转导EB病毒转录的EBERs促进MDCK细胞的恶性化

[目的]通过转导EB病毒(Epstein-Barr Virus，EBV)转录的核内小RNA(EBV encoded small RNAs，EBERs)探讨其对上皮细胞株MDCK细胞生长特性的影响。[方法]用聚合酶链反应(polymerase chain reaction，PCR)技术从EB病毒阳性的鼻咽癌(nasopharyngeal carcinoma，NPC)由来的NPC-KT细胞中扩增出EBERs片段。为了在细胞内大量表达EBERs，利用分子生物学技术，制作了EBERs片段的5次重复排列的5EBERs和10次重复排列的10EBERs。分别插入pCEP4载体质粒，并导入MDCK细胞内。利用Northern Blot法确定细胞内EBERs的表达后，对其在培养中的细胞生长特性和软琼脂中的细胞克隆形成进行了观察。[结果]在培养中大量表达EBERs的MDCK细胞之间丧失了细胞间的相互连接，细胞呈纺锤状，并在软琼脂中形成大量细胞克隆。而非表达EBERs的MDCK细胞的对照组中没有发现细胞生长特性的改变和大量细胞克隆形成。[结论]EBERs的大量表达可以造成MDCK细胞的分散活性和基质非依赖性。提示E-BERs在上皮细胞株MDCK细胞中有癌化作用，从而推论EBERs在同样源于上皮细胞的鼻咽癌中也可能有癌化作用。另外，表达EBERs的MDCK细胞的建立为进一步研究EBERs的作用提供了一个有用的上皮细胞模型。

Hepatocyte transformation and tumor development induced by hepatitis C virus NS3 c-terminal deleted protein

AIM: To study the effect of hepatitis C virus nonstructural protein 3 c-terminal deleted protein (HCV NS3-5') on hepatocyte transformation and tumor development.METHODS: QSG7701 cells were transfected with plasmid pRcHCNS3-5' (expressing HCV NS3 c-terminal deleted protein) by lipofectamine and selected in G418. The expression of HCV NS3 gene and protein was determined by PCR and immunohistochemistry respectively. Biological behavior of transfected cells was observed through cell proliferation assay, anchorage-independent growth and tumor development in nude mice. The expression of HCV NS3 and c-mycproteins in the induced tumor was evaluated by immunohistochemistry.RESULTS: HCV NS3 was strongly expressed in QSG7701 cells transfected with plasmid pRcHCNS3-5' and the positive signal was located in cytoplasm. Cell proliferation assay showed that the population doubling time in pRcHCNS3-5' transfected cells was much shorter than that in pRcCMV and nontransfected cells (24 h, 26 h, 28 h respectively). The cloning ratio of cells transfected with pRcHCNS3-5; pRcCMV and nontransfected cells was 33 %, 1.46 %, 1.11%, respectively,the former one was higher than that in the rest two groups (P＜0.01). Tumor development was seen in nude mice inoculated with pRcHCNS3-5' transfected cells after 15 days.HE staining showed its feature of hepatocarcinoma, and immunohistochemistry confirmed the expressions of HCV NS3and c-mycproteins in tumor tissue. The positive control group inoculated with HepG2 also showed tumor development, while no tumor developed in the nude mice injected with pRcCMV and non-transfected cells after 40 days.CONCLUSION: 1.HCV NS3 c-terminal deleted protein has transforming and oncogenic potential. 2. Human liver cell line QSG7701 may be used as a good model to study HCV NS3 pathogenesis.

Transfection of colorectal cancer cells with chemokine MCP-3(monocyte chemotactic protein-3)gene retards tumor growth and inhibits tumor metastasis

AIM: To evaluate the possibility of the induction of antitumor immune response by transfecting the colorectal cancer cells with chemokine MCP-3 gene. METHODS: Mouse MCP-3 gene was transduced into mouse colorectal cancer cells CMT93 by using of Liposome. G418-resistant clones were selected and the MCP-3 mRNA expression was detected by RT-PCR. The chemotactic activity of MCP-3 in the cell culture supernatant was detected by Chemotaxis assay. The tumorigenicity of wild type CMT93 and CMT93 gene transfectants were detected by in vivo experiments. The immune cell infiltrations in tumor tissue and tumor metastasis were detected histopathologically.RESULTS: MCP-3 mRNA expression was detected by RTPCR in gene-transfected cells (CMT93/MCP-3), but not in control groups. And MCP-3 secreted in the cell culture supernatant possessed chemotatic activity. The results from in vivo experiments showed that the tumorigenicity of CMT93/MCP-3 had not decreased, but the tumors derived from CMT93/MCP-3 cells grew more slowly than those from CMT93 cells (1.021±0.253) cm2 VS(1.769±0.371) cm2,P＜0.05) or CMT93/mock cells (1.021±0.253) cm2 VS(1.680±0.643)cm2, P＜0.05). Histophathological results showed few immune cells infiltrating in the tumor tissue derived from the controls. In the tumor tissue derived from CMT93/MCP-3, infiltrating immune cells increased. In addition, no tumor metastasis was found in all mice inoculated with CMT93/MCP-3 tumor cells. But all mice had tumor metastasis in CMT93 controls and 4 in 5 mice had tumor metastasis in CMT93/mock controls.CONCLUSION: The results suggested that the transfection of chemokine MCP-3 gene could promote the induction of anti-colorectal cancer immunity, but the tumor growth could not be inhibited completely by merely MCP-3 gene transfection.

