Claudins in differential diagnosis between mesothelioma and metastatic adenocarcinoma of the pleura.

AIM: To study the expression of claudins in mesothelioma and metastatic pleural adenocarcinoma. METHODS: Immunohistochemical staining of claudins 1, 2, 3, 4, 5, and 7 was studied in 35 malignant mesotheliomas and the expression compared with 24 cases of pleural metastatic adenocarcinoma. All cases were also immunostained with calretinin. RESULTS: Claudin 1, 2, 3, 4, 5, and 7 expression was seen in 40%, 80%, 18%, 23%, 14%, and 43% of mesotheliomas, respectively, while the corresponding figures for adenocarcinoma were 100%, 88%, 90%, 100%, 50%, and 92%. Claudins 1, 3, 4, 5, and 7 were significantly less positive in mesothelioma than in metastatic adenocarcinoma, while no difference was observed for claudin 2. Claudins 1, 3, 4, 5, and 7 were also inversely associated with calretinin positivity. Sarcomatoid and biphasic mesothelioma subtypes appeared more negative for these claudins than pure epithelioid subtypes. Claudin expression was not associated with survival of patients with malignant mesotheliomas. CONCLUSIONS: The results show that malignant mesotheliomas have a lower expression of claudins 1, 3, 4, 5, and 7 than adenocarcinomas, and their expression could thus be used as an adjunct in differential diagnosis between the two. The difference was most evident for claudins 3 and 4, which were nearly as good as calretinin in mesothelioma detection. Sarcomatoid and biphasic mesotheliomas showed expression of these claudins only occasionally, which could be due to or contribute to their less epithelial appearance.

Activation and relocalization of caspase 3 during the apoptotic cascade of human mesothelioma cells.

Apoptosis plays an important role in cancer biology. We investigated the expression of caspases 3 and 8 in malignant mesothelioma and malignant mesothelioma cell lines and putative changes in their ultrastructural expression prior and after exposure to epirubicin. Further studies were conducted to compare these changes to the localization and expression of the bcl-2 group of proteins bcl-X, bax and mcl-1, and Fas-Fas ligand in the same cells. In the histological samples, caspase 3 and 8 immunoreactivity was seen in 27/37 (73%) and 16/37 (43%) of the mesotheliomas. The immunostaining was cytoplasmic diffuse, granular, and occasionally nuclear. All six mesothelioma cell lines expressed caspases 3 and 8 by immunoblotting. After exposure to epirubicin the extent of apoptosis was increased in all cell lines investigated, being weakest in the most resistant M38K cell line. Immunoelectron microscopy revealed immunogold labeling for caspases 3 and 8 in the mitochondria with the accumulation of caspase 3 in the apoptotic bodies, while the mitochondrial localization of the bcl-2 proteins appeared to be very stable. Fas receptor could be detected by flow cytometry, whereas the most resistant cell line (M38K) lacked Fas ligand when assessed by RT-PCR. These results suggest the importance of caspase 3 during the apoptotic process of mesothelioma cells and indicate that epirubicin-induced apoptosis is independent of the mitochondrial pathway.

MnSOD expression is less frequent in tumour cells of invasive breast carcinomas than in in situ carcinomas or non-neoplastic breast epithelial cells.

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme capable of neutralizing superoxide anion molecules. In previous studies it has been suggested to suppress both tumour proliferation and apoptosis. This study investigated 65 invasive, 50 in situ and 19 benign hyperplastic breast lesions for its immunohistochemical expression. MnSOD expression was also tested with in situ hybridization. To study cell proliferation, apoptosis and their association with MnSOD expression the neoplastic breast lesions were immunostained with a monoclonal antibody to Ki-67 and the extent of apoptosis in them was determined by the TUNEL method. 32/65 (49%) of the invasive ductal carcinomas, 41/50 (82%) of the in situ and 15/19 (79%) of the benign hyperplasias expressed the MnSOD protein. There were significantly more MnSOD positive cases in in situ carcinoma and in benign hyperplasia than in invasive carcinoma (p=0.00016 and p=0.022, respectively). Positivity was also more frequently found in non-neoplastic ductal and acinar epithelial cells than in invasive carcinoma. On the other hand, neoplastic epithelial cells of invasive and in situ carcinoma showed strong positivity more often than the epithelial cells of benign hyperplasia or non-neoplastic epithelium. In breast lesions, MnSOD positivity did not associate with proliferation or apoptosis. The lower frequency of MnSOD positive cases in invasive breast carcinoma suggests that the lack of its expression might contribute to the development of an invasive breast carcinoma phenotype and that it could in this way operate as a tumour suppressor gene, as previously suggested.

