Bernd Werle, Dr. - Academia.edu (original) (raw)
Papers by Bernd Werle, Dr.
PubMed, 2005
Gelatinase B/MMP-9 is a member of matrix metalloproteinases with a major role in extracellular ma... more Gelatinase B/MMP-9 is a member of matrix metalloproteinases with a major role in extracellular matrix degradation, cell proliferation and migration. Its proenzyme form has also been reported in pleural fluids as an inducible species, but its relation to pleural pathology has not yet been fully clarified. The primary goal of this study was to evaluate proMMP-9 as a potential marker for differentiating pleural effusions of both malignant and non-malignant origin. Pleural fluid samples were studied from 194 patients, including tumor etiology in 133 cases, inflammatory disorders in 33, transudates in 12, and unspecified disorders in 16 patients. The concentrations of proMMP-9 were estimated by means of immunoassays and/or by scanning zymography. Samples were also examined for C-reactive protein (CRP). The analysis of proMMP-9 showed significant differences among the etiological groups with the highest concentrations in para-inflammatory exudates, intermediate in para-neoplastic exudates, and the lowest in transudates. However, the analysis of the para-neoplastic group revealed a distinct heterogeneity with a minor portion of fluids reaching values typical for para-inflammatory effusions. A subsequent sorting based on tumor histology showed increased levels particularly in exudates associated with metastatic tumors. Interestingly, proMMP-9 values in general correlated with CRP, a systemic marker of inflammation. Thus, MMP-9 proenzyme appears to complement traditional markers distinguishing pleural fluids of different origin. Yet, the differentiation between paraneoplastic and para-inflammatory exudates must be regarded with caution due to the presence of a high-expressive paraneoplastic sub-population, including effusions associated with metastatic tumors.
Biological Chemistry, Jan 5, 2001
A sandwich-type ELISA has been developed for quantification of the complex between the cysteine p... more A sandwich-type ELISA has been developed for quantification of the complex between the cysteine proteinase cathepsin Β (CB) and its reversible tight-binding inhibitor cystatin C (CC) in normal and pathological sera. The assay is based on a combination of catching Ab (3E1), raised against CB, and a horseradish peroxidase-labelled detection Ab (1A2), raised against CC. Only the CB/CC complex is able to evoke a signal in this assay. The detection limit of the assay was 15.5 ΠΜ and the working range between 31.3-200 nM. The within and between-run coefficients of variance (CV) varied from 4.7% to 9.4% and 11% to 12.8%, respectively, demonstrating satisfactory reproducibility of the method. The concentration of the CB/CC complex was determined in sera from 90 healthy controls, 32 patients with non-cancerous lung diseases, 148 patients with lung and 32 patients with colorectal cancer. The CB/CC complex was significantly less abundant in sera of patients bearing malignant lung tumours than in those with non-cancerous lung diseases or healthy controls (p<0.001). In colorectal cancer sera its level was significantly lower in advanced stages C and D than in early Dukes' stages A and Β (p=0.02). Our results show that the increased levels of CB in malignant sera are not impaired effectively by CC and support the hypothesis of hindered inhibitory capability during cancer progression.
International Journal of Biological Markers, 2000
Cathepsins B, H and L have been shown to participate in processes of tumor growth, vascularizatio... more Cathepsins B, H and L have been shown to participate in processes of tumor growth, vascularization, invasion and metastasis. Their levels in tumor tissue extracts can provide useful clinical information to predict disease-free and overall survival in breast, lung, colorectal, brain and head and neck cancer patients. Recently we have found that both cysteine cathepsins and their endogenous protein inhibitors stefins and cystatin C can also predict prognosis when measured extracellularly. In melanoma and colorectal cancer patients high serum levels of cathepsins B and H correlated with shorter survival. Similarly, increased extracellular levels of stefins A and B and cystatin C correlated significantly with high risk of adverse outcome in cancer patients. However, the cathepsin B/cystatin C complex was found to be less abundant in sera of patients with malignant tumors than in those with benign diseases or in healthy controls, suggesting an imbalance between the enzyme and its inhibitor in cancer patients.
The International Journal of Biological Markers, 2000
Cathepsins B, H and L have been shown to participate in processes of tumor growth, vascularizatio... more Cathepsins B, H and L have been shown to participate in processes of tumor growth, vascularization, invasion and metastasis. Their levels in tumor tissue extracts can provide useful clinical information to predict disease-free and overall survival in breast, lung, colorectal, brain and head and neck cancer patients. Recently we have found that both cysteine cathepsins and their endogenous protein inhibitors stefins and cystatin C can also predict prognosis when measured extracellularly. In melanoma and colorectal cancer patients high serum levels of cathepsins B and H correlated with shorter survival. Similarly, increased extracellular levels of stefins A and B and cystatin C correlated significantly with high risk of adverse outcome in cancer patients. However, the cathepsin B/cystatin C complex was found to be less abundant in sera of patients with malignant tumors than in those with benign diseases or in healthy controls, suggesting an imbalance between the enzyme and its inhibit...
