Tumor tissue inhibitor of metalloproteinases-1 (TIMP-1) in hormone-independent breast cancer might originate in stromal cells, and improves stratification of prognosis together with nodal status.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is shown to be a potential marker for poor prognosis in breast cancer, but the biology of TIMP-1 is only partially understood. In this study, TIMP-1 production was studied in a co-culture model of hormone-independent breast cancer cell lines and mesenchymal stem cells mimicking the stromal components of the tumor. In addition, the prognostic value of TIMP-1 was histologically evaluated in a clinical material of 168 patients with hormone-independent breast tumors. The hormone-independent breast cancer (BC) cell lines MDA-MB-231, M4A4 and NM2C5 did not produce TIMP-1 protein in measureable quantities. Six tested primary mesenchymal stem cell lines all produced TIMP-1. Co-culturing of mesenchymal stem cells and breast cancer cells resulted in positive immunocytochemical diffuse staining for TIMP-1 for both cell types. Culturing breast cancer cells with MSC-conditioned media resulted in a positive cytoplasmic immunoreactivity for TIMP-1, and TIMP-1 protein concentration in cell lysates increased 2.7-fold (range 1.1-4.7). The TIMP-1 mRNA levels remained unaffected in BC cells. This might suggest that breast cancer cells can take up TIMP-1 produced by stromal cells and are thus displaying cellular immunoreactivity. In addition, TIMP-1 was shown to improve stratification of prognosis in clinical material.

Immunohistochemical study of matrix metalloproteinase 9 and tissue inhibitor of matrix metalloproteinase 1 in benign and malignant breast tissue--strong expression in intraductal carcinomas of the breast.

OBJECTIVE: Matrix metalloproteinases (MMPs) are involved in carcinogenesis due to their tissue remodeling capability, and there is convincing evidence linking gelatinase B (MMP-9) with malignant cell invasion. Tissue inhibitor 1 of MMP (TIMP-1) is a strong inhibitor of MMP-9 but has also tumor-enhancing effects. Only few data exist on MMP-9 or TIMP-1 expression in tissue samples of different breast histology. METHODS: MMP-9 and TIMP-1 immunoreactivity was examined in a wide range of breast tissue samples differing in histology from usual ductal hyperplasia (UDH) to fully developed ductal breast carcinoma. Immunohistochemical expression of MMP-9 was studied in 178 samples: 31 UDH samples, 29 atypical ductal hyperplasia (ADH) samples, 28 ductal carcinoma in situ (DCIS) samples and 90 ductal invasive carcinoma samples (30 samples of malignancy grades I, II and III, respectively). TIMP-1 expression was also analyzed in 178 breast tissue samples: 41 UDH, 21 ADH and 34 DCIS lesions, and 82 invasive ductal breast carcinomas (25 in grade I, 30 in grade II and 27 in grade III). RESULTS: A significantly distinctive pattern of MMP-9 protein expression was shown in DCIS samples, where 85.7% of the cases showed moderate or strong positivity and negative staining was rare (p = 0.021). Negative or weakly positive MMP-9 staining was the most prominent finding in UDH (71%), ADH (69%) as well as in invasive carcinoma samples (64.4%). Various degrees of TIMP-1 expression were seen in 86.5% of all cases. DCIS and invasive carcinoma samples revealed similar immunostaining: at least some positivity was seen in 91.1% of the DCIS samples and 91.5% of infiltrative carcinomas. Thus, TIMP-1 negativity (22.2%) was significantly associated with hyperplastic lesions (p = 0.026). CONCLUSIONS: These results suggest that MMP-9 and TIMP-1 overexpression are early markers of breast carcinogenesis preceding tumor invasion. Apparently, DCIS carries the risk to evolve into a malignant phenotype according to these markers. The clinical importance of these findings is discussed.

High serum TIMP-1 correlates with poor prognosis in breast carcinoma - a validation study.

