Effect of NeuroD gene silencing on the migration and invasion of human pancreatic cancer cells PANC-1.

The aim of this study is to investigate the influence of Lenti-EGFP-NeuroD-miR, RNAi lentiviral expression vector, on the expression level of NeuroD and migration, and invasion of PANC-1 cell line. PANC-1 cells were cultured and cotransfected with Lenti-EGFP-NeuroD-miR and Lenti-GFP. The infection rate of lentivirus was determined by fluorescence. The interfering effection by the expression of NeuroD mRNA in PANC-1 cells was analyzed by real-time PCR after transfected. Biological behavior of PANC-1 cells transinfected was observed, and the migration and invasion were studied by transwell assay. Intrapancreatic allografts model in nude mice was established to observe the effects of NeuroD on tumorigenesis, tumor growth, and invasion in vivo. The expression of NeuroD mRNA decreased significantly after RNAi lentivirus transinfecting PANC-1 cell. The cell's migration and invasion ability decreased obviously as soon as down regulate of NeuroD in PANC-1 cells. Comparing with control group, the tumors were smaller in size and the invasiveness was inhibited after 8 weeks intrapancreatic allografts in nude mice. Lenti-EGFP-NeuroD-miR transfected into PANC-1 cells shows a stable, effective, and especial blocking expression of NeuroD in mRNA level. The RNAi of lentiviral vector target NeuroD can reduce the migration and invasion abilities of PANC-1 cells.

Upregulation of miR-194 contributes to tumor growth and progression in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of human cancer worldwide. In the present study, we investigated the diagnostic and biological significance of microRNA-194 (miR-194) in PDAC. miRNA expression profiling of human PDACs and adjacent normal pancreatic tissues identified a total of 16 genes including miR-194 with >1.15-fold expression changes (8 overexpressed and 8 underexpressed). Quantitative real-time polymerase chain reaction (PCR) revealed elevation of serum miR-194 levels were significantly greater in PDAC patients than in duodenal adenocarcinoma patients and healthy controls. Receiver operating characteristic analysis demonstrated that serum miR-194 had a sensitivity of 54.3% and a specificity of 57.5% for discriminating PDAC patients from healthy controls. Combined analysis of the 3 groups yielded a sensitivity of 84.0 and a specificity of 75.0% for the combined detection of miR-192 and miR-194 in the diagnosis of PDAC. Ectopic expression of miR-194 in PANC-1 pancreatic cancer cells enhanced cell proliferation, migration and colony formation, which was coupled with decreased expression of the tumor suppressor DACH1. miR-194 overexpression increased tumor growth and local invasion and suppressed the expression of DACH1 in an orthotopic pancreatic cancer mouse model. In conclusion, upregulation of miR-194 contributes to tumor growth and progression in PDAC, possibly through suppression of DACH1. However, serum miR-194 has a low capacity for detection of PDAC. Combined detection of serum miR-192 and miR-194 levels may serve as a sensitive diagnostic biomarker for PDAC.

Lymphangiogenic and angiogenic microvessel density in chinese patients with gastric carcinoma: correlation with clinicopathologic parameters and prognosis.

The incidence of gastric cancer worldwide, and in particular in developing countries, has shown a marked increase. Poor prognosis of gastric cancer patients occurs due to the rapid metastasis of the disease via the lymphatic and blood vessels. The aim of this study was to investigate the expression and the clinical significance of D2-40 and CD34 in human gastric cancer. D2-40 and CD34 expression wasdetected in 1,072 cases of Chinese patients with gastric carcinoma using immunohistochemistry. The lymphatic vessel density (LVD) and microvessel density (MVD) were calculated and analyzed and the correlation with the clinicopathological factors and prognosis was determined. The LVD and MVD of the gastric cancer cases were significantly higher compared to those of normal tissues (P < 0.05). The expression of D2-40-LVD and CD34-MVD in the malignancies were positively related to the age, tumor size, invasion depth, lymphatic metastasis and pathological tumor-node-metastasis (pTNM) (P < 0.05); However, no statistically significant difference was identified between them with the patient gender (P > 0.05). Up-regulation of D2-40 and CD34 expression was significantly correlated with the poor survival rate in univariate and multivariate analyses. The LVD marked by D2-40 and the MVD marked by CD34 were positively correlated to the clinicopathological factors of the malignancies and may play important role in the development and progression of gastric cancer.

