Redox regulation of proliferation of lens epithelial cells in culture

Both oxidants and antioxidants have been shown to modulate cell proliferation. We studied the effects of hydrogen peroxide and two antioxidants on the rate of proliferation of lens epithelial cells in culture. Hydrogen peroxide at concentrations higher than 32 microM caused a significant inhibition of proliferation. However, in the concentration range of 0.01-0.5 microM, hydrogen peroxide stimulated the rate of proliferation. The effect of hydrogen peroxide was dependent on the amount of cells in an individual culture well, indicating decomposition of hydrogen peroxide by cellular enzymes. In order to eliminate the possibility of decomposition of the dose of hydrogen peroxide given as a bolus, we induced continual production of hydrogen peroxide by adding glucose oxidase to the incubation medium. We found that hydrogen peroxide, generated by 1-50 microU x ml(-1) of glucose oxidase significantly increased the rate of cell proliferation. This effect was most apparent at the beginning of the exponential phase of cellular growth. Glucose oxidase alone (100-500 microU x ml(-1)) did not produce any effect. The effects of pro-oxidative hydrogen peroxide were compared with the effects of two biologically important antioxidants, alpha-tocopherol and retinol. Both antioxidants completely inhibited proliferation at concentrations of 30 microM and higher. In contrast to retinol, the effect of alpha-tocopherol was dependent on the amount of cells, indicating cellular decomposition of alpha-tocopherol. The results document the possibility of redox regulation of cellular proliferation at physiologically relevant reactant concentrations.     

Redox regulation by Keap1 and Nrf2 controls intestinal stem cell proliferation in Drosophila

In Drosophila, intestinal stem cells (ISCs) respond to oxidative challenges and inflammation by increasing proliferation rates. This phenotype is part of a regenerative response, but can lead to hyperproliferation and epithelial degeneration in the aging animal. Here we show that Nrf2, a master regulator of the cellular redox state, specifically controls the proliferative activity of ISCs, promoting intestinal homeostasis. We find that Nrf2 is constitutively active in ISCs and that repression of Nrf2 by its negative regulator Keap1 is required for ISC proliferation. We further show that Nrf2 and Keap1 exert this function in ISCs by regulating the intracellular redox balance. Accordingly, loss of Nrf2 in ISCs causes accumulation of reactive oxygen species and accelerates age-related degeneration of the intestinal epithelium. Our findings establish Keap1 and Nrf2 as a critical redox management system that regulates stem cell function in high-turnover tissues.     

Apoptosis of murine BW 5147 thymoma cells induced by cold shock.

Exposure of thymoma BW 5147 cells to cold (0-2 degrees C) followed by rewarming at 37 degrees C (cold shock) resulted in internucleosomal DNA cleavage. Sensitivity to cold shock-induced cell death was critically dependent on the serum concentration in the medium and limited to serum-deficient medium (2% serum concentration), whereas cells in the complete growth medium (10%) were completely resistant. RNA/protein-synthesis inhibitors (cycloheximide and actinomycin D) had no effect on cold shock-induced DNA cleavage in BW 5147 cells. The DNA fragmentation seems to be independent of increase in the cytosolic Ca2+ level. Moreover, reduction in the calcium content of the external medium by EGTA induced DNA cleavage. Incubation of BW 5147 cells in the presence of colchicine and cytochalasin B led to the apoptosis. The latter suggests that the internucleosomal DNA cleavage induced by cold shock may be concerned with the disruption of some cytoskeletal network caused by cooling. The results are discussed in relation to cell proliferation.     

Redox regulation of cell signaling by selenocysteine in mammalian thioredoxin reductases.

The intracellular generation of reactive oxygen species, together with the thioredoxin and glutathione systems, is thought to participate in redox signaling in mammalian cells. The activity of thioredoxin is dependent on the redox status of thioredoxin reductase (TR), the activity of which in turn is dependent on a selenocysteine residue. Two mammalian TR isozymes (TR2 and TR3), in addition to that previously characterized (TR1), have now been identified in humans and mice. All three TR isozymes contain a selenocysteine residue that is located in the penultimate position at the carboxyl terminus and which is encoded by a UGA codon. The generation of reactive oxygen species in a human carcinoma cell line was shown to result in both the oxidation of the selenocysteine in TR1 and a subsequent increase in the expression of this enzyme. These observations identify the carboxyl-terminal selenocysteine of TR1 as a cellular redox sensor and support an essential role for mammalian TR isozymes in redox-regulated cell signaling.     

