Cell cycle-coupled variation in topoisomerase IIalpha mRNA is regulated by the 3'-untranslated region. Possible role of redox-sensitive protein binding in mRNA accumulation.

Mammalian topoisomerase IIalpha (Topo II) is a highly regulated enzyme essential for many cellular processes including the G(2) cell cycle checkpoint. Because Topo II gene expression is regulated posttranscriptionally during the cell cycle, we investigated the possible role of the 3'-untranslated region (3'-UTR) in controlling Topo II mRNA accumulation. Reporter assays in stably transfected cells demonstrated that, similar to endogenous Topo II mRNA levels, the mRNA levels of reporter genes containing the Topo II 3'-UTR varied during the cell cycle and were maximal in S and G(2)/M relative to G(1). Topo II 3'-UTR sequence analysis and RNA-protein binding assays identified a 177-nucleotide (base pairs 4772-4949) region containing an AUUUUUA motif sufficient for protein binding. Multiple proteins (84, 70, 44, and 37 kDa) bound this region, and the binding of 84- and 37-kDa (tentatively identified as the adenosine- or uridine-rich element-binding factor AUF1) proteins was enhanced in G(1), correlating with decreased Topo II mRNA levels. The binding activity was enhanced in cellular extracts or cells treated with thiol-reducing agents, and increased binding correlated with decreased Topo II mRNA levels. These results support the hypothesis that cell cycle-coupled Topo II gene expression is regulated by interaction of the 3'-UTR with redox-sensitive protein complexes.

Glial cell type-specific responses to menadione-induced oxidative stress.

Glial cell types in the central nervous system are continuously exposed to reactive oxygen species (ROS) due to their high oxygen metabolism and demonstrate differential susceptibility to certain pathological conditions believed to involve oxidative stress. The purpose of the current studies was to test the hypothesis that mtDNA damage could contribute to the differential susceptibility of glial cell types to apoptosis induced by oxidative stress. Primary cultures of rat astrocytes, oligodendrocytes, and microglia were utilized, and menadione was used to produce the oxidative stress. Apoptosis was detected and quantitated in menadione-treated oligodendrocytes and microglia (but not astrocytes) using either positive annexin-V staining or positive staining for 3'-OH groups in DNA. The apoptotic pathway that was activated involved the release of cytochrome c from the intermitochondrial space and activation of caspase 9. Caspase 8 was not activated after exposure to menadione in any of the cells. Using equimolar concentrations of menadione, more initial damage was observed in mtDNA from oligodendrocytes and microglia. Additionally, using concentrations of menadione that resulted in comparable initial mtDNA damage, more efficient repair was observed in astrocytes compared to either oligodendrocytes or microglia. The differential susceptibility of glial cell types to oxidative damage and apoptosis did not appear related to cellular antioxidant capacity, because under the current culture conditions astrocytes had lower total glutathione content and superoxide dismutase activity than oligodendrocytes and microglia. These results show that the differential susceptibility of glial cell types to menadione-induced oxidative stress and apoptosis appears to correlate with increased oxidative mtDNA damage and support the hypothesis that mtDNA damage could participate in the initiation of apoptosis through the enhanced release of cytochrome c and the activation of caspase 9.

A spectrophotometric method for the direct detection and quantitation of nitric oxide, nitrite, and nitrate in cell culture media.

