Apoptosis plays an important role in cancer biology. We investigated the expression of caspases 3 and 8 in malignant mesothelioma and malignant mesothelioma cell lines and putative changes in their ultrastructural expression prior and after exposure to epirubicin. Further studies were conducted to compare these changes to the localization and expression of the bcl-2 group of proteins bcl-X, bax and mcl-1, and Fas-Fas ligand in the same cells. In the histological samples, caspase 3 and 8 immunoreactivity was seen in 27/37 (73%) and 16/37 (43%) of the mesotheliomas. The immunostaining was cytoplasmic diffuse, granular, and occasionally nuclear. All six mesothelioma cell lines expressed caspases 3 and 8 by immunoblotting. After exposure to epirubicin the extent of apoptosis was increased in all cell lines investigated, being weakest in the most resistant M38K cell line. Immunoelectron microscopy revealed immunogold labeling for caspases 3 and 8 in the mitochondria with the accumulation of caspase 3 in the apoptotic bodies, while the mitochondrial localization of the bcl-2 proteins appeared to be very stable. Fas receptor could be detected by flow cytometry, whereas the most resistant cell line (M38K) lacked Fas ligand when assessed by RT-PCR. These results suggest the importance of caspase 3 during the apoptotic process of mesothelioma cells and indicate that epirubicin-induced apoptosis is independent of the mitochondrial pathway.
The aim of the study was to estimate the significance of oxidative/nitrosative damage and expression of antioxidant enzymes in renal cell carcinomas (RCC). For this we investigated immunohistochemically six antioxidant enzymes (AOEs) including MnSOD, ECSOD, thioredoxin, thioredoxin reductase, and gammaglutamyl cysteine synthetase heavy and light chain in 138 RCCs. As an indicator of oxidative/nitrosative damage, sections were stained with an antibody to nitrotyrosine. The extent of apoptosis was evaluated by TUNEL method and proliferation by immunohistochemistry to Ki67. Variable expression of all AOEs could be seen in RCC with expression of MnSOD being strongest. Nitrotyrosine was significantly associated with high grade tumors. MnSOD was associated with tumors of a lower stage. Cases showing ECSOD reactivity had higher and cases expressing thioredoxin lower apoptotic index than other tumors. No association with patient prognosis was observed. According to the results renal cell carcinomas show oxidative/nitrosative damage which, according to nitrotyrosine staining, was higher in high grade tumors. Of AOEs, MnSOD was more abundantly expressed in low stage tumors suggesting that its antioxidant function could play a main role to prevent development of oxidative damage leading to more aggressive tumors.
We investigated apoptosis and the expression of bcl-2, mcl-1, bcl-X, and bax in histological sections from 35 malignant mesotheliomas and 21 metastatic adenocarcinomas. Moreover, the expression of bcl-2, mcl-1, bcl-X, and bax were assessed by Western blotting in nonmalignant human mesothelial cells (Met5A) and seven malignant cell lines. The apoptotic index in mesotheliomas was 1.07+/-1.14%. Patients with mesotheliomas showing a high apoptotic index (> or =0.75%) had a worse prognosis (P = 0.008). bcl-2 positivity was observed in only seven cases, but bcl-X, mcl-1, and bax positivity was seen in all of them. In immunoblotting experiments, all mesothelioma cell lines were negative for bcl-2 but positive for bcl-X, mcl-1, and bax. The apoptotic index in bcl-2-negative mesotheliomas was 1.25+/-1.24% and in bcl-2-positive ones, 0.47+/-0.42% (P = 0.014). The apoptotic index did not significantly associate with bcl-X, mcl-1, or bax expression (P = 0.19, P = 0.25, and P = 0.46, respectively). No significant difference was observed in apoptosis or expression of bcl-2, bcl-X, or bax between malignant mesotheliomas and metastatic adenocarcinomas. The former, however, showed more often weak mcl-1 immunoreactivity (P = 0.01). The results show that the extent of apoptosis may influence patient prognosis. bcl-2 is inversely associated with the apoptotic index but is relatively infrequently expressed in malignant mesotheliomas. Widespread expression of bcl-X, mcl-1, and bax suggests that these proteins may also take part in apoptosis regulation in mesotheliomas.
