Cathepsin B activity in human lung tumor cell lines: ultrastructural localization, pH sensitivity, and inhibitor status at the cellular level.

We investigated the appearance and activity of the cysteine proteinase cathepsin B and its physiological inhibitors, stefins A and B, at the cellular level in human tumor cell lines HS-24, derived from a primary lung tumor (squamous cell), and SB-3, derived from a metastasis (lung adenocarcinoma). In addition to cathepsin B, these tumor cells also expressed the immunologically and functionally related cathepsin L, but not cathepsin H. Stefin A was found in HS-24 but not in SB-3 cells; stefin B was found in both cell types. Using a specific fluorogenic cytochemical assay, the intracellular activity of the enzyme was localized and quantified. Thus, the cellular cathepsin B kinetics for the synthetic substrates Z-Arg-Arg-4M beta NA and Z-Val-Lys-Lys-Arg-4M beta NA, its pH dependence and inhibition by E64, stefins A and B, and cystatin C could be determined. From these measurements it appeared that the enzyme exhibited different cleavage rates for these substrates in the different cell types, showed considerable cleavage activity at neutral pH, which was stable under these conditions for extended time periods, and was highly sensitive to the inhibitors E64 and cystatin C but was considerably less sensitive to stefins, particularly stefin A. By conventional light microscopy, confocal laser scanning microscopy, and electron microscopy the enzymatic activity was localized in lysosomes, as expected, but also in the endoplasmic reticulum, nuclear membrane, and plasma membrane. The endoplasmic reticulum is a site at which only pre-mature enzyme forms exist, which are usually not active. The appearance of enzymatic activity at the plasma membrane confirms earlier biochemical and immunofluorescence microscopic investigations. The different sites of localization within the cells make it likely that different forms of the enzyme are expressed simultaneously, which follow alternate ways of processing and sorting. Taken together, the results support an involvement of the enzyme under extracellular conditions in degradative processes.

Prognostic value of increased lung tumor tissue cathepsin B.

In the study of 65 matched pairs of lung tumor tissue and normal lung parenchyma, cathepsin B (CB) activity was found to be increased about 4.6-fold, when regarding median levels. CB activity was found to be insignificantly higher in adenocarcinoma compared to the other histologic cell types. CB levels did not correlate with tumor stages (TNM) nor with cell differentiation, while increased CB activity was related to shorter survival rates of the patients. It is concluded that lung tumor CB is of some prognostic value for the outcome of the malignant disease.

Cathepsin B and cysteine proteinase inhibitors in bronchoalveolar lavage fluid of lung cancer patients.

The enzymatic activity and the concentration of cathepsin B (CB), determined by ELISA, and total inhibitory activity of cysteine proteinase inhibitors (CPIs) were measured in bronchoalveolar lavage fluid (BALF) of lung tumor patients (n = 49). Significantly higher CB activity and concentration was found in BALF from metastasis (n = 15), when compared to squamous cell carcinoma (SCC; n = 15) and small cell lung carcinoma (SCLC; n = 7). Patients with adenocarcinoma (n = 12) also secreted considerably more CB, about 14- and 3.3-fold, compared to SCC and SCLC patients, respectively. However, only tumor patients without inflammation were considered because the comparison of patients with lung tumors (n = 49) and with other non-neoplastic lung diseases (n = 18) showed no differences due to high CB in BALF of patients with inflammatory conditions, present in some patients from both groups. Immunolabeling of tumor BALF proteins revealed CB immunoreactivity at molecular sizes that corresponded to the size of its precursor and mature forms, as well as to complexes with kininogen(s) and low molecular weight CPIs. Stefins A and B, but not cystatins C and S appeared in the complexes with CB.

Cell interactions and motility in human lung tumor cell lines HS-24 and SB-3 under the influence of extracellular matrix components and proteinase inhibitors.

