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Background: Humans vary in their ability to metabolize endogenous and exogenous compounds. Glutathione S-transferases (GSTs) and N-acetyltransferases (NATs) are enzymes involved in the detoxification of hazardous agents. The GSTM1 and GSTT1 genes exhibit null (i.e., deletion) polymorphisms; in specific individuals, homozygous deletion (i.e., both copies lost) of these genes can be detected. Polymorphism of the NAT2 gene results in slow and fast acetylators of potentially toxic substances. The GSTM1-null and the NAT2 slow-acetylator genotypes have been associated with increased risks for the development of environmentally induced cancers.Purpose: We assessed whether homozygous GSTM1-null or GSTT1-null genotypes or the NAT2 slow-acetylator genotype were associated with increased risks for the development of malignant and nonmalignant asbestos-related pulmonary disorders in a cohort of Finnish construction workers.Methods: The study population consisted of 145 asbestos insulators who were classified as having been exposed to high levels of asbestos; 69 of these individuals had no pulmonary disorders (control subjects), and 76 had either malignant mesothelioma (n = 24) or nonmalignant pulmonary disorders, such as asbestosis and/or pleural plaques (n = 52). Lymphocyte DNA and the polymerase chain reaction were used to determine the GSTM1, GSTT1, and NAT2 genotypes of the study subjects. Odds ratios (ORs) and 95% confidence intervals (CIs) estimating the relative risks of disease associated with specific genotypes were calculated from 2 x 2 tables by use of Fisher's exact method.Results: Risks for the development of asbestos-related pulmonary disorders were not affected significantly by homozygous deletion of the GSTM1 or GSTT1 genes. However, the risk of developing both malignant and nonmalignant pulmonary disorders for individuals with a NAT2 slow-acetylator genotype was more than twice that observed for those with a NAT2 fast-acetylator genotype (OR = 2.3; 95% CI = 1.1-4.7); the risk of developing malignant mesothelioma for NAT2 slow acetylators was increased almost fourfold (OR = 3.8; 95% CI = 1.2-14.3). Individuals who lacked the GSTM1 gene and possessed a NAT2 slow-acetylator genotype had a risk of developing malignant and nonmalignant pulmonary disorders that was approximately fivefold greater than that observed for those who had the GSTM1 gene and a NAT2 fast-acetylator genotype (OR = 5.1; 95% CI = 1.6-17.6); these individuals had a fourfold increased risk of developing nonmalignant pulmonary disorders (OR = 4.1; 95% CI = 1.1-17.2) and an eightfold increased risk of developing malignant mesothelioma (OR = 7.8; 95% CI = 1.4-78.7) when compared with the same reference group.Conclusions: Individuals with homozygous deletion of the GSTM1 gene and a NAT2 slow-acetylator genotype who are exposed to high levels of asbestos appear to have enhanced susceptibility to asbestos-related pulmonary disorders. [ABSTRACT FROM AUTHOR]

Normal human mesothelial cells from individual donors were studied for susceptibility to asbestos-induction of apoptosis and generation of an extended lifespan population. Such populations were generated after death of the majority of cells and arose from a subset of mesothelial cultures (4/16) whereas fibroblastic cells (5/5) did not develop extended lifespan populations after asbestos exposure. All mesothelial cultures were examined for the presence of SV40 T antigen to obtain information on (i) the presence of SV40 T antigen expression in normal human mesothelial cells and (ii) the relationship between generation of an extended lifespan population and expression of SV40 T antigen. Immunostaining for SV40 T antigen was positive in 2/38 normal human mesothelial cultures. These cultures also had elevated p53 expression. However, the two isolates expressing SV40 T antigen did not exhibit enhanced proliferative potential or develop an extended lifespan population. Asbestos-generated extended lifespan populations were specifically resistant to asbestos-mediated but not to α-Fas-induced apoptosis. Deletion of p16Ink4a was shown in 70% of tumor samples. All mesothelioma cell lines examined showed homozygous deletion of this locus which extended to exon 1β. Extended lifespan cultures were examined for expression of p16Ink4a to establish whether deletion was an early response to asbestos exposure. During their rapid growth phase, extended lifespan cultures showed decreased expression of p16Ink4a relative to untreated cultures, but methylation was not observed,, and p16Ink4a expression became elevated when cells entered culture crisis. These data extend the earlier observation that asbestos can generate extended lifespan populations, providing data on frequency and cell type specificity. In addition, this report shows that generation of such populations does not require expression of SV40 T antigen. Extended lifespan cells could represent a population expressing early changes critical for mesothelioma development. Further study of these populations could identify such changes. [ABSTRACT FROM PUBLISHER]

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 cytochromec 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 H[SUB2]O[SUB2]-scavenging mechanisms of mesothelioma cells. [ABSTRACT FROM AUTHOR]

