Proteomic analysis of secreted proteins by human bronchial epithelial cells in response to cadmium toxicity

For years, many studies have been conducted to investigate the intracellular response of cells challenged with toxic metal(s), yet, the corresponding secretome responses, especially in human lung cells, are largely unexplored. Here, we provide a secretome analysis of human bronchial epithelial cells (BEAS‐2B) treated with cadmium chloride (CdCl₂), with the aim of identifying secreted proteins in response to Cd toxicity. Proteins from control and spent media were separated by two‐dimensional electrophoresis and visualized by silver staining. Differentially‐secreted proteins were identified by MALDI‐TOF‐MS analysis and database searching. We characterized, for the first time, the extracellular proteome changes of BEAS‐2B dosed with Cd. Our results unveiled that Cd treatment led to the marked upregulation of molecular chaperones, antioxidant enzymes, enzymes associated with glutathione metabolic process, proteins involved in cellular energy metabolism, as well as tumor‐suppressors. Pretreatment of cells with the thiol antioxidant glutathione before Cd treatment effectively abrogated the secretion of these proteins and prevented cell death. Taken together, our results demonstrate that Cd causes oxidative stress‐induced cytotoxicity; and the differentially‐secreted protein signatures could be considered as targets for potential use as extracellular biomarkers upon Cd exposure.     

Endocrine-like cells of the pulmonary epithelium of the human adult lung

Summary The morphological and histochemical characteristics of endocrinelike cells of the pulmonary epithelium of the right lower lobe of 12 human adult lungs were studied.Few cells were reactive to the argyrophil silver method of Grimelius and of Sevier and Munger and cells with a similar morphology and distribution emitted a green or yellow fluorescence after treatment of the lung epithelium with the amine precursors L-DOPA or L-HTP, respectively. A greater number of cells seems to be demonstrated by electron microscopy. The cells were characterized by small, round secretory granules showing a central dense core and a very thin clear halo between the core and the surrounding membrane.The cells are thought to be related to the endocrine-like cells of the pulmonary epithelium of the human foetal lung and to cells of carcinoids of larger bronchi.     

Proteomic analysis of cellular response to microcystin in human amnion FL cells

Microcystins (MC), the potent inhibitor of protein phosphatase 1 and 2A, are hepatotoxins of increasing importance due to its high acute toxicity and potent tumor promoting activity. So far, the exact mechanisms of MC-induced hepatotoxicity and tumor promoting activity have not been fully elucidated. To better understand the mechanisms underlying microcystin-RR (MC-RR) induced toxicity as well as provide the possibility for the establishment of biomarkers for MC-RR exposure, differential proteome analysis on human amnion FL cells treated by MC-RR was carried out using two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Image analysis of silver-stained 2-dimensional gels revealed that 89 proteins showed significant differential expression in MC-RR treated cells compared with control, and 8 proteins were unique to MC-RR treated cells and 8 proteins were only detected in control cells. Sixty-six proteins were further identified with high confidence by peptide mass fingerprinting. Some of the identified differentially expressed proteins have clearly relationship with the process of apoptosis, signal transduction, and cytoskeleton alteration which are consistent with the literature. The functional implications of alterations in the levels of these proteins were discussed. However, most of which have not been reported previously to be involved in cellular processes responded to MC-RR. Therefore, this work will provide new insight into the mechanism of MC-RR toxicity.     

Adult lung stem cells and their contribution to lung tumourigenesis

The isolation and characterization of lung stem and progenitor cells represent an important step towards the understanding of lung repair after injury, lung disease pathogenesis and the identification of the target cells of transformation in lung carcinogenesis. Different approaches using prospective isolation of progenitor cells by flow cytometry or lineage-tracing experiments in mouse models of lung injury have led to the identification of distinct progenitor subpopulations in different morphological regions of the adult lung. Genetically defined mouse models of lung cancer are offering new perspectives on the cells of origin of different subtypes of lung cancer. These mouse models pave the way to further investigate human lung progenitor cells at the origin of lung cancers, as well as to define the nature of the lung cancer stem cells. It will be critical to establish the link between oncogenic driver mutations recently discovered in lung cancers, target cells of transformation and subtypes of lung cancers to enable better stratification of patients for improved therapeutic strategies.     