Endothelial nitric oxide synthase is strongly expressed in malignant mesothelioma but does not associate with vascular density or the expression of VEGF, FLK1 or FLT1.

AIMS: To investigate endothelial nitric oxide synthase (eNOS) expression in malignant mesothelioma and its association with expression of vascular endothelial growth factor (VEGF), its receptors FLK1 and FLT1, and vascular density. METHODS AND RESULTS: eNOS, VEGF, FLK1 and FLT1 were studied in 36 histological mesothelioma samples by immunohistochemistry. Two mesothelioma (M14K, M38K) and one non-neoplastic mesothelial cell line (MET-5A) were studied for eNOS mRNA expression by reverse transcriptase-polymerase chain reaction (RT-PCR). Vascular density was determined by staining the samples with an antibody to factor VIII. RT-PCR showed that mesothelioma cells synthesize eNOS in vitro. eNOS immunoreactivity was found in 32/36 (89%) tumours. VEGF, FLK1 and FLT1 expression was found in 17 (45%), 24 (69%) and 25 (71%) cases, respectively. FLK1 or FLT1 immunoreactivity was more often seen in epithelioid and biphasic mesotheliomas than in sarcomatoid ones (P=0.007 and P=0.011, respectively). There was a significant association between FLK1 and FLT1 immunoreactivity (P=0.032). No significant association was found between FLK1, FLT1, VEGF and eNOS immunoreactivity and vascular density. CONCLUSIONS: eNOS is strongly expressed in malignant mesothelioma. Since eNOS did not associate with VEGF, FLK1 or FLT1, its synthesis seems not to be regulated through VEGF in malignant mesothelioma as has been shown in non-neoplastic endothelial cells.

Widespread expression of thioredoxin and thioredoxin reductase in non-small cell lung carcinoma.

We investigated the expression of thioredoxin (Trx) and thioredoxin reductase (TrxR) in 89 non-small cell lung carcinomas. Additionally, immortalized human bronchial epithelial cells (BEAS 2B) and four human lung carcinoma cells lines (A549, SK-MES-1, CALU-6, and A427) were studied by Western blot and reverse transcription-PCR for the synthesis of Trx and TrxR protein and mRNA expression in vitro. The histological samples were also studied for immunohistochemical p53 and proliferating cell nuclear antigen expression and apoptosis. In non-neoplastic lung, Trx and TrxR expression was seen in bronchial epithelial cells and alveolar macrophages, metaplastic alveolar epithelial cells, and chondrocytes of the bronchus. In non-small cell lung carcinomas, there was a widespread expression of Trx and TrxR with only three and eight cases negative, respectively. Trx and TrxR expression was located in both cytoplasmic and nuclear compartments of the cells. There was a statistical association between cytoplasmic and nuclear Trx or TrxR expression. Grade I-II tumors showed stronger cytoplasmic and nuclear Trx and TrxR immunoreactivity than grade III tumors. No association was found between p53 and proliferating cell nuclear antigen expression and Trx or TrxR immunoreactivity. However, apoptosis was inversely associated with nuclear Trx and TrxR positivity. In the cell lines studied, both non-neoplastic BEAS 2B cells and all of the carcinoma cell lines expressed Trx and TrxR proteins and mRNA. The results show that these redox-regulating proteins are highly expressed in lung carcinomas taking part in activation of transcriptional factors and regulation of apoptosis in non-small cell lung carcinoma. In high-grade tumors, Trx and TrxR expression is diminished, suggesting loss of redox regulation in tumors with low differentiation.

Expression of caspases-3, -6 and -8 and their relation to apoptosis in non-small cell lung carcinoma.