A method for the spectrophotometric determination of nitric oxide, nitrite, and nitrate in tissue culture media is presented. The method is based on the nitric oxide-mediated nitrosative modification of sulfanilic acid that reacts with N-(1-naphthyl)ethylenediamine dihydrochloride forming an orange-colored product absorbing at 496 nm. Nitric oxide levels were determined in culture media from this absorbance measurement using chemiluminescence standardization. Extinction coefficients of 5400 and 6600 M(-1) cm(-1) were determined for the nitric oxide product in assay solutions containing 0.1 or 100 mM KPO4 buffer (pH 7.4), respectively, with a limit of detection of 1 microM. Acidification of these reactions (pH 2.4) generated a pink-colored product absorbing at 540 nm allowing for quantitation of total nitric oxide/nitrite levels using extinction coefficients of 38,000 and 36,900 M(-1) cm(-1), for the assay solutions described. The limit of detection of this assay was approximately 300 nM. Using the 100 mM KPO4 buffer system, nitrate levels were determined following reduction to nitrite using a copper-coated cadmium reagent with an extinction coefficient of 29,500 M(-1) cm(-1) and a detection limit of 0.5 microM. The utility of these assays was demonstrated in the standardization of nitric oxide-saturated cell culture media, and the release of nitric oxide by the NONOate compound DEA/NO.

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

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.

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.

Metabolic oxidative stress activates signal transduction and gene expression during glucose deprivation in human tumor cells.

The mechanism of glucose deprivation-induced activation of Lyn kinase (Lyn), c-Jun N-terminal kinase 1 (JNK1) and increased expression of basic fibroblast growth factor (bFGF) and c-Myc was investigated in MCF-7/ADR adriamycin-resistant human breast carcinoma cells. Glucose deprivation significantly increased steady state levels of oxidized glutathione content (GSSG) and intracellular prooxidants (presumably hydroperoxides) as well as caused the activation of Lyn, JNK1, and the accumulation of bFGF and c-Myc mRNA. The suppression of GSSG accumulation and prooxidant production by treatment with the thiol antioxidant, N-acetylcysteine, also suppressed all the increases in kinase activation and gene expression observed during glucose deprivation. In addition, glucose deprivation was shown to induce oxidative stress in IMR90 SV40 transformed human fibroblasts, indicating that this phenomena is not limited to the MCF-7/ADR cell line. These and previous observations from our laboratory show that glucose deprivation-induced oxidative stress in MCF-7/ADR cells activates signal transduction involving Lyn, JNK1, and mitogen activated protein kinases (ERK1/ERK2) which results in increased bFGF and c-Myc mRNA accumulation. These results provide support for the hypothesis that alterations in intracellular oxidation/reduction reactions link changes in glycolytic metabolism to signal transduction and gene expression in these human tumor cells.

Genomic instability and catalase gene amplification induced by chronic exposure to oxidative stress.

Chronic exposure (>200 days) of HA1 fibroblasts to increasing concentrations of H2O2 or O2 results in the development of a stable oxidative stress-resistant phenotype characterized by increased cellular antioxidant levels, particularly catalase (D. R. Spitz et al, Arch. Biochem. Biophys., 279: 249-260, 1990; D. R. Spitz et al., Arch. Biochem. Biophys., 292: 221-227, 1992; S. J. Sullivan et al., Am. J. Physiol. (Lung Cell. Mol. Physiol.), 262: L748-L756, 1992). Acutely stressed cells failed to develop a stably resistant phenotype or increased catalase activity, suggesting that chronic exposure is required for the development of this phenotype. This study investigates the mechanism underlying increased catalase activity in the H2O2- and O2-resistant cell lines. In H2O2- and O2-resistant cells, catalase activity was found to be 20-30-fold higher than that in the parental HA1 cells and correlated with increased immunoreactive catalase protein and steady-state catalase mRNA levels. Resistant cell lines also demonstrated a 4-6-fold increase in catalase gene copy number by Southern blot analysis, which is indicative of gene amplification. Chromosome banding and in situ hybridization studies identified a single amplified catalase gene site located on a rearranged chromosome with banding similarities to Z-4 in the hamster fibroblast karyotype. Simultaneous in situ hybridization with a Z-4-specific adenine phosphoribosyltransferase (APRT) gene revealed that the amplified catalase genes were located proximate to APRT on the same chromosome in all resistant cells. In contrast, HA1 cells contained only single copies of the catalase gene that were not located on APRT-containing chromosomes, indicating that amplification is associated with a chromosomal rearrangement possibly involving Z-4. The fact that chronic exposure of HA1 cells to either HO2 or 95% O2 resulted in gene amplification suggests that gene amplification represents a generalized response to oxidative stress, contributing to the development of resistant phenotypes. These results support the hypothesis that chronic exposure to endogenous metabolic or exogenous environmental oxidative stress represents an important factor contributing to gene amplification and genomic instability.