A number of studies have demonstrated that high tumor tissue levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) are associated with a poor prognosis in breast cancer, suggesting that TIMP-1 could be a valid prognostic marker in this disease. Recently, our laboratories have presented results showing that TIMP-1 also carries prognostic information when measured in serum. This is an important finding, since serum is a much more preferable material compared with tumor tissue extracts. The aim of the present study was to validate the previous results concerning the prognostic value of TIMP-1 in serum obtained preoperatively from 68 patients with primary breast cancer. This was done by measuring the same serum samples as in the previous study but in a different laboratory using a different ELISA assay. We confirmed that patients with the highest serum levels of TIMP-1 (> 197.7 ng/ml) had significantly shorter disease-specific survival compared with patients with low serum TIMP-1 levels. In the group of node-negative patients, 53% of the patients with high levels of TIMP-1 survived after 10 years of follow-up compared to 92% of the patients with low levels. This study thus confirms the reproducibility across laboratories of the results concerning the prognostic value of TIMP-1 in serum. We also investigated whether measurements of the specific fraction of uncomplexed TIMP-1 improved the prognostic value of TIMP-1 in serum, as has been shown to be the case for tumor tissue extracts. However, including information of the level of uncomplexed TIMP-1 did not seem to provide additional prognostic information to that already provided by total TIMP-1.

Extracellular superoxide dismutase has a highly specific localization in idiopathic pulmonary fibrosis/usual interstitial pneumonia.

AIMS: Recent studies suggest the importance of oxidant stress in the progression of pulmonary fibrosis. The aim of this study was to investigate extracellular superoxide dismutase (ECSOD), the major antioxidant enzyme of the extracellular matrix of human lung, in biopsy-proven idiopathic pulmonary fibrosis (IPF) related to usual interstitial pneumonia (UIP). METHODS AND RESULTS: Fibrotic areas and fibroblastic foci in UIP lungs were notable for absence of ECSOD by immunohistochemistry. Western blotting showed significantly lowered immunoreactivity of ECSOD in fibrotic compared with non-fibrotic areas of the diseased lung. The only cell type that showed intense ECSOD positivity in UIP was the interstitial mast cell. In order to investigate the mechanism for ECSOD depletion in fibrotic areas, alveolar epithelial cells were exposed to tumour necrosis factor-alpha and transforming growth factor (TGF)-beta1; TGF-beta suggested a trend towards decreased synthesis. Patients with UIP were also assessed to determine whether this disease is associated with a naturally occurring mutation in ECSOD (Arg213Gly) which leads to a loss of tissue binding of ECSOD. No significant differences could be found in the allele or genotype frequencies of this polymorphism between 63 UIP patients and 61 control subjects. CONCLUSION: Overall, consistent with several other antioxidant enzymes, ECSOD is very low in fibrotic areas of UIP, which may further increase the oxidant burden in this disease.

Relationship between histopathological features and the course of idiopathic pulmonary fibrosis/usual interstitial pneumonia.

BACKGROUND: Fibroblastic focus (FF) is the typical histopathological feature of idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia (UIP). A study was undertaken to analyse FF at diagnosis, to analyse the histopathological findings at necropsy, and to examine their association with the course of the disease. METHODS: A retrospective study was made of 76 UIP cases collected over a period of 30 years from one university hospital; 64 had idiopathic IPF. The surface area of one slide of each lung biopsy specimen was defined by image analysis and the total number of FF was quantified. The histological features of necroscopic lung samples were re-analysed in 11 cases. Clinical follow up information was obtained from the registers. RESULTS: Patients with < or =50 FF/cm(2) (n = 34) in the lung biopsy specimen had a median survival of 89 months (95% CI 38 to 140) compared with 49 months (95% CI 36 to 62) in those with >50 FF/cm(2) (n = 42, p = 0.0358). Diffuse alveolar damage (DAD) was detected in 10 necropsy samples and almost prevented the histopathological confirmation of UIP in six cases. Accumulation of neutrophils occurred in nine cases. There was no association between FF at diagnosis and DAD at necropsy, or between FF and exacerbation of the disease before death. CONCLUSIONS: The number of FF in lung samples before death is associated with poor survival but not with DAD, which is a common feature in necropsy specimens of patients with UIP. FF cannot predict an acute exacerbation of IPF.

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

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

Tissue MMP-2 and MMP-9 [corrected] are better prognostic factors than serum MMP-2/TIMP-2--complex or TIMP-1 [corrected] in stage [corrected] I-III lung carcinoma.

