Julia E. Sim

Glucose deprivation-induced oxidative stress in human tumor cells. A fundamental defect in metabolism?

Abstract: Recently, glucose deprivation‐induced oxidative stress has been shown to cause cytotoxicity, activation of signal transduction (i.e., ERK1, ERK2, JNK, and Lyn kinase), and increased expression of genes associated with malignancy (i.e., bFGF and c‐Myc) in MCF‐7/ADR human breast cancer cells. These results have led to the proposal that intracellular oxidation/reduction reactions involving hydroperoxides and thiols may provide a mechanistic link between metabolism, signal transduction, and gene expression in these human tumor cells. The current study shows that several other transformed human cell types appear to be more susceptible to glucose deprivation‐induced cytotoxicity and oxidative stress than untransformed human cell types. In a matched pair of normal and SV40‐transformed human fibroblasts the cytotoxic process is shown to be dependent upon ambient O2 concentration. A theoretical model to explain the results is presented and implications to unifying modern theories of cancer are discussed.

Mitochondrial and H2O2 Mediate Glucose Deprivation-induced Stress in Human Cancer Cells

The hypothesis that glucose deprivation-induced cytotoxicity in transformed human cells is mediated by mitochondrial \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} and H2O2 was first tested by exposing glucose-deprived SV40-transformed human fibroblasts (GM00637G) to electron transport chain blockers (ETCBs) known to increase mitochondrial \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} and H2O2 production (antimycin A (AntA), myxothiazol (Myx), or rotenone (Rot)). Glucose deprivation (2–8 h) in the presence of ETCBs enhanced parameters indicative of oxidative stress (i.e. GSSG and steady-state levels of oxygen-centered radicals) as well as cytotoxicity. Glucose deprivation in the presence of AntA also significantly enhanced cytotoxicity and parameters indicative of oxidative stress in several different human cancer cell lines (PC-3, DU145, MDA-MB231, and HT-29). In addition, human osteosarcoma cells lacking functional mitochondrial electron transport chains (rho(0)) were resistant to glucose deprivation-induced cytotoxicity and oxidative stress in the presence of AntA. In the absence of ETCBs, aminotriazole-mediated inactivation of catalase in PC-3 cells demonstrated increases in intracellular steady-state levels of H2O2 during glucose deprivation. Finally, in the absence of ETCBs, overexpression of manganese containing superoxide dismutase and/or mitochondrial targeted catalase using adenoviral vectors significantly protected PC-3 cells from toxicity and oxidative stress induced by glucose deprivation with expression of both enzymes providing greater protection than was seen with either alone. Overall, these findings strongly support the hypothesis that mitochondrial \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} and H2O2 significantly contribute to glucose deprivation-induced cytotoxicity and metabolic oxidative stress in human cancer cells.

Evaluation of Parameters of Oxidative Stress after In Vitro Exposure to FMCW- and CDMA-Modulated Radiofrequency Radiation Fields

Abstract Hook, G. J., Spitz, D. R., Sim, J. E., Higashikubo, R., Baty, J. D., Moros, E. G. and Roti Roti, J. L. Evaluation of Parameters of Oxidative Stress after In Vitro Exposure to FMCW- and CDMA-Modulated Radiofrequency Radiation Fields. Radiat. Res. 162, 497–504 (2004). The goal of this study was to determine whether radiofrequency (RF) radiation is capable of inducing oxidative stress or affecting the response to oxidative stress in cultured mammalian cells. The two types of RF radiation investigated were frequency-modulated continuous-wave with a carrier frequency of 835.62 MHz (FMCW) and code division multiple access centered on 847.74 MHz (CDMA). To evaluate the effect of RF radiation on oxidative stress, J774.16 mouse macrophage cells were stimulated with γ-interferon (IFN) and bacterial lipopolysaccharide (LPS) prior to exposure. Cell cultures were exposed for 20–22 h to a specific absorption rate of 0.8 W/kg at a temperature of 37.0 ± 0.3°C. Oxidative stress was evaluated by measuring oxidant levels, antioxidant levels, oxidative damage and nitric oxide production. Oxidation of thiols was measured by monitoring the accumulation of glutathione disulfide (GSSG). Cellular antioxidant defenses were evaluated by measuring superoxide dismutase activity (CuZnSOD and MnSOD) as well as catalase and glutathione peroxidase activity. The trypan blue dye exclusion assay was used to measure any changes in viability. The results of these studies indicated that FMCW- and CDMA-modulated RF radiation did not alter parameters indicative of oxidative stress in J774.16 cells. FMCW- and CDMA-modulated fields did not alter the level of intracellular oxidants, accumulation of GSSG or induction of antioxidant defenses in IFN/LPS-stimulated cells. Consistent with the lack of an effect on oxidative stress parameters, no change in toxicity was observed in J774.16 cells after either optimal (with or without inhibitors of nitric oxide synthase) or suboptimal stimulation.

