Antioxidant enzyme levels in oral squamous cell carcinoma and normal human oral epithelium.

BACKGROUND: The antioxidant enzymes (manganese- and copper-zinc-containing superoxide dismutases, catalase and glutathione peroxidase) limit cell injury induced by reactive oxygen species. The purpose of the study was to determine whether human oral squamous cell carcinomas have altered antioxidant enzyme levels. This study is the first to undertake this task in human oral mucosa and squamous cell carcinoma. METHODS: Semiquantitative immunohistochemistry was used to examine 26 archived oral squamous cell carcinoma biopsies. Fourteen well-differentiated and 12 poorly differentiated tumors were examined, as were 12 specimens of oral mucosa. All sections were reviewed by two oral and maxillofacial pathologists, and image analysis of the immunostained sections was performed using NIH Image. Antioxidant enzyme staining intensities were compared in the different groups by Duncan's multiple range test. RESULTS: In general, mucosal basal cells displayed lower antioxidant enzyme levels than spinous cells, and primary tumor cells displayed lower antioxidant enzyme staining intensities than did their normal cell counterparts. Moreover, poorly differentiated tumor cells showed lower antioxidant enzyme staining intensities than well-differentiated tumor cells. Manganese-containing superoxide dismutase staining intensities were, however, higher in well-differentiated oral squamous cell carcinomas than their normal cells of origin. CONCLUSIONS: Detection of antioxidant enzymes may be a useful future marker in the molecular diagnosis of the oral cancer. Moreover, it may be possible to not only monitor the effectiveness of chemopreventive and therapeutic strategies in oral cancer using these enzymes, but to monitor tumor recurrence.

Evidence of oxidative stress following the viral infection of two lepidopteran insect cell lines.

The infection of Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) resulted in increased levels of lipid hydroperoxides and protein carbonyls. In addition, the viral infection resulted in a significant decrease in the reduced glutathione to oxidized glutathione (2GSH/GSSG) ratio. These results are all consistent with an increased level of oxidative stress as a result of the viral infection. It was also observed that the oxidative damage corresponded to reduced cell viability, i.e., the results are consistent with the premise that oxidative damage contributes to cell death. Finally, the measured intracellular activities of most of the antioxidant enzymes, specifically manganese superoxide dismutase (MnSOD), ascorbate peroxidase (APOX), and catalase (CAT, not present in Sf-9 cells), did not significantly decrease following viral infection. In contrast, the measured activity of copper-zinc superoxide dismutase (CuZnSOD) decreased in the Sf-9 and Tn-5B1-4 cells following AcMNPV infection.

V-Ha-Ras overexpression induces superoxide production and alters levels of primary antioxidant enzymes.

Reactive oxygen species have been shown to play important roles in v-Ha-Ras mitogenic signaling. We hypothesized that v-Ha-Ras overexpression would induce superoxide production, and therefore modify expression of the primary antioxidant enzyme system. We have demonstrated that immortal rat kidney epithelial cells stably transduced with constitutively active v-Ha-ras produced significantly larger amounts of superoxide radical than wild-type or vector-transfected control cells. The levels of the primary antioxidant enzymes copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, and glutathione peroxidase were increased in the superoxide-overproducing cells. DNA-binding activities of the transcription factors activator protein-1, activator protein-2, and nuclear factor-kappaB were all enhanced in the superoxide-overproducing cells. These v-Ha-ras transduced cells also had a shortened cell doubling time and higher plating efficiency, and displayed greater constitutive levels of phosphorylated mitogen-activated protein kinases. These data demonstrate that v-Ha-Ras overexpression increases superoxide production and this apparently affects a wide variety of cell signaling and redox systems.

v-Ha-RaS oncogene upregulates the 92-kDa type IV collagenase (MMP-9) gene by increasing cellular superoxide production and activating NF-kappaB.

