Inhibition of cell growth in NIH/3T3 fibroblasts by overexpression of manganese superoxide dismutase: Mechanistic studies

NIH/3T3 mouse fibroblasts were transfected with the cDNA for manganese superoxide dismutase (MnSOD), and two clones overexpressing MnSOD activity were subsequently characterized by comparison with parental and control plasmid-transfected cells. One clone with a 1.8-fold increase in MnSOD activity had a 1.5-fold increase in glutathione peroxidase (GPX) activity (increased GPX-adapted clone), while a second clone with a 3-fold increase in MnSOD activity had a 2-fold decrease in copper, zinc superoxide dismutase (CuZnSOD) activity (decreased CuZnSOD-adapted clone). Increased reactive oxygen species (ROS) levels compared with parental or control plasmid-transfected cells were observed in nonsynchronous cells in the increased GPX-adapted clone, but not in the decreased CuZnSOD-adapted clone. The two MnSOD-overexpressing clones showed different sensitivities to agents that generate oxidative stress. Flow cytometry analysis of the cell cycle showed altered cell cycle progression in both MnSOD-overexpressing clones. During logarithmic growth, both MnSOD-overexpressing clones showed increased mitochondrial membrane potential compared with parental and control plasmid-transfected cells. Both MnSOD-overexpressing clones showed a decrease in mitochondrial mass at the postconfluent phase of growth, suggesting that mitochondrial mass may be regulated by MnSOD and/or ROS levels. Our results indicate that adaptation of fibroblasts to overexpression of MnSOD can involve more than one mechanism, with the resultant cell phenotype dependent on the adaptation mechanism utilized by the cell. J. Cell. Physiol. 175:359–369, 1998. © 1998 Wiley-Liss, Inc.     

Effect of agents changing the intracellular level of reactive oxygen species on the cell cycle phase distribution in 3T3 and 3T3SV40 cell lines

A comparison of cell cycle phase distribution of 3T3 cells and their transformants 3T3SV40 treated with different substances changing the intracellular level of reactive oxygen species (ROS) has been made. In this study the following glutathione synthesis modulating agents were tested: two precursors of intracellular glutathione, antioxidant N-acetyl-L-cysteine (NAC) (-)-2-oxo-4-thiazolidine-carboxylic acid (OTZ), and inhibitor of glutathione synthesis, DL-buthionine-S, R-sulfoximine (BSO). It has been shown that both NAC (10-20 mM) and OTZ (20 mM) decreased the intracellular level of ROS in both cell lines. OTZ was more potent than NAC. However, only NAC caused changes in cell cycle progression of both cell types in dose-dependent manner. These changes differed in 3T3 and 3T3SV40 cells. Flow cytometric analysis of cell cycle phase distribution indicated that NAC (20 mM) blocked cell cycle in the G1 phase. The G1--arrest was completely reversible after removal of NAC from the medium. NAC (10-20 mM) caused a decrease in S and G2/M phases of transformants 3T3SV40. Moreover, a part of the population died apoptoticaly. Different mechanisms of NAC effect on normal and transformed cells are discussed. It is suggested that there is no strong correlation between cell cycle progression and intracellular level of ROS.     

Two distinct mechanisms for inhibition of cell growth in human prostate carcinoma cells with antioxidant enzyme imbalance.

The purpose of the present study was to determine whether manganese superoxide dismutase (MnSOD) overexpression in DU145 human prostate carcinoma cells affected cell reduction-oxidation state (cell redox) and to correlate changes in cell redox status with cell cycle progression and plating efficiency. One MnSOD-overexpressing cell line had no change in other antioxidant enzymes (AEs) (nonadapted clone), whereas a second MnSOD-overexpressing cell line studied had an increase in catalase (CAT) activity (adapted clone). Correlation of biochemical studies with cell cycle studies suggested that heteroploidy observed in the nonadapted MnSOD-overexpressing cell line may be due to increased intracellular peroxides with resultant disruption of the microtubule network, while a decreased mitotic rate was associated with decreased ATP levels in mitosis. In contrast, the decrease in cell growth in the adapted cell line was demonstrated to be due to a decrease in plating efficiency. Our results demonstrate complex effects of AE imbalance on cell growth of DU145 prostate cancer cells.     

