Hepatitis B virus X promotes HepG2 cell cycle progression and growth via downregulation expression of p16 protein / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effects and related mechanisms of hepatitis B virus X (HBx) protein on cell cycle and growth in hepatocellular carcinoma.</p><p><b>METHODS</b>A human hepatocyte HepG2 cell line stably expressing a green fluorescent protein (GFP)-tagged HBx (HepG2/GFP-HBx cells) was used for the experiment, and HepG2 parental and HepG2/GFP cells was used as the controls. Effect of HBx on cell growth was evaluated by the MTT cell proliferation assay and on cell cycle progression by flow cytometry analysis of cells with or without treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR; 5 pmol/L). Effect of HBx expression on promoter methylation status of the p16INK4A tumor-suppressor gene was detected by methylation-specific polymerase chain reaction and on p16 protein level was analyzed with western blotting.</p><p><b>RESULTS</b>The HepG2/GFP-HBx cells showed significantly higher cell proliferation at 72 hrs of culture (3.225+/-0.038 A490) than either control (HepG2: 2.012+/-0.022 A490, t = -46.86, P less than 0.001; HepG2/GFP: 2.038+/-0.029 A490, t = 42.51, P less than 0.001). The HepG2/GFP-HBx cells also showed significantly lower proportion of cells in the G0/G1 phase (16.45%+/-0.45%) than either control (HepG2: 44.81%+/-1.36%, t = -34.202, P less than 0.001; HepG2/GFP: 42.76%+/-1.58%, t = -28.88, P less than 0.001). However, 5-Aza-CdR treatment did lead to a significant amount of HepG2/GFP-HBx cells being arrested in the G0/G1 phase (33.25%+/-0.79%, t = 31.85, P less than 0.001). The p16INK4A promoter was methylated in the HepG2/GFP-HBx cells, and became demethylation after treatment with 5-Aza-CdR. However, no methylation of p16INK4A promoter was observed in both HepG2 and HepG2/GFP cells. The p16 protein level was significantly lower in the HepG2/GFP-HBx (vs. HepG2 and HepG2/GFP cells) and this level increased after treatment with 5-Aza-CdR.</p><p><b>CONCLUSION</b>HBx protein promotes hepatocellular carcinoma cell cycle progression and growth by shortening the G0/G1 phase, and the underlying mechanism may involve inducing p16INK4A promoter methylation and downregulating p16 protein expression.</p>

The comparative study on the regulation of apoptosis by hepatitis B virus X protein between B and C genotype / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the apoptosis regulation on hepatoma cells by HBx between genotype B and C.</p><p><b>METHODS</b>Genotype B and C HBx gene fragments were amplified and inserted into green fluorescent protein (GFP) eukaryotic expression vector pEGFP-C1 to construct recombinant pGFP-XB and pGFP-XC. The pEGFP-C1, pGFP-XB and pGFP-XC were introduced into Bel-7402 cells by Fugene HD to obtain Bel-7402 cells expressing GFP. The transcription and expression of HBx gene were demonstrated by RT-PCR and Western Blot analysis. Bel-7402, Bel-7402/GFP, Bel-7402/GFP-XB, Bel-7402/GFP-XC cells were treated with adriamycin (2.5 microg/ml), and the apoptosis of the cells was determined by trypan blue exclusion, and flow cytometry analysis.</p><p><b>RESULTS</b>RT-PCR and Western Blot analysis showed that HBx genes of genotypes B and C were transcribed and expressed in Bel-7402/GFP-XB, Bel-7402/GFP-XC cells. Trypan blue exclusion showed adriamycin induced time-dependent cell death in Bel-7402, Bel-7402/GFP cells while no significant cell death was observed in Bel-7402/GFP-XB, Bel-7402/GFP-XC cells. Flow cytometry analysis indicated that no significant differences of apoptosis rates of Bel-7402/GFP-XB (3.87%) and of Bel7402/GFP-XC (4.01%) were observed (P > 0.05), moreover, no significant differences of Bel-7402/ GFP-XB (3.87%), Be17402/GFP-XC (4.01%) and of the untreated cells. Apoptosis rates in Bel-7402/GFP-XB (3.87%), Bel-7402/GFP-XC (4.01%) cells were significantly lower than those in Bel-7402 (27.05%) and Bel-7402/GFP (29.14%) cells at 48 hours after the adriamycin treatment (P < 0.01).</p><p><b>CONCLUSIONS</b>Bel-7402 cell lines expressing GFP, GFP-XB and GFP-XC fusion proteins were successfully established. HBV X protein blocks adriamycin-induced apoptosis of Bel-7402 cells. There is no difference between HBx of genotype B and C in inhibiting apoptosis induced by adriamycin.</p>

