INDUCTION OF OXIDATIVE DNA DAMAGE IN U937 CELLS BY TNF OR ANTI-FAS STIMULATION

INDUCTION OF OXIDATIVE DNA DAMAGE IN U937 CELLS BY TNF OR ANTI-FAS STIMULATION

doi:10.1006/cyto.1999.0638, available online at http://www.idealibrary.com on INDUCTION OF OXIDATIVE DNA DAMAGE IN U937 CELLS BY TNF OR ANTI-FAS STIM...

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doi:10.1006/cyto.1999.0638, available online at http://www.idealibrary.com on

INDUCTION OF OXIDATIVE DNA DAMAGE IN U937 CELLS BY TNF OR ANTI-FAS STIMULATION Ilana Nathan,1,5 Miral Dizdaroglu,2 Lori Bernstein,3 Udo Junker,1,6 Chong-kil Lee,1,7 Kathrin Muegge,4 Scott K. Durum1 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.  2000 Academic Press

TNF is a key mediator of inflammation and is produced by many types of cells. Macrophages are major TNF producers in response to stimulation by a variety of agents, including microbial products and other cytokines. TNF induces a broad range of inflammatory responses, such as adhesion and release of secondary mediators. TNF was originally described as a cytotoxic factor for some tumor cell lines.1 The cytotoxic response to TNF has features of both apoptotic and necrotic cell death, and varies between cell types. One trigger of cell death has been attributed to production of oxygen radicals by mitochondria following TNF action.2–6 TNF also induces production of MnSOD7 and glutaFrom the 1Laboratory of Molecular Immunology, National Cancer Institute, Frederick, MD 21702-1201, USA; 2Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA; 3Department of Pathology and Laboratory, Texas A & M Health Science Center, College Station, TX 77843-1114, USA; 4SAIC, National Cancer Institute, Frederick, MD, USA; 5Current adress: Department of Hematology, Soroka Medical Center and Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer Sheva 84105, Israel; 6Current adress: Institute of Clinical Immunology, University of Jena, Jena, Germany; 7Current address: Chungbuk University, Korea Correspondence to: Scott K. Durum; E-mail: [email protected] mail.ncifcrf.gov Received 23 September 1999, accepted for publication 5 November 1999  2000 Academic Press 1043–4666/00/070881+07 $35.00/0 CYTOKINE, Vol. 12, No. 7 (July), 2000: pp 881–887

thione,8 which can serve as a protective mechanism in scavenging the oxygen radicals that are concurrently produced. However, other studies, while verifying a central role for mitochondrial damage in TNF killing, disputed a role of oxygen radicals in mediating this damage.9 The induction of oxygen radicals has been attributed to the ceramide generated by TNF,10 whereas A20, an antiapoptotic protein, has been shown to block oxygen radical production.11 Fas, a member of the TNF receptor family, induces apoptotic cell death following crosslinking by its ligand leading to caspase activation.12 Mitochondrial damage has also been invoked in the Fas killing pathway13–15 and production of oxygen radicals is also associated with this response.16 Because of the potential DNA damage that could result from excessive production of oxygen radicals, we studied whether the TNF and Fas pathways could result in such damage. We provide several lines of evidence that both these pathways induce oxidative DNA damage in U937 cells.

RESULTS We analysed whether TNF treatment resulted in oxidative DNA damage in U937 cells using GC/MS. As shown in Figure 1, TNF induced a two- to five-fold rise in several species of oxidized bases, including 881

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8-OH-Gua µmol/mol DNA

800 600

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0 Time (h)

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Base oxidation induced by TNF and anti-Fas in U937 cells.

Cells were treated with TNF (20 ng/ml, — —) or anti-Fas (0.1 g/ml, — —) for the indicated times. DNA was extracted and analysed for oxidized bases by GC mass spectroscopy.

8-OH-adenosine, Fapy-adenosine, 8-OH-guanine and 2-OH-adenosine. The magnitude of the TNF-induced rise in, for example, 8-OH-adenosine was extremely high, comparable to the level induced by hydrogen peroxide and far exceeding that induced by ionizing radiation.19 TNF is therefore a potent inducer of base oxidation. Anti-Fas induced a smaller rise in some of these species. Significant levels of cell killing were not observed under these conditions of TNF or anti-Fas treatment, indicating that the apoptotic cascade was not initiated, at least not fully. To determine whether the base oxidation induced was genotoxic we used cytogenetic analysis. Together with TNF we also added the drug aphidicolin which blocks some types of DNA repair based on its inhibition of polymerases ,  and . Thus, visualization of single and double strand breaks has been reported to be greatly increased when aphidicolin was combined with various genotoxic agents such as -irradiation,24,25 ultraviolet irradiation,26 dimethylarsinic acid,27 platinum28and melphalan.29 As shown in Table 1, TNF added together with aphidicolin

induced cytogenetic abnormalities. Both single and double stranded DNA breaks would have been involved in generating these types of lesions as shown in the line drawing in Figure 2. TNF alone did not induce significant cytogenetic abnormalities of a magnitude detectable by this type of analysis. This could be due to a rapid repair of oxidized and broken DNA. TABLE 1. cells Treatment

TNF induction of cytogenetic damage in U937 Metaphase spreads

— TNF

75 75

aphidicolin TNF+aphid

75 75

Abnormalities 0 1 (single chromatid break, one arm) 0 10 (5 chromatid breaks, 5 triradiant associates)

*Cells were cultured with TNF or TNF plus aphidicolin for 24 h, washed, then cultured with colcemid for 6 h and examined for chromosomal damage.

