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ABSTRACT: There is currently substantial interest in the identification of human tumor antigens for the diagnosis and immunotherapy of cancer. In our previous study, secretion character and up-regulation of triosephosphate isomerase were observed in lung squamous cell carcinoma, and autoantibodies against triosephosphate isomerase and peroxiredoxin 6 were detected in the sera from over 25% of patients, but in none of the healthy controls. In this study, peroxiredoxin 6 was also found at higher levels in the sera of the patients. Up-regulated triosephosphate isomerase and peroxiredoxin 6 were further validated by enzyme-linked immunosorbent assay in an additional 61 lung squamous cell carcinoma patients, 23 lung adenocarcinoma patients, 56 other types of carcinoma patients, 12 benign lung disease patients, and 59 healthy controls. We found that both triosephosphate isomerase and peroxiredoxin 6 were specifically elevated in lung squamous cell carcinoma sera compared with other groups, with the exception of peroxiredoxin 6 in lung adenocarcinoma patients. Positive correlation between triosephosphate isomerase and distant metastasis was found. At the cut-off point 0.221 (optical density value) on the receiver operating characteristic curve, triosephosphate isomerase could comparatively discriminate lung squamous cell carcinoma from healthy controls with a sensitivity of 65.6%, specificity 84.7%, and total accuracy 75%. For peroxiredoxin 6, at the cut-off point 0.151, it could discriminate the two groups with a sensitivity of 70.5%, specificity 62.7%, and total accuracy 65.8%. With both triosephosphate isomerase and peroxiredoxin 6, discriminant analysis results showed that 68.9% of the lung squamous cell carcinoma and 83.1% of healthy controls were correctly classified. We concluded that triosephosphate isomerase and peroxiredoxin 6 could be markers for lung squamous cell carcinoma.
Cancer Science 09/2009; 100(12):2396-401. · 3.33 Impact Factor
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ABSTRACT: Nasopharyngeal carcinoma (NPC) is a high-incidence malignancy in Southern China and Southeast Asia. Although mutation of p53 tumor-suppressor gene is a rare event in NPC, NPC has a high frequency of overexpressed/accumulated p53 protein, which was reported to be dysfunction or inactivation in most of NPC. We report here a functional characterization of p53 in an undifferentiated NPC cell line CNE2. To elucidate the biological function of p53, we employed the RNA interference (RNAi) approach to knockdown the endogenously expressed p53 in CNE2 cells. Interestingly, suppression of p53 expression in CNE2 cells was associated with significant down-regulation of p21WAF1/CIP1 expression and decreased HDM2 protein level in both steady state and genotoxic stress induced by ionizing radiation (IR). Consistent with these biochemical data were the accelerated cell cycle progression and the increased proliferation rate, suggesting that p53 retained growth inhibitory activity in CNE2 cells. Indeed, down-regulation of p53 in CNE2 enhanced the ability of CNE2 cells to grow anchorage-independently in vitro and to develop tumors in vivo. Together with the radioresistance acquired by CNE2sip53 cells, our data indicate that in contrast to a previous study, p53 in this NPC cell line remains functional, which may have an important therapeutical implication.
International Journal of Oncology 05/2009; 34(4):1017-27. · 2.40 Impact Factor
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ABSTRACT: Latent membrane protein 1 (LMP1) of Epstein-Barr virus has been identified to be crucial in inducing cell transformation. However, the mechanism of LMP1-mediated epithelial cell transformation remains unclear. In this study, nasopharyngeal epithelial cells NP69 were infected with retrovirus with gene encoding wild type LMP1 or mutational LMP1 defective in binding to tumor necrosis factor receptor-associated death domain (TRADD). The NP69-LMP1(TRADD) lost some malignant phenotypes compared with the NP69-LMP1(WT). We performed proteomic approach to gain the differential protein expression profile associated with LMP1-mediated epithelial cell transformation. Furthermore, the differential expressional levels of partial identified proteins were confirmed by Western blot and real-time RT-PCR. Some were known to be related to the development of LMP1-induced transformation, and some were new LMP1-associated proteins. These data are valuable for further study of the mechanism of LMP1 in human nasopharyngeal carcinoma and provide some new clues for investigating other LMP1-associated tumors.