Small interfering RNA targeting to hepatitis B virus X gene and 5-aza-2'-deoxycytidineon inhibited growth of the subcutaneous implanted tumor of hepatocellular carcinoma in nude mice / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the anti-tumor effect of small interfering RNA targeting to HBV X gene (X-siRNA) and 5-aza-2'-deoxycytidine (5-aza-dC) on HBV-related hepatocellular carcinoma.</p><p><b>METHODS</b>X-siRNA and control siRNA were synthesized. HepG2/GFP-HBx cells were treated with X-siRNA, and the levels of HBV X mRNA were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Nude mice were inoculated with HepG2/GFP and HepG2/GFP-HBx cells subcutaneous respectively to establish implant models of hepatocellular carcinoma, and were treated with X-siRNA, 5-aza-dC alone or in combination, and tumor growth was observed. The methylation of p16 gene promoter was detected by methylation specific polymerase chain reaction (MSP).</p><p><b>RESULTS</b>RT-PCR showed the expression of HBV X mRNA in HepG2/GFP-HBx cells was inhibited markedly by X-siRNA. The nude mice experiment showed that the gross tumor volume was much bigger in HepG2/GFP-HBx group than that in HepG2/GFP group (P < 0.05). The growth of palpable tumors in X-siRNA or 5-aza-dC treatment group notably decreased (P < 0.05). MSP analysis showed that p16 gene methylation was observed in HepG2/ GFP-HBx-caused palpable tumors, while no methylation was detected in HepG2/GFP group. However, after treatment with X-siRNA or 5-aza-dC, p16 gene methylation reduced.</p><p><b>CONCLUSIONS</b>HBV X-siRNA and methylation inhibitor can inhibit the growth of hepatoma cells via reversing p16 methylation.</p>

Hepatitis B virus X protein suppresses adriamycin-induced apoptosis of hepatocellular carcinoma cells and expression of p53 and PTEN / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of hepatitis B virus X protein (HBx) on adriamycin-induced apoptosis of hepatocellular carcinoma cells and the expressions of p53 and PTEN.</p><p><b>METHODS</b>HepG2, HepG2/GFP, and HepG2/GFP-HBx cells were treated with adriamycin (2.5 microg/ml), and the apoptotic cell death was determined by observing the morphological changes and flow cytometry. The expressions of p53 and PTEN mRNA in the 3 cells were detected by RT-PCR, and the expressions of p53 and PTEN protein were analyzed by Western blotting.</p><p><b>RESULTS</b>Adriamycin induced significant cell death in HepG2 and HepG2/GFP cells, which became rounded, shrunk, and detached after the treatment; but no significant cell death occurred in HepG2/GFP-HBx cells. Flow cytometry analysis showed that the apoptotic rate was significantly lower in HepG2/GFP-HBx cells (3.94%) than in HepG2 (59.03%) and HepG2/GFP cells (61.38%) at 36 h after the treatment (P<0.001), while no significant difference was observed between HepG2/GFP-HBx (3.94%) and the control cells (2.12%, 2.78%, and 2.55%) (P>0.05). RT-PCR showed lowered expression of PTEN mRNA in HepG2/GFP-HBx cells as compared to that in HepG2 and HepG2/GFP cells, while no significant difference was noted in p53 mRNA. Western blot analysis showed that PTEN protein decreased while p53 protein remain unchanged in HepG2/GFP-HBx cells.</p><p><b>CONCLUSION</b>HBx suppresses adriamycin-induced apoptosis of HepG2 cells and PTEN expression. The inhibitory effect of HBx on the cell apoptosis may be related to the inhibition of p53-PTEN pathway.</p>

Efficacy of the 96-week adefovir dipivoxil therapy in patients with chronic hepatitis B / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the efficacy of the 96-week antiviral therapy with adefovir dipivoxil in patients with chronic hepatitis B.</p><p><b>METHODS</b>80 patients with chronic hepatitis B received the antiviral therapy of adefovir dipivoxil (ADV, 10 mg/d). At the 12th week, 19 cases without early viral response (EVR, HBV DNA drop < 2 log10copies/ml) switched to the therapy of other nucleoside analogues. Aminotransferase (ALT) normalization, HBV DNA negative, HBeAg loss and HBeAg seroconvertion were accessed at the 96th week.</p><p><b>RESULTS</b>At week 96, ALT normalization and HBV DNA negative in 61 patients with ADV therapy were 85.25% (52/61) and 95.08% (58/61); and HBeAg loss and HBeAg seroconvertion were 52.52% (17/33) and 42.42% (14/33) respectively. While for the other 19 patients switching to other nucleoside analogues, ALT normalization and HBV DNA negative came to 57.89% (11/19) and 68.42% (13/19). Both HBeAg loss and HBeAg seroconvertion were 58.33% (7/12).</p><p><b>CONCLUSION</b>Long term ADV antiviral therapy is effective to inhibit HBV DNA replications and benefits patients with chronic hepatits B. Switching to another nucleoside analogue is an optimal alternative if there is no EVR at week 12 in ADV therapy.</p>

The antiviral treatment impacts on clinical outcomes of renal transplantation recipients with hepatitis B virus infection / 中华实验和临床病毒学杂志

