Epithelial A549 Research Articles

Analysis of cytotoxicity induced by proinflammatory cytokines in the human alveolar epithelial cell line A549

Epithelial cell injury under hyperinflammatory conditions is critical in the development of septic acute lung injury (ALI). The aim of the present study is to analyze the cytotoxic effects of a mixture of proinflammatory cytokines in the human alveolar epithelial cell line A549. The cytotoxicity of proinflammatory cytokines were assessed in A549 cells by measuring lactate dehydrogenase released into the culture medium and by crystal violet staining of surviving cells. Activation of the caspase-dependent apoptotic pathway was evaluated by monitoring cleavage of cytokeratin 18 by caspases using enzyme-liked immunosorbent assay (ELISA). To estimate the cytotoxic signaling pathways responsible for epithelial injury, agents with antiinflammatory or antioxidative properties were extensively screened for cytoprotective effects in the inflammation-associated epithelial injury model. The present study revealed that inflammatory cytokines exerted cytotoxicity in A549 cells. A mixture of interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), designated as cytomix, augmented cytotoxicity compared with each individual cytokine. Treatment with glucocorticoid (dexamethasone), tetracycline-derived antiinflammatory antibiotics (minocycline or doxycycline), angiotensin II receptor blockers (losartan or telmisartan), or antioxidants (dimethyl sulfoxide, catalase) attenuated cytomix-induced cytotoxicity, including caspase activation. These results implied that inflammatory cytokines alone could cause alveolar epithelial injury in the pathophysiology of septic ALI. Caspase-dependent apoptosis was speculated to be one mechanism responsible for the cytokine-induced cytotoxicity. Agents with antiinflammatory or antioxidative properties such as glucocorticoid, tetracycline-derived antibiotics, angiotensin II receptor blockers, or direct antioxidants showed substantial effect in attenuating cytokine-induced cytotoxicity and may be candidates for treatment options.

Aqueous Synthesis of Au:CdTe Nanocrystals for Noninvasive Fluorescence Imaging in Living Cells

The gold-doped cadmium telluride (Au:CdTe) nanocrystals were synthesized by aqueous solution route using L-glutathione and L-cysteine as stabilizers. As-prepared Au:CdTe nanocrystals have good monodispersity and a zinc-blende structure. Compared with undoped CdTe nanocrystals, the Au:CdTe nanocrystals exhibited improved photostability, higher cellular affinity, and lower cytotoxicity. The Au:CdTe nanocrystals were used as probes for long-term noninvasive fluorescence imaging in living cells (The human lung epithelial carcinoma A549 cells). They could be endocytic uptaken by A549 cells and stably labeled the cytoplasm for over a week. By transmission electron microscopy (TEM) analysis, the Au:CdTe NCs could be observed in vesicles after being uptaken by A549 cells. Doping semiconductor nanocrystals with gold has the potential to engineer the photostability and biocompatibility for extensive biomedical applications. This work developed a facile aqueous solution route to synthesize gold-doped semiconductor nanocrystals and may assist in the design of doped nanobiomaterials.

Cellular Photodynamic Toxicity of Hematoporphyrin in Various Nanocarrier Systems

In this study, we investigated the cellular uptake of Hematoporphyrin (Hp) encapsulated in nanocarriers by human lung adeno- carcinoma A549 cells and the photodynamic toxic effects on those cells. The nanocarriers included liposomes, micelles, and nanoparti- cles. The Hp was encapsulated in liposomes through a film hydration and extrusion method. Hp was encapsulated in micelles through a film hydration method. The Hp loaded nanoparticles were prepared using an emulsification-diffusion process. The Hp in nanocarriers was characterized by laser light scattering to determine particle size and zeta potential of the surface charge. The cellular uptake of nano- carriers was measured by fluorescence spectrometry. The photodynamic toxicity of various Hp nanocarriers was evaluated in human lung epithelial carcinoma A549 cells using a light-emitting diode (LED) device at a wavelength of 630±5 nm. The averaged size of all nano- carriers was between 112 and 135 nm. The surface charge observed for all formulations, particularly the micelles showed lower negative valued than those of other systems. Cellular uptake and photocytotoxicity of the four Hp preparations in serum free medium were in the following order: micelle-Hp > nanoparticle-Hp > liposome-Hp > free Hp. In the presence of serum protein, the photodynamic toxicity of all formulations significantly decreased.