Molecular and Cellular Biochemistry 08/2008; 314(1-2):73-83. · 2.06 Impact Factor
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ABSTRACT: The human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor (EGFR) family, and it plays an important role in the development of many human adenocarcinomas. The extracellular domain (ECD) of HER2 is an ideal target for therapeutic approaches. In order to obtain large quantities of active HER2 ECD protein for biochemical and structural analysis and for detecting anti-HER2 ECD antibodies in serum, a systematic assessment of optimal parameters for the refolding of the glutathione S-transferase (GST) fusion protein was carried out. After the GST-HER2 ECD inclusion bodies were solubilized with denaturation buffer containing 8M urea, an approach was then used to optimize refolding parameters. This approach utilized dilution of denatured and reduced GST-HER2 ECD into different refolding buffers using orthogonal design method. Optimal refolding was obtained in an alkaline buffer containing reduced and oxidized glutathione, and subsequent incubation at 4 degrees C for 24h. After purification with glutathione Sepharose 4B and PreScission protease cleavage of the fusion protein, 8.9mg of recombinant HER2 ECD was obtained from 1L of Escherichia coli. Rabbit polyclonal antibodies against HER2 ECD were obtained. The purified protein was found to be immunogenic and useful for immunodiagnostic studies of serum HER2 ECD and its antibodies by using enzyme-linked immunosorbent assay (ELISA).
Protein Expression and Purification 07/2007; 53(2):247-54. · 1.59 Impact Factor
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Zhi-Qiang Xiao,
Ying Chen,
Bin Yi,
Mao-Yu Li,
Peng-Fei Zhang,
Hong Yi,
Chao-Jun Duan,
Cui Li,
Jian-Ling Li,
Cen-E Tang, Fang Yang,
Ge-Qin Zhu,
Zhu-Chu Chen
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ABSTRACT: A proteomics-based approach has been used to identify proteins that commonly elicit a humoral immune response in nasopharyngeal carcinoma (NPC). Sera from 19 newly diagnosed NPC patients and 19 healthy individuals were analyzed for IgG autoantibodies against NPC proteins resolved by 2-DE. Protein spots that exhibited selective reactivity with sera from NPC patients were identified by MS. Among nine identified proteins, cytokeratin 19 (CK19), Erb3 binding protein (EBP1), and Rho GDP dissociation inhibitor-beta (Rho-GDI-2) induced autoantibodies in more than 36.8% of NPC patients but not in healthy individuals. Furthermore, Western blot analysis and immunohistochemical staining were performed to determine the expression and localization of CK19, EBP1, and Rho-GDI-2 in NPC and normal nasopharyngeal mucosal tissues. Up-regulated CK19 and EBP1, but not Rho-GDI-2, were observed in NPC vs. normal tissue. Subcellular localization of the three proteins in NPC tissue was same as that in the normal tissue. Thus, overexpression of CK19 and EBP1 may be one of the mechanisms for their autoantibody development in NPC. To validate the findings of a proteomic analysis, occurrence of autoantibodies against these three proteins was detected by immunoprecipitation and Western blot analysis in additional 30 NPC patients, 23 other types of cancer patients and 20 healthy individuals. Results showed that frequency of autoantibodies against CK19, EBP1 and Rho-GDI-2 in NPC patients was significantly higher than that in other types of cancer patients and healthy individuals. We conclude that CK19, EBP1 and Rho-GDI-2 may have utility in NPC screening and diagnosis.
Proteomics. Clinical applications 07/2007; 1(7):688-98. · 1.97 Impact Factor
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ABSTRACT: Metallothioneins (MTs) are a family of low molecular weight, cysteine rich heavy metal binding proteins with multifunction, such as metal detoxification and antioxidation, and are involved in a number of cellular processes including gene expression, apoptosis, proliferation and differentiation. However, high yield expression of human MT in Escherichia coli has not been established effectively. To produce large amounts of human MT protein at low cost, recombinant human metallothionein 2A (MT2A) protein with an N-terminal GST tag was successfully expressed at high levels in soluble form in E. coli and high purification of it was established by affinity chromatography under native conditions. The final yield was about 5mg of the recombinant MT2A per liter of bacterial culture with the purity of 97.9%. Chemical and functional characteristics analysis of the recombinant human MT2A exhibited intact metal binding ability, hydroxyl radical scavenging ability and significant protective role against DNA damage caused by UVC radiation. Establishment of highly purified recombinant human MT2A protein with native characteristics at low cost would improve its function study and wide applications in protecting against oxidative damage and UV radiation.
