatography-mass spectrometry (GC-MS) to analyze oxidized DNA bases.
- Cytogenetic analysis in the presence of aphidicolin (a DNA repair inhibitor).
- Assessment of cell death and the effect of oxygen levels and N-acetyl cysteine (NAC).
Main Results:
- TNF treatment significantly increased oxidized DNA species detected by GC-MS, while anti-Fas showed less oxidative damage.
- In the presence of aphidicolin, TNF induced extensive chromosomal damage.
- Aphidicolin synergized with both TNF and anti-Fas to induce cell death, which was mitigated by reduced oxygen or NAC addition.
Conclusions:
- The TNF and Fas pathways are implicated in inducing significant oxidative DNA damage and activating DNA repair mechanisms.
- Oxygen radical production plays a crucial role in the observed DNA damage and TNF/Fas-mediated cell death.
- These findings suggest that TNF and Fas signaling pathways may contribute to mutagenesis and the aging process.
