Document M306kRMMROJ5d6zdqYBGrBpj

Antioxidant Enzymes and related MECHANISMS IN MALIGNANT pleural mesothelioma Kristiina JArvinen Hospital for Children and Adolescents University of Helsinki Finnish institute of Occupational Health Department of industrial Hygiene and Toxicology Department of internal Medicine University of Oulu academic dissertation To be publicly discussed by permission of the Medical Faculty of the University ofHelsinki, in the Small Lecture Hall BM LSIII ofBiomedicum, Haartmaninkatu 8, Helsinki, on December 5th 2001, at noon. Helsinki 2001 Supervised by: Professor Vuokko Kinnula, M.D., Ph.D. Department of Internal Medicine University of Oulu Oulu, Finland Professor Kari O. Raivio, M.D., Ph.D. Hospital for Children and Adolescents University of Helsinki Helsinki, Finland Reviewed by: Professor Kirsi Vahakangas, M.D., Ph.D. Department of Pharmacology and Toxicology University of Kuopio Kuopio, Finland Dr. Veli-Matti Kosma, M.D., Ph.D. Department of Pathology and Forensic Medicine University of Kuopio Kuopio, Finland ISBN 952-91-4097-5 (nid.) ISBN 952-10-0212-3 (PDF) Yliopistopaino Helsinki 2001 CONTENTS CONTENTS CONTENTS..................................................................................................................................................... 3 ABSTRACT..................................................................................................................................................... 6 LIST OF ORIGINAL PUBLICATIONS........................................................................................................... 8 ABBREVIATIONS........................................................................................................................................... 9 INTRODUCTION.......................................................................................................................................... 11 REVIEW OF THE LITERATURE..................................................................................................................13 Malignant pleural mesothelioma...................................................................................... 13 History........................................................................................................................................................ 13 Epidemiology............................................................................................................................................. 13 Etiology....................................................................................................................................................... 13 Pathogenesis and pathology..................................................................................................................... 15 Clinical features and diagnosis............................................................................................................... 16 Treatment and prognosis.......................................................................................................................... 17 LUNG CANCER........................................................................................................................... 18 Reactive oxygen and nitrogen species............................................................................... 19 Antioxidants.......................................................................................................................... 20 Superoxide dismutases.............................................................................................................................. 21 Glutathione................................................................................................................................................. 23 Enzymes in the glutathione redox cycle: Glutathione peroxidase (GPx) and glutathione reductase (GR)............................................................................................................................................................ 24 Enzymes in glutathione biosynthesis....................................................................................................... 25 Glutathione S-transferases (GSTs).......................................................................................................... 27 Catalase...................................................................................................................................................... 27 Other proteins with antioxidant capacity......................................................................... 27 ATP-DEPENDENT multidrug transporters........................................................................... 28 P-glycoprotein........................................................................................................................................... 28 MRP family................................................................................................................................................. 29 Other mechanisms of cytotoxic drug resistance............................................................. 31 AIMS OF THE STUDY 33 3 CONTENTS MATERIALS..................................................................................................................................................35 Mesothelioma patients......................................................................................................... 35 Tissue specimens......................................................................................................................35 Cell lines................................................................................................................................ 35 Mesothelioma cell lines.............................................................................................................................35 Non-malignant transformed mesothelial cell line - MeT5A.................................................................. 36 Adenocarcinoma cell line - A549 ............................................................................................................ 36 Cell culture conditions....................................................................................................... 36 Antibodies............................................................................................................................... 36 METHODS.................................................................................................................................................... 38 Pretreatments, oxidant and cytotoxic drug exposures.................................................. 38 Northern Blot analysis....................................................................................................... 38 Western blot analysis.......................................................................................................... 39 Enzyme activities...................................................................................................................40 MnSOD........................................................................................................................................................ 40 Glutathione reductase and glutathione peroxidase................................................................................ 40 GST.............................................................................................................................................................. 40 Catalase.......................................................................................................................................................40 Glutathione content............................................................................................................. 40 Immunohistochemistry.......................................................................................................... 41 MnSOD (I)...................................................................................................................................................41 y-glutamylcysteine synthetase (IV,V)....................................................................................................... 41 P-gp, MRP1 and MRP2 (VI)..................................................................................................................... 41 Ki-67 as a marker ofproliferative activity (V, VI).................................................................................42 Light microscopic evaluation ofimmunoreactivity (I, IV, V, VI)......................................................... 42 Assessment of cytotoxicity................................................................................................. 42 Lactate dehydrogenase release (I, II)...................................................................................................... 42 Nucleotide depletion (II, V).......................................................................................................................42 XTT-assay (III, IV)......................................................................................................................................43 Viability, apoptosis and cell proliferation...............................................................................................43 Statistical analysis.............................................................................................................. 44 RESULTS...................................................................................................................................................... 45 Expression of antioxidant enzymes in mesothelioma cells (I)......................................... 45 Protection of mesothelioma cells against hydrogen peroxide and epirubicin (II).......45 Antioxidant mechanisms in mesothelioma cells in comparison to lung adenocarcinoma cells (Ill)................................................................................................... 46 4 CONTENTS Expression of yGCS subunits in lung tumor and mesothelioma biopsies (IV, V)..............46 Expression and possible role of yGCS in mesothelioma cells in vitro (V)........................ 47 The expression of P-glycoprotein and multidrug-resistance proteins 1 and 2 (MRP1 AND MRP2) in mesothelioma tissue biopsies (VI)........................................................................ 47 DISCUSSION.................................................................................................................................................50 MNSOD.................................................................................................................................................. 50 Catalase................................................................................................................................. 52 Glutathione and GSH-related enzymes............................................................................. 52 P-gp and MRP1&2................................................................................................................................ 54 CONCLUSIONS............................................................................................................................................. 56 ACKNOWLEDGEMENTS.............................................................................................................................57 REFERENCES............................................................................................................................................... 59 5 ABSTRACT ABSTRACT Malignant pleural mesothelioma is a rare but fatal tumor caused mainly by asbestos exposure. There is no standard treatment as mesothelioma is primarily resistant to all treatments including chemotherapy. Asbestos-induced oxidative stress is thought to play an essential role in the pathogenesis of mesothelioma in the process possibly increasing the expression of the major antioxidant defense mechanisms of the cells. Both chemo- and radiotherapy act at least partly by provoking reactive oxygen species (ROS) generation suggesting a role for the intracellular antioxidants in drug resistance. Other mechanisms associated with drug resistance include the plasmamembrane drug transporters, of which several are also redox-regulated. In the present study, the expression and possible role of the major antioxidant enzymes (AOEs), i.e. manganese superoxide dismutase (MnSOD), catalase, and mechanisms closely related to glutathione (GSH) metabolism were investigated in the biopsies of malignant mesothelioma and/or cell lines in culture. The methods included Northern Blotting, Western Blotting analysis, immunohistochemistry and measurement of specific enzyme activities. Cell damage after oxidant or cytotoxic drug exposures was analyzed by lactate dehydrogenase release, depletion of highenergy nucleotides and microculture tetrazolium dye assay. MnSOD was highly expressed in mesothelioma tumor biopsies in vivo and cell lines in vitro compared to non-malignant mesothelial cells. Mesothelioma cell line expressing the highest MnSOD (10 fold compared to non-malignant mesothelial cells) levels also had the highest levels of GSH, glutathione S-transferase (GST) and catalase, and was the most resistant cell line to oxidants and cytotoxic drugs. In contrast to mesothelioma cells, lung A549 adenocarcinoma cells, that represent an oxidant and drug resistant cell line, contained similar levels of MnSOD as non-malignant mesothelial cells. They, however, also contained higher intracellular GSH levels and catalase than mesothelioma cells, and also had elevated levels of y-glutamylcysteine synthetase (yGCS), the rate-limiting enzyme in GSH biosynthesis. In contrast to tumor necrosis factor-a (TNFa), cytotoxic drugs failed to induce MnSOD mRNA, protein or activity in A549 cells. Endogenous level of MnSOD or its induction by TNFa did not explain oxidant resistance of these cells. GST could not explain the resistance of adenocarcinoma cells, as the activity of total GST was lower in adenocarcinoma cells than in more sensitive mesothelioma cells. 6 ABSTRACT The role of GSH and catalase was also investigated by treating the mesothelioma cells and A549 adenocarcinoma cells with buthionine sulfoximine (BSO), to block glutathione synthesis, and aminotriazole (ATZ) to inhibit catalase. Both BSO- and ATZ-treatment enhanced H2O2 toxicity in three mesothelioma cell lines, while only the depletion of glutathione increased epirubicin toxicity. BSO treatment also significantly potentiated cisplatin-induced cytotoxicity in mesothelioma and adenocarcinoma cells. Given the obvious importance of GSH in the oxidant and drug resistance of these tumors, altogether 34 mesothelioma tumor biopsies were investigated for both subunits of yGCS. The catalytic, heavy subunit of yGCS was highly expressed in most of the cases, whereas the regulatory, light subunit (yGCSl) expression was weaker. No expression of these proteins could be detected from the non-malignant mesothelium. The integral membrane drug transporter, P-glycoprotein (P-gp), immunopositivity was found in 61 %, and multidrug resistance proteins 1 and 2 (MRP1 and MRP2) immunopositivity in 58 % and 33 % of 36 mesothelioma biopsies. Normal mesothelium did not express these multidrug resistant proteins. There was no significant association between tumor proliferation, apoptosis or patient survival and expression of the multidrug resistant proteins. In conclusion, a simultaneous induction of multiple antioxidant enzymes can occur in human mesothelioma cells. In addition to the high MnSOD activity, H2O2-scavenging antioxidant mechanisms, yGCS, GST and GSH can partly explain the high oxidant and drug resistance of these cells in vitro; the role of catalase during heavy oxidant exposure is possible. MnSOD can be induced by TNFa, but the induction, however, does not provide any protection against repeated oxidant exposures. Many mechanisms contributing to the resistance of mesothelioma remain to be investigated, but yGCS may play important role in the primary drug resistance of this tumor in vivo in maintaining the intracellular glutathione-level. The multidrug resistance proteins P-gp, MRP1 and MRP2 are expressed in mesothelioma cells, but are not likely to be responsible for the primary drug resistance of this malignancy. 7 LIST OF ORIGINAL PUBLICATIONS LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following original publications referred to in the text by their Roman numerals. I. Kahlos, K., Anttila, S., Asikainen, T., Kinnula, K., Raivio, K.O., Mattson, K., Linnainmaa, K., Kinnula, V.L. (1998). Manganese superoxide dismutase in healthy human pleural mesothelium and in malignant pleural mesothelioma. Am J Respir Cell Mol Biol, 18, 570 580. II. Kinnula, K., Linnainmaa, K., Raivio, K.O., Kinnula, V.L. (1998). Endogenous antioxidant enzymes and glutathione-S-transferase in protection of mesothelioma cells against hydrogen peroxide and epirubicin toxicity, Br J Cancer, 77, 1097-1102. III. Jarvinen, K., Pietarinen-Runtti, P., Raivio, K.O., Linnainmaa, K., Kinnula, V.L. (2000). Antioxidant defense mechanisms of human mesothelioma and lung adenocarcinoma cells. Am J Physiol Lung Cell Mol Physiol, 278(4), L696-702. IV. Jarvinen, K., Soini, Y., Kinnula, V.L. y-Glutamylcysteine Synthetase in Lung Cancer; Effect on Cell Viability. Driscoll B (ed) Series in Molecular Medicine: Lung Cancer. In Press. V. Jarvinen, K., Soini, Y., Kahlos, K., Kinnula, V.L. Overexpression of y-glutamylcysteine synthetase in human malignant mesothelioma. Submitted. VI. Soini, Y., Jarvinen, K., Kaarteenaho-Wiik, R., Kinnula, V. The expression of Pglycoprotein and multidrug resistance proteins 1 and 2 (MRP1 and MRP2) in human malignant mesothelioma. Annals of Oncology. In press. 8 ABC AOEs ATZ BSO CAT CPM CuZnSOD ECSOD G6PDH yGCS GSH GPx GR GS GSSG GST ygt HPF Kb Kd LDH LPR MDR MnSOD MRP MT NSCLC NF-kB NO. P-gp ROS RNS SCLC SOD PCR SV40 TGFp ABBREVIATIONS ABBREVIATIONS ATP binding cassette superfamily antioxidant enzymes aminotriazole buthionine sulfoximine catalase counts per minute copper-zinc superoxide dismutase extracellular superoxide dismutase glucose-6-phosphate dehydrogenase y-glutamylcysteine synthetase reduced glutathione glutathione peroxidase glutathione reductase glutathione synthase oxidized glutathione glutathione S-transferase y-glutamyl transpeptidase high power field kilobase kilodalton lactate dehydrogenase lung resistance protein multidrug resistance manganese superoxide dismutase multidrug resistance protein metallothionein non small cell lung cancer nuclear factor -kB nitric oxide P-glycoprotein reactive oxygen species reactive nitrogen species small cell lung cancer superoxide dismutase polymerase chain reaction simian virus 40 transforming growth factor-P 9 TNFa TRX TRXR TS XTT VEGF ABBREVIATIONS tumor necrosis factor-a thioredoxin thioredoxin reductase thymidylate synthase microculture tetrazolium assay vascular endothelial growth factor 10 INTRODUCTION INTRODUCTION Mesothelioma is a tumor derived from the serosal lining of the pleural, peritoneal or pericardial cavities and is most commonly situated in the pleura. Mesotheliomas are rare tumors, accounting for only about 1% of all cancer deaths in the world (Hammar, 1994). Pleural mesothelioma is in approximately 85-90% of cases an asbestos-initiated lethal malignancy (Mossman et al., 1990). The latency period is about 20 to 40 years. Accordingly, the peak in mesothelioma cases is expected in 2010, although the asbestos usage in most industrialized countries has been abolished from the 1980's. The prognosis of mesothelioma is poor, as it is highly invasive and primarily resistant to all treatments including radiotherapy and cytotoxic drugs. A major factor in the pathogenesis has been considered asbestos-induced oxidative stress, which in turn is known to induce several antioxidant mechanisms in the cells (Janssen et al., 1993). Mesothelioma provides an important model for cancer research of a therapy-resistant malignancy in which antioxidant mechanisms may at least partly explain the resistance. Intracellular antioxidants offer protection not only against reactive oxygen species (ROS) but may also modulate the response to different chemotherapeutic drugs that are used in cancer treatment. Manganese superoxide dismutase (MnSOD) that scavenges superoxide radicals has a controversial role in cancer biology. It has been suggested to be a cancer suppressor (Oberley & Oberley, 1997) but on the other hand it offers protection against oxidative stress and thereby may confer resistance against oxidant producing drugs. MnSOD is overexpressed in only some malignant tumors, but its importance in drug resistance is unsolved (Cobbs et al., 1996; Janssen et al., 1998; Nishida et al., 1993). Glutathione has in many studies been linked with drug resistance both for its role as an antioxidant but also for its function in detoxification reactions (Tew, 1994). In most recent studies, attention has been drawn to the enzymes in glutathione biosynthesis and how the cell maintains its glutathione level (Rahman & MacNee, 2000). Studies have also been done with other mechanisms that utilize intracellular glutathione and transport it extracellularly (Borst et al., 2000; Keppler, 1999). Glutathione S-transferases are a family of detoxification enzymes that are often associated with chemoresistance (O'Brien & Tew, 1996; Tew, 1994). However, the activity of these enzymes is unknown in mesothelioma even though polymorphism of GSTM1 has been linked to the development of this disease (Hirvonen et al., 1996). Catalase in addition to glutathione takes part in scavenging excess hydrogen peroxide in the cells. Not many studies link it to drug resistance of malignant cells (Sinha & Mimnaugh, 1990), but its role should be clarified in oxidant and drug resistance of mesothelioma. The classical inducers of multidrug resistance are the drug export pumps in the plasmamembrane that have different substrate specificities. P-glycoprotein has been studied most, but the recently discovered MRP family offers new avenues for investigators in cancer biology (Borst et al., 2000; 11 INTRODUCTION Pastan & Gottesman, 1987). The first members in the MRP family, MRP1 and MRP2, are dependent on intracellular glutathione and they transport glutathione-conjugated substrates. In mesothelioma, these mechanisms have not been thoroughly studied before. This series of studies was designed to systematically investigate the expression of the most important antioxidant pathways and drug transporters in mesothelioma cells in vitro and tumor biopsies in vivo. Besides investigating the expression of these mechanisms, their role in oxidant and chemotherapeutic drug resistance was assessed in vitro. The expression of the AOEs and related proteins was also correlated with tumor growth and patient survival. 