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.     

Proteomic analysis of the response of human lung cells to uranium

The industrial use of uranium and particularly of depleted uranium, has pinpointed the need to review its chemical impact on human health. A proteomic approach was used to evaluate the response of a human lung cell line (A549) to uranium. We established the first 2-D reference map of the A549 cell line, identifying 87 spots corresponding to 81 major proteins. Uranium treatment triggered differential expression of 18 spots, of which 14 corresponded to fragments of cytokeratin 8 (CK8) and cytokeratin (CK18) and 1 to peroxiredoxin 1. We probed several hypotheses regarding CK cleavage, and observed that it did not result from caspase or calpain activity. Furthermore, we showed that the fragments are recognised by an anti-ubiquitin antibody (KM691). These results suggest a regulatory pathway involving CK ubiquitinylation or dysfunction in the proteasome-ubiquitin system in response to uranium exposure in human lung cells.     

Proteomic analysis of total cellular proteins of human neutrophils

Background  

Neutrophils are the most abundant leukocytes in peripheral blood and represent one of the most important elements of innate immunity. Recent subcellular proteomic studies have focused on the identification of human neutrophil proteins in various subcellular membrane and granular fractions. Although there are relatively few studies dealing with the analysis of the total extract of human neutrophils, many biological problems such as the role of chemokines, adhesion molecules, and other activating inputs involved in neutrophil responses and signaling can be approached on the basis of the identification of the total cellular proteins.     

Proteomic analysis of cellular response to osmotic stress in thick ascending limb of Henle's loop (TALH) cells

Epithelial cells of the thick ascending limb of Henle's loop (TALH cells) play a major role in the urinary concentrating mechanism. They are normally exposed to variable and often very high osmotic stress, which is particularly due to high sodium and chloride reabsorption and very low water permeability of the luminal membrane. It is already established that elevation of the activity of aldose reductase and hence an increase in intracellular sorbitol are indispensable for the osmotic adaptation and stability of the TALH cells. To identify new molecular factors potentially associated with the osmotic stress-resistant phenotype in kidney cells, TALH cells exhibiting low or high levels of resistance to osmotic stress were characterized using proteomic tools. Two-dimensional gel analysis showed a total number of 40 proteins that were differentially expressed in TALH cells under osmotic stress. Twenty-five proteins were overexpressed, whereas 15 proteins showed a down-regulation. Besides the sorbitol pathway enzyme aldose reductase, whose expression was 15 times increased, many other metabolic enzymes like glutathione S-transferase, malate dehydrogenase, lactate dehydrogenase, alpha enolase, glyceraldehyde-3-phosphate dehydrogenase, and triose-phosphate isomerase were up-regulated. Among the cytoskeleton proteins and cytoskeleton-associated proteins vimentin, cytokeratin, tropomyosin 4, and annexins I, II, and V were up-regulated, whereas tubulin and tropomyosins 1, 2, and 3 were down-regulated. The heat shock proteins alpha-crystallin chain B, HSP70, and HSP90 were found to be overexpressed. In contrast to the results in oxidative stress the endoplasmic reticulum stress proteins like glucose-regulated proteins (GRP78, GRP94, and GRP96), calreticulin, and protein-disulfide isomerase were down-regulated under hypertonic stress.     

Synthesis of cellular and extracellular glycoproteins by cultured human keratinocytes and their response to retinoids

The glycoproteins synthesized by human keratinocytes cultured on 3T3 feeder layers were studied by metabolic labelling. Keratinocytes freed of feeder cells synthesized a complex pattern of cellular and extracellular glycoproteins that was distinct from that of 3T3 cells, dermal fibroblasts and epidermal melanocytes. The effect of low concentrations of all-trans-retinoic acid and arotinoid ethyl ester on glycoprotein synthesis was examined in keratinocyte cultures depleted of vitamin A. Treatment with either retinoid resulted in a 2-3-fold increase in the amount of D-[3H]glucosamine-labelled material in the culture medium. Gel electrophoresis revealed increased incorporation of D-[3H]glucosamine into extracellular glycoproteins of Mr 245,000, 170,000, 140,000, 130,000, 120,000 and 105,000 as well as into glycosaminoglycans in retinoid-treated cultures. The labelling of extracellular glycoproteins with L-[3H]leucine and L-[35S]methionine was also increased by retinoids suggesting increased synthesis of these components rather than an effect on their glycosylation. The Mr 245 000 glycoprotein was identified as keratinocyte-derived fibronectin by immunoblotting, immunoprecipitation and specific binding to gelatin. The results show that retinoids increase the synthesis of glycoprotein as well as glycosaminoglycan components of the extracellular matrix in human keratinocyte cultures. It is suggested that retinoids select for a population of cells that synthesize relatively large amounts of glycosaminoglycan, fibronectin and other as yet unidentified extracellular glycoproteins.     