Induction of oxidative DNA damage / 883

Figure 2.

TNF+aphidicolin induced chromosomal damage in U937 cells.

Cells were treated with TNF (20 ng/ml) and aphidicolin (0.75 M) and analysed for chromosomal damage as in Table 1. Arrows indicate chromatid breaks and triradial chromosomes. Line drawings to the right depict enlarged visualizations of the indicated chromosomes.

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Figure 3.

Anti-Fas: kinetics of TUNEL-positive cells.

U937 cells were treated with Anti-Fas (0.1 M) for the indicated time. Broken DNA was analysed by TUNEL. A negative control is shown in which polymerase was omitted from the TUNEL assay. (— —, -Fas; — —, Fas () POL; — —, control.)

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0.001 0.003 0.01 0.03 αFas (µg/ml)

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Aphidicolin promotes killing of U937 cells by anti-Fas.

Cells were treated with aphidicolin (0.75 M) for 18 h. Anti-Fas (0.1 M) was added, cells were cultured 18 h cell viability was assessed using WST-1. Cycloheximide (CHX 10 M) was added as a positive control for killing by anti-Fas. (— —), Fos; — —, Fas+APHID; — —, Fas+CHX.)

The TUNEL assay, while widely used for detecting apoptotic DNA has also been used to detect DNA breaks in non-apoptotic cells.30–32 Using the TUNEL assay we detected a peak in DNA breaks 2–3 h following TNF or anti-Fas treatment (Fig. 3), then a decline to baseline by 18 h, presumably because they are repaired. Because the preceding results showed that aphidicolin exacerbated DNA damage by TNF or anti-Fas, we then examined whether this effect was sufficient to kill cells. Indeed, aphidicolin was toxic to U937 cells when combined with either TNF or anti-Fas as shown in Figure 4. Cycloheximide is shown as a positive control for a drug that also synergizes with anti-Fas (or TNF, not shown). Aphidicolin, in addition to blocking DNA repair, also arrests cells in the S-phase of the cell

cycle (based on its inhibition of DNA polymerase  and ), however, normal cells in S-phase were not vulnerable to TNF, as shown in Figure 5A. Even when TNF is combined with cycloheximide, cell death is not restricted to S-phase (Fig. 5C). When TNF was added to aphidicolin, a reduction in S-phase cells was observed (Fig. 5B). This could either result from a selective death of cells in S-phase or from TNF blocking entry into S-phase. It could be argued that aphidicolin augments TNF toxicity because it induces cell cycle arrest. To address this possibility, nocodosol was used (Fig. 5D) to arrest cells in M-phase; there was no promotion of TNF killing by nocodosol, indicating that cell cycle arrest per se is not a contributing factor in TNF toxicity. To examine further whether oxidative DNA damage could account for cell killing by anti-Fas or TNF plus aphidicolin, the effect of reducing atmospheric oxygen was used. As shown in Figure 6, low oxygen levels protected cells from anti-Fas plus aphidicolin, but not from anti-Fas plus cycloheximide. Scavenging oxygen radicals with n-acetyl cysteine also protected cells from death induced by TNF or anti-Fas plus aphidicolin as shown in Table 2. These results support the hypothesis that TNF or anti-Fas induce oxidative DNA damage which cannot be repaired in the presence of aphidicolin, leading to cell death either from an apoptotic mechanism or from a necrotic mechanism due to genotoxic stress.

DISCUSSION We observed that TNF induced extensive base oxidation in U937 cells. Anti-Fas induced a modest level of base oxidation. Extensive cytogenetic damage was detected with TNF combined with aphidicolin, which blocks DNA repair. Cell death was triggered by aphidicolin combined with TNF or anti-Fas, and this death was countered by reducing atmospheric oxygen or addition of oxygen radical scavengers, consistent with it being mediated by oxidative DNA damage. The genotoxicity of the TNF and Fas pathways implicates them as potential mutagens. Oxidative DNA damage is considered a major cause of mutation leading to cancer.33 Moreover, oxidation of mitochondrial DNA is thought to contribute to the aging process.34 Our studies show that these processes could be triggered by the TNF and Fas pathways during normal inflammatory and immune responses. Several types of neoplasia are clearly associated with inflammatory and immune responses. Schistosomula eggs induce chronic inflammation of the bladder and a high incidence of bladder cancer.35 Ulcerative colitis is associated with colon cancer. Liver

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DNA content Figure 5.

