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.

Cathepsin X, a recently discovered lysosomal cysteine protease, shares common structural features and activity properties with cysteine protease cathepsin B. Based on its widespread mRNA distribution in primary tumors and tumor cell lines, a redundant function in tumor progression has been proposed. In this study, we have shown that these two related proteases exhibit different profiles with respect to their protein distribution in cells and tissues and to their possible roles in malignancy. Protein level of cathepsin X did not differ significantly between matched pairs of lung tumor and adjacent lung tissue obtained from patients with lung cancer whereas that of cathepsin B was 9.6-fold higher in tumor compared to adjacent lung tissue. Immunohistochemical analysis of lung tumor cathepsin X revealed very faint staining in tumor cells but positive staining in infiltrated histiocytes, alveolar macrophages, bronchial epithelial cells, and alveolar type II cells. Cathepsin X stained positive also in CD68+ cells in germinal centers of secondary follicles in lymph nodes, corresponding to tingible body macrophages. Two cell lines with proven invasive behavior, MCF-10A neoT and MDA-MB 231, showed positive staining for cathepsin B, but negative for cathepsin X. We showed that the invasive potential of MCF-10A neoT cells can be impaired by specific inhibitor of cathepsin B but not by that of cathepsin X. Cathepsin X was found in large amounts in the pro-monocytic U-937 cell line, in monocytes and in dendritic cells, generated from monocytes in vitro. Our results show that cathepsin X is not involved in degradation of extracellular matrix, a proteolytic event leading to tumor cell invasion and metastasis. Its expression, restricted to immune cells suggests a role in phagocytosis and the regulation of immune response.

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) (5.1-fold), cath B(A7.5) (2.5-fold), cath B(C), (8.5-fold), cath L(C) (6.6-fold), uPA (6.5-fold), PAI-1 (4.2-fold), uPAR (ADI) (2.2-fold), uPAR (HD13) (4.0-fold) and uPAR (IIIF10) (2.6-fold) were higher in tumour tissue compared to the lung parenchyma. Cath B(AT), cath B(A7.5) and cath B(C) in primary tumours correlated with lymph node metastases. Regarding histologies, the concentration of PAI-1 seems to be associated with the histological cell types of NSCLC. We found the highest values of PAI-1 in large cell carcinoma > SCC, AC > carcinoid and lowest values in metastases of primary tumours of other organs. Only PAI-1 was significantly increased in poorly-differentiated cells (G3) compared to well- and moderately- differentiated cells (G1/G2). PAI-1 significantly correlated with cath B(AT) and cath B(A7.5) with uPAR (ADI), uPAR (HD13), uPAR (IIIF10) with uPA, and only weakly with TF, but not with cath B(C) and cath L(C). Significant correlations with overall survival in the total population of NSCLC patients were observed in univariate analysis for cath B(AT), cath B(C), PAI-1, uPAR (ADI), uPAR (HD13), and uPAR (IIIF10). Cath L(C) was not significantly associated with poor prognosis. Regarding the histological tumour type, only in patients with squamous cell carcinomas did cath B(A7.5) and PAI-1 remain significant prognostic factors. In multivariate survival analysis only two proteolytic factors, PAI-1 and uPAR (III101F), stayed significant. In conclusion, among 10 biological parameters evaluated within the same cohort of patients, only PAI-1, uPAR (ADI), uPAR (HD13), uPAR (IIIF10), cath B(AT) and cath B(C) are prognostic factors for overall survival of NSCLC patients. Moreover, PAI-1 and uPAR (IIIF10) add independent prognostic information with regard to established clinical and histomorphological factors in NSCLC.

AIMS:To immunohistochemically investigate the expression of cathepsin B and cathepsin L in various lung cancer cell types in association with the patients' prognosis.MATERIALS AND METHODS:Histological slides obtained from formalin-fixed, paraffin-embedded tissue of 120 potentially curative resected lung cancer specimens (20 cases of each of the following cell types: squamous cell, adeno, large cell anaplastic, small cell anaplastic, intrapulmonary metastases, mesotheliomas) were quantitatively immunohistochemically analysed with an automated image analysing system and correlated with the patients' prognosis and the tumour proliferation rate measured by the expression of Ki-67.RESULTS:The expression of cathepsin B was most frequently present in large cell anaplastic carcinomas and missing in small cell carcinomas. That of cathepsin L was less frequently seen, and mainly expressed in macrophages and adenocarcinoma tumour cells. The structural heterogeneity of cathepsin B expression measured by syntactic structure analysis was greater than that of cathepsin L. The expression of cathepsin B is of prognostic significance in non-small cell lung cancer, and ranges directly after the pN and pT stages in multivariate statistical analysis.CONCLUSION:In concordance with the literature, the expression of cathepsin B in non-small cell lung cancer should be considered as a significant prognostic parameter in contrast to that of cathepsin L, which is not related to the patients' outcome at a statistically significant level.