Synchronized generation of reactive oxygen species with the cell cycle

A possible appearance of reactive oxygen species (ROS) with the normal cell cycle was studied to find how ROS are generated in cells in relation to the cell cycle. The production of ROS in relation to the cell cycle was examined by determining the changes in intracellular ROS concentrations at different phases of the cell cycle by culturing BALB 3T3 cells in the presence and absence of aphidicolin. The amounts of intracellular ROS and the cell population at specific phases (S and G2/M) were determined as the fluorescence of dichlorodihydrofluorescein and propidium iodide taken up simultaneously by the cells, respectively, by flow cytometry. Although intracellular ROS remained at the control levels when the cell growth was arrested with aphidicolin at the G1 phase, they increased when the arrest was released to result in the increase of the cell population at the S phase. Furthermore, ROS was shown to disturb/stop the cell cycle by means of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The cell cycle was regulated through oxidative stress by exposure to hydrogen peroxide and glutathione ethyl ester. The cell cycle was prevented more sensitively in metallothionein-null cells than in the wild type cells. Based on the present observations, we proposed for the first time that ROS are generated synchronously with the normal cell cycle, and that they have to be controlled at certain level for normal progress of the cell cycle.     

Cell cycle-related differences in susceptibility of NIH/3T3 cells to ribonucleases

Microinjection of Onconase or RNase A into NIH/3T3 cells was used to study the intracellular actions of these two proteins. Onconase preferentially killed actively growing cells in both microinjection and cell culture experiments. Moreover, agents that increased the number of cells in S phase such as serum or microinjected signal transduction mediators (Ras, protein kinase C, and mitogen-activated protein kinase) enhanced Onconase cytotoxicity. Conversely, agents that decreased these proliferative pathways (dibutyryl cAMP and protein kinase A) correspondingly diminished Onconase cytotoxicity in microinjection experiments. These results were also mimicked in cell culture experiments since log-phase v-ras-transformed NIH/3T3 cells were more sensitive to Onconase (IC50 of 7 microg/ml) than parental NIH/3T3 fibroblasts (IC50 of 40 microg/ml). Based on those data we postulated that Onconase-mediated cell death in NIH/3T3 cells was related to events occurring at two or more points in the cell cycle preferentially associated with late G1/S and S phases. In contrast, quiescent NIH/3T3 cells were more sensitive to microinjected RNase A than log phase cells and positive mediators of proliferative signal transduction did not enhance RNase A-mediated cytotoxicity. Taken together, these results demonstrate that these two RNases use different pathways and/or mechanisms to elicit cytotoxic responses in NIH/3T3 cells. Predictions formulated from these studies can be tested for relevance to RNase actions in different target tumor cells.     

Inhibition of cell growth by overexpression of manganese superoxide dismutase (MnSOD) in human pancreatic carcinoma

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas26, (2003), 23] and human pancreatic cancer cell lines [Cancer Res.63, (2003), 1297] when compared to normal human pancreas. We hypothesized that stable overexpression of pancreatic cancer cells with MnSOD cDNA would alter the malignant phenotype. MIA PaCa-2 cells were stably transfected with a pcDNA3 plasmid containing sense human MnSOD cDNA or containing no MnSOD insert by using the lipofectAMINE method. G418-resistant colonies were isolated, grown and maintained. Overexpression of MnSOD was confirmed in two selected clones with a 2-4-fold increase in MnSOD immunoreactive protein. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had decreased growth rates, growth in soft agar and plating efficiency in vitro, while in vivo, the MnSOD-overexpressing clones had slower growth in nude mice. These results suggest that MnSOD may be a tumor suppressor gene in human pancreatic cancer.     

