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Published ahead of print on March 23, 2004, doi:10.1165/rcmb.2003-0409OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 147-153, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0409OC

Ultrastructural and Chromosomal Studies on Manganese Superoxide Dismutase in Malignant Mesothelioma

Vuokko L. Kinnula, Tuula Torkkeli, Paula Kristo, Raija Sormunen, Ylermi Soini, Paavo Pääkkö, Tiina Ollikainen, Katriina Kahlos, Ari Hirvonen and Sakari Knuutila

Department of Medicine, Pulmonary Division, University of Helsinki and Helsinki University Hospital, Helsinki; Departments of Pathology and Medical Genetics, Haartman Institute, University of Helsinki and Helsinki University Central Hospital, Laboratory Diagnostics, Helsinki; Departments of Pathology and Internal Medicine, University of Oulu, Biocenter of Oulu, Oulu; and Finnish Institute of Occupational Health, Helsinki, Finland

Address correspondence to: Vuokko Kinnula, Department of Medicine, Pulmonary Division, University of Helsinki and Helsinki University Hospital, Box 22 (Haartmanink 4), 00140 Helsinki, Finland. E-mail: Vuokko.kinnula{at}helsinki.fi


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mesothelioma represents an aggressive tumor type with high resistance to all treatment modalities. Its pathogenesis is strongly associated with exposure to asbestos fibers and probably with free radicals. One of the most important free radical scavenging enzymes, mitochondrial manganese superoxide dismutase (MnSOD), has been shown to be elevated in mesothelioma (K. Kahlos et al., 1998, Am. J. Respir. Cell Mol. Biol. 18:570–580). In the present study, we could detect intense ultrastructural accumulation of MnSOD in the mitochondrial compartment of malignant mesothelioma cells. There was no association between the immunohistochemical reactivity and the most common and functional polymorphic variant of MnSOD, the Ala to Val amino acid change at 9 position (16th amino acid from the beginning of the signal sequence), in the 31 mesothelioma cases investigated. Comparative genomic hybridization and fluorescence in situ hybridization did not reveal any changes in chromosome 6, where the MnSOD gene is located. Sequencing of the MnSOD promoter region in four mesothelioma cell lines showed similar nucleotide variables in the malignant and nonmalignant cells. Therefore, the intense expression of MnSOD in the mitochondria of mesothelioma cells does not appear be associated with any major chromosomal alterations or the polymorphism of MnSOD gene. Association with oxidative/nitrosative stress in mesothelioma using nitrotyrosine immunostaining pointed to a tendency for more intense reactivity in those mesotheliomas with higher MnSOD expression (P = 0.069).

Abbreviations: bovine serum albumin, BSA • comparative genomic hybridization, CGH • fluorescence in situ hybridization, FISH • manganese superoxide dismutase, MnSOD • nuclear factor-{kappa}B, NF-{kappa}B • phosphate-buffered saline, PBS • saline sodium citrate, SSC


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pleural mesothelioma represents an aggressive form of malignancy with resistance to radiation and chemotherapy. Therefore, it can serve as an ideal model for investigating malignant tumors and the mechanisms contributing to invasion and drug resistance of malignant cells. The pathogenesis of mesothelioma is thought to be strongly associated with free radicals. For example, most cases of mesothelioma develop after prolonged exposure to asbestos fibers (1), and asbestos fibers can generate reactive oxygen species (ROS) both directly and indirectly by activating inflammatory cells. Manganese superoxide dismutase (MnSOD), one of the most important antioxidant enzymes in mammalian tissues (2, 3), is induced by asbestos fibers (4, 5) and also by multiple inflammatory cytokines (6, 7). Nonmalignant human pleural mesothelium and cultured mesothelial cells contain almost undetectable MnSOD immunoreactivities and/or activities, whereas human pleural mesothelioma tissues exhibit high MnSOD reactivity (8–10). Several experimental studies have shown that MnSOD transfection evokes increased resistance of malignant cells to oxidants, cytokines, asbestos fibers, and cytotoxic drugs, and conversely antisense technologies have indicated that a deficiency of MnSOD leads to increased oxidant sensitivity and cellular apoptosis (11–15). Thus MnSOD may have multiple effects on the pathogenesis of mesothelioma and also on its resistance to drug therapies and radiation.