The human NSCLC cell lines HS-24 (squamous cell carcinoma) and SB-3 (metastasis derived from an adenocarcinoma) were investigated in respect to cell interactions, motility and invasive properties. HS-24 revealed high self adhesion capacity. Testing the interactions with collagens type I/III or IV, laminin and fibronectin by adhesion, non directional motility and haptotaxis assays, tight interactions and stimulation, particularly with collagen type I/III, was detected. Proteinase inhibitors (E64, Stefin A or leupeptin) revealed a slightly negative influence. Invasion in vitro of lung explants was reduced by leupeptin in a dose dependent manner and slightly increased by plasmin. SB-3 cells revealed low self adhesion. As judged from interaction with fibronectin, these cells have low integrin receptor concentrations and thus reduced adhesiveness to extracellular matrix. Collagen type I/III was inhibitory for undirectional motility and not permissive for haptotaxis. Therefore, it may play a restrictive role during the spread in vivo of these cells. Colonization of lung explants was low and was not influenced by cathepsin B proteinase inhibitors. The results emphasize a particular role of collagens for primary site tumor and metastasis development.

Alpha 1-proteinase inhibitor and mucus proteinase inhibitor in human lung emphysema.

The role of the antiproteases alpha 1-proteinase inhibitor (alpha 1PI) and mucus proteinase inhibitor (MPI) in human lung emphysema was investigated by measuring their amount and functional activity against trypsin, leukocyte elastase, and pancreatic elastase in the bronchoalveolar lavage fluid (BALF). In addition, leukocyte elastase was quantified in the lavage samples by measuring the concentration of the elastase-alpha 1PI-complex. The study population consisted of 38 patients (5 nonsmokers, 8 former smokers, 25 smokers) with acquired emphysema (i.e., emphysema which is not caused by alpha 1PI deficiency), and 44 individuals (16 nonsmokers, 8 former smokers, 20 smokers) without emphysema. No differences were found between patients with and without emphysema in the activities of alpha 1PI and MPI, or in the concentration of alpha 1PI. The concentration of MPI was significantly higher in the BALF of patients with emphysema than in that of patients without emphysema (p = 0.025). A significantly higher concentration of elastase-alpha 1PI complex was found in patients with emphysema than in those without emphysema (p = 0.041). This finding could reflect the higher proteinase burden to which patients with emphysema are exposed. The increase of MPI in lavage fluid of patients with emphysema seems to be the result of increased production in emphysematous lungs. However, it remains unclear why patients develop emphysema while showing an increased content of MPI.

Establishment of an enzyme-linked immuno-sorbent assay for urinary trypsin inhibitor by using a monoclonal antibody.

Monoclonal antibodies against inter-alpha-trypsin-inhibitor (ITI) were produced. One clone showing specificity for urinary trypsin inhibitor (UTI), a proteolytic fragment of ITI, which is excreted into urine, was selected for the establishment of an enzyme-linked immuno-sorbent assay (ELISA). The ELISA for the quantification of UTI was shown to work reproducibly in the range between 0.5 and 10 ng UTI/ml urine. Urines of several patients suffering from different lung diseases were screened for UTI using the established ELISA. Highest UTI levels were found in the urine of patients with lung empyema. A more moderate increase was observed in patients suffering from lung tuberculosis and from secondary and primary lung tumors.

Significance of microbiological and biochemical analyses in empyema thoracis.

Following the indifferent results of a retrospective analysis, a prospective study was undertaken to analyse the causative organisms in 51 cases of empyema. Cultures were positive in 44/51 (= 86.3%) cases. 2 bacterial species were recovered for each empyema. The aerobic gram-positive cocci represented the largest group (57%), followed by aerobic gram-negative bacteria (18.6%), anaerobic bacteria (18.6%), and fungi (5.8%). Polymicrobial empyema accounted for 59.1% of the cases. Anaerobic bacteria were cultured from 36.4% of empyema. Anaerobic bacteria were more frequently isolated from pleural effusions than from other specimens. Swabs were found to be of minor value for anaerobics. Analyses of glucose and pH value in pleural effusions have been reported to be useful in differentiating complicated from uncomplicated effusions in cases where the aspirated fluid is not purulent and is negative on gram stain, but clinical as well as radiological findings point to an empyema. Our results have shown that pH-values less than 7.30 and Glucose less than 60 mg/dl were not absolutely specific for empyema. In contrast, PMN-elastase in pleural effusion and HI-30 in urine showed a statistically significant differentiation of empyema from exudates of other origin.

