Bronchial Epithelial Lung Research Articles

Basal extrusion of single-oncogenic mutant cells induces dome-like structures with altered microenvironments.

At the initial stage of carcinogenesis, oncogenic transformation occurs in single cells within epithelial layers. However, the behavior and fate of the newly emerging transformed cells remain enigmatic. Here, using originally established mouse models, we investigate the fate of RasV12-transformed cells that appear in a mosaic manner within epithelial tissues. In the lung bronchial epithelium, most majority of RasV12-transformed cells are apically extruded, whereas noneliminated RasV12 cells are often basally delaminated leading to various noncell-autonomous changes in surrounding environments; macrophages and activated fibroblasts are accumulated, and normal epithelial cells overlying RasV12 cells overproliferate and form a convex multilayer, which is termed a 'dome-like structure'. In addition, basally extruded RasV12 cells acquire certain features of epithelial-mesenchymal transition (EMT). Furthermore, the expression of COX-2 is profoundly elevated in RasV12 cells in dome-like structures, and treatment with the COX inhibitor ibuprofen suppresses the recruitment of activated fibroblasts and moderately diminishes the formation of dome-like structures. Therefore, basal extrusion of single-oncogenic mutant cells can induce a tumor microenvironment and EMT and generate characteristic precancerous lesions, providing molecular insights into the earlier steps of cancer development.

TITF1 Screening in Human Congenital Diaphragmatic Hernia (CDH).

TITF1 (Thyroid Transcription Factor-1) is a homeodomain-containing transcription factor. Previous studies showed that Titf1 null mice are characterized by failure of tracheo-oesophageal separation and impaired lung morphogenesis resulting in Pulmonary Hypoplasia (PH). In this study, we aim to evaluate the role of TITF1 in the pathogenesis of congenital diaphragmatic hernia (CDH) in humans. We investigated TITF1 expression in human trachea and lungs and performed direct mutation analysis in a CDH population. We studied 13 human fetuses at 14 to 24 weeks of gestation. Five μm sections were fixed in paraformaldehyde and incubated with anti-TITF1 primary antibody. Positive staining was visualized by biotinylated secondary antibody. We also performed TITF1 screening on genomic DNA extracted from peripheral blood of 16 patients affected by CDH and different degrees of PH, searching for mutations, insertions, and/or deletions, by sequencing the exonic regions of the gene. Histochemical studies showed positive brown staining of fetal follicular thyroid epithelium, normal fetal trachea, and normal fetal lung bronchial epithelium. Fetal esophageal wall was immunohistochemically negative. Molecular genetic analysis showed complete identity between the sequences obtained and the Wild Type (WT) form of the gene in all cases. No mutation, insertion and/or deletion was detected. Although TITF1 is expressed in the human fetal lung and has been considered to have a role in the pathogenesis of PH in CDH, the results of our study do not support the hypothesis that TITF1 mutations play a key role in the etiopathogenesis of CDH.

Discrimination of Receptor-Mediated Endocytosis by Surface-Enhanced Raman Scattering.

Cellular energy required for the maintenance of cellular life is stored in the form of adenosine triphosphate (ATP). Understanding cellular mechanisms, including ATP-dependent metabolisms, is crucial for disease diagnosis and treatment, including drug development and investigation of new therapeutic systems. As an ATP-dependent metabolism, endocytosis plays a key role not only in the internalization of molecules but also in processes including cell growth, differentiation, and signaling. To understand cellular mechanisms including endocytosis, many techniques ranging from molecular approaches to spectroscopy are used. Surface-enhanced Raman scattering (SERS) is shown to provide valuable label-free molecular information from living cells. In this study, receptor-mediated endocytosis was investigated with SERS by inhibiting endocytosis with ATP depletion agents: sodium azide (NaN3) and 2-deoxy-d-glucose (dG). Human lung bronchial epithelium (Beas-2b) cells, normal prostate epithelium (PNT1A) cells, and cervical cancer epithelium (HeLa) cells were used as models. First, the effect of NaN3 and dG on the cells were examined through cytotoxicity, apoptosis–necrosis, ATP assay, and uptake inhibition analysis. An attempt to relate the spectral changes in the cellular spectra to the studied cellular events, receptor-mediated endocytosis inhibition, was made. It was found that the effect of two different ATP depletion agents can be discriminated by SERS, and hence receptor-mediated endocytosis can be tracked from single living cells with the technique without using a label and with limited sample preparation.

