Teck Keong Seow

Two-dimensional electrophoresis map of the human hepatocellular carcinoma cell line, HCC-M, and identification of the separated proteins by mass spectrometry

Currently, one of the most popular applications of proteomics is in the area of cancer research. In Africa, Southeast Asia, and China, hepatocellular carcinoma is one of the most common cancers, occurring as one of the top five cancers in frequency. This project was initiated with the purpose of separating and identifying the proteins of a human hepatocellular carcinoma cell line, HCC‐M. After two‐dimensional gel electrophoresis separation, silver staining, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) analyses, tryptic peptide masses were searched for matches in the SWISS‐PROT and NCBI nonredundant databases. Approximately 400 spots were analyzed using this approach. Among the proteins identified were housekeeping proteins such as alcohol dehydrogenase, alpha‐enolase, asparagine synthetase, isocitrate dehydrogenase, and glucose‐6‐phosphate 1‐dehydrogenase. In addition, we also identified proteins with expression patterns that have been postulated to be related to the process of carcinogenesis. These include 14‐3‐3 protein, annexin, prohibitin, and thioredoxin peroxidase. This study of the HCC‐M proteome, coupled with similar proteome analyses of normal liver tissues, tumors, and other hepatocellular carcinoma cell lines, represents the first step towards the establishment of protein databases, which are valuable resources in studies on the differential protein expressions of human hepatocellular carcinoma.

Separation of human erythrocyte membrane associated proteins with one‐dimensional and two‐dimensional gel electrophoresis followed by identification with matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry

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.

Hepatocellular carcinoma: From bedside to proteomics

Hepatocellular carcinoma (HCC or hepatoma) is the most common primary cancer of the liver. It is responsible for approximately one million deaths each year, mainly in underdeveloped and developing countries. The aetiological factors identified in the development of HCC included persistent infection by hepatitis B and hepatitis C viruses, and exposure to aflatoxins. Although immunization can protect individuals from being infected by the hepatitis B virus, the early detection of HCC in those who have been infected by the virus remains a challenge. Thus most HCCs present late and are not suitable for curative treatment. Hence there is a tremendous interest and urgency to identify novel HCC diagnostic marker(s) for early detection, and tumour specific disease associated proteins as potential therapeutic targets in the treatment of HCC. Screening for these HCC proteins has been facilitated by proteomics, a key technology in the global analysis of protein expression and understanding gene function. Present and earlier proteome analyses of HCC have used predominantly experimental in vitro systems. The protein expression profiles of several hepatoma cell lines such as HepG2, Huh7, SK‐Hep1, and Hep3B have been compared with normal liver, and nontransformed cell lines (Chang and WRL‐68), while a comprehensive proteome analysis to create a protein database was carried out for the cell line HCC‐M. In the future, proteome analyses utilizing tumour tissues, which reflect the pathological state of HCC more closely, will be undertaken. This work will complement the gene expression studies of HCC which are already underway. Efforts have also been directed at the proteome analysis of hepatic stellate cells, as these cells play an important role in liver fibrosis. Since liver fibrosis is reversible but not cirrhosis, it is of considerable importance to identify therapeutic targets that can slow its progression.

Proteome analysis of butyrate-treated human colon cancer cells (HT-29)

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.

Proteome database of hepatocellular carcinoma.

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/.

Proteomic investigation of metabolic shift in mammalian cell culture

Mammalian cells, under typical cultivation conditions, produce large quantities of lactate and ammonia that affect cell growth adversely and result in low cell concentration. Controlled nutrient feeding to maintain low concentrations of glucose and glutamine reduces metabolite production drastically, altering the metabolism of the cells. This metabolic shift results in higher cell concentration in continuous cultures and does not affect the specific productivity of the cells. We have taken a proteomics approach to investigate the differential protein expression with metabolic shift. Using two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry (MS), we have found at least eight differentially expressed spots; two proteins were down‐regulated, and the others were up‐regulated with metabolic shift. These included metabolic enzymes, the brain form of phosphoglycerate mutase, which was down‐regulated, and the precursor of the 23 kDa subunit of NADH‐ubiquinone oxidoreductase, which was up‐regulated. Another enzyme, the L1 isozyme of ubiquitin carboxyl‐terminal hydrolase, which is involved in protein turnover and degradation, was also up‐regulated in the metabolically altered cells. The remaining down‐regulated spot had been identified as two isoforms of cytoplasmic actins, while three of the up‐regulated spots were viral GAG polyproteins from various murine viruses. An unidentified protein was also up‐regulated in the cells with altered metabolic state. This study shows the potential of using a proteomics approach in deciphering the intracellular changes in cells with physiological changes such as metabolism shift. The new insight into cell metabolism afforded by this analysis will greatly facilitate process optimization of continuous cell cultures.