Abstract Secretory type 2 cystatins, like cystatins C, E/M and F, are thought to be involved in many pathobiological processes, including vascular amyloidosis, rheumatoid arthritis, Alzheimers disease, osteoporosis, viral and bacterial infections, inflammatory disorders and tumour invasion and metastasis. In order to define the levels of cystatins C, E/M, and F in pleural effusions and to investigate whether these cystatins correlate with diagnostic parameters of pleural and lung diseases, we determined their concentrations in 160 pleural effusions. The median concentration of cystatin C in pleural effusions was 1437 ug/l (95.8 nM), ranging between 18-3967 ug/l. Cystatin C did neither correlate with malignant nor with benign diseases. The concentration of cystatin E/M was significantly higher in effusions of primary pleural tumours (mesotheliomas) compared to secondary pleural tumours and benign diseases. Furthermore, there was a significant correlation between the concentration of cystatin E/M of mesotheliomas and the pleural fluid tumour cell count and of cystatin C. The median values of cystatin F were significantly increased in parapneumonic/ empyema thoracis pleural effusions and tuberculous pleurisy compared to malignant pleural effusions, respectively. The concentration of cystatin F in benign effusions correlated significantly with diagnostic parameters and inflammation (total protein; lactate dehydrogenase; C-reactive protein). Finally, only in the group of parapneumonic/empyema thoracis was there a significant correlation between cystatin F and the neutrophil count. In conclusion, pleural effusions of different origin contain high levels of cystatin C, perhaps constituting the major part of an inhibitor reservoir. The level of cystatin E/M appears to be significantly associated with primary pleural tumours and cystatin F correlates with inflammatory processes of lung disorders.

To assess whether cancer-induced pleurisy is associated with an alteration of nitric oxide (NO)-synthase activity, the levels of nitrate/nitrite (NOx) were measured in blood serum (BS) and pleural effusion (PE) of 35 cancer patients (secondary pleural metastases and mesotheliomas), eight patients with benign lung diseases, and in BS of nine healthy donors. It was found that (1) BS of patients with secondary pleural metastases had an elevated level (P < 0.015) of NOx (59.7 +/- 24.4 mu M, n = 28) in comparison with control level of BS for healthy donors (43.4 +/- 13.5 mu M, n = 9); (2) BS of mesotheliomas (32.1 +/- 12.2 mu M, n = 4) had significantly (P < 0.05) lower level of NOx compared to BS of benign patients (61.2 +/- 28.8, n = 6); (3) differences in mean levels of NOx in BS and same PE of examined patient groups did not reach statistical significance, excepting sub-group of patients with primary mammary carcinoma; (4) significant interindividual differences of NOx in all groups of patients were revealed; (5) fluids from about 11% of cancer patients contained extremely high levels of NOx over 100 muM; (6) a significant elevation of apparent NOx level in BS and PE of patients with secondary pleural metastases in comparison with those in BS of healthy donors was revealed when the native, i.e. protein-contained, samples were managed with Griess reagent.The results described here, point up the diverse role of NOx in cancer patients. Its role is far from being clear but it seems that NOx acts as a signaling mediator during the formation of pleural metastases and might be considered as a non-specific marker in the corresponding PE. Furthermore, NOx could be used to give rationale of proper application of anticancer drugs affecting diversely NO-synthase activity in cells. Besides, a casual effectiveness of NOx measurements in native samples from cancer patients using Griess reagent needs additional elaboration. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