Proteomic analysis of nuclear proteins from proliferative and differentiated human colonic intestinal epithelial cells

Self-renewing tissues such as the intestine contain progenitor proliferating cells which subsequently differentiate. Cell proliferation and differentiation involve gene regulation processes which take place in the nucleus. A human intestinal epithelial cell line model (Caco2/TC7) which reproduces these dynamic processes has been used to perform proteomic studies on nuclear proteins. Nuclei from Caco2/TC7 cells at proliferative and differentiated stages were purified by subcellular fractionation. After two-dimensional gel electrophoresis separation and ruthenium staining, 400 protein spots were detected by image analysis. Eighty-five spots corresponding to 60 different proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry in nuclei from proliferative cells. Comparison of nuclear proteomes from proliferative or differentiated cells by differential display resulted in the identification of differentially expressed proteins such as nucleolin, hnRNP A2/B1 and hnRNP A1. By using Western blot analysis, we found that the expression and number of specific isoforms of these nuclear proteins decreased in differentiated cells. Immunocytochemistry experiments also showed that in proliferative cells nucleolin was distributed in nucleoli-like bodies. In contrast, hnRNPs A2/B1 and A1 were dispersed throughout the nucleus. This study of the nuclear proteome from intestinal epithelial cells represents the first step towards the establishment of a protein database which will be a valuable resource in future studies on the differential expression of nuclear proteins in response to physiological, pharmacological and pathological modulations.     

Proteomic analysis of glutamate-induced toxicity in HT22 cells

In the present study, we have investigated the proteome changes associated with glutamate-induced HT22 cell death, a model system to study oxidative stress-mediated toxicity. Among a number of HT22 proteins exhibiting altered expression, several molecular chaperones demonstrated substantial changes. For example, the levels of Hsp90 and Hsp70 decreased as cell death progressed whereas that of Hsp60 increased dramatically. Interestingly, cytosolic Hsp60 increased more prominently than mitochondrial Hsp60. Concomitantly, the accumulation of poly-ubiquitylated proteins and differential regulation of the peptidase activities and the subunits of 26S proteasomes were observed in glutamate-treated HT22 cells. Our findings that the molecular chaperones and the ubiquitin-proteasome system undergo changes during glutamate-induced HT22 cell death may suggest the importance of a protein quality control system in oxidative damage-mediated toxicity.     

Transforming growth factor beta-1 enhances cytotoxic effect of doxorubicin in human lung adenocarcinoma cells of A549 line

Transforming growth factor beta-1 (TGFbeta-1) is a regulator of cell proliferation, differentiation and apoptosis. Doxorubicin (adriamycin), an anthracycline drug causing double-strand DNA breaks, is widely used in anticancer chemotherapy. Here we demonstrated that TGFbeta-1 enhanced cytotoxic (proapoptotic) action of doxorubicin towards cultured human lung carcinoma A549 cells. Western-blot analysis and immunocytochemistry were used to show that doxorubicin induced PARP degradation in A549 cells, and TGFbeta-1 enhanced that action of the drug. The obtained results suggest a possibility of biomodulating effect of TGFbeta-1 on tumor cell treatment with doxorubicin.     