Synthesis a group of 5(6)‐substituted benzimidazole Zn(II) and Co(II) complexes and investigation their cytotoxic and antimicrobial activities

AbstractA series of 5(6)‐substituted benzimidazole ligand‐zinc(II) and cobalt(II) complexes were synthesized and characterized by1H,13C NMR, IR, CHN analysis techniques. In vitro, anticancer and antimicrobial activities of ligand and complexes were evaluated. The cytotoxic activities of compounds were investigated against the human lung adenocarcinoma (A549) cell lines and healthy human lung bronchial epithelium (BEAS2B) cells. Compounds1,6, and8were observed to exhibit higher cytotoxic activity than the others at the 72nd hour. Also, the compounds showed varying degrees of antimicrobial activity against the microorganisms used. Compounds1,5, and8were most effective against standard strainsPseudomonas aeruginosaATCC 27853 andStaphylococcus aureusATCC 29213. As for the antifungal activity, compounds1,5,6, and8showed high antifungal activity against the yeast type fungiCandida albicansATCC 90028 andCandida tropicalis.

Small-interfering RNA for c-Jun attenuates cell death by preventing JNK-dependent PARP1 cleavage and DNA fragmentation in nitrogen mustard-injured immortalized human bronchial epithelial cells.

Sulfur mustard (a type of vesicant) can directly damage lung bronchial epithelium via aerosol inhalation, and prevalent cell death is an early event that obstructs the respiratory tract. JNK/c-Jun is a stress response pathway, but its role in cell death of the injured cells is not clear. Here, we report that JNK/c-Jun was activated in immortalized human bronchial epithelial (HBE) cells exposed to a lethal dose (20μM) of nitrogen mustard (NM, a sulfur mustard analog). c-Jun silencing using small-interfering RNA (siRNA) rendered the cells resistant to NM-mediated cell death by blocking poly(ADP-ribose) polymerase 1 (PARP1) cleavage and DNA fragmentation. In addition, the transduction of upstream extrinsic (Fasl-Fas-caspase-8) and intrinsic (loss of Bcl-2 and mitochondrial membrane potential, ΔΨm) apoptosis pathways, as well as phosphorylated (p)-H2AX (Ser139), an epigenetic marker contributing to DNA fragmentation and PARP1 activity, was partially suppressed. To mimic the detachment of cells by NM, HBE cells were trypsinized and seeded on culture plates that were pre-coated with poly-HEMA to prevent cell adhesion. The JNK/c-Jun pathway was found to be activated in the detached cells. In conclusion, our results indicate that JNK/c-Jun pathway activation is necessary for NM-caused HBE cell death and further suggest that c-Jun silencing may be a potential approach to protect HBE cells from vesicant damage.

NOTCH inhibition promotes bronchial stem cell renewal and epithelial barrier integrity after irradiation.

Hyperactivity of the NOTCH pathway is associated with tumor growth and radiotherapy resistance in lung cancer, and NOTCH/γ‐secretase inhibitors (GSIs) are a potential therapeutic target. The therapeutic outcome, however, is often restricted by the dose‐limiting toxicity of combined treatments on the surrounding healthy tissue. The NOTCH signaling pathway is also crucial for homeostasis and repair of the normal airway epithelium. The effects of NOTCH/γ‐secretase inhibition on the irradiation of normal lung epithelium are unknown and may counteract antitumor activity. Here we, therefore, investigated whether normal tissue toxicity to radiation is altered upon NOTCH pathway inhibition. We established air‐liquid interface pseudostratified and polarized cultures from primary human bronchial epithelial cells and blocked NOTCH signaling alone or after irradiation with small‐molecule NOTCH inhibitor/GSI. We found that the reduction in proliferation and viability of bronchial stem cells (TP63+) in response to irradiation is rescued with concomitant NOTCH inhibition. This correlated with reduced activation of the DNA damage response and accelerated repair by 24 hours and 3 days postirradiation. The increase in basal cell proliferation and viability in GSI‐treated and irradiated cultures resulted in an improved epithelial barrier function. Comparable results were obtained after in vivo irradiation, where the combination of NOTCH inhibition and irradiation increased the percentage of stem cells and ciliated cells ex vivo. These encourage further use of normal patient tissue for toxicity screening of combination treatments and disclose novel interactions between NOTCH inhibition and radiotherapy and opportunities for tissue repair after radiotherapy.