12 REVIEW OF THE LITERATURE REVIEW OF THE LITERATURE Malignant pleural mesothelioma History In 1767 J. Lieutaud recognized two pleural tumors in a series of autopsies, but Wagner was the first to describe the pathology of a primary malignant pleural tumor in 1870 (reviewed by Browne, 1995). The term mesothelioma was first used by Eastwood and Martin in 1921(Boutin et al., 1998). In 1960 Wagner (Wagner et al., 1960) reported 33 cases of diffuse pleural mesothelioma in South Africa, in an area of crocidolite mining. Of these 33 patients, 32 had a history of asbestos exposure, and this connected mesothelioma with asbestos. The first reports of mesothelioma in Finland are from the 1960's (Karjalainen et al., 1997). Epidemiology Mesothelioma is a rare disease, but its incidence keeps increasing despite the industrial restriction of asbestos usage from 1980's, as the latency period is approximately 20-40 years. About 70 cases of mesothelioma are diagnosed in Finland every year (Mattson et al., 1999). It has been estimated that the peak of mesothelioma incidence in Finland will be around 2010, with approximately 100 cases per year (Karjalainen et al., 1997). The peak incidence has been already achieved in the U.S, but e.g. in Britain the number of cases per year is climbing and is expected to increase to more than 3000 cases per year (Boutin et al., 1998; BTS, 2001). Mesothelioma is more common among men, only about 10% occur in women. In about 80-90% of the male cases an obvious asbestos exposure is known. In females, it has been suggested that only 23% of mesothelioma cases are asbestosrelated (Attanoos & Gibbs, 1997). Sporadic cases among children and infants occur. Etiology Asbestos is the single most important causative agent of mesothelioma, and the exposure to asbestos fibers is usually occupational (Craighead & Mossman, 1982). Other lung diseases are caused by asbestos as well, including asbestosis, lung cancer, pleural plaques, pleural fibrosis, pleural effusions and pseudotumors (Mattson, 2000). Factors determining the risk of mesothelioma include the fiber type, time from exposure, fiber dimensions and fiber surface properties (Jaurand et al., 1987; Mossman et al., 1996). There is evidence that persons with a greater intensity and duration of asbestos exposure have a higher risk for mesothelioma which, however, can develop with minimal exposure. Therefore, the causative role of asbestos is difficult to rule out as most adults in the industrialized world have asbestos in their lungs. Fibers greater than 8 pm in diameter are most commonly associated with mesothelioma (Attanoos & Gibbs, 1997). 13 REVIEW OF THE LITERATURE Asbestos is a commercial term for a variety of naturally occurring hydrated fibrous silicates (Mossman et al., 1996). The material is subdivided into two groups, serpentine fibers and amphiboles. The capacity of different types of asbestos fibers to induce mesothelioma seems to be greatest with amphiboles like amosite ("brown asbestos") and crocidolite ("blue asbestos"), whereas the serpentine fiber chrysotile ("white asbestos") is not as tumorigenic (Boutin et al., 1996). Chrysotile comprises 90% of the asbestos used worldwide. In Finland, however, the main asbestos used has been anthophyllite, which is one of the amphiboles. It is associated with asbestos-induced diseases such as asbestosis and pleural plaques. Mesothelioma cases are rare but some have been reported (Karjalainen et al., 1994). Non-asbestos causes of mesothelioma have not been revealed in epidemiological studies, but theoretically any agent injuring pleura may cause mesothelioma. These include chemical agents, chronic inflammation, viruses and radiation. Smoking does not increase the risk for mesothelioma (Rudd, 1995). Recently a possible connection to Simian Virus 40 (SV40) was suggested (Carbone et al., 1994). In the late 1950's and the early 1960's polio vaccines were contaminated with SV40 and millions of people were exposed. In Finland vaccines were not contaminated and none of the mesothelioma patients in Finland had received a contaminated vaccine. SV40 large T-antigen has been detected in a high proportion of mesothelioma tissue specimens. However, other SV40-like DNA sequences were also found in non-malignant pleural diseases. The role of SV40 is unclear, event though in the U.S. and many parts of Europe the consensus seems to link it to mesothelioma. Genetic susceptibility is associated at least with some detoxification enzyme polymorphisms, including the homozygous deletion of GSTM1 gene or slow acetylation-associated N-acetyl transferase-2 (NAT2) genotype (Hirvonen et al., 1995). 14 REVIEW OF THE LITERATURE ASBESTOS ----- Serpentine v Amphiboles Chrysotile Mg6Si4O10(OH)8 Crocidolite Na2(Fe3+)2(Fe2+)3Si8O22(OH)2 Amosite (Fe,Mg)7Si8O22(OH)2 Tremolite Ca2Mg5Si8O22(OH) 2 Anthophyllite (Mg,Fe)7Si8O22(OH)2 Actinolite Ca2 (Mg,Fe) 5Si8O22(OH) 2 Figure 1. Classification of asbestos fibers (modified from Mossman et al., 1996) Pathogenesis andpathology The inhaled asbestos fibers must be transported to the pleural cavity to reach the target cells (Browne 1995). Parietal pleura is often more extensively involved, but usually it is difficult to determine if mesotheliomas begin in the visceral or parietal pleura (Attanoos & Gibbs, 1997). When inhaled into the respiratory bronchioles and alveoli, chrysotile fibers are usually fragmented by organic acids and cleared by macrophages. Amphiboles are not as easily decomposed and may remain unchanged for years/decades. The asbestos fibers are transported to the pleural cavity via the lymphatic pathway or by penetrating to the visceral pleura. Amphibole fibers concentrate on certain areas of the parietal pleura, called black spots, that are openings of lymph vessels, and at these spots the pleura is exposed for years to the effects of asbestos fibers and toxic reactive oxygen species (ROS) (Boutin et al., 1996). Free radicals and other toxic oxygen metabolites are considered important in the pathogenesis of mesothelioma (Mossman et al., 1989). Fibers themselves have redox properties as they contain ferrous iron which catalyses the reaction forming ROS (Kamp et al., 1992). ROS are also formed indirectly when phagocytic cells meet the fibers; macrophages and neutrophils are known to liberate ROS after asbestos exposure (Klockars & Savolainen, 1992; Hedenborg & Klockars, 1987; Nyberg & Klockars, 1990). These active oxygen intermediates can participate in the oncogenic process by many different mechanisms. Genotoxicity, lipid peroxidation, and oncogene modulation are all possible effects of ROS. The long latency period suggests cumulative genetic, cytotoxic and proliferative events (Janssen et al., 1993). 15 REVIEW OF THE LITERATURE Pleural mesothelioma is divided histologically into three classes (Travis et al., 1999). The epithelial subtype comprises about 54% of all mesothelioma cases. Epithelial mesotheliomas may be predominantly composed of acinar structures, and differential diagnosis from adenocarcinoma is often demanding. Other variants of epithelial mesothelioma also exist. Sarcomatotic mesotheliomas, that histologically resemble fibrosarcomas, represent approximately 20% of the cases, and the rest of the cases fall into biphasic mesotheliomas, representing about 25% of the cases (Hammar, 1994). Clinical features and diagnosis The average age of a patient at the time of diagnosis is approximately 60 years, and there is a strong male predominance (Mossman & Gee, 1989). The first symptoms include chest pain, dyspnea, weakness and cough. Usually the diagnosis is delayed due to the non-specificity of the symptoms. Thoracic radiograph initially shows pleural effusion in 92% of cases, usually on one side. Only in 7 % a multinodular pleural tumor without fluid is seen (Boutin et al., 1998). In early cases of mesothelioma, nodules or plaques of varying size can be detected in the parietal pleura. Serosal thickening and consequent effusion is often marked. The majority of cases are unicavitary. Mesothelioma seldom sends metastasis, but it is highly invasive, e.g. to the pericardium. One of the first diagnostic procedures is cytology of pleural fluid that gives positive results in approximately 30% of cases. Another method used for a diagnostic workup is the computed tomography (CT) scan. The diagnosis is established by biopsy via thoracoscopy in most of the cases. Examination of biopsy of parietal and visceral pleura is the most reliable method for diagnosis (BTS, 2001). Histological diagnosis is, however, difficult because of structural variability between different tumors and even within the same tumor, the main problem being differential diagnosis from metastatic adenocarcinoma of the lung. Other differential diagnostic difficulties arise from bening mesothelial hyperplasia and sarcomas in cases of sarcomatoid mesothelioma. In addition to the typical histopathology, panel of immunohistochemical stains will often suggest the right diagnosis. Many antigens stain positively in adenocarcinoma but remain negative in mesothelioma. The markers used in the diagnostic procedure include the carcinoembryonic antigen (CEA), glycoprotein markers Leu-M1, Ber-EP4 and B72.3, and others like epithelial marker antigen (EMA) and human milk fat globulin-2 (HMFG-2). In epitheloid tumors, diastase resistant neutral mucin is positive in approximately 70% of adenocarcinomas, but usually negative in epithelial mesothelioma. In case of sarcomatoid mesothelioma cytokeratins like CK 5/6 and AE1/AE3 are used, as they are generally positive in sarcomas and negative in sarcomatoid mesothelioma. Calretinin, that reveals the mesothelial origin, is usually positive in mesothelioma and negative in sarcoma and its specificity is over 90% (King & Hasleton, 2001). In differentiating between reactive and neoplastic mesothelium attention should be focused on the degree of cellular atypia and the presence of collagen necrosis that are highly suggestive of malignancy. 16 REVIEW OF THE LITERATURE Table 1. Immunohistochemistry of pleural lesions (modified from Travis et al., 1999). LMW, low molecular weight; HMW, high molecular weight; HMGF, human milk fat globulin; CEA, carcinoembryonic antigen; EMA, epithelial membrane antigen; Vim, vimentin. -, negative; +/- occasionally positive; -/+, usually negative; +, usually positive. Diagnostic problem Mesothelial hyperplasia vs. Keratins LMW/HMW + CEA - B72.3 - LeuM1 - BEREP4 - Epithelial mesothelioma vs. + -/+ -/+ -/+ -/+ Metastatic carcinoma + (adeno ca) ++ + + EMA HMFG-2 -/+ Vim -/+ ++ + -/+ Fibrous pleuritis vs. + Sarcomatoid mesothelioma vs. + Sarcoma (primary or -/+ metastatic) -/+ + -/+ + + Treatment andprognosis Treatment of malignant mesothelioma remains disappointing, and there is no standard treatment (BTS, 2001). As in other malignant tumors, surgery, radiation therapy, chemotherapy, supportive therapy or a combination of different modalities are used. No treatment has so far been shown to offer better survival than supportive therapy alone. Median survival time from diagnosis is less than one year, 5-year survival is less than 5 %. Some factors, however, indicate a more favorable prognosis, including epithelial subtype, age < 65 years, good clinical condition with no weight loss, and absence of visceral pleura involvement (Hammar, 1994). Surgery alone does not improve survival but may be beneficial for palliation. Four different surgical methods are in use: extrapleural pneumonectomy, pleurectomy/decortication, limited pleurectomy and thoracoscopy with talc pleurodesis. Extrapleural pneumonectomy is often used in the combination with radiotherapy. Radiotherapy is also used for palliation, especially in cases with pain. Sometimes the disease may regress, but significant improvement in survival has not been achieved. Radiotherapy is usually given in combination with either surgery or chemotherapy, so the individual effects of the treatment modalities are difficult to document. 17 REVIEW OF THE LITERATURE Many different chemotherapeutic agents have been tested either as single-agent treatment or in combination therapy. In the best clinical series, objective responses are seen after single-agent therapy in about 20-30% of patients, but no significant effect on the overall survival (BTS, 2001). The best results in single-agent treatment have been achieved using anthracyclins, with doxorubicin giving up to a 40% response rate and high-dose cisplatin a response rate of up to 33%. Rather promising results have been achieved also with carboplatin, epirubicin, ifosfamide and mitomycin. High-dose methotrexate treatment resulted in a response rate of 37% in a study of 63 patients (Boutin et al., 1998). Combinations of cisplatin, doxorubicin or an alkylating agent like ifosfamide have been studied, usually two or three drugs are combined. No clear advantage over single-agent therapy has been observed. Combination therapy with cytokines, like interferon-a, has been disappointing despite the promising results in in vitro studies (Boutin et al., 1998). The resistance mechanisms of mesothelioma tumors have been studied only in few publications and therefore remain largely unknown. Some of these studies will be discussed later. Lung cancer Given the difficulties between the differential diagnosis of mesothelioma and lung cancer, this study has included experiments also on the biopsies and cell lines of lung cancer, mainly lung adenocarcinoma. The incidence of lung cancer is increasing due to the habit of tobacco smoking in the world. Over 3 million lung cancer deaths have been estimated worldwide in the year 2000. In Finland 2 075 new lung cancer patients were diagnosed in 1994, after five years only 10% are still alive (Mattson, 2000). Lung cancer is the second most common cancer among men in Finland. The incidence among women is climbing and at present lung cancer is the second most common cause of cancer deaths among women. Tobacco is the most important etiological agent of all four subtypes of lung cancer responsible for approximately 90% of all cases. Other known exogenous risk factors for lung cancer include asbestos, ionizing radiation, and other environmental carcinogens e.g. polycyclic aromatic hydrocarbons, nitrosamines and aromatic amines. The endogenous, host related factors, include immunological factors and genetic predisposition, mainly differences in carcinogen metabolism, DNA repair and altered proto-oncogene and/or tumor suppressor gene expression (Vainio & Husgafvel-Pursiainen, 1996). Lung cancer is divided into two major classes mainly for treatment purposes: small cell lung cancer (SCLC) and non small cell lung cancer (NSCLC). Virtually all cases arise from the epithelial tissue and are bronchogenic carcinoma subtypes. SCLC (30% of all lung cancers) proliferates fast, often sends metastases and is primarily sensitive to anti-cancer drugs. Therefore, the initial treatment is chemotherapy. However, resistance to treatment develops rapidly and many different resistance mechanisms have been speculated. P-glycoprotein cannot solely explain the clinical drug resistance (Lai et al., 1989) and other possible drug resistance mechanisms include multidrug resistance proteins (Wright et al., 1998) and decreased expression of topoisomerase II (Giaccone, 1994). NSCLC comprises three histologically different carcinomas: adenocarcinoma (30-35%), squamous cell carcinoma (30-35%), and large cell anaplastic carcinoma (5%) (Mattson, 2000). The treatment of NSCLC is primarily surgery. Combination chemotherapies are widely used for the treatment of 18 REVIEW OF THE LITERATURE NSCLC since only 10-20% of the cases can be operated. In contrast to SCLC, NSCLC is primarily resistant to single chemotherapeutic agents. In adenocarcinoma, glutathione-related mechanisms have been suggested as potential resistance inducers along with other classical resistance mechanisms (Giaccone et al., 1996; Oguri et al., 1998a; Oguri et al., 1998b; Scagliotti et al., 1999; Sugawara et al., 1995; Zhang et al., 1998). Reactive oxygen and nitrogen species A free radical is defined as a chemical species that has a single unpaired electron in the outer orbital (Fridovich, 1978). In this state the radical is extremely reactive and unstable. The most important radicals are the superoxide radical (O2 ), the hydroxyl radical (OH'), nitric oxide (NO') and peroxynitrite (ONOO-). Reactive oxygen species (ROS) include free radicals and other oxygenrelated reactive compounds, such as hydrogen peroxide (H2O2) (Halliwell, 1991). ROS are generated in normal aerobic metabolism in mitochondria, which are the main site of production of radicals. In the cytosol and plasma membrane, ROS are formed by NADPH oxidase, cytochrome P450 oxidase and xanthine oxidase (see Figure 3). Transitional metals, such as iron and copper, are potential promoters of free radical damage, as they can convert superoxide, which in normal conditions is poorly reactive, into a rapidly reactive and highly toxic hydroxyl radical by Fenton chemistry (Halliwell & Gutteridge, 1985). In Haber-Weiss reaction, hydroxyl radical is generated from O2-. and H2O2. NO. has many useful physiological functions, but in excess amounts is a toxic free radical as well. Many exogenous agents, such as hyperoxia, radiation, asbestos fibers and ozone induce free radical formation in the cell. Asbestos fibers cause oxidant production directly and indirectly, one of the ways being catalysis by the ferrous ion, as asbestos fibers have a high iron content. Inflammatory cells, such as neutrophils and alveolar macrophages, also produce large amounts of ROS when activated, especially when the phagocytosis is incomplete (Kamp et al., 1992). NO. production is also activated via the induction of inducible nitric oxide synthase by TNFa and other cytokines released from the inflammatory cells. Reactive nitrogen species that are formed in reactions of NO. and oxygen/superoxide mediate the harmful effects of NO. (Ohshima & Bartsch, 1994). The pathological effects of ROS are wide-ranging; these toxic products can cause injury practically to all cellular components. Lipid peroxidation of membranes, non-peroxidative mitochondrial damage, lesions in DNA, and cross-linking of proteins are the most relevant reactions of ROS leading to cell injury. ROS are thought to be especially important in lung tissue that is exposed to much higher concentrations of oxygen than most other tissues, but also to cigarette smoke and environmental pollutants. In addition to the toxic effects, ROS are important in non-toxic cellular reactions, including signal transduction (Thannickal & Fanburg, 2000). 19 REVIEW OF THE LITERATURE e 2 ONOO- OH' r* H2O H+ + e OH' Figure 2. The pathway of oxygen reduction and peroxynitrite formation Antioxidants To protect themselves from the harmful effects of oxidants, cells have several antioxidant enzymes and other antioxidant mechanisms. The latter include glutathione (GSH) and numerous GSHdependent enzymes, metal binding proteins, and vitamins. The three main types of antioxidant enzymes are the superoxide dismutases (SODs), catalase (CAT) and peroxidases, of which glutathione peroxidases (GPx) are thought to be the most important (Halliwell and Gutteridge 1989). The SODs dismutate the superoxide radical into H2O2. GPx and CAT reduce H2O2 into water and oxygen. Glutathione redox cycle provides the cell with reduced glutathione (GSH) to act as cosubstrate for the peroxidases but to also participate in detoxification reactions and react nonenzymatically with OH' and peroxynitrite. Other enzymes involved in glutathione metabolism are glutathione reductase, glucose-6-phosphate dehydrogenase, glutathione S-transferases and the enzymes participating in GSH-synthesis: y-glutamylcysteine synthetase (yGCS) and glutathione synthase (GS). Metal-binding proteins ferritin, ceruloplasmin, transferrin, haptoglobin and albumin contribute to the antioxidant system by inactivating catalytic metals. The most important antioxidant vitamins include a-tocopherol, ascorbate, B-carotene and flavonoids, but they will not be discussed in this review. Other enzymes with antioxidant capacity include cysteine-containing proteins such as the families of thioredoxin, glutaredoxin and peroxiredoxin. These may play a role in the resistance of cells against oxidants but also against free radical generating drugs (Holmgren, 2000; Powis et al., 2000; Rhee et al., 1999). 