Potentiation of bacterial killing activity of zinc chloride by pyrrolidine dithiocarbamate

Zinc has antimicrobial activity and zinc salts including zinc chloride (ZnCl₂) have been used for the control of oral malodor. In this study, we hypothesized that pyrrolidine dithiocarbamate (PDTC), a zinc ionophore, may enhance antimicrobial efficacy of ZnCl₂. The bactericidal effectiveness of ZnCl₂ alone (0.5–8 mM) or in combination with PDTC (1 or 10 μM) was evaluated by in vitro short (1 h) time-killing assays against Fusobacterium nucleatum and Porphyromonas gingivalis. Only a slight viability decrease was observed with ZnCl₂ or PDTC alone after 1-h incubation. By contrast, combination of ZnCl₂ and PDTC could achieve a more than 100-fold viability reduction compared with ZnCl₂ or PDTC alone in F. nucleatum and P. gingivalis. Therefore, PDTC greatly enhanced the bactericidal activity of ZnCl₂ against the oral malodor-producing bacteria. These results suggest that use of PDTC may be useful for enhancing bactericidal activity of antimalodor regimens of zinc salts.     

Dual activity of pyrrolidine dithiocarbamate on kappaB-dependent gene expression in U937 cells: II. Regulation by tumour necrosis factor-alpha.

In the human promonocytic U937 cell line, pyrrolidine dithiocarbamate (PDTC) was a potent inhibitor of the nuclear factor-kappaB (NF-kappaB) signalling pathway induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). However, PDTC did not inhibit tumour necrosis factor-alpha (TNF-alpha)-induced NF-kappaB DNA binding activity but potentiated the effect of TNF-alpha on kappaB-dependent gene expression. The stimulatory effect of PDTC with TNF-alpha was not observed with an HIV-1 LTR reporter construct containing two mutated kappaB binding sites or with a construct with a mutation of the activating protein (AP)-2 binding site located between the two kappaB elements. Two distinct signalling pathways, one mediated by TPA and the other by TNF-alpha, were shown to interact, functionally defining a threshold important in the inhibitory or stimulatory effect of PDTC on kappaB-dependent gene expression. Evidence that PDTC induced AP-1 DNA binding and AP-1 reporter gene activity, raised the hypothesis that the effect of PDTC was mediated by an interaction between the AP-1 pathway and p65(RelA). Co-transfection with expression vectors for p65(RelA) and the AP-1 subunits c-Fos and c-Jun resulted in a decrease in the stimulatory effect of PDTC on HIV-1 LTR activity. Co-transfection of p65(RelA) with Tam67, a dominant negative mutant of c-Jun defective in transactivation, stimulated the effect of PDTC on HIV-1 LTR activity. Evidence that the stimulatory effect of Tam67 with PDTC was reduced with c-Jun is consistent with the hypothesis.     

Redox regulation of tumor cell toxicity by flavones from Lethedon tannaensis.

The purpose of the present work has been to seek a correlation of potential predictive value, demonstrating redox control of cytotoxicity toward human nasopharynx carcinoma (KB) cells by seven 5-hydroxy-7-methoxyflavones, together with two glycoside derivatives, all extracted from Lethedon tannaensis. In this approach, redox control is characterized by a physicochemical parameter expressing quantitatively the relative electron-donating power of the flavones, this parameter being the second order rate constant, kdelta, for quenching of singlet oxygen O2 (1deltag). This rate constant kdelta is usually related to the ability of a given molecule D to donate an electron and, thus, with the reduction potential E of the couple (D*+/D). Our results show that the flavone toxicity is linearly correlated with ease of oxidation: the higher the rate constant of reaction with singlet oxygen, the easier the oxidation, the less positive or more negative the reduction potential ((D*+/D), the higher the cytotoxicity. The results suggest new screening strategies to identify and improve potential antitumor drugs.     

Natural killer cell regulation of murine embryonic pulmonary fibroblast survival in vivo

We examined the role of the natural killer (NK) cell in controlling the survival of embryonic pulmonary fibroblasts in vivo. In vitro, both primary embryonic fibroblasts and an embryonic fibroblast line (10T1/2) were lysed by syngeneic C3H/HeN splenocytes threefold more efficiently than primary adult fibroblasts. The membrane phenotype of the effector cells was typical of NK cells. It was asialo GM1+, Lyt2.1-, Lyt 1.1-, Thy 1.2-. The cytotoxicity of the effector cell could be enhanced by IFN-alpha/beta but was deficient in the C3H/HeJ bg/bg mutant. Iododeoxyuridine (131I-dUrd)-labeled embryonic fibroblasts were injected intravenously into syngeneic mice with either enhanced or deficient NK function and their survival in the lung was quantitated. Enhanced fibroblast survival was detected in the NK deficient C3H/HeJ beige (bg/bg) mutant strain compared to its normal littermate C3H/HeJ (bg/+). A second method of NK depletion by pretreatment with rabbit anti-asialo GM1 antiserum also produced a striking increase in fibroblast survival. Poly(I:C) significantly enhanced the elimination of pulmonary fibroblasts from the lung between 4 and 24 hr after injection. Poly(I:C) did not enhance clearance of pulmonary fibroblasts in the C3H/HeJ (bg/bg) mutant, but did so in the normal littermate C3H/HeJ (bg/+). In conclusion, we have shown that the survival of embryonic pulmonary fibroblasts was inversely correlated with in vivo NK activity suggesting a possible role for this cytotoxic cell in the control of fibroblast growth in vivo.     