Investigation into antiproliferative activity and apoptosis mechanism of new arene Ru(ii) carbazole-based hydrazone complexes.

Ruthenium complexes with bioactive ligands are becoming promising substitutes for platinum complexes due to their precise action against various cancers. In the present study, the synthesis of three new arene Ru(ii) complexes containing new carbazole-based hydrazone ligands of general formula [(η6-benzene)Ru(L)Cl] (1-3; L = carbazolone benzhydrazone ligands), and their anticancer properties are described. The structural characterization of the ligands and their ruthenium complexes has been realized with the aid of elemental analysis, IR, UV-vis, NMR and HR-MS techniques. The molecular structures of all three complexes have been elucidated by single crystal X-ray crystallography and reveal the existence of pseudo-octahedral geometry around the ruthenium. The in vitro cancer cell growth inhibition property of the complexes against A549 (lung carcinoma), A2780 (ovarian adenocarcinoma) and non-cancerous 16HBE (human lung bronchial epithelium) cells were examined by MTT assay. All the complexes display good cytotoxicity towards both of these types of cancer cell compared to the standard drug cisplatin, with low IC50 values. Remarkably, complex 3, which contains an electron-donating substituent, induces a significant reduction of viability in A2780 cells. The inhibition capacity of the complexes towards A2780 cells proliferation was further confirmed using 5-ethynyl-2-deoxyuridine (EdU) assay via minimal DNA synthesis. The result of the acridine orange-ethidium bromide (AO-EB) fluorescent staining assay establishes that the cytotoxicity of the complexes was mediated by apoptosis in cancer cells. Furthermore, flow cytometry using Annexin V-FITC/propidium iodide (PI) double staining determines the quantitative discrimination of early apoptosis by the externalization of phosphatidylserine. In addition, cell cycle distribution indicates that the complexes block the cell cycle progression in the S-phase. The outcome of our investigation shows the promising scope and potency of tailored arene ruthenium complexes for precise cancer chemotherapy beyond platinum drugs.

Presence of Stromal Cells Enhances Epithelial-to-Mesenchymal Transition (EMT) Induction in Lung Bronchial Epithelium after Protracted Exposure to Oxidative Stress of Gamma Radiation.

The aim of the study was to investigate the role of a microenvironment in the induction of epithelial-to-mesenchymal transition (EMT) as a sign of early stages of carcinogenesis in human lung epithelial cell lines after protracted low-dose rate γ-radiation exposures. BEAS-2B and HBEC-3KT lung cell lines were irradiated with low-dose rate γ-rays (137Cs, 1.4 or 14 mGy/h) to 0.1 or 1 Gy with or without adding TGF-β. TGF-β-treated samples were applied as positive EMT controls and tested in parallel to find out if the radiation has a potentiating effect on the EMT induction. To evaluate the effect of the stromal component, the epithelial cells were irradiated in cocultures with stromal MRC-9 lung fibroblasts. On day 3 post treatment, the EMT markers: α-SMA, vimentin, fibronectin, and E-cadherin, were analyzed. The oxidative stress levels were evaluated by 8-oxo-dG analysis in both epithelial and fibroblast cells. The protracted exposure to low Linear Energy Transfer (LET) radiation at the total absorbed dose of 1 Gy was able to induce changes suggestive of EMT. The results show that the presence of the stromal component and its signaling (TGF-β) in the cocultures enhances the EMT. Radiation had a minor cumulative effect on the TGF-β-induced EMT with both doses. The oxidative stress levels were higher than the background in both epithelial and stromal cells post chronic irradiation (0.1 and 1 Gy); as for the BEAS-2B cell line, the increase was statistically significant. We suggest that the induction of EMT in bronchial epithelial cells by radiation requires more than single acute exposure and the presence of stromal component might enhance the effect through free radical production and accumulation.

