RESUMO
Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver. Thus there is great interest to identify novel HCC diagnostic markers for early detection of the disease and tumour specific associated proteins as potential therapeutic targets in the treatment of HCC. Currently, we are screening for early biomarkers as well as studying the development of HCC by identifying the differentially expressed proteins of HCC tissues during different stages of disease progression. We have isolated, by reverse transcriptase and polymerase chain reaction (RT-PCR), a 1741bp cDNA encoding a protein that is differentially expressed in HCC. This novel protein was initially identified by proteome analysis and we designate it as Hcc-2. The protein is upregulated in poorly-differentiated HCC but unchanged in well-differentiated HCC. The full-length transcript encodes a protein of 363 amino acids that has three thioredoxin (Trx) (CGHC) domains and an ER retention signal motif (KDEL). Fluorescence GFP tagging to this protein confirmed that it is localized predominantly to the cytoplasm when expressed in mammalian cells. Protein alignment analysis shows that it is a variant of the TXNDC5 gene, and the human variants found in Genbank all show close similarity in protein sequence. Functionally, it exhibits the anticipated reductase activity in the insulin disulfide reduction assay, but its other biological role in cell function remains to be elucidated. This work demonstrates that an integrated proteomics and genomics approach can be a very powerful means of discovering potential diagnostic and therapeutic protein targets for cancer therapy.
Assuntos
Biomarcadores Tumorais/biossíntese , Carcinoma Hepatocelular/enzimologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/enzimologia , Proteínas de Neoplasias/biossíntese , Tiorredoxinas/biossíntese , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Biomarcadores Tumorais/genética , Células CHO , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Diferenciação Celular , Cricetinae , Cricetulus , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Perfilação da Expressão Gênica/métodos , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Estrutura Terciária de Proteína , Proteoma/biossíntese , Proteômica/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de RNA/métodos , Homologia de Sequência de Aminoácidos , Tiorredoxinas/genética , Regulação para CimaRESUMO
In the present study, profiles of protein expression were examined during early development of zebrafish, an increasingly popular experimental model in vertebrate development and human diseases. By 2-DE, an initial increase in protein spots from 6 h post-fertilization (hpf) to 8-10 hpf was observed. There was no dramatic change in protein profiles up to 18 hpf, but significant changes occurred in subsequent stages. Interestingly, 49% of the proteins detected at 6 hpf remained detectable by 1 week of age. To map the protein expression patterns in 2-D gels, MALDI-TOF/TOF MS was employed to identify selected protein spots from early embryos. 108 protein spots were found to match known proteins and they were derived from 55 distinct genes. Interestingly, 11 (20%) of them produced multiple protein isoforms or distinct cleavage products. Although deyolked embryos were used in the analysis, a large number of vitellogenin derivatives remained prominently present in the embryos. Other than these, most of the identified proteins are cytosolic, cytoskeletal and nuclear proteins, which are involved in diversified functions such as metabolism, cytoskeleton, translation, protein degradation, etc. Some of the proteins with interesting temporal expression profiles during development are further discussed.
Assuntos
Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/metabolismo , Animais , Eletroforese em Gel Bidimensional , Embrião não Mamífero/metabolismo , Gástrula/metabolismo , Proteínas de Choque Térmico/biossíntese , Queratinas/biossíntese , Larva , Isoformas de Proteínas/biossíntese , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Vitelogeninas/biossíntese , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Proteome analysis of human hepatocellular carcinoma tissues was conducted using two-dimensional difference gel electrophoresis coupled with mass spectrometry. Paired samples from the normal and tumor region of resected human liver were labeled with Cy3 and Cy5, respectively while the pooled standard sample was labeled with Cy2. After analysis by the DeCyder software, protein spots that exhibited at least a two-fold difference in intensity were excised for in-gel tryptic digestion and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. A total of 6 and 42 proteins were successfully identified from the well- and poorly-differentiated samples, respectively. The majority of these proteins are related to detoxification/oxidative stress and metabolism. Three down-regulated metabolic enzymes, methionine adenosyltransferase, glycine N-methyltransferase, and betaine-homocysteine S-methyltransferase that are involved in the methylation cycle in the liver are of special interest. Their expression levels, especially, methionine adenosyltransferase, seemed to have a major influence on the level of S-adenosylmethionine (AdoMet), a vital intermediate metabolite required for the proper functioning of the liver. Recent work has shown that chronic deficiency in AdoMet in the liver results in spontaneous development of steatohepatitis and hepatocellular carcinoma, and hence the down-regulation of hepatic methionine adenosyltransferase in our hepatocellular carcinoma samples is in line with this observation. Moreover, when a comparison is made between the differentially expressed proteins from our human hepatocellular carcinoma samples and from the liver tissues of knockout mice deficient in methionine adenosyltransferase, there is a fairly good correlation between them.