We analyzed a set of 103 non-small cell lung carcinomas (NSCLCs) for caspase-3, -6 and -8 expression and apoptosis. Additionally, the expression of bcl-2, bax and p53 were studied. Caspase-3 positivity appeared as diffuse, cytoplasmic staining and was restricted to the tumor area. In contrast, the immunoreactivity for caspase-6 was intense, granular and mostly located in single cells or groups of tumor cells showing apoptotic morphology. The caspase-8 expression pattern was a combination of the two other caspases studied, featuring both diffuse and single-cell patterns restricted to the tumor area. No significant differences were seen in caspase -3, -6 and -8 expression between tumors of different histological types or grades. The number of apoptotic cells and bodies was significantly higher in NSCLCs, in which caspase-8 immunostaining was mainly seen in single cells (p = 0.017), whereas caspase -3 and -6 expression had no association with apoptosis. It is apparent that, in lung tissue, up-regulation of caspase expression is a phenomenon associated solely with neoplasia and reflects the readiness of the tumor cells to undergo apoptosis. Interestingly, caspases -3, -6 and -8 each have an individual staining pattern in NSCLC, perhaps reflecting their different position in the caspase hierarchy.

Up-regulation of thioredoxin and thioredoxin reductase in human malignant pleural mesothelioma.

Thioredoxin (Trx) with a redoxactive dithiol together with NADPH and thioredoxin reductase (TrxR) is a major disulfide reductase regulating cellular redox state and cell proliferation and possibly contributing to the drug resistance of malignant cells. We assessed the Trx system in malignant pleural mesothelioma cell lines, in nonmalignant pleural mesothelium and in biopsies of malignant pleural mesothelioma. The mRNA and immunoreactive proteins of Trx and cytosolic and mitochondrial TrxR were positive in all four human mesothelioma cell lines investigated. Six cases of nonmalignant, histologically healthy pleural mesothelium showed no Trx or TrxR immunoreactivity, whereas immunohistochemistry on 26 biopsies of human malignant pleural mesothelioma showed positive Trx in all cases and positive TrxR in 23 (88%) of the cases. Moderate or strong immunoreactivity for Trx or TrxR was detected in 85% (22 cases) and 61% (14 cases) of the mesothelioma cases, respectively. Both Trx and TrxR staining patterns were mainly diffuse and cytoplasmic, but in 39% of the mesothelioma cases prominent nuclear staining could also be detected. Although staining for Trx and TrxR was seen in tumor cells, no significant association could be demonstrated between Trx or TrxR expression and tumor cell proliferation or apoptosis in the biopsies of mesothelioma. There was no significant association between the intensity of Trx or TrxR immunoreactivity and patient survival, which may possibly be related to moderate or intense Trx and TrxR reactivity in most of the cases. Although the Trx system may have an important role in the drug resistance of malignant mesothelioma, these studies also suggest that multiple factors contribute to the promotion, cell proliferation and apoptosis of malignant mesothelioma cells in vivo.

Expression and prognostic significance of catalase in malignant mesothelioma.

BACKGROUND: Free radicals and antioxidant enzymes (AOEs) may play a critical role in cell proliferation and in the resistance of malignant cells against cytotoxic drugs and radiation. Malignant mesothelioma is a resistant tumor with high levels of manganese superoxide dismutase, a central superoxide scavenging AOE. In the current study, the authors assessed the expression and prognostic role of catalase, an important hydrogen peroxide scavenging AOE, in malignant pleural mesothelioma. METHODS: Catalase expression was investigated by immunohistochemistry in 5 cases of nonmalignant healthy pleura and in tumor tissue of 32 mesothelioma patients, and by Western blot in 7 continuous human mesothelioma cell lines. The distribution of catalase in mesothelioma cells was assessed by immunoelectron microscopy. Furthermore, to investigate the effect of catalase inhibition in the drug resistance of these cells in vitro, the authors exposed mesothelioma cells with the highest catalase level to epirubicin with and without aminotriazole pretreatment. RESULTS: Nonmalignant mesothelial cells showed no catalase immunoreactivity whereas most mesothelioma cases (24 of 32, 75%) were catalase positive, 17 cases (53%) showing moderate or high expression. Higher catalase expression in mesothelioma was associated with a better prognosis, mean survival rate from diagnosis being 6 and 24 months for negative/low expression and moderate/high expression, respectively. Furthermore, a coordinately high expression of both manganese-superoxide dismutase (Mn-SOD) and catalase predicted even more favorable outcome of the mesothelioma patients. Catalase also could be detected in all mesothelioma cell lines, the most resistant cell line showing the highest protein expression and compartmentalization of catalase mainly to peroxisomes. Aminotriazole inhibition of catalase had a marginal effect on the toxicity caused by epirubicin. CONCLUSIONS: Catalase may have multifactorial effects in malignant cells; high catalase and/or coordinated high expression of Mn-SOD and catalase may decrease tumor progression by modulating the cellular redox state, but enhanced antioxidant capacity of mesothelioma cells also may protect tumor cells against exogenous oxidants, at least in vitro.

