Fhit regulates invasion of lung tumor cells.

In many types of cancers, the fragile histidine triad (Fhit) gene is frequently targeted by genomic alterations leading to a decrease or loss of gene and protein expression. Fhit has been described as a tumor suppressor gene because of its ability to induce apoptosis and to inhibit proliferation of tumor cells. Moreover, several studies have shown a correlation between the lack of Fhit expression and tumor aggressiveness, thus suggesting that Fhit could be involved in tumor progression. In this study, we explored the potential role of Fhit during tumor cell invasion. We first showed that a low Fhit expression is associated with in vivo and in vitro invasiveness of tumor cells. Then, we showed that Fhit overexpression in Fhit-negative highly invasive NCI-H1299 cells by transfection of Fhit cDNA and Fhit inhibition in Fhit-positive poorly invasive HBE4-E6/E7 cells by transfection of Fhit small interfering RNA induce, respectively, a decrease and an increase in migratory/invasive capacities. These changes in cell behavior were associated with a reorganization of tight and adherens junction molecules and a regulation of matrix metalloproteinase and vimentin expression. These results show that Fhit controls the invasive phenotype of lung tumor cells by regulating the expression of genes associated with epithelial-mesenchymal transition.

['Tired of life': a reason to evaluate physical handicaps]. / Levensmoeheid: een reden om lichamelijke handicaps te evalueren.

A 91-year-old woman was admitted to a nursing home because of functional deterioration: poor vision and hearing, and diminished mobility. She had been widowed for 30 years, had no children and a very small social network. She described herself as 'tired of life', and alluded to the assisted suicide of the former senator Brongersma, where this term was the trigger to the administration of assistance. The Supreme Court however ruled afterwards that a physician has no capacity in 'questions of life and death', but only in requests for help with a medically classifiable underlying disorder. The patient was not assisted in dying and her condition deteriorated further. Some time later she fell and contracted a clinical hip fracture. She refused to be brought to hospital and was given fentanyl plasters. A week later she died. The question on how to respond to the very elderly who seek help because they wish to die, is being discussed in the Netherlands. The term 'tired of life' appears to be rather confusing, as it has no medical connotation. It is recommended that elderly persons who ask for help in dying because they are 'tired of life', based on physical handicaps, are evaluated carefully to determine the severity of the handicaps and the hopelessness and unbearableness of the suffering. This could lead to the patient receiving assistance in dying.

Detection and localization of a Ca2+-ATPase activity in Toxoplasma gondii.

Toxoplasma gondii, the agent causing toxoplasmosis, is an obligate intracellular protozoan parasite. A calcium signal appears to be essential for intracellular transduction during the active process of host cell invasion. We have looked for a Ca2+-transport ATPase in tachyzoites and found Ca2+-ATPase activity (11-22 nmol Pi liberated/mg protein/min) in the tachyzoite membrane fraction. This ATP-dependent activity was stimulated by Ca2+ and Mg2+ ions and by calmodulin, and was inhibited by pump inhibitors (sodium orthovanadate or thapsigargin). We used cytochemistry and X-ray microanalysis of cerium phosphate precipitates and immunolabelling to find the Ca2+, Mg2+-ATPase. It was located mainly in the membrane complex, the conoid, nucleus, secretory organelles (rhoptries, dense granules) and in vesicles with a high calcium concentration. Thus, Toxoplasma gondii possesses Ca2+-pump ATPase (Ca2+, Mg2+-ATPase) as do eukaryotic cells.

Modified basement membrane composition during bronchopulmonary tumor progression.

