Vuokko L. Kinnula

The occurrence of brown adipose tissue in outdoor workers.

SummaryHistochemical reactions and activities of mitochondrial enzymes in adipose tissue around the neck arteries and in pericardium were studied in men who had been outdoor workers in northern Finland. The purpose was to study the occurrence of brown fat in workers having been exposed to cool or cold ambient temperature. Indoor workers of the same age were used as controls.Histochemically, no mitochondrial enzyme reactions were seen in the adipose tissues taken from the indoor workers, whereas some outdoor workers had some multilocular adipose tissue, mostly around the neck arteries. Biochemical parameters also showed increased enzyme activities of aerobic energy metabolism in the adipose tissue of these people.The present results suggest that working in the cold can retain brown adipose tissue in “strategic” places in human adults.

Overexpression of peroxiredoxins I, II, III, V, and VI in malignant mesothelioma

Peroxiredoxins (Prxs) are a recently characterized group of thiol‐containing proteins with efficient antioxidant capacity, capable of consuming hydrogen peroxide in living cells. Altogether six distinct Prxs have been characterized in mammalian tissues. Their expression was investigated in histological samples of mesothelioma and in cell lines established from the tumours of mesothelioma patients. Four cases with histopathologically healthy pleura from non‐smokers were used as controls. Healthy pleural mesothelium was negative or very weakly positive for all Prxs. In mesothelioma, the most prominent reactivity was observed with Prxs I, II, V, and VI. Prx I was highly or moderately expressed in 25/36 cases, the corresponding figures for Prxs II–VI being 27/36 (Prx II), 13/36 (Prx III), 2/36 (Prx IV), 24/36 (Prx V), and 30/36 (Prx VI). Positive staining was observed both in the cytosolic and the nuclear compartment, with the exception of Prx III, which showed no nuclear reactivity. The staining pattern of Prxs III and V was granular. Immunoelectron microscopic localization of Prxs was in accordance with the immunohistochemical findings, showing diffuse cytoplasmic localization for Prxs I, II, IV, and VI and distinct mitochondrial labelling for Prxs III and V. There was no significant association between the extent of staining and different Prxs. It appeared that Prxs may not have prognostic significance, but being prominently expressed in most mesotheliomas these proteins, at least in theory, may play a role in the primary drug resistance of this disease. Copyright

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

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

The signal sequence polymorphism of the MnSOD gene is associated with the degree of carotid atherosclerosis

Redox-state of the cells of vascular walls is an important determinant of atherosclerosis. Manganese superoxide dismutase (MnSOD) is an essential anti-oxidant enzyme working in mitochondria of mammalian cells. A potentially functional amino acid polymorphism (Ala16Val) has been described in the signal sequence of the enzyme. The aim of the current study was to test whether the signal sequence polymorphism of the MnSOD would be associated with the degree of carotid atherosclerosis. The polymorphism was genotyped in a sample of 989 middle-aged hypertensive and control subjects. Carotid atherosclerosis was quantified as intima-media thickness (IMT) by ultrasound. The signal sequence polymorphism was found to be a minor determinant of carotid IMT explaining 1.3% of the overall variation, the Val allele associated with the higher IMT. In women, a significant interaction with plasma levels of low-density lipoprotein (LDL) cholesterol was detected, since LDL cholesterol levels were positively correlated with carotid IMT only in the carriers of the Val allele and the Val allele was associated with higher IMT only in the subjects with highest plasma levels of LDL cholesterol. In conclusion, the signal sequence polymorphism of the MnSOD gene is a minor determinant of carotid IMT pointing out the importance of redox-balance in the atherogenesis.

Induction of mitochondrial manganese superoxide dismutase confers resistance to apoptosis in acute myeloblastic leukaemia cells exposed to etoposide

We investigated the possible roles of mitochondrial manganese superoxide dismutase (MnSOD) and bcl‐2 in etoposide‐induced cell death in acute myeloblastic leukaemia (AML) using two subclones of the OCI/AML‐2 cell line, the etoposide‐sensitive (ES) and the etoposide‐resistant (ER), as models. Cell death after 24 h exposure to 10 μmol/l etoposide was about 60% and 70% in the ES subclone and about 20% and 25% in the ER subclone, when analysed by trypan blue and annexin V respectively. Cytochrome c efflux from mitochondria to cytosol was observed after 4 h of exposure in both subclones, whereas the activation of caspase‐3 was not detectable until after 12 h of exposure in the ES subclone and 24 h of exposure in the ER subclone, using Western blotting. The decrease in mitochondrial membrane potential, when analysed by the JC‐1 probe fluorocytometrically, also appeared to take place later in the ER than in the ES subclone. Both subclones showed evident basal expression of MnSOD and bcl‐2 by Western blotting. Etoposide caused a potent induction of MnSOD, more than 400% at 12 h, in the ER but not in the ES subclone. No significant change in bcl‐2 expression could be observed in either of the subclones during exposure to etoposide when analysed by Western blotting or flow cytometry. In conclusion, we suggest that MnSOD might have a special role in the protection of AML cells against etoposide‐induced cell death. Although unable to influence the cytochrome c efflux to cytosol, MnSOD might prevent the disruption of mitochondrial membrane potential, which evidently leads to cell death by releasing various activators of apoptosis.

Expression of antioxidant enzymes in astrocytic brain tumors.