Protein Expression and Purification 06/2007; 53(1):186-94. · 1.59 Impact Factor
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ABSTRACT: It was evident that nitrosamines can act directly on target tissue and result in carcinogenesis. As has been shown, the carcinogenic activity of nitrosamines relied on its bioactivation by Cytochrome P450 2E1 (CYP2E1). In this study, we investigated the expression of CYP2E1 in Nasopharyngeal carcinoma (NPC) cells, embryonic nasopharyngeal epithelial tissue (ENET) specimens, and NPC biopsies by RT-PCR analysis. CYP2E1 was expressed in all NPC cell lines (6/6, including 7429) and ENET (6/6), and 80% of NPC biopsie (8/10). The fact that Human nasopharynx expresses CYP2E1 suggests that CYP2E1 may play an important role in the course of NPC by indirect carcinogens nitrosamines. To further evaluate the function of CYP2E1, the CYP2E1 was stably expressed in the cell line NIH 3T3/rtTA under a tetracycline-controlled transactivator. The expression of CYP2E1 was tightly regulated in a dose-dependent manner by Doxycycline (Dox) When the catalytic activity of CYP2E1 was assayed, the result showed that the generation of 6-hydroxychlorzoxazone (6-OH-CZ) from chlorzoxazone (CZ) was dose- and time-dependent on Dox addition to the medium. In the presence of 1 microg/ml Dox, the CZ 6-hydroxylase activity of the cell line was found to be 0.986 +/- 0.034 nmol/10(6) cells/h. The metabolic activation of Tet/3T3/2E1-6 cells was also assayed by N,N'-dinitrosopiperazine (DNP) cytotoxicity, and the viability of Tet/3T3/2E1-6 cells treated with Dox was lower than that of untreated cells with a significant difference between them in 80 and 160 microg/ml DNP (P ( 0.05, t test. This cell line will be useful not only to assess the metabolic characteristics of CYP2E1, but also will be useful to investigate the role of CYP2E1 in metabolic activation of carcinogenic nitrosamines in vitro.
Molecular and Cellular Biochemistry 05/2007; 298(1-2):93-100. · 2.06 Impact Factor
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Fang Yang,
Zhi-qiang Xiao,
Xiu-zhi Zhang,
Cui Li,
Peng-fei Zhang,
Mao-yu Li,
Ying Chen,
Ge-qin Zhu,
Yi Sun,
Ying-fu Liu,
Zhu-Chu Chen
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ABSTRACT: Autoantibodies against tumor antigens are promising means for cancer diagnosis and prognosis. In this study, we applied a proteomic approach to identify proteins that commonly elicit humoral response in lung squamous carcinoma (LSC). Sera from 20 newly diagnosed patients with LSC and 20 matched healthy individuals were analyzed for antibody-based reactivity against LSC proteins separated by two-dimensional electrophoresis. Autoantibodies against triosephosphate isomerase (Tim) and superoxide dismutase [Mn] (MnSOD) were detected in sera from over 20% patients with LSC but none from the normal controls. Furthermore, the occurrence of autoantibodies against Tim and MnSOD was evaluated by ELISA in an additional 40 LSC patients, 30 other types of cancer (OTC) patients, and 50 noncancer controls (NC). Results showed that frequency of autoantibody against Tim (27.5%) in LSC patients was significantly higher than that in OTC patients (6.7%, p = 0.027) and in NC (6%, p = 0.005). Likewise, frequency of autoantibody against MnSOD in LSC (20%) patients was significantly higher than that in NC (4%, p = 0.016), however, there was no significant difference when comparing to that in OTC patients (6.7%, p = 0.115). We also observed significantly increased expression and secretion of Tim and MnSOD in LSC, which possibly account for their autoantibody development. Our results indicate that autoantibody and antigen of Tim and MnSOD may be useful for screening and diagnosis of the lung squamous carcinoma.