肺干细胞和肺癌干细胞研究进展

近年来成体干细胞研究进展迅速。肺干细胞和肺癌干细胞在表面标志、分离方法和功能研究等方面也取得了一定进展。在肺组织中,肺干细胞维持着肺上皮的更新和稳定,肺脏不同解剖结构存在不同的干细胞,主要的肺干细胞有气管—支气管干细胞、细支气管干细胞、细支气管肺泡干细胞和肺泡干细胞等,不同干细胞特异表面标志也不同。根据肿瘤干细胞理论,目前研究认为肺癌的发生与肺癌干细胞有关,肺癌干细胞来源于其对应肺干细胞的恶性转化。肺癌干细胞特异标志研究主要集中在侧群细胞、CD133和醛脱氢酶等。与其他成体干细胞相似,肺癌干细胞维持自我更新以及分化能力的信号通路主要有Wnt、Hedgehog和Notch通路等。肺癌干细胞与肺癌的发生、发展、转移、治疗反应及预后关系,也取得了一定的进展。该文对肺干细胞和肺癌干细胞研究进展作简要综述。     

Proteomic map of peripheral blood mononuclear cells

In the field of proteomics extensive efforts have been focused on the knowledge of proteins expressed by different cell types. In particular, enormous progress has been done in the characterization of blood cellular components. In this work, we have established a public 2-DE database for human peripheral blood mononuclear cells (PBMCs) proteins. Two hundred and forty-six spots corresponding to 174 different proteins have been identified on 2-DE gels from PBMCs isolated from six healthy individuals. All the identified proteins have been classified in thirteen categories on the basis of their differential functions or cellular localization and annotated at the http://physiology.unile.it/proteomics. The role of several proteins has been discussed in relation to their biological function. We intend to show the potentiality of PBMCs to investigate the proteomics changes possibly associated with a large number of pathologies such as autoimmune, neurodegenerative and cancer diseases.     

Ascorbate is a pro-oxidant in chromium-treated human lung cells

The human A549 lung cell line is used in this study as a model to evaluate chromium toxicity and mutagenesis since inhalation exposure of this metal gives rise to an epidemiology that indicates the lung as a target organ of chromium toxicity. Hexavalent chromium is considered the carcinogenic form of chromium, however it must be reductively activated following uptake into cells in order to react with intracellular constituents. We have previously established that the fluorescent dyes, dichlorofluorescein (DCF) and dihydrorhodamine, are effective indicators of the reductive activation of chromium and are sensitive measures of the formation of highly reactive chromium species (RCS) intracellularly. In order to examine the role of the two common intracellular reductants, glutathione and ascorbic acid (Vitamin C) in generating RCS intracellularly, we manipulated their intracellular levels through the use of buthionine sulfoximine (BSO) or by the addition of ascorbate into the culture media. We found that the high levels of glutathione in this cancer cell line lowered endogenous oxidation levels markedly, and that, by decreasing intracellular glutathione, BSO not only generated a higher background level of endogenous intracellular oxidation but the chromium-stimulated oxidation also increased markedly. Contrary to it appellation as an anti-oxidant, ascorbic acid stimulated a strong pro-oxidant response upon chromium treatment and this pro-oxidant response was evident regardless of the levels of glutathione in the cells. Based on these results, we conclude that ascorbic acid acts as a pro-oxidant in chromium-treated cells.     

Proteomic signature of human embryonic stem cells

Human embryonic stem cells (hESC) represent a population of undifferentiated pluripotent cells with both self-renewal and multilineage differentiation characteristics. Proteomics provides a powerful approach for studying the characteristics of hESC and discovering molecular markers. We have analyzed proteome profiles of three hESC lines using 2-DE and MALDI TOF-TOF. Out of 844 spots analyzed with MALDI TOF-TOF, 685 proteins were identified of which 60 proteins were classified as the most abundant proteins on 2-D gels. A large number of proteins particularly high abundant ones were identified as chaperones, heat shock proteins, ubiquitin/proteasome, and oxidative stress responsive proteins underscoring the ability of these cells to resist oxidative stress and increase the life span. Several proteins involved in cell proliferation and differentiation were also among the highly expressed proteins. Although overall expression pattern of three hESC were similar, 54 spots changed quantitatively and 14 spots changed qualitatively among the hESC cell lines. Most of these proteins were identified as proteins involved in cell growth, metabolism and signal transduction, which may affect the self-renewal and pluripotency. To our knowledge, this study represents the first proteomic dataset for hESC and provides a better insight into the biology of hESC. Proteome maps of hESC are accessible at http://www.RoyanProteomics.ir.     

Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin

A proteomics method has been developed to purify and identify the specific proteins modified by ubiquitin (Ub) from human cells. In purified samples, Ub and 21 other proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra using SEQUEST. These proteins included several of the expected carriers of Ub including Ub-conjugating enzymes and histone proteins. To perform these experiments, a cell line coexpressing epitope tagged His(6X)-Ub and green fluorescent protein (GFP) was generated by stably transfecting HEK293 cells. Ubiquitinated proteins were purified using nickel-affinity chromatography and digested in solution with trypsin. Complex mixtures of peptides were separated by reversed phase chromatography and analyzed by nano LC-MS/MS using the LCQ quadrupole ion-trap mass spectrometer. Proteins identified from His(6X)-Ub-GFP transfected cells were compared to a list of proteins from HEK293 cells, which associate with nickel-nitrilotriacetic acid (Ni-NTA)-agarose in the absence of His-tagged Ub. In a proof of principle experiment, His(6X)-Ub-GFP transfected cells were treated with As (III) (10 microM, 24 h) in an attempt to identify substrates increasingly modified by Ub. In this experiment, proliferating cell nuclear antigen, a DNA repair protein and known ubiquitin substrate, was confidently identified. This proteomics method, developed for the analysis of ubiquitinated proteins, is a step towards large-scale characterization of Ub-protein conjugates in numerous physiological and pathological states.     

以人皮肤成纤维细胞作为饲养层细胞培养人胚胎干细胞

目的：比较人皮肤成纤维细胞（humandermalfibroblasts,HDFs）与小鼠胚胎成纤维细胞（Mouseembryonicfibroblasts,MEFs）的增殖能力及研究人皮肤成纤维细胞作为饲养层支持人胚胎干细胞（humanembryonicstemcells,hESCs）未分化生长的能力。方法：利用组织贴壁法从人皮肤中分离出HDFs,通过细胞形态的观察和生长曲线的绘制比较HDFs与MEFs的体外增殖能力。将HDFs作为饲养层细胞与hESCs共培养,传代12代后,检测hESCs碱性磷酸酶（AKP）、表面特异性标志及胚胎干细胞特异性转录因子。结果：HDFs可连续传代培养15代以上,10代以下的HDFs增殖迅速,而MEFs自第4代起,增殖能力就明显下降;hESCs在HDFs饲养层上可传代培养12代以上,克隆边界清晰,细胞排列紧密,碱性磷酸酶、表面标志物检测均呈阳性,表达了hESCs特异性转录因子。结论：HDFs比MEFs具有更强的增殖能力;HDFs可作为培养hEscs的饲养层细胞。     

Proteasome inhibition induces inclusion bodies associated with intermediate filaments and fragmentation of the Golgi apparatus

The ubiquitin-proteasome system is involved in a variety of biological processes. Inclusion bodies associated with intermediate filaments (IFs) and ubiquitin are observed in various diseases; however, the precise mechanisms of formation and the pathological significance of inclusion bodies have not been fully understood. We examined the effect of proteasome inhibitors on the structure of IF using anti-cytokeratin antibodies or transfection of green fluorescent protein-fused cytokeratin 18 in a hepatoma cell line, Huh7. Intracellular organelles were visualized by immunofluorescent and electron microscopies. Proteasome inhibitors induced IF inclusions associated with ubiquitin. Electron microscopic examination revealed inclusion bodies surrounded by filamentous structures. Autophagic vacuoles and lysosomes were frequently observed, and the organization of the Golgi apparatus was disrupted in these cells. After the removal of the proteasome inhibitors, the IF network and organization of the Golgi apparatus were restored. The IF inclusions could be induced by inhibition of the proteasome function. IF inclusions induced fragmentation of the Golgi apparatus and might inhibit the function of this important station of membrane traffic. The IF inclusions disappeared by restoring proteasome function, and autophagy and lysosomal degradation might be, at least in part, associated with the elimination of inclusion bodies.