Introduction

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Manganese superoxide dismutase (MnSOD) is a superoxide radical-scavenging mitochondrial enzyme, which is crucial for protecting cells and tissues from oxidant injury and hyperoxia (1, 2). In the lungs of hyperoxia-exposed rats MnSOD is highly induced in pneumocyte II cells, interstitial fibroblasts (3), and along the pleural mesothelium (4), with the major qualitative difference in the mRNA between normal and hyperoxic rat lungs occurring in the mesothelium (4). In cultured mesothelial cells MnSOD is induced by asbestos fibers, tumor necrosis factor  (TNF-), and by their combination (5, 6). Although the regulation of MnSOD and other antioxidant enzymes such as copper-zinc superoxide dismutase (CuZnSOD), catalase, and glutathione peroxidase is relatively well understood in animal models and cell cultures, little is known about their regulation in human lung.

Malignant mesothelioma is a tumor that originates from the mesothelial cells of serous cavities (7), and in most cases is associated with occupational exposure to asbestos fibers (8). Free radicals generated by asbestos fibers are assumed to play a major role in the pathogenesis of mesothelioma and other asbestos-related lung diseases (9, 10). A typical feature of mesothelioma is its resistance to most chemotherapeutic agents and to radiation. Because radiation and several anticancer drugs generate free radicals, antioxidant mechanisms could partly explain the observed resistance (11). We documented highly elevated mRNA levels and specific activities of MnSOD in cultured human mesothelioma cells compared with nonmalignant mesothelial cells in vitro (12). Moreover, the mesothelioma cells with the highest MnSOD levels were more resistant to the exogenous oxidant menadione than were the nonmalignant cells (12). No studies of MnSOD or other antioxidant enzymes in human mesothelial cells or mesothelioma in situ are available.

It has been suggested that a disturbance of the cellular oxidant-antioxidant balance can lead to carcinogenesis (13). Most solid tumors have been reported to express low levels of MnSOD, CuZnSOD, and catalase, whereas the expression of glutathione peroxidase and glutathione reductase appears to be highly variable (reviewed in Ref. 13). Many studies have suggested that the MnSOD gene may be a tumor suppressor gene (14), and that malignant tumors have low MnSOD activity (17). However, several types of tumor cells have been found to contain high levels of MnSOD when compared with their nonmalignant counterparts (12, 18), suggesting that the role of MnSOD in cancer is more complicated than originally assumed.

The role of MnSOD in the resistance of cells and tissues to hyperoxia or oxidant damage, or of tumor cells to cytotoxic drugs and radiation, is also controversial. Some studies suggest that cytokine-induced MnSOD protects cells during oxidant exposure (21), and that tolerance developed during repeated episodes of hyperoxia is associated with SOD induction (24, 25). Transfection of MnSOD can increase resistance of certain cell types to oxidants, cytotoxic drugs, and radiation (26). However, controversial results have been obtained both in transgenic animal models (29) and in TNF-treated cells in vitro (30). In fact, an imbalance between superoxide- and hydrogen peroxide (H₂O₂)-scavenging antioxidant enzymes has been shown to result in increased H₂O₂ production and enhancement of cell death (31).

Our study showed that cultured human mesothelioma cells contain highly elevated MnSOD activities and that these cells are more resistant to the exogenous oxidant menadione than are the nonmalignant mesothelial cells (12). We therefore investigated whether MnSOD is upregulated in human mesothelioma in situ and, if so, whether MnSOD induction explains the high oxidant and drug resistance of mesothelioma cells in vitro.

	Materials and Methods

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Tissue Specimens

Healthy visceral pleural tissue and mesothelioma specimens were obtained from surgical biopsy, lobectomy, or pulmonectomy samples. The samples of healthy visceral pleura, verified by light microscopy examination, were obtained from six individuals who underwent lung surgery for reasons other than malignant mesothelioma or pleural disease. Histopathological diagnosis of pleural mesothelioma was confirmed by the Mesothelioma Panel of Finland. Ethical permission was obtained from the Department of Internal Medicine, University of Helsinki (Helsinki, Finland).