In vitro evaluation of organic extractable matter from ambient PM2.5 using human bronchial epithelial BEAS-2B cells: Cytotoxicity, oxidative stress, pro-inflammatory response, genotoxicity, and cell cycle deregulation

A particular attention has been devoted to the type of toxicological responses induced by particulate matter (PM), since their knowledge is greatly complicated by the fact that it is a heterogeneous and often poorly described pollutant. However, despite intensive research effort, there is still a lack of knowledge about the specific chemical fraction of PM, which could be mainly responsible of its adverse health effects. We sought also to better investigate the toxicological effects of organic extractable matter (OEM) in normal human bronchial epithelial lung BEAS-2B cells. The wide variety of chemicals, including PAH and other related-chemicals, found in OEM, has been rather associated with early oxidative events, as supported by the early activation of the sensible NRF-2 signaling pathway. For the most harmful conditions, the activation of this signaling pathway could not totally counteract the ROS overproduction, thereby leading to critical oxidative damage to macromolecules (lipid peroxidation, oxidative DNA adducts). While NRF-2 is an anti-inflammatory, OEM exposure did not trigger any significant change in the secretion of inflammatory cytokines (i.e., TNFα, IL-1β, IL-6, IL-8, MCP-1, and IFNγ). According to the high concentrations of PAH and other related organic chemicals found in this OEM, CYP1A1 and 1B1 genes exhibited high transcription levels in BEAS-2B cells, thereby supporting both the activation of the critical AhR signaling pathway and the formation of highly reactive ultimate metabolites. As a consequence, genotoxic events occurred in BEAS-2B cells exposed to this OEM together with cell survival events, with possible harmful cell cycle deregulation. However, more studies are required to implement these observations and to contribute to better decipher the critical role of the organic fraction of air pollution-derived PM2.5 in the activation of some sensitive signaling pathways closely associated with G1/S and intra-S checkpoint blockage, on the one hand, and cell survival, on the other hand.

Benzo[a]pyrene activates an AhR/Src/ERK axis that contributes to CYP1A1 induction and stable DNA adducts formation in lung cells

Benzo[a]pyrene (B[a]P), the most extensively studied carcinogen in cigarette smoke, has been regarded as a critical mediator of lung cancer. It is known that B[a]P-mediated Aryl hydrocarbon Receptor (AhR) activation stimulates the mitogen activated protein kinases (MAPK) signaling cascade in different cell models. MAPK pathway disturbances drive alterations in cellular processes, such as differentiation, proliferation, and apoptosis, and the disturbances may also modify the AhR pathway itself. However, MAPK involvement in B[a]P metabolic activation and toxicity in lung tissues is not well understood. Here, we used a non-transformed human bronchial epithelial lung cell line, BEAS-2B, to study the participation of ERK 1/2 kinases in the metabolic activation of B[a]P and in its related genotoxic effects. Our results indicate that B[a]P is not cytotoxic to BEAS–2B cells at relatively low concentrations, but it enhances CYP1A1 gene transcription and protein induction. Additionally, B[a]P promotes Src and ERK 1/2 phosphorylation. Accordingly, inhibition of both Src and ERK 1/2 phosphorylation decreases CYP1A1 protein induction, AhR nuclear translocation and production of B[a]P adducts. Together, these data suggest a crosstalk between AhR and the members of the MAPK pathway, ERK 1/2 mediated by Src kinase. This interaction is important for the adequate AhR pathway signaling that in turn induces transcription and protein induction of CYP1A1 and B[a]P-induced DNA damage in BEAS–2B cells.

GSDMB induces an asthma phenotype characterized by increased airway responsiveness and remodeling without lung inflammation