<p><b>UNLABELLED</b>OBJECTIVE; To investigate the clinic outcomes and the efficacy of antiviral treatment in renal transplantation recipients with hepatitis B viral serum markers positive.</p><p><b>METHODS</b>32 renal transplantation recipients with hepatitis B viral serum markers positive were enrolled. 23 patients in antiviral treatment group have received the lamivudine (19 cases), enticavir (2 cases) and adefovir (1 case). Another 9 patients have not received the antiviral treatment and were as the control group.</p><p><b>RESULTS</b>The biochemical response rate in antiviral treatment group and control group is 82.60% and 22.22%, respectively. 19 of 23 (82.60%) patients in treatment group survived and 1 of 9 (11.11%) patients in control group survived (P < 0.05). 20 of 23 (86.95%) patients in treatment group have the reduction of HBV DNA more than 2 log copies/ml or maintain less than 5 log copies/ml, while 1 of 9 (11.11%) patients in control group has the HBV DNA maintain less than 5 log copies/ml (P < 0.05). The virology rebound was observed in 6 of 19 (31.58%) patients with lamivudine treatment. 2 of them shift to enticavir treatment and 1 of them add adefovir treatment. The three patients survived. Other 3 patients die of liver function failure.</p><p><b>CONCLUSION</b>The antiviral could improve the survival in renal transplantation recipients with hepatitis B viral serum markers positive. When the virology rebound occurs, the add-on with adefovir or the shift to enticavir could be a rescue treatment.</p>

The relationship between the levels of HBV DNA loads and both the clinical characteristics and 48-week prognosis in patients with decompensated cirrhosis due to hepatitis B / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluate the relationship between the levels of HBV DNA loads and both the clinical characteristics and 48-week prognosis in patients with decompensated cirrhosis due to hepatitis B.</p><p><b>METHODS</b>One hundred and forty-three patients with decompensated cirrhosis of hepatitis B virus infection were divided into low level HBV DNA group [HBV DNA < 10(5) copies/ml = (46 cases) and high-level HBV-DNA group (HBV DNA > or = 10(5) copies/ml) (97 cases)]. 21 cases in low-level group and 52 cases in high-level group treated with nucleoside analog.</p><p><b>RESULTS</b>There was no significant difference between the two groups on the demography and the baseline in ALT, ALB, TBil, CHE before treatment, while in AST and HBeAg were statistically different. At 48-week, there was no significant difference between the two groups on the liver function. The mortality rate in low-level group was similar to that in high level group. In the low-level HBV DNA patients, hepatocellular carcinoma, spontaneous peritonitis and gastrointestinal hemorrhage were higer than that in the high-level HBV DNA patients.</p><p><b>CONCLUSION</b>In patients with decompensated cirrhosis due to hepatitis B, those who were in low-level HBV DNA had not got better than that in high-level HBV DNA, which indicated that earlier treatment was also needed in low-level HBV DNA patients.</p>

Hepatitis B virus X protein suppressing adriamycin-induced apoptosis of HepG2 cells / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the effect of hepatitis B virus X protein (HBx) on adriamycin-induced apoptosis of hepatocellular carcinoma cells.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Venous diethylene glycol poisoning in patients with preexisting severe liver disease:a clinical analysis / 中华传染病杂志

Objective To describe the clinical features of venous diethylene glycol poisoning and to identify factors correlating with such kind of poisoning.Methods Retrospective chart review was performed to analyze the epidemiology,clinical presentation,hepatorenal functions,bemodynam- ics and pathological characteristics of 64 patients with severe liver diseases who received intravenous diethylene glycol.Comparative analyses of correlating factors and causes of poisoning were based on the presence or absence of poisoning.Results Fifteen cases of poisoning were reported.After a 5 day incubation period,all poisoned patients displayed acute renal failure and 11 cases with digestive tract symptoms and(or) symptom exacerbations were noted.Neurological system impairment was observed in 10 cases after 2 weeks.Metabolic acidosis developed in 13 cases.Poisoned patients exhibited signif- icantly lower red blood celI(RBC)[(2.32?0.76)?10~(12)/L],hemoglobin(Hb) [(79.5?23.6)g/L] value and higher white blood cell(WBC)[(9.78?3.75)?10~9/L] count.Renal biopsy of poisoned patients revealed acute tubular necrosis and interstitial nephritis.Twelve poisoned patients died.Sig nifieant differences were found between groups regarding preexisting severe hepatitis,ascites,renal disease and diuretic therapy.Prior to diethylene glycol injections,mean values of neutrophil,blood urea nitrogen(BUN),creatinine(Cr) and calcium and phosphorousions differed significantly between groups.Conclusions Features of venous diethylene glycol poisoning include oliguric acute renal fail- ure,metabolic acidosis,digestive symptoms,nervous system impairment and a high probability of anemia and WBC proliferation.Mortality is high.Correlative factors include preexisting severe liver disease,renal disease and infection.