During tumor progression, the extracellular matrix (ECM) and particularly the basement membrane (BM) appear to be dynamic structures that are not only degraded but also deposited around tumor clusters. In this study we examined by immunohistochemistry the localization of three chains of Type IV collagen (alpha1, alpha3 and alpha5), Type VII collagen, and laminin 5 at different stages of bronchopulmonary cancers. In normal tissues, alpha1(IV) chain was detected in all BMs (bronchial, vascular, alveolar, and glandular), alpha5(IV) chain was present only in vascular BM, and laminin 5 and Type VII collagen were co-localized in bronchial and glandular BMs, whereas alpha3(IV) immunolabeling was totally absent from normal bronchi. In well-differentiated carcinomas, alpha3(IV) chain staining was found in some neosynthetized BMs interfacing the tumor cell and the stromal compartment, contrasting with the total absence of labeling in normal tissues. alpha1(IV) chain showed strong reactivity in all BM. Laminin 5 and Type VII collagen were also detected in neosynthetized BM. In poorly differentiated invasive cancers, alpha3(IV) chain and Type VII collagen were not found, whereas laminin 5 and alpha1(IV) chain persisted. The most important modifications in BM composition during tumor progression therefore appear to be the appearance of the alpha3 (IV) chain in well-differentiated carcinomas and its subsequent disappearance in poorly differentiated carcinomas, together with the loss of type VII collagen. alpha5(IV) chain distribution was restricted in vascular BM of well- and poorly differentiated carcinomas. These results show that the composition of BM is modified during the progression of bronchopulmonary tumor, emphasizing that the BM represents a dynamic element in tumor progression and has an important role in tumor cell invasiveness.

Expression of the extracellular matrix metalloproteinase inducer (EMMPRIN) and the matrix metalloproteinase-2 in bronchopulmonary and breast lesions.

Tumor cells interact with stromal cells via soluble or cell-bound factors stimulating the production of matrix metalloproteinases (MMPs), a group of enzymes largely involved in the extracellular matrix (ECM) remodeling in tumor invasion. Among these factors, extracellular matrix metalloproteinase inducer (EMMPRIN) has been shown to stimulate in vitro the fibroblast production of various MMPs such as interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), and gelatinase A (MMP-2). In this study, the EMMPRIN protein was detected by immunohistochemistry prominently in malignant proliferations of the breast and the lung. It was present at the surface of both tumor epithelial and peritumor stromal cells. Because previous studies have reported that stromal cells do not express EMMPRIN mRNAs, it is very likely that EMMPRIN is bound to stromal cells via a specific receptor. Moreover, our observations also demonstrated that the same peritumor stromal cells strongly express MMP-2. Our results show that EMMPRIN is an important factor in tumor progression by causing tumor-associated stromal cells to increase their MMP-2 production, thus facilitating tumor invasion and neoangiogenesis. (J Histochem Cytochem 47: 1575-1580, 1999)

Expression of gelatinase A and its activator MT1-MMP in the inflammatory periprosthetic response to polyethylene.

Wear debris of polyethylene prosthetic components is known to induce a host granulomatous reaction which recruits numerous macrophages and multinucleated giant cells. By releasing cellular mediators of a nonspecific inflammatory reaction, activated phagocytic cells are thought to play a key role in osteolysis leading to aseptic loosening of the prosthesis. Matrix metalloproteinases (MMPs) have been implicated in this destructive process by their ability to degrade extracellular matrix components of bone and adjacent connective tissue. To investigate the roles of gelatinase A, its activator MT1-MMP, and the MMP inhibitors TIMP-1 and TIMP-2 in aseptic loosening of polyethylene prostheses, immunohistochemistry (IHC) and in situ hybridization (ISH) were performed on periprosthetic pseudosynovial interface tissues. Gelatinase A and MT1-MMP were strongly detected immunohistochemically in macrophages and multinucleated giant cells in contact with polyethylene wear debris. In contrast to MT1-MMP, gelatinase A mRNAs were not found in phagocytic cells but in surrounding fibroblasts, thereby suggesting cooperation between macrophages and fibroblasts in this process. While TIMP-1 was expressed essentially in hyperplastic pseudosynoviocytes as assessed by IHC and ISH, TIMP-2, MT1-MMP, and gelatinase A were colocalized in phagocytic cells. These data support the concept of progelatinase A activation involving a trimolecular complex (MT1-MMP-TIMP-2-gelatinase A) mechanism. Thus, this study demonstrated that gelatinase A and its activator might contribute to the aseptic loosening of polyethylene prostheses.