Ubiquitination of heat shock protein 27 is mediated by its interaction with Smad ubiquitination regulatory factor 2 in A549 cells

ABSTRACTSmad ubiquitination regulatory factor 2 (Smurf2) is a crucial part of the ubiquitin-proteasome pathway (UPP) that regulates cellular signal transduction via ubiquitin-dependent degradation of some substrates and receptors. The biological function of Smurf2 in lung diseases, however, is not clear. In this study, the authors found that overexpression of Smurf2 altered the subcellular localization and distribution of heat shock protein 27 (HSP27), and induced a decrease of HSP27 protein levels through HSP27 degradation by the UPP in human lung adenocarcinoma epithelial cell line A549. Colocalized assay using confocal microscopy and coimmunoprecipitated reciprocally by either antibody indicated the interaction between Smurf2 and HSP27, which suggested that Smurf2 mediated ubiquitylation-dependent degradation of HSP27 through their interaction in A549 cells.

Par-4 gene silence inhibited hydrogen peroxide-induced apoptosis in alveolar epithelial cells

Objective To investigate the effects of Par-4 gene silence on hydrogen peroxide-induced apoptosis in alveolar epithelial cells. Method The alveolar epithelial cells A549 were cultured and exposed to hydrogen peroxide. The siRNA sequences targeted Par-4 gene was chemically synthesized and transfected to A549 cells with or without the exposure of hydrogen peroxide. The cells were divided into normal control groups, hydrogen peroxide-treated group(The cells were treated with 0. 1 mmol/L hydrogen peroxide), hydrogen peroxide and Par-4-siRNA-treated group(The cells were treated with 0. 1 mmol/L hydrogen peroxide after transfection of Par-4-siRNA), Non-specific DNA sequence transfection control group. The apoptosis of A549 cells was quantified by flow cytometry. The expression of Smac protein was detected by Western blot.Electrophoretic mobility shift assay was applied for evaluating the change of E2F1 DNA binding activity. Relative activity of Caspase-3 was detected by clolorimetric assay. Results The percent of apoptotic cells in hydrogen peroxide and Par-4-siRNA-treated group was (29.7 ± 2.3) %, which was significantly lower than that of hydrogen peroxide-treated group [(54.2 ± 4.1)%, q= 8.91, P ＜ 0.01)]. Par-4 siRNA could significantly suppress the increase of Smac protein, E2F1 DNA binding activity and caspase-3 activity induced by hydrogen peroxide in A549 cells. Conclusions Par-4 gene silence induced by siRNA might inhibit the apoptosis of alveolar epithelial cells, which might be resulted from suppression of the up-regulation of Smac gene expression, E2F1 DNA binding activity and caspase-3 activity. Key words: Par-4; Small RNA interference; Hydrogen peroxide; Alveolar epithelial cells; Apoptosis

Using a pattern recognition approach to link inorganic chemical fingerprints of ambient PM2.5–0.1 with in vitro biological effects

Atmospheric fine particulates act as prime vehicles for the transport of toxic chemicals into the human respiratory system on a daily basis and adverse human health effects do exist. By examining toxicological differences and chemical composition of ambient fine particles using a novel experimental design and chemometric approach, the present work examines the hypothesis: that it is not clear whether there are significant differences in public health risk from exposure to fine particles in a rural location compared to those in urban locations. In the present study, an investigation into the inorganic chemical characteristics and biological effects of PM2.5–0.1 on human lung epithelial cell line A549 has been performed. Biological responses were evaluated by in vitro tests using equivalent masses of PM2.5–0.1 samples, collected during different seasons at urban and rural locations in Cork, Ireland. The relationship between the biological responses and the chemical composition of the samples were investigated using Principal Component Analysis followed by Partial Least Squares regression analysis. The PM2.5–0.1 samples collected at three contrasting sites in Cork demonstrated the ability to generate reactive oxygen species upon exposure irrespective of season. However, the magnitude of generation was somewhat higher for samples collected in the urban sites, compared to those generated by rural samples. Similarly, metals such as Cu and Mn were found to be present in larger quantities in the urban-based composite samples compared to those for their rural counterparts. The induction of interleukin 6 determined in this study followed a very similar seasonal trend to the measured concentrations of potassium ions in the PM2.5–0.1 samples to which the A549 cells were exposed. The current study provides further support that identifying important chemical components and their sources, with subsequent targeted emission controls, which will likely prove to be a more cost-effective strategy for mitigating toxicity and protecting human health, than current approaches which depend on uncharacterized total particle mass, especially when sophisticated pattern recognition techniques are employed to assess limited airborne datasets.