20 XAO REVIEW OF THE LITERATURE TRX GRX OH / ETC Figure 3. The central intracellular antioxidant mechanisms in mammalian cells. XAO = xanthine oxidase, NADPH-ox = NADPH-oxidase, ETC= mitochondrial electron transport chain, TRX = thioredoxin, GRX = glutaredoxin, PRX = peroxiredoxin, SOD = superoxide dismutase, CAT = catalase, GPx = glutathione peroxidase, GR = glutathione reductase, GSH = glutathione, GSSG = oxidized glutathione, GST = glutathione S-transferase, 7GCS = y-glutamylcysteine synthetase, GS = glutathione synthase, yGT = y-glutamyl traspeptidase, G6PDH = glucose- 6-phosphate dehydrogenase Superoxide dismutases Two main forms of SOD exist intracellularly: a copper-zinc containing superoxide dismutase (CuZnSOD) and a manganese-containing superoxide dismutase (MnSOD) (Fridovich, 1995). CuZnSOD is found in the cytoplasm, and MnSOD in the mitochondria. Extracellular SOD (ECSOD) is located in the extracellular matrix. MnSOD (also known as SOD2) is a homotetramer with a molecular weight of 88 000 and is located in the mitochondrial matrix close to the electron transport chain, where ROS are produced in 21 REVIEW OF THE LITERATURE normal cellular metabolism (Fridovich, 1998). The gene is located in the long arm of chromosome 6 and is transcribed as two distinct mRNAs of 1 kb and 4 kb (Church et al., 1992). MnSOD 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 (Wan et al., 1994; Weisiger & Fridovich, 1973). Two polymorphic variants of MnSOD have been described, one leading to altered mitochondrial targeting of the enzyme and the other possibly to changed MnSOD in vitro activity (Ho & Crapo, 1988; Rosenblum et al., 1996). The importance of MnSOD for normal physiology has been proven with knockout mice lacking the MnSOD gene, who died within 10-20 days of neurological manifestations and cardiotoxicity (Li et al., 1995). Heterozygous mice with half of the MnSOD activity have increased age-related mitochondrial oxidative damage (Williams et al., 1998). Approximately 15% of total intracellular SOD activity is due to MnSOD. In eukaryotic cells, the MnSOD gene regulation is complex. The MnSOD promoter contains binding sites for several transcription factors such as AP1, AP2, SP1 and NF-kB. It has been hypothesized that the oxidative state of the cell is essential in regulating MnSOD expression. MnSOD is induced by the cytokine tumor necrosis factor a (TNFa) (Wong & Goeddel, 1988). TNFa binds to its plasma membrane receptor, which initiates a series of events including intracellular ROS production, activation of NF-kB and induction of the MnSOD gene. The TNFa induction of MnSOD is blocked by the antioxidant N-acetyl cysteine (NAC). Other factors that induce MnSOD are hyperoxia, irradiation, oxidized LDL, interleukin-1, interferon-y, lipopolysaccarides, H2O2 and asbestos fibers (Crapo & Tierney, 1974; Harris et al., 1991; Oberley et al., 1987; Visner et al., 1990; Warner et al., 1996; Wong & Goeddel, 1988). In some studies the MnSOD gene induction is associated with resistance to hyperoxia, which would indicate that oxidant stress induces the enzyme to protect from subsequent oxidant injury (Liochev & Fridovich, 1997; Tsan et al., 1990; Wispe et al., 1992). However, in contrast to many in vivo hyperoxic models, MnSOD is not directly upregulated by high oxygen tension in human bronchial epithelial cells in vitro (Pietarinen-Runtti et al., 1998). In human lung MnSOD is found in type II pneumocytes, bronchial epithelial cells and alveolar macrophages (Coursin et al., 1996; Kinnula et al., 1994; Lakari et al., 1998). High levels of MnSOD are also found from the heart, brain, liver and kidneys (Beyer et al., 1991). In human malignancies, the role of MnSOD is controversial. In carcinogenesis, the antioxidant oxidant imbalance is considered significant. A polymorphism of the MnSOD gene resulting in alteration in the transport of MnSOD into the mitochondria due to conformational change in the protein is a risk factor at least for the development of breast and lung cancers (Wang et al., 2001); Ambrosone et al., 1999). Most studies have shown that MnSOD activity is low in cancer cells, and it has been proposed to be a cancer suppressor gene (Oberley & Oberley, 1997). Transfection studies, in which only the MnSOD gene has been introduced, have shown decreased level of malignancy and transformation of the malignant phenotype to the direction of a non-malignant one. However, interpretation of this study is problematic as transfection creates imbalanced conditions in the cell. On the other hand, at least gliomas, thyroid carcinomas, esophageal carcinomas and colon carcinomas appear to contain high MnSOD levels when compared to the non-malignant tissues. In a study of five samples of lung tumors the activity of total SOD was somewhat lower than in normal lung tissue (Jaruga et al., 1994). In mesothelioma, MnSOD has not been previously 22 REVIEW OF THE LITERATURE studied before our group reported elevated activity of MnSOD in mesothelioma cell lines (Kinnula et al., 1996). It has been reported that MnSOD is not inducible in cancer cells as it is in non-malignant cells (Wong & Goeddel, 1988) but also this issue is controversial. At least human lung adenocarcinoma cells show MnSOD induction by TNFa (Warner et al., 1996). Human A549 lung cells also represent a malignant cell type but appear to show MnSOD induction (Das & White, 1997). CuZnSOD (SOD1) is a homodimer with a molecular weight of 32000 and is localized mainly in the cytosol, but it is also found in the nucleus and peroxisomes (Crapo et al., 1992; Fridovich, 1998). The gene is located in chomosome 21, the gene is transcribed as two mRNAs, 0.9 and 0.7 kb, respectively, the latter being the predominant form (Sherman et al., 1984). In contrast to MnSOD, CuZnSOD-deficient animals and cells are viable but they are sensitive to oxygen toxicity (Huang et al., 1999). Mutation of this gene is associated with familial amyotrophic lateral sclerosis (Rosen et al., 1993). The regulation of CuZnSOD also differs from MnSOD, e.g. its level is constitutive in several animal studies (Clerch & Massaro, 1993) and human lung (Lakari et al., 1998), neither is it induced by hyperoxia (Pietarinen-Runtti et al., 1998; Visner et al., 1990), TNFa or interleukin-6 (reviewed by Kinnula et al., 1995). In healthy human lung, CuZnSOD is found from the bronchial epithelium (Coursin et al., 1996; Lakari et al., 1998). High levels are also found from the liver, erythrocytes, brain and neurons. In a recent study CuZnSOD gene was found to be upregulated in a mesothelioma cell line compared to a non-malignant mesothelial cell line, when assessed in microarray containing over 6900 genes (Rihn et al., 2000). Otherwise its expression and role in human tumors remain unclear. ECSOD is a copper and zinc containing homotetrameric glycoprotein. It is located in the extracellular matrix in all human tissues and its gene is located in chromosome 4 (Fridovich, 1998; Hendrickson et al., 1990). ECSOD is induced by cytokines like TNFa (Stralin & Marklund, 2000), direct oxidant stress does not affect ECSOD like it does MnSOD. In healthy lung, ECSOD is concentrated in pulmonary vessels and airways, and also found from systemic arteries (Oury et al., 1994). Of the pulmonary cell types, it is found from bronchial epithelium, alveolar macrophages and endothelial cells (Oury et al., 1994). Its role and regulation in cancer are unknown. Glutathione Glutathione (L-y-glutamyl- L- cysteinylglycine, GSH) is the predominant intracellular low molecular weight thiol in all mammalian cells (Meister, 1983), usually present in the millimolar range; the intracellular level being approximately 1-8 mM and the extracellular level typically 5-50 p,M (Griffith, 1999). About 99% of the intracellular glutathione is in the reduced form. Approximately 85% of the intracellular glutathione is in the cytosol, about 15% in the mitochondria and a small percentage in the endoplasmic reticulum. The mitochondrial GSH pool is maintained by the activity of a mitochondrial transporter that translocates cytosolic GSH into mitochondria (Rahman & MacNee, 2000). GSH is a central protective antioxidant against free radicals and other oxidants, but it has also an essential role in detoxification reactions. Other cellular events where glutathione is considered 23 REVIEW OF THE LITERATURE valuable are modulation of redox-regulated signal transduction, regulation of cell proliferation, remodeling of extracellular matrix, apoptosis, mitochondrial respiration and a reservoir of cysteine (Rahman & MacNee, 2000). Numerous studies show that resistant human cancer cell lines contain high glutathione levels in vitro and that oxidant induced toxicity can be enhanced by buthionine sulfoximine (BSO) that causes glutathione depletion by inhibiting its synthesis (Tew, 1994; Zhang et al., 1998; Griffith, 1999; O'Brien & Tew, 1996; Sinha & Mimnaugh, 1990). The role of glutathione in oxidant and drug resistance has not been previously investigated in mesothelioma. Enzymes in the glutathione redox cycle: Glutathione peroxidase (GPx) andglutathione reductase (GR) GPx is one of the major enzyme families in removing hydrogen peroxide generated by, e.g., superoxide dismutases. It catalyzes the reaction where GSH is oxidized into GSSG and H2O2 converted into water and oxygen. Four distinct selenoproteins are included in the family of glutathione peroxidases, the classical form being the cytosolic GPx, which is also found in the mitochondria and extracellular matrix. The other three are the gastrointestinal form of GPx (Chu et al., 1993), a non-selenium dependent GPx (Shichi & Demar, 1990) and phospholipid hydroperoxide GPx (Schuckelt et al., 1991). The cytosolic GPx, a tetrameric selenoprotein, has a molecular weight of 85 000. The gene is located in chromosome 3 (Moscow et al., 1994). Recently a polymorphism was found that associated to lung cancer (Ratnasinghe et al., 2000). In normal physiological conditions with low or moderate production of H2O2, GPx has been considered a more important scavenger than catalase, because its Michaelis-Menten constant (Km value) for H2O2 is lower than that of catalase. Selenium is needed in the synthesis of GPx and at least the extracellular GPx is induced by hyperoxia and oxidants (Avissar et al., 1989; Erzurum et al., 1993). GPx is ubiquitously expressed in erythrocytes, kidney and liver. The expression of GPx in malignant tumors is somewhat variable. GPx activity has been suggested to be elevated in adenocarcinoma of the lung (Carmichael et al., 1988; Di Ilio et al., 1987) whereas it was decreased in other lung cancer subtype biopsies when assessed by immunohistochemistry (Coursin et al., 1996; Jaruga et al., 1994). Elevated GPx activity has been linked with chemoresistance of anti cancer drugs, such as adriamycin, that kills cells by releasing free radicals (reviewed by Sinha & Mimnaugh, 1990). Glutathione reductase (GR) converts GSSG back to GSH at the expense of NADPH forming a redox cycle (see Figure 4). Two isoenzymes of GR, one cytosolic and one mitochondrial, are encoded by a single gene located in chromosome 8. It has been postulated that the glutathione conjugates formed in xenobiotic detoxification can inhibit GR thereby accumulating GSSG altering the redox capacity of the cell. The expression of GR in human lung and tumors is unclear. 24 REVIEW OF THE LITERATURE NADPH GSH H2,W0 2 G6PDH GR GPX NADP+ 2HoO Figure 4. The glutathione redox cycle. G6PDH = glucose-6-phosphate dehydrogenase, GR = glutathione reductase, GSSG = oxidized glutathione, GPx = glutathione peroxidase and GSH = glutathione. Enzymes in glutathione biosynthesis y-glutamylcysteine synthetase (yGCS) is the rate-limiting enzyme in GSH biosynthesis (Richman & Meister, 1975). The synthesis requires another ATP-dependent enzyme, glutathione synthase and the amino acids glutamic acid, cysteine and glycine. In general, the activity of yGCS defines the rate of glutathione synthesis and yGCS is feedback-inhibited by the product, GSH. Cysteine is the rate-limiting substrate. Levels of GSH and cysteine are the two factors that regulate the synthesis of glutathione under physiological conditions. The importance of glutathione synthesis was proven in a recent study which showed that homozygous knockout mouse lacking the yGCS heavy subunit gene dies before birth (Dalton et al., 2000). yGCS is a cytosolic heterodimer consisting of a heavy subunit (yGCSh, MW~ 73 000) and a light subunit (yGCSl, MW ~30 000) (Seelig et al., 1984). yGCSh gene is located in chromosome 6 (6p12) and yGCSl gene in chromosome 1 (1p21) and two mRNA transcripts are consistently seen for both subunits (Gipp et al., 1995). yGCSh is the catalytically active subunit; it also binds the feedback inhibitor GSH. It has been suggested that yGCSh alone comprises about half of the enzyme activity when compared with the holoenzyme (Mulcahy et al., 1995). Some studies, however, have concluded that it has no catalytic activity without the light subunit (Lu et al., 1999). yGCSl serves an important regulatory role and reduces the inhibitory effect of GSH. It has been suggested that during GSH depletion, in oxidizing conditions, the enzyme undergoes conformational changes between subunits that allows an increase in the enzyme activity. In normal physiological conditions when abundant amounts of GSH are present, both subunits are needed for the enzyme activity (Huang et al., 1993). yGCS is induced by several agents, including oxidants e.g. H2O2 and menadione, cytokines e.g. TNFa, heavy metals e.g. cadmium and iron, and some chemotherapeutic agents e.g. cisplatin (Lu, 1999). At transcriptional level yGCS subunits are regulated by a number of regulatory signals, including ARE, TRE, AP1 and NF-kB. yGCS activity is also regulated at the post-transcriptional and translational level, and phosphorylation/dephosphorylation may control its activity. Possible 25 REVIEW OF THE LITERATURE inhibitors include glucocorticoids, insulin, prostaglandin E (Rahman & MacNee, 2000), and TGF-0 (Arsalane et al., 1997). Exposure to sublethal doses of oxidants may initiate an adaptive antioxidant response, where the intracellular GSH is first depleted leading to oxidant stress and consequent yGCS upregulation. The role of y-glutamyl transpeptidase (yGT) in regulating yGCS activity indirectly by cleaving extracellular GSH has also been suggested (Hanigan, 1998). The expression of yGCS mRNA varies between different tissues. In healthy human lung, yGCS mRNA has been detected from bronchial epithelial cells (Rahman et al., 1999). There are no previous studies on the expression or distribution of yGCS in malignant tumors. It has been suggested that as chromosome 1 (loss of 1p21-22) is often deleted in malignant mesothelioma, this would predispose an individual to the development of the tumor (Rozet et al., 1998). Elevated levels of yGCS have been detected in many drug-resistant malignant cell lines. Chemoresistance may be associated with accumulation of GSH, which functions as an antioxidant but is also used in detoxification reactions. Glutathione has also been shown to inhibit apoptosis by changing the redox state of the cell (Manna et al., 1998). Apoptosis resistance in turn has been considered important in the drug resistance of malignant cells. Glutathione synthase (GS) is a cytosolic homodimer that catalyses the reaction of L-y-glutamyl-Lcysteine and glycine that forms GSH. GS is composed of two apparently identical subunits (each MV~52 000) and the gene is located in chromosome 20 (Webb et al., 1995). Two forms of glutathione synthetase deficiency have been described. One form is mild, causing hemolytic anemia, but the other more severe form causes 5-oxo-prolinuria with secondary neurological involvement (Webb et al., 1995). The regulation of GS is poorly known. In glutathione biosynthesis, the availability of cysteine is crucial. Cysteine is transported into the cell by a sodium-dependent A system and cystine, an oxidized form of cysteine, by an inducible transporter Xc- (Bannai, 1984). Cystine is then reduced to cysteine that can be used in GSH biosynthesis. The transport of cystine is induced by oxidants, such as hyperoxia and H2O2, contributing to increased GSH levels during oxidative stress (Deneke & Fanburg, 1989). There are no studies on the expression of GS or cysteine transporters in malignant tumors. y-glutamyl transpeptidase (yGT) acts as a salvage enzyme in GSH synthesis. The molecular weight is 50 kD for the heavy and 25 kD for the light subunit (Arai et al., 1995). The gene is located in chromosome 22 (Figlewicz et al., 1993). yGT is located on the plasmamembrane, where it cleaves the y-glutamyl bond in extracellular y-glutamyl cysteinyl-glycine (Hanigan & Ricketts, 1993). The amino acids are returned into the cell and reused for GSH synthesis. yGT is induced by menadione and t-butyl hydroquinone (Liu et al., 1998), suggesting its role in protecting cells during oxidative stress. In addition to other luminal surfaces of the body, lung epithelium contains high levels of this enzyme (Ingbar et al., 1995). There is one study showing that mesothelioma biopsies are negative for this enzyme when assessed by immunohistochemistry (Hanigan et al., 1999). In the same study, strong immunoreactivity is detected from renal cell carcinoma, adenocarcinoma of the prostate and papillary carcinoma of the thyroidea (Hanigan et al., 1999). 26 REVIEW OF THE LITERATURE Glutathione S-transferases (GSTs) GSTs are a superfamily of detoxifying enzymes that have broad substrate specifities (Hayes & Pulford, 1995). Five families of cytosolic GSTs have been identified in human, of which four have been thoroughly characterized: Alpha (a), Mu (p), Pi (n) and Theta (t). The genes for GST-p class are all located in chromosome 1, whereas GST-n gene is in chromosome 11. A polymorphism of GSTM1 (p-class) resulting in dysfunction of the enzyme has proven to be a risk factor for malignant diseases, including mesothelioma (Hirvonen et al., 1996). The GSTs conjugate GSH with compounds containing an electrophilic center and thereby provide critical protection against xenobiotics and products of oxidative stress. Since the GSH-conjugate is transported out of the cell, intracellular GSH is consumed irreversibly in the conjugation and thus maintenance of intracellular GSH levels is essential for the optimal function of GSTs. Many GST enzymes possess GPx activity as well. Many of the substrates of GSTs also induce the expression of the GST genes, suggesting an adaptive response to chemical stress. Carcinogens and alkylating agents may induce GST-n (Zhang et al., 1998). The GST-n family is the predominant GST in human solid tumors and has even been used as a marker in lung, colon, bladder and other human cancers (Zhang et al., 1998). GST activity is often associated with anticancer drug resistance, as the drugs are converted to a less toxic form by the conjugation. Based on one study 77% of mesothelioma cell lines expressed GSTn in immunohistochemistry (Dejmek et al., 1998). Catalase Catalase (CAT) is a tetrameric hemoprotein that catalyses the reaction of decomposition of H2O2 into water and oxygen. It has a molecular weight of 240 000. It is mainly localized in the peroxisomes (Davies et al., 1979) but is also found in the cytoplasm and mitochondria in minor amounts. The gene is localized in chromosome 11. Patients suffering from acatalasemia have a mutation of the CAT gene but are clinically healthy. Catalase has a higher Km than GPx, which suggests a major role for CAT at higher levels of H2O2 but a minor role at physiological levels of H2O2 (Halliwell & Gutteridge). Catalase is not abundantly present in the mitochondria, where the physiological oxidative stress is at its highest. It has been shown to be induced by high oxygen tension in alveolar epithelial cells (Freeman et al., 1986). In other studies, however, no induction could be detected in lung epithelial cell after oxidant or cytokine exposures (Erzurum et al., 1993; Pietarinen-Runtti et al., 1998). There are no systematic studies on catalase in malignant tumors. Some studies have suggested variable catalase expression in lung, breast and colon cancers (Cable et al., 1992; Coursin et al., 1996; el Bouhtoury et al., 1992). One recent study showed that catalase is highly expressed in mesothelioma (Kahlos et al., 2001b). No major role has been suggested to catalase in drug resistance (Sinha & Mimnaugh, 1990). Other proteins with antioxidant capacity Glutaredoxin and peroxiredoxins are cysteine-containing H2O2-scavenging proteins, that have been recently described (Holmgren, 2000; Rhee et al., 1999), but no investigations of these proteins have 27 REVIEW OF THE LITERATURE been conducted in human lung tumors. Thioredoxin is composed of two closely related cysteinecontaining proteins, thioredoxin (TRX) and thioredoxin reductase (TRXR). This group of proteins enhances cell proliferation and increases resistance to apoptosis in several in vitro and in vivo experimental models (Powis et al., 2000). There are two recent studies showing overexpression of TRX and TRXR in lung tumors and mesothelioma (Kahlos et al., 2001a; Soini et al., 2001a). However, the expression of these proteins did not correlate with survival in either tumor. Heme oxygenase (heat shock protein 32) has also been shown to have antioxidative properties. There are no systematic studies on this enzyme in malignant tumors. Based on unpublished studies from the Department of Internal Medicine, University of Oulu, it is not overexpressed in mesothelioma. Metallothioneins (MT) have been proposed as possible inactivators of metal-containing chemotherapeutic agents intracellularly. MT's contain a high level of cysteine and they have the ability to bind heavy metal ions. MT content has been found to correlate with the resistance of SCLC cell lines to cisplatin (Kasahara et al., 1991). MT is expressed in approximately half of mesothelioma tumor biopsies, but does not correlate with patient survival (Isik et al., 2001). ATP-dependent multidrug transporters Drug efflux glycoproteins are the most studied mechanisms in primary/acquired cytotoxic drug resistance (Bellamy & Dalton, 1994; Kartner et al., 1983; Ling, 1992). These pumps are ATPdependent, located in the plasmamembrane, and offer resistance to a wide variety of drugs by decreasing their net cellular accumulation. Two separate families are known, namely Pglycoproteins encoded by the MDR1 gene and the MRP-family of proteins. P-glycoprotein P-glycoprotein is a 170-kDa mammalian ATPase and it belongs to a large superfamily of integral membrane transport proteins, called the ATP Binding Cassette (ABC) superfamily (Doige et al., 1993; Higgins, 1992). It is encoded by the MDR1 gene located in chromosome 7, and two classes (classes I and III) of P-gp exist in humans (Childs & Ling, 1994). P-gp is normally expressed in detectable quantities at least in colon, adrenal cortex, kidney and liver. The blood-brain barrier has a very high expression level of P-gp, which is necessary to restrict the entrance of various drug molecules into the central nervous system. P-gp is the most studied multidrug resistance mechanism in human cancer. Increased rate of drug efflux and in some cases also decreased rate of drug influx result in decreased intracellular drug accumulation. It has been suggested that in about 50% of human cancers the MDR1 gene is expressed at levels that are thought to be significant (Goldstein, 1995). P-gp is known to transport agents such as anthracyclins, vinca-alkaloids, taxol and epipodophylotoxins (Volm, 1998). The MDR1 gene has been shown to be regulated by heat shock, arsenite and cadmium as well as other cytotoxic compounds (Chaudhary & Roninson, 1993; Chin et al., 1990). Different mechanisms of how the pump actually works have been suggested. P-gp has been mostly studied in hematological malignancies because of easy availability of tissue. The general conclusion is that at least in acute 28 REVIEW OF THE LITERATURE myelogenous leukemia, P-gp probably has a role in the development of chemoresistance (Campos et al., 1992). The significance of P-gp in solid tumors is less clear. In kidney, liver and colon carcinomas the expression of P-gp has been associated with shorter survival (Duensing & Slate, 1994; Sinicrope et al., 1992; Soini et al., 1996; Verrelle et al., 1991). In adenocarcinoma of the breast the prognostic value of P-gp screening remains unclear as controversial results have been obtained (Huang et al., 1998; Trock et al., 1997; Verrelle et al., 1991). The role of P-gp in other solid tumors, as in urologic tumors, may be significant (van Brussel & Mickisch, 1998). However, it has been suggested that it may not play role in non-small cell lung carcinoma, as only very low or undetectable levels of the MDR1 gene product are expressed in these tumors (Lai et al., 1989). In mesothelioma, one immunohistochemical study showed that majority of the cases expressed P-gp (Ramael et al., 1992). However, its role in mesothelioma remained unclear as in a study of five doxorubicin resistant mesothelioma cell lines expression of MDR1 gene could not be detected. (Ogretmen et al., 1998). MRP family In addition to P-gp, another family of transporter proteins, multidrug resistance proteins, has been characterized (Cole et al., 1992). While only two genes encode the P-gps, many more genes seem to be related to the MRP family. Currently the family has eight members (Bera et al., 2001; Borst et al., 2000). MRP1 (MW 190 000) was the first member of the family. It shares a 15 % amino acid identity with the P-gp and its gene is located in chromosome 16 (Cole et al., 1992). Physiologically, MRP1 transports leukotriene 4 (Borst et al., 2000). MRP1 knockout mice are viable, but their response to an inflammatory stimulus is impaired (Wijnholds et al., 1997). Preferred substrates for MRP1 are organic anions and drugs conjugated to glutathione, glucuronate or sulfate. MRP1 has proven to be the previously characterized glutathione S-conjugate pump. It has been stated that MRP-mediated export of conjugates represents an indispensable terminal step in detoxification, and the co-ordinate overexpression with the detoxification enzyme GST leads to high level resistance to cytotoxic drugs (Keppler, 1999) (see Figure 4). MRP1 transports vinca alkaloids and anthracyclins conjugated to glutathione, and therefore depletion of intracellular glutathione results in reversal of the drug resistance (Borst et al., 2000). In many cell lines a simultaneous increase in the expression of MRP1 and yGCS is often detected when exposed to pro oxidants such as menadione and heavy metals like cadmium and arsenite (Ishikawa et al., 1996; Kuo et al., 1998; Kuo et al., 1996). MRP1 has also been linked to the oxidative state of the cell, as oxidative stress enhances the expression of MRP1 in cultured cells (Yamane et al., 1998). Cisplatin resistance has not been seen in MRP1 overexpressing cells. MRP1 is found ubiquitously in the human body; in non-malignant lung tissue MRP1 has been detected in bronchial epithelium and hyperplastic pneumocyte II cells; (Wright et al., 1998) (Thomas et al., 1994). Most histological subtypes of NSCLC, but not SCLC, have detectable levels of MRP, when assessed with immunohistochemistry; (Nooter et al., 1998; Sugawara et al., 1995; Wright et al., 1998). In cancers such as breast cancer (Nooter et al., 1997), gastric cancer (Endo et al., 1996), 29 REVIEW OF THE LITERATURE retinoblastoma (Chan et al., 1997), NSCLC (Young et al., 2001) and neuroblastoma (Norris et al., 1996) MRP1 expression has been associated with drug resistance or poor patient outcome. In five mesothelioma cell lines, MRP1 was found to be overexpressed when compared to non-malignant mesothelial cells (Ogretmen et al., 1998). MRP1 tissue expression has not been previously studied in mesothelioma. MRP2 was previously known as the canalicular multispecific organic anion transporter that is in normal physiological conditions found in the canalicular membrane of hepatocytes (Borst et al., 2000; Paulusma et al., 1996). It has a 45 % amino acid identity with MRP1 and the gene is located in chromosome 10 (Borst et al., 2000). Patients with Dubin-Johnson syndrome have inactivating mutations in their MRP2 gene and are deficient in their bilirubin-glucuronide secretion (Paulusma et al., 1996). In addition to liver, MRP2 is found from kidney and gastrointestines. MRP2 in known to handle a similar range of GSH conjugates as MRP1. The expression of MRP2 in tumor tissues is at present unclear. MRP2 expression in lung cancer cells has not been thoroughly studied; in colorectal cancer the mRNA levels of MRP2 correlated with resistance to cisplatin (Hinoshita et al., 2000). MRP2 is also expressed in gastric tumor cell lines (Narasaki et al., 1997) . MRP2 mRNA seems to be present in cultured cell lines as well as samples of patient tissues with no difference between NSCLC and SCLC (Young et al., 1999). In transfected cells, overexpression of MRP2 results in resistance to methotrexate, cisplatin, etoposide, doxorubicin, epirubicin and mitoxantrone (Borst et al., 2000; Cui et al., 1999; Konig et al., 1999). 