1.Hepatitis B virus X protein suppressing adriamycin-induced apoptosis of HepG2 cells.
Hong-mei FAN ; Lin YANG ; Qi-feng XIE ; Xiao-yan HAN ; Meng WU ; Fu-cheng ZHANG ; Chun-lan YAO ; Gang LI ; Zhi-liang GAO
Chinese Journal of Hepatology 2008;16(1):25-28
OBJECTIVESTo investigate the effect of hepatitis B virus X protein (HBx) on adriamycin-induced apoptosis of hepatocellular carcinoma cells.
METHODSHBx gene fragment was amplified from subtype adr HBV plasmid by PCR, and inserted into Hind III and Kpn I sites of green fluorescent protein (GFP) eukaryotic expression vector pEGFP-C1 to construct recombinant pGFP/HBx. The pEGFP-C1 and pGFP-HBx were introduced into HepG2 cells by Lipofectamine 2000 to obtain HepG2 cells expressing GFP. GFP-HBx fusion protein was selected using G418. The expression of HBx gene was demonstrated by RT-PCR analysis. HepG2, HepG2/GFP and HepG2/GFP-HBx cells were treated with adriamycin (2.5 microg/ml), and apoptosis of the cells was determined by their morphological changes, trypan blue exclusion, and flow cytometry analysis.
RESULTSUnder a fluorescence microscope, visible expression of GFP and GFP-HBx fusion proteins were observed in HepG2/GFP and HepG2/GFP-HBx cells, even after growing over 70 generations. RT-PCR analysis showed that HBx gene was expressed in HepG2/GFP-HBx cells. Trypan blue exclusion showed adriamycin induced time-dependent cell death in HepG2 and HepG2/GFP cells while no significant cell death was observed in HepG2/GFP-HBx cells. Flow cytometry analysis showed that apoptosis rates in HepG2/GFP-HBx (3.94%) cells were significantly lower than those in HepG2 (59.03%) and HepG2/GFP cells (61.38%) at 36 hours after the adriamycin treatment (P < 0.01). No significant differences of apoptosis rates of HepG2/GFP-HBx (3.94%) and of the untreated cells (2.12%, 2.78%, 2.55%) (P > 0.05) were observed.
CONCLUSIONA HepG2 cell line expressing GFP and GFP-HBx fusion proteins was successfully established. HBV X protein blocks adriamycin-induced apoptosis of these HepG2 cells.
Apoptosis ; drug effects ; Doxorubicin ; pharmacology ; Hep G2 Cells ; Humans ; Plasmids ; Trans-Activators ; genetics
2.Expression of CIITA Gene in five human cell lines and its significance.
Wen-Li ZUO ; Yong-Ping SONG ; Rong GUO
Journal of Experimental Hematology 2008;16(5):1158-1161
The objective of study was to investigate the relationship between expressions of CIITA and MHC molecules in five human cell lines. The expressions of MHC molecules and CIITA protein were detected by Western blot, immunohistochemistry and flow cytometry. The expression of CIITA gene was measured by RT-PCR. The results indicated that the expression of MHC-II molecules in 5 human cell lines was consistent with expression of CIITA. The cell lines constitutively expressed CIITA also expressed MHC-II molecules, the expression of MHC-II molecules in cell lines expressed CIITA after induction with IFN-gamma also recovered; the cell lines unexpressed CIITA after induction with IFN-gamma did not respond to IFN-gamma-promoting expression of MHC-II molecules. It is concluded that some cell lines cannot express MHC-II molecules which may be related with deficiency of CIITA expression. It suggest that CIITA participates in regulation of MHC-II molecule expression, which may plays a certain role in escape from carcinogenesis under surveillance of immune system.
Cell Line
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Genes, MHC Class II
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Humans
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Interferon-gamma
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pharmacology
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Nuclear Proteins
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metabolism
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Trans-Activators
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metabolism
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Tumor Cells, Cultured
4.Hepatic cell apoptosis was triggerred by HBx accumulation and independent on verapamil.
Haiping WANG ; Xiaoping CHEN ; Xiangjun BAI
Journal of Huazhong University of Science and Technology (Medical Sciences) 2004;24(3):281-283
In order to study the roles of HBx and calcium inhibitor verapamil in apoptosis of human normal hepatic cells, L02-off, a pTet-off stably integrated human hepatic cell line was established, in which HBx expression was tightly induced by Doxycycline. The effect of different amounts of HBx and verapamil on apoptosis of human normal hepatic cells was detected. The study showed that apoptosis was triggered by accumulation of intracellular HBx, while verapamil had no effects on the apoptotic process. It was concluded that apoptosis mediated by HBx was dose-dependent but calcium-independent.