BACKGROUND AND OBJECTIVE: All-trans retinoic acid (ATRA) induces growth arrest and apoptosis in acute myeloblastic leukemia (AML) cells. Since cellular redox state regulates these events, we were interested in studying whether it has any role in the responsiveness of AML cells to ATRA. DESIGN AND METHODS: Two human AML cell lines, the ATRA-sensitive OU-AML-3, and the ATRA-resistant OU-AML-7, were used as models. Clonogenic cell culture assay, annexin V method, and measurement of mitochondrial membrane potential were used for the determination of cell growth and apoptosis. Peroxide formation was analyzed by flow cytometry, glutathione and g-glutamylcysteine synthetase (g-GCS) activity was determined spectrophotometrically, and the expression of manganese superoxide dismutase (MnSOD) by Western blotting. RESULTS: ATRA inhibited clonogenic cell growth and induced apoptosis particularly in OU-AML-3 cells. The OU-AML-7 cells had a higher basal level of glutathione and g-GCS activity than the OU-AML-3 cells. ATRA enhanced the generation of peroxides after 24h exposure, which was more prominent in the sensitive than the resistant cell line and was not preventable by N-acetyl-L-cysteine. ATRA also increased the activity of g-GCS, which was associated with increased intracellular glutathione in the resistant cell line, while the glutathione level was maintained in the sensitive cell line. During ATRA exposure, MnSOD was induced in the sensitive cell line, but not until after 72 h. Buthionine sulfoximine significantly increased the inhibitory effect of ATRA on colony formation in both cell lines, but only marginally enhanced the effect of ATRA on the induction of apoptosis. INTERPRETATION AND CONCLUSIONS: The balance between oxidative and antioxidative actions of ATRA, as well as the basal redox state of the cells seem to have a definite influence on the responsiveness of AML cells to ATRA.
BACKGROUND: Chlamydia pneumoniae infection and immune response to the C. pneumoniae heat shock protein 60 (CpHsp60) have been suggested to be associated with asthma. OBJECTIVES: To study whether a slightly elevated C-reactive protein (CRP) level as a marker of low-grade systemic inflammation has a role in this association, we collected serum and sputum samples from 103 asthma patients with disease severity ranging from mild to moderate and from 30 healthy volunteers. METHODS: IgA and IgG antibodies to C. pneumoniae elementary bodies (CpEB) and CpHsp60 were measured by enzyme immunoassay. Serum CRP levels were measured with a rapid two-site ultra-sensitive assay based on time-resolved immunofluorometry. RESULTS: The asthma patients, especially those with moderate asthma, had higher serum IgA antibody levels to CpHsp60 than the healthy controls (test for trend, p = 0.05), whereas antibody levels to CpEB antigen did not differ between the study groups. CRP levels were higher in both asthma groups compared to the control group and moreover, the patients with moderate asthma had higher CRP levels than those with mild asthma (test for trend, p or =1.8 mg/l, had higher CpEB IgA (p = 0.001), CpEB IgG (p = 0.008) and CpHsp60 IgA (p = 0.023) antibody levels in serum compared to the subjects with lower CRP levels. CONCLUSIONS: Slightly elevated CRP levels as a marker of low-grade systemic inflammation may be associated with C. pneumoniae infection in asthma patients.
Tenascin-C is an extracellular matrix glycoprotein that is spatially expressed during organogenesis, in inflammatory and fibrotic disorders, and in neoplasms. The aim of this study was to analyze its expression in developing human lung tissues during pseudoglandular, canalicular, saccular, and alveolar periods corresponding to Weeks 12 to 40. Lung tissues were obtained at autopsy from 34 nonmalformed cases. An immunohistochemical analysis and a messenger RNA (mRNA) in situ hybridization method combined with light microscopy were used. The extent of tenascin-C immunoreactivity was scored as absent, low, moderate, or strong in and around different types of pulmonary cells. The immunohistochemical expression for tenascin-C was strong beneath the airway epithelium, especially at the sites of airway subdivision during Weeks 12 to 23, whereas its expression was moderate or weak underneath alveolar and bronchiolar epithelia between Weeks 24 and 40. The expression for tenascin-C was strong in the intima of veins, especially in the canalicular period, i.e., Weeks 17 to 28. A moderate or strong immunoreactivity for tenascin-C was also observed around chondrocytes in every case studied during all periods. The increased expression of tenascin-C mRNA was most often seen in the cells below the airway epithelium. Taken together, tenascin-C is expressed in human lung during all developmental periods, and its expression is especially strong below the airway epithelium at the sites of airway subdivision.