Quantitative immunolocalization of a P29 protein (GRA7), a new antigen of toxoplasma gondii.

Ultrastructural localization of a P29 protein of Toxoplasma gondii was examined on thin sections by an immunogold technique using a P29 antigen-specific monoclonal antibody (5-241-178). Immunolocalization of the P29 protein in extracellular tachyzoites demonstrated that this antigen was present in the dense granules. Thus, we have identified this P29 antigen as the seventh protein (GRA7) to be localized to the dense granules of T. gondii. P29 immunolocalization in intracellular tachyzoites demonstrated association of this antigen with the parasite membrane complex, tubular elements of the intravacuolar network, and with the parasitophorous vacuolar membrane. Our immunolabeling data suggest trafficking of the P29 (GRA7) antigen from the dense granule via the intravacuolar network to the parasitophorous vacuolar membrane on invasion of the tachyzoite into the host cell. (J Histochem Cytochem 46:1411-1421, 1998)

Interferon-gamma signal transduction during parasite infection: modulation of MAP kinases in the infection of human monocyte cells (THP1) by Toxoplasma gondii.

We assayed mitogen-activated protein (MAP) kinase phosphorylation in a human monocyte cell line (THP1) during their infection by Toxoplasma gondii. In addition, we tested the effect of specific MAP kinase inhibitors (PD098059 and SB203580) on parasite invasion. MAP kinase phosphorylation was increased in the cytosol and membrane fractions of THP1 infected with T. gondii. The MAP kinase phosphorylation of uninfected THP1 cells was not significantly modified by incubation for 20 h with 1000 U/ml of IFN-gamma. However, IFN-gamma treatment of infected cells significantly reduces the increase in phosphorylation caused by parasite infection. There was also MAP kinase activity in the cytosol and membrane fractions of extracellular T. gondii tachyzoites. IFN-gamma altered the distribution of activity in subcellular fractions of extracellular T. gondii tachyzoites. This indicates that IFN-gamma directly affects parasite MAP kinase activity. The results provide evidence that MAP kinase pathways participate in the infection by T. gondii and that the decrease in MAP kinase activity in infected cells caused by IFN-gamma may be involved in mediating their protective signals.

Involvement of calcium and calmodulin in Toxoplasma gondii tachyzoite invasion.

The tachyzoite of Toxoplasma gondii must successfully invade a host cell before it can replicate. Depletion of the Ca2+ in the external medium (EGTA) reduced tachyzoite invasion, suggesting that the initial tachyzoite-host cell interaction is Ca2+ dependent. The interaction of tachyzoites with host cells was also inhibited by Ca2+ channel blockers (verapamil) and calmodulin antagonists (trifluoperazine, calmidazolium). The calmodulin concentrated at the apical end of the tachyzoite could be involved in cytoskeleton movement and conoid extrusion. Invasion also depends on changes in tachyzoite cytosolic calcium. Depletion of Ca2+ with A23187+EGTA and release of Ca2+ from intratachyzoite pools (nuclear and perinuclear areas) inhibited invasion. In contrast, Ca-ionophore and thapsigargin which increase host cell cytosolic Ca2+, significantly decreased tachyzoite invasion. We therefore suggest that the effect of the drug is significantly different from the localized Ca2+ signal that is produced after parasite attachment to its host cell receptors and leads to its internalization into the host cell.

The elastase-induced expression of secretory leukocyte protease inhibitor is decreased in remodelled airway epithelium.