Mesothelioma Patients

The clinical data of eight patients are presented in Table 1 (34). All patients were male, 43-72 yr old, and all had evidence of occupational exposure to asbestos as established from their work histories and the fiber content of the lung tissue samples. Four patients had tumors with epithelial histology and four patients had tumors with mixed histology. Survival was 5-20 mo from the diagnosis of mesothelioma. The samples were obtained before any cancer therapy, except in the case of one patient (M9), who had received chemotherapy (mitoxantrone).

Cell Cultures

Simian virus 40 (SV40)-transformed human pleural mesothelial cells (Met5A) were kindly provided by Dr. C. C. Harris (Laboratory of Human Carcinogenesis, National Cancer Institute, Bethesda, MD). These cells are near-diploid nontumorigenic cells exhibiting typical mesothelial cell characteristics (35). Two continous mesothelioma cell lines (M14K and M38K) were used. These cell lines had the highest (M38K) and lowest (M14K) MnSOD activity of the four cell lines established from the original tumors of our untreated mesothelioma patients (12). In the case of M14K, the original tumor was unavailable. The cytogenetic and histological characterization of these two cell lines has been reported (34). The cells were grown to confluency in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.03% L-glutamine (all from LTI Life Technologies, Paisley, Scotland) at 37°C in 5% CO₂ atmosphere. For immunocytochemistry the cells were grown on microscopic slides (Slidechambers; Nunc, Roskilde, Denmark), using the same medium and atmosphere as mentioned previously.

Immunohistochemistry

Tissue sections, which had been fixed by inflation-fixation with 10% phosphate-buffered formalin, were cut (4 µm thick), placed on slides coated with 0.5% gelatin-0.05% chrome alum solution, air dried, and incubated overnight at 38°C. The sections were dewaxed with xylene, dehydrated through a series of alcohol solutions, and incubated in 0.3% (v/v) hydrogen peroxide in absolute methanol for 30 min to quench endogenous peroxidase activity. The sections were then incubated in goat serum (1:30) for 30 min at 20°C, and subsequently overnight with a 1:1,000 dilution of rabbit antibody to recombinant human MnSOD (a gift from Dr. J. D. Crapo, Duke University Medical Center, Durham, NC) in Tris-bovine serum albumin (36). Nonspecific binding was blocked by incubation in rabbit sera. Biotinylated secondary antibody was applied for 30 min, followed by an avidin-biotin complex treatment for 30 min (Vector Laboratories, Burlingame, CA). After each step, excluding the incubation with normal serum, the sections were washed with 0.05 M Tris buffer (pH 7.6). The sections were then exposed to a reaction solution containing the chromogen diaminobenzidine (Sigma, St. Louis, MO) and counterstained with hematoxylin.

Cultured cells growing on slides (Met5A, M14K, and M38K) were air dried and heat fixed (38°C, 1 h), and the same avidin-biotin complex method that was used to stain the paraffin sections was applied. The reaction solution contained the chromogen 3-amino-9-ethylcarbazole (Sigma). Replacement of the primary antibody by the Tris-bovine serum albumin buffer was used as a negative control in each series. Immunoreactivity was independently assessed by two of the authors. The staining was assessed by grading the average staining intensity of the tumor cells in comparison with the negative controls and nonmalignant mesothelial cells.

Northern Blot Analysis

For RNA isolation, cells were scraped into 4 M guanidine thiocyanate buffer, and samples were immediately frozen at 70°C. Total RNA was isolated using the acid phenol- chloroform method of Chomczynski and Sacchi (37). Denaturated RNA samples were electrophoresed on 1% agarose gels containing 0.36 M formaldehyde. After ethidium bromide staining and ultraviolet (UV) examination to confirm loading homogeneity, the RNA was transferred onto Hybond-N nylon filters (Amersham, Arlington Heights, IL) and cross-linked to the filters by UV illumination. Filters were prehybridized at 58.5°C for more than 1 h in a buffer containing 50% deionized formamide, 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 50 mM sodium phosphate (pH 6.5), 5× Denhardt's reagent, and 100 µg/ml herring sperm DNA.