Neoplasma, 2005
Gelatinase B/MMP-9 is a member of matrix metalloproteinases with a major role in extracellular ma... more Gelatinase B/MMP-9 is a member of matrix metalloproteinases with a major role in extracellular matrix degradation, cell proliferation and migration. Its proenzyme form has also been reported in pleural fluids as an inducible species, but its relation to pleural pathology has not yet been fully clarified. The primary goal of this study was to evaluate proMMP-9 as a potential marker for differentiating pleural effusions of both malignant and non-malignant origin. Pleural fluid samples were studied from 194 patients, including tumor etiology in 133 cases, inflammatory disorders in 33, transudates in 12, and unspecified disorders in 16 patients. The concentrations of proMMP-9 were estimated by means of immunoassays and/or by scanning zymography. Samples were also examined for C-reactive protein (CRP). The analysis of proMMP-9 showed significant differences among the etiological groups with the highest concentrations in para-inflammatory exudates, intermediate in para-neoplastic exudates...
International Journal of Oncology, Jul 1, 1994
Cancer 12 (1995) 113-160 investigated 109 lung adenocarcinomas, mostly small, peripheral, stage I... more Cancer 12 (1995) 113-160 investigated 109 lung adenocarcinomas, mostly small, peripheral, stage I tumours (X1/109) for presence of K-ras gene mutations at codons 12 and 13. Mutations were detected by denaturing gradient gel ekctrophoresis analysis of specitic sequences amplified by polymerase chain reaction from DNA extracted from archival pathological material. Thirty-three of 109 (30.3%) tumours showed mutations at codon 12 (28/33,84.8%) or 13 (5/33, 15.2%) ofthe gene. Mutations and type of nucleotide substitutions were differently distributed among cytological subtypes, being more prevalent among less differentiated (G2 and G3) tumours and among bronchial than bronchiole-alveolar type adenocarcinomas. Survival analysis showed an adverse effect of K-ras mutation on survival, restricted to stage I tumours. Median survival for 81 stage I patients was 30 months for non-mutated tumours versus 20 months for mutated tumours @ = 0.016). Muhivariate analysis showed that age of patient @ = 0.001) and K-ras mutation status @ = 0.04) were the only independent factors influencing survival signiBcanUy. These data strengthen the hypothesis that K-ras gene mutations may be useful in identifying a subgroup of patients with poor outcome.
Electronic journal of pathology and histology, 2001
Le procede et le dispositif decrits servent a determiner l'activite d'enzymes dans des li... more Le procede et le dispositif decrits servent a determiner l'activite d'enzymes dans des liquides de maniere largement automatique. Le dispositif de mise en oeuvre de ce procede comprend une colonne (1) qui contient un substrat chromatographique pour traiter un echantillon. Le substrat est melange avec une substance qui se lie a des inhibiteurs d'au moins un enzyme presents dans l'echantillon. Un dispositif d'amenee (2) des echantillons est associe a une extremite de la colonne (1). Un agencement pompe/soupape (7, 11, 14, 15) monte en aval de la colonne (1) dans le sens d'ecoulement de l'echantillon permet de remplir au moins une eprouvette (5) avec un substrat et au moins une partie de l'echantillon. Le substrat est decompose en produits de clivage par l'enzyme. Au moyen d'un detecteur, on determine l'augmentation de la concentration en au moins un produit de clivage du substrat par unite de temps pendant une periode d'incubation. En co...
British Journal of Cancer, 2001
Cathepsins have been shown to participate in dissolution and remodelling of connective tissue and... more Cathepsins have been shown to participate in dissolution and remodelling of connective tissue and basement membranes in the processes of tumour growth, invasion and metastasis (Sloane et al, 1994; Kos and Lah, 1998). Increased levels of Cats B and L in tumours and some extracellular fluids are associated with shorter disease-free and overall survival periods and may serve as prognostic factors for cancer patients (Kos and Lah, 1998). The role of another cysteine proteinase, Cat S in tumour progression is less understood. In contrast to Cats B and L, which are active at acidic pH, Cat S retains most of its enzymatic activity at pH 7.0-7.5. Another distinct feature is the substantial elastolytic activity it exhibits at neutral pH (Chapman et al, 1997). Cat S exhibits restricted tissue expression and is present in higher amounts in lymph nodes (Turenšek et al, 1975), spleen and antigen-presenting cells (APCs), including macrophages, dendritic cells and B lymphocytes (Shi et al, 1994; Morton et al, 1995). Its expression was found to be induced by cytokines, such as interferon γ and interleukin 1β (Chapman et al, 1997). Cat S activity appears to be essential in the MHC class II antigen presentation pathway. It specifically degrades the invariant chain (Ii), a MHC class II chaperone, prior to its removal from the MHC class II peptide-binding cleft. This facilitates the loading of antigenic peptides to MHC class II αβ-dimers and subsequent transportation of the complex to the cell surface to initiate MHC class II restricted CD4 + T-cell recognition (Chapman et al, 1997; Pierre and Mellman, 1998). Besides participation in the immune response some other specific functions, associated with high elastolytic activity of Cat S have been