Effects of HBV X gene and arsenic trioxide on the expression of p53 in cultured HepG2 cells.
BACKGROUND
Hepatitis 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.


METHODS
Cell 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.


RESULTS
Total 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.


CONCLUSIONS
As2O3 up-regulates p53 protein expression and enhance its activity. HBV X up-regulates As2O3 induced-p53 protein expression while suppresses its activity.
Lei
Jian-hua
JH
Liver Disease Research Center, Second Xiangya Hospital, Central South University, Changsha 410011, China.
He
Xing-e
XE
Yang
Xu
X
Zhang
Min
M
Lian
Jun
J
Luo
Hong-Yu
HY
Wang
Wen-long
WL
D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D010087:Oxides / Q000494:pharmacology*; D034622:RNA Interference; D015534:Trans-Activators / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000032:analysis*; D054334:Viral Regulatory and Accessory Proteins
D001152:Arsenicals; D010087:Oxides; D015534:Trans-Activators; D016159:Tumor Suppressor Protein p53; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D000077237:Arsenic Trioxide