All cDNA probes (MnSOD, CuZnSOD, catalase, and glutathione peroxidase) were kindly provided by Dr. Y.-S. Ho (Wayne State University, Detroit, MI). ³²P-labeled cRNA probes were transcribed from full-length MnSOD, CuZnSOD, catalase, and glutathione peroxidase cDNAs, all cloned into the pSP65 vector. Probes were purified using NucTrap columns (Stratagene, La Jolla, CA), added to prehybridization solution at 1 × 10⁶ cpm/ml, and hybridized overnight at 58.5°C with shaking. After hybridization, the filters were washed for 20 min in 2× SSC at room temperature, and twice for 20 min in 0.2× SSC-0.1% sodium dodecyl sulfate (SDS) at 65°C. After washing, the filters were exposed to Kodak (Rochester, NY) X-Omat AR photographic film at 80°C. Following autoradiography, the same filters were hybridized using the same procedures with a -actin control probe transcribed from p-TRI--actin plasmid (Ambion, Austin, TX). mRNA expression of these enzymes was compared quantitatively with the -actin mRNA expression by scanning densitometry, using the 300A Computing Densitometer and Image Quant Software v3.0 Fast Scan (Molecular Dynamics, Sunnyvale, CA).

Western Blot

The cells (Met5A, M14K, and M38K) were grown to confluency, detached with trypsin, centrifuged, and washed with phosphate-buffered saline (PBS). The cell pellets were resuspended in sterile water, mixed with the electrophoresis sample buffer, and boiled for 5 min at 95°C. The protein concentration of the samples was measured using the Bio-Rad protein assay (Bio-Rad, Hercules, CA) (38), and 50 µg of cell protein was applied per lane to a 12% sodium dodecyl sulfate-polyacrylamide gel (39). The gel was electrophoresed for 1.5 h (90 V), and the protein transferred (45 min, 100 V) onto Hybond ECL nitrocellulose membranes (Amersham) in a Mini-PROTEAN II Cell (Bio-Rad). The blotted membrane was incubated with rabbit antibody to recombinant human MnSOD (dilution, 1:10,000), followed by donkey anti-rabbit antibody conjugated to horseradish peroxidase (Amersham) (dilution, 1:30,000). MnSOD was detected using the enhanced chemiluminescence (ECL) system (Amersham), and the luminol excitation was imaged on X-ray film. The same membranes were stripped of bound antibodies, and reprobed using a monoclonal anti-actin antibody (1:2,500) followed by sheep anti-mouse antibody conjugated to horseradish peroxidase (1:3,000) (Amersham). -Actin expression was detected and imaged as described previously for MnSOD. The amount of MnSOD immunoreactive protein is shown relative to the corresponding -actin protein as described for Northern blots.

Enzyme Activities

For the enzyme assays, the cells were pelleted and immediately frozen at 70°C until analysis. The cells were resuspended in phosphate-buffered saline and treated with 1% Triton X-100 to disrupt the cellular organelles. Total SOD activity was measured spectrophotometrically using the method of McCord and Fridovich (40). MnSOD activity was distinguished from CuZnSOD activity by its resistance to 1 mM potassium cyanide. Glutathione peroxidase activity was analyzed by following NADPH oxidation in the presence of t-butylhydroperoxide (41). Glutathione reductase activity was determined by following the oxidation of NADPH in the presence of oxidized glutathione (41). The activity of catalase was analyzed using a Clark oxygen electrode as described earlier (42). Enzyme activities were expressed as units per milligram protein, and the protein concentration was measured according to the method of Lowry and co-workers (43).

Oxidant and Drug Exposures

The cells were exposed to 25-75 µM menadione for 4 h or to 10 µM menadione for 48 h. In other experiments, the cells were exposed to 0.1-0.2 µg/ml (0.17-0.34 µM) epirubicin for 48 h. These concentrations were chosen because our previous studies have shown that these concentrations cause minimal to significant injury to Met5A cells (12, 44, 45).

Adenine Nucleotide Measurements

Cells were preincubated with 0.1 mM [¹⁴C]adenine (specific activity, 51-55 mCi/mmol; Amersham International), starting 1 d before the exposures. After labeling, the cells were washed, and then exposed to menadione or epirubicin as described previously. After exposures, the medium was removed, and the cells were extracted with 0.4 N perchloric acid (PCA). Adenine nucleotides in the cell extract, and the nucleosides and bases in the medium, were separated by thin-layer chromatography and counted as described elsewhere (46). The results are expressed as the percentage distribution of radioactivity (counts per minute, cpm) between PCA-soluble compounds of interest in the cells (ATP, ADP, and AMP) and the medium (intact nucleotides and the sum of the catabolic products hypoxanthine, xanthine, and uric acid).