We examined gap junctional intercellular communication (GJIC) of primary human mesothelial cells and cell lines of asbestos-associated human pleural mesotheliomas, and the effect of asbestos and other mineral fibres on these cells. In homologous cultures, the GJIC capacity of six out of seven tumour cell lines was markedly less than for primary mesothelial cells. This defect in GJIC appeared not to be at the expression level of mRNA and protein of the gene encoding the 43 kDa gap junction protein. In heterologous cocultures of tumour cells and primary mesothelial cells, however, 80–90% of the tumour cell/normal cell contacts were functional. Exposure of primary mesothelial cells to TPA, a phorbol ester tumour promoter, resulted in marked inhibition of GJIC, being an action common to numerous tumour promoters. Such an effect though was not observed with the carcinogenic mesothelioma-inducing mineral fibres chrysotile and amosite, neither with glass wool. These results suggest that a permanent defect in GJIC capacity is a common feature of human mesothelioma cells, but how mineral fibres are involved in the process of mesotheliomagenesis is still unclear. [ABSTRACT FROM PUBLISHER]

Seven mesothelioma cell lines were established from clinical specimens from five patients with asbestmrelated malignant pleural mesothelioma. The cells in culture show either epithelial or mixed epithelial/fibrosarcomatous growth with an average doubling time of 30 h. Giant multinucleated cells are common in all the cell Lines, as well as long thin microvllli on the cell surfaces. All cell lines were cytokerath pitive and they stained negatively for monocyte—macrophage markers. AU seven cell lines and one long-term tissue culture from a sixth mesothelioma patient were characterized cytogenetically. hyotype analyses revealed complex structural and numerical abnormalities, primarily involving chromosome 1, 4, 5, 6, 7, 8, 9, 11, 12, 13 and 22. An excess of chromosome material of the short arm of chromosome 5 was seen consistently in six cell lines and in the long-term culture. In cell lines from four patients, changes in chromosome 13, mainly monasomy 13, were observed. The marker chromosomes observed in the e d y passages were conserved and few additional changes appeared in later passages. Six of the cell lines tested for tumorigenicity in athymic nude mice were weakly positive. [ABSTRACT FROM PUBLISHER]

Gap junctional intercellular communication (GJIC) has been reported to be markedly reduced in human mesothelloma tumour cell lines compared with primary mesothelial cells. Iminunofluorescence stainings have shown that the gap junction protein connexin43 (Cx43) is expressed In both malignant and normal mesothelial cells. In this study the mRNA expression of Cx43 and three different connexlns—Cx37, Cx40 and Cx45, which are highly expressed in lung tissue—was investigated in eight human mesothelioma cell lines, and in human primary mesothellal cells from several donors. The expression of the intercellular adhesion molecules A-CAM (N-cadherln) and L-CAM (E-cadherin) was studied at the protein level. No mRNA expression of Cx37, Cx40 or Cx45 in either mesothelioma tumour cells or the primary mesothelial cells was detected. Cx43 was expressed at both the mRNA and the protein level, in seven out of eight mesothelloma cell lines, as well as in all the primary mesothellal cell cultures. The intercellular adhesion molecule A-CAM was expressed at the cell—cell borders In six out of seven mesothelioma cell lines, as well as in normal mesothellal cells. No expression of L-CAM was observed in these cells. The results suggest that Cx43 and A-CAM are the major proteins in gap and adherens Junctions respectively in human mesothellal cells. Most mesothelioma tumour cell lines with markedly reduced GJIC still express both Cx43 and A-CAM. Only one of our mesothelloma tumour cell lines severely deficient in GJIC lacks both the gap junction protein Cx43 and the cell adhesion molecule A-CAM. [ABSTRACT FROM PUBLISHER]

Fecapentaenes are mutagens found in human feces and may play a role in the pathogenesis of colon carcinoma. However, the genotoxk effects of fecapentaenes have not been previously studied in mammalian cells. We now report that fecapenta-ene-12 (fec-12), a prototype for these compounds, causes DNA single strand breaks, sister chromatid exchanges and mutations in cultured human fibroblasts. These results indicate that fec-12 is a potent genotoxic agent in human cells. [ABSTRACT FROM PUBLISHER]

The two hexachloroethane (HC)-based smoke formulations studied consisted of HC/Zn/2,4,6-trinitrotoluene (TNT) and HC/Zn. In the in vitro tests, human bronchial epithelial cells were exposed to the smokes at various concentrations. The responses studied were acute toxicity (viability of cells, trypan blue exclusion method) and genotoxicity (DNA single-strand breaks, COMET assay). The tests were conducted in a laboratory-scale chamber (V = 150 L) and in a container (V = 55 m3). Both smoke formulations appeared to be acutely toxic and genotoxic. For the 0.5- and 1-g burning experiments the responses were more pronounced with HC/Zn/TNT than with HC/Zn smoke. To study the irritation potency of the smokes, the mouse bioassay according to ASTM E 981-84 was applied. The respiratory parameters measured were tidal volume (VT), airflow during expiration at 0.5 VT (VD), time of pause after expiration (TP), time of breaking after inspiration (TB), and the respiratory frequency (BPM; breaths per minute). In the single-exposure experiments, HC/Zn/TNT smoke induced concentration-dependent sensory irritation in mice and the occupational exposure limit (TLV) was estimated to be 4 mg/m3. In the repeated-exposure experiments, HC/Zn/TNT smoke induced sensory irritation at the beginning of the exposure. Pulmonary irritation tended to dominate when the exposures were repeated. With HC/Zn smoke we were unable to generate sufficient high exposure concentrations. In the repeated-exposure experiments, indications of sensory and pulmonary irritation were seen at concentrations used. No evidence of apoptotic cell death was found in caspase-3-like protease activity assay.