Proteomic analysis of rice endosperm cells in response to expression of hGM-CSF

The accumulation of significant levels of transgenic products in plant cells is required not only for crop improvement, but also for molecular pharming. However, knowledge about the fate of transgenic products and endogenous proteins in grain cells is lacking. Here, we utilized a quantitative mass spectrometry-based proteomic approach for comparative analysis of expression profiles of transgenic rice endosperm cells in response to expression of a recombinant pharmaceutical protein, human granulocyte-macrophage colony stimulation factor (hGM-CSF). This study provided the first available evidence concerning the fate of exogenous and endogenous proteins in grain cells. Among 1883 identified proteins with a false positive rate of 5%, 103 displayed significant changes (p-value < 0.05) between the transgenic and the wild-type endosperm cells. Notably, endogenous storage proteins and most carbohydrate metabolism-related proteins were down-regulated, while 26S proteasome-related proteins and chaperones were up-regulated in the transgenic rice endosperm. Furthermore, it was observed that expression of hGM-CSF induced endoplasmic reticulum stress and activated the ubiquitin/26S-proteasome pathway, which led to ubiquitination of this foreign gene product in the transgenic rice endosperm.     

Proteomic analysis of the resistance to aplidin in human cancer cells

Aplidin (plitidepsin) is an antitumoral agent that induces apoptosis via Rac1-JNK activation. A proteomic approach using 2D-DIGE technology found 52 cytosolic and 39 membrane proteins differentially expressed in wild-type and Aplidin-resistant HeLa cells, of which 39 and 27 were identified by MALDI-TOF mass spectrometry and database interrogation. A number of proteins involved in apoptosis pathways were found to be deregulated. Alterations in Rab geranylgeranyltransferase, protein disulfide isomerase (PDI), cystathionine gamma-lyase, ezrin, and cyclophilin A (CypA) were confirmed by immunoblotting. Moreover, the role of PDI and CypA in Aplidin resistance was functionally confirmed by using the inhibitor bacitracin and overexpression, respectively. These deregulated proteins are candidates to mediate, at least partially, Aplidin action and might provide a route to the cells to escape the induction of apoptosis by this drug.     

Proteomic characterization of skin and epidermis in response to environmental agents

The skin and its outer epidermis layer in particular, prevent access of various environmental agents including potential allergens, irritants, carcinogens, ultraviolet radiation and microbes. Cells in the epidermis make a significant contribution to innate as well as adaptive immune reactions in skin. The skin immunity thus provides a biologic defense in response to hazardous environmental agents. Although proteomics has been utilized to establish skin proteomes and investigate skin responses to some environmental agents, it has not been extensively used to address the complexity of skin responses to various environments. This review summarizes cutaneous genes and proteins that have been characterized as related to skin exposure to environmental agents. In parallel, this review emphasizes functional proteomics and systems biology, which are believed to be an important future direction toward characterizing the skin proteome-environmental interaction and developing successful therapeutic strategies for skin diseases caused by environmental insults.     

Proteomic characterization of skin and epidermis in response to environmental agents

The skin and its outer epidermis layer in particular, prevent access of various environmental agents including potential allergens, irritants, carcinogens, ultraviolet radiation and microbes. Cells in the epidermis make a significant contribution to innate as well as adaptive immune reactions in skin. The skin immunity thus provides a biologic defense in response to hazardous environmental agents. Although proteomics has been utilized to establish skin proteomes and investigate skin responses to some environmental agents, it has not been extensively used to address the complexity of skin responses to various environments. This review summarizes cutaneous genes and proteins that have been characterized as related to skin exposure to environmental agents. In parallel, this review emphasizes functional proteomics and systems biology, which are believed to be an important future direction toward characterizing the skin proteome–environmental interaction and developing successful therapeutic strategies for skin diseases caused by environmental insults.     