Lactate Dehydrogenase Measurements

Lactate dehydrogenase (LDH) release into the medium was measured by spectrophotometry using pyruvic acid as the substrate (47). The total cellular LDH was measured in cell lysates obtained by 1% Triton X-100 treatment.

Statistical Analysis

The results are expressed as the mean ± SD of three to seven experiments. The groups were compared by analysis of variance and Scheffe's post hoc test. P < 0.05 was considered to be significant.

	Results

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MnSOD in Healthy Visceral Pleural Tissue and Tumor Biopsies

Visceral pleural mesothelial cells showed no MnSOD immunoreactivity in five of the six samples of microscopically normal pleura (Figure 1a; Table 2). In one case the mesothelial surface of the visceral pleura showed a granular layer of immunoreactivity. In this particular case no signs of inflammation were detected, but some mesothelial cell proliferation and weak immunostaining in alveolar epithelial cells could be seen (not shown).

View larger version (160K): [in this window] [in a new window]   Figure 1.   MnSOD immunoreactivity in healthy pleura and pleural mesothelioma. Healthy human visceral pleura (arrowheads) shows no MnSOD immunoreactivity (a). Epithelial-type mesothelioma (M91) shows moderate immunoreactivity (b), and mixed-type mesothelioma (M38) shows intense immunoreactivity (c). Bar: 25 µm.

All seven mesothelioma tissue samples showed moderate or intense immunoreactivity in the malignant cells (Figures 1b and 1c; Table 2). These samples did not contain healthy mesothelium. Granular positive staining was restricted in the cytoplasm of the malignant cells, and the intensity of staining ranged from intense to moderate in different regions of the same biopsy sample. Intense immunoreactivity was detected regardless of the fiber content of the lung. Immunostaining for MnSOD appeared to be more intense in the mixed histology tumors than in the epithelial tumors (Table 2). No sarcomatous samples were available for evaluation. Inflammatory cells were seen occasionally in the tumor tissue samples, but their numbers did not appear to correlate with the intensity of the immunoreactivity of the sample (not shown). The immunoreactivity of individual inflammatory cells was variable, and is not analyzed in any detail here.

MnSOD in Cultured Nonmalignant Mesothelial Cells and Mesothelioma Cell Lines

In the nonmalignant Met5A cells positive immunoreactivity was found in the cell clusters, but otherwise only weak if any immunostaining could be detected (Figure 2a). Both mesothelioma tumor cell lines (M14K and M38K) were positive for MnSOD immunoreactivity, and the proportion of positive cells was much higher and the staining more intense than in Met5A cells (Figure 2; Table 2). As with the tumor biopsies, the immunoreactivity appeared as granular and heterogeneous staining in the cytoplasm of the cells. The staining pattern was intense in the M38K cells. This cell line was derived from the M38 mesothelioma tissue, which also showed intense MnSOD immunoreactivity (Figures 1c and 2c).

View larger version (86K): [in this window] [in a new window]   Figure 2.   Immunocytochemical staining for MnSOD in the cell lines. Nonmalignant Met5A mesothelial cells show weak MnSOD immunoreactivity (a). Both M14K (b) and M38K (c) mesothelioma cell lines show intense immunoreactivity. Bar: 25 µm.

Figure 3a shows a representative Western blot of Met5A, M14K, and M38K cells. The amount of MnSOD protein in relation to -actin was significantly higher in the mesothelioma cell lines (M14K and M38K) than in the nonmalignant mesothelial Met5A cells (Figure 3b). When compared with Met5A cells, Northern blot analysis indicated significant upregulation of the 1- and 4-kb transcripts of MnSOD in M38K cells and the 4-kb transcript in M14K cells (Figures 4a and 4b). MnSOD activity was also significantly higher in M38K cells than in M14K cells or Met5A cells (Figure 4c).

