[Effects of hepatitis B virus X gene on p21(WAF1) expression through p53-dependent and p53-independent pathways].
OBJECTIVE
It was reported that the hepatitis B virus X could inhibit the function of p53 and have contrary effects on p21(WAF1), a downstream gene of p53, but the mechanism is not clear up to now. So this study was designed to investigate HBx's effect on p21(WAF1) gene.


METHODS
After co-transfection of sense or antisense wild type p53 gene (wtp53) with HBx into SMMC-7721 and HBx alone transfection into other hepatoma cell lines with different endogenous status of p53, we evaluated the luciferase expression level under the p21(WAF1) promoter by detecting the luciferase activity and alteration of cell cycle in these transfected cell lines by flow cytometry. Expressions of p53 and p21 (WAF1) in hepatoma cells after transfected with HBx were also estimated by Western blot analysis.


RESULTS
The luciferase activity (1.007+/-0.098) in SMMC-7721 cells cotransfected with HBx and sense-wtp53 was higher than that in cells with wtp53 gene alone (0.490+/-0.012, P< 0.05), and also was depressed in the other experimental groups transfected with HBx (P< 0.05). Western blot analysis showed that after transfected with HBx, p53 expression was elevated in all hepatoma cell lines with different endogenous status of p53, and the expression of p21(WAF1) and luciferase activity under the p21(WAF1) promoter (0.053+/-0.010 vs. 0.094+/-0.013, P< 0.05) were both decreased in SMMC-7721 cells with low expression of p53, but relatively increased in HepG2 cell line with high expression of p53 (1.252+/-0.052 vs. 0.767+/-0.031, P< 0.05). Flow cytometry showed that fewer SMMC-7721 (42.31%) and Hep3B (36.96%) cells were arrested in G(0)/G(1) phase in transiently transfected HBx groups than in the control groups (47.10% and 42.90%), which was the opposite case in HepG2 cell line (63.62% vs. 57.42%). Moreover, after stably transfected with pcDNA3HBx, HepG2 cells reduced in G(0)/G(1) phase in compared with the control group (57.31% vs 61.49%).


CONCLUSIONS
HBx may not only increase the expression of p21(WAF1) by introducing the accumulation of p53 in cytoplasm but also inhibit the transcriptional activity of p21(WAF1) promoter in a p53-independent manner.
Lin
Jing
J
Department of Pathology, Changhai Hospital, The Second Military Medical University, Shanghai, 200433, P.R.China.
Zhu
Ming-Hua
MH
Qu
Jian-Hui
JH
Li
Fang-Mei
FM
Ni
Can-Rong
CR
D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000096:biosynthesis* / Q000235:genetics; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016373:DNA, Antisense / Q000235:genetics; D006511:Hepatitis B Antigens / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D008156:Luciferases / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000235:genetics*; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis* / Q000235:genetics; D054334:Viral Regulatory and Accessory Proteins
C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016373:DNA, Antisense; D006511:Hepatitis B Antigens; D015534:Trans-Activators; D016159:Tumor Suppressor Protein p53; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D008156:Luciferases