Alterations of cellular characteristics of a human ovarian teratocarcinoma cell line after in vitro treatment with retinoids

Differentiation of the human teratocarcinoma derived cell line. PA-1, with retinoids was examined at concentrations (10(-6)-10(-8) M) that did not exhibit an antiproliferative effect during log-phase growth. Treatment with naturally occurring retinoic acid or certain synthetic retinoids (13-cis retinoic acid, Ro10-9359, and Ro13-7410), while not significantly altering the log-phase growth rate, decreased the saturation cell density and mitotic indices after confluence. Retinoid treatment also induced changes in cell morphology, which appear to be related to reorganization of microtubules and microfilaments. Following retinoid treatment, the expression of cell glycoproteins (of 162 kDa, 152 kDa, 143 kDa. and 51 kDa) was altered. Treated cells also exhibited decreased expression of alkaline phosphatase, as well as an increased capacity for intercellular communication as evidenced by gap-junctional transfer of the phosphorylated toxic intermediate of 6-thioguanine to HPRT- cells. Treatment with retinoic acid dramatically reduced the quantity of shed plasma membrane material and altered its composition.     

Proteomic analysis of iron overload in human hepatoma cells

Iron-mediated organ damage is common in patients with iron overload diseases, namely, hereditary hemochromatosis. Massive iron deposition in parenchymal organs, particularly in the liver, causes organ dysfunction, fibrosis, cirrhosis, and also hepatocellular carcinoma. To obtain deeper insight into the poorly understood and complex cellular response to iron overload and consequent oxidative stress, we studied iron overload in liver-derived HepG2 cells. Human hepatoma HepG2 cells were exposed to a high concentration of iron for 3 days, and protein expression changes initiated by the iron overload were studied by two-dimensional electrophoresis and mass spectrometry. From a total of 1,060 spots observed, 21 spots were differentially expressed by iron overload. We identified 19 of them; 11 identified proteins were upregulated, whereas 8 identified proteins showed a decline in response to iron overload. The differentially expressed proteins are involved in iron storage, stress response and protection against oxidative stress, protein folding, energy metabolism, gene expression, cell cycle regulation, and other processes. Many of these molecules have not been previously suggested to be involved in the response to iron overload and the consequent oxidative stress.     

Proteomic analysis of cancer stem cells in human prostate cancer cells

Results from recent studies support the hypothesis that cancer stem cells (CSCs) are responsible for tumor initiation and formation. Here, we applied a proteome profiling approach to investigate the mechanisms of CSCs and to identify potential biomarkers in the prostate cancer cell line DU145. Using MACS, the DU145 prostate cancer cell line was isolated into CD44+ or CD44− cells. In sphere culture, CD44+ cells possessed stem cell characteristics and highly expressed genes known to be important in stem cell maintenance. In addition, they showed strong tumorigenic potential in the clonogenic assay and soft agar colony formation assay. We then analyzed and identified proteins that were differentially expressed between CD44+ and CD44− using two-dimensional gel electrophoresis and LC-MS/MS. Cofilin and Annexin A5, which are associated with proliferation or metastasis in cancer, were found to be positively correlated with CD44 expression. These results provide information that will be important to the development of new cancer diagnostic tools and understanding the mechanisms of CSCs although a more detailed study is necessary to investigate the roles of Cofilin and Annexin A5 in CSCs.     

Calcium-independent growth of human ovarian carcinoma cells

Evidence in the literature suggests that cancer cell growth in vitro is generally not sensitive to external calcium. A human ovarian carcinoma cell line (SKOV3) retained 60% of its normal growth in Dulbecco modified Eagle's medium (DME) when the calcium concentration was reduced from 3 mM to 10 microM. Chinese hamster ovary cells (CHO) were growth-arrested in media containing less than 500 microM calcium. In low-calcium (10 microM) DME, 10 microM of a calmodulin antagonist W7 inhibited the growth of SKOV3 cells by more than 90%, while 100 microM of its inactive analog W5 was mildly inhibitory (20%). The growth inhibition by W7 was antagonized by increasing calcium concentrations in the culture media, while the inhibition by W5 was calcium-independent. The phorbol ester TPA was also effective in antagonizing W7's growth inhibition in low-calcium DME, suggesting that the W7 effect is mediated via protein kinase C inhibition. SKOV3 total cellular protein kinase C activity was 1.6 times higher than CHO cells when incubated in normal DME. When incubated in low-calcium DME, a large drop in protein kinase C activity in the CHO cells was observed while the enzyme activity was unchanged in the SKOV3 cells. Our data suggest that these human ovarian tumor cells have altered cellular calcium regulatory processes associated with the defective down-regulation of protein kinase C. This defect may confer these cells the ability to proliferate independently of the external calcium concentration. Targeting the cellular signal transduction components may be useful in cancer chemotherapy.     