Cell cycle analysis of U937 cells treated with (A) TNF, (B) aphidicolin, (C) cycloheximide and (D) nocodosol.

Cell viability (% of control)

Cells were pre-treated for 18 h with aphidicolin or nocodosol. TNF and cycloheximide were added and 8 h later analysed for cell cycle by flow cytometry.

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Low oxygen atmosphere vs cell killing by anti-Fas, aphidicolin and cycloheximide.

Cells were maintained in low 0.5% O2 (0–0) or normal 20% O2 (0–0) atmosphere 2 h before aphidicolin was added. Viability was measured using WST in a normal atmosphere. Left panel: Fas+aphidicolin. Right panel: Fas+cycloheximide.

cancer is associated with the chronic inflammation of viral hepatitis. Myeloma in mice is induced by inflammatory oil in the peritoneal cavity. A number of studies have shown that TNF induces mitochondrial damage.2–5 Mitochondria were required to mediate the TNF death signal as shown in cells rendered deficient, then reconstituted with mito-

chondria.36 TNF-induced generation of oxygen radicals by mitochondria has been suggested to result from a blocking of electron transfer, which also results in respiratory failure.9 Increased activity of the mitochondrial enzyme PLA-2 may trigger the later mitochondrial changes such as permeability.37 The leakage of cytochrome c from mitochondria (which is blocked by

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TABLE 2. Protection by n-acetyl cysteine from cell death induced by TNF or anti-Fas plus aphidicolin Treatment — Fas — Fas TNF TNF

— — aphid aphid — aphid

NAC

+NAC

100* 78.8 100 38.5 97 52.4

100 65.7 100 103 76 101

*Cells were cultured with TNF, anti-Fas and aphidicolin and assayed for cell viability. Data are expressed as percentage of control.

the anti-apoptotic agent Bcl-2) has recently been implicated as a general apoptotic mechanism leading to the activation of the caspase cascade.14,38 There has been much progress in elucidating the structure of the TNF–receptor complex.39 However, the pathway leading from the crosslinked receptor to mitochondrial damage has not been clearly defined. One candidate is ceramide, which was found to induce the mitochondrial permeability transition following TNF treatment of cells.40 Ceramide is generated by sphingomyelinase, which could occur in the cytosol in the vicinity of the crosslinked TNF receptor. Although Fas is related to the TNF receptors, and the Fas pathway has been shown to induce mitochondrial damage in a number of studies, the mechanisms may differ from that of TNF. It has recently been shown that a caspase 1-like enzyme is involved in the Fas pathway leading to mitochondrial disruption41 and to production of oxygen radicals16 (which we presume mediate DNA damage in our study). Bcl-2, which protects mitochondria from some types of agents such as pro-oxidants and ceramide, did not protect them from damage induced by caspase-1. Downstream of mitochondrial damage, caspase 3 was activated leading to nuclear apoptosis.41 The oxidative DNA damage induced by TNF or anti-Fas did not lead to cell death (unless DNA repair was blocked), suggesting that mitochondrial damage was not sufficient to fully trigger an apoptotic response. Cell death was observed in the presence of aphidicolin, which blocks nuclear, but not mitochondrial42 DNA repair. This cell death was not mediated by p53, which is one known pathway following single and double strand DNA breaks,43 as the U937 cell line used in these studies lacks p53.44 Perhaps DNA damage is sufficiently extensive that it disrupts normal cellular functions leading to death.

MATERIALS AND METHODS Cell culture U937 cells (obtained from ATCC) were grown in RPMIcontaining 10% fetal bovine serum (2 mM),

1640

L-glutamine (2 mM), penicillin (100 units/ml) and streptomycin (100 g/ml). Cells were treated with TNF (Peprotech), anti-Fas (Santa Cruz Biotechnologies) and aphidicolin (Sigma) at the indicated concentrations for the indicated times. Hypoxic culture was performed in 1% O2. Cells were harvested and analysed for viability using WST-1 dye (4-3(4iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzene disulfonate).17

Gas chromatography-mass spectrometry (GC/MS) Analyses for oxidized bases was performed by GC/MS as previously described.18,19

Cytogenetic analysis U937 cells were treated in mid-log phase with TNF (20 ng/ml) with or without aphidicolin (0.25 g/ml) and cultured for 18 h. Cells were washed, cultured for an additonal 6 h, colcemid (25 ng/ml, Gibco BRL) was added and the cells cultured for a further hour. Cells were harvested and cytogenetic analysis performed20 by H and W Cytogenetic Services (Lovettsville, VA), analysing 75 chromosomal spreads for each sample.

Cell cycle analysis Cell cycle analyses were performed by analysing cells for DNA content by flow cytometry analyses using propidium iodide staining as described.21,22

TUNEL analysis To detect broken DNA in whole cells, TUNEL assays were performed as described (23) and analysed by flow cytometry.

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