Karyotypic characterization of established cell lines derived from a squamous cell carcinoma and an adenocarcinoma of human lung cancers.

Two non-small cell carcinoma cell lines from the major histopathologic groups of human lung cancers have been karyotyped: HS-24 was established from a squamous cell carcinoma, and SB-3 was obtained from a metastasis of a poorly differentiated adenocarcinoma. Endoreduplication is characteristic for both cell lines. Subsequent loss of chromosomes led finally to hypotetraploid karyotypes with modal chromosome numbers of 66-68 and 70-72 for HS-24 and SB-3, respectively. The structural analysis was performed by G- and C-banding. Stable overrepresentation of chromosomes 7, 8, 12, and 16 was found. Both cell lines developed a characteristic set of disomic and stable markers. Chromosomes involved in markers were 1, 2, 5, 6, 10, 11, 16, and 17. Consistent numerical and structural normality for chromosomes 4, 18, and 21 was observed.

[Clinical aspects, results of diagnostic pulmonary function tests and biochemical parameters in children with homozygote alpha-1-antitrypsin deficiency]. / Klinik, lungenfunktionsdiagnostische und biochemische Parameter bei Kindern mit homozygotem Alpha-1-Antitrypsinmangel.

The clinical course and pulmonary function tests of individuals with severe Alpha-1-Antitrypsin (Alpha-1-AT) deficiency reveal a marked interindividual variability. 4 patients with PI type ZZ and 2 patients with PI type SZ had been identified by neonatal cholestasis. None had pulmonary symptoms at a mean age of 13 (range 9-16) years. Lung function tests disclosed signs of incipient pulmonary emphysema in one girl. Bronchial hyperreactivity had led to the diagnosis of Alpha-1 AT-deficiency (PI type ZZ) in another girl at the age of 7 years. The biochemical analysis of the serum of three children (two PI ZZ and one PI SZ) revealed additional protease inhibitors that may be effective as compensatory mechanisms. The lack of those protective factors as well as the presence of environmental hazards may contribute to the increased risk of emphysema in individual patients.

Localization of cathepsin B in two human lung cancer cell lines.

We demonstrated the cysteine proteinase cathepsin B in two human lung tumor cell lines by cytochemical and immunocytochemical methods. The cell lines were derived from a squamous cell carcinoma of the lung (HS-24) and a metastasis to the adrenal gland from an adenocarcinoma of the lung (SB-3). For comparison and control, normal human lung fibroblasts cells (Wi-38) were also investigated. Intracellular cathepsin B activity was detected in all three cell lines. SB-3 and the normal fibroblast cells showed almost equal cathepsin B activity, which was considerably stronger than that in the HS-24 cells. Specific inhibitors for cathepsin B (E64, leupeptin, antipain) suppressed its activity completely. Stefin A, the physiological cathepsin B inhibitor, was less effective; this might depend on its limited penetrability into living cells. Localization of the cathepsin B was performed by conventional immunofluorescence microscopy and laser scanning microscopy. With specific anti-cathepsin B antibodies, the enzyme was localized in HS-24, SB-3, and Wi-38 fibroblast cells within perinuclear granules representing the lysosomal compartment. In the SB-3 cells, we additionally localized a minor fraction of the enzyme bound to the plasma membrane in a speckled distribution, accessible to the antibodies from the outside. This direct demonstration of cathepsin B distribution supports biochemical data about the dual localization of the enzyme in tumor cells. It also supports the possibility of a direct involvement of cathepsin B in the degradation of the extracellular matrix, and thus a contribution of the enzyme in invasion and metastasis.