Gasdermin B (GSDMB) on chromosome 17q21 demonstrates a strong genetic linkage to asthma, but its function in asthma is unknown. Here we identified that GSDMB is highly expressed in lung bronchial epithelium in human asthma. Overexpression of GSDMB in primary human bronchial epithelium increased expression of genes important to both airway remodeling [TGF-β1, 5-lipoxygenase (5-LO)] and airway-hyperresponsiveness (AHR) (5-LO). Interestingly, hGSDMBZp3-Cre mice expressing increased levels of the human GSDMB transgene showed a significant spontaneous increase in AHR and a significant spontaneous increase in airway remodeling, with increased smooth muscle mass and increased fibrosis in the absence of airway inflammation. In addition, hGSDMBZp3-Cre mice showed increases in the same remodeling and AHR mediators (TGF-β1, 5-LO) observed in vitro in GSDMB-overexpressing epithelial cells. GSDMB induces TGF-β1 expression via induction of 5-LO, because knockdown of 5-LO in epithelial cells overexpressing GSDMB inhibited TGF-β1 expression. These studies demonstrate that GSDMB, a gene highly linked to asthma but whose function in asthma is previously unknown, regulates AHR and airway remodeling without airway inflammation through a previously unrecognized pathway in which GSDMB induces 5-LO to induce TGF-β1 in bronchial epithelium.

The occurrence of polycyclic aromatic hydrocarbons and their derivatives and the proinflammatory potential of fractionated extracts of diesel exhaust and wood smoke particles

Exposure to combustion emissions, including diesel engine exhaust and wood smoke particles (DEPs and WSPs), has been associated with inflammatory responses. To investigate the possible role of polycyclic aromatic hydrocarbons (PAHs) and PAH-derivatives, the DEPs and WSPs methanol extracts were fractionated by solid phase extraction (SPE), and the fractions were analyzed for more than ∼120 compounds. The pro-inflammatory effects of the fractionated extracts were characterized by exposure of bronchial epithelial lung cells (BEAS-2B). Both native DEPs and WSPs caused a concentration-dependent increase in IL-6 and IL-8 release and cytotoxicity. This is consistent with the finding of a rather similar total content of PAHs and PAH-derivatives. Yet, the samples differed in specific components, suggesting that different species contribute to the toxicological response in these two types of particles. The majority of the IL-6 release and cytotoxicity was induced upon exposure to the most polar (methanol) SPE fraction of extracts from both samples. In these fractions hydroxy-PAHs, carboxy-PAHs were observed along with nitro-amino-PAHs in DEP. However, the biological effects induced by the polar fractions could not be attributed only to the occurrence of PAH-derivatives. The present findings indicate a need for further characterization of organic extracts, beyond an extensive analysis of commonly suspected PAH and PAH-derivatives. Supplemental materials are available for this article. Go to the publisher's online edition of Journal of Environmental Science and Health, Part A, to view the supplemental file.

Role of size and surface area for pro-inflammatory responses to silica nanoparticles in epithelial lung cells: Importance of exposure conditions

The present study compared non-crystalline silica particles of nano (50nm)- and submicro (500nm)-size (Si50 and Si500) for the potential to induce cytokine responses in bronchial epithelial lung cells (BEAS-2B). The cell cultures were exposed to equal mass and surface area concentrations of the two particles in different exposure media; LHC-9 and DMEM:F12. The state of agglomeration was different in the two media; with marked agglomeration in LHC-9 and nearly no agglomeration in DMEM:F12. On a mass basis, Si50 was more potent than Si500 in inducing cytokine responses in both exposure media. In contrast, upon exposure to similar surface area concentrations, Si500 was more potent than Si50 in DMEM:F12. This might be due to different agglomeration/sedimentation properties of Si50 versus Si500 in the two media. However, influence of differences in particle reactivity or particle uptake cannot be excluded. The data indicated no qualitative changes in the cytokine gene-expression patterns induced by the two particles, suggesting effects through similar mechanisms. These aspects might be of importance for interpretation of in vitro studies of nanomaterials.

Clinic significance of microRNA‐99a expression in human lung adenocarcinoma

The purpose of this study was to investigate the clinical significance of microRNA-99a expression in lung adenocarcinoma. qRT-PCR assay was performed to detect miR-99a expression in lung adenocarcinoma cells or tissues. The correlations of miR-99a expression with clinicopathological factors and prognosis of lung adenocarcinoma patients were analyzed. The effects of miR-99a expression on growth and apoptosis of lung adenocarcinoma cell line and its potential target gene were determined by MTT, flow cytometry, luciferase reporter, and Western blot assays. The relative miR-99a expression in lung adenocarcinoma cells was significantly lower than that in normal lung bronchial epithelium cell line. Also, miR-99a expression in lung adenocarcinoma tissues was significantly lower than that in corresponding nontumor tissues. Low miR-99a expression was found to be closely correlated with advanced clinical stage and lymph node metastasis. Kaplan-Meier survival and Cox regression analyses showed that the status of miR-99a expression was an independent prognosis factor for lung adenocarcinoma patients. Functional analyses showed that upregulation of miR-99a could inhibit growth and induce apoptosis in lung adenocarcinoma cells by targeting mTOR. Low MiR-99a expression was a poor prognostic factor for patients with lung adenocarcinoma, and miR-99a functions as a tumor suppressor by targeting mTOR.