View larger version (30K): [in this window] [in a new window]   Figure 3.   Representative Western blot analyses showing MnSOD immunoreactive protein (21 kD) in the nonmalignant mesothelial cells (Met5A) and in the two mesothelioma cell lines (M14K and M38K) (a). MnSOD protein levels were lowest in Met5A cells and significantly elevated in M14K and M38K cells (b). -Actin (arrow) expression was detected to confirm loading homogeneity and to assess the relative amount of MnSOD immunoreactive protein. The optical density of MnSOD protein relative to that of -actin is expressed as relative intensity. Values are means ± SD (n = 3). *P < 0.05 compared with Met5A cells.

View larger version (18K): [in this window] [in a new window]   Figure 4.   MnSOD expression in the nonmalignant Met5A mesothelial cells and mesothelioma cell lines M14K and M38K. Northern blot for MnSOD mRNA indicates upregulation of the 4-kb transcript in M14K cells and both the 1- and 4-kb transcripts in M38K cells when compared with Met5A cells (a). The optical density of MnSOD mRNA relative to that of -actin is expressed as relative intensity (n = 3) (b). MnSOD activity was significantly higher in M38K cells (n = 5) than in M14K (n = 7) or in Met5A (n = 7) cells (c). Values are means ± SD. *P < 0.05 compared with Met5A cells.

Cytotoxicity Studies

Met5A mesothelial cells, M14K mesothelioma cells (low MnSOD activity), and M38K mesothelioma cells (high MnSOD activity) were exposed to menadione and to epirubicin to assess the role of MnSOD in the oxidant resistance and to compare oxidant resistance with the resistance to epirubicin in these cells. Acute exposure to menadione (25 µM for 4 h) resulted in a significant LDH release from Met5A cells but not from M38K cells (Figure 5a). Additional experiments with cellular nucleotides (ATP, ADP, and AMP) and nucleotide catabolites (xanthine, hypoxanthine, and uric acid) indicated that under the same exposure conditions the cellular energy state was best maintained in M38K cells, intermediate in M14K cells, and lowest in Met5A cells (Figures 5b and 5c). Similarly, 48-h exposure to menadione (10 µM) caused significant cell injury only in Met5A cells as assessed either by LDH release or adenine nucleotide depletion (not shown).

View larger version (15K): [in this window] [in a new window]   Figure 5.   LDH release (a), adenine nucleotide depletion (b), and accumulation of the nucleotide catabolite products (c) in menadione-exposed cells. Menadione (25 µM) caused significantly higher LDH release from Met5A mesothelial cells than from mesothelioma cells M14K and M38K. LDH release from the exposed cells compared with the control cells is expressed as relative LDH release. M38K cells were also most resistant and Met5A most sensitive to menadione-induced cell injury when assessed by high-energy nucleotide depletion or by nucleotide catabolite accumulation. Cellular nucleotide changes are expressed as percentage of the total counts per minute (CPM). Values are means ± SD (n = 4). *P < 0.05 compared with unexposed control cells. P < 0.05 compared with M38K cells. P < 0.05 compared with M14K and M38K cells.

When the cells were exposed to epirubicin (0.1 µg/ml, 48 h), Met5A cells released more LDH than did M14K or M38K cells, and the difference was even more pronounced at an epirubicin concentration of 0.2 µg/ml (Figure 6a). In addition, epirubicin caused the most significant reduction in adenine nucleotides and accumulation of nucleotide catabolites in Met5A cells. M38K cells were completely resistant to epirubicin at this concentration (Figures 6b and 6c).

View larger version (17K): [in this window] [in a new window]   Figure 6.   LDH release (a), adenine nucleotide depletion (b), and accumulation of the nucleotide catabolite products (c) in epirubicin-exposed cells. Met5A cells were sensitive and mesothelioma cells completely resistant to epirubicin exposure (0.1 µg/ml, 4 h) when assessed by LDH release, high-energy nucleotide depletion, or nucleotide catabolite accumulation. A higher concentration (0.2 µg/ml, 4 h) caused significant LDH release from Met5A cells and M14K mesothelioma cells, but again M38K cells were most resistant and Met5A cells were most sensitive. Values are means ± SD (n = 4). *P < 0.05 compared with unexposed control cells. P < 0.05 compared with M14K and M38K cells.