The MnSOD gene is localized to chromosome 6q25.3 and the enzyme is synthesized in the cytoplasm as a precursor molecule containing a leader signal, which is removed during the transport of the molecule to the mitochondria (16). The gene is composed of 5 exons and 4 introns, the promoter region does not have any TATA or CAAT boxes within the 708 bp of the putative transcription initiation site. However, this site does contain GC-rich sequences immediately upstream from the transcription initiation site which is preceded by several potential binding sites for transcription factors like SP1 and AP2. Moreover the flanking region of the MnSOD gene contains a nuclear factor (NF)-{kappa}B consensus sequence, a potential regulatory element in MnSOD gene expression (17). The most common polymorphism of MnSOD, a mutation having also functional consequences, results in an Alanine (Ala) to Valine (Val) amino acid change at –9 position from the first amino acid of the mature protein, the 16th amino acid from the beginning of the signal sequence. Indirect evidence has indicated that this produces a conformational change in the helical structure of the protein, which may impair the transport of the protein into the mitochondria (16). So far, there is no experimental evidence supporting the effects of this polymorphism on the MnSOD transport in vivo. Several recent studies have, however, pointed to the importance of the functional polymorphism of MnSOD in malignant tumors; the Ala-allele appears to pose an increased risk for breast cancer (18, 19), whereas Val-allele has been linked to an increased lung cancer risk (20). Although activity analyses suggest that MnSOD expression is mainly attributable to the mitochondrial fraction in mesothelioma cells (21), this has not been visually demonstrated.

The aim of this study was to use immunoelectron microscopy to confirm the high expression of MnSOD in the mitochondrial compartment of malignant mesothelioma cells. There are numerous potential reasons for the high MnSOD level in the mesothelioma cells. They may be related to extra chromosome numbers or other chromosomal alterations, resulting in increased copy numbers of MnSOD in the cells, changes in the regulatory zones of the MnSOD gene, or to MnSOD variants associated with increased MnSOD transport to the mitochondria. Therefore, we also investigated a possible correlation between the numbers of chromosome 6 and genetic alterations in malignant mesothelioma cell lines by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). MnSOD is a highly regulated gene, harboring multiple positive and negative regulatory motifs in its promoter region. To detect any changes in this region that could possibly account for the observed high expression of MnSOD in mesothelioma, promoter regions from these cells and normal blood samples were sequenced and compared. Moreover, the occurrence of the MnSOD polymorphism was compared with the expression of MnSOD in the specimens of malignant mesothelioma. Because it is known that MnSOD is highly induced by oxidant stress in vivo and it has been demonstrated to be overexpressed in several aggressive tumors including mesothelioma (22), we also assessed a marker of oxidative/nitrosative stress in mesothelioma and correlated this parameter with MnSOD expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Thirty-one biopsies of histopathologically confirmed malignant pleural mesothelioma were retrieved from the files of the Department of Pathology, Oulu University Hospital. The biopsy material was fixed in 10% neutral formalin, and embedded in paraffin. Mesotheliomas were classified into epithelial, sarcomatoid, or biphasic subtypes according to the method of Travis and coworkers (23). They were distinguished from metastatic adenocarcinomas on the basis of the presence of intra- or extracellular hyaluronic acid in mesothelioma. In problematic cases, diagnostic electron microscopy and immunohistochemistry were also performed (24). The mesothelioma patients did not receive any anticancer therapy before biopsy, except for four patients who were treated with intrapleural or systemic anticancer drugs or radiation therapy. The majority of these biopsies have been published earlier in a study on MnSOD expression (25). In addition, 10 frozen mesothelioma tissues were used for MnSOD immunohistochemistry.

Mesothelial and Mesothelioma Cells in Culture
Nonmalignant Met5-A mesothelial cells were obtained from American Tissue Culture Collection (Teddington, UK). The mesothelioma cell lines—the M14K, M24K, M25K, and M38K—were originally established from the tumor tissue of untreated patients with mesothelioma (26). They were grown in RPMI 1640 cell culture 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, UK) at 37°C in 5% CO2 atmosphere.