Bcl-2 and Bcl-xL in peroxide-resistant A549 and U87MG cells.

Overexpression of the bcl-2 and the related bcl-xL protooncogene proteins enhance cell survival by inhibiting apoptosis induced by many agents including oxidants. Whether these proteins contribute to survival in oxidant-resistant cells is not known. The current study assessed the expression of bcl-2 and bcl-xL proteins in human glioblastoma U87MG cells and human lung adenocarcinoma A549 cells selected for resistance to 0, 50, 100, 200, and 400 microM H2O2 by exposure to this oxidant one time each passage for 9 months. When examined 7 to 32 days after cessation of peroxide exposure (times when peroxide resistance was maintained), bcl-2 protein levels were significantly increased in most peroxide-resistant U87MG cells. However, the increase was not dose dependent and was not accompanied by an increase in mRNA levels. A549 cells did not express significant levels of bcl-2 protein, although bcl-2 mRNA was detectable. A549 cells expressed large amounts of bcl-xL and immunohistochemistry demonstrated extensive localization of this protein around the nucleus. However, expression of this protein was not altered in peroxide-resistant lines nor was bcl-2 protein increased to a measurable level. U87MG cells also expressed bcl-xL but it was not altered in peroxide-resistant cells. Although the increased bcl-2 protein in peroxide-resistant U87MG cells may contribute to their oxidant tolerance, the lack of a dose-response relationship, the failure to induce bcl-xL protein, and the absence of any bcl-2 or bcl-xL protein induction in peroxide-resistant A549 cells suggest these genes are not primary factors in oxidant resistance.

Increased lipid peroxidation and impaired antioxidant enzyme function is associated with pathological liver injury in experimental alcoholic liver disease in rats fed diets high in corn oil and fish oil.

Increased hepatic oxidative stress with ethanol administration is hypothesized to be caused either by enhanced pro-oxidant production or decreased levels of antioxidants or both. We used the intragastric feeding rat model to assess the relationship between hepatic antioxidant enzymes and pathological liver injury in animals fed different dietary fats. Male Wistar rats (5 per group) were fed ethanol with either medium-chain triglycerides (MCTE), palm oil (PE), corn oil (CE), or fish oil (FE). Control animals were fed isocaloric amounts of dextrose instead of ethanol with the same diets. The following were evaluated in each group: liver pathology, lipid peroxidation, manganese superoxide dismutase (MnSOD) levels, copper-zinc SOD (CuZnSOD) levels, glutathione peroxidase (GPX) levels, and catalase (CAT) levels. All enzymes were evaluated using activity assays and immunoblots. Rats fed FE showed the most severe pathology (fatty liver, necrosis, and inflammation), those fed CE showed moderate changes, those fed PE showed fatty liver only, and those fed MCTE were normal. Parameters indicative of lipid peroxidation (conjugated dienes and thiobarbituric acid-reactive substances) were also greater in rat livers from animals fed the diets high in polyunsaturated fatty acids (CE and FE). CuZnSOD, GPX, and CAT activities showed an inverse correlation (r=-.92, P < .01) with severity of pathological injury, with the lowest levels for both enzymes found in FE-fed rats. Decreased enzyme activity in CE- and FE-fed rats was accompanied by similar decreases in immunoreactive protein. Ethanol administration did not cause significant decreases in enzyme activity in groups that showed no necroinflammatory changes (MCTE and PE). MnSOD activity showed no significant change in any ethanol-fed group. Our results show that decreases in CuZnSOD, GPX, and CAT occur in rats showing pathological liver injury and also having the highest levels of lipid peroxidation. These results suggest that feeding dietary substrates that enhance lipid peroxidation can exacerbate both ethanol-induced oxidative damage as well as necroinflammatory changes. The decrease in activity of antioxidant enzymes observed in animals fed diets high in polyunsaturated fatty acids and ethanol could possibly increase the susceptibility to oxidative damage and further contribute to ethanol-induced liver injury.