Determination of F2-isoprostanes in cultured human lung epithelial cells after exposure to metal oxide and silica nanoparticles by high-performance liquid chromatography/tandem mass spectrometry

The environmental impact of nanotechnology has caused a great concern. Many in vitro studies showed that many types of nanoparticles were cytotoxic. However, whether these nanoparticles caused cell membrane damage was not well studied. F2-isoprostanes are specific products of arachidonic acid peroxidation by nonenzymatic reactive oxygen species and are considered as reliable biomarkers of oxidative stress and lipid peroxidation. In this article, we investigated the cytotoxicity of different nanoparticles and the degree of cellular membrane damage by using F2-isoprostanes as biomarkers after exposure to nanoparticles. The human lung epithelial cell line A549 was exposed to four silica and metal oxide nanoparticles: SiO2 (15 nm), CeO2 (20 nm), Fe2O3 (30 nm), and ZnO (70 nm). The levels of F2-isoprostanes were determined by using high-performance liquid chromatography/mass spectrometry. The F2-isoprostanes’ peak was identified by retention time and molecular ion m/z at 353. Oasis HLB cartridge was used to extract F2-isoprostanes from cell medium. The results showed that SiO2, CeO2, and ZnO nanoparticles increased F2-isoprostanes levels significantly in A549 cells. Fe2O3 nanoparticle also increased F2-isoprostanes level, but was not significant. This implied that SiO2, CeO2, ZnO, and Fe2O3 nanoparticles can cause cell membrane damage due to the lipid peroxidation. To the best of our knowledge, this is the first report on the investigation of effects of cellular exposure to metal oxide and silica nanoparticles on the cellular F2-isoprostanes levels.

Abstract 81: Differential metabolic and viability responses of the A549 and MRC-5 cell lines upon exposure to selective organic inhibitors of glycolysis

Abstract Lung cancer is a one of the most prevalent and deadly cancers in United States. Research has shown that cancer cells exhibit higher glycolytic rates than normal cells. Studies on the differential role of the interference with glycolysis/energy generation by cancer cells through the use natural glycolytic inhibitors were limited in the literature. Therefore, we hypothesize that exposure of lung cancer cells to natural inhibitors of glycolysis will negatively influence their metabolic activities as well as their cell viabilities due to the inhibition of substrate-level ATP generation, while differentially not affecting the normal lung phenotype. The human lung fibroblast cell line (MRC-5) was selected to represent the normal human lung and the human alveolar epithelial cell line A549 was selected to represent lung cancer in vitro. These cells were maintained and exposed to eleven different organic reagents including fructose diphosphate (FDP), sodium citrate, ascorbic acid, crude honey, sodium bicarbonate, D-glucose, oxalic acid, glycerol, zinc acetate, pyruvic acid, and sodium ascorbate at concentration levels ranging from 31.3-2,000 ug/ml for 24-72 hours in 5% CO2 and 96 well plates using MTT, Alamar blue and the T4 cellometer assays as well as phase-contrast photo-imaging. Our results indicate that exposure of A549 cells to these organics resulted in concentration dependent differential cell destruction of the A549 cell line. Nine of the eleven organics used showed statistically significant (p<0.05) differential negative effects (impaired metabolic activity and cell death) on the A549 cell line in comparison to its control as well as to their effects on the MRC-5 cell line using MTT, Alamar blue and T4 cell viability assays. Data from the MTT assay showed limitations in detecting the true cell viability in comparison to the T4 data. Viability using the T4 cellometer ranged between 60-76% for the A549 compared to 96-100% for its control as well as the exposed MRC-5 cell line. LC50 using MTT and Alamar blue assays ranged between 161-1041 ug/ml. None of the eleven chemicals used allowed for cell proliferation of the A549 cell line while showing no or minor effects on the MRC-5 cell line. We conclude that nine of the tested organics impaired glycolysis, which is crucial to the generation of cellular energy and survival of the A549 cell line. Supported by NIH-RCMI grant# G12RR13459 and NIH-RISE grant # 63228. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 81.

