Cathepsin B and cysteine proteinase inhibitors in human lung cancer cell lines.

Cell lines derived from human squamous cell (EPCL), large cell (LCLC), and small cell lung cancer (SCLC) lines were investigated for the expression of cathepsin B (Cat B) and cysteine proteinase inhibitors (CPIs). The EPLC and LCLC lines expressed 5- to 50-fold more Cat B activity and contained more mature Cat B of M(r) 27-29 kDa (> 2.5 microg/mg total protein) than the SCLC lines (< 1.0 microg/mg total protein). The LPLC lines also secreted the highest amounts of Cat B precursor of M(r) about 46 kDa. Inhibitory activities against Cat B and papain were associated with high molecular mass (HMM) and low molecular mass (LMM) inhibitory proteins, both in cell extracts and in media. About 75% of the inhibitory activity was associated with HMM inhibitors, the majority of which were kininogens (M(r) > or = 67 kDa). The LMM inhibitors of M(r) 10-15 kDa were cystatin C and stefins A and B, which were quantitated by ELISA: stefins A and B were present in cell extracts and medium in similar concentrations (5-200 ng/10(6) cells), while 80-99% of the cystatin C was released in the medium (10-195 ng/10(6) cells). Phorbol ester (PMA), which induces protein-kinase C mediated signal transduction and enhances cellular differentiation in many non-small cell lung cancer (NSCLC) cell lines, increased intracellular Cat B activity and Cat B protein as well as its secretion in some cell lines but not in others, regardless of their histological type. PMA significantly (P < 0.049) decreased intracellular stefin A concentrations in two EPLC lines and non-significantly in two LCLC lines. PMA decreased secretion of stefin A in all EPLC lines, but not in LCLC lines, while IGF-I significantly increased stefin B secretion in both SCLC lines. These data showed that lung tumor cells produce both cysteine proteinases and cystatins. As the antagonistic molecules are regulated differently in histologically different types of lung tumor cells, it is possible that an imbalance between the proteinases and their specific inhibitors plays a role in progression of certain types of lung tumors in vivo.

Tissue factor is the only activator of coagulation in cultured human lung cancer cells.

It is a long-known principle that tumour cells tend to exploit the host's physiologic systems in order to get support in terms of, for example, nutrition, growth or metastasis. One of these physiologic systems is the blood coagulation cascade, which has been found activated in many tumour patients. The mechanisms of the activation of coagulation have been assessed in numerous animal and in vitro experiments, and the results appeared to point to several distinct activators. The present study used a large panel of different cultivated human lung cancer cell lines and experimental systems involving normal plasma, plasmas deficient of factors V, VII or X, purified coagulation factors II and X, recombinant tissue factor (TF), and specific inhibitory antibodies against factor VII and TF. The results provide strong evidence that there is no activator of coagulation besides TF in the wide array of lung cancer cells examined. However, this work reveals a striking variability of TF content among the cell lines. This might explain ambiguous results of clinical trials of anticoagulation as an adjunct to antineoplastic therapy in lung cancer. By sensitive diagnostic tools like the plasma thrombin-antithrombin complex levels it might be possible to select patients with activated coagulation, who might benefit from anticoagulation.

Transition from suspension to adherent growth is accompanied by tissue factor expression and matrix metalloproteinase secretion in a small cell lung cancer cell line.

Small cell lung cancer (SCLC) is a very malignant tumor known to grow aggressively and to metastasize early. It is well established that metastasis generally involves both tumor cell adhesion and proteolytic degradation of the extracellular matrix. However, SCLC cells cultured in vitro, such as the classic SCLC cell line NCI-H69, grow in floating aggregates and express only negligible proteolytic activity. In this report, we show that NCI-H69 cells can be selected for adherent growth. In contrast to parental suspension cells, the adherent cells were found to express tissue factor as well as gelatinolytic activity, attributable to matrix metalloproteinases 2 and 9. Such a switch of tumor cell characteristics, if it could occur in SCLC patients, might add to the understanding of the steps involved in the spreading of this highly metastatic type of lung cancer.

Studies on tumor-cell-induced platelet aggregation in human lung cancer cell lines.

We investigated the ability of human lung cancer cells of different histological subtypes to cause platelet aggregation. Tumor-cell-induced platelet aggregation (TCIPA) was studied in vitro in 13 human lung cancer cell lines [small-cell lung cancer (SCLC), squamous-cell lung cancer, large-cell lung cancer, adenocarcinoma and alveolar-cell lung cancer]. Three tumor cell lines failed to aggregate platelets in platelet-rich plasma, whereas platelet aggregation was induced by 12 cell lines when added to washed platelets and minimal amounts of platelet-poor plasma (0.5% v/v). The thrombin antagonist hirudin inhibited TCIPA in non-small-cell lung cancer cell lines (NSCLC). In SCLC, TCIPA was fully abolished only when the ADP scavenger apyrase was added to hirudin. Thus ADP and thrombin generation by these tumor cell lines are responsible for platelet aggregation. The ability to activate platelets independently of coagulation factors VII and X was demonstrated for 8 cell lines. Electron-microscopically, direct tumor-cell/platelet contact was found to be the initiating mechanism of TCIPA in SCLC, whereas tumor-cell/platelet contacts in NSCLC could only be observed at the peak of the aggregation curve. Lung cancer cells activate platelets in vitro by generation of thrombin and/or ADP.