Expression of tumor related gene NAG6 in gastric cancer and restriction fragment length polymorphism analysis

AIM: NAG6 gene is a novel tumor related gene identified recently. This study was designed to examine the expression of this gene in gastric cancer and corresponding normal tissues, and to investigate its role in the occurrence and development of gastric cancer, also to study if the genetic structure of NAG6 was altered in gastric cancer.METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), Northern blot analysis and dot hybridization were used to compare the expression level of NAG6 gene in 42 cases of gastric cancer tissues with their corresponding normal tissues of the same patients respectively. In addition,restriction fragment length polymorphism (RFLP) analysis was adopted to study if the genetic structure of NAG6 was altered in gastric carcinomas.RESULTS: The expression of NAG6 in 57.1% gastric cancer tissues (25/42) was absent by RT-PCR analysis. The down-regulation rate of NAG6 in gastric cancer tissues was significantly higher than that in corresponding normal tissues(P<0.01). However no correlation between the down-regulation of NAG6 and lymph-node and/or distance metastasis was found in this study (P>0.05). Dot hybridization confirmed the results of RT-PCR. Furthermore,the results of EcoRI RFLP analysis of NAG6 gene demonstrated that 3 of 7 cases of gastric cancer showed loss of 5 kb fragment in comparison with their corresponding normal tissues.CONCLUSION: NAG6 gene is significantly down regulated in gastric cancer. The loss of genetic materials may be the cause of down-regulation of NAG6 expression. This seems to suggest that NAG6 may represent a candidate of putative tumor suppressor gene at 7q31-32 loci associated with gastric carcinoma. The down-regulation of this gene may play a role in occurrence and development of this disease, however it may not be associated with lymph node and/or distance metastasis.

CONSTRUCTION OF HU-PBL/SCID CHIMERAS AND DEVELOPMENTOF EBV-RELATED LYMPHOMAS

Objective To construct hu-PBL/SCID chimeras and to investigate the development of lymphoma and oncogenicity of the Epstein-Barr virus (EBV). Methods Human peripheral blood lymphocytes (PBLs) were isolated from healthy adult donors and transplanted intraperitoneally into severe combined immunodeficient(SCID) mice. Mice with hu-PBL engraftment from healthy EBV seronegative donors were injected intraperitoneally with EBV-containing supernatant from suspension culture of B95-8 cell line (active infection), whereas mice receiving lymphocytes from healthy EBV seropositive donors were not re-infected with B95-8 derived EBV (latent infection). Pathological examination and molecular analysis were performed on experime-ntal animals and induced neoplasms. Results In the early stage of this experiment, 12 mice died of acute graft-versus-host disease, mortality was 34.3% (12/35 mice) with an average life span of 17.5 days. In 19 survival hu-PBL/SCID chimeric recipients from 12 healthy donors, tumor incidence was 84.2% (16/19 mice). The average survival time of tumor-bearing mice was 65.5 days. EBV-related neoplasms in SCID mice were nodular tumors with aggressive and fatal features. Histological morphology of tumors exhibited diffuse large cell lymphomas. Immunohistochemistry revealed that LCA (CD45) and L26 (CD20) were positive, but both PS1 (CD3) and UCHL-1 (CD45RO) were negative, and EBV products ZEBRA, LMP1, and EBNA2 were expressed in a small number of tumor cells. EB virus particles were seen in the nuclei of some tumor cells by electron microscopy, and EBV DNA could be amplified in the tumor tissues by PCR. In situ hybridization indicated that the nuclei of tumor cells contained human-specific Alu sequence. Conclusions EBV-induced tumors were human B-cell malignant lymphomas. We obtained direct causative evidence dealing with EBV-associated tumor deriving from normal human cells.