Matrix metalloproteinases (MMPs) are involved in tumor growth and spreading. Here, we investigated the tumor immunoreactive protein of MMP-2, MMP-9 and TIMP-1 as well as the levels of circulating total TIMP-1 and MMP-2/TIMP-2-complex as prognostic factors in lung cancer patients. The material included 59 patients, 30 with a squamous cell carcinoma, 21 with an adenocarcinoma and eight with other histology. Circulating antigens were measured by ELISA assay and the protein expression in primary tumors was analyzed by streptavidin-biotin immunohistochemical staining using specific monoclonal antibodies. The strong positivity for MMP-2 or MMP-9 in tumor predicted poor prognosis. The 5-year survival rates were 83 or 85% in patients negative for MMP-2 or MMP-9, respectively. Only 17% of the patients with a tumor highly positive for MMP-2 and 43% of those with a high positivity for MMP-9 survived at that time (Cox regression P=0.042 for MMP-2 and log rank P=0.046 for MMP-9). On the contrary, strong tissue positivity for TIMP-1 demonstrated a tendency for a favorable survival, although the difference did not reach statistical significance. In patients with a squamous cell carcinoma Stage I, low serum TIMP-1 (or=300 ng/ml) associated with an increased survival rate, the 5-year survival being 81 versus 34% (log rank P=0.069) in patients with high or low serum levels for MMP-2/TIMP-2-complex, respectively. Tissue MMP-2 correlated to high expression of MMP-9 immunoreactive protein (P=0.003), but the serum levels of MMP-2/TIMP-2-complex or TIMP-1 did not correlate to the immunostaining of the corresponding tumors. We conclude that in lung carcinoma the best prognostic value is achieved by using immunohistochemistry for MMP-2 and MMP-9. In early disease, however, serum TIMP-1 or MMP-2/TIMP-2-complex could offer some further prognostic value.

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

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

Manganese superoxide dismutase is increased in the airways of smokers' lungs.

Oxidant stress is a key mechanism for smoking-induced chronic obstructive pulmonary disease (COPD). Smoking has been shown to upregulate several antioxidant enzymes, with potential effects on the prevention of the disease and/or its progression. Superoxide dismutases (SOD)s are the only enzymes capable of consuming superoxide radicals. The purpose of the present study was to investigate SODs in the lungs of nonsmokers, smokers and COPD patients. Manganese superoxide dismutase (MnSOD), copper zinc SOD (CuZnSOD), and extracellular SOD (ECSOD), were investigated by immunohistochemistry in the airways of 13 nonsmokers, 20 smokers and 22 COPD patients with mild-to-moderate disease. Lung tissue homogenates of three nonsmokers and four smokers were used for Western blot and enzyme activity analysis. The expression of MnSOD was higher in the central bronchial epithelium of smokers with COPD and in the alveolar epithelium of smokers without or with COPD than innonsmokers. Lung MnSOD immunoreactivity, evaluated by Western blotting and specific activity, were 33% and 51% higher, respectively, in smokers than in nonsmokers. No major changes could be observed in lung CuZnSOD or ECSOD immunoreactivities. Manganese superoxide dismutase is elevated in the alveolar epithelium of cigarette smokers, probably due to the increased oxidant burden in smokers' lungs.

Glutamate-L-cysteine ligase in breast carcinomas.

AIMS: To investigate the immunohistochemical expression of the catalytic and regulatory subunits of gamma-glutamyl cysteine synthetase, i.e. glutamate-L-cysteine ligase (GLCL) in 274 invasive and in-situ breast carcinomas. GLCL is the rate-limiting enzyme in glutathione synthesis, which is one of the most important intracellular antioxidants participating in the detoxification reactions of several cytotoxic drugs. METHODS AND RESULTS: In the tumour cells GLCL reactivity was observed in 50% and 44% of the cases for the catalytic and the regulatory subunits, respectively. There was a statistically significant association between their expression (P = 0.002). Lobular invasive carcinomas expressed the catalytic and regulatory subunits more often than other tumours (P = 0.050 and P = 0.046, respectively). Also in-situ carcinomas expressed the catalytic subunit more often (P = 0.005). Tumours showing no immunoreactivity for the catalytic subunit had axillary metastases significantly more often (P = 0.013). Patients with tumours showing positivity for either subunit or both had a better survival (P = 0.037). No difference in survival could be observed between GCLC-positive or -negative cases in the subgroup receiving chemotherapy. CONCLUSIONS: Expression of the catalytic and regulatory subunits of GLCL is found in a substantial number of breast carcinomas and their expression is more pronounced in lobular invasive and in-situ carcinomas. Even though the overall expression of GLCL was associated with improved survival, no such effect was observed separately in the group receiving chemotherapy.

Matrix metalloproteinase-2 (MMP-2) is associated with survival in breast carcinoma.