H₂O₂-Scavenging Antioxidant Enzymes

Because oxidants and chemotherapeutic agents generate both superoxide and H₂O₂, one possible explanation for the resistance of M38K cells is a coordinated increase in several intracellular antioxidant enzymes. Additional experiments were therefore conducted to assess the expression and activity of CuZnSOD, catalase, glutathione peroxidase, and glutathione reductase in the three cell types. The mRNA level of CuZnSOD was significantly higher in M38K cells than in Met5A cells but the activity of this enzyme did not differ between the three cell lines (Figure 7). The activity of catalase was higher in M38K cells than in M14K cells or in Met5A cells, and in M38K cells the mRNA level of catalase was significantly higher compared with M14K cells and marginally elevated compared with Met5A cells (Figure 8). The mRNA level of glutathione peroxidase was slightly but not significantly (+24%) elevated in M38K cells when compared with Met5A cells, and there was also a nonsignificant tendency toward an increase in glutathione peroxidase activity in M38K cells, whereas the activities of glutathione reductase in these three cell lines were similar (not shown).

View larger version (15K): [in this window] [in a new window]   Figure 7.   CuZnSOD in Met5A, M14K, and M38K cells. CuZnSOD mRNA was significantly upregulated in M38K cells compared with Met5A and M14K cells (a and b). CuZnSOD activity did not differ significantly between the cell lines (Met5A and M14K, n = 7; M38K, n = 5) (c). The optical density of CuZnSOD mRNA relative to that of -actin is expressed as relative intensity. Values are means ± SD (n = 4). *P < 0.05 compared with Met5A cells.

View larger version (14K): [in this window] [in a new window]   Figure 8.   Catalase in Met5A, M14K, and M38K cells. In M38K cells, catalase mRNA was significantly upregulated compared with the other two cell lines (a and b). Catalase activity was significantly higher in M38K cells (n = 7) than in M14K (n = 9) and Met5A cells (n = 6) (c). The optical density of catalase mRNA relative to that of -actin mRNA is expressed as relative intensity. Values are means ± SD (n = 3). *P < 0.05 compared with Met5A and M14K cells.

	Discussion

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We have shown that MnSOD reactivity in human pleural mesothelial cells is low, and that human malignant pleural mesothelioma cells express high levels of MnSOD in situ when compared with healthy human pleural mesothelial cells. Furthermore, the results of Northern blotting, Western blotting, immunohistochemistry, and the specific activity of MnSOD were consistent with our previous finding of high MnSOD mRNA and specific activity in human mesothelioma cells (12). The M38K mesothelioma cell line was the most resistant cell line to both oxidant and epirubicin exposure, and this same cell line had not only the highest MnSOD activity but also the highest catalase activity.

It has been assumed that tumor cells have low levels of MnSOD (17), and that the MnSOD gene could be a tumor suppressor gene (14). On the other hand, Marklund and co-workers (48) investigated several different tumors and cell lines from tumors, and found that cultured mesothelioma cells were the only type of malignant cells to show high levels of MnSOD activity. However, in that study as well as in many others, inadequate controls were used. Conclusions have often been drawn from activity measurements taken from plasma samples, which do not have the same enzyme levels as do tumor cells (reviewed in Ref. 13). The same is true for the studies in which the enzyme activity has been measured in tissue homogenates, which often contain various cell types and therefore do not reflect the enzyme activity of any particular cell type. This is also true of mesothelioma, which is a tumor with a high proportion of stromal tissue relative to malignant cells. Observations of cultured tumor cells may not be representative of the situation in vivo, but we found that MnSOD levels were highly elevated in both the M38K tumor and in the cell line established from this tumor. We also found that MnSOD was highly elevated in the tumor cells of the original tumor compared with nonmalignant human mesothelial cells.

All our mesothelioma patients had received significant occupational exposure to asbestos and the tumor cells showed intense immunoreactivity for MnSOD, but there was no tendency for higher fiber content in the samples to correlate with greater MnSOD reactivity. Healthy pleural mesothelium from our mesothelioma patients was not available, but if the induction of MnSOD in the tumors from these patients is due to their previous exposure to asbestos, adjacent healthy pleural mesothelium might also show MnSOD induction. We did not find any correlation between the stage of the tumor and the level of MnSOD expression, either. Although the role of MnSOD in the carcinogenesis of mesothelioma remains unclear, MnSOD induction in mesothelioma may be due to the oxidant stress and genotoxicity induced by asbestos exposure and inflammation (5, 6, 49).