Nitric oxide enhances the manganese superoxide dismutase-dependent suppression of proliferation in HT-1080 fibrosarcoma cells.

The overexpression of manganese superoxide dismutase (MnSOD), an enzyme that catalyzes the removal of superoxide (O2*-) from the mitochondria, has been shown to be closely associated with tumor regression in vivo and loss of the malignant phenotype in vitro. To investigate the mechanism by which MnSOD overexpression mediates this reversal, we have established 29 independent, clonal MnSOD-overexpressing HT-1080 fibrosarcoma cells. MnSOD activity is inversely correlated with cell proliferation in our cell lines. Incubating cells in 3% oxygen can prevent the inhibition of cellular proliferation mediated by MnSOD, suggesting that oxygen is a prerequisite component of the MnSOD-dependent proliferative inhibition. Confocal laser microscopy was used in combination with the oxidant-sensitive fluorescent dyes dihydrorhodamine-123, dihydroethidium, and 2',7'-dichlorodihydrofluorescein diacetate to determine the oxidizing capacity of the MnSOD-overexpressing cells. When compared with parental or control cell lines, there was a significant decrease in the rate of oxidation of the fluorophores in the MnSOD-overexpressing cell lines. Thus, an increase in the oxidizing capacity of the cells does not appear to mediate the inhibition of proliferation associated with MnSOD overexpression. Superoxide dismutase has also been shown to enhance the cytotoxic activity of NO* toward tumor cells. In this study, we have shown that MnSOD overexpression enhances the cytostatic action of the NO* donors, sodium nitroprusside, 3-morpholinosydnonomine, and (Z)-1-[2-aminethyl)-N-(2-ammonioethyl)amino]diazen-1-+ ++ium-1,2-diolate in a dose-dependent manner. In addition, the NO* toxicity is blocked by oxyhemoglobin, a NO* scavenger. Our findings suggest that NO* may play a role in the reversal of tumorigenicity associated with MnSOD overexpression.     

Inhibition of Cell Growth by Overexpression of Manganese Superoxide Dismutase (MnSOD) in Human Pancreatic Carcinoma

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas26, (2003), 23] and human pancreatic cancer cell lines [Cancer Res.63, (2003), 1297] when compared to normal human pancreas. We hypothesized that stable overexpression of pancreatic cancer cells with MnSOD cDNA would alter the malignant phenotype. MIA PaCa-2 cells were stably transfected with a pcDNA3 plasmid containing sense human MnSOD cDNA or containing no MnSOD insert by using the lipofectAMINE method. G418-resistant colonies were isolated, grown and maintained. Overexpression of MnSOD was confirmed in two selected clones with a 2-4-fold increase in MnSOD immunoreactive protein. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had decreased growth rates, growth in soft agar and plating efficiency in vitro, while in vivo, the MnSOD-overexpressing clones had slower growth in nude mice. These results suggest that MnSOD may be a tumor suppressor gene in human pancreatic cancer.     

Cyclin D1 production in cycling cells depends on ras in a cell-cycle-specific manner.

BACKGROUND: Cellular Ras and cyclin D1 are required at similar times of the cell cycle in quiescent NIH3T3 cells that have been induced to proliferate, but not in the case of cycling NIH3T3 cells. In asynchronous cultures, Ras activity has been found to be required only during G2 phase to promote passage through the entire upcoming cell cycle, whereas cyclin D1 is required through G1 phase until DNA synthesis begins. To explain these results in molecular terms, we propose a model whereby continuous cell cycle progression in NIH3T3 cells requires cellular Ras activity to promote the synthesis of cyclin D1 during G2 phase. Cyclin D1 expression then continues through G1 phase independently of Ras activity, and drives the G1-S phase transition. RESULTS: We found high levels of cyclin D1 expression during the G2, M and G1 phases of the cell cycle in cycling NIH3T3 cells, using quantitative fluorescent antibody measurements of individual cells. By microinjecting anti-Ras antibody, we found that the induction of cyclin D1 expression beginning in G2 phase was dependent on Ras activity. Consistent with our model, cyclin D1 expression during G1 phase was particularly stable following neutralization of cellular Ras. Finally, ectopic expression of cyclin D1 largely overcame the requirement for cellular Ras activity during the continuous proliferation of cycling NIH3T3 cells. CONCLUSIONS: Ras-dependent induction of cyclin D1 expression beginning in G2 phase is critical for continuous cell cycle progression in NIH3T3 cells.     