Adjuvant therapy is one of the major advances in the treatment of breast carcinoma patients - but do all patients need it? New predictive markers, which are able to save breast carcinoma patients from the most toxic adjuvant therapies, are still needed. The expression of matrix metalloproteinases (MMP-2) has been previously linked to invasiveness of carcinoma cells. In this study, we explored the role of MMP-2 as a prognostic factor in breast carcinoma in a large series to be able to show the favourable effect of MMP-2 negativity in poor prognosis subgroup of hormone receptor-negative patients. The MMP-2 immunoreactive protein was evaluated from primary adenocarcinoma of the breast in 453 cases by using a specific monoclonal antibody in immunohistochemical stainings. The MMP-2 protein found in breast carcinoma tumour cells was here shown to be associated with a shortened recurrence-free survival or relative overall survival (P=0.03). It was shown here that MMP-2 negativity is significantly linked to favourable prognosis in patients considered to be at risk due to their hormone receptor negativity. In the patient group presenting with a progesterone receptor-negative tumour, the survival rate of the MMP-2-positive cases was 58% while it was 95% in MMP-2-negative cases after 10 years of follow-up (P=0.005). The present data shows for the first time that MMP-2 negativity could serve as a marker for favourable prognosis in breast carcinoma patients with a hormone receptor-negative tumour usually associated with high risk. MMP-2 is also shown to correlate to shortened survival independent of major prognostic indicators in patients with primary breast carcinoma.

Apoptosis and regression of embryonic mesenchyme in the development of the middle ear spaces.

Apoptosis was studied using temporal bones from three fetuses representing different times of gestation and from three neonates. Paraffin-embedded sections 20-microm thick were studied using the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling method based on 3'-end-labeling of fragmented DNA. Phenotyping of the immune cells was performed using regular monoclonal antibodies. In the bone marrow the granulocyte series dominated and the number of cells in the macrophage series was noticeably fewer, with apoptotic cells occurring in both. In the embryonic mesenchyme, solitary apoptotic cells occurred in all locations in both the fetuses and neonates. Apoptosis is a basic factor in the regression of embryonal mesenchyme, but may not be preprogrammed. Basic scientific data obtained from modified tissue cultures show that mechanical forces cause cells to switch between different genetic programs. It is suggested that the act of swallowing causes periodic changes in the amniotic fluid pressure and provides the necessary force for regression of the mesenchyme by apoptosis.

Over-expression of tenascin-C in malignant pleural mesothelioma.

AIMS: Tenascin-C is an extracellular matrix glycoprotein known to have anti-adhesive characteristics and to be expressed in various human malignant neoplasms. We hypothesized that the expression of tenascin-C would be increased in human malignant pleural mesothelioma, and its accumulation associated with the prognosis of the patients with this disease. METHODS AND RESULTS: Thirty-seven cases of mesothelioma were studied by immunohistochemically using a monoclonal antibody against tenascin-C, and with a semiquantitative scoring system for tenascin-C in different areas of the tumours. In 10 selected cases tenascin-C mRNA in-situ hybridization was also analysed. Since transforming growth factor-beta (TGF-beta) is known to induce both the synthesis of tenascin-C and the growth of mesotheliomas, an immunohistochemical analysis of TGF-beta 1, -beta 2 and -beta 3 was also performed. Normal pleura (n = 7) and metastatic pleural adenocarcinomas (n = 7) were used as controls. Tenascin-C protein was expressed in every histological subtype of malignant mesothelioma, being most prominent in the fibrotic stroma of a tumour, around tumour cells and at the invasive border, whereas tenascin-C mRNA was scarce in tumour cells. The patients with less immunohistochemical expression for tenascin-C tended to live longer (P = 0.028 by Fishers' exact probability test). All mesotheliomas showed positivity for at least one isoform of TGF-beta. CONCLUSIONS: In conclusion, high expression of tenascin-C protein in malignant pleural mesotheliomas may play a role in its invasive growth, and might serve as a prognostic marker of the disease.

Expression of antioxidant enzymes in bronchial metaplastic and dysplastic epithelium.