Proteomic analysis of the molecular response of Raji cells to maslinic acid treatment

Maslinic acid, a natural pentacyclic triterpene has been shown to inhibit growth and induce apoptosis in some tumour cell lines. We studied the molecular response of Raji cells towards maslinic acid treatment. A proteomics approach was employed to identify the target proteins. Seventeen differentially expressed proteins including those involved in DNA replication, microtubule filament assembly, nucleo-cytoplasmic trafficking, cell signaling, energy metabolism and cytoskeletal organization were identified by MALDI TOF-TOF MS. The down-regulation of stathmin, Ran GTPase activating protein-1 (RanBP1), and microtubule associated protein RP/EB family member 1 (EB1) were confirmed by Western blotting. The study of the effect of maslinic acid on Raji cell cycle regulation showed that it induced a G1 cell cycle arrest. The differential proteomic changes in maslinic acid-treated Raji cells demonstrated that it also inhibited expression of dUTPase and stathmin which are known to induce early S and G2 cell cycle arrests. The mechanism of maslinic acid-induced cell cycle arrest may be mediated by inhibiting cyclin D1 expression and enhancing the levels of cell cycle-dependent kinase (CDK) inhibitor p21 protein. Maslinic acid suppressed nuclear factor-kappa B (NF-κB) activity which is known to stimulate expression of anti-apoptotic and cell cycle regulatory gene products. These results suggest that maslinic acid affects multiple signaling molecules and inhibits fundamental pathways regulating cell growth and survival in Raji cells.     

Proteomic analysis of human epithelial ovarian cancer xenografts in immunodeficient mice exposed to chronic psychological stress

This work aims at comparing alterations in the proteomes of human epithelial ovarian cancer xenografts between stressed and non-stressed immunodeficient mice as well as exploring the molecular mechanisms linking chronic psychological stress to ovarian cancer oncogenesis and progression. SK-OV-3 cells were injected subcutaneously into nude mice. The stress group was subjected to a chronic physical restraint protocol for 6 h on 35 consecutive days, while the control group was unrestrained. All mice were sacrificed on day 36 after SK-OV-3 cell injection, and tumors were excised. Tumor tissues were processed for 2D gel electrophoresis, mass spectrometry (nanoUPLC-ESI-MS/MS) and Western blotting. The expression of 20 proteins was found to be significantly altered between the stress and control groups, of which 14 were up-regulated, five were down-regulated, and one protein was found only in the stress group. All proteins were identified by UPLC-ESI-MS/MS, and Western blotting results were consistent with those of proteomic methods. The present results provide new evidence relating to the molecular mechanism underlying the relationship between psychological stress and tumor progression.     

Proteomic analysis of cisplatin resistance in human ovarian cancer using 2-DE method

Platinum-based chemotherapy, such as cisplatin, is the primary treatment for human ovarian cancer. However, overcoming drug resistance has become an important issue in cancer chemotherapy. In this study, we performed 2-DE and ESI-Q-TOF MS/MS analysis to identify differential proteins expression between cisplatin-sensitive (A2780S) and cisplatin-resistant (A2780-CP) ovarian cancer cell lines. Of the 14 spots identified as differentially expressed (±over twofold, P < 0.05) between the two cell lines, ten spots (corresponding to ten unique proteins) were positively identified by ESI-Q-TOF MS/MS analysis. These proteins include capsid glycoprotein, fructose-bisphosphate aldolase C, heterogeneous nuclear ribonucleoproteins A2/B1, putative RNA-binding protein 3, Ran-specific GTPase-activating protein, ubiquitin carboxyl-terminal hydrolase isozyme L1, stathmin, ATPSH protein, chromobox protein homolog3 and phosphoglycerate kinase 1. The proteins identified in this study would be useful in revealing the mechanisms underlying cisplatin resistance and also provide some clues for further research.     