Secretion of a latent, acid activatable cathepsin L precursor by human non-small cell lung cancer cell lines.

Secretion of pro-cathepsin L, the precursor of a lysosomal cysteine proteinase, has been described for ras-transfected mouse fibroblasts and several human cancer cell lines. The secretion of a latent but stable precursor might be a means for tumor cells to involve this proteinase in extracellular matrix breakdown. Since lung cancer is the leading cause of cancer death in the industrialized countries, we therefore studied the secretion of pro-cathepsin L in 11 human non-small cell lung cancer cell lines (EPLC 32M1, NCI H157, EPLC 272H, U1752, LCLC 103H, LCLC 97TM1, U 1810, NCI H661, NCI H23, NCI H125, and NCI H596) and 8 human small cell lung cancer cell lines (SCLC 22H, NCI H60, NCI H82, NCI H526, NCI H146, NCI H841, NCI H510, and DMS 79). Immunoblot analysis of cell conditioned media showed that latent pro-cathepsin L (M(r) 42 kDa) was secreted in all 11 non-small cell lung cancer cell lines. Three of these cell lines secreted an additional inactive form of cathepsin L of M(r) 24 kDa. In contrast, the 8 small cell lung cancer cell lines did not secrete any detectable cathepsin L-immunoreactive material. Phorbol-12-myristate-13-acetate increased the secretion of pro-cathepsin L in 6 of the non-small cell lung cancer cell lines. The cathepsin L precursor could be activated in vitro at pH 3, accompanied by a shift in molecular mass to 34 kDa. Chicken egg white cystatin prevented the acid activation. Specific antibodies against a synthetic peptide from the pro-sequence of cathepsin L reacted with the nonsmall cell lung cancer cathepsin L precursor. Extracellular pro-cathepsin L may be important in the tumor biology of non-small cell lung cancer and would be a good target for novel diagnostic and therapeutic approaches, since the majority of physiological lysosomal proteinases are contained in intracellular compartments only.

The endogenous cyclic AMP antagonist, cyclic PIP: its ubiquity, hormone-stimulated synthesis and identification as prostaglandylinositol cyclic phosphate.

This report shows that the cyclic AMP antagonist cyclic PIP is present in all organs and tissues of the rat so far examined: brain, heart, lung, intestine, kidney, liver, spleen, skeletal muscle and fat. The synthesis of cyclic PIP is stimulated by insulin or noradrenaline (alpha-adrenergic action) in a dose-dependent fashion. Increasing cyclic PIP synthesis with increasing insulin concentrations matches the insulin receptor binding curves. Cyclic PIP levels in blood serum remain low after hormonal stimulation and no cyclic PIP can be detected in urine. As an indication of its ubiquity, cyclic PIP was even detected in yeast. Prostaglandin E (as shown by incorporation of [3H]PGE into cyclic PIP and demonstration of a constant specific activity), myo-inositol (as shown by acid hydrolysis of the dephosphorylated cyclic PIP and mass spectrometric identification of the products) and one phosphate (as shown by the ionic nature of cyclic PIP and its inactivation by phosphodiesterase plus phosphatase) are components of cyclic PIP. Chemical derivatization experiments of cyclic PIP suggest the phosphate to be bound to myo-inositol and the myo-inositol phosphate to the prostaglandin E by its C15-hydroxyl group.

Insulin resistance, a result of reduced synthesis of prostaglandylinositol cyclic phosphate, a mediator of insulin action? Regulation of cyclic PIP synthetase activity by oral antidiabetic and antihypertensive drugs.

Reduced ability or failure to stimulate cyclic adenosinemonophosphate (AMP) synthesis on a second addition of hormone 30 min after a first stimulation was taken as an indirect indication of the synthesis of the cyclic AMP antagonist prostaglandylinositol cyclic phosphate (cyclic PIP). In diabetic rats, because of an increased possibility of restimulating cyclic AMP synthesis, the formation of cyclic PIP should be reduced. Additionally, severalfold increased basal cyclic AMP synthesis can be observed in diabetic hepatocytes in comparison with controls. Upon measuring cyclic PIP levels after hormonal stimulation in all organs of diabetic rats, it was found that stimulation of cyclic PIP synthesis by insulin decreased gradually in a time-dependent manner. Plasma membranes were prepared from diabetic Ksj db/db mice and from spontaneously hypertensive rats (SHR), and in a subsequent assay for cyclic PIP synthetase, an up to 60% decrease of enzyme activity was found. Cyclic PIP synthetase can be completely inhibited by preincubation with protein kinase A. It is most likely that this serine phosphorylation reaction by which the enzyme is inhibited also in vivo is a result of increased cyclic AMP levels. The addition of 10(-5)-10(-4) M sulfonylureas to the enzyme assay of liver plasma membrane causes full inhibition, and the addition of 10(-5)-10(-4) M biguanides, a two- to fourfold activation of the enzyme. Activation of cyclic PIP synthetase by biguanides can also be demonstrated in intact cells. It is a fast reaction and additive with respect to the activation by fluoride or guanylyl-imidodiphosphate (GMP-PNP), and it is most likely the effect with which the biguanides produce the correcting changes in metabolism. Furthermore, antihypertensive drugs like captopril, guanethidine, and dihydralazine also activate cyclic PIP synthetase. In contrast to the activation by the biguanides, this effect is not additive to the activation by fluoride. It appears that essential hypertension and type 2 diabetes are connected with or may be the result of a reduction in synthesis of the intracellular messenger cyclic PIP, whose synthesis is stimulated by hormones like insulin and noradrenaline (alpha-adrenergic action).