Apoptosis
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drug effects
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Calcium
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pharmacology
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Calcium Channel Blockers
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pharmacology
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Cells, Cultured
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Dose-Response Relationship, Drug
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Hepatitis B Antigens
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pharmacology
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Hepatocytes
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cytology
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Humans
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Trans-Activators
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pharmacology
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Verapamil
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pharmacology
5.Hepatic cell apoptosis was triggerred by HBx accumulation and independent on verapamil.
Haiping, WANG ; Xiaoping, CHEN ; Xiangjun, BAI
Journal of Huazhong University of Science and Technology (Medical Sciences) 2004;24(3):281-3
In order to study the roles of HBx and calcium inhibitor verapamil in apoptosis of human normal hepatic cells, L02-off, a pTet-off stably integrated human hepatic cell line was established, in which HBx expression was tightly induced by Doxycycline. The effect of different amounts of HBx and verapamil on apoptosis of human normal hepatic cells was detected. The study showed that apoptosis was triggered by accumulation of intracellular HBx, while verapamil had no effects on the apoptotic process. It was concluded that apoptosis mediated by HBx was dose-dependent but calcium-independent.
Apoptosis/*drug effects
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Calcium/pharmacology
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Calcium Channel Blockers/pharmacology
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Cells, Cultured
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Dose-Response Relationship, Drug
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Hepatitis B Antigens/pharmacology
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Hepatocytes/*cytology
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Trans-Activators/*pharmacology
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Verapamil/*pharmacology
6.Study of effects of HBV X gene and As2O3 on expression and activity of p53 in HepG2 cells with shRNA.
Xing-E HE ; Jian-Hua LEI ; Xu YANG ; Wen-Long WANG ; Hong-Yu LUO ; Jun LIANG
Chinese Journal of Hepatology 2006;14(10):757-761
OBJECTIVETo delineate the effects of HBV X gene and of As(2)O(3) on p53 expression and activity in HepG2 cells by shRNA-mediated RNA interference (RNAi).
METHODSHepG2 cells and cells with stable expression of HBV X gene, HepG2-X, were treated with 2 micromol/L As(2)O(3), and the corresponding untreated cells were used as controls. Cell and nuclear lysates were extracted. Total level and the relative activity absorbance of p53 were detected by modified ELISA. HBV X gene sequence-specific shRNA expression vectors, Xi-S1 and Xi-S2, and sequence-unrelated control Xi-S3 were transfected into HepG2-X. The effect of As(2)O(3) on p53 expression and activity were retested.
RESULTSTotal p53 level was up-regulated and its relative activity ratio was enhanced by As(2)O(3) in HepG2 and HepG2-X cells. The total p53 level induced by As(2)O(3) was further up-regulated by HBX expression, while its relative activity was significantly suppressed. The suppression was removed after HBX expression was suppressed by shRNA.
CONCLUSIONAs(2)O(3) could up-regulate p53 expression and enhance its activity. shRNA-mediated RNA interference is conveniently being used in studies on the effect of HBV X gene expression on p53 expression and activity. HBV X expression could up-regulate p53 gene expression level induced by As(2)O(3), while it suppressed the activity of p53.
Apoptosis ; Arsenicals ; pharmacology ; Gene Expression ; Hep G2 Cells ; Hepatitis B virus ; genetics ; Humans ; Oxides ; pharmacology ; RNA, Small Interfering ; Trans-Activators ; genetics ; Tumor Suppressor Protein p53 ; genetics ; metabolism
8.Effects of HBV X gene and arsenic trioxide on the expression of p53 in cultured HepG2 cells.
Jian-hua LEI ; Xing-e HE ; Xu YANG ; Min ZHANG ; Jun LIAN ; Hong-Yu LUO ; Wen-long WANG
Chinese Medical Journal 2007;120(24):2181-2184
BACKGROUNDHepatitis B virus (HBV) X protein (HBx) and p53 could mutually down-regulate at transcriptional level and HBx could bind with p53 protein within its transactivation domain and inhibit the function of p53 protein. In recent years, effects of arsenic trioxide (As2O3) on the expression of p53 protein have been widely studied, while little is known about the activity of p53 protein. This study was undertaken to delineate the effect of HBV X gene and As2O3 on p53 protein expression (level and activity) in HepG2 cells by small hairpin RNA (shRNA)-mediated RNA interference (RNAi) technique.