proposed. For example, Cat S has been suggested to promote motility of the cilia of conducting airway cells in the lung (Chapman et al, 1997). Additionally, it was found to participate in vascular matrix remodelling and in the formation of the atherosclerotic intima (Sukhova et al, 1998). In rat, Cat S is expressed in thyroid tissue, where a role in processing thyroglobulin and release of thyroid hormone has been proposed (Petancheska and Devi, 1992). Increased expression of Cat S has also been associated with the pathogenesis of Alzheimer disease (Lemere et al, 1995). The aim of the present study was to evaluate the role this enzyme might have in the progression of lung cancer. For this purpose a quantitative method for measuring the level of Cat S in tissue extracts and sera of lung cancer patients was developed and the levels have been tested for their relationship to clinical features, considering especially the correlation with the survival rate for cancer patients. Additionally, the cell types, expressing Cat S in tumours, lung parenchyma and lymph nodes, have been evaluated by IHA. MATERIALS, METHODS AND PATIENTS Antigen, antibodies Human pro-Cat S was produced in E. coli using an inducible T7based expression system (Kopitar et al, 1996
British Journal of Cancer, 1997
In this study we examined both the pH dependence of cathepsin B (cath B) activity and its stabili... more In this study we examined both the pH dependence of cathepsin B (cath B) activity and its stability at physiological pH of 7.5 in lung tumours and normal lung tissue by means of fluorogenic assays with Z-Arg-Arg-AMC as specific substrate. Specificity was verified with the cath B blocking inhibitors E-64 and CA-074. With respect to pH dependence of activity, we found a deviation from a normal-shaped pHactivity curve. Besides the typical activity peak at pH 6.0, there were shoulders at pH 4.5-5.5 and at pH 7.0-7.5. This heterogeneity was found in both tumour and normal tissue. To test the stability of cath B at physiological pH of 7.5, homogenates were kept at pH 7.5 for 60 min. Altogether, 82-100% of residual cath B activity was found at pH 5.0-5.5, whereas activity in the range between 5.5 and 7.4 dropped drastically to 26-42%. At pH 7.5, there was still 20-34% residual cath B activity detectable. To test the hypothesis whether the cath B fraction active at pH 7.5 is more abundant in tumour tissues compared with the normal counterparts, we determined this fraction in 91 pairs of lung tumour and normal lung tissue. We found a 2.3-fold increase of median cath B fraction active at pH 7.5 in tumour tissue, although this fraction represented only a small part (about 16%) of the native, acidic (pH 6.0) cath B activity. However, in contrast to native cath B at 6.0, the cath B fraction active at pH 7.5 was related to post-operative probability of survival in curatively operated patients, since activity values higher than 292 (,uEU mg-' protein) were significantly associated with poor prognosis in patients with squamous cell carcinomas (n = 33, P = 0.04). It is concluded that in lung tumour and in normal lung tissue, cath B activity can be divided into at least three fractions with stability optima at different pH values, indicating various forms of cath B. The cath B fraction active at pH 7.5 provides prognostic information in patients with squamous cell carcinoma.
British Journal of Cancer, 1999
In order to evaluate the possible role of the proteolytic enzyme cathepsin B (cath B) in human no... more In order to evaluate the possible role of the proteolytic enzyme cathepsin B (cath B) in human non-small cell lung cancer (NSCLC) we examined cath B concentrations (cath B c) and activities (cath B A) in homogenates of 127 pairs of lung tumour tissues and corresponding nontumourous lung parenchyma. Total cath B activity (cath B AT) and enzymatic activity of the fraction of cath B, which is stable and active at pH 7.5 (cath B A7.5) were determined by a fluorogenic assay using synthetic substrate Z-Arg-Arg-AMC. The immunostaining pattern of cath B was determined in 239 lung tumour tissue sections, showing the presence of the enzyme in tumour cells (cath B T-I) and in tumour-associated histiocytes (cath B H-I). The median levels of cath B AT , cath B A7.5 and cath B C were 5.6-, 3.2-and 9.1-fold higher (P < 0.001), respectively, in tumour tissue than in non-tumourous lung parenchyma. Out of 131 tissue sections from patients with squamous cell carcinoma (SCC), 59.5% immunostained positively for cath B, while among the 108 adenocarcinoma (AC) patients 48.2% of tumours showed a positive reaction. There was a strong relationship between the levels of cath B AT , cath B A7.5 , cath B C and cath B T-I in the primary tumours and the presence of lymph node metastases. Significant correlation with overall survival was observed for cath B T-I and cath B A7.5 (P < 0.01 and P < 0.05, respectively) in patients suffering from SCC. In these patients positive cath B in tumour cells (cath B T-I) and negative cath B in histiocytes (cath B H-I) indicated significantly shorter survival rate compared with patients with negative cath B T-I and positive cath B H-I (P < 0.0001). In contrast, in AC patients, both, positive cath B T-I and positive cath B H-I , indicated poor survival probability (P < 0.014). From these results we conclude that the proteolytic enzyme cath B is an independent prognostic factor for overall survival of patients suffering from SCC of the lung.