Spontaneous assembly and active disassembly balance adherens junction homeostasis

The homeostasis of adherens junctions was studied using E-cadherin and its two mutants tagged by the photoconvertible protein Dendra2 in epithelial A-431 cells and in CHO cells lacking endogenous cadherin. The first mutant contained point mutations of two elements, Lys738 and the dileucine motif that suppressed cadherin endocytosis. The second mutant contained, in addition, an extensive truncation that uncoupled the mutant from beta-catenin and p120. Surprisingly, the intact cadherin and its truncated mutant were recruited into the junctions with identical kinetics. The full-size cadherin was actively removed from the junctions by a process that was unaffected by the inactivation of its endocytic elements. The cadherin's apparent half-residence time in the junction was about 2 min. Cadherin clusters made of the truncated mutant exhibited much slower but ATP-independent junctional turnover. Taken together, our experiments showed that adherens junction homeostasis consists of three distinctive steps: cadherin spontaneous recruitment, its lateral catenin-dependent association, and its active release from the resulting clusters. The latter process, whose mechanism is not clear, may play an important role in various kinds of normal and abnormal morphogenesis.

Synthesis and Evaluation of the Analogues for Galactosyl Donors as Inhibitors of β-1,4-Galactosyltransferase

The design and synthesis of analogs of galactosyl donors as potent and selective inhibitors of β-1,4-galactosyltransferase (β-1,4-GalT) are described. We employed a new strategy by incorporating 5-fluorouracil and an appropriate amino acid to galactose to prepare selective glycosyltransferase inhibitors. The in vitro cell assay showed that these compounds exhibited high cytotoxicity against the human leukemia cell line HL-60, human lung epithelial cell line A-549, and human liver cancer cell line BEL-7402, but low cytotoxicity against the murine leukemia cell line P388.

Abstract C47: 21-OH 6,19-epoxyprogesterone: A new dissociated glucocorticoid with anti-inflammatory properties but without antiapoptotic effects on chemotherapeutically treated mouse mammary tumor cells

Abstract Glucocorticoids (GCs) are widely used as coadyuvants in the treatment of solid tumors with chemotherapics such as paclitaxel (PXL) and doxorubicin (DOXO). However, clinical evidence supports the idea that GCs may induce resistance to these antitumor therapies. To improve cancer treatment quality, intense efforts are being made to design new dissociated steroids (steroid receptor glucocorticoid modulators (SRGMs). These compounds should mimic only the beneficial aspects of glucocorticoid therapy without inducing the undesired side effects in cancer patients. We have previously shown that dexamethasone (Dex), a well-known synthetic GC, inhibits PXL or DOXO induced apoptosis in breast tumor cells through a mechanism involving Bcl-XL and Bcl-2 induction. Here, our goal was to compare Dex effects with those triggered by 21-OH-6,19-epoxyprogesterone (21-OH-6,19-OP), which is a rigid steroid considered a passive GC antagonist. Molecular dynamic simulation studies predicted that 21-OH-6,19OP is unable to induce GR/21-OH-6,19-OP complex homodimerization and/or binding to coactivators, making these complex incapable to directly activate gene transcription (Alvarez et al J Med Chem. 2008. 13:1352). The effects of 21-OH-6,19-OP on apoptosis induced by DOXO or PXL in the epithelial mouse mammary tumor cell line LM3 was determined by analyzing Caspase-3 activity and gene and protein expression of antiapoptotic members of the Bcl family, i.e. Bcl-2 and Bcl-XL. The results show that, contrary to Dex, 21-OH-6,19-OP cannot reverse apoptosis induced by DOXO or PXL in LM3 cells. In fact, 21-OH-6,19-OP and Dex differentially regulate Bcl-2 and Bcl-XL expression. Dex's reversion on chemotherapic induced apoptosis (Dex+DOXO o Dex+PXL) correlates with an increase in Bcl-XL levels, while 21-O-H-6, 19-OP does not affect neither cell death nor Bcl-XL levels. The relevance of Bcl-XL involvement in DOXO resistance of cells treated with Dex was confirmed by knocking-down Bcl-XL with specific siRNA. Silencing Bcl-XL expression directly implicated loss of antiapoptotic GC effects. On the other hand, 21-OH-6,19-OP antiinflammatory action was also evaluated on the lung epithelial tumor cell line A549 by determining Interleukin-8 (IL-8) and Cyclooxygenase-2 (COX-2) expression in the presence of the Tumor Necrosis Factor-alpha (TNF-α). Expression levels of IL-8 and COX-2, measured by qRT-PCR, indicate that 21-OH-6,19-OP may have antiinflammatory effects as it reverses these inflammation markers expression induced by TNF-α in lung epithelial cells. Together, these findings suggest that 21-OH-6,19-OP is a SRGM that keeps glucocorticoid antiinflammatory properties without antiapoptotic effects, thus becoming a novel candidate for treatment of solid tumors as breast cancer. Citation Information: Cancer Res 2009;69(23 Suppl):C47.