Assuntos
Carcinoma Hepatocelular/química , Eletroforese em Gel Bidimensional , Neoplasias Hepáticas/química , Espectrometria de Massas , Proteoma/análise , Carcinoma Hepatocelular/patologia , Humanos , Processamento de Imagem Assistida por Computador , Neoplasias Hepáticas/patologia , Coloração pela Prata , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/farmacologiaRESUMO
The metabolic state of hybridoma cells in continuous culture varies with the cultivation condition from which the culture is initiated. At a metabolically shifted state, cells have markedly reduced glucose and other nutrient consumption and lactate production as compared to cells in batch culture or in continuous culture without a metabolic shift. Taking a combined genomics and proteomics approach, we investigated the molecular mechanism of metabolic shift. Cells from continuous cultures at two different steady states with a glucose consumption to lactate production molar ratio (DeltaL/DeltaG) of 0.08 and 1.4 were studied. Affymetrix GeneChips as well as cDNA microarrays were employed to identify differentially expressed mRNA transcripts, and the differentially expressed proteins were identified using the 2D gel electrophoresis-mass spectrometry approach. The decrease in glucose metabolism upon metabolic shift is accompanied by a decrease in gene expression of a number of genes involved in its metabolism. However, the number of genes differentially expressed and the extent of differential expression upon metabolic shift are relatively moderate. The change in the expression of metabolic genes at the transcriptional level was confirmed by real time PCR. The results suggest that metabolic shift is a combined effect of both biochemical events at reaction level and gene expression at transcription and translation level. This approach of integrating transcriptional profiling, proteomic techniques and biochemical analysis provides a more global view of the metabolism of mammalian cells in culture.
Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/fisiologia , Hibridomas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteoma/genética , Proteoma/metabolismo , Animais , Linhagem Celular , Eletroforese em Gel Bidimensional/métodos , Glucose/metabolismo , Ácido Láctico/biossíntese , Camundongos , Proteômica/métodos , Transcrição Gênica/fisiologiaRESUMO
A classical proteomic analysis was used to establish a reference map of proteins associated with healthy human erythrocyte ghosts. Following osmotic lysis and differential centrifugation, ghost proteins were separated by either one-dimensional gel electrophoresis (1-DE) or two-dimensional gel electrophoresis (2-DE). Selected protein bands or spots were excised and trypsinized before mass spectrometric analyses and data mining was performed using the SWISS-PROT and NCBI nonredundant databases. A total of 102 protein spots from a 2-D gel were successfully identified. These corresponded to 59 distinct polypeptides with the remaining 43 being isoforms. As for the 1-D gel, 44 polypeptides were identified, of which 19 were also found on the 2-D gel. Most of the 19 common polypeptides were membrane cytoskeletal proteins that are often referred to as the "band" proteins. The remaining 25 polypeptides that were found exclusively on 1-D gels were proteins with high hydrophobicity (e.g., sorbitol dehydrogenase and glucose transporter) and high molecular mass (e.g., Kell blood group glycoprotein and Janus-kinase 2). A higher number of signaling proteins was also identified on 1-D gels compared to 2-D gels. These included Ras, cAMP dependent protein kinase and TGF-beta receptor type 1 precursor.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Eritrócitos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Centrifugação , Citoesqueleto/metabolismo , Bases de Dados como Assunto , Membrana Eritrocítica/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Osmose , Peptídeos/química , Proteínas/análise , Transdução de Sinais , Tripsina/farmacologiaRESUMO
Hepatocellular carcinoma (HCC or hepatoma) is the most common primary cancer of the liver. Persistent viral infection by the hepatic B or C virus is probably the most important cause of HCC worldwide. It is responsible for approximately one million deaths each year, predominantly in the underdeveloped and developing countries, but its incidence is also on the rise in the developed countries. For most patients suffering from HCC, long-term survival is rare, as they are presented late and are often unsuitable for curative treatment. Thus there is great interest to identify novel HCC diagnostic markers for early detection of the disease, and tumour specific associated proteins as potential therapeutic targets in the treatment of HCC. Proteome analyses of HCC cell lines and liver tumour tissues should facilitate the screening and discovery of these HCC proteins. The creation of a comprehensive HCC proteome database would be an important first step towards achieving this goal. This review presents an update of the two-dimensional electrophoresis proteome database of the cell line, HCC-M, which is also now freely accessible through the World Wide Web at http://proteome.btc.nus.edu.sg/hccm/.