Radiology and Oncology, Jan 6, 2002
Advances in Experimental Medicine and Biology, 1997
British Journal of Cancer, 2000
In order to evaluate the role of cysteine peptidase cathepsin H (Cath H) in human lung cancer its... more In order to evaluate the role of cysteine peptidase cathepsin H (Cath H) in human lung cancer its protein levels were determined in 148 pairs of lung tumour tissue and adjacent non-tumourous lung parenchyma using the enzyme-linked immunosorbent assay technique. Additionally, Cath H levels were determined in sera of 171 patients with malignant tumours, 34 patients with benign lung diseases and 47 healthy controls. The median level of Cath H in tumour tissue was 0.64 times that in the corresponding lung parenchyma. Relating tumour levels with histological type we found higher Cath H levels in small-cell and adenocarcinomas and lower levels in squamous cell carcinoma, large-cell carcinoma and secondary tumours. A significant difference in Cath H level between lung tumour tissue and non-tumourous lung parenchyma was associated with the group of cigarette smokers (156 vs 263 ng mg-1 protein, P < 0.001). For this group of patients Cath H tumour levels correlated with the survival rate, while for the entire patient population this was not the case. Smokers with high tumour levels of Cath H experienced poor survival. Cath H was significantly higher in sera of patients with malignant and benign lung diseases than in control sera (P < 0.001). The increase was significant for all histological types, being the highest in small-cell and squamous cell carcinomas. Our study reveals that in lung tumours there is different behaviour of Cath H compared with other cysteine peptidases, e.g. cathepsin B and cathepsin L. Variations between tissue and serum levels of Cath H indicate either reduced expression or enhanced secretion of this enzyme in lung tumours.
Biological Chemistry Hoppe-Seyler, 1995
In a series of pairs of lung tumor tissue and non-tumor lung parenchyma from 50 patients, the act... more In a series of pairs of lung tumor tissue and non-tumor lung parenchyma from 50 patients, the activity of cathepsin L was measured with Z-Phe-Arg-AMC using the inhibitor CA-074 to delimitate from cathepsin B activity also present in the tissue extracts. Cathepsin B was assessed in the same samples with its specific substrate Z-Arg-Arg-AMC. It was found that in tumor tissue the median activities of cathepsin L and cathepsin B were increased 1.6-fold and 4.9-fold, respectively. The levels of activity of both enzymes did not correlate with TNM stages nor with cell differentiation of bronchial carcinomas. Cathepsin L activity was found to be insignificantly higher in adenocarcinoma compared to squamous cell carcinoma, while cathepsin B activity did not vary across the histologies. The activities of both enzymes were low in pulmonary carcinoids, which are known to be low-grade malignant neoplasms. The amount of cathepsin B activity exceeded by far that of cathepsin L activity as proven by measurement with Z-Phe-Arg-AMC in the presence of the inhibitor Z-Phe-Phe-CHN 2 : 95-98% of cathepsin B activity vs 2-5% of cathepsin L activity were determined. By SDS-PAGE separation and immunoblot analysis, it could be demonstrated that significant amount of cathepsin L is complexed with the cysteine proteinase inhibitor kininogen. This explains the rather low cathepsin L activity values in the tissue extracts.
Anticancer research
To evaluate the possible role of cysteine proteases and serine proteases, as well as their respec... more To evaluate the possible role of cysteine proteases and serine proteases, as well as their respective inhibitors and receptors, as new prognostic factors in NSCLC, we examined, for the first time, 10 biological parameters related to three proteolytic systems within a homogeneous collective of 147 cases of NSCLC. Activities (cath B(AT), cath B(A7.5)) and protein levels of cath B(C), cath L(C), uPA, PAI-1, uPAR [measured by three different assays uPAR (ADI), uPAR (HD13), uPAR (IIIF10)] and TF were measured in homogenates of lung tumour tissue and corresponding non-malignant lung parenchyma. Total cath B activity (cath B(AT)) and enzymatic activity of the fraction of cath B, which is stable and active at pH 7.5 (cath B(A7.5)), were determined by a fluorogenic assay using synthetic substrate Z-Arg-Arg-AMC. The concentrations of cath B(C), cath L(C), uPA, PAI-1, uPAR and TF were determined by ELISAs. uPAR was determined using three different ELISA formats. The median levels of cath B(AT)...
Anticancer research
Secreted pro-cathepsin B of HS-24 human lung-tumour cells, human alveolar macrophages and Wi-38 h... more Secreted pro-cathepsin B of HS-24 human lung-tumour cells, human alveolar macrophages and Wi-38 human lung-fibroblast cells was pre-purified by ion exchange chromatography and investigated by 2D gel electrophoresis. Four (Wi-38), six (HS-24) and ten (alveolar macrophages) polypeptides differing in charge, but with the same molecular mass of 45 kDa, were found. The isoelectrical points of these polypeptides ranged from 5.43 to 6.57. Deglycosylation reduced the mass (7 kDa) but did not change the charge pattern. This investigation established cell type-specific patterns of secreted pro-cathepsin B-forms, but only parts of these may be cell type-specific forms depending on differentiated expression of mRNA and post-translational modification.