AIMS: To investigate endothelial nitric oxide synthase (eNOS) expression in malignant mesothelioma and its association with expression of vascular endothelial growth factor (VEGF), its receptors FLK1 and FLT1, and vascular density. METHODS AND RESULTS: eNOS, VEGF, FLK1 and FLT1 were studied in 36 histological mesothelioma samples by immunohistochemistry. Two mesothelioma (M14K, M38K) and one non-neoplastic mesothelial cell line (MET-5A) were studied for eNOS mRNA expression by reverse transcriptase-polymerase chain reaction (RT-PCR). Vascular density was determined by staining the samples with an antibody to factor VIII. RT-PCR showed that mesothelioma cells synthesize eNOS in vitro. eNOS immunoreactivity was found in 32/36 (89%) tumours. VEGF, FLK1 and FLT1 expression was found in 17 (45%), 24 (69%) and 25 (71%) cases, respectively. FLK1 or FLT1 immunoreactivity was more often seen in epithelioid and biphasic mesotheliomas than in sarcomatoid ones (P=0.007 and P=0.011, respectively). There was a significant association between FLK1 and FLT1 immunoreactivity (P=0.032). No significant association was found between FLK1, FLT1, VEGF and eNOS immunoreactivity and vascular density. CONCLUSIONS: eNOS is strongly expressed in malignant mesothelioma. Since eNOS did not associate with VEGF, FLK1 or FLT1, its synthesis seems not to be regulated through VEGF in malignant mesothelioma as has been shown in non-neoplastic endothelial cells.
We analyzed a set of 103 non-small cell lung carcinomas (NSCLCs) for caspase-3, -6 and -8 expression and apoptosis. Additionally, the expression of bcl-2, bax and p53 were studied. Caspase-3 positivity appeared as diffuse, cytoplasmic staining and was restricted to the tumor area. In contrast, the immunoreactivity for caspase-6 was intense, granular and mostly located in single cells or groups of tumor cells showing apoptotic morphology. The caspase-8 expression pattern was a combination of the two other caspases studied, featuring both diffuse and single-cell patterns restricted to the tumor area. No significant differences were seen in caspase -3, -6 and -8 expression between tumors of different histological types or grades. The number of apoptotic cells and bodies was significantly higher in NSCLCs, in which caspase-8 immunostaining was mainly seen in single cells (p = 0.017), whereas caspase -3 and -6 expression had no association with apoptosis. It is apparent that, in lung tissue, up-regulation of caspase expression is a phenomenon associated solely with neoplasia and reflects the readiness of the tumor cells to undergo apoptosis. Interestingly, caspases -3, -6 and -8 each have an individual staining pattern in NSCLC, perhaps reflecting their different position in the caspase hierarchy.
In this study we investigated the immunohistochemical expression of inducible nitric oxide synthase (iNOS) in a set of normal pleural mesothelial tissues, malignant mesotheliomas, mesothelioma cell lines and metastatic pleural adenocarcinomas. Furthermore, the expression of mRNA was assessed in four malignant mesothelioma cell lines in culture. Apoptosis and vascular density in malignant mesotheliomas was assessed by the TUNEL method and by immunohistochemistry with an antibody against FVIII-related antigen. Immunohistochemically mesothelial cells in non-neoplastic healthy pleural tissues were mostly negative for iNOS. Positivity for iNOS was observed in 28/38 (74%) and 24/25 (96%) of malignant mesotheliomas and metastatic pleural adenocarcinomas, respectively. Epithelial and mixed mesotheliomas expressed more often strong iNOS immunoreactivity compared to the sarcomatoid subtype (P = 0.023). Moreover, metastatic adenocarcinomas expressed more often iNOS positivity than mesotheliomas (P = 0.021). Experiments with the cell lines confirmed that malignant mesothelioma cells are capable of synthesizing iNOS. No significant association was found between iNOS expression and apoptosis or vascular density in malignant mesotheliomas. The higher expression of iNOS in the epithelial subtype of mesothelioma and pleural metastatic adenocarcinoma might be due to an increased sensitivity of these cell types to cytokine-mediated iNOS upregulation. The strong expression of iNOS suggests a putative role for NO in the growth and progression of these tumours.
BACKGROUND: Malignant mesothelioma is a malignancy with a primary resistance to chemo- and radiotherapies for reasons which are still unclear. Multidrug resistance proteins might explain the observed resistance, but no studies have assessed their expression in mesothelioma. PATIENTS AND METHODS: Immunohistochemical expression of P-glycoprotein (P-gp), and the multidrug resistance proteins 1 and 2 (MRP1 and MRP2) were investigated in 36 cases of malignant mesothelioma and in samples from normal mesothelium. RESULTS: P-gp immunopositivity was found in 61%, MRP1 immunopositivity in 58% and MRP2 positivity in 33% of the cases. Normal mesothelium did not express these multidrug-resistant proteins. There was a significant association between P-gp and MRP2 (P = 0.022) expression. No or weak P-gp, MRP1 or MRP2 immunostaining was significantly more frequent in sarcomatoid mesothelimas than in epithelial or biphasic mesotheliomas (P = 0.031, P = 0.034 and P = 0.024, respectively). There was no significant association between patient survival and expression of the multidrug-resistant proteins. CONCLUSIONS: The results show that P-gp, MRP1 and MRP2 are induced and expressed in malignant mesothelial cells. Regardless of their expression no association with survival of the patients was seen, suggesting that the primary resistance of malignant mesotheliomas is not solely dependent on their expression or function.