Assuntos
Carcinoma Hepatocelular/metabolismo , Bases de Dados de Proteínas , Neoplasias Hepáticas/metabolismo , Proteoma , Eletroforese em Gel Bidimensional , HumanosRESUMO
Butyrate, a 4-carbon fatty acid, has been shown to cause growth arrest and apoptosis of cancer cells in vitro and in vivo. The signaling pathways leading to changes in cell growth are unclear. We used a functional proteomics approach to delineate the pathways and mediators involved in butyrate action in HT-29 cells at 24 hr posttreatment. Using 2-dimensional gel electrophoresis, we showed that butyrate treatment resulted in alterations in the proteome of HT-29 cells. MALDI-TOF mass spectrometry was used to identify butyrate-regulated spots. First, our results revealed that the expression of various components of the ubiquitin-proteasome system was altered with butyrate treatment. This suggests that, in addition to the regulation of gene expression through the histone deacetylase pathway, proteolysis could be a means by which butyrate may regulate the expression of key proteins in the control of cell cycle, apoptosis and differentiation. Second, we found that both proapoptotic proteins (capase-4 and cathepsin D) and antiapoptotic proteins (hsp27, antioxidant protein-2 and pyruvate dehydrogenase E1) were simultaneously upregulated in butyrate-treated cells. Western blotting was carried out to confirm butyrate regulation of the spots. Both cathepsin D and hsp27 showed a time-dependent increase in expression with butyrate treatment in HT-29 cells. However, in HCT-116 cells, which were 5-fold more sensitive to butyrate-induced apoptosis, the upregulation of cathepsin D with time was not accompanied by a similar increase in hsp27 levels. Thus, the simultaneous upregulation of both proapoptotic and antiapoptotic proteins in HT-29 cells may account for their relative resistance to butyrate-induced apoptosis.
Assuntos
Butiratos/farmacologia , Células HT29/efeitos dos fármacos , Proteoma/análise , Eletroforese em Gel Bidimensional , Células HT29/metabolismo , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
In the last few years, the number of biologics produced by mammalian cells have been steadily increasing. The advances in cell culture engineering science have contributed significantly to this increase. A common path of product and process development has emerged in the last decade and the host cell lines frequently used have converged to only a few. Selection of cell clones, their adaptation to a desired growth environment, and improving their productivity has been key to developing a new process. However, the fundamental understanding of changes during the selection and adaptation process is still lacking. Some cells may undergo irreversible alteration at the genome level, some may exhibit changes in their gene expression pattern, while others may incur neither genetic reconstruction nor gene expression changes, but only modulation of various fluxes by changing nutrient/metabolite concentrations and enzyme activities. It is likely that the selection of cell clones and their adaptation to various culture conditions may involve alterations not only in cellular machinery directly related to the selected marker or adapted behavior, but also those which may or may not be essential for selection or adaptation. The genomic and proteomic research tools enable one to globally survey the alterations at mRNA and protein levels and to unveil their regulation. Undoubtedly, a better understanding of these cellular processes at the molecular level will lead to a better strategy for 'designing' producing cells. Herein the genomic and proteomic tools are briefly reviewed and their impact on cell culture engineering is discussed.