PubMed, 2005
Gelatinase B/MMP-9 is a member of matrix metalloproteinases with a major role in extracellular ma... more Gelatinase B/MMP-9 is a member of matrix metalloproteinases with a major role in extracellular matrix degradation, cell proliferation and migration. Its proenzyme form has also been reported in pleural fluids as an inducible species, but its relation to pleural pathology has not yet been fully clarified. The primary goal of this study was to evaluate proMMP-9 as a potential marker for differentiating pleural effusions of both malignant and non-malignant origin. Pleural fluid samples were studied from 194 patients, including tumor etiology in 133 cases, inflammatory disorders in 33, transudates in 12, and unspecified disorders in 16 patients. The concentrations of proMMP-9 were estimated by means of immunoassays and/or by scanning zymography. Samples were also examined for C-reactive protein (CRP). The analysis of proMMP-9 showed significant differences among the etiological groups with the highest concentrations in para-inflammatory exudates, intermediate in para-neoplastic exudates, and the lowest in transudates. However, the analysis of the para-neoplastic group revealed a distinct heterogeneity with a minor portion of fluids reaching values typical for para-inflammatory effusions. A subsequent sorting based on tumor histology showed increased levels particularly in exudates associated with metastatic tumors. Interestingly, proMMP-9 values in general correlated with CRP, a systemic marker of inflammation. Thus, MMP-9 proenzyme appears to complement traditional markers distinguishing pleural fluids of different origin. Yet, the differentiation between paraneoplastic and para-inflammatory exudates must be regarded with caution due to the presence of a high-expressive paraneoplastic sub-population, including effusions associated with metastatic tumors.
Biological Chemistry, Jan 5, 2001
A sandwich-type ELISA has been developed for quantification of the complex between the cysteine p... more A sandwich-type ELISA has been developed for quantification of the complex between the cysteine proteinase cathepsin Β (CB) and its reversible tight-binding inhibitor cystatin C (CC) in normal and pathological sera. The assay is based on a combination of catching Ab (3E1), raised against CB, and a horseradish peroxidase-labelled detection Ab (1A2), raised against CC. Only the CB/CC complex is able to evoke a signal in this assay. The detection limit of the assay was 15.5 ΠΜ and the working range between 31.3-200 nM. The within and between-run coefficients of variance (CV) varied from 4.7% to 9.4% and 11% to 12.8%, respectively, demonstrating satisfactory reproducibility of the method. The concentration of the CB/CC complex was determined in sera from 90 healthy controls, 32 patients with non-cancerous lung diseases, 148 patients with lung and 32 patients with colorectal cancer. The CB/CC complex was significantly less abundant in sera of patients bearing malignant lung tumours than in those with non-cancerous lung diseases or healthy controls (p<0.001). In colorectal cancer sera its level was significantly lower in advanced stages C and D than in early Dukes' stages A and Β (p=0.02). Our results show that the increased levels of CB in malignant sera are not impaired effectively by CC and support the hypothesis of hindered inhibitory capability during cancer progression.
International Journal of Biological Markers, 2000
Cathepsins B, H and L have been shown to participate in processes of tumor growth, vascularizatio... more Cathepsins B, H and L have been shown to participate in processes of tumor growth, vascularization, invasion and metastasis. Their levels in tumor tissue extracts can provide useful clinical information to predict disease-free and overall survival in breast, lung, colorectal, brain and head and neck cancer patients. Recently we have found that both cysteine cathepsins and their endogenous protein inhibitors stefins and cystatin C can also predict prognosis when measured extracellularly. In melanoma and colorectal cancer patients high serum levels of cathepsins B and H correlated with shorter survival. Similarly, increased extracellular levels of stefins A and B and cystatin C correlated significantly with high risk of adverse outcome in cancer patients. However, the cathepsin B/cystatin C complex was found to be less abundant in sera of patients with malignant tumors than in those with benign diseases or in healthy controls, suggesting an imbalance between the enzyme and its inhibitor in cancer patients.
The International Journal of Biological Markers, 2000
Cathepsins B, H and L have been shown to participate in processes of tumor growth, vascularizatio... more Cathepsins B, H and L have been shown to participate in processes of tumor growth, vascularization, invasion and metastasis. Their levels in tumor tissue extracts can provide useful clinical information to predict disease-free and overall survival in breast, lung, colorectal, brain and head and neck cancer patients. Recently we have found that both cysteine cathepsins and their endogenous protein inhibitors stefins and cystatin C can also predict prognosis when measured extracellularly. In melanoma and colorectal cancer patients high serum levels of cathepsins B and H correlated with shorter survival. Similarly, increased extracellular levels of stefins A and B and cystatin C correlated significantly with high risk of adverse outcome in cancer patients. However, the cathepsin B/cystatin C complex was found to be less abundant in sera of patients with malignant tumors than in those with benign diseases or in healthy controls, suggesting an imbalance between the enzyme and its inhibit...