Characterization of redox state of two human prostate carcinoma cell lines with different degrees of aggressiveness

We characterized the redox profiles in two different human prostate carcinoma cell lines (LNCaP vs PC3) that are known to exhibit varying degrees of invasiveness/metastatic ability. We confirmed that PC3 cells were more invasive than LNCaP cells through an in vitro analysis. The present study documented higher 8-hydroxy-2'-deoxyguanosine levels in PC3 cells than in LNCaP cells. The levels of lipid peroxidation were higher in LNCaP cells than in PC3 cells. The reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio increased to a greater extent during cell growth in PC3 cells than in LNCaP cells, whereas both reduced GSH and GSSG levels were higher in the medium of PC3 cells than in that of LNCaP cells. The levels of reactive oxygen (ROS) and reactive nitrogen species (RNS), both intracellularly and in the medium, were higher for LNCaP cells than for PC3 cells during cell growth. In addition, our results demonstrated higher ROS/RNS levels in LNCaP cells than in PC3 cells in S and G(2)/M phases of the cell cycle during logarithmic growth. Each cell type showed distinct cytotoxic responses to low-molecular-weight redox-modulating compounds. Our results document that human prostate cancer cell lines of varying degrees of aggressive behavior have distinct redox properties, findings that could lead to novel therapeutic interventions.     

Second-site changes affect viability of amphotropic/ecotropic chimeric enveloped murine leukemia viruses

Chimeras were previously generated between the ecotropic (Moloney-MuLV) and amphotropic (4070A) SU and TM proteins of murine leukemia virus (MuLV). After passage in D17 cells, three chimeras with junctions in the C terminus of SU (AE5, AE6, and AE7), showed improved kinetics of viral spreading, suggesting that they had adapted. Sequencing of the viruses derived from the D17 cell lines revealed second-site changes within the env gene. Changes were detected in the receptor binding domain, the proline-rich region, the C terminus of SU, and the ectodomain of TM. Second-site changes were subcloned into the parental DNA, singly and in combination, and tested for viability. All viruses had maintained their original cloned mutations and junctions. Reconstruction and passage of AE7 or AE6 virus with single point mutations recovered the additional second-site changes identified in the parental population. The AE5 isolate required changes in the VRA, the VRC, the VRB-hinge region, and the C terminus of SU for efficient infection. Passage of virus, including the parental 4070A, in D17 cells resulted in a predominant G100R mutation within the receptor binding domain. Viruses were subjected to titer determination in three cell types, NIH 3T3, canine D17, and 293T. AE6 viruses with changes in the proline-rich region initially adapted for growth on D17 cells could infect all cell types tested. AE6-based chimeras with additional mutations in the C terminus of SU could infect D17 and 293T cells. Infection of NIH 3T3 cells was dependent on the proline-rich mutation. AE7-based chimeras encoding L538Q and G100R were impaired in infecting NIH 3T3 and 293T cells.     

Activation of matrix metalloproteinase-2 by overexpression of manganese superoxide dismutase in human breast cancer MCF-7 cells involves reactive oxygen species