Journal of Proteome Research 03/2007; 6(2):751-8. · 5.11 Impact Factor
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Yi Sun,
Hong Yi,
Peng-Fei Zhang,
Mao-Yu Li,
Cui Li,
Feng Li,
Fang Peng,
Xue-Ping Feng,
Yi-Xuan Yang, Fang Yang,
Zhi-Qiang Xiao,
Zhu-Chu Chen
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ABSTRACT: Although mutation of p53 tumor-suppressor gene is rare in nasopharyngeal carcinoma (NPC), NPC has a high frequency of overexpression of p53 protein. There seem to be complex mechanisms of inactivation and stabilization of p53 in NPC. To detect proteins associated with the function of p53 in high throughout screening, we succeeded in establishing p53 knockdown human NPC CNE2 cell line (CNE2sip53) using stable RNA interference, and compared the proteomic changes between CNE2sip53 and control cell line CNE2/pSUPER using two-dimensional gel electrophoresis. Twenty-two differentially expressed proteins between the two cell lines were identified by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization tandem mass spectrometry, some of which are known to be associated with the p53 function (HSP27, hnRNP K, 14-3-3sigma, etc.), and others may be novel proteins associated with p53 function (eIF4B, TPT1, hnRNP H3, SFRS1 etc.). Furthermore, several differential proteins including HSP27, HSP70, GRP75 and GRP78 were verified as p53 interacting proteins in NPC by immunoprecipitation and Western blot analysis, and the suppression of HSP27 expression by HSP27 antisense oligonucleotides could decrease the p53 protein level. Our data suggest that these differential proteins may be associated with the function of p53 in NPC, and provide new clues to elucidate the mechanisms of inactivation and stabilization of p53 in NPC.
FEBS Letters 02/2007; 581(1):131-9. · 3.54 Impact Factor
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ABSTRACT: To express and purify human MT-2a in prokaryotic cells and to prepare the MT-2a-specific rabbit antiserum.
GST-MT-2a fusion protein was expressed after IPTG induction and further purified with Glutathione Sepharose 4B. Then the purified GST-MT-2a fusion protein was used to immunize New Zealand rabbits. The titer and specificity of rabbit antiserum were evaluated by double immunodiffusion, ELISA and Western blot.
GST-MT-2a fusion protein was highly expressed. The final yield of the pure GST-MT-2a was about 38 mg per liter of bacterial culture. Its antiserum with high specificity and potency was also obtained.
The successful expression of GST-MT-2a fusion protein in E. coli and the preparation of MT-2a specific rabbit antiserum will be valuable for the study on the function of human MT-2a.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 08/2006; 22(4):530-2.
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ABSTRACT: A novel gene, laryngeal carcinoma-related gene 1 (LCRG1), had the characteristics of tumor-suppressor genes. It was cloned in our laboratory. The objective was to find and characterize the proteins related to LCRG1 and to elucidate the molecular mechanism of LCRG1.
We used the established cell lines of Hep-2/LCRG1 (Hep-2 cells transfected by recombinant plasmid pcDNA3.1[+]/LCRG1) and Hep-2/pcDNA3.1(+) (Hep-2 cells transfected by control vector pcDNA3.1[+]) as cell models.
Two-dimensional gel electrophoresis (2-DE) technology was performed to separate the proteins of Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cell lines, respectively. The differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization-quadruple time-of-flight MS/MS (ESI-Q-TOF MS/MS). Then the differential expression levels of partial identified proteins were determined by Western blotting analysis and quantitative real-time reverse transcriptase-polymerase chain reaction.
The results showed the attained 2-DE patterns of the two cell lines were well-resolved and reproducible. There were 1075+/-43 and 1027+/-23 protein spots observed in Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cell lines, respectively. The average matching rate of the two cell lines was 91%. Twenty-six differentially expressed protein spots were identified (twenty spots for MALDI-TOF-MS, six spots for ESI-Q-TOF MS/MS). Most of the characterized proteins were characterized as the members of enzymes (phosphoglycerate mutase, manganese superoxide dismutase, and so on), transcription proteins (rho gdp dissociation inhibitor), and so on. Those identified proteins might contribute to the tumor-suppressive function of LCRG1. The differential expression levels of the partial proteins were confirmed by real-time polymerase chain reaction and Western blotting.
We tentatively proposed those differentially expressed proteins were involved in the tumor-suppressive process related to LCRG1. These data will be helpful to elucidate the molecular mechanism of LCRG1.
The Laryngoscope 03/2006; 116(2):224-30. · 1.75 Impact Factor
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ABSTRACT: To purify the extracellular domain of HER2 in vitro and improve its prokaryotic expression abundance, the cDNA fragment encoding extracellular domain of HER2 was obtained by PCR and cloned into the expression vector pGEX-6P-1. After transforming it into Escherichia coli BL21, we instituted an investigation of different inducing conditions to try out the optimal condition for expressing soluble fusion protein. As for insoluble inclusion bodies, they were dissolved in 8 M Urea and refolded in refolding buffer. The soluble protein and the refolded protein were purified with Glutathione Sepharose 4B, respectively. The results showed that both the soluble and insoluble protein existed in Escherichia coli, but the majority was insoluble. It is beneficial to the expression of soluble fusion protein by induction at lower temperature (30 degrees C) and higher optical density (A600= 1.8) with the use of certain additive in medium. By purification of the supernatant of the lysate and refolded protein, the yield of the fusion protein was about 1.23 mg per liter culture. As a result, we have obtained the maximum soluble extracellular domain of HER2 protein, and thus have laid a foundation for further work on functional study and antibody preparation for HER2.