Expression of fibroblast activation protein in human pancreatic adenocarcinoma and its clinicopathological significance.

AIM: To examine fibroblast activation protein (FAP) expression in pancreatic ductal adenocarcinoma (PDAC) and to analyze its relationship with the clinicopathology of PDAC. METHODS: FAP expression was examined in 134 PDAC specimens by immunohistochemistry, and in four pancreatic cancer cell lines (SW1990, Miapaca-2, AsPC-1 and BxPC-3) by Western blotting assay. We also analyzed the association between FAP expression in PDAC cells and the clinicopathology of PDAC patients. RESULTS: The results showed that the FAP was ex-pressed in both stromal fibroblast cells (98/134, 73.1%) and carcinoma cells (102/134, 76.1%). All 4 pancreatic cancer cell lines expressed FAP protein at different levels. Protein bands corresponding to the proteolytically active 170-kDa seprase dimer and its 88-kDa seprase subunit were identified. Higher FAP expression in carcinoma cells was associated with tumor size (P < 0.001), fibrotic focus (P = 0.003), perineural invasion (P = 0.009) and worse clinical outcome (P = 0.0085). CONCLUSION: FAP is highly expressed in carcinoma cells and fibroblasts in PDAC tissues, and its expression is associated with desmoplasia and worse prognosis.

Genetic alterations of tumor suppressor ING1 in human non-small cell lung cancer.

The aim of this study was to investigate the function of the ING1 gene in lung carcinoma. To detect the inhibitory effect of ING1 in human lung cancer, recombinant ING1b plasmids were transfected into two lung cancer cell lines with different p53 status, A549 with wild-type p53 (wtp53) and SK-MES-1 with mutant p53. Apoptosis, cell cycle, growth rate and the expression of downstream gene p21waf1 were analyzed. In addition, the complex of p33ING1b and p53 was analyzed with coimmunoprecipitation. To detect the gene alteration and the expression of ING1, 70 cases of fresh-frozen lung carcinomas and 217 cases of formalin-fixed, paraffin-embedded specimens were examined for loss of heterozygosity (LOH) and p33ING1b protein expression by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and immunohistochemistry using tissue microarrays, respectively. Overexpression of ING1b inhibited the cell growth of A549 and SK-MES-1, induced cell cycle arrest and apoptosis. p21waf1 was up-regulated and a complex of p33ING1b and wtp53 was found after transfection of ING1b in the wtp53-positive lung cancer cell. High LOH frequency was found in lung carcinomas (55.7%) and p33ING1b expression was lost in 115 of 217 carcinomas (53.0%). Furthermore, there was a highly significant inverse correlation between expression and LOH frequency (P<0.05). ING1 can inhibit the growth of lung cancer cell lines through the induction of cell cycle arrest and apoptosis by forming a complex with wtp53 and up-regulating p21waf1. In human lung cancer, expression of the ING1 gene was reduced or lost and high LOH frequency of ING1 microsatellites was found. The LOH of microsatellites may down-regulate p33ING1b and/or affect its function, thereby, contributing to lung cell carcinogenesis.

[Effects of moxibustion on the expression of IL-1beta, IL-2, IL-6 mRNA and protein in the cerebral cortex in tumor-bearing mice].