Matrix metalloproteinases (MMPs) participate in cell migration and remodeling processes by affecting the extracellular matrix. MMP-2 is thought to be involved in cancer cell invasiveness. It has been proposed that the activity of MMP-2 can be modulated by intracellular reactive oxygen species (ROS)/reactive nitrogen species. We hypothesized that manganese superoxide dismutase (MnSOD) could mediate MMP-2 activity by changing the intracellular ROS level and that nitric oxide ((.)NO) may be involved in this process. Human breast cancer MCF-7 cells were stably transfected with plasmids containing MnSOD cDNA. A 2-30-fold increase of MnSOD protein and activity was observed in four clones. Our data demonstrated that overexpression of MnSOD stimulated the activation of MMP-2 with a corresponding elevation of ROS. A decrease in ROS by ebselen, a glutathione peroxidase mimetic, or by transduction of adenovirus containing human catalase or glutathione peroxidase cDNA abolished the effect of MnSOD on MMP-2 activation. Treatment of MCF-7 cells with antimycin A or rotenone increased intracellular ROS production and MMP-2 activation simultaneously. Our data also showed a suppression of endothelial nitric-oxide synthase expression that was accompanied by decreased (.)NO production in MnSOD-overexpressing cells. However, the changes in endothelial nitric-oxide synthase and (.)NO did not correlate with the MnSOD activity. Corresponding changes of MMP-2 activity after the addition of a NOS inhibitor (N(G)-amino-l-arginine) or a (.)NO donor ((Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate) to the cells suggested the possibility that (.)NO may be involved in the MnSOD-mediated MMP-2 activation pathway. These results indicate that MnSOD induces MMP-2 activity by regulation of intracellular ROS and imply that signaling pathways involving (.)NO may also be involved in the MnSOD mediation of MMP-2 activity.     

MnSOD drives neuroendocrine differentiation, androgen independence, and cell survival in prostate cancer cells

An increase in neuroendocrine (NE) cell number has been associated with progression of prostate tumor, one of the most frequent cancers among Western males. We previously reported that mitochondrial manganese superoxide dismutase (MnSOD) increases during the NE differentiation process. The goal of this study was to find whether MnSOD up-regulation is enough to induce NE differentiation. Several human prostate cancer LNCaP cell clones stably overexpressing MnSOD were characterized and two were selected (MnSOD-S4 and MnSOD-S12). MnSOD overexpression induces NE morphological features as well as coexpression of the NE marker synaptophysin. Both MnSOD clones exhibit lower superoxide levels and higher H(2)O(2) levels. MnSOD-overexpressing cells show higher proliferation rates in complete medium, but in steroid-free medium MnSOD-S12 cells are still capable of proliferation. MnSOD up-regulation decreases androgen receptor and prevents its nuclear translocation. MnSOD also induces up-regulation of Bcl-2 and prevents docetaxel-, etoposide-, or TNF-induced cell death. Finally, MnSOD-overexpressing cells enhance growth of androgen-independent PC-3 cells but reduce growth of androgen-dependent cells. These results indicate that redox modulation caused by MnSOD overexpression explains most NE-like features, including morphological changes, NE marker expression, androgen independence, inhibition of apoptosis, and enhancement of cell growth. Many of these events can be associated with the androgen dependent-independent transition during prostate cancer progression.     

Delaying the onset of M phase in NIH 3T3 cells blocked in early S phase occurs via accumulating cyclin B1 and tyrosine-phosphorylated p34cdc2 in the nucleus

An affinity-purified antibody (anti-Cdc2C) raised against the carboxy terminal sequence LDNQIKKM of p34^cdc2 uncovered in NIH 3T3 cells a protein subpopulation, the location and the level of accumulation of which evolve during progression through the cell cycle: it first emerges inside the nucleus in late G1/early S phase and continues to build up principally in this location throughout S phase; a cytoplasmic expression then becomes apparent near the end of S phase, develops during G2 and sometimes prevails over the nuclear expression; it finally relocates to the nucleus in early prophase. We propose that a major part of this subpopulation would represent p34^cdc2 molecules existing inside a complex with cyclin B1. NIH 3T3 cells arrested in early S phase with aphidicolin do not commit prematurely to mitosis which indicates that the regulatory pathway involved in preserving the temporal order of S and M phases is functioning in these conditions. Conjugated Western blot analysis and immunofluorescence microscopy showed that cyclin A, cyclin B1 and tyrosine-phosphorylated p34^cdc2 continue to build up predominantly in the nucleus of the arrested cells. After release from the block, the cells rapidly reenter S and G2 phases and, concomitantly, cyclin B1 and tyrosine-phosphorylated p34^cdc2 relocate to the cytoplasm before redistributing again in the nucleus in early prophase. These data would suggest that delaying the onset of M phase in NIH 3T3 cells in which the rate of DNA replication is reduced, is first ensured by a mechanism that prevents the cytoplasmic relocation of inactive p34^cdc2/cyclin B1 complexes continually forming in the nucleus once the G1 period of mitotic cyclin instability is over.     

