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. 2003 Jan 7;100(1):143-8.
doi: 10.1073/pnas.0237083100. Epub 2002 Dec 23.

Nitric oxide-induced cellular stress and p53 activation in chronic inflammation

Lorne J Hofseth  1 , Shin'ichi Saito, S Perwez Hussain, Michael G Espey, Katrina M Miranda, Yuzuru Araki, Chamelli Jhappan, Yuichiro Higashimoto, Peijun He, Steven P Linke, Martha M Quezado, Irit Zurer, Varda Rotter, David A Wink, Ettore Appella, Curtis C Harris
Affiliations

Nitric oxide-induced cellular stress and p53 activation in chronic inflammation

Lorne J Hofseth et al. Proc Natl Acad Sci U S A. .

Abstract

Free radical-induced cellular stress contributes to cancer during chronic inflammation. Here, we investigated mechanisms of p53 activation by the free radical, NO. NO from donor drugs induced both ataxia-telangiectasia mutated (ATM)- and ataxia-telangiectasia mutated and Rad3-related-dependent p53 posttranslational modifications, leading to an increase in p53 transcriptional targets and a G(2)M cell cycle checkpoint. Such modifications were also identified in cells cocultured with NO-releasing macrophages. In noncancerous colon tissues from patients with ulcerative colitis (a cancer-prone chronic inflammatory disease), inducible NO synthase protein levels were positively correlated with p53 serine 15 phosphorylation levels. Immunostaining of HDM-2 and p21(WAF1) was consistent with transcriptionally active p53. Our study highlights a pivotal role of NO in the induction of cellular stress and the activation of a p53 response pathway during chronic inflammation.

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Figures

Figure 1
Figure 1
(a) DNA damage is induced in MCF-7 cells after exposure to 0.5 mM SPER/NO. Cells were exposed for 4 h as indicated, then processed for the alkaline comet assay. At least 50 comets were quantified per treatment. (b) Time-course increase in p53 posttranslational modifications and p53 levels after exposure to 0.5 mM SPER/NO. Equal amounts of protein were immunoprecipitated, and Western blot assays were performed. HCT 116 cells genetically engineered to be p53−/− were used as a negative control. UV treatment (25 J/m2, 24 h) was used as a positive control. Purified p53 from baculovirus infected cells was loaded onto the gel to identify the specific p53 band (not shown). (c) p53 accumulated and was posttranslationally modified in MCF-7 cells cocultured with NO-releasing ANA-1 macrophages. MCF-7 cells were either unexposed or exposed to unactivated or activated ANA-1 macrophages as described in Methods. Appropriate controls used are indicated. After 8 h of coincubation, cells were lysed, and Western blot assays were performed. Lane 1, MCF-7 cells only; lane 2, MCF-7 cells + unstimulated ANA-1 cells; lane 3, MCF-7 cells + stimulated ANA-1 cells; lane 4, unstimulated ANA-1 cells only; lane 5, stimulated ANA-1 cells only; lane 6, MCF-7 cells + cytokine stimulation; lane 7, MCF-7 cells + cytokine stimulation + l-NMMA (250 μM); lane 8, MCF-7 cells + stimulated ANA-1 cells + l-NMMA (250 μM); lane 9, HCT 116 p53−/− cells; lane 10, MCF-7 cells + UV. Relative nitrate plus nitrite levels are also shown.
Figure 2
Figure 2
(a) ATM partially mediates NO-induced P-Ser-18 in MEFs. ATM+/+ or ATM−/− MEFs were exposed to 0.5 mM SPER/NO, 5 Gy γ-irradiation, or 25 J/m2 UV for indicated time points (hr). Cells were lysed, and Western blot assays were performed. (b) Bar graphs representing quantitative densitometry of Western blot bands shown in a. (c) ATM and ATR mediate NO-induced P-Ser-15 in human cells. Human lymphoblastoid cells from a healthy individual (C3ABR) or an individual with ataxia telangiectasia (AT) were exposed to 0.5 mM SPER/NO ± caffeine (1 mg/ml) for indicated time points (hr). The calpain inhibitor, ALLN (20 μM), which inhibits the proteasome, was used as a negative control for posttranslational modifications; MCF-7 cells exposed to 25 J/m2 UV were used as a positive antibody control. Cells were lysed, and Western blot assays were performed. (d) Bar graphs representing quantitative densitometry of Western blot bands shown in c.
Figure 3
Figure 3
(a) Specific proteins are induced in a p53-dependent manner after exposure of colon cancer cells to 1 mM SPER/NO. HCT 116 and HCT 116 p53−/− cells were exposed to the 0.5 mM SPER/NO for indicated time points (hr), then lysed. Western blot assays were performed. Numbers above the bands indicate densitometry values as a ratio relative to control values. (b) There is a p53- and p21-dependent G2/M arrest in colon cancer cells exposed to NO. HCT 116 cells (●), HCT 116 p53−/− (○), or HCT 116 p21−/− (▴) cells were exposed to 1 mM SPER/NO for indicated time periods (hr). Cells were harvested at the same time, then FACS analysis was performed. Data are presented as a percentage relative to untreated cells. Gating was done according to Fig. 7e.
Figure 4
Figure 4
(a) iNOS, P-Ser-15, and p53 protein levels are elevated in colon tissues from UC patients. Two samples (A and B) were taken from surgical specimens derived from each of 11 UC patients. Results from four patients (eight samples) are shown here. Results from the other seven patients are shown in Fig. 8b. Representative colon postmortem tissues from five donors without disease (control samples) also were included (two samples are shown). (b) iNOS and P-Ser-15 levels, and P-Ser-15 and p53, are correlated in UC colon tissues. After densitometry, the Spearman rank correlation coefficient was calculated to examine the relationship among iNOS levels, p53 levels, and levels of p53 posttranslational modifications. (c) iNOS, P-Ser-15, and p53 downstream protein levels are elevated in UC patients. Serial tangential sections of crypts of Leiberkuhn were exposed to indicated antibodies, as described in Methods. (Left) Normal colon. (Top to Bottom) Anti-iNOS, antiphosphoserine-15, anti- p21WAF1, anti-HDM-2. (Right) UC colon. (Top to Bottom) Anti-iNOS (*, a lymphocytic aggregate, consistent with an inflammatory infiltrate; E, epithelial; and S, stromal cells of the UC tissue), antiphosphoserine-15, anti-p21WAF1, anti-HDM-2. Antibody controls are shown in Fig. 8b. All are ×100; Inset is ×400.
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