TheBCG1619cgene is not essential for invasion and intracellular persistence ofMycobacterium bovisBCG in human THP-1 and A549 cell lines

The BCG1619c gene of Mycobacterium bovis bacillus Calmette-Guérin (BCG) encodes for a 24 kDa invasin-like protein and is identical to the Rv1566c gene of Mycobacterium tuberculosis. To assess whether this protein was necessary for entry and (or) intracellular persistence in professional phagocytes and (or) in lung epithelial cells, a BCG1619c knockout mutant of M. bovis BCG was generated and compared with the parental BCG strain for its ability to infect and multiply in human monocyte derived THP-1 cells and in the lung epithelial cell line A549. No significant difference between the mutated and the parental BCG strain was observed in either of these in vitro infection systems, indicating that the BCG1619c gene is not essential for cell invasion and intracellular growth of BCG.

Model cell systems representing expression regulation and signal transduction of thymic stromal lymphopoietin (TSLP) in the development of asthmatic symptoms

Thymic stromal lymphopoietin (TSLP) is a novel interleukin-7-like cytokine, which triggers dendritic cell-mediated inflammatory responses ultimately executed by Th2 helper cells. TSLP is a central player in the development of allergic, especially asthmatic symptoms, and has been suggested to represent a missing link in the information flow from antigen-exposed epithelial cells via dendritic cells (DCs) to T helper cells. We have challenged the lung epithelial cell line A549 with various allergens such as extracts from dust mites, pollen and fungi, and could show by real time PCR for the first time an allergen-dependent specific transcriptional upregulation of TSLP. To approach the as yet poorly defined TSLP-induced signal transduction in dendritic cells, we established a cellular model system on the basis of the murine pro-B cell line Ba/F3. The heterodimeric human TSLP receptor (hTSLPR) consisting of the novel TSLP receptor chain and the IL-7 receptor alpha chain was functionally reconstituted in factor-dependent Ba/F3 cells. We could demonstrate by phosphotyrosine protein analysis and by STAT type-specific reporter gene assays, that the ligand-stimulated TSLPR triggers activation of STAT3 and STAT5 as well as of STAT1. Employment of specific inhibitors proved the functional involvement of Janus kinases (JAKs) in TSLP-dependent, receptor-mediated cellular responses. These achievements provide tools for research towards a better molecular understandimg of atopic asthma and for the future development of new therapeutics interfering with TSLP function.

Evaluation of Direct-Oxidative DNA Damage on Human Lung Epithelial Cells Exposed to Urban Airborne Particulate Matter

Airborne particulate matter (PM) extracts were investigated for their content of organic compounds and for the direct and oxidative DNA damage induced on lung epithelial cells A549. PM10 was seasonally collected at two monitoring sites (Stations 1 and 2), characterized by different traffic loads. The cells were exposed for 30 min to extracts of PM10 diluted at 0.025%, 0.05%, and 0.1% for summer samples, and at 0.05%, 0.1%, and 0.15% for winter samples. Oxidative and direct DNA damage were evaluated by formamidopyrimidine glycosylase (fpg) comet assay analyzing tail moment (TM) values from fpg-enzyme-treated cells (TMenz) and enzyme untreated cells (TM) respectively and by comet percentage analysis. Measurements relating to Station 2 showed higher levels of polycyclic aromatic hydrocarbons (PAHs), and their methyl-(methyl-PAHs) and nitro-(nitro-PAHs) derivatives in both the seasons. Nitro-PAH concentrations were higher in summer than in winter at both the stations. We found a significant increase of comet percentages at the highest dose of extract from both stations in summer and from Station 2 in winter. The TM and TMenz values relative to the summer sampling showed an early oxidative DNA damage induction also followed by direct DNA damage more evident at Station 2, that seems to correlate with the presence of higher nitro-PAH concentrations during the warm season. At both monitoring stations, the results from winter sampling campaign showed a direct DNA damage induction at 0.1% of extract and oxidative-direct DNA damage at the highest dose (0.15%).