The meeting was held in Heidelberg in July, 2001 and provided the basis for a European cooperation in molecular research focused on translational research in lung cancer. During this meeting, molecular and immunological analysis was discussed as the basis for the development of successful future cancer treatment strategies.R. Rosell, M. Monzó and M. Taron reported on new biotechnological and molecular approaches in a European framework for lung cancer management.Novel methods for diagnosis and management of cancer are being developed today and the most rapidly evolving field is oncogenomics. Oncogenic transformation confers resistance to chemotherapy through a variety of mechanisms including suppression of apoptosis, increased drug metabolism and modification of target proteins. Extensive characterization of genetic alterations in lung cancer can provide rapidly assessable repertoires that can be used for clinical stratification in phase II–III studies. For the sake of simplicity, these repertoires can be classified in five categories: allelic imbalance, aberrant promoter methylation, gene RNA overexpression, microtubule alterations and polymorphisms. Interest at the meeting was centred on ERCC1 mRNA levels as a direct mechanism of cisplatin resistance that could be exploited in customised chemotherapy. European collaboration in molecular research will rely on two simple principles: serial measurements of serum/plasma tumour DNA burden at baseline and after three and six cycles of chemotherapy and very importantly, the isolation of RNA, not only from paraffin-embedded tissue, but also from serum and plasma. Some ongoing Spanish studies are looking at serum RNA. We have successfully met the first challenge of sending blood samples for RNA extraction from geographically distant institutions. Now, we are performing RNA assessment of potential key markers, such as ERCC1, SXR (steroid xenobiotic receptor), ribonucleotide reductase M2 subunit and α- and β-tubulin, which can provide some clues to the prediction of chemotherapy response in individual patients.J. Niklinski, L. Chyczewski, J. Laudanski and W. Niklinska presented the state of advancement of the ongoing projects in Bialystok, Poland.They first described the organisation of the multidisciplinary team, comprising surgeons, molecular biologists, pathologists, pneumonologists and oncologists. Dr Niklinski then presented the method of sample collection, basically from patients treated by surgery. The samples consisted of tissue specimens, serum and nucleated cells (lymphocytes) from the blood; the collection is performed in conditions allowing analyses at both DNA and RNA levels. He then presented a short review of their evaluation of critical genes involved in lung cancer; he also provided information about their different possibilities to analyse p53 consisting of immunohistochemistry, ELISA for p53 antibodies, indirect and direct sequencing of the gene and finally a yeast functional assay. Dr Niklinski commented that some approaches have a significant number of limitations; presently it seems that direct sequencing is the most adequate way to study p53, not only the region comprised between exons 5 and 8, but also exons 4 and 11, in which a significant number of mutations can be determined. A promising new approach seems to be the development of a functional yeast assay, since some preliminary reports mention a 70% sensitivity; however, more information and complete follow-up data are needed, especially in relation to prognosis. Dr Niklinski then spoke about the alteration, both genetic and epigenetic, of the p16 gene. Point mutations appear to be rather rare events, but LOH and methylation of the promoter region (determined by MS-PCR) are rather common in established lung cancer and during its premalignant stages. They should be taken into consideration for the multicenter evaluation. He then talked about ras evaluation, emphasising that its clinical significance is not clear today. Sensitive approaches based on the enriched PCR technique are available and by using this sensitive technique, it would be worth a multiple evaluation of the gene. Finally, Dr Niklinski gave a short presentation about molecular evaluations in premalignant conditions, in order to obtain a clear definition of premalignant changes prone to evolve to malignancy. It seems that laser microdissection techniques are the most correct to study molecular alterations in premalignant changes of the lung.H. Lahm and J.R. Fischer presented their program on immunological and molecular markers for the assessment of genetic and epigenetic alterations in lung cancer.Alterations of the DNA by mutations, deletions or epigenetic modification, as well as dysregulation of gene expression determine the biological behavior and the response of neoplastic lung cells significantly. The Immunology-Molecular Biology Laboratory (IML) at the Thoraxklinik Heidelberg gGmbH undertakes efforts to determine these effects both in the periphery and at the tumour site. Plasma/serum DNA serves as a source to detect mutations (K-RAS, β-tubulin, p53) and hypermethylated promoter regions (RARβ, p16INK4a, DAPK, MGMT, FHIT, RASSF1A). In addition, the immunological status of the patients will be assessed by determination of cytokine secretion (IL-2, IL-6, IL-10, TNFα) by peripheral blood lymphocytes via ELISA. Earlier investigations of this laboratory have established the predominant prognostic significance of impaired cytokine secretion for survival of patients with SCLC and NSCLC. Several lines of evidence suggest that this cytokine suppression is caused by immunosuppressive factors secreted by tumour cells. Furthermore, investigation of tumour material will include an analysis of mutations, the status of hypermethylation, a quantitative assessment of gene expression (IL-10, COX-2, ERCC1, TGFβ1, telomerase) by means of a real-time RT-PCR analysis. A careful comparison with the results obtained in the periphery will be performed. All results will be stored in a central database, which will enable a correlation with any relevant parameters of the patients (therapy, survival). A particular interest will be given to the combination of several factors with respect to the patients’ situation. The work is ultimately focussed on obtaining an individual pattern of molecular and immunological markers, which may serve as the basis to propose a customised and improved therapy for each patient.N. van Zandwijk, L. van't Veer, P. Baas and W.J. Mooi proposed a program for analysis of prognostic factors for non-small cell lung cancer by micro-array techniques in a joint project.For more than 30 years, lung cancers for therapeutic purposes have been subdivided into two groups: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The majority (±80%) of lung cancers are of the NSCLC group, which comprises carcinomas with squamous, adeno- and some without a specific differentiation (large cell carcinomas). Although there is evidence that squamous cell lung cancers have a somewhat different metastatic pattern when compared with those lacking squamous differentiation, the histological subdivisions within the heterogenous group of non-small cell lung cancers bear no major prognostic significance. Taking also into account the inability to predict a successful outcome of surgery—only ≈50% of patients, who currently undergo surgery for NSCLC with curative intent will be alive after 5 years—there is an urgent need for prognostic factors that can reliably predict long term survival and/or the emergence of metastatic disease.