An integrated approach in the discovery and characterization of a novel nuclear protein over-expressed in liver and pancreatic tumors

An integrated approach in protein discovery through the use of multidisciplinary tools was reported. A novel protein, Hcc‐1, was identified by analysis of the hepatocellular carcinoma (HCC)‐M cell proteome. The assembled EST sequence of the 210 amino acid novel protein was subsequently confirmed by rapid amplification of cDNA ends (RACE). A total of 687 bp at the 5′ untranslated region of Hcc‐1 was identified. Promoter activity and several upstream open reading frames (uORFs) were demonstrated at this region. Bioinformatics prediction showed that the first 42 amino acids of the protein is a SAP domain with sequence matches to hnRNP from various vertebrate species. The Hcc‐1 protein was localized to the cell nucleus while the gene was localized to chromosome 7q22.1. Hcc‐1 cDNA level was increased in pancreatic adenocarcinoma. The level was also increased in well‐differentiated hepatocellular carcinoma but decreases as the carcinoma progressed to a poorly differentiated stage.

Proteome analysis of a human heptocellular carcinoma cell line, HCC-M: An update

Recently, we reported the proteome analysis of a human hepatocellular carcinoma cell line, HCC‐M (Electrophoresis 2000, 21, 1787–1813), using two‐dimensional gel electrophoresis (2‐DE) and matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS). From a total of 408 unique spots excised from the 2‐DE gel, 301 spots yielded good MALDI spectra. Out of these, 272 spots had matches returned from the database search leading to the identification of these proteins. Here, we report the results on the identification of the remaining 29 spots using nanoelectrospray ionization‐tandem mass spectrometry (nESI‐MS/MS). First, “peptide tag sequencing” was performed to obtain partial amino acid sequences of the peptides to search the SWISS‐PROT and NCBI nonredundant protein databases. Spots that were still not able to find any matches from the databases were subjected to de novopeptide sequencing. The tryptic peptide sequences were used to search for homologues in the protein and nucleotide databases with the NCBI Basic Local Alignment Search Tool (BLAST), which was essential for the characterization of novel or post‐translationally modified proteins. Using this approach, all the 29 spots were unambiguously identified. Among them, phosphotyrosyl phosphatase activator (PTPA), RNA‐binding protein regulatory subunit, replication protein A 32 kDa subunit (RP‐A) and N‐acetylneuraminic acid phosphate synthase were reported to be cancer‐related proteins.*

Identification of a serine protease inhibitor homologue in Bird's Nest by an integrated proteomics approach.

For centuries, the edible nests of Collocalia spp. (“Birds Nests”) have been used as a Chinese delicacy that had been claimed to be an effective health‐giving tonic. However, clinical studies indicated that in Singapore, Birds Nest is the most common cause of food‐induced anaphylaxis in children, which could lead to potentially life‐threatening allergenic reactions. The purpose of this study was to characterize the major allergens in Birds Nest by using the combined technologies of two‐dimensional gel electrophoresis (2‐DE), immunochemistry, N‐terminal protein sequencing, and mass spectrometry. Results from the immunostaining of the Western blots of the Birds Nest 2‐DE separated proteins with the sera from allergic patients indicated the presence of a major allergen of 66 kDa. Initial searches of the matrix assisted laser desorption/ionization – time of flight – mass spectrometry (MALDI‐TOF‐MS) tryptic peptide masses of the allergen in the SWISS‐PROT and NCBI nonredundant databases revealed that this protein was novel. Based on the partial protein sequence information obtained from N‐terminal microsequencing and nanoelectrospray‐tandem MS, the 66 kDa immunoreactive allergen was found to be homologous to ovoinhibitor, a Kazal‐type serine protease inhibitor, which is one of the dominant allergens found in chicken egg white.

Hcc-2, a novel mammalian ER thioredoxin that is differentially expressed in hepatocellular carcinoma.

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 1741 bp 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.