Cyclin D1 Expression Mediated by Phosphatidylinositol 3-Kinase through mTOR-p70S6K-Independent Signaling in Growth Factor-Stimulated NIH 3T3 Fibroblasts

Phosphatidylinositol (PI) 3-kinase is required for G₁ to S phase cell cycle progression stimulated by a variety of growth factors and is implicated in the activation of several downstream effectors, including p70^S6K. However, the molecular mechanisms by which PI 3-kinase is engaged in activation of the cell cycle machinery are not well understood. Here we report that the expression of a dominant negative (DN) form of either the p110α catalytic or the p85 regulatory subunit of heterodimeric PI 3-kinase strongly inhibited epidermal growth factor (EGF)-induced upregulation of cyclin D1 protein in NIH 3T3(M17) fibroblasts. The PI 3-kinase inhibitors LY294002 and wortmannin completely abrogated increases in both mRNA and protein levels of cyclin D1 and phosphorylation of pRb, inducing G₁ arrest in EGF-stimulated cells. By contrast, rapamycin, which potently suppressed p70^S6K activity throughout the G₁ phase, had little inhibitory effect, if any, on either of these events. PI 3-kinase, but not rapamycin-sensitive pathways, was also indispensable for upregulation of cyclin D1 mRNA and protein by other mitogens in NIH 3T3 (M17) cells and in wild-type NIH 3T3 cells as well. We also found that an enforced expression of wild-type p110 was sufficient to induce cyclin D1 protein expression in growth factor-deprived NIH 3T3(M17) cells. The p110 induction of cyclin D1 in quiescent cells was strongly inhibited by coexpression of either of the PI 3-kinase DN forms, and by LY294002, but was independent of the Ras-MEK-ERK pathway. Unlike mitogen stimulation, the p110 induction of cyclin D1 was sensitive to rapamycin. These results indicate that the catalytic activity of PI 3-kinase is necessary, and could also be sufficient, for upregulation of cyclin D1, with mTOR signaling being differentially required depending upon cellular conditions.     

Regulation of NIH-3T3 cell G1 phase transit by serum during exponential growth

The proliferation rate of mammalian cells is regulated normally in the G₁ phase of the cell cycle. During this phase, it is convenient to assign positive and negative roles to the molecular programs that regulate the duration of G₁ and the phase transition from G₁ to S phase. Density-dependent inhibition of cellular proliferation results in an increase in the duration of G₁. This form of regulation is due to both secreted factors and cell—cell contact. Serum is mitogenic to a variety of mammalian cell types. Because quiescent cells enter S phase as a result of serum addition to culture media, serum is usually regarded as a source of positive regulatory growth factors. We have measured the length of the G₁, S and G₂+ M phases of NIH 3T3 cells during exponential growth as a function of cell density and serum concentration. The G₁ length increases during exponential growth as a function of density while S and G₂+ M are relatively constant. Further, this increase in G₁ phase time, or density mediated negative regulation, is inhibited by increasing serum concentration. This phenotype is saturable between 10% to 20% serum. Serum concentrations above 2.5% are able to increase the rate of cell cycling (decrease the G₁ phase time) by inhibiting density dependent negative regulation of NIH 3T3.     

