Paul Amstad

The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-κB

BACKGROUND The inducible, higher eukaryotic transcription factor NF-kappa B is activated by a variety of stimuli. Several lines of evidence have suggested that reactive oxygen intermediates (ROIs) serve as messengers for most if not all of these stimuli. To identify the relevant ROI species and to gain more direct evidence for an involvement of ROIs as messengers, we investigated whether changes in the levels of enzymes that control intracellular ROI levels affect the activation of NF-kappa B. RESULTS Cell lines stably overexpressing the H2O2-degrading enzyme catalase were deficient in activating NF-kappa B in response to tumor necrosis factor alpha (TNF) or okadaic acid. The catalase inhibitor aminotriazol restored NF-kappa B induction. In contrast, stable overexpression of cytoplasmic Cu/Zn-dependent superoxide dismutase (SOD), which enhances the production of H2O2 from superoxide, potentiated NF-kappa B activation. The level of cytoplasmic NF-kappa B-I kappa B complex was unchanged, indicating that synthesis of NF-kappa B was not affected. CONCLUSIONS Our data show that one ROI species, H2O2 acts as a messenger in the TNF- and okadaic acid-induced post-translational activation of NF-kappa B. Superoxide is only indirectly involved, as a source for H2O2. These data explain the inhibitory effects of many antioxidative compounds on the activation of NF-kappa B and its target genes. H2O2 is overproduced in response to various stimuli, and normal levels of catalase appear insufficient to remove it completely. H2O2 can therefore accumulate and act as an intracellular messenger molecule in the response to pathogens.

p53-Induced Up-Regulation of MnSOD and GPx but not Catalase Increases Oxidative Stress and Apoptosis

p53-mediated apoptosis may involve the induction of redox-controlling genes, resulting in the production of reactive oxygen species. Microarray expression analysis of doxorubicin exposed, related human lymphoblasts, p53 wild-type (WT) Tk6, and p53 mutant WTK1 identified the p53-dependent up-regulation of manganese superoxide dismutase (MnSOD) and glutathione peroxidase 1 (GPx). Consensus p53 binding sequences were identified in human MnSOD and GPx promoter regions. A 3-fold increase in the MnSOD promoter activity was observed after the induction of p53 in Li-Fraumeni syndrome (LFS) fibroblast, TR9-7, expressing p53 under the control of a tetracycline-regulated promoter. An increased protein expression of endogenous MnSOD and GPx also positively correlated with the level of p53 induction in TR9-7 cells. However, catalase (CAT) protein expression remained unaltered after p53 induction. We also examined the expression of MnSOD, GPx, and CAT in a panel of normal or LFS fibroblasts, containing either WT or mutant p53. We found increased MnSOD enzymatic activity, MnSOD mRNA expression, and MnSOD and GPx protein in LFS fibroblasts carrying a WT p53 allele when compared with homozygous mutant p53 isogenic cells. The CAT protein level was unchanged in these cells. We observed both the release of cytochrome C and Ca2+ from the mitochondria into the cytoplasm and an increased frequency of apoptotic cells after p53 induction in the TR9-7 cells that coincided with an increased expression of MnSOD and GPx, and the level of reactive oxygen species. The increase in apoptosis was reduced by the antioxidant N-acetylcysteine. These results identify a novel mechanism of p53-dependent apoptosis in which p53-mediated up-regulation of MnSOD and GPx, but not CAT, produces an imbalance in antioxidant enzymes and oxidative stress.