Matrix metalloproteinase 9 (MMP-9) degrades basement membrane type IV collagen and is expressed during cellular migration and invasion. Here we show that v-Ha-Ras overexpression in rat kidney epithelial cells (REC) caused upregulation of MMP-9 gene expression in part by increasing cellular oxidant levels. v-Ha-Ras mediated the production of superoxide in Ras-transfected cells, which was associated with upregulated MMP-9 gene expression. Conversely, v-Ha-Ras expression decreased steady-state levels of mRNAs from tissue inhibitor of metalloproteinase 1 (TIMP-1), an inhibitor of MMP-9; plasminogen activator inhibitor 1 (PAI-1), which indirectly activates MMP-9 by increasing plasmin levels; and collagen IV, a substrate of MMP-9 and a major component of basement membrane. Gel mobility shift assays demonstrated that Ras overexpression enhanced NF-kappaB, but not AP-1 DNA binding to motifs in the MMP-9 gene promoter. The Ras-induced increase in NF-kappaB DNA binding could be inhibited by treatment with the antioxidants N-acetyl-L-cysteine and glutathione monoester, suggesting that intracellular oxidant levels can mediate MMP-9 transcription. Our findings identify an important role for Ras in the regulation of MMP-9 expression, and suggest that increased superoxide production can upregulate MMP-9 expression and thus contribute to malignant conversion.

Constitutive activation of transcription factor AP-2 is associated with decreased MnSOD expression in transformed human lung fibroblasts.

Activator protein-2 (AP-2) is a transcription factor with transactivating and transrepressing potential in different promoter contexts. AP-2 contains seven cysteines, and its in vitro DNA binding activity is redox-sensitive. Superoxide dismutase-2 (SOD2), which encodes the antioxidant enzyme manganese superoxide dismutase (MnSOD), is a putative tumor suppressor gene whose loss of expression is associated with the malignant phenotype. SOD2 promoter mutations that generate new AP-2 sites are associated with loss of MnSOD expression in cancer cells. In the current study, we have identified an inverse expression pattern between AP-2 and MnSOD in normal versus transformed human cells. MRC5 cells are a normal human lung fibroblast cell strain that is mortal and senesces after a certain number of passages in vitro. MRC5-VA is a simian virus transformed variant of MRC5. We determined the levels of expression of MnSOD and AP-2 in these two cell types at the levels of mRNA, protein, and activity. Our results indicated that MnSOD expression was significantly decreased in MRC5-VA cells compared with MRC5 cells at each level of investigation, whereas AP-2 showed an opposing pattern of expression and DNA binding activity. These results suggest that AP-2 may participate in the mechanism(s) underlying decreased expression of SOD2 in transformed cells.

Anticancer therapy by overexpression of superoxide dismutase.

Cancer cells are in general low in the enzymatic activities of both manganese-containing (MnSOD) and copper- and zinc-containing superoxide dismutase. We have hypothesized that part of the tumor cell phenotype is due to this loss of enzymatic activity. To test this hypothesis, we have overexpressed MnSOD via plasmid and adenovirus transfection in various cancer cell types and have shown tumor suppression. This tumor suppression is via a noncytotoxic mechanism and probably occurs due to cell-cycle perturbations. We have also shown that MnSOD overexpression causes the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to have increased cytotoxicity. Our hypothesis for the mechanism of action of this combination is that overexpression of MnSOD leads to increased peroxide levels and that BCNU inhibits peroxide removal. We currently are investigating the use of adenovirus MnSOD plus BCNU in the treatment of cancer. Results thus far are consistent with the idea that we can use the alterations in antioxidant enzymes observed in cancer cells to therapeutic advantage.

Redox modulation of the pro-fibrogenic mediator plasminogen activator inhibitor-1 following ionizing radiation.