Sheng wu yi xue gong cheng xue za zhi = Journal of biomedical engineering = Shengwu yixue gongchengxue zazhi 03/2006; 23(1):136-41.
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ABSTRACT: The cDNA encoding the rabbit metallothionein-I was amplified by RT-PCR from the rabbit liver induced by cadmium and cloned into prokaryotic fusion expression vector pQE40. Then it was transformed into Escherichia coli M15. Positive expression clones were detected by colony blotting. Target protein solubility was determined by Western blotting analysis. The optimal induction condition of the level of protein expression with IPTG induction was established by SDS-PAGE electrophoresis and ImageMaster VDS software analysis. The fusion protein can be purified from lysates with Ni-NTA agarose. We found that the fusion protein with apparent molecular weight 32 KD existed in two ways: soluble and insoluble in Escherichia coli. After 1 mM IPTG induction, the level of expression of the fusion protein increased with the prolongation of induction time and reached a peak in 9 h by ImageMaster VDS software analysis, accounting for 57.4% of all the insoluble protein. The purified fusion protein was obtained by Ni-NTA affinity chromatography. This fusion protein can be used in further studies on the preparation of MT-I protein and development of protein product.
Sheng wu yi xue gong cheng xue za zhi = Journal of biomedical engineering = Shengwu yixue gongchengxue zazhi 03/2004; 21(1):76-80.
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ABSTRACT: To express and purify human metallothionein-1E (MT-1E) fusion protein in vitro.
The cDNA encoding human MT-1E was amplified by RT-PCR and cloned into prokaryotic expressing vector pQE40. After transforming it into Escherichia Coli M15, we determined the solubility of target protein by western blotting and investigated the IPTG inducing condition. The target protein was purified from lysates with Ni-NTA agarose column.
Western blotting analysis suggested that both the soluble and insoluble fusion protein existed in Escherichia Coli, but the insoluble was the main expression form. Induced by 1 mM IPTG, the expression of target protein increased with the prolongation of induction time. In our study, after being induced for 8h, the target protein accounted for about 32% of the total bacterial protein. Purified protein was obtained by affinity chromatography.
We have obtained purified human MT-1E fusion protein, which lays the foundation for the antibody preparation and further functional study.
Hunan yi ke da xue xue bao = Hunan yike daxue xuebao = Bulletin of Hunan Medical University 01/2004; 28(6):583-6.
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ABSTRACT: To investigate the role of CYP2E1 gene in chemical carcinogen-induced nasopharyngeal carcinogenesis and to provide new evidence about etiology and pathogenesis of nasopharyngeal carcinoma.
RT-PCR was used to clone the CYP2E1 gene in human embryonic nasopharyngeal epithelial (HENE) cell, the transformed nasopharyngeal epithelial cell line (7,429), and the nasopharyngeal carcinoma cell line (HNE1). The cloned segments were inserted into pGEM-T Easy vector to sequence by DNA recombination technique.
In comparison with HENE-2E1 cDNA, there were two point mutations at positions 846 (A to T) and 901 (A to G) in 7,429-2E1 cDNA as well as only one point mutation at position 901 (A to G) in HNE1-2E1 cDNA. In comparison with human (adult, ethanol-inducible) liver CYP2E1 gene (GenBank NO. J02843), HENE-2E1 cDNA had one point mutation at position 901 (G to A). All these point mutations didn't affect the amino acid sequence. But no base change was found in HNE1-2E1 cDNA.
There are a few of base substitutions among HENE-2E1 cDNA, 7,429-2E1 cDNA, and HNE1 cDNA sequences. All these point mutations are synonymous mutation. The study reconfirms that the human CYP2E1 gene is relatively well conserved.
Hunan yi ke da xue xue bao = Hunan yike daxue xuebao = Bulletin of Hunan Medical University 05/2003; 28(2):107-10.