Comparative toxicity of air pollution particulate matter (PM2.5) sampled under urban, industrial or rural influence in human bronchial epithelial lung cells (BEAS-2B)

Comparative toxicity of air pollution particulate matter (PM2.5) sampled under urban, industrial or rural influence in human bronchial epithelial lung cells (BEAS-2B)

Structure-Activity Relationship of Capsaicin Analogs and Transient Receptor Potential Vanilloid 1-Mediated Human Lung Epithelial Cell Toxicity

Activation of intracellular transient receptor potential vanilloid-1 (TRPV1) in human lung cells causes endoplasmic reticulum (ER) stress, increased expression of proapoptotic GADD153 (growth arrest- and DNA damage-inducible transcript 3), and cytotoxicity. However, in cells with low TRPV1 expression, cell death is not inhibited by TRPV1 antagonists, despite preventing GADD153 induction. In this study, chemical variants of the capsaicin analog nonivamide were synthesized and used to probe the relationship between TRPV1 receptor binding, ER calcium release, GADD153 expression, and cell death in TRPV1-overexpressing BEAS-2B, normal BEAS-2B, and primary normal human bronchial epithelial lung cells. Modification of the 3-methoxy-4-hydroxybenzylamide vanilloid ring pharmacophore of nonivamide reduced the potency of the analogs and rendered several analogs mildly inhibitory. Correlation analysis of analog-induced calcium flux, GADD153 induction, and cytotoxicity revealed a direct relationship for all three endpoints in all three lung cell types for nonivamide and N-(3,4-dihydroxybenzyl)nonanamide. However, the N-(3,4-dihydroxybenzyl)nonanamide analog also produced cytotoxicity through redox cycling/reactive oxygen species formation, shown by inhibition of cell death by N-acetylcysteine. Molecular modeling of binding interactions between the analogs and TRPV1 agreed with data for reduced potency of the analogs, and only nonivamide was predicted to form a "productive" ligand-receptor complex. This study provides vital information on the molecular interactions of capsaicinoids with TRPV1 and substantiates TRPV1-mediated ER stress as a conserved mechanism of lung cell death by prototypical TRPV1 agonists.

Increased Angiotensin II AT 1 receptor mRNA and binding in spleen and lung of AT 2 receptor gene disrupted mice

To clarify the relationship between Angiotensin II AT 1 and AT 2 receptors, we studied AT 1 receptor mRNA and binding expression in tissues from AT 2 receptor gene disrupted (AT 2 −/−) female mice, where AT 2 receptors are not expressed in vivo, using in situ hybridization and quantitative autoradiography. Wild type mice expressed AT 1A receptor mRNA and AT 1 receptor binding in lung parenchyma, the spleen, predominantly in the red pulp, and in liver parenchyma. In wild type mice, lung AT 2 receptors were expressed in lung bronchial epithelium and smooth muscle, and were not present in the lung parenchyma, the spleen or the liver. This indicates that AT 1 and AT 2 receptors were not expressed in the same cells. In AT 2 −/− mice, we found higher AT 1A receptor mRNA and AT 1 receptor binding in lung parenchyma and in the red pulp of the spleen, but not in the liver, when compared to littermate wild type controls. Our results suggest that impaired AT 2 receptor function upregulates AT 1 receptor transcription and expression in a tissue-specific manner and in cells not expressing AT 2 receptors. AT 1 upregulation explains the increased sensitivity to Angiotensin II characteristic of the AT 2 −/− phenotype, consistent with enhanced AT 1 receptor activation in a number of tissues.