Immunohistochemical Stainings
Tissue biopsies, fixed in 10% neutral formalin and embedded in paraffin, were deparaffinized in xylene and rehydrated in a descending ethanol series. A polyclonal antibody for MnSOD (generous gift from J. D. Crapo, National Jewish Medical Center, Denver, CO) was used (1:1,000) for the detection of MnSOD as described earlier (25). Frozen sections were processed using 5-µm-thick sections, which were air dried and washed in phosphate-buffered saline (PBS). Subsequently, the sections were incubated for 2 h at room temperature with a 1:1,000 concentration of the primary MnSOD antibody. The immunostaining was performed using the Histostain-Plus Bulk Kit (Zymed Laboratories Inc., South San Francisco, CA) and employing the AEC chromogen (Zymed Laboratories Inc.). Oxidative/nitrosative stress in the tissues was evaluated using nitrotyrosine. Nitrotyrosine rabbit polyclonal antibody was purchased from Upstate (Lake Placid, NY). The immunostaining was performed using the Histostain-Plus Bulk Kit (Zymed Laboratories Inc.), and the chromogen used was AEC (Zymed Laboratories Inc.).

The sections were lightly counterstained with hematoxylin and mounted with Eukitt (Kindler, Freiburg, Germany). Replacement of the primary antibody with PBS at pH 7.2 and isotype control were used as the negative controls.

The immunostainings for MnSOD and nitrotyrosine were evaluated semiquantitatively and divided into four groups as follows: –, no immunostaining; +, weak immunostaining; ++, moderate immunostaining; and +++, strong immunostaining. Nitrotyrosine immunoreactivity was also seen in tumor stroma; semiquantitative analysis was based on both cytoplasmic and stromal staining in tumor tissue.

Western Blot
The cells were grown to confluence, detached with trypsin, centrifuged, and washed with PBS. Fifty micrograms of cell protein (Bio-Rad, Hercules, CA) (27) was applied per lane to a 12% sodium dodecyl sulfate-polyacrylamide gel. The blotted membrane was incubated with rabbit antibody to recombinant human MnSOD (1:10,000 dilution), followed by donkey anti-rabbit antibody conjugated to horseradish peroxidase (Amersham, Arlington Heights, IL) (1:30,000 dilution). MnSOD was detected using the enhanced chemiluminescence system (Amersham) where 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 dilution) followed by sheep anti-mouse antibody conjugated to horseradish peroxidase (1:3,000 dilution; Amersham). The amount of MnSOD immunoreactive protein was related to the corresponding amount of ß-actin protein. The intensities were assessed quantitatively by scanning densitometry using 300A Computing Densitometer and ImageQuant Software v3.0 Fast Scan (Molecular Dynamics, Sunnyvale, CA).

Electron Microscopy and Immunoelectron Microscopy
Transmission electron microscopy was performed to assess the morphology of mitochondria in mesothelioma cells and in nonmalignant Met-5A cells. Cells were fixed in 2.5% glutaraldehyde and in 0.1 M phosphate buffer, pH 7.4, and detached from the cell culture flasks by using a rubber policeman. The cells were fixed for 1 h, postfixed with OsO4 in 0.1 M phosphate buffer, pH 7.4, for 1 h, dehydrated in acetone, and embedded in Epon LX 112. Thin sections were cut with Reichert Ultracut E-ultramicrotome and examined in a Philips CM 100 transmission electron microscope, by using an acceleration voltage of 80 kV.

For immunoelectron microscopy, the cultured cells were detached from the Petri dishes with a cell scraper fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 for 1 h. The cell pellet was immersed in 2.3 M sucrose and frozen in liquid nitrogen. Thin cryosections were cut with a Leica Ultracut UCT microtome. For the immunolabeling, the sections were first incubated in 5% bovine serum albumin (BSA) containing 0.1% CWFS gelatin (Aurion, Wageningen, The Netherlands) in PBS. Antibodies and gold conjugate were diluted in 0.1% BSA-C (Aurion) in PBS. All washings were performed in 0.1% BSA-C in PBS. Sections were then incubated with rabbit anti-MnSOD antibody for 60 min. After washings, sections were exposed to protein A-gold complex (size 10 nm) for 30 min, made according to Slot and Geuze (28). The controls were prepared by carrying out the labeling procedure without primary antibody. The sections were embedded in methylcellulose and examined in Philips CM100 transmission electron microscope.