Mesothelioma is induced almost exclusively by exposure to asbestos fibers. We have investigated whether the induction of DNA damage in human bronchial epithelial BEAS 2B cells and human mesothelial MeT 5A cells by crocidolite asbestos (2 microg/cm2) requires the presence of asbestos fibers in the cells. DNA damage was measured microscopically by the Comet assay, and the presence of fibers in the same cells was assessed using bright-field illumination. After treatment times of 6-72 hr, damage levels were, on the average, two times higher in cells with fibers than in cells without fibers. It was further found that DNA damage decreased with time in BEAS 2B cells both with and without fibers. No decrease in damage with time was seen in MeT 5A cells, suggesting that these mesothelial cells repair the initial damage poorly, lack induction of protective systems, or constantly produce high levels of damaging species. Our results indicate that crocidolite-treated human mesothelial MeT 5A and bronchial epithelial BEAS 2B cells show an elevated level of DNA damage if they contain a fiber. In comparison with epithelial BEAS 2B cells, mesothelial MeT 5A cells have more DNA damage after the crocidolite treatment and the damage is more persistent.

Human malignant mesothelioma (MM) is a highly aggressive neoplasm related to occupational asbestos exposure and characterised by a long latency period between the exposure and onset of disease. Previous studies indicate that losses at different genomic regions are present in MM. We examined allele loss at three known tumour suppressor gene regions (22q/NF2 gene, 9p/p16 gene, and 3p/FHIT gene) and at two other frequently deleted areas (14q and 6q) in MM. Loss of heterozygosity (LOH) was investigated in cell cultures and primary tumours with several highly polymorphic markers for each site. To study if LOH of the NF2 gene is a consistent feature in MM, we performed a more detailed analysis of chromosome 22q that included a NF2 marker (NF2CA3). We observed a high frequency of LOH occurring simultaneously at multiple loci. In particular, 100% of the cultured MM cells exhibited LOH at the NF2 gene region. From the other chromosomal sites analysed, recurrent allele loss was detected at 9p (5/7; 71%), 3p (4/7; 57%), 14q (3/7; 43%), and 6q (3/7; 43%). Of the 32 tumours, even those trimmed to exclude normal tissue, few showed LOH, suggesting consielment by normal cells within MM tumours, whereas tumour cells in primary cultures showed LOH already in passages 1-2. In conclusion, our present LOH data indicate that MM cells exhibit allele losses at multiple tumour suppressor gene sites concurrently, involving NF2 gene preferentially. This supports the view that the accumulation of multiple genetic hits is characteristic to malignant transformation of MM cells.

Manganese superoxide dismutase (MnSOD) activity is highly elevated in the biopsies of human asbestos-associated malignant mesothelioma. We therefore examined if polymorphism in the mitochondrial targeting sequence of the MnSOD gene modified individual susceptibility to this malignancy or related asbestos-associated pulmonary disorders. The study population consisted of 124 male Finnish asbestos insulators who were all classified as having been exposed to high levels of asbestos; 63 of the workers had no pulmonary disorders and 61 either had malignant mesothelioma or the non-malignant pulmonary disorders asbestosis and/or pleural plaques. No significant associations were found between the MnSOD genotypes and these ill-health. This study therefore suggest no major modifying role for the MnSOD polymorphism in development of asbestos-associated pulmonary disorders.