We investigated immunohistochemical expression of manganese superoxide dismutase (MnSOD) and three hydrogen peroxide (H(2)O(2)) scavenging pathways, i.e. catalase (CAT), gamma-glutamyl cysteine synthetase (gammaGCS) and thioredoxin (Trx) system in normal bronchial epithelium, bronchial metaplasia and dysplasia and correlated their expression with NF-kappaB activation (p50) and proliferation (Ki67). Normal bronchial epithelium was positive for MnSOD, heavy and light subunits of gammaGCS, CAT and Trx and TrxR. Metaplastic epithelium showed strongest expression of gammaGCSh and Trx, whereas dysplastic epithelium expressed most prominently MnSOD and CAT. There was a significant correlation between expression of gammaGCSh and gammaGCSl (P=0.034) and Trx and TrxR (P=0.037). Trx expression also correlated with gammaGCSh (P<0.001) and gammaGCSl (P=0.012) and TrxR with gammaGCSh (P<0.001) but not with gammaGCSl immunoreactivity (P=0.744). Expression of p50 was highest in metaplastic epithelium while Ki67 was highest in dysplastic lesions. Expression of Trx and gammaGCSh correlated inversely with age of the patients (R=-0.6038, P<0.001 for Trx and R=-0.6162, P<0.001 for gammaGCSh). Changes in the expression of these enzymes in bronchial lesions might be due to alterations of antioxidative mechanisms due to irritation via exogenous toxins and activation of reactive oxygen species (ROS) known to be associated with induction of metaplasia and dysplasia in the bronchial tree.

Apoptotic activity is increased in brain cortex infarct after hypothermic circulatory arrest in a porcine model.

OBJECTIVE: It has been shown that apoptosis contributes to neuronal cell death after ischemia, and we evaluated the degree of apoptotic activity occurring in brain cortex of pigs after hypothermic circulatory arrest (HCA). DESIGN: Thirty-one pigs underwent 75 min of HCA at 20 degrees C. Histological examination of the brain was performed, and slides of brain cortex were evaluated for apoptotic activity by the TUNEL method. RESULTS: Ten animals died during the first postoperative day and 21 survived until the seventh postoperative day. Brain cortex infarcts were found in animals that survived 7 days and these were included in this study. The median histopathological score among animals that died on the first postoperative day was 3.0 (range, 2-4), whereas it was 4.0 (range, 2-4) among survivors (p = 0.019). The apoptotic index was particularly high in the area of the infarct, whereas only a few TUNEL-stained cells were observed in noninfarcted areas. The apoptotic index was nil in all pigs that died in the first postoperative period, whereas it was 2.0 (range, 0-6) among the animals that survived until the seventh postoperative day (p < 0.0001). CONCLUSION: The apoptotic index was significantly increased in brain cortex infarcts of animals that survived 7 days after HCA, whereas only a few apoptotic cells were observed in noninfarcted areas of these animals as well as in animals that died on the first postoperative day. Further studies are required to elucidate the timing of development of brain infarction after HCA and whether neuroprotective strategies targeting the apoptotic process may mitigate brain damage.

Prospective population-based study on the survival of patients with lung cancer.

The purpose of this study was to evaluate the change, over 20 yrs, in the survival of lung cancer patients in a population-based study. Information on all patients with lung cancer in a defined geographical area during 1990-1992 (n=602) was prospectively gathered. The survival of these patients was assessed and also compared with the results of a similar study in the same area during the years 1968-1971 (n=446). The 5-yr survival had improved during 20 yrs from 4% to 12%. The 5-yr survival of the patients with squamous cell carcinoma had increased from 6% to 16%, and adenocarcinoma from 4% to 19%, whereas the survival of small cell carcinoma had remained the same (2% and 3%, respectively). Even though the recent patients were older than those of the earlier series the proportion of surgically treated patients had remained the same (16% and 20%), but the 5-yr survival of patients who had been operated on had increased significantly from 23% to 48%. The differences in survival in the second cohort (1990-1992) between histological types (Chi-squared logrank=59.2), tumour, node, metastasis stages (Chi-squared logrank=199.6), symptomatic stages (Chi-squared logrank=120, p<0.001) and treatment (Chi-squared logrank=277) were significant. Based on this study the independent prognostic factors for better survival of lung cancer patients are tumour, node, metastasis stages I and II, surgical treatment and Feinstein's symptomatic stages I and II.

Cell specific expression of peroxiredoxins in human lung and pulmonary sarcoidosis.