OBJECTIVE: To investigate the effect of moxibustion on the expression of IL-1beta, IL-2 and IL-6 proteins and mRNA in the cerebral cortex in tumor-bearing mice so as to study its mechanism underlying immunomodulation. METHODS: Forty Balb/c mice were randomly divided into control, tumor-bearing, non-acupoint moxibustion (N-AM) and acupoint-moxibustion (AM) groups (n = 10/group). Moxibustion was applied to "Dazhui" (GV 14), once every other day for 6 times. The expression of IL-1beta mRNA, IL-2 mRNA and IL-6 mRNA was detected by in situ hybridization, and the immunoactivity of IL-1beta, IL-6 and IL-2 determined by immunohistochemistry. RESULTS: Compared to the control group, the expression levels of IL-1beta mRNA and IL-2 mRNA, IL-1beta and IL-2 in the cerebral cortex of the tumor-bearing group were down-regulated significantly (P < 0.05, P < 0.01), while those of IL-6 mRNA and IL-6 up-regulated significantly (P < 0.05). Compared to the tumor-bearing group, the expression of IL-1beta mRNA and IL-2 mRNA, IL-1beta and IL-2 in the cerebral cortex in AM group were increased considerably (P < 0.05, P < 0.01); while cortical IL-6 immunoactivity in N-AM group was decreased significantly (P < 0.05), and IL-6 mRNA had no significant change in N-AM group (P > 0.05). Comparison between AM and N-AM groups showed that the expression levels of cortical IL-1beta mRNA and IL-2 mRNA, and IL-1beta and IL-2 proteins of the former group were obviously higher than those of the later group (P < 0.05, P < 0.01); while the immunoactivity of cortical IL-6 of AM group was significantly lower than that of N-AM group (P < 0.05). No significant difference between AM and N-AM groups in the expression of IL-6 mRNA (P > 0.05). CONCLUSION: Moxibustion treatment can up-regulate the expression of cortical IL-1beta mRNA, IL-2 mRNA, IL-1beta and IL-2 proteins, and down-regulate the expression of IL-6 mRNA and IL-6 in tumor-bearing mice, which may contribute to its effect in improving the immunosuppressing state under tumor conditions.

[Hepatitis B virus X protein inhibits hepatoma cell growth in vitro through p14(ARF)-dependent and p14(ARF)-independent pathways].

OBJECTIVE: To explore the effects of hepatitis B virus X protein (HBx) on hepatoma cell growth through p14(ARF)-dependent and p14(ARF)-independent pathways. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Experimental study of peripheral nerve grafts for repairing of chronic spinal cord injury in adult rats].

OBJECTIVE: To explore the pathological mechanism in the repair of chronic spinal cord injury with free grafting of autoperipheral nerve tissues in rats. METHODS: The SD rats were used to establish SCI model with modified Allen method. The rats were divided into two groups at 12 weeks after the injury, each group had 20 rats. In the experimental group, the sural nerves were removed epineurium and transplanted into SCI lesion by using microsurgical technique; and in the control group, the rats were treated without any operation. The survival and differentiation of the grafts, and the ability of repairing host spinal cord were observed under the light microscope at the postoperative 4th and 12th week. Regeneration rates of nerve tracts in spinal cord were evaluated by using HRP tracing technique at the postoperative 4th and 12th week. The morphological changes were observed at section of spinal cord and the motor functions of both hind legs of rats were detected. RESULTS: In the control group, spinal cord exhibited degeneration with cicatrices and cavitates. In the experimental group, peripheral nerve was almost survived, fused with the spinal tissue and axons could regrow into or span the place of injured spinal cord. Higher number of labeled nerve tracts in spinal cord were observed in experimental group, there was significant difference when compared with the control group. Motor function of hind legs of rats recovered significantly in the treatment group. CONCLUSION: Autoperipheral nerve graft tissues transplantation could survive and integrate with the host and have repairing effects on chronic spinal cord injury in rats.

[Maturation of dendritic cells and activation of B-lymphocytes in spleens of ICR mice infected with chloroquine-resistant Plasmodium berghei].