Neoplasma, 2005
Gelatinase B/MMP-9 is a member of matrix metalloproteinases with a major role in extracellular ma... more Gelatinase B/MMP-9 is a member of matrix metalloproteinases with a major role in extracellular matrix degradation, cell proliferation and migration. Its proenzyme form has also been reported in pleural fluids as an inducible species, but its relation to pleural pathology has not yet been fully clarified. The primary goal of this study was to evaluate proMMP-9 as a potential marker for differentiating pleural effusions of both malignant and non-malignant origin. Pleural fluid samples were studied from 194 patients, including tumor etiology in 133 cases, inflammatory disorders in 33, transudates in 12, and unspecified disorders in 16 patients. The concentrations of proMMP-9 were estimated by means of immunoassays and/or by scanning zymography. Samples were also examined for C-reactive protein (CRP). The analysis of proMMP-9 showed significant differences among the etiological groups with the highest concentrations in para-inflammatory exudates, intermediate in para-neoplastic exudates...
International Journal of Oncology, Jul 1, 1994
Cancer 12 (1995) 113-160 investigated 109 lung adenocarcinomas, mostly small, peripheral, stage I... more Cancer 12 (1995) 113-160 investigated 109 lung adenocarcinomas, mostly small, peripheral, stage I tumours (X1/109) for presence of K-ras gene mutations at codons 12 and 13. Mutations were detected by denaturing gradient gel ekctrophoresis analysis of specitic sequences amplified by polymerase chain reaction from DNA extracted from archival pathological material. Thirty-three of 109 (30.3%) tumours showed mutations at codon 12 (28/33,84.8%) or 13 (5/33, 15.2%) ofthe gene. Mutations and type of nucleotide substitutions were differently distributed among cytological subtypes, being more prevalent among less differentiated (G2 and G3) tumours and among bronchial than bronchiole-alveolar type adenocarcinomas. Survival analysis showed an adverse effect of K-ras mutation on survival, restricted to stage I tumours. Median survival for 81 stage I patients was 30 months for non-mutated tumours versus 20 months for mutated tumours @ = 0.016). Muhivariate analysis showed that age of patient @ = 0.001) and K-ras mutation status @ = 0.04) were the only independent factors influencing survival signiBcanUy. These data strengthen the hypothesis that K-ras gene mutations may be useful in identifying a subgroup of patients with poor outcome.
Electronic journal of pathology and histology, 2001
Le procede et le dispositif decrits servent a determiner l'activite d'enzymes dans des li... more Le procede et le dispositif decrits servent a determiner l'activite d'enzymes dans des liquides de maniere largement automatique. Le dispositif de mise en oeuvre de ce procede comprend une colonne (1) qui contient un substrat chromatographique pour traiter un echantillon. Le substrat est melange avec une substance qui se lie a des inhibiteurs d'au moins un enzyme presents dans l'echantillon. Un dispositif d'amenee (2) des echantillons est associe a une extremite de la colonne (1). Un agencement pompe/soupape (7, 11, 14, 15) monte en aval de la colonne (1) dans le sens d'ecoulement de l'echantillon permet de remplir au moins une eprouvette (5) avec un substrat et au moins une partie de l'echantillon. Le substrat est decompose en produits de clivage par l'enzyme. Au moyen d'un detecteur, on determine l'augmentation de la concentration en au moins un produit de clivage du substrat par unite de temps pendant une periode d'incubation. En co...
British Journal of Cancer, 2001
Cathepsins have been shown to participate in dissolution and remodelling of connective tissue and... more Cathepsins have been shown to participate in dissolution and remodelling of connective tissue and basement membranes in the processes of tumour growth, invasion and metastasis (Sloane et al, 1994; Kos and Lah, 1998). Increased levels of Cats B and L in tumours and some extracellular fluids are associated with shorter disease-free and overall survival periods and may serve as prognostic factors for cancer patients (Kos and Lah, 1998). The role of another cysteine proteinase, Cat S in tumour progression is less understood. In contrast to Cats B and L, which are active at acidic pH, Cat S retains most of its enzymatic activity at pH 7.0-7.5. Another distinct feature is the substantial elastolytic activity it exhibits at neutral pH (Chapman et al, 1997). Cat S exhibits restricted tissue expression and is present in higher amounts in lymph nodes (Turenšek et al, 1975), spleen and antigen-presenting cells (APCs), including macrophages, dendritic cells and B lymphocytes (Shi et al, 1994; Morton et al, 1995). Its expression was found to be induced by cytokines, such as interferon γ and interleukin 1β (Chapman et al, 1997). Cat S activity appears to be essential in the MHC class II antigen presentation pathway. It specifically degrades the invariant chain (Ii), a MHC class II chaperone, prior to its removal from the MHC class II peptide-binding cleft. This facilitates the loading of antigenic peptides to MHC class II αβ-dimers and subsequent transportation of the complex to the cell surface to initiate MHC class II restricted CD4 + T-cell recognition (Chapman et al, 1997; Pierre and Mellman, 1998). Besides participation in the immune response some other specific functions, associated with high elastolytic activity of Cat S have been