Identification of Hydrogen Peroxide as the Relevant Messenger in the Activation Pathway of Transcription Factor NF-κB

The inducible higher eukaryotic transcription factor NF-κB is activated by a large variety of distinct simuli [1–3]. In unstimulated cells, this factor resides in a latent form in the cytoplasm [4]. Latency is achieved by association of the DNA-binding NF-κB dimer with an inhibitory subunit, called IκB [5]. IΚB suppresses DNA-binding and nuclear transport of NF-κB. Upon stimulation of cells, IκB is phosphorylated and proteolytically degraded [6–9]. Both reactions are required for activation [10]. The released NF-κB is then translocated to the nucleus where it initiates transcription of target genes. Among the numerous proteins which are induced by a concerted action of NF-kB with other transcription factors are cytokines, chemokines, cell adhesion molecules, hematopoetic growth factors and receptors, histocompatibility antigens and acute phase proteins [1–3]. While NF-κB may be indispensable as inducer of many immediate-early inflammatory and immune reactions, the transcription factor is likely to play a fatal role in certain diseases and syndromes that involve an abberrant expression of inflammatory cytokines [22–24].

Oxidant Carcinogenesis and Antioxidant Defensea

Growth promotion by oxidants is observed with cultured human and mouse fibroblasts as well as epidermal cells. It is expected to play a role in inflammation, fibrosis, and tumorigenesis. Indeed, oxidants trigger (patho)physiological reactions that resemble those induced by growth and differentiation factors. For example, active oxygen activates protein kinases. causes DNA breakage, and induces the growth competence‐related protooncogenes c‐fos and c‐myc.

Cellular and Molecular Studies of Growth, Differentiation and Neoplastic Transformation of Human Bronchial Epithelial Cells in Vitro

Normal human cells in vitro appear to retain many normal phenotypic properties, remain diploid, eventually undergo senescence and rarely, if ever “spontaneously” transform to malignant cells. Retained properties may include synthesis of classes of proteins associated with specific cell types such as collagens, keratins, or melanin; responsiveness to hormones; and antigenic specificity. In addition, human cells with abnormal phenotypes such as either enzymatic deficits or malignant properties frequently maintain these phenotype in vitro. Human cells cultured in vitro have thus proven to be extremely useful to scientists studying the molecular and biochemical aspects of human carcinogenesis. Such studies have been facilitated by the recent development of improved methods for culturing normal human epithelial tissues and cells1. Chemically defined media have been developed for culturing many of these tissues and cells from normal organs, including those with a high rate of cancer in humans. Serum-free media have several advantages in studies of cultured human cells, including: (a) less experimental variability compared to serum-containing media; (b) selective growth conditions for normal cells of different types (e.g. epithelial versus fibroblastic) or for normal versus malignant cells; (c) ease of identification of growth factors, inhibitors of growth, and inducers of differentiation; and (d) ease of isolating and analyzing secreted cellular products. Advances in cell biology, including the delineation of biochemical and morphological markers of specific cell types, have also facilitated the identification of cells in vitro (including keratins as markers for epithelial cells and collagen types I and III for identifying fibroblasts). These advances have created experimental approaches to answering critical questions in human cell carcinogenesis1,2.

Repair and Expression of Aflatoxin B1-Induced DNA Damage

The chemical properties, cellular repair and biological expression of aflatoxin B1 (AFB1)-DNA adducts were investigated in mouse embryo fibroblasts 10T1/2, human epithelioid lung cells A549 and human skin fibroblasts (NF). These studies were complicated by the inherent chemical instability of AFB1-DNA adducts. In order to distinguish enzymatic reactions from spontaneous decomposition it was necessary to compare routinely the reactions occurring intracellularly to those of free AFB1-DNA in vitro and of AFB1-DNA in situ in excision repair deficient Xeroderma pigmentosum (XPA) fibroblasts. Following treatment of very actively metabolizing 10T1/2 cells with the procarcinogen AFB1 or of NF and A549 with microsome activated AFB1, approximately 90% of the adducts corresponded to 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-AFB1 (AFB1-N 7 -Gua). AFB1-DNA adducts were introduced preferentially in nucleosomal-linker relative to — core DNA both after cellular metabolic activation of AFB1 and exogenous activation by microsomes. For the determination of the nucleosomal distribution, the primary adducts, AFB1-N7-Gua, were transformed into the chemically more stable secondary products 2,3-dihydro-2(N5-formyl–2′,5′,6′-triamino-4′-oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1 (AFB1-triamino-Py) by a short exposure of the AFB1-treated cells to pH 9.5 medium.