OBJECTIVE: To investigate the relation between activation of B-cells and maturation of dendritic cells (DC) in the spleens of ICR mice infected with chloroquine-resistant (RC) or chloroquine-sensitive (N) strain of Plasmodium berghei. METHODS: Spleens were taken after the mice were infected with N or RC strains of P. berghei and attained certain degree of parasitemia. Changes of B-cells and DCs were examined by pathological method, immunohistochemistry and immunofluorescence methods, transmission electron microscopy (TEM) and flow cytometry technology. RESULTS: Proliferation of white pulps in the spleen of mice infected with RC strain was found as compared to that with N strain. The percentage of cluster of differentiation (CD) 45R/B220, CD19 cells increased in the spleen cells, number of medium and small lymphocytes increased in the germinal centers, the immature and mature plasma cells also increased in the red pulps of spleen in RC strain-infected mice. On the contrary, in the N strain-infected mice spleen, the white pulps were reduced and the red pulps were filled with parasite-infected red blood cells; less small lymphocytes, immature and mature plasma cells were observed in red pulps. The number of CD11c DCs increased, especially in the periarteriolar lymphoid sheath, T cell area; the expression of major histocompatibility complex II (MHC II) on DC was up-regulated in RC strain-infected mice as compared to that in N strain-infected mice. TEM showed that the DCs in RC strain-infected mice spleens were more active than that in N strain-infected mice. CONCLUSION: Infection of RC strain P. berghei increases mature DCs in the spleen, which induces the proliferation of B cells and immune response.

Ketanserin-induced baroreflex enhancement in spontaneously hypertensive rats depends on central 5-HT(2A) receptors.

1. Ketanserin may influence baroreflex function by blocking 5-HT(2A) receptors and/or alpha(1)-adrenoceptors through central and/or peripheral mechanisms. 2. In the present study, we tested the hypothesis that the baroreflex sensitivity (BRS)-enhancing effects of ketanserin are mediated by central 5-HT(2A) receptors in spontaneously hypertensive rats (SHR). 3. Using a conjugate of a monoclonal antibody to the serotonin reuptake transporter (SERT) and the toxin saporin (anti-SERT-SAP), which specifically eliminates the neurons that express SERT, the effects of ketanserin (0.3 and 3.0 mg/kg, i.g.) on BRS, blood pressure (BP), heart period (HP) and blood pressure variability (BPV) were compared between conscious intact SHR and SHR pretreated with anti-SERT-SAP. 4. Immunochemistry showed that, 2 weeks after intracerebroventricular injection of the toxin, 5-HT expression was strikingly attenuated in the brain, whereas values of BRS, BPV and BP were similar to those in the sham group. In intact SHR, 0.3 mg/kg ketanserin significantly improved BRS (191% control) and reduced BPV without affecting BP; at 3.0 mg/kg, ketanserin significantly increased BRS (197% control) and decreased BPV and BP. In toxin-pretreated SHR, only the high dose of ketanserin improved BRS (132% control), neither of the ketanserin doses reduced BPV, but both significantly decreased BP. 5. We conclude that the BRS-enhancing effects of ketanserin are mediated largely by central 5-HT(2A) receptors, whereas the antihypertensive effect of ketanserin persists even after destruction of serotonergic neurons in the central nervous system.

[The expression and role of Fas/FasL in infantile hemangioma].

OBJECTIVE: To investigate the expression of Fas/FasL in infantile hemangiomas and discuss the role of Fas/FasL in the pathologic evolution of infantile hemangioma. METHOD: The EnVision immunohistochemical stain and RT-PCR technique was used to examine the expression of Fas/FasL protein and mRNA in the infantile hemangiomas. RESULTS: (1) In the early and middle proliferating stage, a number of infantile hemangioma cells expressed Fas. In the late proliferating stage, the number of positive cells increased obviously and the expression of Fas mRNA was reaching the strongest level. In the early regressing stage the Fas still existed in some cells and after that the expression decreased quickly. (2) Up to the middle proliferating stage, there were a few of FasL(+) cells foound. In the late proliferating stage, the number of FasL(+) cells increased significantly. From the early regressing stage, the number of FasL(+) cells decreased rapidly and disappeared. CONCLUSION: There may exist significant correlation between the expression of Fas/FasL and the development of the infantile hemangioma cells. The apoptosis of the infantile hemangioma cells mediated by Fas/ FasL may be the major reason of the spontaneous involution of infantile hemangioma.

[Expressions of nm23, P53 and S100A4 proteins and their relationships with metastasis potential in gastric carcinoma].