P120-Catenin Is a Key Component of the Cadherin–γ-Secretase Supercomplex

In this work, we show several previously unknown features of p120-catenin in a cadherin-catenin complex that are critical for our understanding of cadherin-based adhesion and signaling. We show that in human epithelial A-431 cells, nearly all p120 molecules engage in high-affinity interaction with E-cadherin-catenin complexes located at the cellular surface. p120 is positioned in proximity to alpha-catenin in the complex with cadherin. These findings suggest a functional cooperation between p120 and alpha-catenin in cadherin-based adhesion. A low level of cadherin-free p120 molecules, in contrast, could facilitate p120-dependent signaling. Finally, we present compelling evidence that p120 is a key linker cementing the E-cadherin-catenin complex with the transmembrane protease gamma-secretase. The cell-cell contact location of this supercomplex makes it an important candidate for conducting different signals that rely on gamma-secretase proteolytic activity.

Differentiation potential of human pancreatic stem cells for epithelial- and endothelial-like cell types

Pancreatic stem cells (PSC) have proved their high plasticity by differentiating into cell types of all three germ layers after the formation of organoid bodies. Motivated by this high differentiation potential this study focused on the immanent stem cell, endothelial and epithelial characteristics of PSC to elucidate whether PSC are a possible source for a stem cell-based in vitro model for screening of pharmaceutical substances. Furthermore, it was investigated whether marker expression was influenced by application of protocols for inducing endothelial or epithelial differentiation originating from research with mesenchymal stem cells or by cultivation on extracellular matrices (ECM). PSC showed expression of relevant stem cell markers CD 45, nestin, Oct 4 and c-kit. As endothelial characteristics they displayed the markers endoglin, VCAM 1, VEGFR 1 and vWF as well as the formation of vessel-like structures. Those endothelial properties were not further intensified by application of protocols employing vascular endothelial growth factor (VEGF), basic fibroblastic growth factor (bFGF) and endothelial cell growth medium. As typical epithelial characteristics PSC showed expression of keratin 7, 8, 18 and 19, ZO-1 and catenin beta1. In order to induce epithelial differentiation PSC were cocultured with the lung epithelial cell lines A549 and Calu-3 either by the usage of conditioned medium or by cultivation of PSC on fixed epithelial cells. Depending on the applied coculture system and epithelial cell type used expression of the epithelial marker cadherin-1 was altered compared to control. Cultivation on different ECM changed expression of all investigated markers only marginally. These results confirm that PSC possess endothelial and epithelial properties. Furthermore, epithelial characteristics represented by expression of CDH 1 were altered by coculture with epithelial cell lines.

INFLAMMATORY-ACTIVATED MICROVASCULAR ENDOTHELIAL CELLS REGULATE INTERLEUKIN-8 AND MONOCYTE CHEMOATTRACTANT PROTEIN-1 EXPRESSION OF A549 CELLS IN A PARACRINE FASHION

Increased levels of interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 have been found in bronchoalveolar lavage fluid of acute respiratory distress syndrome (ARDS) patients. The authors investigated whether inflammatory-activated microvascular pulmonary endothelial cells (HMVECL) regulate expression of IL-8 and MCP-1 in the alveolar epithelial cell line A549 and studied the effects of hypoxia (5% O2) and hyperoxia (85% O2). The authors observed significant up-regulation of IL-8 and MCP-1 expression in A549 cells that was independent from the IL-1 receptor pathway but was modified by oxygen tension. These data show that the pulmonary microvascular endothelium is able to regulate epithelial cell chemokine expression in a paracrine fashion.