Fibrosis is a common form of normal tissue damage after exposure to a wide variety of insults believed to involve oxidative stress. Plasminogen activator inhibitor-1 (PAI-1) is thought to play a major role in the development of progressive fibrosis via the inhibition of extracellular matrix degradation. Because radiation causes oxidative injury, which has been shown to trigger fibrogenic responses, the present study was designed to test the hypothesis that PAI-1 expression is redox-regulated after irradiation. Irradiating rat kidney tubule epithelial cells (NRK52E) with 1-20 Gy gamma-rays led to dose-dependent increases in steady-state levels of PAI-1 mRNA and immunoreactive protein within 24 and 48 h, respectively. Enhancement of intracellular soluble thiol pools after incubation with N-acetylcysteine (2.5 mM), from 3.27 +/- 0.27 nM/mg protein to 5.34 +/- 0.52 nM/mg protein in cells incubated with N-acetylcysteine 30 min before and assessed 4 h after irradiation, abolished the radiation-induced up-regulation of PAI-1. In addition, overexpression of catalase inhibited radiation-induced increases in PAI-1 expression, suggesting a mechanistic role for hydrogen peroxide (H2O2) in regulating PAI-1 expression after oxidative insult. In support of this notion, incubating NRK52E cells with H2O2 (100 microM) also led to a nearly 3-fold increase in PAI-1 gene expression. These results demonstrate that PAI-1 is redox-regulated after exposure to ionizing radiation or H2O2 and suggest that H2O2 scavenging might represent a fundamental mechanism for modulating fibrogenic disease via inhibition of the induction of profibrogenic mediators after acute or chronic oxidative stress.

H(2)O(2)-induced O(2) production by a non-phagocytic NAD(P)H oxidase causes oxidant injury.

Non-phagocytic NAD(P)H oxidases have been implicated as major sources of reactive oxygen species in blood vessels. These oxidases can be activated by cytokines, thereby generating O(2), which is subsequently converted to H(2)O(2) and other oxidant species. The oxidants, in turn, act as important second messengers in cell signaling cascades. We hypothesized that reactive oxygen species, themselves, can activate the non-phagocytic NAD(P)H oxidases in vascular cells to induce oxidant production and, consequently, cellular injury. The current report demonstrates that exogenous exposure of non-phagocytic cell types of vascular origin (smooth muscle cells and fibroblasts) to H(2)O(2) activates these cell types to produce O(2) via an NAD(P)H oxidase. The ensuing endogenous production of O(2) contributes significantly to vascular cell injury following exposure to H(2)O(2). These results suggest the existence of a feed-forward mechanism, whereby reactive oxygen species such as H(2)O(2) can activate NAD(P)H oxidases in non-phagocytic cells to produce additional oxidant species, thereby amplifying the vascular injury process. Moreover, these findings implicate the non-phagocytic NAD(P)H oxidase as a novel therapeutic target for the amelioration of the biological effects of chronic oxidant stress.

Antioxidant defense systems of two lipidopteran insect cell lines.

Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines were found to contain unique assemblages of antioxidant enzymes. Specifically, the Sf-9 insect cell line contained Manganese and Copper-Zinc superoxide dismutase (MnSOD and CuZnSOD) for reducing the superoxide radical (O(2)(*-)) to hydrogen peroxide (H(2)O(2)) and ascorbate peroxidase (APOX) for reducing the resulting H(2)O(2) to H(2)O. Approximately one third of the total SOD activity was found to be MnSOD. The Tn-5B1-4 cells were also found to contain MnSOD (approximately two thirds of the total SOD activity), CuZnSOD and APOX activities. However, the Tn-5B1-4 cell line, in contrast to the Sf-9 cell line, contained catalase (CAT) activity for reducing H(2)O(2) to H(2)O. Both the Sf-9 and Tn-5B1-4 cell lines contained glutathione reductase and dehydroascorbic acid reductase activities for regenerating the reduced forms of glutathione and ascorbic acid, respectively. In addition, both cell lines contained glutathione S-transferase peroxidase activity towards hydroperoxides other than H(2)O(2). Finally, neither cell line contains the glutathione peroxidase activity that is ubiquitous in mammalian cells.