Modulation of cell and DNA damage by poly(ADP)ribose polymerase in lung cells exposed to H(2)O(2) or asbestos fibres.

Poly(ADP)ribose polymerase (PARP) may participate in cell survival, apoptosis and development of DNA damage. We investigated the role of PARP in transformed human pleural mesothelial (MeT-5A) and alveolar epithelial (A549) cells exposed from 0.05 to 5mM hydrogen peroxide (H(2)O(2)) or crocidolite asbestos fibres (1-10 microg/cm(2)) in the presence and absence of 3-aminobenzamide (ABA), a PARP inhibitor. The cells were investigated for the development of cell injury, DNA single strand breaks and depletion of the cellular high-energy nucleotides. Compared to H(2)O(2), fibres caused a minor decrease in cell viability and effect on the cellular high-energy nucleotide depletion, and a marginal effect on the development of DNA strand breaks when assessed by the single cell gel electrophoresis (the Comet assay). Inhibition of PARP transiently protected the cells against acute H(2)O(2) related irreversible cell injury when assessed by microculture tetrazolium dye (XTT) assay and potentiated oxidant related DNA damage when assessed by the Comet assay. However, PARP inhibition had no significant effect on fibre-induced cell or DNA toxicity with the exception of one fibre concentration (2 microg/cm(2)) in MeT-5A cells. Apoptosis is often associated with PARP cleavage and caspase activation. Fibres did not cause PARP cleavage or activation of caspase 3 further confirming previous results about relatively low apoptotic potential of asbestos fibres. In conclusion, maintenance of cellular high-energy nucleotide pool and high viability of asbestos exposed cells may contribute to the survival and malignant conversion of lung cells exposed to the fibres.

Expression of inducible nitric oxide synthase in healthy pleura and in malignant mesothelioma.

In this study we investigated the immunohistochemical expression of inducible nitric oxide synthase (iNOS) in a set of normal pleural mesothelial tissues, malignant mesotheliomas, mesothelioma cell lines and metastatic pleural adenocarcinomas. Furthermore, the expression of mRNA was assessed in four malignant mesothelioma cell lines in culture. Apoptosis and vascular density in malignant mesotheliomas was assessed by the TUNEL method and by immunohistochemistry with an antibody against FVIII-related antigen. Immunohistochemically mesothelial cells in non-neoplastic healthy pleural tissues were mostly negative for iNOS. Positivity for iNOS was observed in 28/38 (74%) and 24/25 (96%) of malignant mesotheliomas and metastatic pleural adenocarcinomas, respectively. Epithelial and mixed mesotheliomas expressed more often strong iNOS immunoreactivity compared to the sarcomatoid subtype (P = 0.023). Moreover, metastatic adenocarcinomas expressed more often iNOS positivity than mesotheliomas (P = 0.021). Experiments with the cell lines confirmed that malignant mesothelioma cells are capable of synthesizing iNOS. No significant association was found between iNOS expression and apoptosis or vascular density in malignant mesotheliomas. The higher expression of iNOS in the epithelial subtype of mesothelioma and pleural metastatic adenocarcinoma might be due to an increased sensitivity of these cell types to cytokine-mediated iNOS upregulation. The strong expression of iNOS suggests a putative role for NO in the growth and progression of these tumours.