Detection of cathepsin B, plasminogen activators and plasminogen activator inhibitor in human non-small lung cancer cell lines.

Human non-small lung cancer cell lines HS-24 (established from a primary squamous cell carcinoma) and SB-3 (established from a metastasis of a primary adenocarcinoma of the lung into the adrenal gland) were analysed for the proteinases tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator inhibitor (PAI-1). The proteinases were characterized by activity measurements, inhibition studies, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis. Cell-associated proteinases were determined in cell lysates, secreted proteinases in cell conditioned culture media. Both cell lines were found to secrete uPA and PAI-1, whereas tPA could be detected only in HS-24 conditioned media. No cathepsin B activity could be detected in media of both cell lines. However, activation experiments and western blot analysis showed, that at least HS-24 secrete an inactive precursor. Cell lysates of HS-24 and SB-3 show PA activity, but on a low level. Cathepsin B activity was also found to be low in HS-24 lysates. However, SB-3 lysates show high cathepsin B activity. Further characterization of the proteinases by their sensitivity against several inhibitors suggests that they are similar to the corresponding proteinases of normal, nonmalignant cells.

[Trypsin and pancreatic elastase-inhibitory capacity of serum and bronchoalveolar lavage fluid of smokers, ex-smokers and nonsmokers]. / Trypsin- und Pankreaselastase-inhibitorische Kapazität von Serum und bronchoalveolärer Lavageflüssigkeit bei Rauchern, Exrauchern und Nichtrauchern.

Smoking habits have no differentiating effect on the functional activity of protease inhibitor (alpha 1PI) in the serum and bronchoalveolar lavage fluid (BALF). In the BALF, the trypsin-inhibitory capacity TIC/alpha 1PI ratio is significantly greater in patients with emphysema than in non-emphysematous subjects. PIC/alpha 1PI ratios that are greater than 1 demonstrate the existence of additional antiproteases in the lower respiratory tract.

[Clinical aspects, diagnostic lung function and biochemical parameters in children with homozygous alpha 1-antitrypsin deficiency]. / Klinik, lungenfunktionsdiagnostische und biochemische Parameter bei Kindern mit homozygotem alpha 1-PI-Mangel.

Both the clinical course of the homozygous alpha 1-PI deficiency and also the pulmonary function and measured clinical parameters in these children revealed very marked inter-individual fluctuations. In one child, the lung function revealed certain signs of incipient pulmonary emphysema. The biochemical parameters of two children revealed TIC/alpha 1-PI- and PEIC/alpha 1-PI ratios greater than 1. This indicates that, in addition to alpha 1-PI, further inhibitors may develop a compensatory effect.

[Functional activity of the alpha1-proteinase inhibitor in serum and bronchoalveolar lavage fluid in congenital lung emphysema]. / Funktionelle Aktivität des alpha 1-Proteinaseinhibitors in Serum und bronchoalveolärer Lavageflüssigkeit beim erworbenen Lungenemphysem.