OBJECTIVE: To investigate the expression levels of nm23, P53, and S100A4 in gastric carcinoma and their relationships with clinicopathologic parameters and metastasis potential. METHODS: Pathological specimens from gastric carcinoma,matched para-tumor tissues, metastatic lymph node and distant metastatic tissues were examined for the expression levels of nm23, P53, and S100A4 proteins by tissue microarray technique and immunohistochemistry. RESULTS: The expression levels of P53 and S100A4 were upregulated (P< 0.01), while the expression of nm23 downregulated (P< 0.05) in gastric carcinoma compared with non-tumor tissues. S100A4 expression was significantly higher in distant metastatic tissues, while nm23 lower in metastatic lymph nodes than those in cancer tissues. Upregulating expression levels of nm23, P53, and S100A4 were significantly correlated with some malignant behaviour of gastric cancer. The expression rates of nm23+/P53+, P53+/S100A4+, and nm23+/S100A4+ immunohistochemical phenotypes were 48/74 (64.9%), 50/74 (67.6%), and 39/74 (52.7%). P53+/S100A4+, nm23+/S100A4+, and nm23+/P53+/S100A4+ phenotypes were associated with high metastasis potential of gastric cancer. CONCLUSIONS: Alteration of nm23, P53, and S100A4 expression may contribute to the development of gastric carcinoma. Nm23 and S100A4 proteins play a critical role in tumor metastasis. Co-detection of the expression of P53, nm23, and/or S100A4 can be used to evaluate high metastasis potential of gastric cancer.

Age-related effects of estrogen on the expression of estrogen receptor alpha and beta mRNA in the ovariectomized monkey hypothalamus.

In the present study, we reported distribution of ER alpha and ER beta mRNAs in the hypothalamus of young and old ovariectomized (OVX) rhesus macaques. The ER alpha were detected in all six major vestiblular nuclei which included arcuate nucleus (ARC) , paraventricularis nucleus (PVN) , periventricular nucleus (PeriV) , supraoptic nucleus (SON) , medial prioptic nucleus (MPN) and lateral hypothalamus area (LHA). However, the ER beta mRNA can also detected in those nuclei excerpt SON, but the signals of ER beta mRNA were weaker than those of ER alpha mRNA. We observed that the degree of expression of ERs mRNA were different in most nucleus of old and young monkeys. The ER alpha mRNAs were highly expressed in ARC and SON in young monkeys compared with old monkeys. Moderate amount of ERalpha mRNAs hybridization signals and weak signals were observed in LHA, and MPN both in young and old monkeys. In contrast, only lower level of ER alpha hybridization signal were observed in PVN and PeriV in young monkeys, and the signals of ER alpha were very low in those nucleus of old monkeys. In general, the expression of ER beta mRNA were weaker than that of ER alpha mRNA in above nucleus excerpt LHA. The relatively higher density of ER beta hybridization signals have been observed in the LHA in young monkey compared with old monkeys. Low amount of ER beta mRNA hybridization signals were observed in the ARC, PVN and MPN, and no age differences were seen in PVN and MPN of those monkeys. In PeriV, we observed some signals in young monkey and a few signals in old monkeys. It was different from the rodent in which we did not found ER beta hybridization signal in SON. This study showed that both of the two estrogen receptors not only had the same pattern of expression but also had many different patterns of expression. The different expression of ER alpha and ER beta mRNAs in the young and old monkey brain may imply diverse functions in different regions of the monkey brain.

Effects of hepatitis B virus on p53 expression in hepatoma cell line SMMU-7721.

AIM: To investigate the contribution of HBV in the development of hepatocarcinoma by examining the effects of HBV on p53 function in SMMU-7721 cell line. METHODS: Plasmid pCMVp53 was transfected or cotransfected with pCMVHBVa (wild-type HBV) or PCMVHBVb (mutation type HBV) into the hepatoma cell line SMMU-7721 by lipofectamine. Apoptosis cells were labeled with annexin V-FITC and confirmed by flow cytometry. Reporter plasmid PG13-CAT or p21-luc was cotransfected, respectively, into each group to determine the transactivation activity of p53 and its effect on p21 promoter. Western blot was performed to observe p53 expression in hepatoma cell line of each group. RESULTS: The group transfected with pCMVp53 alone exhibited higher luciferase activity and higher apoptosis rate, otherwise, the p53 expression and reporter activity of PG13-CAT or P21-luc as well as cell apoptosis rate were obviously higher in the group cotransfected of pCMVp53 with pCMVHBVa, but not in the other cotransfected group. CONCLUSION: Transient transfection of HBV into the SMMU-7721 cell line can enhance p53 expression and its effects on development of hepatocarcinoma.