Dominant-negative Jun N-terminal protein kinase (JNK-1) inhibits metabolic oxidative stress during glucose deprivation in a human breast carcinoma cell line.

Signal transduction pathway involved in glucose deprivation-induced oxidative stress were investigated in human breast carcinoma cells (MCF-7/ADR). In MCF-7/ADR, glucose deprivation-induced prolonged activation of c-Jun N-terminal kinase (JNK1) as well as cytoxicity and the accumulation of oxidized glutathione. Glucose deprivation also caused significant increases in total glutathione, cysteine, gamma-glutamylcysteine, and immunoreactive proteins corresponding to the catalytic as well as regulatory subunits of gamma-glutamylcysteine, and immunoreactive proteins corresponding to the catalytic as well as regulatory subunits of gamma-glutamylcysteine synthetase, suggesting that the synthesis of glutathione increased as an adaptive response. Expression of a catalytically inactive dominant negative JNK1 in MCF-7/ADR inhibited glucose deprivation- induced cell death and the accumulation of oxidized glutathione as well as altered the duration of JNK activation from persistent (> 2 h) to transient (30 min). In addition, stimulation of glutathione synthesis during glucose deprivation was not observed in cells expressing the highest levels of dominant negative protein. Finally, a linear dose response suppression of oxidized glutathione accumulation was noted for clones expressing increasing levels of dominant negative JNK1 during glucose deprivation. These results show that expression of a dominant negative JNK1 protein was capable of suppressing persistent JNK activation as well as oxidative stress and cytotoxicity caused by glucose deprivation in MCF-7/ADR. These findings support the hypothesis that JNK signaling pathways may control the expression of proteins contributing to cell death mediated by metabolic oxidative stress during glucose deprivation. Finally, these results support the concept that JNK signaling-induced shifts in oxidative metabolism may provide a general mechanism for understanding the diverse biological effects seen during the activation of JNK signaling cascades.

Alterations in heat-induced radiosensitization accompanied by nuclear structure alterations in Chinese hamster cells

This paper examined heat-induced radiosensitization in two Chinese hamster heat-resistant cell lines, HR-1 and OC-14, that were isolated from the same wild-type HA-1 cell line. It found a reduction of the magnitude of heat-induced radiosensitization after exposure to 43°C in both HR-1 and OC-14 cells and a similar reduction after exposure to 45°C in HR-1 cells, but not in OC-14 cells. The effect of heat exposure on a class of ionizing radiation-induced DNA damage that inhibits the ability of nuclear DNA to undergo super-coiling changes was also studied using the fluorescent halo assay in these three cell lines. Wild type cells exposed to either 43 or 45°C before irradiation had a DNA rewinding ability that was intermediate between control and unheated cells, a phenomenon previously described as a masking effect. This masking effect was significantly reduced in HR-1 cells exposed to either 43 or 45°C or in OC-14 cells exposed to 43°C under conditions that heat-induced radiosensitization was reduced. In contrast, the masking effect was not altered in OC-14 cells exposed to 45°C, conditions under which heat-induced radiosensitization was similar to that observed in wild-type HA-1 cells. These results suggest that a reduction in the masking effect is associated with a reduction of the magnitude of heat-induced radiosensitization in the HR-1 and OC-14 heat-resistant cell lines. The reduction of the masking effect in the cell lines resistant to heat-induced radiosensitization was associated with neither a reduction in the magnitude of the heat-induced increase in total nuclear protein content nor major differences in the protein profiles of the nucleoids isolated from heated cells.