Hepatitis B virus X protein inhibits hepatoma cell growth in vitro through p14(ARF)-dependent and p14(ARF)-independent pathways / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effects of hepatitis B virus X protein (HBx) on hepatoma cell growth through p14(ARF)-dependent and p14(ARF)-independent pathways.</p><p><b>METHODS</b>HBx and p14(ARF) were transfected either separately or in combination into HepG2 cells containing wt-p53 but not expressing p14(ARF). The cells were divided into 4 groups, namely pcDNA3 (control), pcDNA3HBx, pcDNA3p14(ARF), and pcDNA3HBx + pcDNA3p14(ARF) groups. Flow cytometry was used to examine the apoptosis rates and cell cycle progression of HepG2 cells in different groups. The expression of p14(ARF), MDM2, p53, and p21(WAF1) proteins were investigated by detecting the activity of p21(WAF1) promoter-luciferase and using Western blotting.</p><p><b>RESULTS</b>The apoptosis rates of HepG2 cells in pcDNA3HBx and pcDNA3p14(ARF) groups were significantly higher than that in the control group (14.11%, 13.72% vs 10.66%). Compared with the control group, pcDNA3HBx and pcDNA3p14(ARF) groups also showed significantly higher cell percentages arrested at G(0)/G(1) phase (63.62%, 61.75% vs 57.42%), luciferase activity of p21 promoter (1.25-/+0.05, 1.09-/+0.06 vs 0.77-/+0.03) and expressions of p53 and p21(WAF1). The cell apoptosis rate, percentage of cells in G(0)/G(1) phase and expression level of p14(ARF) were even higher in pcDNA3HBx+pcDNA3p14(ARF) group (18.61%, 66.74%, and 3.53-/+0.43, respectively) than in either p14(ARF) or HBx group.</p><p><b>CONCLUSION</b>HBx induces p53 expression through p14(ARF)-dependent and independent pathways to activate p21(WAF1) promoter, leading to G(0)/G(1) arrest and apoptosis of HepG2 cells.</p>

Influence of Smad4-independent pathway of transforming growth factor beta1 on the biological activity of pancreatic cancer cells / 中华病理学杂志

<p><b>OBJECTIVE</b>To study effects of the expression of transforming growth factor (TGF)-beta1 on the growth of Smad4-null pancreatic cancer cells.</p><p><b>METHODS</b>TGF-beta1 eukaryotic expression vector was transfected into pancreatic cancer cell line BxPC3. Effects of the expressison of TGF-beta1 was studied by growth curve analysis and flow cytometry. Cell motility was monitored by wound-healing assay. Western blot was used to estimate the expression level of p21(WAF/CLIP1), a cyclin-dependent kinase inhibitor.</p><p><b>RESULTS</b>Transfection of TGF-beta1 changed the morphology of BxPC3 into spindle shaped cells. The growth rate of BxPC3 began to decrease after the fourth day of TGF-beta1 transfection, compared with the control groups. Flow cytometry showed that the percentages of cells in the S phase were (27.53 +/- 0.02)%, (26.32 +/- 0.01)% and (17.01 +/- 0.03)% in naïve BxPC3, vector-control group and TGF-beta1 transfection group respectively. Lesser cells entered the S phase after TGF-beta1 transfection (P < 0.01), but no difference was seen between the BxPC3 and vector groups (P > 0.05). The expression of p21(WAF/CLIP1) increased upon the expression of TGF-beta1.</p><p><b>CONCLUSION</b>The Smad4-independent pathway of TGF-beta1 not only induces epithelial-mesenchymal transition in pancreatic cancer BxPC3, but also inhibits its growth through the up-regulation of p21(WAF/CLIP1).</p>