[Mucinous noncystic (colloid) adenocarcinoma of the pancreas].

OBJECTIVE: To determine the clinicopathologic characteristics and the relationship between related gene expression and pathobiologic behavior of pancreatic mucinous noncystic adenocarcinoma. METHODS: Among the 249 pancreatic carcinoma cases from the department files, 6 tumors were identified to meet the pathologic criteria of colloid carcinoma. Envision immunohistochemical staining technique was used to detect expression of p21(ras), p21(WAF1), p16, p33(ING1), p53, ATM, MDM2, PCNA, Cyclins (D1, D3, A, B and E). Intra- and extra- cellular mucin production were determined by AB-PAS staining. Clinically, all of 6 cases were followed to June, 2003. RESULTS: In all 6 cases, the tumors were located in the head of the pancreas and all displayed similar microscopic findings. Duodenal invasion was seen in 4 cases and perineural invasion was seen in 1 case. Tumor metastasis in the liver was seen in 2 cases and in the regional lymph nodes in 2 cases. Positive immunostaining was seen in 5 cases with p21(ras), 3 cases with p21(WAF1), 1 case with p16, 4 cases with p33(ING1), 2 cases with p53, 3 cases with ATM, 3 cases with MDM2, 6 cases with PCNA, 3 cases with cyclinA, 3 cases with cyclinD1, 4 cases with cyclinD3, 4 cases with cyclinB and 6 cases with cyclinE. Both extracellular and intracellular mucin was strongly positive for AB-PAS staining. Clinical follow-up found that 2 patients died of their tumors at 14 and 20 months. Three patients were alive after 28, 49 and 87 months of follow-up. One case were lost contact. CONCLUSIONS: Pancreatic mucinous noncystic adenocarcinoma has distinct morphologic features and biologic behavior. Multiple gene products including many cyclins may be involved in the pathogenesis of pancreatic colloid carcinoma. The tumor has an aggressive behavior with a high frequency of invasion and metastases, though the prognosis could be better than that of ordinary ductal adenocarcinoma of pancreas.

Inhibitory effect of tumor suppressor p33(ING1b) and its synergy with p53 gene in hepatocellular carcinoma.

AIM: To investigate the inhibitory effect of tumor suppressor p33(ING1b) and its synergy with p53 gene in hepatocellular carcinoma (HCC). METHODS: Recombinant sense and antisense p33(ING1b) plasmids were transfected into hepatoma cell line HepG2 with lipofectamine. Apoptosis, G0/G1 arrest, cell growth rate and cloning efficiency in soft agar of HepG2 were analyzed after transfection. In three hepatoma cell lines with different endogenous p53 gene expressions, the synergistic effect of p33(ING1b) with p53 was analyzed by flow cytometry and luciferase assay was performed to detect the activation of p53 downstream gene p21(WAF1/CIP1). In addition, the expression and mutation rates of p33(ING1b) in HCC tissues were measured by immunohistochemistry and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). RESULTS: Overexpression of p33(ING1b) inhibited cell growth of HepG2, induced more apoptosis and protected cells from growth in soft agar. Combined transfer of p33(ING1b) and p53 gene promoted hepatoma cell apoptosis, G0/G1 arrest and elevated expression of p21(WAF1/CIP1). Immunostaining results showed co-localized P33(ING1b) with P53 protein in HCC tissues and there was a significant relation between protein expression rates of these two genes (P<0.01). Among 28 HCC samples, p33(ING1b) presented a low gene mutation rate (7.1%). CONCLUSION: p33(ING1b) collaborates with p53 in cell growth inhibition, cell cycle arrest and apoptosis in HCC. Loss or inactivation of p33(ING1b) normal function may be an important mechanism for the development of HCC retaining wild-type p53.

[Hepatitis B virus and deletion of its COOH-terminal forty amino acids: proliferative impact on hepatoma cell line SMMC-7721].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.