Naringin prevents ultraviolet‐B radiation‐induced oxidative damage and inflammation through activation of peroxisome proliferator‐activated receptor γ in mouse embryonic fibroblast (NIH‐3T3) cells

The present study, we investigate the preventive role of naringin, a dietary flavonoid, against ultraviolet‐B (UVB) radiation (280‐320 nm) induced oxidative damage and inflammatory responses in mouse embryonic fibroblast cell lines (NIH‐3T3). In this study, 20 mJ/cm ² of UVB radiation induces cell cytotoxicity, reactive oxygen species (ROS) generation, DNA damage, and antioxidants depletion in NIH‐3T3 cells. Treatment with naringin (60 µM) prior UVB exposure prevented the cell cytotoxicity, ROS generation, DNA damage, and antioxidants depletion in NIH‐3T3 cells. Furthermore, naringin prevents UVB‐induced mitogen‐activated protein kinase families and nuclear factor‐κB (NF‐κB)‐mediated activation of inflammatory factors, that is TNF‐α, IL‐6, IL‐10, and COX‐2 in NIH‐3T3 cells. Peroxisome proliferator‐activated receptor γ (PPARγ) is an anti‐inflammatory agent and it suppressed the UVB‐mediated oxidative and inflammatory responses. In this study, naringin activates PPARγ and prevents inflammatory biomarkers in NIH‐3T3 cells. Thus, naringin prevents UVB‐mediated inflammation and oxidative damage in NIH‐3T3 cells probably over controlling NF‐κB expression and activation of PPARγ.     

Manganese superoxide dismutase gene dosage affects chromosomal instability and tumor onset in a mouse model of T cell lymphoma

Increased reactive oxygen species (ROS) such as superoxide have been implicated as causal elements of oncogenesis. A variety of cancers have displayed changes in steady-state levels of key antioxidant enzymes, with the mitochondrial form of superoxide dismutase (MnSOD) being commonly implicated. Increasing MnSOD expression suppresses the malignant phenotype in various cancer cell lines and suppresses tumor formation in xenograft and transgenic mouse models. We examined the impact of MnSOD expression in the development of T cell lymphoma in mice expressing proapoptotic Bax. Lck-Bax38/1 transgenic mice were crossed to mice overexpressing MnSOD (Lck-MnSOD) as well as MnSOD+/- mice. The effects of MnSOD on apoptosis, cell cycle, chromosomal instability (CIN), and lymphoma development were determined. The apoptotic and cell cycle phenotypes observed in thymocytes from control and Bax transgenic mice were unaffected by variations in MnSOD levels. Remarkably, increased gene dosage of MnSOD significantly decreased aneuploidy in premalignant thymocytes as well as the onset of tumor formation in Lck-Bax38/1 mice. The observed effects of MnSOD support a role for ROS in CIN and tumor formation in this mouse model of T cell lymphoma.     

Contrasting Antioxidant and Cytotoxic Effects of Peroxiredoxin I and II in PC12 and NIH3T3 Cells

We examined the impact of peroxiredoxin-I (Prx-I) and peroxiredoxin-II (Prx-II) stable transduction on oxidative stress in PC12 neurons and NIH3T3 fibroblasts and found variability depending on cell type and Prx subtype. In PC12 neurons, Prx-II suppressed reactive oxygen species (ROS) generation by 36% (p < 0.01) relative to vector-infected control cells. However, in NIH3T3 fibroblasts, Prx-II overexpression resulted in a 97% (p < 0.01) increase in ROS generation. Prx-I transduction elevated ROS generation in PC12 cells. The effect of Prx-I on PC12 cells was potentiated in the presence of menadione, and suppressed by an inhibitor of nitric oxide synthetase. Prx-II transduction resulted in 25–35% lower levels of glutathione (GSH) in both cell types, while Prx-I transduction increased GSH levels in neurons and decreased GSH and caspase-3 activity in fibroblasts. Prx-I and Prx-II also had differing effects on cell viability. These results suggest that Prx-I and Prx-II can either increase or decrease intracellular oxidative stress depending on cell type or experimental conditions, particularly conditions affecting nitric oxide levels.Equivalent contributions were made by each author