[Hepatitis B virus X protein suppressing adriamycin-induced apoptosis of HepG2 cells].
OBJECTIVE
To investigate the effect of hepatitis B virus X protein (HBx) on adriamycin-induced apoptosis of hepatocellular carcinoma cells.


METHODS
HBx 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.


RESULTS
Under 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.


CONCLUSIONS
A HepG2 cell line expressing GFP and GFP-HBx fusion proteins was successfully established. HBV X protein blocks adriamycin-induced apoptosis of these HepG2 cells.
Fan
Hong-mei
HM
Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University, Key Laboratory of Hepatology of Guangdong Province, Guangzhou 510630, China.
Yang
Lin
L
Xie
Qi-feng
QF
Han
Xiao-yan
XY
Wu
Meng
M
Zhang
Fu-cheng
FC
Yao
Chun-lan
CL
Li
Gang
G
Gao
Zhi-liang
ZL
D017209:Apoptosis / Q000187:drug effects*; D004317:Doxorubicin / Q000494:pharmacology*; D056945:Hep G2 Cells; D006801:Humans; D010957:Plasmids; D015534:Trans-Activators / Q000235:genetics*; D054334:Viral Regulatory and Accessory Proteins
D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D004317:Doxorubicin