During airway inflammation, proteinases such as human leukocyte elastase are actively secreted. Secretory leukocyte protease inhibitor is a major serine proteinase inhibitor, secreted by bronchial, bronchiolar and lung epithelial cells. We recently identified secretory leukocyte protease inhibitor in human nasal epithelium, exclusively in remodelled areas of the surface epithelium. We now investigated the influence of remodelling and inflammation of the nasal tissue on the in vitro capacity of these cells to respond to human leukocyte elastase. Primary cultures of surface epithelial cells were established from various nasal polyp samples. At confluency, cell cultures were exposed to different human leukocyte elastase concentrations. The secretory leukocyte protease inhibitor immunocytolocalisation, expression and secretion were then investigated. Immunocytochemistry, showed a human leukocyte elastase dose-dependent increase of secretory leukocyte protease inhibitor containing cells and a basal extracellular localization of secretory leukocyte protease inhibitor after incubation with 100 microg/ml human leukocyte elastase. The relative amount of secretory leukocyte protease inhibitor mRNA transcripts increased with respect to the human leukocyte elastase concentration. Nevertheless, the potential stimulation of secretory leukocyte protease inhibitor secretion by human leukocyte elastase was lower in the more remodelled and inflamed tissue. Our results suggest that the contribution of the surface epithelial cells of poorly remodelled tissues to the protection against the deleterious effect of neutrophil proteinases is severely decreased in highly remodelled and inflamed tissues.

[Extrapyramidal disorders following sertraline use; experiences with a new selective antidepressant]. / Extrapiramidale stoornissen na sertralinegebruik; ervaringen met een nieuw selectief antidepressivum.

In five patients, two women aged 40 and 65 years and three men aged 42, 70 and 84 years, extrapyramidal symptoms such as rigidity and tremors, were considered to be caused by the use of the antidepressant drug sertraline, a serotonin reuptake inhibitor. After discontinuation of the medication the symptoms disappeared.

Glandular-like morphogenesis and secretory activity of human tracheal gland cells in a three-dimensional collagen gel matrix.

The extracellular matrix has been demonstrated to affect the differentiation of epithelial cells. We present evidence that in a three-dimensional (3-D) type I collagen gel matrix, isolated human adult tracheal gland (HTG) cells are capable of reconstructing new functional gland-like tubules in vitro. During the first two weeks in culture, HTG cells developed globular epithelial cell aggregates in which lumina is absent. By the third week in culture, the tubulogenesis and the formation of branching structures became evident with a polarized morphology, which in many aspects resembles the in vivo morphology. A central lumen was lined by polarized secretory epithelial cells exhibiting well-developed microvilli and apical secretory granules. Furthermore, we showed that the capacity of in vitro tracheal gland differentiation was associated with the basal deposition of laminin and type IV collagen around the gland-like tubules. A cell-associated 72 kDa type IV collagenase was expressed in developing tubule cells, as shown by immunocytochemistry. The secretion of the antileucoprotease (ALP), a protein marker of tracheal gland serous cells, was bidirectional in gland-like tubules, since up to 65% of released ALP was in the basolateral direction. Taken together, these observations indicate that isolated HTG cells in a 3-D collagen matrix form functional tracheal gland-like tubules and suggest that similar new tracheobronchial gland formations may occur during the human normal gland development and remodeling.

Toxoplasma gondii-structure variations of the antigen P30.

The major surface immunodominant antigen (P30) of Toxoplasma gondii was purified by two methods (i) SDS-PAGE and (ii) immunoaffinity chromatography. The secondary elements within this protein were assessed by circular dichroism and spectra obtained were compared to those proposed by Manavalan & Johnson (1983). The results allowed us to determine an all beta protein status for this antigen. This experimental result was in agreement with the predicted secondary structures deduced from the P30 primary sequence. Modifications in conformation according to pH and temperature were recorded without any change in immunoactivity. The epitope, which was always recognized by a monoclonal antibody against P30, could be a linear epitope.

Studies on human porin. IX. Immunolocalization of porin and CFTR channels in human surface respiratory epithelium.