Phospholipid hydroperoxide glutathione peroxidase protects against singlet oxygen-induced cell damage of photodynamic therapy.

Phospholipid hydroperoxide glutathione peroxidase (PhGPx) is an important enzyme in the removal of lipid hydroperoxides (LOOHs) from cell membranes. Cancer treatments such as photodynamic therapy (PDT) induce lipid peroxidation in cells as a detrimental action. The photosensitizers used produce reactive oxygen species such as singlet oxygen ((1)O(2)). Because singlet oxygen introduces lipid hydroperoxides into cell membranes, we hypothesized that PhGPx would provide protection against the oxidative stress of singlet oxygen and therefore could interfere with cancer treatment. To test this hypothesis, human breast cancer cells (MCF-7) were stably transfected with PhGPx cDNA. Four clones with varying levels of PhGPx activity were isolated. The activities of other cellular antioxidant enzymes were not influenced by the overexpression of PhGPx. Cellular PhGPx activity had a remarkable inverse linear correlation to the removal of lipid hydroperoxides in living cells (r = -0.85), and correlated positively with cell survival after singlet oxygen exposure (r = 0.94). These data demonstrate that PhGPx provides significant protection against singlet oxygen-generated lipid peroxidation via removal of LOOH and suggest that LOOHs are major mediators in this cell injury process. Thus, PhGPx activity could contribute to the resistance of tumor cells to PDT.

A family of AP-2 proteins down-regulate manganese superoxide dismutase expression.

Manganese superoxide dismutase (Mn-SOD) is a primary antioxidant enzyme whose expression is essential for life in oxygen. Mn-SOD has tumor suppressor activity in a wide variety of tumors and transformed cell systems. Our initial observations revealed that Mn-SOD expression was inversely correlated with expression of AP-2 transcription factors in normal human fibroblasts and their SV-40 transformed counterparts. Thus we hypothesized that AP-2 may down-regulate Mn-SOD expression. To examine the functional role of AP-2 on Mn-SOD promoter transactivation we cotransfected AP-2-deficient HepG2 cells with a human Mn-SOD promoter-reporter construct and expression vectors encoding each of the three known AP-2 family members. Our results indicated that AP-2 could significantly repress Mn-SOD promoter activity, and that this repression was both Mn-SOD promoter and AP-2-specific. The three AP-2 proteins appeared to play distinct roles in Mn-SOD gene regulation. Moreover, although all three AP-2 proteins could repress the Mn-SOD promoter, AP-2alpha and AP-2gamma were more active in this regard than AP-2beta. Transcriptional repression by AP-2 was not a general effect in this system, because another AP-2-responsive gene, c-erbB-3, was transactivated by AP-2. Repression of Mn-SOD by AP-2 was dependent on DNA binding, and expression of AP-2B, a dominant negative incapable of DNA binding, relieved the repression on Mn-SOD promoter and reactivated Mn-SOD expression in the AP-2 abundant SV40-transformed fibroblast cell line MRC-5VA. These results indicate that AP-2-mediated transcriptional repression contributes to the constitutively low expression of Mn-SOD in SV40-transformed fibroblasts and suggest a mechanism for Mn-SOD down-regulation in cancer.

Mechanisms of circulatory and intestinal barrier dysfunction during whole body hyperthermia.