30 REVIEW OF THE LITERATURE YX Figure 5. Model showing the relationship between MRP and glutathione. Some drugs (X) conjugated to glutathione by GSTs are then transported by MRP. Others (Y) are cotransported with glutathione. In both cases, transport is glutathione dependent and can be blocked with BSO. BSO = buthionine sulfoximine, yGCS = y-glutamylcysteine synthetase, GS = glutathione synthase, GSH = glutathione, GST = glutathione S-transferase, GS-X = glutathione conjugate, MRP = multidrug resistance protein Other mechanisms of cytotoxic drug resistance Resistance to chemotherapy represents a major source of failure in cancer treatment. Resistance can be roughly divided into two classes: primary (intrinsic) resistance, as in mesothelioma and NSCLC, and acquired resistance that develops rapidly during treatment, as in SCLC. The mechanisms behind these two classes are, however, overlapping. The resistance mechanisms naturally vary between drugs that have different mechanisms of action. Most of the information on drug resistance has been obtained from anthracyclins and alkylating agents. Anthracyclins are redox-active antibiotic chemotherapeutics and form quinone-hydroquinone structures, when activated. The most commonly used anthracyclins are epirubicin, daunorubicin and adriamycin. Menadione and 31 REVIEW OF THE LITERATURE mitomycin C are also quinones that are used in anticancer treatments and form ROS. The use of anthracyclins is often limited in clinical practice because of difficult side effect including cardiotoxicity. Alkylating agents include cisplatin, carboplatin and ifosfamide. These agents bind to DNA interfering with proliferation of the cell. Cisplatin forms intrastand cross-links in DNA, especially in-between neighboring guanines. Cisplatin causes renal failure as a side effect due to necrosis of tubular cells and is highly emetic. The most studied drug-resistance mechanism is, as already mentioned, P-gp. However, other mechanisms besides P-gp, MRPs and glutathione conjugation may be involved. Recently, one mechanism of interest has been resistance to apoptosis, and the expression of the bcl-2 group of proteins such as bcl-2, bcl-XL, mcl-1 and bax are widely investigated. The expression and significance of various bcl-2 group proteins are, however, unclear in many cancers, but it is known that bcl-2 is associated with decreased and bax with increased apoptosis. Mesothelioma has low or undetectable bcl-2 expression and weak to moderate expression of bcl-X, mcl-1 and bax. However, the expression levels do not appear to correlate with apoptosis of the tumor or prognosis of this disease (Soini et al., 1999). Another oncogene, p53, is associated with enhanced apoptosis and widely investigated in malignant tumors (Thompson, 1995). The expression of p53 does not correlate with survival of mesothelioma patients, either (Esposito et al., 1997). Chemotherapy has been generally agreed to be most successful in rapidly proliferating tumors, and therefore many cell cycle proteins have been thought to be involved in drug resistance (Drewinko et al., 1981). Topoisomerase II is a ubiquitous nuclear enzyme that is important in DNA functioning, replication, recombination and transcription. Decreased activity confers "atypical" resistance to e.g. anthracyclins and epipodophyllotoxins in human leukemic cells (Beck et al., 1993). A recent study with five mesothelioma cell lines suggested that mutations in topoisomerase IIa can be associated with resistance of mesothelioma to etoposide (McLaren et al., 2001). Lung resistance protein (LPR), localized in the perinuclear area, is one of four vault proteins which form barrel-like structures and its overexpression has been associated with decreased drug response at least in acute myeloid leukemia and ovarian carcinoma (Scheffer et al., 1995). In mesothelioma its expression has not been studied. Thymidylate synthase (TS) plays a central role in DNA biosynthesis and is the target of many chemotherapeutic drugs. TS expression has been connected to doxorubicin resistance in lung cancer (Volm et al., 1979) but also to resistance to cisplatin (Xu et al., 1991). No studies have been conducted on TS expression in mesothelioma. Many solid tumors contain areas of hypoxic cells that are more resistant to drugs than the cells in normoxia (Teicher, 1994). This can partly be explained by poor vascularisation and hence decreased influx of anti-cancer agents into the hypoxic area. However, it has been found that tumors with poor vascularisation can express at least GSTn, TS, MT and P-gp (Koomagi et al., 1995). At present the most rapidly growing area of cancer research is undoubtedly angiogenesis of solid tumors (Saaristo et al., 2000). For instance, a recent study on mesothelioma reported the expression of several angiogenic cytokines, such as vascular endothelial growth factor (VEGF), transforming growth factor (TGF0) and fibroblast growth factor (FGP), in this disease. These cytokines may contribute to the high invasiveness but not to the primary drug resistance of mesothelioma (Kumar-Singh et al., 1999). 32 AIMS OF THE STUDY AIMS OF THE STUDY Asbestos fibers provoke oxidative stress, which is thought to play an essential part in the pathogenesis of mesothelioma. Mesothelioma is primarily resistant to chemo- and radiotherapy, both of which act at least partly by provoking ROS generation in the cells. It can be hypothesized that in mesothelioma antioxidant mechanisms may play an important role in the pathogenesis and drug resistance and in part explain the poor prognosis. These mechanisms have not been previously studied in this disease, neither have the potential mechanisms of primary drug resistance, such as the drug export pumps, been explored in mesothelioma. Comparison with lung adenocarcinoma gives further information on the specificity of these mechanisms in malignant disease. The specific aims were: 1. To study the expression and localization of MnSOD in mesothelioma tissue biopsies compared to non-malignant mesothelium in vivo and to assess the expression of this enzyme in mesothelioma cell lines in vitro. 2. To investigate in vitro the inducibility and role of MnSOD in mesothelioma cells in comparison with adenocarcinoma cells with low MnSOD activity. 3. To investigate the activities of catalase and glutathione-associated pathways in mesothelioma cells and to evaluate their importance in oxidant and drug resistance. 4. To study yGCS expression in lung and mesothelioma tumor biopsies and cell lines, and to assess its significance in primary drug resistance of mesothelioma. 5. To investigate the expression of the multidrug resistance proteins, P-gp, MRP1 and MRP2, in mesothelioma tumor biopsies and to evaluate their role in tumor growth and aggressiveness. 33 MATERIALS Table 2. Characteristics of the mesothelioma patients Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 AGE/SEX 42/F 56/M 57/F 58/M 63/M 74/M 66/M 70/F 70/M 50/M 68/M 63/M 52/M 33/F 71/M 73/M 67/M 59/M 54/M 67/M 78/M 57/M 57/M 73/M 78/M 79/M 63/M 66/M 72/M 73/F 68/M 69/M 69/M 77/M 52/F 67/F 62/M 43/M 72/M 44/M 52/M 46/M 54/M Diagnosis Sarcomatoid Epithelial Biphasic Sarcomatoid Epithelial Epithelial Epithelial Epithelial Biphasic Epithelial Biphasic Epithelial Sarcomatoid Sarcomatoid Sarcomatoid Epithelial Epithelial Epithelial Epithelial Sarcomatoid Epithelial Biphasic Epithelial Sarcomatoid Epithelial Epithelial Epithelial Epithelial Epithelial Epithelial Sarcomatoid Epithelial Epithelial Epithelial Epithelial Epithelial Epithelial Epithelial Epithelial Biphasic Biphasic Biphasic Biphasic Survival (months) 3 9 21 nk 1 124 0 2 14 7 2 9 2 96 4 24 8 8 1 4 21 21 3 0 1 1 5 6 34 40 5 2 7 11 13 * 13 7 20 5 14 13 6 Asbestos exposure nk + nk nk nk + + + nk + nk nk + nk + + + + nk nk + + + + + + + + nk + + + + + + + nk= not known, *= still alive Smoking history + nk + + + + + + + + + + + + nk + + + + + nk + + nk + + + + + + - 34 MATERIALS MATERIALS Mesothelioma patients Altogether 43 biopsies of mesotheliomas were included, 7 biopsies were obtained from the Finnish Institute of Occupational Health (I) and 36 from the Department of Pathology, Oulu University Hospital (V, VI). The clinical data on the mesothelioma patients were obtained from the patient records and are presented in Table 2. The survival time was defined as time from diagnosis until death. Tissue specimens Healthy visceral pleural tissue and mesothelioma specimens were obtained from surgical biopsy, lobectomy or pulmonectomy samples. The samples of healthy visceral pleura, verified by light microscopic examination, were obtained from individuals who underwent surgery for reasons other than malignant mesothelioma or other pleural disease. Histopathologic diagnosis of malignant mesothelioma was confirmed, when necessary, in the Mesothelioma Panel of Finland and the tumors were classified into epithelial, sarcomatoid or biphasic according to the criteria from WHO (Travis et al, 1999). Cell lines Mesothelioma cell lines Seven mesothelioma cell lines (M10K, M14K, M24K, M25K, M28K, M33K and M38K) were originally established from the tumor tissue of untreated mesothelioma patients, with the exception of M10K which was established from a mesothelioma metastasis (Pelin-Enlund et al., 1990). This particular patient had received both radio- and chemotherapy prior to surgery. With the exception of M25K, all the patients were known to have been exposed to asbestos. The data on these patients are presented in Table 3. The tumor biopsy was processed not more than two hours after surgery. The specimen was freshly minced with a scalpel and cultured as described by Pelin-Enlund et al (Pelin-Enlund et al., 1990). The continuous cell lines were characterized cytogenetically and the tumorigenity of some cell lines was tested on experimental mice. In order to confirm the karyotype of the cell lines after culturing up to passage 53, the cells were re-characterized to reveal that no major changes had occurred, but the cell lines had remained cytogenetically relatively stable. 35 MATERIALS Table 3. Characteristics of the tumors from which the mesothelioma cell lines originated. Based on Pelin et al, 1994 1 per gram dry weight 2 duration of experiment 52-71 weeks, wit M28K 31 weeks. Cell line M10K M14K M24K M25K M28K M33K M38K Histological subtype biphasic epithelial biphasic biphasic epithelial biphasic biphasic Fiber content in lung' 13.0 x 106 26.0 x 106 11.0 x 106 no data 150 x 106 4.0 x 106 6.2 x 106 Number of chromosomes 38-46 41-45 no data no data no data no data no data Tumor incidence in nude mice2 2/5 2/5 no data no data 0/7 2/5 no data Non-malignant transformed mesothelial cell line - MeT5A MeT5A is a non-malignant pleural mesothelial cell line from American Type Culture Collection (Rockville, MD, USA). The cell line was originally established by transfecting mesothelial cells with a plasmid-containing Simian Virus (SV40) DNA. MeT5A cells therefore express the SV40 large T antigen, but are non-tumorigenic with typical mesothelial morphology (Ke et al., 1989). Adenocarcinoma cell line - A549 A549 cells, which are human lung adenocarcinoma cells, were obtained from American Type Culture Collection (Rockville, MD, USA). The cell line was initially established from human alveolar cell carcinoma, and since it has many pneumocyte II characters (Lieber et al., 1976), it has been used both as a model for adenocarcinoma (Das & White, 1997) and type II pneumocyte (Rahman et al., 1999). Cell culture conditions The mesothelioma cells and Met5A cells were cultured in monolayer in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 pg/ml streptomycin and 0.03% L-glutamine at 37 C in 5% CO2 atmosphere. A549 cells were grown in monolayer in F12 nutrient mixture supplemented with 15% FCS. Antibodies The antibodies that were used in immunohistochemistry and Western blots are listed in Table 4. 36 MATERIALS Table 4. The primary and secondary antibodies used in immunohistochemistry (IH) and Western blot (WB). Abbreviations: MnSOD = manganese superoxide dismutase, yGCSh = the heavy subunit of y-glutamylcysteine synthetase, yGCSl = the light subunit of y-glutamylcysteine synthetase, P-gp = P-glycoprotein, MRP = multidrug resistance protein, GST-n = glutathione Stransferase-n Antibody MnSOD Source Polyclonal, rabbit anti-human (Dr.J.D.Crapo) Concentration 1:1000 IH 1:10 000 WB Secondary antibody Swine anti-rabbit 1:2000 Swine anti-rabbit 1:2000 yGCSh Polyclonal, rabbit anti-human (Dr. T.Kavanagh) 1:1000 IH 1:40 000 WB Anti-rabbit (Zymed Kit) Donkey anti-rabbit1:50 000 yGCSl P-gP MRP1 MRP2 Ki-67 Caspase 3 GST-n Polyclonal, rabbit anti-human (Dr. T.Kavanagh) Monoclonal, mouse anti-human (JSB1, Alexis Biochemicals) Monoclonal, mouse anti-human (MRPm6, Alexis Biochemicals) Monoclonal, mouse anti-human (Alexis Biocemicals) Monoclonal, mouse anti-human (Zymed) Polyclonal, rabbit anti-human Polyclonal rabbit anti-human (BioPrep) 1:1000 IH 1:20 000 WB 1:20 IH 1:50 IH 1:50 IH 1:50 IH 1:2000 WB 1:5000 WB Anti-rabbit (Zymed Kit) Donkey anti-rabbit1:50 000 Anti-mouse (Zymed Kit) Anti-mouse (Zymed Kit) Anti-mouse (Zymed Kit) Sheep anti-mouse 1:300 Sheep anti-mouse Donkey anti-rabbit 1:30 000 37 METHODS METHODS Pretreatments, oxidant and cytotoxic drug exposures Buthionine sulfoximine (BSO) is a widely used inhibitor of yGCS for the purpose of depleting intracellular glutathione (Buckley et al., 1991). The final concentration of BSO varied between 0.2 mM (MeT5A, M14K) and 1mM (M38K). These concentrations were tested in preliminary studies in which cell survival and intracellular GSH concentrations were analyzed. The pretreatment time was overnight (16-18h). Aminotriazole (ATZ) inactivates cellular catalase (Margoliash et al, 1960) Cells were pretreated with 20 (II) or 30 (III) mM ATZ for 60 min. These concentrations were selected on the basis of previous studies (Kinnula et al., 1991) and also in the preliminary experiments of this study (II). TNFa is a cytotoxic cytokine released from inflammatory cells. It causes NF-kB activation and ROS generation in cells and its effects are numerous (reviewed by Vanden Berghe et al., 2000). The concentration of TNFa was 10 ng/ml and the exposure time 24-48 h. Menadione is a quinone that creates continuous oxidant stress by forming a redox cycle intracellularly (Frei et al., 1986). The continuous generation of oxidants is considered to provide a better model of physiological/pathological conditions in the cell than a bolus of oxidants. H2O2, on the other hand, causes a relatively short oxidant stress and is quickly decomposed intracellularly. The concentrations used were 10-100 pM for menadione (4-48h) (I, II, III) or 0.01-1 mM for H2O2 (1-48h) (II, III). The cytotoxic drugs used in these experiments (I, II, III, IV, V) were epirubicin, cisplatin, vindesin and methotrexate. Epirubicin, an anthracyclin, is activated to free radical state and, in addition, it interacts with molecular oxygen to generate ROS and thus causes oxidant-mediated DNA damage (Sinha & Mimnaugh, 1990). Epirubicin was chosen since it is widely used in the treatment of mesothelioma. The concentrations varied between 0.005 pg/ml- 0.5 pg/ml (0.01-1 pM). Cisplatin is an alkylating agent and has a platinum complex in the cis-position. Glutathione-related mechanisms have been linked to resistance against cisplatin (Zhang et al., 1998). In addition to other pulmonary malignancies, cisplatin is also used in the treatment of mesothelioma. Cisplatin concentrations varied between 0.02-2 pM in (III), 20-250 pM (IV) and 10-100 pM (V). Methotrexate is a dihydrofolic acid reductase inhibitor but has been shown to alter also yGCS and GPx activity (Kussmann et al., 1993). The concentration varied between 0.01-1 pM (III). Vindesin interferes with microtubule formation in the mitotic spindle, and mainly P-gp has been suggested to explain resistance against it. The concentration varied between 0.007-1 pM. All exposure times were 48h. Northern Blot analysis The cells were scraped into 4 M guanine thiocyanate buffer and the samples were immediately frozen at -70 C. Total RNA was isolated with the acid phenol-chloroform method of 38 METHODS Chomczynski and Sacchi (Chomczynski & Sacchi, 1987). Denaturated RNA samples (10-15 p,g) were electrophoresed on 1% agarose gel with 0.36 M formaldehyde. The RNA was transferred onto Hybond-N nylon filters after ethium bromide staining and UV examination to check the loading homogeneity, and cross-linked to the filters by UV illumination. The filters were prehybridised at 58.5 C for more than one hour in a buffer containing 50% deionized formamide, 5 x saline-sodium citrate (SSC), 50 mM sodium phosphate at pH 6.5, 5 x Denhardt's reagent, and 100 p,g/ml herring sperm DNA. The cDNA probes for MnSOD, CuZnSOD, catalase and glutathione peroxidase were kindly provided by Dr. Y.-S. Ho from Wayne State University, USA. Filters were hybridized with 32Plabeled cRNA probes representing human MnSOD (Ho & Crapo, 1988), human CuZnSOD (Halliwell & Gutteridge, 1985), human catalase (Quan et al., 1986) or rat GPx (Ho et al., 1988), each cloned into the pSP65 vector. The probes were purified by NucTrap columns (Stratagene) and added to the prehybridization solution at 2x106 cpm/ml. Hybridization was carried out overnight at 58.5 C with shaking. The filters were washed thoroughly and autoradiography was performed at 80 C using Kodak BioMax MR film (Eastman Kodak Co, Rochester, NY, USA). After autoradiography, the filters were hybridized with a P-actin control probe transcribed from pTRI-Pactin plasmid (Ambion, Austin, TX, USA). The enzyme mRNA expressions were quantified relative to actin expression by an X-rite 331 Transmission densitometer. Western blot analysis The cells were detached with trypsin, centrifuged, and washed with phosphate buffered saline (PBS), then mixed with the electrophoresis sample buffer and boiled for 5 min at 95 C. Cell protein was measured by using the Bio-Rad method, and 50 p,g (I), 30 p,g (II) and 70 p,g (V) of cell protein were applied per lane in a 12% sodium dodecyl sulphate-polyacrylamide gel. The gel was electrophoresed for 1.5 h (90 V) at room temperature and the protein was transferred (60 min, 100V) onto Hybond ECL nitro cellulose membranes (Amersham, Buckinghamshire, UK) in a Mini-PROTEAN II Cell (Bio-Rad, Hercules, CA). The blotted membrane was incubated with rabbit antibodies to MnSOD (dilution 1:10 000), GST-n (dilution 1:5000), yGCSh and yGCSl (dilution 1:40 000, 1:20 000) or to caspase 3 (dilution 1:2000), followed by a secondary antibody conjugated to horseradish peroxidase (Amersham) (dilution 1:30 000 (I, II); 1:50 000(V, IV)). MnSOD, GST-n, yGCS and caspase 3 protein were detected by an enhanced chemiluminescense system (ECL, Amersham), and the luminol excitation was imaged on X-ray film (Biomax MR, XOmat, USA). P-actin expression was analyzed in MnSOD, GST-n and yGCS Western blots in order to confirm loading homogeneity. The membranes were first stripped of antibodies and reprobed using a monoclonal anti-actin antibody and secondary antibody that was conjugated to horseradish peroxidase. P-actin was detected and imaged as described above. 39 METHODS Enzyme activities The activities of the antioxidant enzymes were evaluated in MeT5A, M14K, M38K and A549 cells. For the assays the cells were detached with trypsin, pelleted and frozen immediately at -70 C until analysis. The cells were resuspended in PBS and treated with 1% Triton X-100 to disrupt the cellular organelles. MnSOD Total superoxide dismutase activity was assessed with some modifications using the method of McCord and Fridovitch (McCord & Fridovich, 1969). The activity of the enzyme was measured following the decrease in the rate of reduction, in other words the absorbancy change rate at 550 nm, of 12.8 |jM cytochrome c in the presence of 0.5 mM xanthine and xanthine oxidase. MnSOD activity was distinguished from CuZnSOD activity by its resistance to 1 mM potassium cyanide. The results are expressed as units per mg of total cellular protein. Glutathione reductase andglutathione peroxidase Glutathione peroxidase activity was assessed by measuring NADPH oxidation in the presence of tbutylhydroperoxide, glutathione reductase and glutathione according to Beutler (Beutler et al., 1975). Glutathione reductase activity was determined by measuring the oxidation of NADPH in the presence of oxidized glutathione GSSG (Beutler et al., 1975). Enzyme activities are expressed as units per mg of total cellular protein. GST Glutathione-S-transferase was measured spectrophotometrically using 1mM 1-chloro-2,4dinitrobenzene and 1mM glutathione (Habig & Jakoby, 1981). Enzyme activity is expressed as U/mg protein. Catalase Catalase activity was assessed according to Kinnula et al (Kinnula et al., 1992). The enzyme activity was measured polarographically by following oxygen production with a Clark oxygen electrode fitted into a stirred chamber. The cells were sonicated in Tris-EDTA (pH 7.4) and the reaction was started by adding the cells into the buffer containing 0.5 mM H2O2 equilibrated with nitrogen. The effect of catalase inhibition was tested with ATZ. The activity is expressed as units per mg of total cellular protein. Glutathione content Total glutathione content was determined spectrophotometrically following the reduction of 5,5'dithiobis (2-nitrobenzoic) acid by NADPH in the presence of glutathione reductase (Beutler et al., 1975, Akerboom et al., 1981). Glutathione content is expressed as nmol/ mg protein. Proteins were analyzed by the micromethod of BioRad (Hercules, Ca, USA). 