We investigated the role of glutathione and nitric oxide synthase (NOS) in fiber-induced cell and DNA toxicity using alkaline (pH 13) single-cell gel electrophoresis (the Comet assay). Transformed cultured human pleural mesothelial (MeT-5A) cells and alveolar epithelial cells (A549) were exposed to crocidolite asbestos fibers (1-10 microg/cm(2)) in the presence of buthionine sulfoximine (BSO) or L-arginine-methyl ester (L-NAME). BSO inhibits gamma-glutamylcysteine synthetase (gamma-GCS) and causes glutathione depletion, and L-NAME inhibits nitric oxide generation. Studies were also conducted to assess the expression of the heavy and light subunits of gamma-GCS in human pleural mesothelium and bronchial epithelium in vivo and the induction of inducible NOS (iNOS) by asbestos fibers. Asbestos fibers caused DNA single-strand breaks, and the process was significantly enhanced by BSO (69% compared to the non-treated cells). A549 cells had a 3.5-fold glutathione content compared to MeT-5A cells, which was consistent with the higher resistance of these cells against oxidants and fibers. Flow cytometry of iNOS showed no change of iNOS by the fibers in either cell type in vitro. L-NAME had no effects on the DNA single-strand breaks in the Comet assay, either. Studies on lung biopsies showed that the immunoreactivities of both gamma-GCS subunits were very low in healthy human mesothelium in vivo. We conclude that glutathione may play an essential role in protecting intact cells against fiber-induced oxidative DNA alterations, and low gamma-GCS reactivity in pleural mesothelium may be associated with the high sensitivity of mesothelial cells to fiber-induced toxicity.

To reveal genes relevant for malignant mesothelioma (MM), we carried out cDNA array experiments on 4 MM cell lines and 2 primary mesothelial cell cultures established from pleural fluid of non-cancer patients. Human cancer gene filters including 588 genes were used for the cDNA array experiments. Our study revealed 26 over-expressed genes that play a role in the regulation of cell fate, cell cycle, cell growth and DNA damage repair and 13 under-expressed genes encoding growth factors, receptors and proteins involved in cell adhesion, motility and invasion to be common to 3 or 4 MM cell lines. We confirmed the cDNA array results using RT-PCR for 5 of the over-expressed genes and for 3 of the under-expressed genes. Our study presents gene expression profiles in MM cell lines and shows the involvement of several genes, such as those encoding JAGGED1, ser/thr protein kinase NIK, Ku80 and cyclin D2, novel in MM., (Copyright 2001 Wiley-Liss, Inc.)

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.

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., (Copyright 2000 Wiley-Liss, Inc.)

The development of drug resistance of tumors is multifactorial and still poorly understood. Some cytotoxic drugs generate free radicals, and, therefore, antioxidant enzymes may contribute to drug resistance. We investigated the levels of manganese superoxide dismutase (Mn SOD), its inducibility, and its protective role against tumor necrosis factor-alpha and cytotoxic drugs (cisplatin, epirubicin, methotrexate, and vindesin) in human pleural mesothelioma (M14K) and pulmonary adenocarcinoma (A549) cells. We also studied other major antioxidant mechanisms in relation to oxidant and drug resistance of these cells. A549 cells were more resistant than M14K cells toward both oxidants (hydrogen peroxide and menadione) and all the cytotoxic drugs tested. M14K cells contained higher basal Mn SOD activity than A549 cells (28.3 +/- 3.4 vs. 1.8 +/- 0.3 U/mg protein), and Mn SOD activity was significantly induced by tumor necrosis factor-alpha only in A549 cells (+524%), but the induction did not offer any protection during subsequent oxidant or drug exposure. Mn SOD was not induced significantly in either of these cell lines by any of the cytotoxic drugs (0.007-2 microM, 48 h) tested when assessed by Northern blotting, Western blotting, or specific activity. A549 cells contained higher catalase activity than M14K cells (7.6 +/- 1.3 vs. 3.6 +/- 0.5 nmol O(2). min(-1). mg protein(-1)). They also contained twofold higher levels of glutathione and higher immunoreactivity of the heavy subunit of gamma-glutamylcysteine synthetase than M14K cells. Experiments with inhibitors of gamma-glutamylcysteine synthetase and catalase supported our conclusion that mechanisms associated with glutathione contribute to the drug resistance of these cells.

We chose to treat malignant pleural mesothelioma with a combination of docetaxel and irinotecan (CPT-11), because there have been preliminary reports that CPT-11 is active against mesothelioma, and docetaxel and CPT-11 were the most active agents in our in vitro experiments in human mesothelioma cell lines. Fifteen previously untreated patients with pleural mesothelioma (IMIG Stage III-IV) were given docetaxel 60 mg/m2 followed by CPT-11 190 mg/m2 on day 1, repeated every 3 weeks. All the patients were evaluable for toxicity and 13 patients were evaluated for response. No objective responses (complete or partial) were achieved, but there were two minor responses (overall response rate 15%) each of a duration of 4 months. Three patients had stable disease (23%); median time to progression was 7 months. Median survival in all the patients was 8.5 months from the first chemotherapy cycle and 11 months from diagnosis. Toxicity was severe with seven of 15 patients suffering neutropenic fever and six of 15 patients grade 3-4 diarrhea. The trial was discontinued because of toxicity and lack of activity. We do not recommend the combination of docetaxel and CPT-11 using the schedule presented here for further investigation in malignant mesothelioma. However, CPT-11 and docetaxel, individually, still warrant further study in this disease, especially in combination with cisplatin.