This work tested the hypotheses that splanchnic oxidant generation is important in determining heat tolerance and that inappropriate.NO production may be involved in circulatory dysfunction with heat stroke. We monitored colonic temperature (T(c)), heart rate, mean arterial pressure, and splanchnic blood flow (SBF) in anesthetized rats exposed to 40 degrees C ambient temperature. Heating rate, heating time, and thermal load determined heat tolerance. Portal blood was regularly collected for determination of radical and endotoxin content. Elevating T(c) from 37 to 41.5 degrees C reduced SBF by 40% and stimulated production of the radicals ceruloplasmin, semiquinone, and penta-coordinate iron(II) nitrosyl-heme (heme-.NO). Portal endotoxin concentration rose from 28 to 59 pg/ml (P < 0.05). Compared with heat stress alone, heat plus treatment with the nitric oxide synthase (NOS) antagonist N(omega)-nitro-L-arginine methyl ester (L-NAME) dose dependently depressed heme-.NO production and increased ceruloplasmin and semiquinone levels. L-NAME also significantly reduced lowered SBF, increased portal endotoxin concentration, and reduced heat tolerance (P < 0.05). The NOS II and diamine oxidase antagonist aminoguanidine, the superoxide anion scavenger superoxide dismutase, and the xanthine oxidase antagonist allopurinol slowed the rates of heme-.NO production, decreased ceruloplasmin and semiquinone levels, and preserved SBF. However, only aminoguanidine and allopurinol improved heat tolerance, and only allpourinol eliminated the rise in portal endotoxin content. We conclude that hyperthermia stimulates xanthine oxidase production of reactive oxygen species that activate metals and limit heat tolerance by promoting circulatory and intestinal barrier dysfunction. In addition, intact NOS activity is required for normal stress tolerance, whereas overproduction of.NO may contribute to the nonprogrammed splanchnic dilation that precedes vascular collapse with heat stroke.

Genes regulated in human breast cancer cells overexpressing manganese-containing superoxide dismutase.

The mitochondrial antioxidant enzyme manganese-containing superoxide dismutase (MnSOD) functions as a tumor suppressor gene. Reconstitution of MnSOD expression in several human cancer cell lines leads to reversion of malignancy and induces a resistant phenotype to the cytotoxic effects of TNF and hyperthermia. The signaling pathways that underlie these phenotypic changes in MnSOD-overexpressing cells are unknown, although alterations in the activity of several redox-sensitive transcription factors, including AP-1 and NF-kappaB, have been observed. To determine the downstream signaling molecules involved in MnSOD-induced cell resistant phenotype, in the present study we analyzed the expression profile of several groups of genes related to stress response, DNA repair, and apoptosis, in a human breast cancer MCF-7 cell line overexpressing MnSOD (MCF+SOD). Of 588 genes examined, 5 (0.85%) were up-regulated (2-42-fold), and 11 (1.9%) were down-regulated (2-33-fold) in the MCF+SOD cells compared to the parental MCF-7 cells. The five up-regulated genes were MET, GADD153, CD9, alpha-catenin and plakoglobin. The genes with the most significant down-regulation included: vascular endothelial growth factor receptor 1, TNF-alpha converting enzyme, and interleukin-1beta. GADD153 (involved in the repair of DNA double strand breaks) showed a 33-fold increase in microarray analysis and these results were confirmed by RT-PCR. To further determine the specificity in MnSOD-induced gene regulation, MCF+SOD cells were stably transfected with an antisense MnSOD sequence whose expression was controlled by a tetracycline-inducible regulator. Expression of three up-regulated genes was measured after induction of antisense MnSOD expression. Interestingly, expression level of GADD153 but not MET or CD9 was reduced 24 h after antisense MnSOD induction. Together, these results suggest that reconstitution of MnSOD in tumor cells can specifically modulate the expression of down-stream effector genes. GADD153 and other elements observed in the MCF+SOD cells may play a key role in signaling the MnSOD-induced cell phenotypic change.

Gene transfer of extracellular superoxide dismutase increases superoxide dismutase activity in cerebrospinal fluid.