The expression of the voltage-dependent anion channel (VDAC) "Porin 31 HL" and its cellular and subcellular immunocytochemical localization in the human respiratory epithelium were studied with monoclonal and polyclonal antibodies using immunofluorescence and immunogold labelling with light (LM) and transmission electron microscopy (TEM), respectively. Porin was identified in the apical domain of the ciliated cells and in the basal cells of the respiratory epithelium. Immunogold labelling was present in the apical plasma membrane and subapical vesicles of the ciliated cells. In pre-embedded freshly dissociated surface epithelial cells, porin could also be identified with TEM at the outer part of the plasma membrane of basal cells. By LM double immunolabelling, both porin and cystic fibrosis transmembrane conductance regulator (CFTR) were identified in the apical domain of ciliated cells but not in basal cells where CFTR was never identified. On Western blots of solubilized total membrane protein preparations from the same frozen surface epithelial respiratory cells, the antibodies recognized a group of 3 proteins of 31, 60 and 130-140 kDa with a strong reactivity for a 31 kDa protein, corresponding to the porin and a protein of 170 kDa which is consistent with mature CFTR. These results suggest that porin might be part of a multi-component chloride channel complex which could interact with CFTR.

Role of acidic subcellular compartments in the degradation of internalized insulin and in the recycling of the internalized insulin receptor in liver cells: in vivo and in vitro studies.

Upon interaction with liver cells, insulin is internalized along with its receptor into nonlysosomal endocytic structures termed endosomes. In this work, the biochemical evidence supporting the role of endosomal acidity in the degradation of internalized insulin and in the recycling of the internalized insulin receptor is described. Treatment of rats by chloroquine and/or quinacrine, two acidotropic drugs, increases by 5-10 fold the amount of endogenous insulin associated with endosomal fractions and, in rats injected by 125I-labeled or native insulin, the endosomal uptake of these ligands at late times after injection. With 125I-insulin, these drugs inhibit the degradation of internalized hormone as judged on physical, biological and immunological criteria. Chloroquine and quinacrine treatment also increases the insulin receptor content of endosomal fractions and, in rats injected by native insulin, the ligand-induced accumulation of receptors in endosomal fractions at late times after injection. Subfractionation of endosomal fractions on Percoll gradients shows that chloroquine treatment shifts the distribution of both insulin and the insulin receptor towards higher densities, the receptor shift being slightly more pronounced in insulin-injected rats. Incubation of isolated endosomes containing internalized insulin at 30-37 degrees C results in a rapid degradation of this ligand, with a maximal at pH 5-6. Addition of ATP, by decreasing the endosomal pH, stimulates insulin degradation above pH 7, whereas addition of chloroquine and quinacrine, by elevating endosomal pH, exerts opposite effects. These data indicate that endosomal acidity is required for optimum degradation of internalized insulin within endosomes and recycling of the internalized receptor.

Mechanisms of up-regulation of the liver insulin receptor in chronically hypoinsulinemic rats: assessment of receptor endocytosis.

Chronic hypoinsulinemic states in rodents are known to cause an increase in the number of insulin receptors at the hepatocyte surface. To assess whether this change results from a reduced endocytosis of the receptors, the effects of streptozotocin treatment and fasting on the number and the subcellular distribution of hepatic insulin receptors have been evaluated in the rat. In streptozotocin-treated rats, insulin receptor number was increased by 25-40% in plasma membrane and total cellular membrane fractions, and by 60-130% in the light Golgi-endosomal (GE) fraction. In contrast, receptor number was unaffected in the intermediate GE fraction and decreased by 25-35% in the heavy GE fraction. Such changes were detectable at 12 h in GE fractions and at 2 days in other subcellular fractions, and lasted for at least 8 days. Streptozotocin treatment also led to a 3- to 4-fold decrease in the insulin content of GE fractions, indicating reduced hormone endocytosis. Fasting for 16 h elicited changes in receptor and ligand concentration in cell fractions comparable to those induced by streptozotocin. It is concluded that, although endocytosis of hepatic insulin receptors is reduced in chronic hypoinsulinemic states, changes in receptor synthesis and/or degradation also occur in these states.