In this study, we used four human mesothelioma cell lines (M14K, M24K, M25K and M38K), one transformed human mesothelial cell line (MeT-5A) and one primary mesothelial culture (UPL) to test for in vitro sensitivity to docetaxel, paclitaxel, SN-38 [an active metabolite of irinotecan (CPT-11)] and gemcitabine, as single agents. Subconfluent cell cultures were treated with 2x10(-9), 5x10(-9), 10(-8), 2x10(-8) and 5x10(-8) M concentrations of each drug for 48 h. The sensitivity was measured in terms of cell viability using the Trypan blue exclusion method. All four drugs were potent inhibitors of mesothelioma cell growth, but cell lines from different patients diverged in their sensitivity to the individual agents. In most cases docetaxel, paclitaxel and SN-38 were more potent killers of mesothelioma cells than gemcitabine. The induction of DNA damage was investigated using the Comet assay; cells from two cell lines (M14K and M25K) were treated with subtoxic 10(-8) M concentrations of each drug for 4, 24 and 48 h. Each of the agents caused a slight increase in DNA single-strand breaks at a concentration of 10(-8) M.

We investigated apoptosis and the expression of bcl-2, mcl-1, bcl-X, and bax in histological sections from 35 malignant mesotheliomas and 21 metastatic adenocarcinomas. Moreover, the expression of bcl-2, mcl-1, bcl-X, and bax were assessed by Western blotting in nonmalignant human mesothelial cells (Met5A) and seven malignant cell lines. The apoptotic index in mesotheliomas was 1.07+/-1.14%. Patients with mesotheliomas showing a high apoptotic index (> or =0.75%) had a worse prognosis (P = 0.008). bcl-2 positivity was observed in only seven cases, but bcl-X, mcl-1, and bax positivity was seen in all of them. In immunoblotting experiments, all mesothelioma cell lines were negative for bcl-2 but positive for bcl-X, mcl-1, and bax. The apoptotic index in bcl-2-negative mesotheliomas was 1.25+/-1.24% and in bcl-2-positive ones, 0.47+/-0.42% (P = 0.014). The apoptotic index did not significantly associate with bcl-X, mcl-1, or bax expression (P = 0.19, P = 0.25, and P = 0.46, respectively). No significant difference was observed in apoptosis or expression of bcl-2, bcl-X, or bax between malignant mesotheliomas and metastatic adenocarcinomas. The former, however, showed more often weak mcl-1 immunoreactivity (P = 0.01). The results show that the extent of apoptosis may influence patient prognosis. bcl-2 is inversely associated with the apoptotic index but is relatively infrequently expressed in malignant mesotheliomas. Widespread expression of bcl-X, mcl-1, and bax suggests that these proteins may also take part in apoptosis regulation in mesotheliomas.

Occupational asbestos exposure can be demonstrated in 80% of mesothelioma cases. A possible role of simian virus 40 (SV40) in the etiology of mesothelioma was raised because several studies reported the presence and expression of SV40-like DNA sequences in human mesotheliomas. It is also known that expression of SV40 large T antigen inhibits cellular Rb and p53. This suggests that SV40 might render infected cells more susceptible to asbestos carcinogenicity. The SV40-like sequences are suggested to have arisen from contaminated polio vaccines. Millions of people in the United States and most European countries were inoculated with SV40-contaminated polio vaccine in 1955-1963. However, in Finland, where polio vaccination started in 1957, no SV40-contaminated vaccine was used. We used a polymerase chain reaction-based method to test for the presence of SV40-like sequences in DNA extracted from the frozen tumor tissues of 49 Finnish mesothelioma patients, most of whom had been occupationally exposed to asbestos. All of the Finnish tumor tissues tested negative for SV40-like sequences. The results suggest that the SV40-like sequences detected in mesothelioma tissue in some previous studies may indeed originate from SV40-contaminated polio vaccines. It is a matter of speculation whether the absence of SV40 infection has contributed to the relatively low incidence of mesothelioma in Finland (1/10(5) in 1990-1995)., (Copyright 1999 Wiley-Liss, Inc.)

Exposure to the carcinogen asbestos is a major factor in the development of malignant mesothelioma. However, not all mesotheliomas are associated with asbestos exposure, and only a small minority of people exposed to asbestos develop mesothelioma. Therefore, the identification of the cofactors that render certain individuals more susceptible to asbestos or that cause mesothelioma in people not exposed to asbestos has been a major priority of the International Mesothelioma Interest Group. The possible association of SV40 with mesothelioma was recently discussed in a special session at the Fourth International Mesothelioma Interest Group Conference, and it was decided to conduct a multi-institutional study to independently verify the presence of this tumor virus in mesotheliomas. We report the results of this investigation: (a) DNA and protein analyses revealed SV40 sequences and SV40 large T antigen expression in 10 of 12 mesotheliomas tested (83%); and (b) electron microscopy demonstrated variable amounts of asbestos fibers in 5 (71%) of 7 corresponding lung tissues available for analysis. Our results demonstrate that SV40 DNA is frequently present and expressed in mesotheliomas in the United States. Because our data demonstrate that some patients test positive for both SV40 and asbestos, the possibility that these two carcinogens interact should be investigated in future studies.