HBV X PROTEIN(HBX) INTERACTS WITH GENERAL TRANSCRIPTION FACTOR TFIIB BOTH INVITRO AND IN VIVO

Objective.In order to demonstrate the binding of HBV X protein (HBX) with the general transcription factor TFIIB. Methods.In vitro glutathion S transferase (GST) resin Pull Down assay and Far Western Blotting assay, in vivo Co immunoprecipition assay were used. Results.The X199(51 99) domain of HBX is reponsible for HBX binding to TFIIB. While the d10 domain (125 295) of TFIIB is required for TFIIB binding to HBX. When the two basic amino acids(K) at position 178 and 189 of TFIIB were substituted by neutral amino acids(L), the binding of TFIIBK178L and K189L to HBX was siginificantly reduced. When the the basic amino acids were substituted by the acidic amino acids(E),the binding of TFIIB K178E and K189E to HBX were almost lost. In vitro results of HBX binding to TFIIB were further confirmed by in vivo co immunoprecipitation assay. Our results also indicated that the Woodchuck hepatitis virus X protein (WHX) interacts with TFIIB. Conclusion.These results suggested that the communication between HBX and general transcription factor TFIIB is one of the mechanisms which account for its transcriptional transactivation.

TNFα as a Physiological Regulator of Hematopoietic Progenitor Cells:Increase of Early Hematopoietic Progenitor Cells in TNFR55--/-mice in vivo and Potent Inhibition of Progenitor Cell Proliferation by TNFα in Vitro

Murine lineage marker (Lin)^--Sca-1^+c-kit^+and Lin^-Sca-1^-c-kit^+ cens represent the primitive hematopoietic stem cells (HSC)and coommitted hematopoietic progenitor cells(HPC),respectively. The number of Lin^-Sca-1^+c-kit^+HSCs in bone marrow was significantly increased (2.9folds) in tumor necrosis factor(TNF)-receptor-55(TNF-R55)-deficient mice compared with that of wildtype ones without marked change in cellularity of bone marrow.

Effect of Human Interleukin-8(IL-8) on Highighly Purified Murine Hematopoietic Progenitor Cells

Gene targeting of mouse homolog of interleukin-8 receptor (IL-8R) resulted in the increase of myeloid cells in the bone marrow and peripheral blood, sugesting a potential ro le of IL-8 mregulating hematopoiesis of the myeloid lineage.In order to clarify the functions of IL-8 inhematopoiesis,

Proteomic Comparison of Two-Dimensional Gel Electrophoresis Profiles from Human Lung Squamous Carcinoma and Normal Bronchial Epithelial Tissues

Differential proteome profiles of human lung squamous carcinoma tissue compared to paired tumor-adjacent normal bronchial epithelial tissue were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorp-tion/ionization time of flight mass spectrometry (MALDI-TOF-MS). The results showed that well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained under the condition of 0.75-mg protein-load. The average deviation of spot position was 0.733±0.101 mm in IEF direction, and 0.925±0.207 mm in SDS-PAGE direction. For tumor tissue, a total of 1241±88 spots were detected, 987±65 spots were matched with an average matching rate of 79.5%. For control, a total of 1190±72 spots were detected, and 875±48 spots were matched with an average matching rate of 73.5%. A total of 864±34 spots were matched between tumors and controls. Forty-three differential proteins were characterized: some proteins were related to oncogenes, and others involved in the regulation of cell cycle and signal transduc-tion. It is suggested that the differential proteomic approach is valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis. These data will be used to establish human lung cancer proteome database to further study human lung squamous carcinoma.

Regulation of c-Jun/JunB heterodimers mediated by Epstein-Barr virus encoded latent membrane protein 1 on p16

Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) is considered as the major oncogenic protein of EBV encoded proteins, which could transactivate many transcription factors including activator protein 1 (AP-1). Transcription factor plays its role in biological effects through binding to the target gene promoter,transactivating the transcription of target gene, regulating the target gene expression, etc. Recently, we found that LMP1 could mediate a new heterodimer form of c-Jun and JunB, which could bind to AP1 DNA sequence. In this report,we confirmed p16 as a putative target gene of c-Jun/JunB using bioinformatics. We used Tet-on-LMP1 HNE2 cell line as a cell model, which is a dual-stable LMP1 integrated HNE2 cell line and the expression of LMP1 could be regu-lated by the Tet system, and we wanted to explore whether c-Jun/JunB heterodimers mediated by LMP1 could regulate p16. Data demonstrated that c-Jun/Jun B heterodimers me-diated by LMP1 downregulated both the promoter activity and p16 expression, and accelerated the cell cycle progression. These findings established a new direct connection between the AP-I singnal pathway and the cell cycle, and provided a new model for the carcinogenesis mechanism.

PRODUCTION AND CHARACTERIZATION OF CC-1 SECOND GENERATION MONOCLONAL ANTIBODIES BY INTRASPLENIC DEPOSITED IMMUNIZATION WITH MINUTE AMOUNTS OF ANTIGEN

McAb CC-1 （IgM） could react with 45％ of the human colon carcinomas, its antigenic determinant is extended LeYOur purpose of producing CC-1 second generation McAbs is to select more effective McAbs in clinical application, and to find out the structure and excretion of glycolipid antigen so as to understand the mechanism of originality and development of tumors?