MnSOD Genotyping
A quantity of 100 ng of lymphocyte DNA extracted by standard techniques was used as a template in a PCR-based restriction fragment length polymorphism assay performed essentially as described earlier (18). Briefly, a 107-bp PCR product was amplified using specific primers (5'-ACC AGC AGG CAG CTG GCG CCG G-3' and 5'-GCG TTG ATG TGA GGT TCC AG-3'), which create a restriction cut site for NgoM IV (New England Biolabs, Beverly, MA) in the Ala-allele. Positive and negative controls were used within each batch of PCR amplification performed unaware of the case-control status. After NgoM IV digestion, the resulting digested (89 bp and 18 bp) and uncut (107 bp) fragments were separated on 3% agarose gels. The EtBr-stained gels were subsequently photographed under ultraviolet light, after which two independent readers interpreted the gel images. All samples with ambiguous results, and a random selection of 10% of all samples were assessed a second time to ensure laboratory quality control.

FISH
Chromosome preparations of mesothelioma and nonmalignant control cells for FISH analyses were made as described by Pelin-Enlund and colleagues (26) with slight modifications. The negative controls were prepared from lymphocyte cultures of five healthy young adults and the positive control from an acute myelogenous leukemia patient with a 6;9-translocation: t(6;9)(p13;q34). FISH analyses of chromosome 6 aberrations were performed using a human DNA probe specific for chromosome 6, according to the manufacturer's instructions (Cambio, Cambridge, UK). Unstained chromosome slides from five cell lines, five negative controls, and one positive control were dehydrated through an ethanol series (70, 96, and 100%, each for 4 min) and air-dried. The dried slides were denatured by incubating in 70% formamide at 65°C for 2 min, after which 15 µl prewarmed hybridization solution was pipetted onto the slides, and the slides were covered with coverslips. The edges of coverslips were sealed with a rubber solution and incubated overnight at 42°C. Then the coverslips were removed and the slides were washed once in 2 x saline sodium citrate (SSC) (3 M sodium chloride and 0.3 M sodium citrate, pH 7.0) at 45°C for 5 min, twice for 5 min in 50% formamide/2 x SSC at 45°C for 5 min, and twice in 2 x SSC at 45°C for 5 min. One hundred microliters of pre-incubated detection reagent F1 were pipetted onto the slides, which were covered with coverslips and incubated at 42°C for 15 min. Then the slides were washed three times in wash solution (250 µl Tween detergent in 4 x SSC) at 45°C for 5 min. One hundred microliters of pre-incubated detection reagent were pipetted onto the slides, and the same procedure was performed as above using detection reagent F1 followed by dehydration for 5 min in an ethanol series (70, 96, 100%) at room temperature for 5 min. Eighteen microliters of counterstain (DAPI and Propium Iodide) mounting mixture was added to the slides, which were covered with coverslips. Fifteen metaphases were detected in each cell line.

CGH
DNAs from healthy volunteers (one female and one male) were used as negative CGH controls, and the positive control was from a malignant fibrous histiocytoma tumor with numerous DNA copy number changes, including a gain at 6q. The hybridizations were performed as described by El-Rifai and coworkers (29) with slight modifications. The tumor DNA was labeled with fluorescein isothiocyanate (FITC)-dUTP (DuPont NEN Products, Boston, MA) and the normal DNA, extracted from peripheral blood of healthy donors, with Texas Red-5-dUTP (DuPont). Equal amounts of labeled test and reference DNA were denatured at 75°C for 5 min and applied to normal metaphase slides. Before hybridization, metaphase slides were dehydrated in a series of 70, 80, and 100% ethanol and denatured at 65°C in a formamide solution (70% formamide/2 x SSC) for 2 min. The hybridization was performed in a moist chamber at 37°C for 48 h. After hybridization the slides were washed three times in 50% formamide/2 x SSC, twice in 2 x SSC, three times in 0.1 x SSC at 45°C, for 10 min each, and once in 4 x SSC/0.2% Tween at room temperature for 5 min. The slides were counterstained with DAPI (Sigma, St. Louis, MO) and analyzed using the ISIS CGH analysis system (MetaSystems GmbH, Altlussheim, Germany).