Fibronectin (Fn) and tenascin (Tn) are two major extracellular matrix (ECM) glycoproteins that may have important roles both in fibrotic lung diseases and in lung tumors. The significance of Fn and Tn in human pleural mesothelial cells and pleural diseases is unclear. Transformed human pleural mesothelial cells (Met5A), primary cultures of mesothelial cells, and cultured mesothelioma cell lines were investigated for Fn and Tn immunoreactivity. Mesothelial cells were exposed for 48 to 96 h to transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), amosite asbestos fibers, or oxidants (H2O2 and menadione, a compound that auto-oxidizes to produce superoxide). Immunofluorescence and Western blotting with monoclonal anti-Fn and anti-Tn antibodies, and Northern blotting with a complementary DNA (cDNA) probe for Tn showed that mesothelial cells are capable of producing Fn and Tn. The mRNA level and immunoreactivity of Tn was enhanced by TGF-beta and TNF-alpha, whereas Fn was intensified only by TGF-beta. A wide range of amosite, H2O2, or menadione concentrations had no clear effect on Fn or Tn reactivity. Fn and Tn were present at low or undetectable concentrations in five of six mesothelioma cell lines, whereas the organization of Fn immunoreactivity in these cell lines was variable. Furthermore, results obtained with the tumor tissue of these same mesothelioma patients suggested that Fn and Tn expressions do not necessarily parallel either each other or results obtained with the cultured cells.

The comet assay (single cell gel electrophoresis) is a novel method to assess DNA strand breaks in single cells. We studied the oxidant sensitivity of cultured primary and transformed (MeT-5A) human pleural mesothelial cells, as well as primary and transformed (BEAS 2B) human bronchial epithelial cells, and compared the results obtained with the Comet assay to other markers of oxidant effects on cells, such as depletion of intracellular high-energy nucleotides (ATP, ADP, AMP), accumulation of products of nucleotide catabolism (xanthine, hypoxanthine, uric acid), and release of lactate dehydrogenase (LDH). The cells were exposed for 5 min to 4 h to 50-500 microM H2O2 or to 5-50 microM menadione. Significant tail moment increase, which is a marker of DNA strand breaks in the Comet assay, and intracellular nucleotide depletion occurred simultaneously in MeT-5A and BEAS 2B cells during the first 30-60 min of exposure to H2O2 and menadione. In the Comet assay variation between the individual cells could be detected. LDH release, a marker of cell injury, showed that mesothelial cells were far more sensitive than epithelial cells to oxidant-induced lytic cell injury. MeT-5A and BEAS 2B cells contained similar intracellular antioxidant enzyme activities, which may explain their similar oxidant sensitivity in the Comet assay. A significant increase (164%) in the tail moment was detectable in MeT-5A cells exposed to 50 microM H2O2 for 30 min. This returned to control level during the 4 h of continuing exposure. A 30 min exposure of 25 microM menadione caused a 61% increase in the mean tail moment but, unlike with H2O2, the change was irreversible during the following 4 h incubation. We conclude that human pleural mesothelial cells and bronchial epithelial cells show similar oxidant sensitivity when assessed by the Comet assay, but various oxidants differ in their potency in causing DNA breaks in these cells.

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.

The in vitro cytotoxicity and the induction of micronuclei of two ultrafine titanium dioxide (TiO(2)) samples was assessed in a rat liver epithelial cell (RLE) assay. Pigmentary TiO(2) was used as a control particle, and mitomycin C, a potent inducer of chromosome damage, was used as a positive control agent in the micronucleus experiments. Since photoexcitation of TiO(2) particles has been reported to increase the cell-killing effect of the dust, a duplicate series of experiments was carried out by irradiating the TiO(2) exposed cells with near-UV light. Neither of the ultrafine TiO(2) samples was toxic to the cells at the concentration range of 5-200 mug/cm(2). The UV treatment had no significant effect on the results. The induction of micronuclei was tested in three concentrations (5, 10 and 20 mug/cm(2)). None of the TiO(2) samples, either ultrafine or pigmentary, increased the numbers of micronuclei in the RLE cells. By contrast, all three samples had a slight decreasing effect on the frequency of micronuclei at the lowest treatment concentration of 5 mug/cm(2), both in the absence and in the presence of UV irradiation. The results suggest that ultrafine particles, similar to pigmentary TiO(2), have no direct clastogenic potential.