BACKGROUND AND PURPOSE: Copper-zinc superoxide dismutase (CuZnSOD) is expressed intracellularly, while extracellular SOD (EC-SOD) is released from cells. The purpose of this study was to determine whether gene transfer of CuZnSOD increases SOD activity predominantly in tissues, and gene transfer of EC-SOD increases SOD activity in cerebrospinal fluid (CSF). We also determined whether heparin or dextran sulfate releases EC-SOD into CSF. METHODS: We injected recombinant adenoviruses expressing EC-SOD (AdEC-SOD), CuZnSOD (AdCuZnSOD), or beta-galactosidase (Adbeta-gal) into the cisterna magna of rabbits. RESULTS: Total SOD activity in CSF was 39+/-11 U/mL (mean+/-SE) before virus injection. Three days later, total SOD activity in CSF increased to 148+/-22 U/mL after AdEC-SOD and 92+/-10 U/mL after AdCuZnSOD (P:<0.05 versus AdEC-SOD), with no change after Adbeta-gal (49+/-5 U/mL). EC-SOD protein was detected in CSF after AdEC-SOD but not AdCuZnSOD or Adbeta-gal. Injection of heparin or dextran sulfate into the cisterna magna increased total SOD activity 27-fold and 32-fold over basal values, respectively, in CSF of rabbits that received AdEC-SOD. In contrast to effects in CSF, total SOD activity in basilar artery and meninges was significantly higher after AdCuZnSOD and tended to be higher after AdEC-SOD than after Adbeta-gal. CONCLUSIONS: -We have developed a method for intracranial gene transfer of CuZnSOD and EC-SOD. After gene transfer, CuZnSOD was expressed mainly in tissues, and EC-SOD was released into the CSF, especially after injection of heparin or dextran sulfate. Gene transfer of different isoforms of SOD may be useful in studies of cerebral vascular physiology and pathophysiology.

Measurement of MnSOD and CuZnSOD activity in mammalian tissue homogenates.

Three basic forms of mammalian SODs exist and they are distinguished by their sizes and locations. SOD enzyme activity is not easily monitored by direct measurement because the substrate disappearance is very rapid at physiological pH. Activity can be measured as described in this unit by a number of indirect competitive inhibition assays based on the principle that the superoxide anion radical will reduce an inhibitory substrate [such as nitroblue tetrazolium (NBT) or cytochrome c] and SOD activity will reduce the rate of reduction in a competitive fashion. The SOD-mediated inhibition of the indicator substrate reduction can then be quantitated and plotted as a function of the quantity of protein added to the reaction to construct an inhibition curve.

Pixel analysis of MR perfusion imaging in predicting radiation therapy outcome in cervical cancer.

The purpose of this study was to assess heterogeneity of tumor microcirculation determined by dynamic contrast-enhanced magnetic resonance (MR) imaging and its prognostic value for tumor radiosensitivity and long-term tumor control using pixel-by-pixel analysis of the dynamic contrast enhancement. Sixteen patients with advanced cervical cancer were examined with dynamic contrast-enhanced MR imaging at the time of radiation therapy. Pixel-by-pixel statistical analysis of the ratio of post- to precontrast relative signal intensity (RSI) values in the tumor region was performed to generate pixel RSI distributions of dynamic enhancement patterns. Histogram parameters were correlated with subsequent tumor control based on long-term cancer follow-up (median follow-up 4.6 years; range 3.8-5.2 years). The RSI distribution histograms showed a wide spectrum of heterogeneity in the dynamic enhancement pattern within the tumor. The quantity of low-enhancement regions (10th percentile RSI < 2.5) significantly predicted subsequent tumor recurrence (88% vs. 0%, P = 0.0004). Discriminant analysis based on both 10th percentile RSI and pixel number (reflective of tumor size) further improved the prediction rate (100% correct prediction of subsequent tumor control vs. recurrence). These preliminary results suggest that quantification of the extent of poor vascularity regions within the tumor may be useful in predicting long-term tumor control and treatment outcome in cervical cancer. J. Magn. Reson. Imaging 2000;12:1027-1033.

The role of cellular glutathione peroxidase redox regulation in the suppression of tumor cell growth by manganese superoxide dismutase.