Prostaglandylinositol cyclic phosphate, an antagonist to cyclic AMP.

The existence of a low molecular weight intracellular regulator has been demonstrated. It is composed of PGE, myoinositol, and one phosphate and has been named cyclic PIP. Its synthesis is stimulated by hormones such as insulin in a dose-dependent manner, it is therefore suggested that cyclic PIP is a second messenger equivalent to cyclic AMP in its potency but antagonistic in its action.

Differentiation capacity of human non-small-cell lung cancer cell lines after exposure to phorbol ester.

Three cell lines of squamous-cell carcinoma and 3 of large-cell carcinoma origin were investigated for the expression of differentiation markers and functional parameters (proliferation, morphology, cornified envelope formation, involucrin staining, transglutaminase activity, adhesiveness and migration) under normal cell culture conditions and after treatment with the tumor promoter phorbol-12-myristate-13-acetate (PMA). Although all original tumors had been described as poorly differentiated by histological grading, we found significant heterogeneity in the expression of differentiation markers in cell culture. A systematic grading of the cell lines became possible only after PMA stimulation. PMA generally increased expression of differentiation markers in cell lines of comparably low grades of differentiation, as indicated by dose-dependent inhibition of proliferation and cloning efficiency, induction of squamous markers, and decreased adhesiveness and cell motility. In contrast, cell lines of apparently higher differentiation by these criteria showed little response to PMA. The results presented show that the assessment of differentiation capacity by comparison of differentiation markers under normal cell culture and PMA-stimulated conditions in established NSCLC cell lines allows for a refined cell culture grading, which might advance the classification and characterization of such cell lines which, otherwise, appear to be very heterogeneous. It may also help to correlate cellular functions with various states of differentiation in vitro.

Control of proteinase expression by phorbol-ester- and Fos-dependent pathways in human non-small-cell lung-cancer cells.

Activation of protein kinase C- (PKC) and Fos/Jun-dependent signal transduction pathways are thought to be major effects of oncogene action in different tumor systems including human non-small-cell lung carcinoma (NSCLC). We have previously shown that the phorbol ester analogue phorbol-myristate-acetate (PMA), which is a potent activator of PKC, can induce squamous-type cellular differentiation and the expression of proteinases, such as plasminogen activators and pro-cathepsin L, in several NSCLC cell lines. To investigate the PMA-dependent effect on proteinase secretion in more detail, we have now analysed the role of a downstream transmitter of PKC activity in this process, namely Fos, which is part of the AP-1 transcription factor in the nucleus. We transfected a cell line derived from an undifferentiated squamous-cell lung carcinoma with different chimeric fos-estrogen receptor constructs (fos-ER) which makes selective activation of this transcription factor possible. The resulting clones were treated either with PMA as activator of PKC, or with diethylstilbestrol (DES), an estrogen analogue binding to and thereby activating preformed Fos-ER molecules. We show that cells treated with either substance undergo similar phenotypic changes (change from cuboidal to spindle-cell type) and decrease their doubling rates and cloning efficiencies. This is paralleled by the induction of several proteinase genes such as t-PA, urokinase, and pro-cathepsins B and L. Contrary to activated PKC, Fos in this system seems to be unable to initiate terminal squamous-cell differentiation, as assessed by the production of cornified envelopes. It is, however, efficient in the stimulation of neutral or lysosomal proteinase secretion as determined by Western-blot analysis and zymography. This Fos-ER expressing system thus seems to be a valuable tool in the molecular dissection of pathways that lead to the activation and secretion of proteinases in NSCLC cells.

Plasma quality after whole-blood filtration depends on storage temperature and filter type.

The study evaluated the quality of plasma obtained after whole-blood filtration with four different polyester filters and one polyurethane filter. The activities of coagulation factors and proteinase inhibitors were not or only negligibly affected by filtration, in all experiments. Filtration did not increase markers of clotting and fibrinolysis. Only a strong neutrophil and complement activation was observed, which depended on the type of filter and whole-blood storage conditions. However, as neutrophil elastase-specific degradation products did not increase and the complement-derived anaphylatoxin C3a was found in its inactivated form, C3a-desArg, these filtration-dependent changes apparently have little impact on the therapeutic quality of whole-blood-filtered fresh frozen plasma for transfusion.