MnSOD has been shown to be highly inducible in rat mesothelium and it has been suggested that mesothelial cells may be particularly rich in more highly inducible MnSOD mRNA species (4). The present study showed that both the 4- and 1-kb mRNA transcripts of MnSOD are upregulated in mesothelioma cells. We and others have also found that TNF and asbestos fibers cause upregulation of both these transcripts in cultured human mesothelial cells (5, 6). On the other hand, in human lung tissue and hepatoma cells, the 1-kb transcript is the major band of hybridization (50), and in rat lung and liver asbestos fibers and interleukin 6 (IL-6) result in upregulation of the 1-kb transcript (51, 52). The 4-kb transcript has faster turnover and a shorter half-life than the 1-kb transcript (53), but no systematic studies are available on the relative role of these two transcripts in human lung.

Met5A cells, which were used here to represent nonmalignant cultured mesothelial cells, are nontumorigenic cells that have typical mesothelial cell morphology. These cells were originally established from normal human mesothelial cells by transfection with a plasmid containing SV40 early region DNA, and they express SV40 T antigen (35). Met5A cells form densely packed colonies in culture, which are not found in primary cultured mesothelial cells (35). Interestingly, SV40 T antigen-related DNA has been detected in tissue samples of a high proportion of mesothelioma cases in the United States and in Europe, and an etiopathogenetic link between SV40 exposure and mesothelioma has been suggested (54, 55). In the present study MnSOD immunoreactivity in Met5A cells was localized in cell clusters, and only weak if any immunoreactivity for MnSOD was found in other Met5A cells. The reason for MnSOD induction in these cell colonies remains unclear, but theoretically it could be associated with the SV40 transformation.

Anticancer drug resistance in mesothelioma probably involves several mechanisms, one of which might be the antioxidant capacity of the tumor cells (11). High levels of MnSOD may significantly potentiate resistance of these cells to cytotoxic drugs, because anthracyclins, for example, produce superoxide radicals as part of their antitumor activity (11, 56). Because MnSOD scavenges superoxide into hydrogen peroxide, the coordinated balance of various antioxidant enzymes, in particular the balance between MnSOD and enzymes scavenging hydrogen peroxide, may be more important for cell protection than any individual enzyme. In fact, we found that simultaneous induction of superoxide-scavenging MnSOD and hydrogen peroxide-scavenging catalase in M38K mesothelioma cells was associated with the highest resistance to both menadione and epirubicin. Whether marginally increased CuZnSOD or glutathione peroxidase, or other glutathione-dependent detoxification enzymes (such as glutathione-S-transferases, on which we did not focus here), play any role in the resistance of these cells is unclear.

Relatively little is known about the H₂O₂-scavenging antioxidant enzymes in human solid tumors. Glutathione peroxidase and glutathione reductase levels are usually low but can vary between different malignant tumors (13). Coursin and colleagues (57) reported that glutathione peroxidase is low and catalase is undetectable or low in lung cancer. Catalase has not been investigated earlier in human mesothelioma cells, but it has been shown to be low in rat mesothelium in situ (44). Catalase reactivity and specific activity are rapidly decreased during the first days of culture in airway epithelial cells (36) and in alveolar epithelial type II cells (42). We found that M38K cells contained a higher activity of catalase than did nonmalignant mesothelial cells. Although upregulation of catalase may not be a typical finding in mesothelioma, we found that catalase levels were significantly elevated in those mesothelioma cells that were most resistant to menadione and epirubicin.

In conclusion, MnSOD immunoreactivity is low in healthy human pleural mesothelium, and high in human malignant mesothelioma. Mesothelioma cells containing highly elevated MnSOD were the cell type most resistant to menadione and epirubicin in vitro. MnSOD induction may be a typical feature of asbestos-related mesothelioma, but whether it has clinical implications in the diagnosis of mesothelioma or plays a role in the drug resistance of this tumor in vivo remains to be investigated. In addition to MnSOD induction, coordinated balance of multiple antioxidant pathways may be responsible for the high oxidant and drug resistance of mesothelioma cells in vitro and possibly also in vivo.