METHODSCell line HepG2 and cells with stable expression of HBV X gene (HepG2-X) were treated with 2 micromol/L As2O3, with corresponding untreated cells serving as controls. Cell lysates and nuclear extracts were extracted. Total level and the relative activity of p53 protein were detected by modified enzyme-linked immunosorbent assay (ELISA). HBV X gene sequence-specific shRNA expression vector (pXi-1 and pXi-2) and sequence-unrelated control (pXi-3) were transfected into HepG2-X. Single cell clone with stable expression of shRNA was selected and exposed to propagating culture. The effect of As2O3 on p53 protein expression and activity was re-observed.
RESULTSTotal p53 protein level was up-regulated and its relative activity ratio was enhanced by As2O3 in HepG2 and HepG2-X cells. The total p53 protein level induced by As2O3 was up-regulated by HBV X gene expression, while its relative activity was significantly suppressed. The suppression was removed after HBV X gene expression was repressed by shRNA.
CONCLUSIONSAs2O3 up-regulates p53 protein expression and enhance its activity. HBV X up-regulates As2O3 induced-p53 protein expression while suppresses its activity.
Arsenicals ; pharmacology ; Cell Line, Tumor ; Enzyme-Linked Immunosorbent Assay ; Humans ; Oxides ; pharmacology ; RNA Interference ; Trans-Activators ; genetics ; Tumor Suppressor Protein p53 ; analysis
9.Intracellular Antibody Fragment Against Hepatitis B Virus X Protein Does Not Inhibit Viral Replication.
Young Hee JIN ; Seung Ho HONG ; Kyongmin KIM ; Ho Joon SHIN ; Sun PARK
Yonsei Medical Journal 2006;47(5):721-728
Replication of the hepatitis B virus is suppressed by deficiency of the X protein. Although several molecules that block cellular targets of X protein reduce the production of hepatitis B virus progeny, the effect of a specific inhibitor of X protein on viral replication has not been investigated. To block X protein specifically, we adopted an intracellular expression approach using H7 single chain variable fragment (H7scFv), an antibody fragment against X protein. We previously demonstrated that cytoplasmic expression of H7scFv inhibits X protein-induced tumorigenicity and transactivation. In this study, intracellular H7scFv expression inhibits reporter gene transactivation but not viral replication determined by endogenous hepatitis B virus polymerase activity assay and real-time PCR. Our findings imply that intracellular expression of antibody fragment against X protein may not be an alternative therapeutic modality for inhibition of hepatitis B virus replication.
Virus Replication/*drug effects
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Trans-Activators/*antagonists & inhibitors/immunology
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Immunoglobulin Variable Region/genetics/metabolism/*pharmacology
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Hepatitis B virus/*drug effects/physiology
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Hepatitis B e Antigens/metabolism
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Cell Line
10.Overexpression of CIITA in T Cells Aggravates Th2-Mediated Colitis in Mice.
Tae Woon KIM ; Hyo Jin PARK ; Eun Young CHOI ; Kyeong Cheon JUNG
Journal of Korean Medical Science 2006;21(5):877-882
The MHC class II transactivator (CIITA) is the master transcriptional regulator of genes involved in MHC class II restricted antigen presentation. Previously we suggested another role of CIITA in Th1/Th2 balance by demonstrating that forced expression of CIITA in murine T cells repressed Th1 immunity both in vitro and in vivo. However, the results were contradictory to the report that CIITA functioned to suppress the production of Th2 cytokine by CD4+T cells in CIITA deficient mice. In this study, we investigated the influence of constitutive expression of CIITA in T cells on Th2 immune response in vivo using murine experimental colitis model. In the dextran sodium sulfate-induced acute colitis, a disease involving innate immunity, CIITA transgenic mice and wild type control mice showed similar progression of the disease. However, the development of oxazolone-induced colitis, a colitis mediated by predominantly Th2 immune response, was aggravated in CIITA-transgenic mice. And, CD4+T cells from the mesenteric lymph node of CIITA-transgenic mice treated with oxazolone exhibited a high level of IL-4 secretion. Together, these data demonstrate that constitutive expression of CIITA in T cells skews immune response to Th2, resulting in aggravation of Th2-mediated colitis in vivo.
Trans-Activators/*physiology
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Th2 Cells/*immunology
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T-Lymphocytes/*metabolism
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Oxazolone/pharmacology
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Nuclear Proteins/*physiology
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Mice, Transgenic
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Mice, Inbred C57BL
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Mice
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Interleukin-4/biosynthesis
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Colitis/*etiology
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Animals