Manganese-containing superoxide dismutase (MnSOD) is an essential primary antioxidant enzyme that converts superoxide radical to hydrogen peroxide and molecular oxygen within the mitochondrial matrix. Cytosolic glutathione peroxidase (GPX) converts hydrogen peroxide into water. MnSOD is reduced in a variety of tumor types and has been proposed to be a new kind of tumor suppressor gene, but the mechanism(s) by which MnSOD suppresses malignancy is unclear. According to the enzymatic reactions catalyzed by MnSOD and cytosolic GPX, change in the cellular redox status, especially change attributable to accumulation of hydrogen peroxide or other hydroperoxides, is a possible reason to explain the suppression of tumor growth observed in MnSOD-overexpressing cells. To test this possible mechanism, we transfected human cytosolic GPX cDNA into human glioma cells overexpressing MnSOD. The results showed that GPX overexpression not only reversed the tumor cell growth inhibition caused by MnSOD overexpression but also altered the cellular contents of total glutathione, reduced glutathione, oxidized glutathione, and intracellular reactive oxygen species. Overexpression of GPX also inhibited degradation of the inhibitory subunit alpha of nuclear factor-KB. These results suggest that hydrogen peroxide or other hydroperoxides appear to be key reactants in the tumor suppression by MnSOD overexpression, and growth inhibition correlates with the intracellular redox status. This work suggests that manipulations that inhibit peroxide removal should enhance the tumor suppressive effect of MnSOD overexpression.

Localization of antioxidant enzymes and oxidative damage products in normal and malignant prostate epithelium.

BACKGROUND: The risk for prostate cancer seems to be reduced by certain antioxidant compounds (vitamins E and A, and selenium). METHODS: Antioxidant enzymes and oxidative damage products were localized in normal prostatic epithelium and malignant glands in primary and metastatic prostatic adenocarcinomas, using well-characterized antibodies and immunoperoxidase techniques. RESULTS: Antioxidant enzymes and four markers of oxidative damage were compared in basal and secretory cells of normal prostatic epithelium and prostate adenocarcinoma cells, and each cell type had unique patterns of enzymes and oxidative damage products. One marker of oxidative damage, a fluorophore derived from 4-hydroxy-2-nonenal-lysine adduction, was found in secretory cells of normal but not malignant epithelium, demonstrating a different oxidative metabolism in normal vs. malignant prostate epithelium. Metastatic lesions from primary prostate cancer had higher levels of manganese superoxide dismutase and nuclear oxidative damage products than did primary tumors. CONCLUSIONS: Antioxidant enzymes and oxidative damage products are modulated in metastatic compared to primary prostate cancer.

Immunolocalization and adenoviral vector-mediated manganese superoxide dismutase gene transfer to experimental oral tumors.

The anti-oxidant enzyme system protects cellular macromolecules against damage from reactive oxygen species. One component of this system, manganese superoxide dismutase (MnSOD), has also been shown to display tumor suppressor gene-like activity. The purpose of this study was to examine changes in MnSOD expression during hamster cheek pouch carcinogenesis, and the effects of MnSOD overexpression using an adenoviral vector. Tumor induction was carried out using 7,12-dimethylbenz[alpha]anthracene. Animals were killed at periodic intervals, and cheek pouch tissues were excised and examined for MnSOD expression by immunohistochemistry and digital image analysis. We observed a reduction in MnSOD expression as early as 2 weeks after the start of carcinogen application. Low MnSOD expression persisted until the end of the 23-week experimental period. Solid hamster cheek pouch carcinoma xenografts were then established in nude mice. An adenoviral vector encoding the human MnSOD gene was delivered to the xenografts by direct injection. We observed high, immediate expression of MnSOD in the xenografts that persisted for 10 days following cessation of viral construct delivery. Delivery of the MnSOD construct resulted in a maximal 50% reduction in tumor growth compared with untreated controls. Our results suggest that MnSOD may be a tumor suppressor gene in the hamster cheek pouch model system.