Chemical Knockdown of Protein-tyrosine Phosphatase 1B by 1,2-Naphthoquinone through Covalent Modification Causes Persistent Transactivation of Epidermal Growth Factor Receptor

1,2-Naphthoquinone (1,2-NQ), an atmospheric contaminant, causes the contraction of guinea pig trachea through the activation of epidermal growth factor receptor (EGFR) by inhibiting protein-tyrosine phosphatases (PTPs). Phosphorylation of EGFR is negatively regulated by PTPs, but details of the mechanism by which 1,2-NQ inhibits PTPs have not been elucidated. Results described in this report demonstrate that 1,2-NQ forms covalent bonds with PTP1B after exposure to human epithelial A431 cells. In this study, a concentration-dependent phosphorylation of EGFR was found to be coupled to the reduction of PTP activity in the cells. The reduction in PTP activity was due to the irreversible modification of PTP1B, and when PTP1B was overexpressed by the cells, the 1,2-NQ-mediated EGFR phosphorylation was suppressed. Studies with purified PTP1B and 1,2-NQ showed that the reduction in enzyme activity was due to a nucleophilic attack by the quinone on the enzyme, to form covalent bonds. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry analysis and mutation experiments revealed that PTP1B inactivation was primarily due to covalent attachment of the quinone to Cys-121 of the enzyme, with binding to His-25 and Cys-215 as well. Collectively, the results show that covalent attachment of 1,2-NQ to PTP1B is at least partially responsible for the reduction of PTP activity, which leads to prolonged transactivation of EGFR in the cells.

Lysophosphatidic Acid Decreases Epidermal Growth Factor Receptor Binding in Airway Epithelial Cells

We showed previously that treatment of human airway smooth muscle cells and lung fibroblasts with lysophosphatidic acid (LPA) increases the binding of epidermal growth factor (EGF) to EGF receptors (EGFRs). The purpose of this study was to determine whether LPA also regulates EGFR binding in airway epithelial cells. Airway epithelial cells were incubated in the absence or presence of 10 microM LPA for increasing times, and binding of 125I-EGF to intact cells on ice was measured. Exposure to LPA for only 15 min caused a 30 to 70% decrease in EGFR binding in a dose-dependent manner, depending on the cell line. This decrease in binding was sustained to at least 18 h in BEAS-2B and primary human bronchial epithelial cells. In contrast, the LPA-induced decrease in binding reversed rapidly in two lung cancer epithelial cell lines, H292 and A549, returning to control levels within 3 h. LPA increased phosphorylation of the EGFR in BEAS-2B cells, and this phosphorylation was inhibited by both 4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline (AG1478; EGFR tyrosine kinase inhibitor) and N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide (GM6001; matrix metalloproteinase inhibitor) but not by CRM197 (heparin-binding EGF inhibitor). AG-1478 and GM6001 also inhibited the LPA-induced decrease in EGFR binding but only by 50%, suggesting only partial involvement of EGFR transactivation in the decrease in EGFR binding. In summary, LPA stimulates a decrease in EGFR binding in airway epithelial cells that is sustained in normal cells but that rapidly reverses in cancer cells. LPA-induced transactivation of EGFRs occurs and contributes to the decrease in EGFR binding, but additional pathway(s) may also be involved.

Development and Characterization of a Specific Anti-Caveolin-1 Antibody for Caveolin-1 Functional Study in Human, Goat and Mouse

Caveolin-1 of the caveolin family of proteins regulates mammary gland development and has been shown to play a contradictory role in breast tumor progression. A specific anti-Caveolin-1 antibody will be useful for functional study of Caveolin-1 in different tissues. In this study, we generated a rabbit polyclonal antibody that specifically recognizes the N-terminal amino acids 50-65 of Caveolin-1. This polyclonal antibody specifically reacted with Caveolin-1 extracted from cells of different species, including human epithelial A431 cells, goat primary mammary epithelial cells and mice fibroblast NIH 3T3 cells, by Western blotting. Endogenous Caveolin-1 protein expressing in cells and normal human tissues was detected by this polyclonal antibody using immunocytofluorescent and immunohistochemical staining, respectively. Furthermore, an apparent decrease in Caveolin-1 expression in tumorous breast and colon tissues was detected by this polyclonal antibody. In conclusion, we have identified amino acids 50-65 of Caveolin-1, which contains an epitope that is specific to Caveolin-1 and is conserved in the human, goat and mouse. In future, this anti-Caveolin-1 antibody can be used to examine the progression of breast and colon cancers and to study functions of Caveolin-1 in human, goat and mouse cells.