The molecular basis of malignant mesothelioma is poorly known. We examined genetic changes in 11 mesothelioma specimens by comparative genomic hybridization (CGH). Five DNA specimens originated from uncultured tumor tissues and six from cell lines established from the same patients. Findings from the classical karyotypic characterization of both primary tumors and cell lines have been reported previously. In the CGH analyses the most common genetic alterations in the 11 mesothelioma specimens were losses of chromosomal regions in 1p, 8p, 14q, and 22q and gains of 5p, 6p, 8q, 15q, 17q, and 20. The cell lines had on average a much higher total number of genetic changes than the uncultured tumor specimens. Clonal relationship between the cell lines and the uncultured tissue specimens could not usually be demonstrated even though they originated from the same patient. The observed differences may partly be due to high frequency of chromosomal rearrangements, which CGH cannot detect, partly due to contamination of tumor specimens with normal tissue, and partly due to genetic evolution in tumor cell lines.

While the accumulation of genetic changes in a somatic cell is considered essential for the genesis of a cancer, it has become clear that not all carcinogens are genotoxic, suggesting that some carcinogens indirectly participate in the generation of genetic changes during carcinogenesis. A European project funded by the European Community was thus conceived to study mechanisms of nongenotoxic aspects of carcinogenesis. Two main strategical approaches were adapted: (i) to study whether and how Syrian hamster embryo (SHE), Syrian hamster dermal (SHD) and BALB/c 3T3 cell transformation systems simulate in vivo carcinogenesis, and to examine whether they can detect nongenotoxic carcinogens; (ii) to study, refine and validate mechanisms-based end-points for detection of nongenotoxic carcinogens. For mechanisms-based research, the proposed end-points included gap junctional intercellular communication (GJIC) inhibition, altered expression of critical genes, immortalization and aberrant cell proliferation. We also selected model compounds commonly usable for various endpoints. Our major results can be summarized as follows: (1) SHE and BALB/c 3T3 transformation systems reflect both genotoxic and nongenotoxic carcinogenic events; they detect not only genotoxic but also many although not all, nongenotoxic carcinogens. This is further supported by the fact that both genotoxic and nongenotoxic carcinogens were able to immortalize SHD cells. (2) Many nongenotoxic carcinogens, although not all, inhibit GJIC in vitro as well as in vivo. Mechanistic studies suggest an important role of blocked GJIC in carcinogenesis and that different mechanisms are involved in inhibition of the communication by different agents used. However, inhibition of GJIC is not a prerequisite for the enhancement (or induction) of transformation of SHE or BALB/c 3T3 cells. (3) Among compounds examined, there was a good correlation between induction of micronuclei and cell transformation in SHE cells while no such correlation was found between the induction of cell transformation and ornithine decarboxylase activity. (4) Two transgenic mouse mutation assays (lacI and lacZ) were established and validated. The genotoxin dimethylnitrosamine was shown to be mutagenic to the liver in both assays. Ortho-anisidine, a bladder-specific carcinogen that was inactive in standard rodent genetic toxicity assays was uniquely mutagenic to the bladder of the transgenic mice. The peroxisome proliferator methyl clofenipate was established as nonmutagenic to the liver of both transgenic mice. That eliminated DNA damage as a cause of the liver tumours produced by this chemical and weakened the idea that induced cell division leads to mutation induction. (5) With an in vitro DNA replication model, it was found that DNA damage induced by genotoxic agents can be responsible for inhibition of DNA replication, while certain nongenotoxic agents such as phorbol esters increase DNA replication. (6) An attempt to use structure-activity relationship for subfamilies of nongenotoxic carcinogens, e.g., receptor-mediated carcinogens, has been initiated with some promising results. Our results support the idea that there are multiple nongenotoxic mechanisms in carcinogenesis, and that working hypothesis-oriented approaches are encouraged rather than simple screening of chemicals in developing test systems for the detection of nongenotoxic carcinogens.

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.

The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters alpha and beta of the linear quadratic model (LQ-model) and mean inactivation dose (D(MID)) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean alpha value was 0.26 (range 0.48 - 0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The alpha/beta ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h.

Besides asbestos exposure, the factors that determine susceptibility to malignant mesothelioma are unknown. We evaluated the risk of GSTM1 null genotype and slow acetylation-associated NAT2 genotype for malignant mesothelioma in relation to asbestos exposure. Both the GSTM1 null genotype and the NAT2 slow acetylator genotype placed individuals at about 2-fold increased risk of developing malignant mesothelioma [odds ratio (OR) = 1.8, 95% confidence interval (CI) = 1.0-3.5 and OR = 2.1, 95% CI = 1.1-4.1, for the GSTM1 and NAT2 genes, respectively]. When the patients were divided into low/moderate and high exposure groups according to their asbestos exposure histories, the effect of the at-risk genotypes was mostly attributable to the high exposure groups (OR = 2.3, 95% CI = 1.0-5.6 and OR = 3.7, 95% CI = 1.3-10.2, for the GSTM1 and NAT2 genes, respectively). The individuals with combined GSTM1 and NAT2 defects had about a 4-fold risk of developing malignant mesothelioma compared to those with the GSTM1 gene and NAT2 fast acetylator genotype (OR = 3.6; 95% CI = 1.3-9.6). Moreover, the risk among subjects highly exposed to asbestos with the double at-risk genotype was more than 7-fold greater compared to those with the more beneficial genotypes of both GSTM1 and NAT2 genes (OR = 7.4; 95% CI = 1.6-34.0).