Pulmonary emphysema is thought to be due to an elastase-antielastase imbalance which leads to the destruction of alveolar walls. It is generally agreed that cigarette smoking is the major cause of acquired emphysema although many smokers fail to develop overt disease. Cigarette smoke inactivates alpha 1-proteinase inhibitor (alpha 1PI) which is believed to be the major antielastase in the lower respiratory tract. There is, however, controversy regarding the activity of alpha 1PI in smokers compared with nonsmokers. We, therefore, investigated the trypsin inhibitory capacity (TIC), the pancreatic elastase inhibitory capacity (PEIC), and the amount of immunoreactive alpha 1PI in serum and bronchoalveolar lavage fluid (BALF) of 24 individuals (15 smokers, 5 former smokers, 4 non-smokers) with clinical signs of emphysema and 32 persons (15 smokers, 6 former smokers, 11 non-smokers) without emphysema. Pancreatic elastase is known to be inhibited only by non-oxidised alpha 1PI whereas trypsin is inhibited by both native and oxidised alpha 1PI. Serum values of TIC/alpha 1PI and PEIC/alpha 1PI did not differ between the groups of subjects with emphysema (TIC/alpha 1PI: 0.71 +/- 0.12; PEIC/alpha 1PI: 0.44 +/- 0.06) and without emphysema (TIC/alpha 1PI: 0.68 +/- 0.13; PEIC/alpha 1PI: 0.41 +/- 0.07). Both serum-inhibitory capacities were found to be smaller than unit (= 1) indicating inactivation of alpha 1PI. BALF values of TIC/alpha 1PI showed a wide variation. TIC/alpha 1PI was greater than unity in 84% of subjects with emphysema (2.06 +/- 1.28) compared to 61% without emphysema (1.14 +/- 1.07) providing evidence for the presence of additional inhibitor(s) in those lavage fluids.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of leukocyte elastase-inhibitor complexes and leukocyte neutral proteinase inhibitor by enzyme immunoassays. Leukocyte elastase-inhibitor complexes in porcine blood, III.

Sensitive enzyme immunoassays for the determination of total leukocyte neutral proteinase inhibitor and polymorphonuclear elastase-leukocyte neutral proteinase inhibitor complexes are described. The usable ranges of the standard curves were from 80 ng/l to 10 micrograms/l. The relative intra-assay coefficients of variation of the tests were between 2 and 4%, and the inter-assay coefficients of variation between 4 and 10%. In vitro and in vivo studies were performed with septic pigs and isolated leukocytes. The results show that leukocyte neutral proteinase inhibitor can be used in pigs as a parameter for the development of a septicaemia.

Proteolytic activity of human tumor cell lines deriving from bronchial squamous cell carcinoma, pulmonary metastasis of rhabdomyosarcoma and pleural metastasis of mesothelioma.

The proteolytic activities of human tumor cell lines deriving from bronchial squamous cell carcinoma, a lung metastasis of an embryonal rhabdomyosarcoma and a pleural mesothelioma were measured by use of chromogenic substrates. N-acetyl-alanine aminopeptidase activity, plasminogen activator activity, H-D-Ile-Pro-Arg-p-NA splitting activity as well as plasmin-like activity, cathepsin G-like activity and plasma-kallikrein-like activity were found in cell lysates. The enzymatic activity of N-acetyl-alanine aminopeptidase, plasminogen activator and H-D-Ile-Pro-Arg-p-NA splitting activity changed during culturing. Plasminogen activator and H-D-Ile-Pro-Arg-p-NA splitting activity decreased to very low values, whereas N-acetyl-alanine aminopeptidase activity leveled at 1 x 10(-5) mU/cell. Unlike other proteolytic activities, plasminogen activator was released into the medium. Plasminogen activator activity could be measured in culture medium which contained no fetal calf serum.

Neutral proteinase inhibitors in PMN leukocytes. II. Isolation and characterization of a neutral proteinase inhibitor from porcine PMN leukocytes.

An inhibitor of neutral proteinases was purified from porcine PMN leukocytes by gel filtration on Sephadex G-75 superfine and ion-exchange chromatography on Mono S. Thus an inhibitor preparation with a specific inhibitory activity against chymotrypsin of 10 IU/mg was obtained. In dodecyl sulfate gel electrophoresis a single protein band with an apparent molecular mass of 40 kDa was found under reducing conditions. Under non-reducing conditions the inhibitor forms higher molecular mass aggregates. On isoelectric focusing several protein bands with isoelectric points between pH 7.0 and 7.5 could be separated. The amino-acid composition of the inhibitory protein was determined. The inhibition mechanism was studied and association rate constants (kon) were measured and calculated for the reaction with chymotrypsin as well as leukocyte and pancreatic elastase. In Western blot analysis and in enzyme immunoassay studies crossreactivity between antibodies directed against porcine leukocyte neutral proteinase inhibitor and the corresponding inhibitor of bovine PMN leukocytes could be demonstrated.