proposed. For example, Cat S has been suggested to promote motility of the cilia of conducting airway cells in the lung (Chapman et al, 1997). Additionally, it was found to participate in vascular matrix remodelling and in the formation of the atherosclerotic intima (Sukhova et al, 1998). In rat, Cat S is expressed in thyroid tissue, where a role in processing thyroglobulin and release of thyroid hormone has been proposed (Petancheska and Devi, 1992). Increased expression of Cat S has also been associated with the pathogenesis of Alzheimer disease (Lemere et al, 1995). The aim of the present study was to evaluate the role this enzyme might have in the progression of lung cancer. For this purpose a quantitative method for measuring the level of Cat S in tissue extracts and sera of lung cancer patients was developed and the levels have been tested for their relationship to clinical features, considering especially the correlation with the survival rate for cancer patients. Additionally, the cell types, expressing Cat S in tumours, lung parenchyma and lymph nodes, have been evaluated by IHA. MATERIALS, METHODS AND PATIENTS Antigen, antibodies Human pro-Cat S was produced in E. coli using an inducible T7based expression system (Kopitar et al, 1996
British Journal of Cancer, 1997
In this study we examined both the pH dependence of cathepsin B (cath B) activity and its stabili... more In this study we examined both the pH dependence of cathepsin B (cath B) activity and its stability at physiological pH of 7.5 in lung tumours and normal lung tissue by means of fluorogenic assays with Z-Arg-Arg-AMC as specific substrate. Specificity was verified with the cath B blocking inhibitors E-64 and CA-074. With respect to pH dependence of activity, we found a deviation from a normal-shaped pHactivity curve. Besides the typical activity peak at pH 6.0, there were shoulders at pH 4.5-5.5 and at pH 7.0-7.5. This heterogeneity was found in both tumour and normal tissue. To test the stability of cath B at physiological pH of 7.5, homogenates were kept at pH 7.5 for 60 min. Altogether, 82-100% of residual cath B activity was found at pH 5.0-5.5, whereas activity in the range between 5.5 and 7.4 dropped drastically to 26-42%. At pH 7.5, there was still 20-34% residual cath B activity detectable. To test the hypothesis whether the cath B fraction active at pH 7.5 is more abundant in tumour tissues compared with the normal counterparts, we determined this fraction in 91 pairs of lung tumour and normal lung tissue. We found a 2.3-fold increase of median cath B fraction active at pH 7.5 in tumour tissue, although this fraction represented only a small part (about 16%) of the native, acidic (pH 6.0) cath B activity. However, in contrast to native cath B at 6.0, the cath B fraction active at pH 7.5 was related to post-operative probability of survival in curatively operated patients, since activity values higher than 292 (,uEU mg-' protein) were significantly associated with poor prognosis in patients with squamous cell carcinomas (n = 33, P = 0.04). It is concluded that in lung tumour and in normal lung tissue, cath B activity can be divided into at least three fractions with stability optima at different pH values, indicating various forms of cath B. The cath B fraction active at pH 7.5 provides prognostic information in patients with squamous cell carcinoma.
British Journal of Cancer, 1999
In order to evaluate the possible role of the proteolytic enzyme cathepsin B (cath B) in human no... more In order to evaluate the possible role of the proteolytic enzyme cathepsin B (cath B) in human non-small cell lung cancer (NSCLC) we examined cath B concentrations (cath B c) and activities (cath B A) in homogenates of 127 pairs of lung tumour tissues and corresponding nontumourous lung parenchyma. Total cath B activity (cath B AT) and enzymatic activity of the fraction of cath B, which is stable and active at pH 7.5 (cath B A7.5) were determined by a fluorogenic assay using synthetic substrate Z-Arg-Arg-AMC. The immunostaining pattern of cath B was determined in 239 lung tumour tissue sections, showing the presence of the enzyme in tumour cells (cath B T-I) and in tumour-associated histiocytes (cath B H-I). The median levels of cath B AT , cath B A7.5 and cath B C were 5.6-, 3.2-and 9.1-fold higher (P < 0.001), respectively, in tumour tissue than in non-tumourous lung parenchyma. Out of 131 tissue sections from patients with squamous cell carcinoma (SCC), 59.5% immunostained positively for cath B, while among the 108 adenocarcinoma (AC) patients 48.2% of tumours showed a positive reaction. There was a strong relationship between the levels of cath B AT , cath B A7.5 , cath B C and cath B T-I in the primary tumours and the presence of lymph node metastases. Significant correlation with overall survival was observed for cath B T-I and cath B A7.5 (P < 0.01 and P < 0.05, respectively) in patients suffering from SCC. In these patients positive cath B in tumour cells (cath B T-I) and negative cath B in histiocytes (cath B H-I) indicated significantly shorter survival rate compared with patients with negative cath B T-I and positive cath B H-I (P < 0.0001). In contrast, in AC patients, both, positive cath B T-I and positive cath B H-I , indicated poor survival probability (P < 0.014). From these results we conclude that the proteolytic enzyme cath B is an independent prognostic factor for overall survival of patients suffering from SCC of the lung.