Proliferation, apoptosis, and manganese superoxide dismutase in malignant mesothelioma.

Proliferation and apoptotic indices of tumour cells may have important prognostic significance. Manganese superoxide dismutase (MnSOD), an important anti-oxidant enzyme, has been shown to decrease proliferation of malignant cells transfected with the MnSOD gene. The aim of the present study was to investigate the indices of cell proliferation and apoptosis and their prognostic significance in human mesothelioma and to assess the effect of MnSOD on the proliferation and apoptosis of the mesothelioma cells expressing high constitutive MnSOD activity. Tissue sections from 35 subjects with malignant pleural mesothelioma were studied for cell proliferation by Ki-67 immunohistochemistry and for apoptosis by the TUNEL assay. In additional experiments, 2 mesothelioma cell lines expressing either low (M14K) or high (M38K) MnSOD levels were assessed for proliferative and apoptotic responses to epirubicin. The median proliferation and apoptotic indices of the mesothelioma tissue were 8.2% and 0.75%, respectively. Patients with a high proliferation (>8%) or apoptotic index (>0.75%) showed a worse prognosis (p < 0.001). MnSOD expression was inversely correlated with cell proliferation (p = 0.02). Our cell line experiments indicated that cells expressing high MnSOD levels were more resistant to apoptosis and showed lower proliferation when exposed to epirubicin in vitro. These findings show that high proliferation and apoptosis are associated with a poor prognosis of mesothelioma and that a high MnSOD level is associated with low proliferation of tumour cells. Furthermore, experiments with cultured mesothelioma cells suggest the importance of MnSOD in the proliferation and apoptosis caused by drug exposure.

Inducible nitric oxide synthase expression, apoptosis, and angiogenesis in in situ and invasive breast carcinomas.

In this investigation, we studied the expression of inducible nitric oxide synthase (iNOS) and its association to apoptosis and angiogenesis in 43 in situ and 68 invasive breast carcinomas. Its expression was studied immunohistochemically using a polyclonal iNOS antibody, and the staining was evaluated both in tumor and stromal cells. Apoptosis was detected by 3' end labeling of fragmented DNA (terminal deoxynucleotidyl transferase-mediated nick end labeling method). Vascularization was detected immunohistochemically using an antibody to the FVIII-related antigen, and calculated microvessel densities were determined. In addition to strong iNOS expression in stromal cells, iNOS positivity was observed in tumor cells in 46.5% of in situ and 58.8% of invasive carcinomas. In invasive carcinomas, there were more cases with iNOS positivity both in tumor and stromal cells compared to in situ carcinomas (0.007). The proportion of cases with iNOS-positive tumor cells increased in in situ carcinomas from grade I to III (20.0%, 46.2%, and 73.3%). In invasive ductal carcinomas, there were more cases with iNOS-positive tumor cells than with in situ carcinomas (P = 0.04). Carcinomas with both iNOS-positive tumor and stromal cells had a higher apoptotic index (P = 0.02) and a higher calculated microvessel densities index (P = 0.02). A high number of iNOS-positive stromal cells associated with metastatic disease (P = 0.05). The results show that breast carcinoma cells, in addition to stromal cells, express iNOS and are capable of producing NO. Carcinomas with iNOS-positive tumor and stromal cells have a higher apoptotic indices and increased vascularization, suggesting that iNOS contributes to promotion of apoptosis and angiogenesis in breast carcinoma. The association of the number of iNOS-positive stromal cells with metastatic disease might be attributable to stimulation of angiogenesis, resulting in a higher vascular density and consequently a higher probability for tumor cells to invade.

Manganese superoxide dismutase as a diagnostic marker for malignant pleural mesothelioma.