Sequencing of MnSOD Promoter Region
For sequencing analyses, chromosomal DNA was extracted from four malignant mesothelioma cell lines and 12 normal lymphocytes (EDTA-venous blood samples obtained from working-age blood donors to the Finnish Red Cross Transfusion Service) using High Pure PCR Template Preparation Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to standard protocols. The sequencing primers were designed by using Gene Runner software program (Hastings Software Inc., Moraga, CA). Overlapping PCR amplicons covering the region –1820 to +410 of MnSOD gene from the genomic DNAs were synthesized in stringent conditions and purified for sequencing by using QIAquick PCR Purification Kit (QIAGEN Inc., Hilden, Germany). The amplicons were sequenced both in forward and reverse orientations using cycle sequencing with Big Dye Terminator kit (version 3.0) supplied by Applied Biosystems (ABI, Foster City, CA) and the reactions were run on ABI 3,100 capillary sequencer (ABI) according to the manufacturer's instructions. The primary sequencing data was compared with the MnSOD sequence published in the genomic data bank annotated by NCBI (http://www.ncbi.nlm.nih.gov/entrez/viewer). The sequence comparisons were performed using NCBI's BLAST software program (http://www.ncbi.nlm.nih.gov/BLAST/). The starting point for the numbering of nucleotides was set in the translation start site (A(+1)TG) in all of our sequencing data.

Statistical Analyses
The correlations between the MnSOD immunoreactivities and genotype and between the MnSOD level and nitrotyrosine reactivities were assessed by Fisher's exact probability test using SPSS for Windows (10.1; SPSS, Chicago, IL). The survival of the mesothelioma patients in relation to MnSOD immunoreactivity was assessed by the Log Rank test. Probability values P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunohistochemistry and the MnSOD Polymorphism
In the series of 31 patients, the immunoreactivity of MnSOD was high or moderate in 24 cases (Figure 1, Table 1). None of the biopsies were negative for MnSOD reactivity. The prognosis of the patients was poor (mean 18 mo, median 5 mo, range 0–124 mo), and there was no significant association between the MnSOD immunoreactivity and patient survival (P = 0.1696 by Log Rank test). Frozen sections available from 10 specimens showed high MnSOD reactivity in five cases, moderate in three cases, and weak in two cases. In our recent study the occurrence of AlaAla, AlaVal, and ValVal genotypes of MnSOD in the controls were 19%, 57.1%, and 23.9%, respectively, and no significant associations were found between this polymorphism and the development of mesothelioma (30). No significant association could be observed when the immunoreactivity of MnSOD in the mesothelioma specimens was correlated here with the AlaVal polymorphism (P = 0.30) (Table 1).



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  		Figure 1. Light micrograph shows prominent immunostaining of MnSOD in malignant mesothelioma (Patient 1 in Table 1). The sections were lightly counterstained with hematoxylin; no immunostaining could be detected in the negative controls.

 

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  		TABLE 1. Mesothelioma cases, their histopathological typing, MnSOD and nitrotyrosine expressions by immunohistochemistry, and the MnSOD polymorphisms in the same samples

 
In further studies, oxidative/nitrosative stress in the mesothelioma tissues was analyzed via nitrotyrosine, which is known to reflect both oxidative and nitric oxide mediated reactions (31). Nitrotyrosine immunoreactivity was diffuse and cytoplasmic. There was a tendency toward an association between nitrotyrosine and MnSOD positivity, but this did not achieve statistical significance (Table 1) (P = 0.069).

Electron Microscopy and Immunoelectron Microscopy
The cell line studies confirmed previous results (8) that the MnSOD levels were higher in mesothelioma cell lines compared with nonmalignant mesothelial MeT-5A cells (Figure 2). The mitochondria of mesothelioma cells (Figure 3A) were dense and abundant, and the cristae were malformed and hardly recognizable. The endoplasmic reticulum was often abundant in the vicinity of the mitochondria. Immuno EM was conducted on three cell lines, i.e., M38K mesothelioma cells with high, M14K mesothelioma cells with moderate, and nonmalignant MeT-5A cells with low MnSOD levels (8, 10). The analyses showed that high levels of MnSOD immunoreactivity were localized to the mitochondria of mesothelioma cells; no MnSOD protein could be detected in the cytoplasmic compartment of the cells (Figure 3B). It was difficult to detect any MnSOD immunoreactivity in the MeT-5A mesothelial cells (data not shown).