Cysteine proteinase cathepsin S (Cat S) is expressed mainly in lymphatic tissues and has been characterised as a key enzyme in major histocompatibility complex class II (MHC-II) mediated antigen presentation. Cat S has been measured in tissue cytosols of lung parenchyma, lung tumours and lymph nodes and in sera of patients with lung tumours and of healthy controls, by specific enzyme-linked immunosorbent assay (ELISA). A difference in Cat S level was found between tumour and adjacent control tissue cytosols of 60 lung cancer patients (median 4.3 vs. 2.8 ng mg –1 protein). In lymph nodes obtained from 24 patients of the same group, the level of Cat S was significantly higher than in tumours or lung parenchyma ( P < 0.001). Additionally, significantly higher levels were found in non-infiltrated than in infiltrated lymph nodes (median 16.6 vs 7.5 ng mg –1 protein). Patients with low levels of Cat S in tumours and lung parenchyma exhibited a significantly higher risk of death than those with high levels of Cat S ( P = 0.025 – tumours; P = 0.02 – parenchyma). Immunohistochemical analysis (IHA) of lung parenchyma revealed a staining reaction in alveolar type II cells, macrophages and bronchial epithelial cells. In regional lymph node tissue, strong staining of Cat S was found in lymphocytes and histiocytes. Nevertheless, Cat S was detected also in tumour cells, independently of their origin. Our results provide evidence that Cat S may be involved in malignant progression. Its role, however, differs from that of the related Cats B and L and could be associated with the immune response rather than with remodelling of extracellular matrix. © 2001 Cancer Research Campaign http://www.bjcancer.com

A sandwich-type ELISA has been developed for quantification of the complex between the cysteine proteinase cathepsin B (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 nM 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 B (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.

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.

BACKGROUND. Tumor cells require specific proteolytic enzymes for invasion and metastasis, including lysosomal peptidases-cathepsins. Cathepsin B is a lysosomal cysteine peptidase, which appears to play a major role in invasion and metastasis of human turners. In this study, the authors focused on the possible role of cathepsin B in lymphogenic metastasis by investigating the enzyme localization and its activity in lung tumors and corresponding tumor-infiltrated lymph nodes.METHODS. Cathepsin B activity was determined in lung turners, lung parenchyma, and tumor cell-infiltrated and noninfiltrated regional lymph nodes of the same patient. The authors investigated 35 cancer patients suffering from nonsmall cell lung carcinoma. Cathepsin B throughout activity was measured by cleavage of the fluorogenic substrate Z-Arg-Arg-AMC at pH 6.0.RESULTS. The median specific cathepsin B activity was highest in tumors, followed by the infiltrated lymph nodes, noninfiltrated lymph nodes, and lung parenchyma. The authors showed a significant 1.8-fold increase in cathepsin B activity in turner-infiltrated lymph nodes compared with noninfiltrated regional lymph nodes and a 4.5-fold increase in lung tumor tissue compared with lung parenchyma. High cathepsin B activity, both in tumors and tumor cell-infiltrated lymph nodes, indicated poor prognosis for overall survival. Immunohistochemical analysis showed the presence of cathepsin B in histiocytes and tumor cells but not in lymphocytes of lymph node tissue.CONCLUSIONS. The authors' findings on higher cathepsin B levels in tumor cell-infiltrated lymph nodes show that increased level of cathepsin B activity is characteristic of the invasive tumor cell phenotype. This corroborates the hypothesis, that tumor cell associated cathepsin B may play a role in lymphogenic metastasis. The authors' results support the use of lymph node associated cathepsin B as a prognostic factor for survival of patients with lung carcinoma. Cancer 2000;89: 2282-91. (C) 2000 American Cancer Society.