Glucose deprivation-induced cytotoxicity and alterations in mitogen-activated protein kinase activation are mediated by oxidative stress in multidrug-resistant human breast carcinoma cells.

We previously observed that glucose deprivation induces cell death in multidrug-resistant human breast carcinoma cells (MCF-7/ADR). As a follow up we wished to test the hypothesis that metabolic oxidative stress was the causative process or at least the link between causative processes behind the cytotoxicity. In the studies described here, we demonstrate that mitogen-activated protein kinase (MAPK) was activated within 3 min of being in glucose-free medium and remained activated for 3 h. Glucose deprivation for 2-4 h also caused oxidative stress as evidenced by a 3-fold greater steady state concentration of oxidized glutathione and a 3-fold increase in pro-oxidant production. Glucose and glutamate treatment rapidly suppressed MAPK activation and rescued cells from cytotoxicity. Glutamate and the peroxide scavenger, pyruvate, rescued the cells from cell killing as well as suppressed pro-oxidant production. In addition the thiol antioxidant, N-acetyl-L-cysteine, rescued cells from glucose deprivation-induced cytotoxicity and suppressed MAPK activation. These results suggest that glucose deprivation-induced cytotoxicity and alterations in MAPK signal transduction are mediated by oxidative stress in MCF-7/ADR. These results also support the speculation that a common mechanism of glucose deprivation-induced cytotoxicity in mammalian cells may involve metabolic oxidative stress.

Differences in basal and hyperoxia-associated HO expression in oxidant-resistant hamster fibroblasts.

Heme oxygenase (HO) is the rate-limiting enzyme in the production of bilirubin from heme, and HO-1 is its inducible isoenzyme. In the metabolic pathway of HO a potential oxidant, heme, is degraded, a potential antioxidant, bilirubin, is generated, and a potent sequestering agent of redox active iron, ferritin, is thought to be coinduced. Therefore, the sum of the reactions of HO may be useful in antioxidant defense. To explore the role of HO in protection against oxidative stress, we examined HO-1 expression in Chinese hamster fibroblasts (HA-1) as well as stable hydrogen peroxide (H2O2)-resistant (OC-14) and 95% O2-resistant (O2R95) variant cell lines derived from HA-1, after exposure to 72 h of hyperoxia (95% O2-5% CO2). Total HO activity, HO-1 protein, and HO-1 mRNA steady-state levels were assessed before exposure and daily during exposure to hyperoxia. Controls were exposed to 95% air-5% CO2. Confluent monolayers of O2R95 and OC-14 cells had increased basal immunoreactive HO-1 protein levels relative to HA-1 cells when the cells were grown in normoxia, and O2R95 had higher total basal HO activity. When exposed to hyperoxia for up to 3 days, O2R95 cells, which were resistant to oxygen-induced killing, did not show induction of HO-1 mRNA or increased immunoreactive protein, whereas OC-14 and HA-1, which were relatively more sensitive than O2R95 to oxygen-related cytotoxicity, demonstrated significant increases in HO-1 expression during exposure to hyperoxia. Basal ferritin protein levels were highest in the O2R95 cells, intermediate in OC-14, and lowest in HA-1, but ferritin protein did not increase further, with hyperoxic exposure, in any of the cell lines. We conclude that increased constitutive HO-1 expression is associated with resistance to hyperoxia, whereas induction of HO-1 mRNA is an index of oxidative injury, since it only occurs after cells have sustained cytotoxic injury. We also conclude that increased ferritin expression does not necessarily accompany increased HO-1 expression in oxidant stress. We speculate that HO-1 plays a role in protection against hyperoxic damage.