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  		Figure 2. Western blotting analysis shows low MnSOD immunoreactivity in non-malignant MeT5A mesothelial cells and variable to high MnSOD immunoreactivity in the four mesothelioma cell lines.

 



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  		Figure 3. Electron microscopy (A) and immunoelectron microscopy (B) show abundant and malformed mitochondria with high levels of intramitochondrial MnSOD in M38K mesothelioma cells; no labeling could be detected in other cell compartments.

 
FISH and CGH
FISH of mesothelioma cell lines (M14K, M25K, and M38K) detected two normal copies of chromosome 6 (15 metaphases in each cell line and the control were studied). The analyses did not reveal any translocation, nor did DAPI chromosome banding show any cross-chromosome aberration in chromosome 6. The positive control with the 6;9-translocation revealed the change in each of the 15 cells studied. Comparative genomic hybridization (CGH) did not show any copy number changes in chromosome 6 in any of the cell lines, confirming the FISH results. However, many other chromosomes also contained DNA copy number gains and losses (not reported here). Normal DNAs showed no changes in chromosome 6 nor in any of the other chromosomes, whereas the positive control revealed the gain at 6q as well as the other aberrations known to be present in the tumor.

Sequencing of MnSOD Promoter
The nucleotide changes detected in the promoter region of MnSOD ranging from –1,820 to +410 (A of ATG being 1) in the mesothelioma cell lines (M14K, M24K, M25K, and M38K) are shown in Table 2. The same changes were seen in the lymphocyte DNAs of the 12 blood donors that were sequenced as controls (data not shown). The promoter sequences of malignant mesothelioma cell lines did not contain insertions or deletions larger than one nucleotide (Table 2), and all of these changes could also be seen in the non-malignant cells (data not shown).


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  		TABLE 2. The polymorphic sites detected in the genomic DNA isolated from the four mesothelioma cell lines

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows for the first time a clear ultrastructural accumulation of MnSOD protein in the mitochondria of cancer cells, and this could have potential effects on tumor cell survival and resistance. MnSOD immunoreactivity in the tumor biopsies was not associated with any functional polymorphism of the MnSOD gene. Neither the alterations in chromosome numbers nor the translocations, as assessed by CGH or FISH, in chromosome 6 could explain the overexpression of MnSOD in mesothelioma. Furthermore, it is most unlikely that any sequence variable or mutation in the promoter of MnSOD gene is the cause of the unusually high level of MnSOD in malignant mesothelioma cells either. Assessment of nitrotyrosine revealed a nonsignificant tendency for an association between elevated oxidative/nitrosative stress and MnSOD expression in mesothelioma.

The isolated overexpression of MnSOD in vitro has been connected to decreased proliferation and growth of malignant cells, and changes of the cellular phenotype toward higher differentiation indicate that MnSOD is an antitumor gene (32–34). On the other hand, several aggressive human tumors appear to overexpress MnSOD moreover in some of these tumors, high levels of MnSOD have correlated with poor prognosis (22, 35–40). The previous and present findings have also shown that MnSOD is low or virtually nondetectable in nonmalignant human pleural mesothelium, but it is clearly elevated in the mitochondrial compartment of human pleural mesothelioma cells (8–10). Because mesothelioma represents one of the most resistant and aggressive human tumors, MnSOD probably plays an important role in the resistance of this tumor to cytotoxic drugs and oxidant-induced apoptosis. Nearly all of our specimens showed moderate to strong MnSOD reactivity. We could not show any significant association between the MnSOD immunoreactivity and patient survival, but the prognosis of these patients, in general was very poor.

The differences between the cell culture studies using transfected MnSOD (32, 33) and the current in vivo biopsy findings can most likely be explained by the net changes in the cellular redox-state of the cells. Introducing a single enzyme, such as MnSOD into cultured cells could well lead to an imbalanced milieu for cell survival, whereas simultaneous overexpression of several antioxidant enzymes has been observed to occur in resistant and invasive tumors (22). One possible reason for the overexpression of MnSOD in mesothelioma cells is the high oxidant or metabolic state of the cell leading to the induced state of gene expression. We therefore assessed the immunoreactivity of nitrotyrosine as a marker of oxidative/nitrosative stress in the tissue specimens of malignant mesothelioma and correlated this parameter with the MnSOD reactivity. Even though the association was not significant (P = 0.069), this result may hint at a meaningful association.