40 METHODS Immunohistochemistry The biopsy material was first fixed in 10% neutral formalin, dehydrated and embedded in paraffin. Four micron thick sections were cut from a representative paraffin block, the sections were deparaffinized in xylene, and rehydrated in through a series of ethanol solutions. Endogenous peroxidase was consumed by incubating the sections in hydrogen peroxide in absolute methanol (0.3% for 30min, I; 0.1% for 10 minutes IV, V, VI). To enhance the immunoreactivity, the sections were incubated in 10 mM citrate buffer (pH 6.0) and boiled in a microwave oven for 2 min at 850 W and 8 min in 350 W. The paraffin blocks were then incubated with 2% milk powder to diminish background staining. For immunocytochemistry (I), cells (MeT5A, M14K and M38K) were grown on microscopic slides (slidechambers, Nalge Nunc) using the same culture conditions as already described. The cells were air-dried and heat-fixed at 38C for 1 h before further processing. A modification of immunocytochemistry was applied to evaluate proliferation and apoptosis. In V the cell pellets from cell cultures were fixed in 10% neutral formalin overnight, after which formalin was removed, and melted 2% agar was laid over the pellets. The agar blocks were further embedded in paraffin. Four micron thick sections were cut from the cell blocks and processed further as described above. MnSOD (I) The polyclonal rabbit antibody for human MnSOD was a generous gift from Professor James D. Crapo (National Jewish Medical Center, Denver, Colorado). After blocking the endogenous peroxidase activity, the sections were incubated with the primary antibody overnight at room temperature. After incubation with a biotinylated swine anti-rabbit secondary antibody for 30 min, the avidin-biotin complex treatment for 30 min was performed. Diaminobenzine was used as a chromogen. The sections were lightly counterstained with haematoxylin and mounted with Eukitt. PBS or normal rabbit serum were used instead of primary antibody in negative controls. y-glutamylcysteine synthetase (IV, V) The polyclonal rabbit antibodies for human yGCSh and yGCSl were kindly provided by Dr. Terrance Kavanagh (University of Washington, Seattle, Washington, USA). The sections were incubated with the primary antibodies for yGCSh and yGCSl, the immunostaining was done using the Histostain-Plus Kit (Zymed Laboratories Inc, South San Francisco, CA) and the chromogen was aminoethyl carbazole (AEC) (Zymed Laboratories Inc.) In negative controls the primary antibodies were substituted with PBS or non-immune rabbit serum. P-gp, MRP1 and MRP2 (VI) A mouse monoclonal IgG1 antibody to human P-gp (JSB1, Alexis Biochemicals), MRP1 (MRPm6, Alexis Biochemicals) and MRP2 (Alexis Biochemicals) were used in incubation of the tumor sections. The immunostaining was done as with yGCS immunohistochemistry, except that the primary antibody was replaced by non-immune mouse or rat serum when assessing negative control stainings. 41 METHODS Ki-67 as a marker ofproliferative activity (V, VI) Tumor cell proliferation was studied with a monoclonal mouse anti-human Ki-67 antibody (Zymed, San Francisco, CA, USA). Ki-67 was developed using diaminobenzidine as a chromogen, and the sections were lightly counterstained with haemotoxylin and mounted with Eukitt as with MnSOD. Light microscopic evaluation ofimmunoreactivity (I, IV, V, VI) A semi-quantitative assessment of the immunohistochemistry of yGCS, P-gp, MRP1 and MRP2 was employed by grading the staining as (0) negative, (1) < 33%, (2) 33-66%, (3) 67-100% of tumor cells showing positive staining. In MnSOD immunohistochemistry the staining was classified into five groups as (0) negative, (1) <25%, (2) 25-50%, (3) 51-75% and (4) >75%. The intensity of the immunostaining was graded as (0) negative, (1) weak, (2) moderate, and (3) strong (V), with the multidrug resistance proteins also (4), very strong, was used (VI). The qualitative and quantitative immunostaining scores were added in order to get a combined score for the immunoreactivity. The score was divided into four main groups (V, VI); - = no immunostaining; score 0 + = weak immunostaining; scores 1-2 ++ =moderate immunostaining, scores 3-4 +++ =strong immunostaining, scores 5-8 Two authors did the evaluation of immunostaining separately. In immunocytochemistry, the staining of MnSOD was assessed by grading the average staining intensity of the tumor cells in comparison with the negative controls and non-malignant mesothelial cells (I). The results for Ki-67 immunostaining were also divided into groups as follows. Weak staining= less than 5 % of cell nuclei positive Moderate staining= 5-10 % of cell nuclei positive Strong staining= 10-50 % of cell nuclei positive Very strong staining= over 50 % of cell nuclei positive Assessment of cytotoxicity Lactate dehydrogenase release (I, II) Lactate dehydrogenase (LDH) release was measured from MeT5A, M14K and M38K cells in order to evaluate lytic cell damage. The cells were first exposed to menadione or epirubicin (I) or H2O2 and epirubicin (II). LDH was measured spectrophotometrically using pyruvic acid as the substrate (Bergmayer & Bernt, 1974). Total cellular LDH was measured from cell lysates obtained by 1% Triton X-100 treatment. The results are expressed as percent of LDH released into the medium from the total LDH amount from the cells and the medium . Nucleotide depletion (II, V) Depletion of high-energy nucleotides was used because of the sensitivity of this method in the assessment of cell injury. The cells were incubated for 16 h with 0.1 mM [14C] adenine (specific 42 METHODS activity 51-55 mCi mmol -1) to prelabel the high-energy nucleotides in intact cells. After labeling, the cells were washed three times and then exposed to menadione, epirubicin (I) or cisplatin (V). After the exposure the medium was collected and the cells were extracted with 0.4 M perchloric acid. Thin-layer chromatography was used to separate the purine nucleotides (ATP, ADP and AMP) both in the cell extract and the medium and the nucleotide catabolic products (xanthine, hypoxanthine, uric acid) in the medium. The results are expressed as per cent distribution of radioactivity (counts per min, cpm) between nucleotides in the cells, nucleotides leaked to the medium and catabolic products in the medium. XTT-assay (III, IV) In selected experiments a cell proliferation kit, the microculture tetrazolium dye calorimetric assay (XTT assay) was used to assess cell damage. This method was conducted using a commercial kit according to the instructions of the manufacturer (Boehringer, Mannheim, Germany). The cells were grown in a 96-well microtiter plate and exposed to cisplatin for 48 hours. After the exposure, 50 pl of labeling mixture was added to each well and the absorbance was measured with the microculture plate reader (Victor; 1420 Wallac Inc, Turku, Finland). Viability of the cells is expressed as relative absorbance. Viability, apoptosis and cellproliferation Viable cells were counted under the microscope after pretreatment with TNFa, exposure to epirubicin (III) and exposure to various concentrations of cisplatin in cell cultures pretreated with BSO (V). Apoptosis was assessed by the ApopTag (Oncor, Gaithesburg, MD, USA) in situ detection kit both in tissue biopsies and cultured cells. ApopTag detects the 3'-ends of the DNA fragments generated by apoptosis-associated endonucleases. The sections were dewaxed in xylene, rehydrated in ethanol and incubated with 20 pg/ml proteinase K at room temperature for 15 min. Endogenous peroxidase activity was blocked by 2% H2O2. After this, the slides were exposed to terminal transferase enzyme and digoxigenin-labeled nucleotides. The slides were then incubated with anti-digoxigenin antibody labeled with peroxidase. The color was developed with diaminobenzine-H2O2 and the samples were lightly counterstained with haematoxylin. Cells were defined as apoptotic, if the whole nuclear area of the cell labeled positively. Apoptotic bodies were defined as small positively labeled globular bodies in the cytoplasm of the tumor cells that could be found either singly or in groups. To estimate the apoptotic index (the percentage of apoptotic events in a given area), apoptotic cells and bodies were counted in 10 high power fields (HPFs) and this figure was divided by the number of tumor cells in the same HPFs. In addition apoptosis was assessed by Western blot analysis where caspase 3 cleavage was detected as already described. Caspase 3 is a cysteine protease acting as an executor enzyme in the late phase of apoptosis (Nunez et al., 1998). Ki-67 was used in the assessment of tumor cell proliferation. The antibody reacts with Ki-67 nuclear antigen associated with cell proliferation (Table 4). The antigen is present throughout the cell cycle except in the resting cells in phase G0. The results for Ki-67 immunostaining were evaluated by counting the percentage of positive nuclei out of the total tumor cell population. 43 METHODS Statistical analysis SPSS (7.5) for Windows was used for the statistical analysis. Comparisons between two independent groups were performed using independent samples t-test or Mann-Whitney U-test. With dependent groups paired samples t-test was used. Analysis of variance and Kruskal-Wallis test or Scheffe's post hoc test were used when comparing several independent groups. The significance of association was determined by Fisher's exact probability test, correlation analysis and two-tailed t-test. The survival analysis was performed by the Kaplan-Meyer curve and the survival in relation to the histochemical findings was assessed by the log rank, Breslow tests and Tarone-Ware tests. In the immunoreactivity evaluation, correlation coefficient obtained from the results of two investigators was established according to Cohen's kappa statistics (IV, V, VI) (Silcocks, 1983). P-value less than 0.05 was considered statistically significant. 44 RESULTS RESULTS Expression of antioxidant enzymes in mesothelioma cells (I) In human healthy visceral pleura no MnSOD immunoreactivity could be detected in five of the six biopsies (Fig 1a, I). In one case the mesothelial surface of the visceral pleura showed a granular layer of immunoreactivity. All seven biopsies from mesotheliomas showed moderate or intense immunoreactivity in the malignant cells (Fig 1b, 1c, I). The immunoreactivity ranged from moderate to intense within the same sample. MnSOD was also detected in the mesothelial cell line MeT5A and the mesothelioma cell lines M14K and M38K. MeT5A cells, however, showed positive immunoreactivity only in cell clusters, whereas M38K cells showed intense staining in all cells. The protein expression of MnSOD was verified with Western blot -assay. In addition to MnSOD, CuZnSOD, catalase and glutathione peroxidase were assessed in the cell lines by Northern blot analysis and specific activity measurement. These results indicated that M38K cells had a higher level of these enzymes when compared to MeT5A and M14K cells that they were also more resistant to menadione and epirubicin [ Fig 7, Fig 8, (I) Table 1,(II)] Protection of mesothelioma cells against hydrogen peroxide and epirubicin (II) Total GST activity and the levels of GSH were higher in M38K cells than in MeT5A and M14K cells (Table 1, II ). M38K cells were also most resistant to hydrogen peroxide, as judged both by LDH-release and by depletion of high-energy nucleotides (Fig 1, II). Aminotriazole (ATZ) to inactivate catalase and buthionine sulfoximine (BSO) to inhibit yGCS, the rate limiting enzyme in GSH synthesis, were used to study the importance of the hydrogen peroxide scavenging mechanisms catalase and glutathione redox cycle in oxidant and drug resistance. After ATZ pretreatment, the activity of CAT decreased by 85% in MeT5A, 82% in M14K and 89% in M38K cells. Pretreatment with 0.2 mM BSO decreased glutathione to undetectable levels in MeT5A and M14K cells. In M38K after 0.2 mM BSO pretreatment 15% of baseline glutathione remained, and the concentration of 1 mM BSO was chosen for further experiments (7% of baseline GSH left). ATZ or BSO alone in these concentrations did not cause nucleotide depletion in any of the cell lines and were considered non-toxic. Pretreated MeT5A and M14K cells were exposed to 0.1 mM and M38K cells to 0.5 mM H2O2 for 4 h. In M14K cells only BSO pretreatment potentiated nucleotide depletion, whereas in MeT5A and M38K cells both ATZ and BSO pretreatment enhanced adenine nucleotide catabolism in H2O2 exposed cells (Fig 2, II). 45 RESULTS Similar experiments were conduced with epirubicin. MeT5A and M14K cells were exposed to 0.1 pg/ml, and M38K to 0.5 pg/ml epirubicin for 48 h. BSO pretreatment potentiated epirubicin toxicity in all three cell lines when assessed by nucleotide depletion, whereas ATZ pretreatment had no effect on the cellular nucleotide levels in any of these cell lines (Fig 3, II). We also found a higher level of GST-n protein in M38K cells compared to MeT5A and M14K cells, suggesting that this isoenzyme may be essential in the drug resistance of these cells (Fig 4, II) Antioxidant mechanisms in mesothelioma cells in comparison to lung adenocarcinoma cells (III) The inducibility of MnSOD was investigated in the mesothelioma cell line M14K and in the human lung adenocarcinoma cell line A549. A549 cells contained lower basal level of MnSOD than M14K cells, the activity being 1.8 + 0.6 and 28.3 + 5.8 U/mg protein, respectively. In A549 cells all the cytotoxic drugs tested with the exception of methotrexate caused a modest increase in the mRNA level of the 4-kb transcript of MnSOD, but TNF-a a much higher increase in both 1 kb and 4 kb transcripts (Fig 3, III). TNFa increased the level of MnSOD protein in both cell types (Fig 1, III), but none of the cytotoxic drugs tested (cisplatin, epirubicin, methotrexate or vindesin) had any effect. The specific activity of MnSOD was induced significantly by TNFa only in A549 cells (+524%) (Fig 2, III) but the cytotoxic drugs had no inducing effect on the specific activity in either cell type. A549 cells were more resistant against menadione and epirubicin than M14K cells (Fig 4, III). The hypothesis that MnSOD induction may increase oxidant and drug resistance was tested in A549 cells. TNFa pretreated cells were exposed to either menadione (16 h) or epirubicin (48 h) at concentrations that caused the loss of approximately 50% of the control cells. MnSOD induction did not protect these cells against either of these exposures (Fig 5, III). A549 cells were also more resistant against exogenous H2O2 than M14K cells, which suggested the importance of the protective role of catalase and/or glutathione. The specific activity of CAT was approximately twice as high in A549 cells than M14K cells, and total glutathione levels were approximately 2.5 times higher (Fig 7, III). The depletion of CAT (with ATZ) or glutathione (with BSO) made A549 cells more vulnerable to oxidant injury caused by H2O2 but only BSO pretreatment had an effect on the toxicity caused by epirubicin. The hypothesis that glutathionerelated mechanisms are important for the resistance of A549 cells was supported by Western blot studies showing that yGCSh was prominently expressed in A549 but not in M14K cells (Fig 7, III). Furthermore, depletion of GSH with BSO enhanced the injury in A549 cells when exposed to cisplatin (Fig 1, IV). The activity of total GST was somewhat lower in A549 than in M14K cells, suggesting that GST may not be involved in the oxidant resistance of these adenocarcinoma cells. Expression of ^GCS subunits in lung tumor and mesothelioma biopsies (IV, V) Antibodies for the heavy and light subunits of yGCS were used in staining lung tumor biopsies and cell lines. These antibodies were shown to be applicable in the histochemical assessment of paraffin embedded tissues, and yGCSh and yGCSI subunits were expressed in lung malignancies (Fig 1, IV). 46 RESULTS Both subunits of yGCS were investigated in five samples of non-malignant mesothelium and 34 mesothelioma tumor biopsies. Non-malignant mesothelium showed no expression of yGCSh or yGCSl in any of the samples (Fig 1, V). Most mesotheliomas (29/34) showed strong intensity of immunoreactivity for YGCSh. The combined qualitative and quantitative scores of immunostaining are shown in Table 1(V). The intensity of yGCSI immunoreactivity was considerably weaker. Of the 34 cases, 12 cases showed no immunoreactivity with the light subunit antibody, strong/moderate staining was seen in 15 cases. The histologic type of mesothelioma had no statistically significant effect on yGCS reactivity even though the epithelial subtypes had a tendency to show stronger immunoreactivity. There was no correlation between either YGCSh or yGCSl immunoreactivity and patient survival. Expression and possible role of 7GCS in mesothelioma cells in vitro (V) Seven mesothelioma cell lines were investigated for the expression of both yGCS subunits with Western blot analysis. Both subunits were detectable in all cell lines (Fig 2, V). The cell line most resistant to oxidants, M38K, contained the highest level of YGCSh. In the nucleotide depletion assay, M38K cells were more resistant to cisplatin than M14K cells. Pretreatment with BSO to deplete intracellular glutathione potentiated the cisplatin-induced toxicity significantly (Fig 4, V). Caspase 3 cleavage was used as an indicator of the apoptotic process. M14K cells exposed to 50 pM cisplatin showed caspase 3 cleavage within 48 h, which was not potentiated by BSO pretreatment. In M38K cells no apoptosis could be seen when assessed by caspase 3 cleavage after cisplatin exposure without or with BSO (Fig 5, V). Apoptosis was also assessed morphologically after the same exposures. Again cisplatin caused apoptosis in M14K cells but not in M38K cells, and apoptosis was not enhanced by BSO. Apoptotic index in M38K cells was very low (between 2.4% in controls and 4.5% in cisplatin exposed BSO treated cells). The expression of P-glycoprotein and multidrug-resistance proteins 1 and 2 (MRP1 and MRP2) in mesothelioma tissue biopsies (VI) Positive staining with the P-gp antibody was seen in 61 % of the mesothelioma tumor biopsies (n=36): 13 cases showed strong, five moderate, and four weak staining (Table 1, VI). The reactivity was mainly localized to the plasmamembrane. Epithelial and biphasic subtypes showed significantly more often strong immunoreactivity than did the sarcomatoid subtype (p=0.031). No positive staining was observed in non-malignant mesothelial cells. Positive immunoreactivity for MRP1 was seen in 58 % of the cases. The staining was both membrane-bound and cytoplasmic. Equal incidences of strong, moderate and weak immunoreactivity for MRP1 were seen. MRP1 reactivity was weaker in sarcomatoid than epithelial or biphasic mesotheliomas (p=0.034). No immunostaining was seen in samples of non-neoplastic mesothelium (Fig 1, VI). 47 RESULTS MRP2 antibody detected non-specific nuclear immunostaining in addition to membrane-bound immunoreactivity, but only membrane-bound staining was considered significant. 33 % cases showed positive reactivity and the staining was usually weak. Significant association was detected between MRP2 and moderate or strong P-gp staining (p=0.021), but no association was found between MRP2 and MRP1 reactivity (p=0.41). Sarcomatoid mesotheliomas showed significantly more often negative MRP2 staining than epithelial and biphasic ones (p=0.024). Non-neoplastic mesothelium was not stained with MRP2 (Fig 1, VI). No significant association was found with patient survival and expression of any of the multidrug resistance proteins studied (p=0.135, p=0.09 and p=0.88 for P-gp, MRP2 and MRP1, respectively). There was no association between these glycoproteins and tumor proliferation or apoptosis. We also looked for an association between expression of yGCS and that of MRP1 or MRP2. There was a significant correlation between yGCSh and MRP2 expression (p=0.048), whereas the correlation between yGCSl and MRP2 was not significant (p=0.35). No correlation was seen between MRP1 and yGCSh (p=0.67) or yGCSl (p=0.50), either. 