Radiology and Oncology, Jan 6, 2002
Advances in Experimental Medicine and Biology, 1997
British Journal of Cancer, 2000
In order to evaluate the role of cysteine peptidase cathepsin H (Cath H) in human lung cancer its... more In order to evaluate the role of cysteine peptidase cathepsin H (Cath H) in human lung cancer its protein levels were determined in 148 pairs of lung tumour tissue and adjacent non-tumourous lung parenchyma using the enzyme-linked immunosorbent assay technique. Additionally, Cath H levels were determined in sera of 171 patients with malignant tumours, 34 patients with benign lung diseases and 47 healthy controls. The median level of Cath H in tumour tissue was 0.64 times that in the corresponding lung parenchyma. Relating tumour levels with histological type we found higher Cath H levels in small-cell and adenocarcinomas and lower levels in squamous cell carcinoma, large-cell carcinoma and secondary tumours. A significant difference in Cath H level between lung tumour tissue and non-tumourous lung parenchyma was associated with the group of cigarette smokers (156 vs 263 ng mg-1 protein, P < 0.001). For this group of patients Cath H tumour levels correlated with the survival rate, while for the entire patient population this was not the case. Smokers with high tumour levels of Cath H experienced poor survival. Cath H was significantly higher in sera of patients with malignant and benign lung diseases than in control sera (P < 0.001). The increase was significant for all histological types, being the highest in small-cell and squamous cell carcinomas. Our study reveals that in lung tumours there is different behaviour of Cath H compared with other cysteine peptidases, e.g. cathepsin B and cathepsin L. Variations between tissue and serum levels of Cath H indicate either reduced expression or enhanced secretion of this enzyme in lung tumours.
Biological Chemistry Hoppe-Seyler, 1995
In a series of pairs of lung tumor tissue and non-tumor lung parenchyma from 50 patients, the act... more In a series of pairs of lung tumor tissue and non-tumor lung parenchyma from 50 patients, the activity of cathepsin L was measured with Z-Phe-Arg-AMC using the inhibitor CA-074 to delimitate from cathepsin B activity also present in the tissue extracts. Cathepsin B was assessed in the same samples with its specific substrate Z-Arg-Arg-AMC. It was found that in tumor tissue the median activities of cathepsin L and cathepsin B were increased 1.6-fold and 4.9-fold, respectively. The levels of activity of both enzymes did not correlate with TNM stages nor with cell differentiation of bronchial carcinomas. Cathepsin L activity was found to be insignificantly higher in adenocarcinoma compared to squamous cell carcinoma, while cathepsin B activity did not vary across the histologies. The activities of both enzymes were low in pulmonary carcinoids, which are known to be low-grade malignant neoplasms. The amount of cathepsin B activity exceeded by far that of cathepsin L activity as proven by measurement with Z-Phe-Arg-AMC in the presence of the inhibitor Z-Phe-Phe-CHN 2 : 95-98% of cathepsin B activity vs 2-5% of cathepsin L activity were determined. By SDS-PAGE separation and immunoblot analysis, it could be demonstrated that significant amount of cathepsin L is complexed with the cysteine proteinase inhibitor kininogen. This explains the rather low cathepsin L activity values in the tissue extracts.
Anticancer research
To evaluate the possible role of cysteine proteases and serine proteases, as well as their respec... more To evaluate the possible role of cysteine proteases and serine proteases, as well as their respective inhibitors and receptors, as new prognostic factors in NSCLC, we examined, for the first time, 10 biological parameters related to three proteolytic systems within a homogeneous collective of 147 cases of NSCLC. Activities (cath B(AT), cath B(A7.5)) and protein levels of cath B(C), cath L(C), uPA, PAI-1, uPAR [measured by three different assays uPAR (ADI), uPAR (HD13), uPAR (IIIF10)] and TF were measured in homogenates of lung tumour tissue and corresponding non-malignant lung parenchyma. Total cath B activity (cath B(AT)) and enzymatic activity of the fraction of cath B, which is stable and active at pH 7.5 (cath B(A7.5)), were determined by a fluorogenic assay using synthetic substrate Z-Arg-Arg-AMC. The concentrations of cath B(C), cath L(C), uPA, PAI-1, uPAR and TF were determined by ELISAs. uPAR was determined using three different ELISA formats. The median levels of cath B(AT)...
Anticancer research
Secreted pro-cathepsin B of HS-24 human lung-tumour cells, human alveolar macrophages and Wi-38 h... more Secreted pro-cathepsin B of HS-24 human lung-tumour cells, human alveolar macrophages and Wi-38 human lung-fibroblast cells was pre-purified by ion exchange chromatography and investigated by 2D gel electrophoresis. Four (Wi-38), six (HS-24) and ten (alveolar macrophages) polypeptides differing in charge, but with the same molecular mass of 45 kDa, were found. The isoelectrical points of these polypeptides ranged from 5.43 to 6.57. Deglycosylation reduced the mass (7 kDa) but did not change the charge pattern. This investigation established cell type-specific patterns of secreted pro-cathepsin B-forms, but only parts of these may be cell type-specific forms depending on differentiated expression of mRNA and post-translational modification.