The MnSOD genotypes have been linked to the pathogenesis of lung and breast cancer (18–20). Our recent study on Finnish asbestos insulators found a non-significant tendency for elevated risk of malignant mesothelioma in those individuals carrying the MnSOD AlaVal and ValVal genotypes with respective ORs of 1.38 (95% Cs I 0.30–6.34) and 2.01 (95% CI 0.34–11.9) (30). The effect of this functional polymorphism on MnSOD expression in vivo has not been earlier investigated. In the present study there was no association between the intensity of MnSOD staining in the mesothelioma biopsies and the MnSOD AlaVal genotypes.

Experimental studies suggesting MnSOD to be an antitumor gene have concluded that the low level of this enzyme in malignant cells is not probably due to any defect in the primary structure of the MnSOD protein. Furthermore, it is not explained by a change in the dosage of the MnSOD gene or by any decrease in the stability of MnSOD mRNA in tumor cells (41). Several heterozygous mutations around the G-C–rich promoter region of human MnSOD have, however, been observed (3 mutations in 14 different malignant cell lines). These mutations change the binding pattern of AP-2, reducing the MnSOD transciptional activity (42, 43). Thus this repression is mediated by decreased DNA binding to a methylated AP2 cis-regulatory element present in the MnSOD promoter (42, 44). However, in contrast to mesothelioma, this alteration results in a decreased MnSOD level in cancer cells. We employed CGH and FISH to assess changes in chromosome 6, where the MnSOD gene is located, to evaluate the cause for the increased activity of the mitochondrial MnSOD in human mesothelioma patients compared with control subjects, but no such abnormalities could be detected that would explain high MnSOD immunoreactivity in these cells. Previous cytogenetic studies on primary tumors and cell lines conducted by us and others have indicated copy number losses at 6 q, but rarely copy number gains in mesothelioma (45, 46). Even though our results did not reveal any gross genetic changes in chromosome 6, molecular changes, such as duplication of the gene or a part of it, cannot be ruled out by the methods we employed.

MnSOD is highly induced by a variety of intracellular and environmental compounds including tumor necrosis factor-{alpha}, interferon-{gamma}, interleukins, lipopolysaccharide, oxidative/nitrosative stress, asbestos fibers and cigarette smoke (6, 47). Induction has been found to be mediated by various transcription factors such as NF-{kappa}B, c-fos, c-jun, AP1, and CREB-1/ATF-1 like factor (47–50) and there are multiple binding sites for these factors immediately upstream from the transcription initiation site (17, 51). For example, the cytokine inducible enhancer containing binding sites for NF-{kappa}B, C/EBP, and NF-1 have been localized to the 236 sequence within intron 2 of the MnSOD gene (52). Sp1- and AP2-binding sites for the equivalent transcription factors in the proximal promoter of human MnSOD are important regulators of MnSOD; Sp1 positively promotes transcription and AP2 represses promoter activity. Some of the SNPs detected in the present study are encompassed within the area of the known or suggested regulatory elements. However, it is unlikely that they have any major role in the enhancement of gene expression in the malignant mesothelioma cell lines, because the control samples showed similar variations, but nonetheless exhibited low MnSOD expression. Oxidants and cytokines are potent inducers of MnSOD, but they also act as inhibitors of several signaling pathways. Overall, aggressive tumors can eventually contribute both to the induction, suggested here, and/or to the stability of MnSOD mRNA in the cells. Disentangling the exact mechanism underlying this phenomena would be of fundamental importance for the development of new treatment strategies for highly aggressive, invasive tumors like mesothelioma.


   Acknowledgments
 
This study has been partly supported by the Finnish Antituberculosis Association Foundation, Juselius Foundation and Cancer Society of Finland. The antibody against MnSOD was provided by Professor James D. Crapo, National Jewish Medical Center, Denver, CO. The technical assistance of Raija Sirviö and Manu Tuovinen is acknowledged. The authors also thank Juha Tuukkanen for helping us in image processing of the immunoelectron microscopy.

Received in original form November 13, 2003

Received in final form March 11, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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