We studied the expression of antioxidant enzymes (AOEs) and related proteins manganese superoxide dismutase (MnSOD), thioredoxin (Trx), thioredoxin reductase (TrxR), and the catalytic (GLCL‐c) and regulatory (GLCL‐r) subunits of glutamate cysteine ligase (γ‐glutamylcysteinesynthetase) in 433 astrocytomas. Expression of MnSOD was found in 91%, Trx in 46%, TrxR in 66%, GLCL‐c 73% and GLCL‐r in 89% of the cases. Diffuse astro‐cytomas showed more intense staining for Trx (p= 0.002), TrxR (p=0.004), GLCL‐c (p=0.001), GLCL‐r (p=0.04) and MnSOD (p=0.01) than pilocytic astrocytomas. Within diffuse astrocytomas only Trx (p= 0.0001) and TrxR (p=0.04) significantly associated with increased malignancy grade. Necrotic tumors were more often immunopositive for Trx (p=0.001) and TrxR (p=0.02) and AOE expression was generally higher in mitotically active tumors. Expression of Trx and lack of MnSOD expression was associated with a worse prognosis in diffuse astrocytomas. None of the AOEs had any prognostic value in pilocytic grade I astrocytomas. Familial astrocytomas, which included 23 of the cases studied, did not differ in their expression of MnSOD from sporadic ones. The results show that MnSOD and Trx may influence the biological behaviour of astrocytomas, possibly by modulating cell proliferation and necrosis in these tumors.

Proliferation, apoptosis, and manganese superoxide dismutase in malignant mesothelioma

Proliferation and apoptotic indices of tumour cells may have important prognostic significance. Manganese superoxide dismutase (MnSOD), an important anti‐oxidant enzyme, has been shown to decrease proliferation of malignant cells transfected with the MnSOD gene. The aim of the present study was to investigate the indices of cell proliferation and apoptosis and their prognostic significance in human mesothelioma and to assess the effect of MnSOD on the proliferation and apoptosis of the mesothelioma cells expressing high constitutive MnSOD activity. Tissue sections from 35 subjects with malignant pleural mesothelioma were studied for cell proliferation by Ki‐67 immunohistochemistry and for apoptosis by the TUNEL assay. In additional experiments, 2 mesothelioma cell lines expressing either low (M14K) or high (M38K) MnSOD levels were assessed for proliferative and apoptotic responses to epirubicin. The median proliferation and apoptotic indices of the mesothelioma tissue were 8.2% and 0.75%, respectively. Patients with a high proliferation (>8%) or apoptotic index (>0.75%) showed a worse prognosis (p < 0.001). MnSOD expression was inversely correlated with cell proliferation (p = 0.02). Our cell line experiments indicated that cells expressing high MnSOD levels were more resistant to apoptosis and showed lower proliferation when exposed to epirubicin in vitro. These findings show that high proliferation and apoptosis are associated with a poor prognosis of mesothelioma and that a high MnSOD level is associated with low proliferation of tumour cells. Furthermore, experiments with cultured mesothelioma cells suggest the importance of MnSOD in the proliferation and apoptosis caused by drug exposure. Int. J. Cancer 88:37–43, 2000.

DNA single strand breaks induced by asbestos fibers in human pleural mesothelial cells in vitro

The mechanisms of the cellular effects and DNA damage caused by asbestos fibers in human mesothelial cells are not well understood. We exposed transformed human pleural mesothelial cells to 1–4 μg/cm2 crocidolite and to 10–100 ng/ml tumor necrosis factor alpha for up to 48 hr and studied the induction of DNA damage using the Comet assay. As a positive control, 100 μM H2O2 was used. The DNA single strand breaks were assessed as the mean tail moments and as distributions of the tail DNA in the cell. The Comet assay showed significant but reversible increases in the mean tail moments, but not in the distribution of Comet tails in the histograms in cells exposed to 1 μg/cm2 crocidolite for 6 hr. At higher concentrations of asbestos fibers all the indices in the Comet assay showed significant and irreversible change. All the doses of TNF‐α caused marginal increase in the mean tail moments. The mean tail moments were highest in the cells with concurrent treatment to TNF‐α and crocidolite. In the cells pretreated with inhibitors of antioxidant enzymes (aminotriazole for catalase and buthionine sulfoximine for γ‐glutamylcysteine synthetase) asbestos fibers slightly increased oxidant‐related fluorescence of dichlorofluorescein (DCFH) but did not cause any further increases in the mean tail moments. This study shows that asbestos fibers cause DNA single strand breaks in human mesothelial cells. Since the inhibition of antioxidant enzymes did not have an effect on the DNA damage caused by the fibers, other mechanisms than free radicals seem to be involved in the induction of DNA damage by mineral fibers. Environ. Mol. Mutagen. 33:153–160, 1999

Expression and prognostic significance of catalase in malignant mesothelioma

Free radicals and antioxidant enzymes (AOEs) may play a critical role in cell proliferation and in the resistance of malignant cells against cytotoxic drugs and radiation. Malignant mesothelioma is a resistant tumor with high levels of manganese superoxide dismutase, a central superoxide scavenging AOE. In the current study, the authors assessed the expression and prognostic role of catalase, an important hydrogen peroxide scavenging AOE, in malignant pleural mesothelioma.

The expression of P-glycoprotein and multidrug resistance proteins 1 and 2 (MRP1 and MRP2) in human malignant mesothelioma

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.