Dual activity of pyrrolidine dithiocarbamate on kappa B-dependent gene expression in U937 cells: I. Regulation by the phorbol ester TPA.

Pyrrolidine dithiocarbamate (PDTC) has been widely used as an inhibitor of the nuclear factor-kappa B, (NF-kappa B) signalling pathway. Here, we show that kappa B-dependent reporter gene expression induced by low concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA) is potentiated by PDTC in the human pro-monocytic U937 cell line. The stimulatory effect of PDTC on kappa B-dependent gene expression was shown with a 4 x kappa B chloramphenicol acetyltransferase construct and required an intact kappa B element in the human immunodeficiency virus long terminal repeat (HIV-1 LTR). Unexpectedly, an HIV-1 LTR construct with a mutation of the activator protein 2 (AP-2) binding site located between the two kappa B elements was unresponsive to the stimulatory effect of PDTC with TPA. The stimulation or inhibition of kappa B-dependent gene expression was dependent on PDTC pre-treatment and the concentration of TPA. No stimulatory effect on HIV-1 LTR activity was observed with the metal chelator dipyridyl or the anti-oxidant N-acetyl-L-cysteine. These results are consistent with the hypothesis that PDTC treatment potentiated kappa B-dependent gene expression in a manner dependent on the concentration of TPA.     

Redox regulation in plant programmed cell death

Programmed cell death (PCD) is a genetically controlled process described both in eukaryotic and prokaryotic organisms. Even if it is clear that PCD occurs in plants, in response to various developmental and environmental stimuli, the signalling pathways involved in the triggering of this cell suicide remain to be characterized. In this review, the main similarities and differences in the players involved in plant and animal PCD are outlined. Particular attention is paid to the role of reactive oxygen species (ROS) as key inducers of PCD in plants. The involvement of different kinds of ROS, different sites of ROS production, as well as their interaction with other molecules, is crucial in activating PCD in response to specific stimuli. Moreover, the importance is stressed on the balance between ROS production and scavenging, in various cell compartments, for the activation of specific steps in the signalling pathways triggering this cell suicide process. The review focuses on the complexity of the interplay between ROS and antioxidant molecules and enzymes in determining the most suitable redox environment required for the occurrence of different forms of PCD.     

Redox regulation of matrix metalloproteinase gene family in small cell lung cancer cells

It has been implicated that reactive oxygen species (ROS) play important roles in modulating tumor progression. However, the mechanisms by which redox-regulated tumor progression are largely unknown. We previously demonstrated that reduced intracellular redox conditions could be achieved in stably transfected small cell lung cancer cells with gamma-glutamylcysteine synthetase (gamma-GCSh) cDNA which encodes a rate-limiting enzyme in the biosynthesis of glutathione (GSH), a major physiological redox regulator. In the present study, using DNA microarray analyses, we compared the expression profiles between the gamma-GCSh-transfected cells and their nontransfected counterpart. We observed downregulation of several matrix metalloproteinases (MMPs), i.e., MMPI and MMP3, and MMP10 in the transfected cells. Dot blot and Northern blot hybridizations confirmed that, among the 18 MMP gene family members and four tissue inhibitors of matrix metalloprotein family (TIMP) analyzed, the expression levels of these three MMPs were consistently reduced. Transiently increased gamma-GCSh expression using tetracycline-inducible gamma-GCSh adenoviral expression system also showed down-regulation of MMP3 and MMP10, but not MMP1. Our results demonstrated that redox regulation of MMP1, MMP3 and MMP10 expression depend upon different modes of redox manipulation. These results bear implication that antioxidant modulation of antitumor progression may be contributed at least in part by the downregulation of a subset of metrix metalloproteins.     

Apoptosis of murine BW 5147 thymoma cells induced by dexamethasone and gamma-irradiation

The mode and the kinetics of the death of T-thymoma cells upon dexamethasone treatment and gamma-irradiation (10Gy) have been studied using flow cytometry and biochemical analysis. It has been shown that the hormone and gamma-irradiation induce cell death by apoptosis. In both cases the cells are initially blocked in G2/M and die only after overcoming the blockage and cytokinesis. A short exposure to dexamethasone results in a cytostatic effect, whereas a cytotoxic effect is absent. Reducing serum concentration to 2% causes more rapid death both following gamma-irradiation and dexamethasone. These results are discussed in relation to cell death and proliferation.     

Toxoplasma gondii: selective killing of extracellular parasites by oxidation using pyrrolidine dithiocarbamate

Extracellular Toxoplasma parasites are sensitive to pyrrolidine dithiocarbamate (PDTC) at low micromolar concentrations. Loss of parasite viability following PDTC treatment is shown to be mediated by oxidation, which is reminiscent of PDTC killing in mammalian cells. Intracellular parasites, by contrast, are resistant to PDTC killing, although treatment does cause reversible growth arrest. In addition to the possible implications relative to the biology of the parasite, these observations suggest that PDTC could be of use in eliminating undesired extracellular parasites during assays and selections in vitro.