Calcium-dependent proapoptotic effect of Taenia solium metacestodes annexin B1 on human eosinophils: a novel strategy to prevent host immune response

Annexins are a family of calcium-dependent phospholipid-binding proteins that have been proposed to be involved in a wide range of important biological processes. At present, only a few annexins have been identified in parasites, and the physiological roles of these annexins are obscure. Earlier, we cloned a novel annexin (annexin B1) from Taenia solium metacestodes and found that annexin B1 was detectable in the surrounding host-derived layer with granulomaous infiltration. The objective of this study was to investigate the secretion and physiological function of annexin B1. We expressed a green fluorescent protein-tagged annexin B1 (GFP-anxB1) in living SiHa cells and showed that it was secreted upon stimulation with dexamethasone (Dex). This secretion was not inhibited by brefeldin A but was blocked by pre-treatment with the intracellular calcium-specific chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Furthermore, we describe for the first time that annexin B1 can bind to the extracellular surface of human eosinophils and produce Ca(2+)-influx. The Ca(2+)-influx induced apoptosis in eosinophils, which was inhibited by pre-loading the Ca(2+) channel blocker 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-metho-xyphenethyl]-1H-imidazole, HCl (SKF-96365). In conclusion, these findings represent direct and substantial evidence for the secretion of annexin B1 by living cells; the apoptosis in eosinophil induced by annexin B1 might be a novel strategy for T. solium metacestodes to prevent the host's immune attack.     

Proteomic analysis of the cellular responses induced in uninfected immune cells by cell-expressed X4 HIV-1 envelope

HIV-1 envelope gp120 and gp41 glycoproteins (Env), expressed at the cell surface, induce uninfected CD4 T-cell death, but the molecular mechanisms leading to this demise are still largely unknown. To better understand these events, we analyzed by a proteomic approach the differential protein expression profile of two types of uninfected immune cells after their coculture for 1-3 days with cells that express, or not, Env. First, umbilical cord blood mononuclear cells (UCBMCs) were used to approach the in vivo situation, i.e., blood uninfected naive cells that encounter infected cells. Second, we used the A2.01/CD4.403 T-cell line expressing wild type CXCR4 and a truncated form of CD4 that still undergoes Env-mediated apoptosis, independently of CD4 signaling. After coculture with cells expressing Env, 35 and 39 proteins presenting an altered expression in UCBMCs and the A2.01/CD4.403 T-cell line, respectively, were identified by mass-spectrometry. Whatever the cell type analyzed, the majority of these proteins are involved in degradation processes, redox homeostasis, metabolism and cytoskeleton dynamics, and linked to mitochondrial functions. This study provides new insights into the events that sequentially occur in bystander T lymphocytes after contact with HIV-infected cells and leading, finally, to apoptotic cell death.     

Increased sensitivity to cis-diamminedichloroplatinum(II) in human ovarian carcinoma cells in response to treatment with 12-O-tetradecanoylphorbol 13-acetate

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) enhanced sensitivity to cis-diamminedichloroplatinum(II) (DPP) in human ovarina carcinoma 2008 cells by a factor of 2.53 +/- 0.74 fold (S.D.). Sensitization was maximum 3 h after a 1-h exposure to TPA and had disappeared completely by 7 h after treatment. An equivalent degree of sensitization was produced in a 2008 variant selected for 10-fold resistance to DDP. TPA neither increased nor decreased cellular accumulation of DDP. Phorbol, a TPA analog which does not activate protein kinase C, did not cause sensitization. This synergistic interaction between TPA and DDP was completely inhibited by pretreatment with staurosporine, a protein kinase C inhibitor. Cellular cAMP was not altered by TPA stimulation. Furthermore, cycloheximide, a potent protein synthesis inhibitor, did not block the TPA-induced enhancement of drug sensitivity. These results strongly suggest that DDP sensitivity can be modulated by protein kinase C and regulated by phosphorylation of a protein kinase C substrate in both intrinsically sensitive and DDP-resistant cells.