Although several immunohistochemical markers are available, differential diagnosis between mesothelioma and metastatic adenocarcinoma of the pleura is difficult. We have found that the immunoreactivity of manganese superoxide dismutase (MnSOD), an important antioxidant enzyme, is high in mesothelioma compared to healthy pleural mesothelium. The aim of the present study was to investigate whether MnSOD can be used in the differential diagnosis of malignant mesothelioma and metastatic adenocarcinoma of the pleura. MnSOD expression was assessed by using immunohistochemistry in biopsies of malignant mesothelioma (n = 35) and metastatic adenocarcinoma of the pleura (n = 21). MnSOD immunoreactivity was assessed semiquantitatively with and without microwave pretreatment. Fifteen of the 35 malignant mesotheliomas showed moderate or strong MnSOD expression without and 23 with microwave pretreatment, the corresponding figures for metastatic adenocarcinoma of the pleura being 1 and 2 out of 21 (P = 0.002 and P < 0.001, respectively by Fisher's exact test). Only mesothelioma biopsies showed strong MnSOD reactivity, and it was never negative in mesothelioma, whereas one-third of the adenocarcinomas showed no MnSOD reactivity. In conclusion, MnSOD immunoreactivity can, combined with other markers, aid the differential diagnosis between malignant mesothelioma and metastatic adenocarcinoma of the pleura.

Generation of reactive oxygen species by human mesothelioma cells.

Malignant mesothelioma cells contain elevated levels of manganese superoxide dismutase (MnSOD) and are highly resistant to oxidants compared to non-malignant mesothelial cells. Since the level of cellular free radicals may be important for cell survival, we hypothesized that the increase of MnSOD in the mitochondria of mesothelioma cells may alter the free radical levels of these organelles. First, MnSOD activity was compared to the activities of two constitutive mitochondrial enzymes; MnSOD activity was 20 times higher in the mesothelioma cells than in the mesothelial cells, whereas the activities of citrate synthase and cytochrome c oxidase did not differ significantly in the two cell lines. This indicates that the activity of MnSOD per mitochondrion was increased in the mesothelioma cells. Superoxide production was assayed in the isolated mitochondria of these cells using lucigenin chemiluminescence. Mitochondrial superoxide levels were significantly lower (72%) in the mesothelioma cells compared to the mesothelial cells. Oxidant production in intact cells, assayed by fluorimetry using 2',7'-dichlorodihydrofluorescein as a fluorescent probe, did not differ significantly between these cells. We conclude that mitochondrial superoxide levels are lower in mesothelioma cells compared to nonmalignant mesothelial cells, and that this difference may be explained by higher MnSOD activity in the mitochondria of these cells. Oxidant production was not different in these cells, which may be due to the previously observed increase in H2O2-scavenging mechanisms of mesothelioma cells.

Type III and type I procollagen markers in fibrosing alveolitis.

Deposition of types I and III collagen is a typical feature in the development of pulmonary fibrosis. We assessed the propeptides of these procollagens as prognostic markers in 18 patients with fibrosing alveolitis. We analyzed the amino-terminal propeptide of type III procollagen (PIIINP) and the carboxy-terminal propeptide of type I procollagen (PICP) from samples of bronchoalveolar lavage fluid (BALF) and serum, and also estimated their concentrations in epithelial lining fluid (ELF) by the urea method. The level of PIIINP in serum (p < 0.05), BALF (p < 0.05), and ELF (p < 0.05), and the levels of PICP in BALF (p < 0.001) and ELF (p < 0.001) but not in serum, were significantly increased in the patients with fibrosing alveolitis as compared with 17 controls who had been investigated for minor respiratory symptoms. In the BALF and ELF of patients with fibrosing alveolitis, PICP but not PIIINP had significant negative correlations with the specific diffusion coefficient for carbon monoxide (DLCO/ VA). The amino-terminal propeptide of type III procollagen and the carboxy-terminal propeptide of type I procollagen in BALF correlated significantly with one another. During the follow-up period of 6 yr, seven of the 18 patients with fibrosing alveolitis died of the disease, 3 others died of malignancy, and one patient died from an unknown cause. DLCO (p < 0.05) differed significantly between the surviving patients and those who died of fibrosing alveolitis, and detectable PIIINP in BALF predicted death from fibrosing alveolitis (p = 0.05). In conclusion, these results show that PIIINP in BALF, ELF, and serum, and PICP in BALF and ELF, are increased in patients with fibrosing alveolitis. A high level of PICP in BALF, and especially in ELF, suggests a chronic process and increased synthesis of type I collagen in the lungs, whereas PIIINP in BALF and ELF suggests active disease and a poor prognosis.