The ability of amosite asbestos fibers to induce chromosomal aberrations in human primary mesothelial cells obtained from pleural effusions of 10 noncancerous patients was investigated. The glutathione S-transferase M1 (GSTM1) genotypes of the patients were determined, since the GSTM1 null genotype has been associated with increased susceptibility to lung cancer and chemically induced cytogenetic damage. Four of the patients represented the GSTM1 null genotype, and six the GSTM1 positive genotype. Successful chromosome aberration analyses were obtained from six cases, three of them with the GSTM1 null genotype. The level of aberrant cells in unexposed cultures ranged from 2.0% to 7.5%. Statistically significant increases (2.3-3.0-fold compared to controls) in the number of aberrant cells were observed in two cases only: in one case treated with 1 microgram/cm2 of amosite, and in another treated with 2 micrograms/cm2 of amosite. Cell cultures from four individuals showed minor or no increases in the numbers of aberrant cells in the doses tested (1 and 2 micrograms/cm2). Chromosome breaks were the major type of aberration. The amosite exposed cells with significantly increased aberrations were from patients with GSTM1 positive genotypes. Two cases that showed no cytogenetic response to asbestos fibers were of the GSTM1 null genotype. Thus, our results suggest that the lack of the GSTM1 gene does not render human mesothelial cells more susceptible to chromosomal damage induced by asbestos. GSTM1 null cells appeared, however, to be more sensitive to the growth inhibitory effects of asbestos than did GSTM1 positive cells. Variation in the cytogenetic response of human primary mesothelial cells to asbestos fibers was observed to exist, but the fibers do not appear to be potent inducers of structural chromosomal aberrations in these cells. It remains to be established whether individual sensitivity to asbestos fibers, due to specific genetic traits, exists.

This study investigates reactive oxygen species generation and oxidant-related cytotoxicity induced by amosite asbestos fibers and polymorphonuclear leucocytes (PMNs) in human mesothelial cells and human bronchial epithelial cells in vitro. Transformed human pleural mesothelial cells (MET 5A) and bronchial epithelial cells (BEAS 2B) were treated with amosite (2 micrograms/cm2) for 48 h. After 24 h of incubation, the cells were exposed for 1 h to nonactivated or amosite (50 micrograms) activated PMNs, washed, and incubated for another 23 h. Reactive oxygen species generation by the PMNs and the target cells was measured by chemiluminescence. Cell injury was assessed by cellular adenine nucleotide depletion, extracellular release of nucleotides, and lactate dehydrogenase (LDH). Amosite-activated (but also to a lesser degree nonactivated) PMNs released substantial amounts of reactive oxygen metabolites, whereas the chemiluminescence of amosite-exposed mesothelial cells and epithelial cells did not differ from the background. Amosite treatment (48 h) of the target cells did not change intracellular adenine nucleotides (ATP, ADP, AMP) or nucleotide catabolite products (xanthine, hypoxanthine, and uric acid). When the target cells were exposed to nonactivated PMNs, significant adenine nucleotide depletion and nucleotide catabolite accumulation was observed in mesothelial cells only. In separate experiments, when the target cells were exposed to amosite-activated PMNs, the target cell injury was further potentiated compared with the amosite treatment alone or exposure to nonactivated PMNs. In conclusion, this study suggests the importance of inflammatory cell-derived free radicals in the development of amosite-induced mesothelial cell injury.

Mesothelioma is a malignant tumor of the serous surfaces in the thorax and abdomen, which has proved exceptionally resistant to treatment. A recent phase II trial of a high-dose methotrexate regime on 63 Norwegian patients has, however, achieved a response rate of 37%. Some responses have also been achieved using interferon (IFN)-gamma administered intrapleurally or recombinant (r) IFN-alpha administered subcutaneously. Our earlier in vitro sensitivity studies of mesothelioma cell lines showed that IFN augments the response to chemotherapeutic agents in mesothelioma. The aim of this study was to assess the response of four mesothelioma cell lines, derived from diffuse asbestos-related pleural malignant mesothelioma, to methotrexate alone and in combination with recombinant IFN-alpha and IFN-gamma. Anti-proliferative effects were assayed by vital dye exclusion. A combination of IFN-alpha and IFN-gamma consistently augmented the response of the cell lines to methotrexate, by as much as 75% for one cell line, although the response to the individual IFNs was variable. We were also able to compare the effects of natural IFN-beta with those of IFN-alpha and IFN-gamma. The IFN-beta sensitivity profile for each of the four cell lines was similar to that of IFN-alpha. In two cell lines, the combination of IFN-beta and IFN-gamma produced a similar effect to the IFN-alpha and IFN-gamma combination.