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–1protein, 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. © 2000 Cancer Research Campaign

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 non-tumourous 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.

We established a continuous semi-microassay, and for large-scale studies both a stopped and a continuous microtiter plate assay for the fluorometric determination of cathepsin L and cathepsin S activities in body fluids, tissues or cell extracts in the presence of cathepsin B. For the detection of enzymatic activities we used the synthetic substrate Z-Phe-Arg-AMC, and for discrimination between cathepsin L, S and cathepsin B the specific inhibitor CA-074 for blocking interfering cathepsin B activities was applied. Furthermore, we took advantage of the stability of cathepsin S at pH 7.5 for further differentiation between cathepsin L and cathepsin S activities. The kinetic assays were characterized in terms of imprecision, analytical sensitivity, accuracy and substrate concentration. The within-run coefficients of variation were found to be 4.9%-7.2% for the continuous semi-microassay, 10.3%-11.7% for the stopped, and 4.5%-11.8% for the continuous microtiter plate assay. The between-days coefficients of variation for the continuous semi-microassay were 8.1%-8.9%, while for the stopped and continuous microtiter plate assays the coefficients were 11.2%-13.5% and 5.8%-12.2%, respectively. Compared to the continuous semi-microassay, the stopped and the continuous microtiter plate assays showed 3-fold and 11-fold higher sensitivity, respectively. Comparison between the continuous enzyme activity assays at substrate concentrations of 40 microM and 200 microM demonstrated a significant correlation of r = 0.97 and r = 0.99, respectively. The newly developed microtiter plate assay will allow efficient, sensitive and high precision determination of cathepsin L and cathepsin S activities in large-scale studies of cysteine-cathepsin dependent diseases.

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 pH- activity 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 (muEU mg(-1) 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.

In human lung tumor tissue specimen (n=73) concentrations of stefins A and B were found to be increased 2.0-fold (p<0.01) and 1.3-fold (p<0.01), respectively, as compared to matched normal tissue. Stefin A and B concentrations were higher in primary tumors than in secondary tumors, i.e. metastases from other organs to the lung (p<0.01; p<0.05, respectively). Cystatin C concentrations were rather low and did not differ between tumor and normal tissue. Both concentrations of stefins did not correlate with TNM stages. Stefin A was higher in squamous cell carcinoma than in adenocarcinoma (p<0.01), while stefin B did not show such a difference. At investigation of a relationship between survival probability of patients with primary tumors it was found that increased stefin B concentrations and total cysteine-protease-inhibitory activities but not stefin A concentrations were positively correlated with survival probability.It is concluded that stefins A and B are major contributors to the cysteine protease inhibitory activity in primary lung tumors. Stefin B proved to be a prognostic factor, especially in squamous cell carcinoma.

The levels of cathepsins were determined in 77 matched pairs of human lung tumors and normal lung parenchyma. Cathepsin B and cathepsin L activities were assayed using fluorogenic substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC, respectively. The concentrations of the cathepsins were measured by ELISAs. Activities and concentrations of both cathepsins were significantly increased (p<0.001) in primary and secondary tumors. However, the levels, of the cathepsins did not correlate with established indicators of malignancy, i.e. pTNM stages and cell differentiation. There was also no difference in cathepsin levels between squamous cell carcinomas and adenocarcinomas. In respect of cathepsin expression and overall survival probability only cathepsin L concentration was related to survival time, since increased values were significantly associated with poor prognosis (p=0.01). From this study of lung tumor tissues it is concluded that in spite of overexpression of both cathepsins B and L, an association with tumor progression can only be established for cathepsin L concentration.

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.