Manganese superoxide dismutase in healthy human pleural mesothelium and in malignant pleural mesothelioma.

We hypothesized that manganese superoxide dismutase (MnSOD), known to be induced in rat mesothelial cells by asbestos fibers, cytokines, and hyperoxia, may also be induced in asbestos-related pleural diseases such as mesothelioma. MnSOD was assessed in healthy human pleural mesothelium (n = 6), in biopsy samples of human pleural mesothelioma (n = 7), in transformed nonmalignant human mesothelial cells (Met5A), and in two human mesothelioma cell lines (M14K and M38K) established from the tumor tissue of mesothelioma patients. There was no MnSOD immunoreactivity in five of the six samples of healthy pleural mesothelium, whereas MnSOD immunoreactivity was high in the tumor cells in all the mesothelioma samples. Northern blotting, immunohistochemistry, Western blotting, and specific activity measurements showed lower MnSOD in the nonmalignant Met5A mesothelial cells than in the M14K and M38K mesothelioma cells. In additional experiments the mesothelial and mesothelioma cells were exposed to menadione, which generates superoxide intracellularly, and to epirubicin, a cytotoxic drug commonly used to treat mesothelioma. The M38K mesothelioma cells were most resistant to menadione and epirubicin when assessed by LDH release or by adenine nucleotide (ATP, ADP, and AMP) depletion. These same cells showed not only the highest MnSOD levels, but also the highest mRNA levels and activities of catalase, whereas glutathione peroxidase and glutathione reductase levels did not differ significantly. We conclude that MnSOD expression is low in healthy human pleural mesothelium and high in human malignant mesothelioma. The most resistant mesothelioma cells contained coordinated induction of MnSOD and catalase.

Manganese superoxide dismutase in human pleural mesothelioma cell lines.

Mesothelioma is a malignant pleural or intraperitoneal tumor attributable to asbestos exposure in more than 80% of the cases. Manganese superoxide dismutase (MnSOD), a mitochondrial superoxide radical scavenging enzyme, is low in most tumors but is known to be induced by asbestos fibers and certain cytokines. Induction of MnSOD may be associated in asbestos-related pulmonary diseases in vivo. We investigated here MnSOD specific activity and MnSOD mRNA level using healthy human lung tissue, SV40-transformed human pleural mesothelial cells (Met5A), and six human malignant mesothelioma cell line cells. Total SOD (CuZnSOD + MnSOD) and MnSOD activities were 20.0 +/- 4.8 U/mg protein and 3.2 +/- 1.2 U/mg protein in healthy human lung tissue, and 25.6 +/- 10.7 U/mg and 3.8 +/- 1.0 U/mg in Met5A cells, respectively. In four mesothelioma cell lines MnSOD activity was significantly elevated, the highest activity (30.1 +/- 8.2 U/mg) was almost 10-fold compared to the activity in Met5A cells. The steady state mRNA level of MnSOD was low in Met5A cells and markedly higher in all mesothelioma cell lines roughly in proportion with enzyme activities. Cytotoxicity experiments, which were conducted in four cell lines, indicated that cells containing high MnSOD mRNA level and activity were resistant to the mitochondrial superoxide-producing agent menadione. In conclusion, our results suggest that human mesothelioma may express high levels of MnSOD, which is associated with high oxidant resistance of these cells.

Volatile constituents of wild and in vitro cultivated Gloeophyllum odoratum.

The brown-rot fungus Gloeophyllum odoratum was collected from spruce stumps in southern Finland. The volatiles in the fruiting body and fungal cultures grown in malt extract and liquid medium were investigated. Chitin, chitosan and D-(+)-glucosamine at a concentration of 450 mgl-1 medium were used as elicitors. Chitosan completely inhibited growth in the solid medium. The main volatile(s) according to GC and GC-MS analysis were either linalool, citronellol, geraniol and methyl p-methoxyphenylacetate or drimenol depending on the culture type and elicitor. The composition of volatiles in the natural fungus differed slightly from that of the cultivated fungus since the major compound was methyl p-methoxyphenylacetate. The volatile oils were toxic to larvae of the brine shrimp, Artemia salina, indicating that they may possess insecticidal and cytotoxic activity.