48 RESULTS Table 5. The expression of antioxidant enzymes and related mechanisms in mesothelial cell line (MeT5A), mesothelioma cell lines (M14K, M38K), lung adenocarcinoma cell line (A549), nonmalignant mesothelium and mesothelioma biopsies published by us or other laboratories. The results have been assessed at the level of mRNA (Northern blot, NB), protein (Western blot, WB), immunohistochemistry (IH), and/or the specific activity (SA) or concentration (c), when available. Nd = not done. Antioxidant/ related mechanism Mesothelial cell line (MeT5A) MnSOD CuZnSOD Catalase + (NB,WB,IH,SA) + (NB,SA) + (NB,SA) GSH GPx + (C) + (NB,SA) GST + (WB,SA) YGCSh YGCSl MRP1 MRP2 P-gp TRX + (WB) + (WB) nd nd nd nd PRXI-VI (+, Kinnula, 2001,WB) Mesothelioma cell lines (M14K/ M38K) +++ / +++ (NB,WB,IH,SA) ++ / ++ (NB,SA) ++/ +++ (NB,SA) Lung adenocarcinoma cell line (A549) (+) (NB,WB,SA) nd +++ (SA) +/ +++ (C) + (+)/ +(+) (NB,SA) ++/ +++ (WB,SA) +++ (C) nd + (SA) ++/ +++ (WB) +/ + (WB) nd nd nd (++/++, Kahlos, 2001 NB,WB) (+ - ++, Kinnula, 2001, WB) +++ (WB) + (WB) nd nd nd (++, Soini, 2001, WB) nd Nonmalignant mesothelium Mesothelioma tissue samples neg (IH) nd (neg, Kahlos, 2001, IH) nd nd +++ (IH) nd (+++, Kahlos, 2001, IH) nd nd (++, Dejmek, 98, Segers, 96, IH) neg (IH) neg (IH) neg (IH) neg (IH) neg (IH) (neg, Kahlos, 2001, IH) (neg, Kinnula, 2001, IH) (++ (+), Dejmek, 98, Segers, 96, IH) +++ (IH) ++ (IH) ++ (IH) + (IH) ++ (IH) (+++, Kahlos, 2001, IH) (+-+++, Kinnula, 2001, IH) 49 DISCUSSION DISCUSSION MnSOD The role of MnSOD in cancer biology is controversial. In the literature, it has been stated repeatedly that MnSOD activity is diminished in cancer cells and regaining the activity will eventually decrease the state of malignancy (Oberley & Oberley). However, increased MnSOD activities have been documented in some cancer types, at least glioma (Cobbs et al., 1996), renal carcinoma (Oberley et al., 1994) colorectal cancers (Janssen et al., 1998) and thyroid malignancies (Nishida et al., 1993). In some of these cancers, increased MnSOD level has been linked with poor survival (Janssen et al., 1998; Ria et al., 2001). Many problems arise, however, when investigating the expression and role of MnSOD in cancer cells. The studies that have found increased MnSOD levels have been criticized by arguing that they have not included adequate controls or that they have not measured the specific activity of the enzyme (Oberley&Oberley). Then again studies evaluating the role of MnSOD in malignancy have often been done using gene transfection. The problem with transfection is that it does not mimic the actual in vivo situation, in which many antioxidants may be simultaneously induced. Transfection of the MnSOD cDNA alone results in accumulation of H2O2 and subsequent toxicity, which could lead to conclusions based on false assumptions. The balance of antioxidants is crucial in protecting the cell against various exogenous and endogenous oxidants (de Haan et al., 1996; Li et al., 2000; Thannickal & Fanburg, 2000). Conclusions have also been drawn from activity measurements directly in plasma samples from patients. Also tumor homogenates have been used to measure enzyme activities. Neither of these methods describes enzyme activity in a cancer cell. Tumor homogenates contain various cell types that are not always cancerous, like stroma. The problem with continuous cell lines is that the levels of AOEs usually decline in culture and selection of tumor cells also takes place during culture. The activity of an enzyme such as MnSOD in cultured cells may also be dependent on factors such as plating density, stage of cell growth, and constituents of the culture medium. It has to be noted that MnSOD is essential to living cells; knockout mice lacking the MnSOD gene are not viable (Li et al., 1995). Furthermore, transgenic animals expressing elevated lung MnSOD activity are more resistant to high oxygen tension than the control animals (Tsan, 2001). In this study, the level of MnSOD was found to be significantly higher in mesothelioma cells than in healthy mesothelium or non-malignant MeT5A mesothelial cells in vivo and in vitro. In addition to immunohistochemistry, in which tumor samples where compared with non-malignant mesothelium, MnSOD was further investigated using northern blot and western blot analysis, and 50 DISCUSSION the specific activity of the enzyme was assessed. All these results confirm the hypothesis that MnSOD is overexpressed in human pleural mesothelioma. In the present study MeT5A was used as a model of a non-malignant cell line. It, however, does not represent a primary mesothelial cell line, as MeT5A has been transformed with SV40 and it has qualities of a reactive mesothelium. MeT5A cells are nevertheless non-cancerous with typical mesothelial characteristics. Due to the difficulties in obtaining healthy mesothelium for culture of human mesothelial cell, MeT5A cells have been widely used for this purpose. Since reactive mesothelium in vivo and also the cell clusters of MeT5A cells showed positive immunoreactivity (Kahlos et al., 2000a), MnSOD cannot be used in the differential diagnosis between reactive mesothelium and mesothelioma. The differential diagnosis between mesothelioma and adenocarcinoma in pleura is difficult. Further studies have shown that MnSOD is higher in primary malignant mesothelioma than in other tumors metastasized to pleura (Kahlos et al., 2000a). Thus MnSOD may play role in the differential diagnosis of mesothelioma. Studies on mesothelioma cell lines revealed that the cell line with the highest MnSOD activity (M38K) contained also the highest level of other antioxidants, like catalase and glutathione. M38K also turned out to be the most resistant of the mesothelioma cell lines. This supports the hypothesis that a simultaneous increase of many AOEs offers best protection. However, many mechanisms other than AOEs may also be overexpressed in this cell line, for example the apoptotic protein BclX (Soini et al., 1999), DNA repair mechanisms, or even the drug efflux pumps. It has been suggested that MnSOD cannot be induced in cancer cells (Wong & Goeddel, 1988). The present study, nevertheless, found that MnSOD protein and activity were increased by TNFa in both a mesothelioma cell line (M14K) and a pulmonary adenocarcinoma cell line (A549). This result is also in line with some other studies showing MnSOD induction in malignant cells at least by TNFa (Warner et al., 1996). In the present study MnSOD was induced more in A549 cells, in which the baseline MnSOD was low. Some of the cytotoxic drugs tended to increase MnSOD mRNA levels, but induction failed to reach significance. Induction in vivo is, however, possible. Other investigators (Akashi et al., 1996; Das et al., 1998) have shown that MnSOD mRNA was upregulated after anticancer drug treatment in cultured cells and that NF-kB is indeed induced by anticancer agents without TNFa involvement (Das & White, 1997). The induction in vivo is dependent on the drug concentration at the site of the tumor and time of exposure and these are difficult to mimic in cell cultures. Induced MnSOD level has been suggested to offer protection against oxidant stress such as radiotherapy (Wong & Goeddel, 1988). Pulmonary adenocarcinoma cells treated with TNFa had an elevated level of MnSOD and increased resistance to paraquat (Warner et al., 1996). Its role in anticancer drug resistance has however, remained unclear and controversial. In this study, MnSOD induction failed to offer extra protection against either oxidants or the anti-cancer drug epirubicin. If anything, the cells were more susceptible to oxidant injury. The effects of TNFa are, however, numerous and TNFa has been shown to induce the apoptotic pathways by activating NF-kB (Darnay & Aggarwal, 1997). There is evidence that higher MnSOD level offers protection against apoptosis by blocking the effects of TNFa and the same effect has been ascribed to yGCS that inhibits the activation of NF-kB and prevents the apoptotic cascade (Manna et al., 1998; Morales et al., 1997; Obrador et al., 1997). 51 DISCUSSION Several studies on cell cultures and experimental models and MnSOD transfected cells have suggested that MnSOD reduces tumor cell proliferation (Oberley&Oberley). Kahlos et al reported that high MnSOD in mesothelioma correlates with decreased proliferation. However, there was no relationship between proliferation or spontaneous apoptosis and patient survival (Kahlos et al., 2000b). Based on previous and the present results, MnSOD is elevated in mesothelioma and may possibly be used as an additional marker in the differential diagnosis of this disease. MnSOD induction does not protect malignant cells against oxidant and drug exposure, and besides MnSOD, H2O2 scavenging AOEs may play an important role both in primary and acquired drug resistance. Catalase There are only a few studies on catalase in malignant tumors, and no major role has been proposed for it in anti-cancer drug resistance (Sinha & Mimnaugh, 1990; Tome et al., 2001). The present study showed that the mRNA and the specific activity of catalase were higher in the most resistant mesothelioma cell line compared to the non-malignant mesothelial cell line. In addition, a recent study confirmed the overexpression of catalase in mesothelioma. In that study a coordinate expression of catalase and MnSOD predicted a better survival of mesothelioma patients (Kahlos et al., 2001b). However, in cultured mesothelioma cells, inactivation of catalase with ATZ did not have significant effect on the toxicity of epirubicin, although it increased the susceptibility to H2O2induced toxicity. This would imply a function for catalase during heavy oxidant exposure, which is in line with its physiological role (Halliwell&Gutteridge). On the other hand, ATZ does not completely inactivate catalase, so these studies may underestimate its role. Besides MnSOD and CAT, high oxidant resistance of mesothelioma may thus involve a simultaneous induction of many other antioxidant mechanisms most importantly the glutathione system. Glutathione and GSH-related enzymes The level of glutathione has often been linked with anti-cancer drug resistance (O'Brien & Tew, 1996; Tew, 1994; Zhang et al., 1998). Glutathione can confer resistance to cytotoxic drugs that cause ROS generation through the classical antioxidant pathway, the glutathione redox cycle. It is also irreversibly consumed in detoxification reactions catalyzed by GSTs. Also some of the plasmamembrane drug efflux pumps, mainly MRP1 and MRP2, are dependent on glutathione (Borst et al., 2000). Resistance to apoptosis has been suggested to be redox-regulated as well (Manna et al., 1998; Susin et al., 1998). Therefore, the synthesis and maintenance of the intracellular GSH level and the GSH:GSSG ratio are relevant for optimal defense against cytotoxic drugs. M38K, the most resistant mesothelioma cell line, contained not only the highest level of MnSOD and catalase, but also of glutathione, glutathione peroxidase, glutathione S-transferase and YGCSh. Depletion of intracellular glutathione resulted in reversal of the resistance to epirubicin and cisplatin. Additional studies with the adenocarcinoma cell line A549 also emphasized the importance of the glutathione system both in the oxidant and drug resistance. A549 had a higher 52 DISCUSSION level of glutathione and 7GCS compared to M14K. Inhibition of yCCS with BSO also enhanced the oxidant and drug (cisplatin) sensitivity of these cells significantly. A549 cell line is a resistant cell line to oxidants and oxidant producing drugs, but in these cells the MnSOD level was very low whereas GSH content was high. In light of these results, glutathione depletion with BSO could possibly have therapeutic implications in lung cancer and mesothelioma. One way of reversing the resistance could be injection of BSO intrapleurarly to avoid the systematic problems of glutathione depletion. In fact, phase I trials have been conducted with BSO and melphalan, and the continuousinfusion BSO was relatively nontoxic and resulted in depletion of tumor glutathione (Bailey et al., 1997). The capacity to synthesize GSH is essential in protection and detoxification reactions of nonmalignant and malignant cells. In addition to cysteine availability, the expression and activity of yCCS is of major importance in maintaining adequate levels of GSH. Most articles regarding 7GCS have been studies on YGCSh and its regulation, lately some studies have been published also on the light subunit (Galloway et al., 1997; Shi et al., 1994; Tipnis et al., 1999; Zhang et al., 1998). Both subunits of 7GCS have been connected with resistance against melphalan, doxorubicin and cisplatin (Iida et al., 1999). No studies have, however, been done with the actual expression and distribution of the enzyme subunits in malignant tumors. There is a recent microarray study comparing a mesothelioma cell line with a non-malignant mesothelioma cell line showing increased expression of yGCS in the malignant cell (Rihn et al., 2000). Yet only mRNA, not protein, was detected from these cells. In the present study all mesothelioma cell lines expressed both subunits of 7GCS. The highest level of YGCSh, which is responsible for the catalytic activity, was found, not surprisingly, in the M38K cells. In immunohistochemistry most mesotheliomas expressed both subunits of yGCS. Healthy mesothelium was negative, but by immunohistochemistry the presence of small amounts of the enzyme in healthy tissue cannot be excluded. No correlation between patient survival and yGCS expression was observed. On the other hand, the overall prognosis of mesothelioma is poor and the survival time is not always reliable as the time of diagnostic biopsy within the time span of the disease varies uncontrollably between patients. Results from a recent immunohistochemical study with lung cancer biopsies also showed that both subunits of yGCS are abundantly expressed in lung cancer cells (Soini et al., 2001b). In these samples a positive correlation was found between high apoptotic tendency and low yGCS expression. These results are in line with the previous report that yGCS is antiapoptotic as it changes the redox-state of the cell possibly by inhibiting the proapoptotic signals caused by e.g. TNFa (Manna et al., 1998). This same result was not achieved with the mesothelioma biopsies. In the present study BSO pretreatment of mesothelioma cell lines that was followed by cisplatin exposure did not enhance apoptosis in M38K cells, indicating strong resistance towards apoptosis but also other mechanisms than the redox state in controlling the apoptotic pathway. GST-n has been reported to be overexpressed in neoplastic tissues (O'Brien & Tew, 1996). Also the relationship between the overexpression of GSTs and the sensitivity of tumor cells has been documented (Hao et al., 1994, reviewed by Zhang et al., 1998). The present study found a higher 53 DISCUSSION expression of GST-n in the M38K cell line than the more sensitive mesothelioma cell line and the non-malignant cell line. In the adenocarcinoma cell line the activity of total GST was considerably lower compared to the mesothelioma cell lines. On the other hand, A549 cells were more resistant to oxidants and cytotoxic drugs and had a significantly higher GSH-level than mesothelioma cells. These results are in line with others showing that GSH and its re-synthesis capacity explain the resistance of A549 cells to cytotoxic drugs (al-Kabban et al., 1990), cadmium (Hatcher et al., 1995) and oxidants (Arsalane et al., 1997; Rahman et al., 2001). On the other hand, there are studies showing that lung cancer cell lines can express several GSTs and GPx without a GSH increase (Hao et al., 1994). Multiple detoxification mechanisms may thus be involved and non-enzymatic detoxification by glutathione conjugation may take place as well. The resistance mechanisms must also differ between different drugs since their way of killing the cancer cells is diverse. Anthracyclins that cause ROS formation are possibly detoxified in part by AOEs. On the other hand, cisplatin may not cause oxidative stress and thus it may be conjugated to GSH and exported from the cell; the role of MnSOD and other antioxidants in cisplatin resistance may therefore be minimal. The induction of the salvage enzyme yGT has been reported during the development of drug resistance. In a study analyzing 451 human cancers, all 10 mesothelioma samples were negative for yGT immunoreactivity (Hanigan et al., 1999). This suggests that intracellular glutathione levels are maintained with de novo synthesis probably by yGCS. To conclude, the present study emphasized the importance of GSH-related mechanisms in the intrinsic resistance of mesothelioma, and these findings were supported by studies conducted with lung adenocarcinoma cells. High expression of yGCS is of major importance both in the detoxification pathway and the antioxidant function of these cells. P-gp and MRP1&2 The plasmamembrane pumps have been extensively studied, and the emphasis has been on P-gp (Pastan & Gottesman, 1987). One study on cultured mesothelioma cell lines suggested that the resistance to vindesin and doxorubicin is P-gp dependent. In that study, however, the baseline expression of P-gp was almost undetectable, and the expression increased when exposing the cells continuously to the cytotoxic drugs (Licht et al., 1991; Licht et al., 1995) Therefore, P-gp is not likely to account for the primary resistance of the tumor. In an immunohistochemical study on 33 mesothelioma tumor biopsies, P-gp was expressed in the majority of tumors (Ramael et al., 1992). In another study on the expression of P-gp, MRP1 and yGCS in five mesothelioma cell lines the conclusion was that MRP1 and yGCS may be co-ordinately overexpressed in doxorubicin-resistant cells (Ogretmen et al., 1998). The present study found the expression level of P-gp to line up with findings from other tumors, such as kidney, colon and liver (Duensing & Slate, 1994; Sinicrope et al., 1992; Soini et al., 1996). MRP1 was expressed in the majority of the biopsies, which is in agreement with other tumors such as colon and gastric cancers (Endo et al., 1996; Fukushima et al., 1999). MRP2 was expressed in only 33% of mesotheliomas, and the staining was usually weak. It has been detected in unselected lung, gastric and colorectal tumor cell lines (Kool et al., 1997; Taniguchi et al., 1996). None of 54 DISCUSSION these proteins correlated with patient survival so the expression of these efflux proteins cannot be used as a prognostic factor. A recent study has suggested that even a low expression of these transport proteins may cause clinical drug resistance, as the maximum dose of a cytotoxic drug tolerated by the patient is often barely sufficient to kill a useful percentage of the tumor cells (Allen et al., 2000). Therefore relatively small increases in the drug resistance in tumor cells are sufficient to make the drug clinically ineffective. Our results show that these transporters cannot be used for the assessment of primary resistance, as most patients did not receive chemotherapy and the efflux pumps are not known to cause proliferation of the malignant cells or progression of the disease. In agreement with this neither tumor proliferation nor apoptosis showed any association with the expression of the multidrug resistance proteins. yGCS expression correlated with MRP2, but not with MRP1. A connection has been made with yGCS and MRP1 in a number of studies (Ishikawa et al., 1996; Kuo et al., 1996) but no corresponding studies on MRP2 are available. MRP2 has not, however, been studied as much and even its expression in different malignancies is largely unknown. Cisplatin has been shown to cause upregulation of MRP2, and MRP2 to cause resistance against cisplatin (Borst et al., 2000; Konig et al., 1999). Most of our patients had not received chemotherapy so the immunoreactivity of MRP2 describes primary, not cisplatin induced, expression of the protein. (Ishikawa et al., 1996. It remains to be investigated whether this correlation has any true significance in resistance of mesothelioma, as the expression of MRP2 is weak altogether and present only in 33% of the biopsies. In contrast of trying to find significant associations with patient survival, the expression of these mechanisms might be considered as tumor characteristics that can be used when trying to understand the natural course of this malignancy. In fact the actual role of these efflux proteins in the clinical primary resistance of any malignant tumor is far from clear. For example, new members to the MRP family have been discovered (Bera et al., 2001) and their contribution to clinical drug resistance has not yet been studied. 55 CONCLUSIONS CONCLUSIONS Malignant pleural mesothelioma shows a simultaneous high expression of several antioxidant enzymes and related proteins. These mechanisms may play an important role in the overall poor prognosis, and in the drug resistance of this disease. The following conclusions can be drawn from the present study: 1. The level of MnSOD is significantly higher in malignant mesothelioma cells than in non- malignant mesothelial cells both in vitro and in vivo. High MnSOD can be detected at the level of mRNA, immunoreactive protein and specific activity. 2. MnSOD in mesothelioma cells can be induced by TNFa but not by the cytotoxic drugs. Induction of MnSOD, however, does not provide any protection against repeated oxidant or drug exposures. Additional studies with lung adenocarcinoma cells further suggest the importance of high glutathione content in the oxidant and drug resistance of these cells. 3. The resistance of mesothelioma cells in vitro is partly associated with glutathione and glutathione S-transferase whereas the significance of catalase is limited only to heavy oxidant exposure. 4. In addition to MnSOD, yGCS, the rate-limiting enzyme in glutathione synthesis, is expressed at high quantities in most mesotheliomas. yGCS may play role in the overall primary drug resistance of malignant lung cells, since treatment of mesothelioma and lung adenocarcinoma cells with BSO, to inhibit yGCS, significantly potentiates drug-induced cytotoxicity. 5. Variable expression of P-gp, MRP1 and MRP2 can be detected in malignant mesothelioma, suggesting that the primary resistance of mesothelioma is not solely dependent on their expression or function. As tumor growth, apoptosis or patient survival do not associate with these membrane glycoproteins, they cannot be used as a prognostic factor in mesothelioma. 56 ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS This work was carried out at the Research Laboratory of the Hospital for Children and Adolescents, University of Helsinki; Finnish Institute of Occupational Health, Helsinki; and the Department of Internal Medicine, University of Oulu, Finland. I wish to express my sincere gratitude to the Heads of these departments for the excellent research facilities. I am most grateful to my supervisor Professor Vuokko Kinnula for pushing me into this project and patiently teaching me about pulmonary malignancies, antioxidants and science in general. Without her encouragement this thesis would not have been completed. I am also indebted to my other supervisor Professor Kari Raivio for his critical evaluation of my work. I deeply admire his knowledge and passionate attitude towards science. I am very grateful to Professor Kirsi Vahakangas and Dr. Veli-Matti Kosma for carefully reviewing this thesis and giving valuable comments. I warmly thank all the collaborators from the original articles. I am indebted to Dr. Ylermi Soini for his help and patience and I admire his vast knowledge in tumor pathology. I also wish to thank Dr. Katriina Kahlos for her collaboration and indispensable help during this project. Dr. Kaija Linnainmaa deserves a thank you for the support and also for providing the cell lines used in the original papers. The collaboration from Dr. Petra Pietarinen-Runtti, Dr. Tiina Asikainen, and Dr. Riitta Kaarteenaho-Wiik, is gratefully acknowledged. I also wish to thank Professor Karin Mattson from the University Hospital of Helsinki, Dr. Sisko Anttila from the Finnish Institute of Occupational Health, and acknowledge the support from Dr. Paavo Paakko from the Department of Pathology, University of Oulu. I am grateful to my American collaborators Dr. Terrance Kavanagh and Dr. Cecil Krejsa for the yGCS antibody and Professor James Crapo for providing the MnSOD antibody. I wish to acknowledge the support from Dr. Kai Savolainen from the Finnish Institute of Occupational Health. I am also grateful to the senior scientists Dr. Hannu Norppa, Dr. Kirsi Husgafel-Pursiainen, and Dr. Ari Hirvonen from the Laboratory of Molecular and Cellular Toxicity for making me feel as one of the group. The "junior" scientists Tiina Ollikainen, Harriet Gullsten, Ghita Falck and Lea Pylkkanen deserve a warm and special thank you, but also everyone else from the corridors and coffee room! I deeply appreciate Raija Sirvio for teaching me laboratory work in Oulu and for the wonderful discussions concerning life in general. I would also like to thank Manu Tuovinen for his enormous role in completing the immunohistochemistry. The technical assistance from Satu Suhonen and Sari Linden is also acknowledged. 