Hepatitis B virus and deletion of its COOH-terminal forty amino acids: proliferative impact on hepatoma cell line SMMC-7721 / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore the biological impact of 40 amino acid deletion at the C-terminal of hepatitis B virus X on the proliferation of hepatoma cells.</p><p><b>METHODS</b>Cells of SMMC-7721 hepatoma cell line were transfected with HBx and its derivative HBx3'-40, harboring the 40 amino acid deletion at the distal C-terminal region. Cell growth curve, colony formation in soft agar plate and tumorigenesis assay in nude mice were used to observe the alterations induced by the transfection of HBx and HBx3'-40. The expression level of PCNA in tumor cells was also investigated.</p><p><b>RESULTS</b>The growth rates of the cells transfected with HBx and HBx3'-40 were markedly increased as compared with that of the control group. The colony formation rates were enhanced in the cells transfected with HBx(48.7 +/- 8.1) and HBx3'-40 (82.8+/-6.0), comparing with the control (26.9 +/- 3.5) %. In the tumorigenic assay, the size and weight of tumors were significantly increased in the cells transfected with HBx (0.412 +/- 0.212, 0.395 +/- 0.159) % and HBx3'-40 (1.476 +/- 0.232, 0.987 +/- 0.279) %, as compared with the control group (0.051 +/- 0.024, 0.033 +/-0.004) %. The expression level of PCNA in tumors was increased in both HBx (59.00 +/- 2.58) % and HBx3'-40 (69.25 +/- 3.77) % transfected cells, comparing with the control (37.67 +/- 2.52) %. Overall, the cells transfected with HBx3'-40 demonstrated the highest proliferative capacity.</p><p><b>CONCLUSION</b>The deletion of 40 amino acids in the C-terminal of HBx is correlated with an enhanced proliferation of hepatoma cells and may play an important role in the malignant transformation of the liver.</p>

Expression of proteins in p53 (p14ARF-mdm2-p53-p21WAF/CIP1) pathway and their significance in exocrine pancreatic carcinoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the expression of p14(ARF), p53, mdm2 and p21(WAF/CIP1) proteins and their relationship in exocrine pancreatic carcinoma.</p><p><b>METHODS</b>Specimens of pancreatic carcinoma, adjacent non-neoplastic pancreatic tissue and pancreatic benign lesions were examined for p14(ARF), p53, mdm2 and p21(WAF/CIP1) protein expression by tissue microarray technique and immunohistochemistry.</p><p><b>RESULTS</b>The expression of p14(ARF), p53, mdm2 and p21(WAF/CIP1) proteins in pancreatic carcinoma were 35.3% (59/167), 57.5% (96/101), 64.1% (107/167) and 39.5% (66/167) respectively. The expression of p53 proteins was increased in pancreatic carcinoma (P < 0.01), while the expression of p14(ARF) and p21(WAF/CIP1) proteins was reduced (P < 0.05), as compared with that in non-neoplastic pancreatic tissue. p21(WAF/CIP1) protein expression in pancreatic carcinoma significantly correlated with the age of patients and perineural invasion (P < 0.05). p53 protein expression correlated significantly with tumor differentiation, lymph node metastasis and perineural invasion (P < 0.05). Mdm2 protein expression correlated significantly with tumor differentiation (P < 0.05), while p14(ARF) protein expression correlated significantly with the age of patients and metastasis (P < 0.05). There was also statistic correlation between the expression of these four genes (P < 0.05).</p><p><b>CONCLUSIONS</b>Overexpression of p53 and mdm2 and loss of p14(ARF) and p21(WAF/CIP1) expression may contribute to the pathogenesis of pancreatic carcinoma. These proteins play a critical role in cell cycle arrest and apoptosis after DNA damage through p14(ARF)-mdm2-p53-p21(WAF/CIP1) pathway. Detection of p53 and Mdm2 protein overexpression may be useful in evaluation of the aggressiveness of pancreatic carcinoma.</p>

Interaction effect of p53 with HBV in hepatoma cell line SMMC-7721 / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To study the interaction of hepatitis virus B (HBV) and tumor suppressor p53.</p><p><b>METHODS</b>Plasmid pCMVp53 was transfected or cotransfected with pCMVHBVa (wild type HBV) or PCMVHBVb (mutant type HBV) into the hepatoma cell line SMMC-7721 by lipofectamine. Apoptosis cells were labeled by annexin V-FITC and confirmed by flow cytometry. Reporter plasmids PG13-CAT or p21-luc were cotransfected respectively in each group to indicate transactivation activity of p53 and it's effect on p21 promoter. Western blot was performed to observe p53 expression in each group.</p><p><b>RESULTS</b>The group transfected by pCMVp53 alone exhibit higher luciferase activity and higher apoptosis rate, otherwise, p53 expression, enzyme activity of PG13-CAT or p21- luc and cell apoptosis rate were much higher in the group cotransfected by pCMVp53 and pCMVHBVa, but not in the other cotransfected group; HBV replication was enhanced in p53 cotransfected group.</p><p><b>CONCLUSION</b>p53 expression and effects could be enhanced by HBV and p53 had positive regulation effect on HBV replication.</p>