H-REV107-1 Stimulates Growth in Non-Small Cell Lung Carcinomas via the Activation of Mitogenic Signaling

H-REV107-1, a known member of the class II tumor suppressor gene family, is involved in the regulation of differentiation and survival. We analyzed H-REV107-1 in non-small cell lung carcinomas, in normal lung, and in immortalized and tumor-derived cell lines. Sixty-eight percent of lung tumors revealed positive H-REV107-1-specific staining. Furthermore, survival analysis demonstrated a significant association of cytoplasmic H-REV107-1 with decreased patient survival. This suggested that H-REV107-1, known as a tumor suppressor, plays a different role in non-small cell lung carcinomas. Knock-down of H-REV107-1 expression in lung carcinoma cells inhibited anchorage-dependent and anchorage-independent growth whereas overexpression of H-REV107-1 induced tumor cell proliferation. Consistent with results of the survival analysis, cytoplasmic localization of the protein was essential for this growth-inducing function. Analysis of signaling pathways potentially involved in this process demonstrated that overexpression of H-REV107-1 stimulated RAS-GTPase activity, ERK1,2 phosphorylation, and caveolin-1 expression in the cell lines analyzed. These results indicate that H-REV107-1 is deficient in its function as a tumor suppressor in non-small cell lung carcinomas and is required for proliferation and anchorage-independent growth in cells expressing high levels of the protein, thus contributing to tumor progression in a subset of non-small cell lung carcinomas.

Peribronchial innervation of the rat lung.

Mammalian peribronchial tissue is supplied by several peptide-containing nerve fibers. Although it is well established that different neuropeptides exert significant effects on bronchial and vascular tone in the lungs, the role played by some neuromediators on the general regulation, differentiation and release of locally active substances is still controversial. We studied the innervation of rat peribronchial tissue by immunohistochemical techniques. The immunoperoxidase method with nickel amplification was applied to detect the distribution of nerve fibers using antibodies against the general neuronal marker PGP 9.5 (neuron-specific cytoplasmic protein), while the cholinacetyltransferase immunoreactivity was studied by immunohistochemistry. A slight immunoreactivity for NT receptors is observed in lung bronchial epithelium. There is increasing evidence that NTs may act with a paracrine mechanism regulating functional activity of neuronal and non-neuronal structures. A specific immunoreactivity for NTs and NT receptors was also demonstrated within different layers of large, medium and small sized intrapulmonary arteries and veins, according to a recent study of our group. Moreover our data describe the expression of NTs and NT receptors in lymphoid aggregates of the lung (BALT) in which both lymphocytes and macrophages express TrkA receptor and synthesize NTs. Our results show the presence of an extensive network of innervation in the rat peribronchial tissue, confirming a morphological basis for a possible neural modulation of the respiratory mucosa and the physiological/pathophysiological mechanisms of the lung.

Acetylcholine is an autocrine or paracrine hormone synthesized and secreted by airway bronchial epithelial cells.

The role of acetylcholine (ACh) as a key neurotransmitter in the central and peripheral nervous system is well established. However, the role of ACh may be broader because ACh may also function as an autocrine or paracrine signaling molecule in a variety of nonneuronal tissues. To begin to establish ACh of nonneuronal origin as a paracrine hormone in lung, we have examined neonatal and adult monkey bronchial epithelium for the components involved in nicotinic cholinergic signaling. Using immunohistochemistry and RT-PCR, we have demonstrated in lung bronchial epithelial cells (BECs) expression of choline acetyltransferase, the vesicular ACh transporter, the choline high-affinity transporter, alpha7, alpha4, and beta2 nicotinic ACh receptor (nAChR) subunits, and the nAChR accessory protein lynx1. Confocal microscopy demonstrates that these factors are expressed in epithelial cells and are clearly distinct from neighboring nerve fibers. Confirmation of RNA identity has been confirmed by partial sequence analysis of PCR products and by cDNA cloning. Primary culture of BECs confirms the synthesis and secretion of ACh and the activity of cholinesterases. Thus, ACh meets all the criteria for an autocrine/paracrine hormone in lung bronchial epithelium. The nonneuronal cholinergic signaling pathway in lung provides a potentially important target for cholinergic drugs. This pathway may also explain some of the effects of nicotine on fetal development and also provides additional mechanisms by which smoking affects lung cancer growth and development.