57 ACKNOWLEDGEMENTS I wish to thank all my friends outside of work for keeping my feet on the ground and bringing some perspective into my little world. My parents deserve the warmest thank you for always emphasizing the importance of meaningful and rewarding work and also for the true interest towards my thesis. Finally, I express a special thanks to my dear husband Juha who has tried to focus my attention to other things than medicine (and succeeded!) but also encouraged me to finish the work I have started. This work was supported by grants from the Finnish Antituberculosis Association Foundation, Cancer society of Finland, The Ida Montin Foundation, Paulo Research Foundation, The Juselius Foundation and The Ahokas Foundation, which I gratefully acknowledge. Helsinki, October, 2001 Kristiina Jarvinen 58 REFERENCES REFERENCES Akashi, M., Takagi, S. & Hachiya, M. (1996). Anti-cancer agent OK432 induces manganese superoxide dismutase in human granulocytes. Int J Cancer, 68, 384-90. al-Kabban, M., Stewart, M.J., Watson, I.D. & Reglinski, J. (1990). The effect of doxorubicin on the glutathione content and viability of cultured human lung cancer cell lines A549 and GLC4 210. Clin Chim Acta, 194, 121-9. Allen, J.D., Brinkhuis, R.F., van Deemter, L., Wijnholds, J. & Schinkel, A.H. (2000). Extensive contribution of the multidrug transporters P-glycoprotein and Mrp1 to basal drug resistance. Cancer Res, 60, 5761-6. Ambrosone, C.B., Freudenheim, J.L., Thompson, P.A., Bowman, E., Vena, J.E., Marshall, J.R., Graham, S., Laughlin, R., Nemoto, T. & Shields, P.G. (1999). Manganese superoxide dismutase (MnSOD) genetic polymorphisms, dietary antioxidants, and risk of breast cancer. Cancer Res, 59, 602-6. Arai, K., Sumi, S.H., Yoshida, K. & Komoda, T. (1995). A precursor form of human kidney gammaglutamyl transferase in normal and cancerous tissues, and its possible post-translational modification. Biochim Biophys Acta, 1253, 33-8. Arsalane, K., Dubois, C.M., Muanza, T., Begin, R., Boudreau, F., Asselin, C. & Cantin, A.M. (1997). Transforming growth factor-beta1 is a potent inhibitor of glutathione synthesis in the lung epithelial cell line A549: transcriptional effect on the GSH rate-limiting enzyme gamma-glutamylcysteine synthetase. Am JRespir Cell Mol Biol, 17, 599-607. Attanoos, R.L. & Gibbs, A.R. (1997). Pathology of malignant mesothelioma. Histopathology, 30, 403-18. Avissar, N., Whitin, J.C., Allen, P.Z., Wagner, D.D., Liegey, P. & Cohen, H.J. (1989). Plasma selenium- dependent glutathione peroxidase. Cell of origin and secretion. J Biol Chem, 264, 15850-5. Bailey, H.H., Ripple, G., Tutsch, K.D., Arzoomanian, R.Z., Alberti, D., Feierabend, C., Mahvi, D., Schink, J., Pomplun, M., Mulcahy, R.T. & Wilding, G. (1997). Phase I study of continuous-infusion L-S,Rbuthionine sulfoximine with intravenous melphalan. JNatl Cancer Inst, 89, 1789-96. Bannai, S. (1984). Transport of cystine and cysteine in mammalian cells. Biochim Biophys Acta, 779, 289 306. Beck, W.T., Danks, M.K., Wolverton, J.S., Granzen, B., Chen, M., Schmidt, C.A., Bugg, B.Y., Friche, E. & Suttle, D.P. (1993). Altered DNA topoisomerase II in multidrug resistance. Cytotechnology, 11, 115-9. Bellamy, W.T. & Dalton, W.S. (1994). Multidrug resistance in the laboratory and clinic. Adv Clin Chem, 31, 1-61. Bera, T.K., Lee, S., Salvatore, G., Lee, B. & Pastan, I.H. (2001). Mrp8, a new member of abc transporter superfamily, identified by est database mining and gene prediction program, is highly expressed in breast cancer. Mol Med, 7, 509-16. Bergmayer, H.U. & Bernt, E. (1974). Lactate dehydrogenase UV-assay with pyruvate and NADH. In Methods of Enzymatic Analysis, Bergmayer, H.U. (ed), Vol. 2. pp. 574-579. Academic press: New York. Beutler, E., Beutler, B. & Matsumoto, J. (1975). Glutathione peroxidase activity of inorganic selenium and seleno-DL- cysteine. Experientia, 31, 769-70. Beyer, W., Imlay, J. & Fridovich, I. (1991). Superoxide dismutases. Prog Nucleic Acid Res Mol Biol, 40, 221-53. 59 REFERENCES Borst, P., Evers, R., Kool, M. & Wijnholds, J. (2000). A family of drug transporters: the multidrug resistance-associated proteins. JNatl Cancer Inst, 92, 1295-302. Boutin, C., Dumortier, P., Rey, F., Viallat, J.R. & De Vuyst, P. (1996). Black spots concentrate oncogenic asbestos fibers in the parietal pleura. Thoracoscopic and mineralogic study. Am J Respir Crit Care Med, 153, 444-9. Boutin, C., Schlesser, M., Frenay, C. & Astoul, P. (1998). Malignant pleural mesothelioma. Eur Respir J, 12, 972-81. Browne, K. (1995). Asbestos-related disorders. In Occupational lung disorders, Parkers, W.R. (ed) pp. 465 504. Buttenford-Heineman: Oxford. BTS. (2001). Statement on malignant mesothelioma in the United Kingdom. Thorax, 56, 250-65. Buckley, B.J., Kent, R.S. & Whorton, A.R. (1991). Regulation of endothelial cell prostaglandin synthesis by glutathione. J Biol Chem, 266, 16659-66. Cable, S., Keller, J.M., Colin, S., Haffen, K., Kedinger, M., Parache, R.M. & Dauca, M. (1992). Peroxisomes in human colon carcinomas. A cytochemical and biochemical study. Virchows Arch B Cell Pathol Incl Mol Pathol, 62, 221-6. Campos, L., Guyotat, D., Archimbaud, E., Calmard-Oriol, P., Tsuruo, T., Troncy, J., Treille, D. & Fiere, D. (1992). Clinical significance of multidrug resistance P-glycoprotein expression on acute nonlymphoblastic leukemia cells at diagnosis. Blood, 79, 473-6. Carbone, M., Pass, H.I., Rizzo, P., Marinetti, M., Di Muzio, M., Mew, D.J., Levine, A.S. & Procopio, A. (1994). Simian virus 40-like DNA sequences in human pleural mesothelioma. Oncogene, 9, 1781 90. Carmichael, J., Forrester, L.M., Lewis, A.D., Hayes, J.D., Hayes, P.C. & Wolf, C.R. (1988). Glutathione Stransferase isoenzymes and glutathione peroxidase activity in normal and tumour samples from human lung. Carcinogenesis, 9, 1617-21. Chan, H.S., Lu, Y., Grogan, T.M., Haddad, G., Hipfner, D.R., Cole, S.P., Deeley, R.G., Ling, V. & Gallie, B.L. (1997). Multidrug resistance protein (MRP) expression in retinoblastoma correlates with the rare failure of chemotherapy despite cyclosporine for reversal of P-glycoprotein. Cancer Res, 57, 2325-30. Chaudhary, P.M. & Roninson, I.B. (1993). Induction of multidrug resistance in human cells by transient exposure to different chemotherapeutic drugs. JNatl Cancer Inst, 85, 632-9. Childs, S. & Ling, V. (1994). The MDR superfamily of genes and its biological implications. Important Adv Oncol, 21-36. Chin, K.V., Tanaka, S., Darlington, G., Pastan, I. & Gottesman, M.M. (1990). Heat shock and arsenite increase expression of the multidrug resistance (MDR1) gene in human renal carcinoma cells. J Biol Chem, 265, 221-6. Chomczynski, P. & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction. Anal Biochem, 162, 156-9. Chu, F.F., Doroshow, J.H. & Esworthy, R.S. (1993). Expression, characterization, and tissue distribution of a new cellular selenium-dependent glutathione peroxidase, GSHPx-GI. J Biol Chem, 268, 2571-6. Church, S.L., Grant, J.W., Meese, E.U. & Trent, J.M. (1992). Sublocalization of the gene encoding manganese superoxide dismutase (MnSOD/SOD2) to 6q25 by fluorescence in situ hybridization and somatic cell hybrid mapping. Genomics, 14, 823-5. Clerch, L.B. & Massaro, D. (1993). Tolerance of rats to hyperoxia. Lung antioxidant enzyme gene expression. J Clin Invest, 91, 499-508. Cobbs, C.S., Levi, D.S., Aldape, K. & Israel, M.A. (1996). Manganese superoxide dismutase expression in human central nervous system tumors. Cancer Res, 56, 3192-5. Cole, S.P., Bhardwaj, G., Gerlach, J.H., Mackie, J.E., Grant, C.E., Almquist, K.C., Stewart, A.J., Kurz, E.U., Duncan, A.M. & Deeley, R.G. (1992). Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science, 258, 1650-4. Coursin, D.B., Cihla, H.P., Sempf, J., Oberley, T.D. & Oberley, L.W. (1996). An immunohistochemical analysis of antioxidant and glutathione S- transferase enzyme levels in normal and neoplastic human lung. Histol Histopathol, 11, 851-60. 60 REFERENCES Craighead, J.E. & Mossman, B.T. (1982). The pathogenesis of asbestos-associated diseases. N Engl J Med, 306, 1446-55. Crapo, J.D., Oury, T., Rabouille, C., Slot, J.W. & Chang, L.Y. (1992). Copper,zinc superoxide dismutase is primarily a cytosolic protein in human cells. Proc Natl Acad Sci U S A, 89, 10405-9. Crapo, J.D. & Tierney, D.F. (1974). Superoxide dismutase and pulmonary oxygen toxicity. Am J Physiol, 226, 1401-7. Cui, Y., Konig, J., Buchholz, J.K., Spring, H., Leier, I. & Keppler, D. (1999). Drug resistance and ATPdependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells. Mol Pharmacol, 55, 929-37. Dalton, T.P., Dieter, M.Z., Yang, Y., Shertzer, H.G. & Nebert, D.W. (2000). Knockout of the mouse glutamate cysteine ligase catalytic subunit (Gclc) gene: embryonic lethal when homozygous, and proposed model for moderate glutathione deficiency when heterozygous. Biochem Biophys Res Commun, 279, 324-9. Darnay, B.G. & Aggarwal, B.B. (1997). Early events in TNF signaling: a story of associations and dissociations. JLeukoc Biol, 61, 559-66. Das, K.C., Guo, X.L. & White, C.W. (1998). Protein kinase Cdelta-dependent induction of manganese superoxide dismutase gene expression by microtubule-active anticancer drugs. J Biol Chem, 273, 34639-45. Das, K.C. & White, C.W. (1997). Activation of NF-kappaB by antineoplastic agents. Role of protein kinase C. JBiol Chem, 272, 14914-20. Davies, P., Drath, D.B., Engel, E.E. & Huber, G.L. (1979). The localization of catalase in the pulmonary alveolar macrophage. Lab Invest, 40, 221-6. de Haan, J.B., Cristiano, F., Iannello, R., Bladier, C., Kelner, M.J. & Kola, I. (1996). Elevation in the ratio of Cu/Zn-superoxide dismutase to glutathione peroxidase activity induces features of cellular senescence and this effect is mediated by hydrogen peroxide. Hum Mol Genet, 5, 283-92. Dejmek, A., Brockstedt, U. & Hjerpe, A. (1998). Immunoreactivity of pleural malignant mesotheliomas to glutathione S- transferases. Apmis, 106, 489-94. Deneke, S.M. & Fanburg, B.L. (1989). Regulation of cellular glutathione. Am J Physiol, 257, L163-73. Di Ilio, C., Del Boccio, G., Casaccia, R., Aceto, A., Di Giacomo, F. & Federici, G. (1987). Selenium level and glutathione-dependent enzyme activities in normal and neoplastic human lung tissues. Carcinogenesis, 8, 281-4. Doige, C.A., Yu, X. & Sharom, F.J. (1993). The effects of lipids and detergents on ATPase-active Pglycoprotein. Biochim Biophys Acta, 1146, 65-72. Drewinko, B., Patchen, M., Yang, L.Y. & Barlogie, B. (1981). Differential killing efficacy of twenty antitumor drugs on proliferating and nonproliferating human tumor cells. Cancer Res, 41, 2328-33. Duensing, T.D. & Slate, D.L. (1994). Intracellular expression of P-glycoprotein in a human colon tumor cell line. Anticancer Res, 14, 13-9. el Bouhtoury, F., Keller, J.M., Colin, S., Parache, R.M. & Dauca, M. (1992). Peroxisomal enzymes in normal and tumoral human breast. JPathol, 166, 27-35. Endo, K., Maehara, Y., Kusumoto, T., Ichiyoshi, Y., Kuwano, M. & Sugimachi, K. (1996). Expression of multidrug-resistance-associated protein (MRP) and chemosensitivity in human gastric cancer. Int J Cancer, 68, 372-7. Erzurum, S.C., Danel, C., Gillissen, A., Chu, C.S., Trapnell, B.C. & Crystal, R.G. (1993). In vivo antioxidant gene expression in human airway epithelium of normal individuals exposed to 100% O2. JApplPhysiol, 75, 1256-62. Esposito, V., Baldi, A., De Luca, A., Claudio, P.P., Signoriello, G., Bolognese, A., Centonze, P., Giordano, G.G., Caputi, M., Baldi, F. & Giordano, A. (1997). p53 immunostaining in differential diagnosis of pleural mesothelial proliferations. Anticancer Res, 17, 733-6. Figlewicz, D.A., Delattre, O., Guellaen, G., Krizus, A., Thomas, G., Zucman, J. & Rouleau, G.A. (1993). Mapping of human gamma-glutamyl transpeptidase genes on chromosome 22 and other human autosomes. Genomics, 17, 299-305. Freeman, B.A., Mason, R.J., Williams, M.C. & Crapo, J.D. (1986). Antioxidant enzyme activity in alveolar type II cells after exposure of rats to hyperoxia. Exp Lung Res, 10, 203-22. 61 REFERENCES Frei, B., Winterhalter, K.H. & Richter, C. (1986). Menadione- (2-methyl-1,4-naphthoquinone-) dependent enzymatic redox cycling and calcium release by mitochondria. Biochemistry, 25, 4438-43. Fridovich, I. (1978). The biology of oxygen radicals. Science, 201, 875-80. Fridovich, I. (1995). Superoxide radical and superoxide dismutases. Annu RevBiochem, 64, 97-112. Fridovich, I. (1998). The trail to superoxide dismutase. Protein Sci, 7, 2688-90. Fukushima, Y., Oshika, Y., Tokunaga, T., Hatanaka, H., Tomisawa, M., Kawai, K., Ozeki, Y., Tsuchida, T., Kijima, H., Yamazaki, H., Ueyama, Y., Tamaoki, N., Miura, S. & Nakamura, M. (1999). Multidrug resistance-associated protein (MRP) expression is correlated with expression of aberrant p53 protein in colorectal cancer. Eur JCancer, 35, 935-8. Galloway, D.C., Blake, D.G., Shepherd, A.G. & McLellan, L.I. (1997). Regulation of human gammaglutamylcysteine synthetase: co-ordinate induction of the catalytic and regulatory subunits in HepG2 cells. Biochem J, 328, 99-104. Giaccone, G. (1994). Small cell lung cancer and topoisomerases. Anticancer Res, 14, 269-76. Giaccone, G., van Ark-Otte, J., Rubio, G.J., Gazdar, A.F., Broxterman, H.J., Dingemans, A.M., Flens, M.J., Scheper, R.J. & Pinedo, H.M. (1996). MRP is frequently expressed in human lung-cancer cell lines, in non- small-cell lung cancer and in normal lungs. Int J Cancer, 66, 760-7. Gipp, J.J., Bailey, H.H. & Mulcahy, R.T. (1995). Cloning and sequencing of the cDNA for the light subunit of human liver gamma-glutamylcysteine synthetase and relative mRNA levels for heavy and light subunits in human normal tissues. Biochem Biophys Res Commun, 206, 584-9. Goldstein, L.J. (1995). Clinical reversal of drug resistance. CurrProbl Cancer, 19, 65-124. Griffith, O.W. (1999). Biologic and pharmacologic regulation of mammalian glutathione synthesis. Free Radic Biol Med, 27, 922-35. Habig, W.H. & Jakoby, W.B. (1981). Assays for differentiation of glutathione S-transferases. Methods Enzymol, 77, 398-405. Halliwell, B. (1991). Reactive oxygen species in living systems: source, biochemistry, and role in human disease. Am J Med, 91, 14S-22S. Halliwell, B. & Gutteridge, J.M. (1985). The importance of free radicals and catalytic metal ions in human diseases. Mol Aspects Med, 8, 89-193. Hammar, S.P. (1994). Pleural diseases. In Pulmonary Pathology, Dail, D.H. & Hammar, S.P. (eds) pp. 1463-1579. Springer-Verlag: New York. Hanigan, M.H. (1998). gamma-Glutamyl transpeptidase, a glutathionase: its expression and function in carcinogenesis. Chem Biol Interact, 111-112, 333-42. Hanigan, M.H., Frierson, H.F., Jr., Swanson, P.E. & De Young, B.R. (1999). Altered expression of gammaglutamyl transpeptidase in human tumors. Hum Pathol, 30, 300-5. Hanigan, M.H. & Ricketts, W.A. (1993). Extracellular glutathione is a source of cysteine for cells that express gamma-glutamyl transpeptidase. Biochemistry, 32, 6302-6. Hao, X.Y., Bergh, J., Brodin, O., Hellman, U. & Mannervik, B. (1994). Acquired resistance to cisplatin and doxorubicin in a small cell lung cancer cell line is correlated to elevated expression of glutathionelinked detoxification enzymes. Carcinogenesis, 15, 1167-73. Harris, C.A., Derbin, K.S., Hunte-McDonough, B., Krauss, M.R., Chen, K.T., Smith, D.M. & Epstein, L.B. (1991). Manganese superoxide dismutase is induced by IFN-gamma in multiple cell types. Synergistic induction by IFN-gamma and tumor necrosis factor or IL-1. J Immunol, 147, 149-54. Hatcher, E.L., Chen, Y. & Kang, Y.J. (1995). Cadmium resistance in A549 cells correlates with elevated glutathione content but not antioxidant enzymatic activities. Free Radic Biol Med, 19, 805-12. Hayes, J.D. & Pulford, D.J. (1995). The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol, 30, 445-600. Hedenborg, M. & Klockars, M. (1987). Production of reactive oxygen metabolites induced by asbestos fibres in human polymorphonuclear leucocytes. J Clin Pathol, 40, 1189-93. Hendrickson, D.J., Fisher, J.H., Jones, C. & Ho, Y.S. (1990). Regional localization of human extracellular superoxide dismutase gene to 4pter-q21. Genomics, 8, 736-8. Higgins, C.F. (1992). ABC transporters: from microorganisms to man. Annu Rev Cell Biol, 8, 67-113. 62 REFERENCES Hinoshita, E., Uchiumi, T., Taguchi, K., Kinukawa, N., Tsuneyoshi, M., Maehara, Y., Sugimachi, K. & Kuwano, M. (2000). Increased expression of an ATP-binding cassette superfamily transporter, multidrug resistance protein 2, in human colorectal carcinomas. Clin Cancer Res, 6, 2401-7. Hirvonen, A., Pelin, K., Tammilehto, L., Karjalainen, A., Mattson, K. & Linnainmaa, K. (1995). Inherited GSTM1 and NAT2 defects as concurrent risk modifiers in asbestos-related human malignant mesothelioma. Cancer Res, 55, 2981-3. Hirvonen, A., Saarikoski, S.T., Linnainmaa, K., Koskinen, K., Husgafvel-Pursiainen, K., Mattson, K. & Vainio, H. (1996). Glutathione S-transferase and N-acetyltransferase genotypes and asbestosassociated pulmonary disorders. JNatl Cancer Inst, 88, 1853-6. Ho, Y.S. & Crapo, J.D. (1988). Isolation and characterization of complementary DNAs encoding human manganese-containing superoxide dismutase. FEBS Lett, 229, 256-60. Ho, Y.S., Howard, A.J. & Crapo, J.D. (1988). Nucleotide sequence of a rat glutathione peroxidase cDNA. Nucleic Acids Res, 16, 5207. Holmgren, A. (2000). Antioxidant function of thioredoxin and glutaredoxin systems. Antioxid Redox Signal, 2, 811-20. Huang, C.I., Kohno, N., Ogawa, E., Adachi, M., Taki, T. & Miyake, M. (1998). Correlation of reduction in MRP-1/CD9 and KAI1/CD82 expression with recurrences in breast cancer patients. Am J Pathol, 153, 973-83. Huang, C.S., Chang, L.S., Anderson, M.E. & Meister, A. (1993). Catalytic and regulatory properties of the heavy subunit of rat kidney gamma-glutamylcysteine synthetase. J Biol Chem, 268, 19675-80. Huang, T.T., Carlson, E.J., Raineri, I., Gillespie, A.M., Kozy, H. & Epstein, C.J. (1999). The use of transgenic and mutant mice to study oxygen free radical metabolism. Ann N Y Acad Sci, 893, 95 112. Iida, T., Mori, E., Mori, K., Goto, S., Urata, Y., Oka, M., Kohno, S. & Kondo, T. (1999). Co-expression of gamma-glutamylcysteine synthetase sub-units in response to cisplatin and doxorubicin in human cancer cells. Int J Cancer, 82, 405-11. Ingbar, D.H., Hepler, K., Dowin, R., Jacobsen, E., Dunitz, J.M., Nici, L. & Jamieson, J.D. (1995). gammaGlutamyl transpeptidase is a polarized alveolar epithelial membrane protein. Am J Physiol, 269, L261-71. Ishikawa, T., Bao, J.J., Yamane, Y., Akimaru, K., Frindrich, K., Wright, C.D. & Kuo, M.T. (1996). Coordinated induction of MRP/GS-X pump and gamma-glutamylcysteine synthetase by heavy metals in human leukemia cells. JBiol Chem, 271, 14981-8. Isik, R., Metintas, M., Gibbs, A.R., Metintas, S., Jasani, B., Oner, U., Harmanci, E., Demircan, S. & Isiksoy, S. (2001). p53, p21 and metallothionein immunoreactivities in patients with malignant pleural mesothelioma: correlations with the epidemiological features and prognosis of mesotheliomas with environmental asbestos exposure. RespirMed, 95, 588-93. Janssen, A.M., Bosman, C.B., Sier, C.F., Griffioen, G., Kubben, F.J., Lamers, C.B., van Krieken, J.H., van de Velde, C.J. & Verspaget, H.W. (1998). Superoxide dismutases in relation to the overall survival of colorectal cancer patients. Br J Cancer, 78, 1051-7. Janssen, Y.M., Van Houten, B., Borm, P.J. & Mossman, B.T. (1993). Cell and tissue responses to oxidative damage. Lab Invest, 69, 261-74. Jaruga, P., Zastawny, T.H., Skokowski, J., Dizdaroglu, M. & Olinski, R. (1994). Oxidative DNA base damage and antioxidant enzyme activities in human lung cancer. FEBS Lett, 341, 59-64. Jaurand, M.C., Fleury, J., Monchaux, G., Nebut, M. & Bignon, J. (1987). Pleural carcinogenic potency of mineral fibers (asbestos, attapulgite) and their cytotoxicity on cultured cells. J Natl Cancer Inst, 79, 797-804. Kahlos, K., Paakko, P., Kurttila, E., Soini, Y. & Kinnula, V.L. (2000a). Manganese superoxide dismutase as a diagnostic marker for malignant pleural mesothelioma. Br J Cancer, 82, 1022-9. Kahlos, K., Soini, Y., Paakko, P., Saily, M., Linnainmaa, K. & Kinnula, V.L. (2000b). Proliferation, apoptosis, and manganese superoxide dismutase in malignant mesothelioma. Int J Cancer, 88, 37 43. 63 REFERENCES Kahlos, K., Soini, Y., Saily, M., Koistinen, P., Kakko, S., Paakko, P., Holmgren, A. & Kinnula, V.L. (2001a). Up-regulation of thioredoxin and thioredoxin reductase in human malignant pleural mesothelioma. Int J Cancer, 95, 198-204. Kahlos, K., Soini, Y., Sormunen, R., Kaarteenaho-Wiik, R., Paakko, P., Linnainmaa, K. & Kinnula, V.L. (2001b). Expression and prognostic significance of catalase in malignant mesothelioma. Cancer, 91, 1349-57. Kamp, D.W., Graceffa, P., Pryor, W.A. & Weitzman, S.A. (1992). The role of free radicals in asbestosinduced diseases. Free Radic Biol Med, 12, 293-315. Karjalainen, A., Meurman, L.O. & Pukkala, E. (1994). Four cases of mesothelioma among Finnish anthophyllite miners. Occup Environ Med, 51, 212-5. Karjalainen, A., Pukkala, E., Mattson, K., Tammilehto, L. & Vainio, H. (1997). Trends in mesothelioma incidence and occupational mesotheliomas in Finland in 1960-1995. Scand J Work Environ Health, 23, 266-70. Kartner, N., Riordan, J.R. & Ling, V. (1983). Cell surface P-glycoprotein associated with multidrug resistance in mammalian cell lines. Science, 221, 1285-8. Kasahara, K., Fujiwara, Y., Nishio, K., Ohmori, T., Sugimoto, Y., Komiya, K., Matsuda, T. & Saijo, N. (1991). Metallothionein content correlates with the sensitivity of human small cell lung cancer cell lines to cisplatin. Cancer Res, 51, 3237-42. Ke, Y., Reddel, R.R., Gerwin, B.I., Reddel, H.K., Somers, A.N., McMenamin, M.G., LaVeck, M.A., Stahel, R.A., Lechner, J.F. & Harris, C.C. (1989). Establishment of a human in vitro mesothelial cell model system for investigating mechanisms of asbestos-induced mesothelioma. Am J Pathol, 134, 979-91. Keppler, D. (1999). Export pumps for glutathione S-conjugates. Free Radic Biol Med, 27, 985-91. King, J.E. & Hasleton, P.S. (2001). Immunohistochemistry and the diagnosis of malignant mesothelioma. Histopathology, 38, 471-6. Kinnula, V.L., Chang, L., Everitt, J.I. & Crapo, J.D. (1992). Oxidants and antioxidants in alveolar epithelial type II cells: in situ, freshly isolated, and cultured cells. Am J Physiol, 262, L69-77. Kinnula, V.L., Crapo, J.D. & Raivio, K.O. (1995). Generation and disposal of reactive oxygen metabolites in the lung. Lab Invest, 73, 3-19. Kinnula, V.L., Everitt, J.I., Whorton, A.R. & Crapo, J.D. (1991). Hydrogen peroxide production by alveolar type II cells, alveolar macrophages, and endothelial cells. Am J Physiol, 261, L84-91. Kinnula, V.L., Pietarinen-Runtti, P., Raivio, K., Kahlos, K., Pelin, K., Mattson, K. & Linnainmaa, K. (1996). Manganese superoxide dismutase in human pleural mesothelioma cell lines. Free Radic Biol Med, 21, 527-32. Kinnula, V.L., Yankaskas, J.R., Chang, L., Virtanen, I., Linnala, A., Kang, B.H. & Crapo, J.D. (1994). Primary and immortalized (BEAS 2B) human bronchial epithelial cells have significant antioxidative capacity in vitro. Am JRespir Cell Mol Biol, 11, 568-76. Klockars, M. & Savolainen, H. (1992). Tumour necrosis factor enhances the asbestos-induced production of reactive oxygen metabolites by human polymorphonuclear leucocytes (PMN). Clin Exp Immunol, 90, 68-71. Konig, J., Nies, A.T., Cui, Y., Leier, I. & Keppler, D. (1999). Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance. Biochim Biophys Acta, 1461, 377-94. Kool, M., de Haas, M., Scheffer, G.L., Scheper, R.J., van Eijk, M.J., Juijn, J.A., Baas, F. & Borst, P. (1997). Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines. Cancer Res, 57, 3537-47. Koomagi, R., Mattern, J. & Volm, M. (1995). Up-regulation of resistance-related proteins in human lung tumors with poor vascularization. Carcinogenesis, 16, 2129-33. Kumar-Singh, S., Weyler, J., Martin, M.J., Vermeulen, P.B. & Van Marck, E. (1999). Angiogenic cytokines in mesothelioma: a study of VEGF, FGF-1 and -2, and TGF beta expression. J Pathol, 189, 72-8. Kuo, M.T., Bao, J., Furuichi, M., Yamane, Y., Gomi, A., Savaraj, N., Masuzawa, T. & Ishikawa, T. (1998). Frequent coexpression of MRP/GS-X pump and gamma-glutamylcysteine synthetase mRNA in 64 REFERENCES drug-resistant cells, untreated tumor cells, and normal mouse tissues. Biochem Pharmacol, 55, 605 15. Kuo, M.T., Bao, J.J., Curley, S.A., Ikeguchi, M., Johnston, D.A. & Ishikawa, T. (1996). Frequent coordinated overexpression of the MRP/GS-X pump and gamma- glutamylcysteine synthetase genes in human colorectal cancers. Cancer Res, 56, 3642-4. Kussmann, M., Wiehr, D., Knepper, T. & Przybylski, M. (1993). Synthesis, structural and biochemical characterization of cytostatic methotrexate-gamma-glutamyl-glutathione conjugates. Adv Exp Med Biol, 338, 453-6. Lai, S.L., Goldstein, L.J., Gottesman, M.M., Pastan, I., Tsai, C.M., Johnson, B.E., Mulshine, J.L., Ihde, D.C., Kayser, K. & Gazdar, A.F. (1989). MDR1 gene expression in lung cancer. J Natl Cancer Inst, 81, 1144-50. Lakari, E., Paakko, P. & Kinnula, V.L. (1998). Manganese superoxide dismutase, but not CuZn superoxide dismutase, is highly expressed in the granulomas of pulmonary sarcoidosis and extrinsic allergic alveolitis. Am JRespir Crit Care Med, 158, 589-96. Li, S., Yan, T., Yang, J.Q., Oberley, T.D. & Oberley, L.W. (2000). The role of cellular glutathione peroxidase redox regulation in the suppression of tumor cell growth by manganese superoxide dismutase. Cancer Res, 60, 3927-39. Li, Y., Huang, T.T., Carlson, E.J., Melov, S., Ursell, P.C., Olson, J.L., Noble, L.J., Yoshimura, M.P., Berger, C., Chan, P.H. & et al. (1995). Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. Nat Genet, 11, 376-81. Licht, T., Fiebig, H.H., Bross, K.J., Herrmann, F., Berger, D.P., Shoemaker, R. & Mertelsmann, R. (1991). Induction of multiple-drug resistance during anti-neoplastic chemotherapy in vitro. Int J Cancer, 49, 630-7. Licht, T., Lubbert, M., Martens, C., Bross, K.J., Fiebig, H.H., Mertelsmann, R. & Herrmann, F. (1995). Modulation of vindesine and doxorubicin resistance in multidrug- resistant pleural mesothelioma cells by tumor necrosis factor-alpha. Cytokines Mol Ther, 1, 123-32. Lieber, M., Smith, B., Szakal, A., Nelson-Rees, W. & Todaro, G. (1976). A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int J Cancer, 17, 62-70. Ling, V. (1992). Charles F. Kettering Prize. P-glycoprotein and resistance to anticancer drugs. Cancer, 69, 2603-9. Liochev, S.I. & Fridovich, I. (1997). How does superoxide dismutase protect against tumor necrosis factor: a hypothesis informed by effect of superoxide on "free" iron. Free Radic Biol Med, 23, 668-71. Liu, R.M., Shi, M.M., Giulivi, C. & Forman, H.J. (1998). Quinones increase gamma-glutamyl transpeptidase expression by multiple mechanisms in rat lung epithelial cells. Am J Physiol, 274, L330-6. Lu, S.C. (1999). Regulation of hepatic glutathione synthesis: current concepts and controversies. Faseb J, 13, 1169-83. Lu, S.C., Bao, Y., Huang, Z.Z., Sarthy, V.P. & Kannan, R. (1999). Regulation of gamma-glutamylcysteine synthetase subunit gene expression in retinal Muller cells by oxidative stress. Invest Ophthalmol Vis Sci, 40, 1776-82. Manna, S.K., Zhang, H.J., Yan, T., Oberley, L.W. & Aggarwal, B.B. (1998). Overexpression of manganese superoxide dismutase suppresses tumor necrosis factor-induced apoptosis and activation of nuclear transcription factor-kappaB and activated protein-1. JBiol Chem, 273, 13245-54. Mattson, K. (2000). Thorax-alueen kasvaimet. In Keuhkosairaudet, Kinnula, V., Laitinen, L. & Tukiainen, P. (eds) pp. 514-518. Duodecim: Helsinki. Mattson, K., Kyosola, K., Ojala, A. & Knuuttila, A. (1999). Mesoteliooma. In Syopataudit, Joensuu, H. (ed) pp. 262-7. Duodecim: Helsinki. McCord, J.M. & Fridovich, I. (1969). Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem, 244, 6049-55. McLaren, B.R., Whitaker, D., Robinson, B.W. & Lake, R.A. (2001). Expression and integrity of DNA topoisomerase II isoforms does not explain generic drug resistance in malignant mesothelioma. Cancer Chemother Pharmacol, 48, 1-8. Meister, A. (1983). Selective modification of glutathione metabolism. Science, 220, 472-7. 65 REFERENCES Morales, A., Garcia-Ruiz, C., Miranda, M., Mari, M., Colell, A., Ardite, E. & Fernandez-Checa, J.C. (1997). Tumor necrosis factor increases hepatocellular glutathione by transcriptional regulation of the heavy subunit chain of gamma- glutamylcysteine synthetase. JBiol Chem, 272, 30371-9. Moscow, J.A., Schmidt, L., Ingram, D.T., Gnarra, J., Johnson, B. & Cowan, K.H. (1994). Loss of heterozygosity of the human cytosolic glutathione peroxidase I gene in lung cancer. Carcinogenesis, 15, 2769-73. Mossman, B.T., Bignon, J., Corn, M., Seaton, A. & Gee, J.B. (1990). Asbestos: scientific developments and implications for public policy. Science, 247, 294-301. Mossman, B.T. & Gee, J.B. (1989). Asbestos-related diseases. NEngl JMed, 320, 1721-30. Mossman, B.T., Hansen, K., Marsh, J.P., Brew, M.E., Hill, S., Bergeron, M. & Petruska, J. (1989). Mechanisms of fibre-induced superoxide release from alveolar macrophages and induction of superoxide dismutase in the lungs of rats inhaling crocidolite. IARC SciPubl, 90, 81-92. Mossman, B.T., Kamp, D.W. & Weitzman, S.A. (1996). Mechanisms of carcinogenesis and clinical features of asbestos- associated cancers. Cancer Invest, 14, 466-80. Mulcahy, R.T., Bailey, H.H. & Gipp, J.J. (1995). Transfection of complementary DNAs for the heavy and light subunits of human gamma-glutamylcysteine synthetase results in an elevation of intracellular glutathione and resistance to melphalan. Cancer Res, 55, 4771-5. Narasaki, F., Oka, M., Nakano, R., Ikeda, K., Fukuda, M., Nakamura, T., Soda, H., Nakagawa, M., Kuwano, M. & Kohno, S. (1997). Human canalicular multispecific organic anion transporter (cMOAT) is expressed in human lung, gastric, and colorectal cancer cells. Biochem Biophys Res Commun, 240, 606-11. Nishida, T., Sugiyama, T., Kataoka, A., Tashiro, M., Yakushiji, M. & Ishikawa, M. (1993). Serum manganese superoxide dismutase (MnSOD) and histological virulence of ovarian cancer. Asia Oceania J Obstet Gynaecol, 19, 427-31. Nooter, K., de la Riviere, G.B., Klijn, J., Stoter, G. & Foekens, J. (1997). Multidrug resistance protein in recurrent breast cancer. Lancet, 349, 1885-6. Nooter, K., Kok, T., Bosman, F.T., van Wingerden, K.E. & Stoter, G. (1998). Expression of the multidrug resistance protein (MRP) in squamous cell carcinoma of the oesophagus and response to pre operative chemotherapy. Eur J Cancer, 34, 81-6. Norris, M.D., Bordow, S.B., Marshall, G.M., Haber, P.S., Cohn, S.L. & Haber, M. (1996). Expression of the gene for multidrug-resistance-associated protein and outcome in patients with neuroblastoma. N Engl JMed, 334, 231-8. Nunez, G., Benedict, M.A., Hu, Y. & Inohara, N. (1998). Caspases: the proteases of the apoptotic pathway. Oncogene, 17, 3237-45. Nyberg, P. & Klockars, M. (1990). Measurement of reactive oxygen metabolites produced by human monocyte- derived macrophages exposed to mineral dusts. Int J Exp Pathol, 71, 537-44. Oberley, L.W. & Oberley, T.D. (1997). Role ofantioxidant enzymes in the cancer phenotype. Oxygen, Gene Expression and Cellular Function (Lung Biology in Health and Disease). Marcel Dekker Inc: New York. Oberley, L.W., St Clair, D.K., Autor, A.P. & Oberley, T.D. (1987). Increase in manganese superoxide dismutase activity in the mouse heart after X-irradiation. Arch Biochem Biophys, 254, 69-80. Oberley, T.D., Sempf, J.M., Oberley, M.J., McCormick, M.L., Muse, K.E. & Oberley, L.W. (1994). Immunogold analysis of antioxidant enzymes in human renal cell carcinoma. Virchows Arch, 424, 155-64. Obrador, E., Navarro, J., Mompo, J., Asensi, M., Pellicer, J.A. & Estrela, J.M. (1997). Glutathione and the rate of cellular proliferation determine tumour cell sensitivity to tumour necrosis factor in vivo. Biochem J, 325, 183-9. O'Brien, M.L. & Tew, K.D. (1996). Glutathione and related enzymes in multidrug resistance. Eur J Cancer, 32A, 967-78. Ogretmen, B., Bahadori, H.R., McCauley, M.D., Boylan, A., Green, M.R. & Safa, A.R. (1998). Co ordinated over-expression of the MRP and gamma-glutamylcysteine synthetase genes, but not MDR1, correlates with doxorubicin resistance in human malignant mesothelioma cell lines. Int J Cancer, 75, 757-61. 66 REFERENCES Oguri, T., Fujiwara, Y., Isobe, T., Katoh, O., Watanabe, H. & Yamakido, M. (1998a). Expression of gammaglutamylcysteine synthetase (gamma-GCS) and multidrug resistance-associated protein (MRP), but not human canalicular multispecific organic anion transporter (cMOAT), genes correlates with exposure of human lung cancers to platinum drugs. Br J Cancer, 77, 1089-96. Oguri, T., Fujiwara, Y., Ochiai, M., Fujitaka, K., Miyazaki, M., Takahashi, T., Yokozaki, M., Isobe, T., Ohune, T., Tsuya, T., Katoh, O. & Yamakido, M. (1998b). Expression of lung-resistance protein gene is not associated with platinum drug exposure in lung cancer. Anticancer Res, 18, 4159-62. Ohshima, H. & Bartsch, H. (1994). Chronic infections and inflammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis. MutatRes, 305, 253-64. Oury, T.D., Chang, L.Y., Marklund, S.L., Day, B.J. & Crapo, J.D. (1994). Immunocytochemical localization of extracellular superoxide dismutase in human lung. Lab Invest, 70, 889-98. Pastan, I. & Gottesman, M. (1987). Multiple-drug resistance in human cancer. NEngl J Med, 316, 1388-93. Paulusma, C.C., Bosma, P.J., Zaman, G.J., Bakker, C.T., Otter, M., Scheffer, G.L., Scheper, R.J., Borst, P. & Oude Elferink, R.P. (1996). Congenital jaundice in rats with a mutation in a multidrug resistanceassociated protein gene. Science, 271, 1126-8. Pelin-Enlund, K., Husgafvel-Pursiainen, K., Tammilehto, L., Klockars, M., Jantunen, K., Gerwin, B.I., Harris, C.C., Tuomi, T., Vanhala, E., Mattson, K. & et al. (1990). Asbestos-related malignant mesothelioma: growth, cytology, tumorigenicity and consistent chromosome findings in cell lines from five patients. Carcinogenesis, 11, 673-81. Pietarinen-Runtti, P., Raivio, K.O., Saksela, M., Asikainen, T.M. & Kinnula, V.L. (1998). Antioxidant enzyme regulation and resistance to oxidants of human bronchial epithelial cells cultured under hyperoxic conditions. Am JRespir Cell Mol Biol, 19, 286-92. Powis, G., Mustacich, D. & Coon, A. (2000). The role of the redox protein thioredoxin in cell growth and cancer. Free Radic Biol Med, 29, 312-22. Quan, F., Korneluk, R.G., Tropak, M.B. & Gravel, R.A. (1986). Isolation and characterization of the human catalase gene. Nucleic Acids Res, 14, 5321-35. Rahman, I., Antonicelli, F. & MacNee, W. (1999). Molecular mechanism of the regulation of glutathione synthesis by tumor necrosis factor-alpha and dexamethasone in human alveolar epithelial cells. J Biol Chem, 274, 5088-96. Rahman, I. & MacNee, W. (2000). Oxidative stress and regulation of glutathione in lung inflammation [In Process Citation]. Eur Respir J, 16, 534-54. Rahman, I., Mulier, B., Gilmour, P.S., Watchorn, T., Donaldson, K., Jeffery, P.K. & MacNee, W. (2001). Oxidant-mediated lung epithelial cell tolerance: the role of intracellular glutathione and nuclear factor-kappaB. Biochem Pharmacol, 62, 787-94. Ramael, M., van den Bossche, J., Buysse, C., van Meerbeeck, J., Segers, K., Vermeire, P. & van Marck, E. (1992). Immunoreactivity for P-170 glycoprotein in malignant mesothelioma and in non-neoplastic mesothelium of the pleura using the murine monoclonal antibody JSB-1. J Pathol, 167, 5-8. Ratnasinghe, D., Tangrea, J.A., Andersen, M.R., Barrett, M.J., Virtamo, J., Taylor, P.R. & Albanes, D. (2000). Glutathione peroxidase codon 198 polymorphism variant increases lung cancer risk. Cancer Res, 60, 6381-3. Rhee, S.G., Kang, S.W., Netto, L.E., Seo, M.S. & Stadtman, E.R. (1999). A family of novel peroxidases, peroxiredoxins. Biofactors, 10, 207-9. Ria, F., Landriscina, M., Remiddi, F., Rosselli, R., Iacoangeli, M., Scerrati, M., Pani, G., Borrello, S. & Galeotti, T. (2001). The level of manganese superoxide dismutase content is an independent prognostic factor for glioblastoma. Biological mechanisms and clinical implications. Br J Cancer, 84, 529-34. Richman, P.G. & Meister, A. (1975). Regulation of gamma-glutamyl-cysteine synthetase by nonallosteric feedback inhibition by glutathione. JBiol Chem, 250, 1422-6. Rihn, B.H., Mohr, S., McDowell, S.A., Binet, S., Loubinoux, J., Galateau, F., Keith, G. & Leikauf, G.D. (2000). Differential gene expression in mesothelioma. FEBSLett, 480, 95-100. Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hentati, A., Donaldson, D., Goto, J., O'Regan, J.P., Deng, H.X. & et al. (1993). Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature, 362, 59-62. 67 REFERENCES Rosenblum, J.S., Gilula, N.B. & Lerner, R.A. (1996). On signal sequence polymorphisms and diseases of distribution. Proc Natl Acad Sci U S A, 93, 4471-3. Rozet, J.M., Gerber, S., Perrault, I., Calvas, P., Souied, E., Chatelin, S., Viegas, P., Molina-Gomez, D., Munnich, A. & Kaplan, J. (1998). Structure and refinement of the physical mapping of the gammaglutamylcysteine ligase regulatory subunit (GLCLR) gene to chromosome 1p22.1 within the critically deleted region of human malignant mesothelioma. Cytogenet Cell Genet, 82, 91-4. Rudd, R.M. (1995). Asbestos-related diseases. In Respiratory medicine, Brewis, R.A.L. (ed) pp. 557-569. WB Saunders: London. Saaristo, A., Karpanen, T. & Alitalo, K. (2000). Mechanisms of angiogenesis and their use in the inhibition of tumor growth and metastasis. Oncogene, 19, 6122-9. Scagliotti, G.V., Novello, S. & Selvaggi, G. (1999). Multidrug resistance in non-small-cell lung cancer. Ann Oncol, 10, S83-6. Scheffer, G.L., Wijngaard, P.L., Flens, M.J., Izquierdo, M.A., Slovak, M.L., Pinedo, H.M., Meijer, C.J., Clevers, H.C. & Scheper, R.J. (1995). The drug resistance-related protein LRP is the human major vault protein. Nat Med, 1, 578-82. Schuckelt, R., Brigelius-Flohe, R., Maiorino, M., Roveri, A., Reumkens, J., Strassburger, W., Ursini, F., Wolf, B. & Flohe, L. (1991). Phospholipid hydroperoxide glutathione peroxidase is a selenoenzyme distinct from the classical glutathione peroxidase as evident from cDNA and amino acid sequencing. Free Radic Res Commun, 14, 343-61. Seelig, G.F., Simondsen, R.P. & Meister, A. (1984). Reversible dissociation of gamma-glutamylcysteine synthetase into two subunits. J Biol Chem, 259, 9345-7. Sherman, L., Levanon, D., Lieman-Hurwitz, J., Dafni, N. & Groner, Y. (1984). Human Cu/Zn superoxide dismutase gene: molecular characterization of its two mRNA species. Nucleic Acids Res, 12, 9349 65. Shi, M.M., Kugelman, A., Iwamoto, T., Tian, L. & Forman, H.J. (1994). Quinone-induced oxidative stress elevates glutathione and induces gamma- glutamylcysteine synthetase activity in rat lung epithelial L2 cells. J Biol Chem, 269, 26512-7. Shichi, H. & Demar, J.C. (1990). Non-selenium glutathione peroxidase without glutathione S-transferase activity from bovine ciliary body. Exp Eye Res, 50, 513-20. Silcocks, P.B. (1983). Measuring repeatability and validity of histological diagnosis--a brief review with some practical examples. J Clin Pathol, 36, 1269-75. Sinha, B.K. & Mimnaugh, E.G. (1990). Free radicals and anticancer drug resistance: oxygen free radicals in the mechanisms of drug cytotoxicity and resistance by certain tumors. Free Radic Biol Med, 8, 567 81. Sinicrope, F.A., Dudeja, P.K., Bissonnette, B.M., Safa, A.R. & Brasitus, T.A. (1992). Modulation of Pglycoprotein-mediated drug transport by alterations in lipid fluidity of rat liver canalicular membrane vesicles. J Biol Chem, 267, 24995-5002. Soini, Y., Kahlos, K., Napankangas, U., Kaarteenaho-Wiik, R., Saily, M., Koistinen, P., Paaakko, P., Holmgren, A. & Kinnula, V.L. (2001a). Widespread expression of thioredoxin and thioredoxin reductase in non- small cell lung carcinoma. Clin Cancer Res, 7, 1750-7. Soini, Y., Kinnula, V., Kaarteenaho-Wiik, R., Kurttila, E., Linnainmaa, K. & Paakko, P. (1999). Apoptosis and expression of apoptosis regulating proteins bcl-2, mcl-1, bcl-X, and bax in malignant mesothelioma. Clin Cancer Res, 5, 3508-15. Soini, Y., Napankangas, U., Jarvinen, K., Kaarteenaho-Wiik, R., Paakko, P. & Kinnula, V.L. (2001b). Expression of g-glutamylcysteine synthetase in non-small cell lung carcinoma. Cancer, In press. Soini, Y., Virkajarvi, N., Raunio, H. & Paakko, P. (1996). Expression of P-glycoprotein in hepatocellular carcinoma: a potential marker of prognosis. J Clin Pathol, 49, 470-3. Stralin, P. & Marklund, S.L. (2000). Multiple cytokines regulate the expression of extracellular superoxide dismutase in human vascular smooth muscle cells. Atherosclerosis, 151, 433-41. Sugawara, I., Yamada, H., Nakamura, H., Sumizawa, T., Akiyama, S., Masunaga, A. & Itoyama, S. (1995). Preferential expression of the multidrug-resistance-associated protein (MRP) in adenocarcinoma of the lung. Int J Cancer, 64, 322-5. 68 REFERENCES Susin, S.A., Zamzami, N. & Kroemer, G. (1998). Mitochondria as regulators of apoptosis: doubt no more. Biochim Biophys Acta, 1366, 151-65. Taniguchi, K., Wada, M., Kohno, K., Nakamura, T., Kawabe, T., Kawakami, M., Kagotani, K., Okumura, K., Akiyama, S. & Kuwano, M. (1996). A human canalicular multispecific organic anion transporter (cMOAT) gene is overexpressed in cisplatin-resistant human cancer cell lines with decreased drug accumulation. Cancer Res, 56, 4124-9. Teicher, B.A. (1994). Hypoxia and drug resistance. Cancer Metastasis Rev, 13, 139-68. Tew, K.D. (1994). Glutathione-associated enzymes in anticancer drug resistance. Cancer Res, 54, 4313-20. Thannickal, V.J. & Fanburg, B.L. (2000). Reactive oxygen species in cell signaling. Am JPhysiol Lung Cell Mol Physiol, 279, L1005-28. Thomas, G.A., Barrand, M.A., Stewart, S., Rabbitts, P.H., Williams, E.D. & Twentyman, P.R. (1994). Expression of the multidrug resistance-associated protein (MRP) gene in human lung tumours and normal tissue as determined by in situ hybridisation. Eur J Cancer, 11, 1705-9. Thompson, C.B. (1995). Apoptosis in the pathogenesis and treatment of disease. Science, 267, 1456-62. Tipnis, S.R., Blake, D.G., Shepherd, A.G. & McLellan, L.I. (1999). Overexpression of the regulatory subunit of gamma-glutamylcysteine synthetase in HeLa cells increases gamma-glutamylcysteine synthetase activity and confers drug resistance. Biochem J, 337, 559-66. Tome, M.E., Baker, A.F., Powis, G., Payne, C.M. & Briehl, M.M. (2001). Catalase-overexpressing thymocytes are resistant to glucocorticoid- induced apoptosis and exhibit increased net tumor growth. Cancer Res, 61, 2766-73. Travis, W.D., Colby, T.V., Corrin, B., Shimosato, Y. & Brambilla, E. (1999). Histological Typing of Lung and Pleural Tumors. World Health Organization. International Histological Classification of Tumours. Springer: Berlin. Trock, B.J., Leonessa, F. & Clarke, R. (1997). Multidrug resistance in breast cancer: a meta-analysis of MDR1/gp170 expression and its possible functional significance. J Natl Cancer Inst, 89, 917-31. Tsan, M.F. (2001). Superoxide dismutase and pulmonary oxygen toxicity: lessons from transgenic and knockout mice (Review). Int J Mol Med, 7, 13-9. Tsan, M.F., White, J.E., Santana, T.A. & Lee, C.Y. (1990). Tracheal insufflation of tumor necrosis factor protects rats against oxygen toxicity. JApplPhysiol, 68, 1211-9. Vainio, H. & Husgafvel-Pursiainen, K. (1996). Mechanisms of carcinogenesis and molecular epidemiology. Br J Hosp Med, 56, 162-70. van Brussel, J.P. & Mickisch, G.H. (1998). Circumvention of multidrug resistance in genitourinary tumors. Int J Urol, 5, 1-15. Vanden Berghe, W., Vermeulen, L., De Wilde, G., De Bosscher, K., Boone, E. & Haegeman, G. (2000). Signal transduction by tumor necrosis factor and gene regulation of the inflammatory cytokine interleukin-6. Biochem Pharmacol, 60, 1185-95. Verrelle, P., Meissonnier, F., Fonck, Y., Feillel, V., Dionet, C., Kwiatkowski, F., Plagne, R. & Chassagne, J. (1991). Clinical relevance of immunohistochemical detection of multidrug resistance P-glycoprotein in breast carcinoma. J Natl Cancer Inst, 83, 111-6. Visner, G.A., Dougall, W.C., Wilson, J.M., Burr, I.A. & Nick, H.S. (1990). Regulation of manganese superoxide dismutase by lipopolysaccharide, interleukin-1, and tumor necrosis factor. Role in the acute inflammatory response. J Biol Chem, 265, 2856-64. Volm, M. (1998). Multidrug resistance and its reversal. Anticancer Res, 18, 2905-17. Volm, M., Wayss, K., Kaufmann, M. & Mattern, J. (1979). Pretherapeutic detection of tumour resistance and the results of tumour chemotherapy. Eur J Cancer, 15, 983-93. Wagner, J.C., Slegss, C.A. & Marchand, P. (1960). Diffuce pleural mesothelioma and asbestos exposure in the North Westerne Cape Province. Br J Ind Med, 17, 260-271. Wan, X.S., Devalaraja, M.N. & St Clair, D.K. (1994). Molecular structure and organization of the human manganese superoxide dismutase gene. DNA Cell Biol, 13, 1127-36. Wang, L.I., Miller, D.P., Su, L., Wain, J.C., Luynch, T.J. & Christiani, D.C. (2001). Manganese superoxide dismutase (MnSOD) Ala9Val polymorphism and lung cancer risk. In American Association for Cancer Research; 92nd Annual Meeting. New Orleans, U.S.A. 69 REFERENCES Warner, B.B., Stuart, L., Gebb, S. & Wispe, J.R. (1996). Redox regulation of manganese superoxide dismutase. Am JPhysiol, 271, L150-8. Webb, G.C., Vaska, V.L., Gali, R.R., Ford, J.H. & Board, P.G. (1995). The gene encoding human glutathione synthetase (GSS) maps to the long arm of chromosome 20 at band 11.2. Genomics, 30, 617-9. Weisiger, R.A. & Fridovich, I. (1973). Mitochondrial superoxide simutase. Site of synthesis and intramitochondrial localization. J Biol Chem, 248, 4793-6. Wijnholds, J., Evers, R., van Leusden, M.R., Mol, C.A., Zaman, G.J., Mayer, U., Beijnen, J.H., van der Valk, M., Krimpenfort, P. & Borst, P. (1997). Increased sensitivity to anticancer drugs and decreased inflammatory response in mice lacking the multidrug resistance-associated protein. Nat Med, 3, 1275-9. Williams, M.D., Van Remmen, H., Conrad, C.C., Huang, T.T., Epstein, C.J. & Richardson, A. (1998). Increased oxidative damage is correlated to altered mitochondrial function in heterozygous manganese superoxide dismutase knockout mice. J Biol Chem, 273, 28510-5. Wispe, J.R., Warner, B.B., Clark, J.C., Dey, C.R., Neuman, J., Glasser, S.W., Crapo, J.D., Chang, L.Y. & Whitsett, J.A. (1992). Human Mn-superoxide dismutase in pulmonary epithelial cells of transgenic mice confers protection from oxygen injury. JBiol Chem, 267, 23937-41. Wong, G.H. & Goeddel, D.V. (1988). Induction of manganous superoxide dismutase by tumor necrosis factor: possible protective mechanism. Science, 242, 941-4. Wright, S.R., Boag, A.H., Valdimarsson, G., Hipfner, D.R., Campling, B.G., Cole, S.P. & Deeley, R.G. (1998). Immunohistochemical detection of multidrug resistance protein in human lung cancer and normal lung. Clin Cancer Res, 4, 2279-89. Xu, H.J., Hu, S.X., Cagle, P.T., Moore, G.E. & Benedict, W.F. (1991). Absence of retinoblastoma protein expression in primary non-small cell lung carcinomas. Cancer Res, 51, 2735-9. Yamane, Y., Furuichi, M., Song, R., Van, N.T., Mulcahy, R.T., Ishikawa, T. & Kuo, M.T. (1998). Expression of multidrug resistance protein/GS-X pump and gamma- glutamylcysteine synthetase genes is regulated by oxidative stress. J Biol Chem, 273, 31075-85. Young, L.C., Campling, B.G., Cole, S.P., Deeley, R.G. & Gerlach, J.H. (2001). Multidrug resistance proteins MRP3, MRP1, and MRP2 in lung cancer: correlation of protein levels with drug response and messenger RNA levels. Clin Cancer Res, 7, 1798-804. Young, L.C., Campling, B.G., Voskoglou-Nomikos, T., Cole, S.P., Deeley, R.G. & Gerlach, J.H. (1999). Expression of multidrug resistance protein-related genes in lung cancer: correlation with drug response. Clin Cancer Res, 5, 673-80. Zhang, K., Mack, P. & Wong, K.P. (1998). Glutathione-related mechanisms in cellular resistance to anticancer drugs. Int J Oncol, 12, 871-82. 70