The role of hepatitis B virus X gene and p53 on hepatocellular carcinoma cell growth / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore the effects of hepatitis B virus X gene and p53 on hepatocellular growth.</p><p><b>METHODS</b>Two kinds of plasmids containing sense and antisense human wild p53 gene respectively were constructed. SMMU-7721 cells were transfected with HBx, sense-wtp53 antisense-wtp53 separately or cotransfected with either HBx and sense-wtp53 or HBx and antisense-wtp53. Flow cytometry was adopted to measure the apoptosis rates and the effects of HBx on cell cycle progression. The activity of p21(Waf1) promoter-luciferase construct was detected. Growth curves for SMMU-7721 stably transfected with pcDNA3 and pcDNA3HBx were analyzed.</p><p><b>RESULTS</b>After doxorubicin administration, HBx was noticed able to initiate apoptosis of the liver cells. The apoptosis rate was 5.32% in the pcDNA3 transfected and 12.66% in the pcDNA3HBx transfected groups respectively. HBx could also abrogate p53-mediated apoptosis. The apoptosis rate in groups transfected with pcDNA3, pcDNA3wtp53 and pcDNA3HBx + pcDNA3wtp53 was 5.32%, 11.72% and 4.67% respectively. In compared with the normal group, the number of cells in transiently HBx-expressed group and HBx-transfected group decreased 4.79% and 10.25% respectively. HBx inhibited the activity of p21(Waf1) promoter-luciferase constructed (P < 0.05) and promoted cell growth. The growth rate of HBx expression cells was faster.</p><p><b>CONCLUSION</b>Under DNA damage, HBx reduced expression of p21(Waf1) by repressing the activity of p53 protein, followed by disturbing the regulation of G(0)-G(1) cell cycle checkpoint, and promoted the growth rate of hepatoma cells.</p>

Immunohistochemical demonstration of cyclins A, B, D1, D3 and E in hepatocellular carcinomas using tissue microarrays / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the expression of five kinds of cyclins in hepatocellular carcinoma (HCC) and their association with degree of tumor differentiation, metastasis and infection of hepatitis B virus (HBV).</p><p><b>METHODS</b>The HCC tissue microarrays were composed of those from 273 cases of HCC tissues, 144 surrounding-tumor liver tissues and 10 normal liver tissues obtained from autopsy. The diameter of each specimens on tissue microarrays was 2.0 mm. Immunohistochemistry was used to detect the expression of cyclin A, cyclin B, cyclin D1, cyclin D3 and cyclin E on HCC tissue microarrays. The association of the expression of these cyclins with the infection rate of HBV was also analyzed.</p><p><b>RESULTS</b>Three paraffin-embedded HCC tissue microarrays were successfully constructed, including 136, 143 and 148 tissue spots, respectively. The positive rates of cyclins in 273 cases of HCC were cyclin A 52.7%, cyclin B 45.4%, cyclin D1 35.9%, cyclin D3 44.3% and cyclin E 23.1%; while the figures in 144 surrounding-tumor tissues were 8.3%, 5.6%, 4.9%, 6.3% and 1.4%, respectively. In 10 normal liver tissues these cyclins exhibited negative staining, with the exception that cyclin D1 was positive in one case of normal liver tissue. The positive rate of cyclins in HCC were significant higher than those in surrounding-tumor liver tissues (P < 0.01), in HCC tissues with histological grade II and III, the cyclins expression were stronger than that in grade I (P < 0.05). The positive rates of cyclins, except cyclin A in HCC with portal vein invasion were higher than those without portal vein invasion (P < 0.01). Infection of HBV did not have significant relationship with the expression of cyclins (P > 0.05).</p><p><b>CONCLUSION</b>Cyclins in different cell cycles overexpressed at varied levels in hepatocellular carcinoma, and the increased expression of cyclins may shorten the tumor cell cycle phase, accelerate cell proliferation, and have a close relationship with HCC aggressiveness.</p>