BACKGROUND: Six proteins of the peroxiredoxin (Prx) family have recently been characterised which have the capacity to decompose hydrogen peroxide in vivo and in vitro. These proteins may have an important role in the protection of human lung against endogenous and exogenous oxidant stress. However, the expression and distribution of these proteins in healthy human lung and diseased lung tissue is unknown. METHODS: The cell specific expression of Prxs in healthy lung tissue from four non-smokers and in parenchymal tissue from 10 subjects with pulmonary sarcoidosis was investigated by immunohistochemistry, and expression of these proteins in various cultured lung cells and cells of bronchoalveolar lavage (BAL) fluid of controls and patients with sarcoidosis was assessed by Western blot analysis. RESULTS: All six Prxs could be synthesised in cultured human lung cells. The bronchial epithelium showed moderate to high expression of Prxs I, III, V and VI, the alveolar epithelium expressed mainly Prxs V and VI, and alveolar macrophages expressed mainly Prxs I and III. Granulomas of subjects with sarcoidosis expressed mainly Prxs I and III. Samples of BAL fluid from controls and from subjects with sarcoidosis had very similar findings, except that Prxs II and III had a tendency for increased immunoreactivity in sarcoidosis tissue. CONCLUSIONS: Prxs I, III, V, and VI, in particular, have prominent and cell specific expression in human lung tissue. High expression of Prxs I and III in granulomas and alveolar macrophages of sarcoidosis parenchyma may have a significant effect on the oxidant burden and the progression of lung injury in this disease.

Expression of gamma-glutamyl cysteine synthetase in nonsmall cell lung carcinoma.

BACKGROUND: The purpose of this study was to investigate expression of gamma glutamyl cysteine synthetase (gamma GCS), the rate-limiting enzyme in glutathione synthesis in nonsmall cell lung carcinoma (NSCLC). METHODS: Eighty-five samples of NSCLC were studied using immunohistochemistry with polyclonal antibodies to the heavy and light subunits of gamma GCS (gamma GCS-h, gamma GCS-l), and the expressions were correlated with apoptosis and patients survival. Further studies were conducted in cultured cells also to investigate the effects of gamma GSC inhibition with buthionine sulfoximine on the cell survival. RESULTS: In the biopsies, gamma GCS-h positivity was found in 71% and gamma GCS-l positivity in 67% of NSCLCs, and they were expressed in all cell lines studied. There was a strong association between the expression of the heavy and light subunits of gamma GCS in NSCLC (P = 0.003). Strong or moderate gamma GCS-h expression was found significantly more often in squamous cell carcinomas (P = 0.00013) and in Grade 1-2 tumors (P = 0.008). There was a significantly higher extent of apoptosis in tumors with a low gamma GCS-h expression (P = 0.016). A similar tendency was observed with gamma GCS-l (P = 0.073). No association was found between patient survival and high or low expression of gamma GCS-l or gamma GCS-h in NSCLCs (P = 0.34 and P = 0.47, respectively). CONCLUSIONS: The results show that gamma GCS is strongly expressed in NSCLCs and probably takes part in the defense of the tumor cells against oxidative damage. This is reflected by the lower extent of apoptosis in tumors with a high gamma GCS expression. Because expression of gamma GCS has been connected with chemoresistance, downregulation of its activity by inhibitors in NSCLC might have putative therapeutic potential in the treatment of lung carcinoma.

Expression and regulation of hemeoxygenase 1 in healthy human lung and interstitial lung disorders.

Hemeoxygenase 1 (HO-1) has been implicated in the protection of lung tissue against exogenous oxidant exposure. However, the expression and cellular distribution of HO-1 in human lung continue to be poorly characterized. The localization of HO-1 was studied in histopathologically healthy lung from nonsmoking patients with a carcinoid or other lung tumor (5 cases), pulmonary sarcoidosis (13 cases), and chronic interstitial pneumonias (9 cases, usual interstitial pneumonia; 10 cases, desquamative interstitial pneumonia). Immunostaining was graded from 0 (no immunoreactivity) to +++ (intense immunoreactivity). In healthy lung, HO-1 was localized to alveolar macrophages with reactivity varying from moderate to intense, and in bronchial epithelium, alveolar epithelium, endothelium, and interstitium, the immunoreactivity was not detectable or was very low. Sarcoidosis and interstitial pneumonias showed intense HO-1 immunoreactivity in alveolar macrophages in most of the cases and weak to intense immunoreactivity in the granulomas of sarcoidosis. The immunoreactivity of interstitium was negative or weak in the fibrotic areas of the lung and also in the samples of bronchoalveolar lavage fluid obtained from the patients with UIP. Western blotting indicated that HO-1 is up-regulated by exposure of monocytes to formylated peptide, fMLP, which causes respiratory burst in the cells, and that inhibition of HO-1 by tin protoporphyrin potentiates the injury of fMLP-exposed cells. In conclusion, these data show differential distribution of HO-1 in human lung cells and strongly suggest the importance of HO-1, especially in the defense of alveolar macrophages in normal human lung and in the inflammatory, but not in the fibrotic, stage of interstitial lung disorders.