High-Wettability Poly(dimethylsiloxane) Substrate for Ultrastable Conductive Three-Dimensional Woven Ag Nanowire Grids.

Three-dimensional (3D) woven Ag nanowire (AgNW) grids have great potential for enhancing the mechanical stabilities, conductivity, and transmittance of flexible transparent electrodes (FTEs). However, it is a great challenge to control the formation of 3D woven AgNW grids on various substrates, especially the poly(dimethylsiloxane) (PDMS) substrate. This work presents a microtransfer-printing method for preparing a high-wettability poly(dimethylsiloxane) (PDMS) substrate to control the formation of 3D woven AgNW grids. The as-prepared PDMS substrate shows a high wettability performance. The surface structures of the PDMS substrate can control the sharp shrinkage of the ink membrane to give rise to a uniform liquid membrane evaporation behavior, which is the key factor for preparing a uniform 3D woven nanowire network. A thin uniform 3D woven AgNW network with a low sheet resistance of 24.3 Ω/□ and high transmittance of 92% was coated on the PDMS substrate. The networks directly coated the surface of the replicated PDMS, which simplified the peeling process and protected the networks from peeling strain and mechanical deformations. Moreover, the increment of resistance retained a small value (∼5%) when bending cycles reached 9,000. An alternating current electroluminescent (ACEL) device was prepared, and the uniform electroluminescence implies that a defect-free electrode has been fabricated. These results indicate that the as-prepared FTEs have excellent mechanical performance and great potential for flexible optoelectronic applications.

High Expression Levels of Long Noncoding RNA Small Nucleolar RNA Host Gene 18 and Semaphorin 5A Indicate Poor Prognosis in Multiple Myeloma.

BACKGROUND: The aim of this study was to detect the expression of long noncoding RNA small nucleolar RNA host gene 18 (SNHG18) andsemaphorin 5A (SEMA5A) genes in multiple myeloma (MM) patients and to explore the correlation of the expression of these genes with the clinical characteristics and prognosis of MM patients. METHODS: Forty-seven newly diagnosed MM, 18 complete remission MM, 13 refractory/relapse MM, and 22 iron deficiency anemia (serving as control) samples were extracted at the Department of Hematology, Second Affiliated Hospital of Xian Jiaotong University between January 2015 and December 2016. The clinical features of the MM patients are summarized. Real-time quantitative PCR was performed to analyze the relative expression levels of the SNHG18 and SEMA5Agenes. The clinical characteristics and overall survival (OS) of the MM patients were statistically analyzed while measuring different levels of SNHG18 and SEMA5Agene expression. At the same time, the correlation between the expression of SNHG18 and SEMA5A was also analyzed. RESULTS: The analysis confirmed that SNHG18 and its possible target gene SEMA5A were both highly expressed in newly diagnosed MM patients. After analyzing the clinical significance of SNHG18 and SEMA5A in MM patients, we found that the expression of SNHG18 and SEMA5A was related to the Durie-Salmon (DS), International Staging System (ISS), and Revised International Staging System (R-ISS) classification systems, and the Mayo Clinic Risk Stratification for Multiple Myeloma (mSMART; p < 0.05). Moreover, we observed a significant difference in OS between the SNHG18/SEMA5A high expression group and the low expression group. We found a positive correlation between SNHG18 and SEMA5A expression (r = 0.709, p < 0.01). Surprisingly, the expected median OS times of both the SNHG18 and SEMA5Ahigh expression groups were significantly decreased, which was in contrast to those of both the SNHG18 and SEMA5Alow expression groups and the single-gene high expression group (p < 0.05). CONCLUSION: High expression of both SNHG18 and SEMA5A is associated with poor prognosis in patients with MM.

[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.

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.

[Influence of Smad4-independent pathway of transforming growth factor beta1 on the biological activity of pancreatic cancer cells].

OBJECTIVE: To study effects of the expression of transforming growth factor (TGF)-beta1 on the growth of Smad4-null pancreatic cancer cells. METHODS: 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. RESULTS: 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. CONCLUSION: 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).

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.

[Expression and clinical significance of P53 pathway-associated proteins in pancreatic carcinoma].

BACKGROUND & OBJECTIVE: P53 pathway plays a critical role in carcinogenesis of pancreatic carcinoma. However, its trigger and function mechanisms have seldom been reported. This study was to investigate the expression and clinical significance of P53 pathway-related proteins ATM, P53, Mdm2, and P21(WAF/CIP1) in pancreatic carcinoma. METHODS: The expression of ATM, P53, Mdm2, and P21(WAF/CIP1) proteins in 167 specimens of pancreatic carcinoma and 112 specimens of non-cancer pancreatic tissues was detected by tissue microarray and immunohistochemistry. RESULTS: The positive rates of P53 and Mdm2 were higher in pancreatic carcinoma than in non-tumor pancreatic tissues (57.5% vs. 6.3%, 64.1% vs. 5.4%, P < 0.01), while the positive rates of ATM and P21(WAF/CIP1) were lower in pancreatic carcinoma than in non-tumor pancreatic tissues (67.7% vs. 82.1%, 39.5% vs. 71.4%, P < 0.05). ATM expression in pancreatic carcinoma was related to patients' age (P < 0.05). P53 expression was related to tumor differentiation, lymph node metastasis, and nerve involvement (P < 0.05). Mdm2 expression was related to tumor differentiation (P < 0.05). P21(WAF/CIP1) expression was related to patients' age and nerve involvement (P < 0.05). There were statistical correlations between these 4 proteins (P < 0.05). CONCLUSIONS: Overexpression of P53 and Mdm2 and loss of ATM and P21(WAF/CIP1) expression may contribute to the tumorigenesis and development of pancreatic carcinoma. The 4 proteins may affect cell transformation and tumorigenesis through ATM-Mdm2-P53-P21(WAF/CIP1) pathway. Co-detection of P53 and Mdm2 can be used to evaluate the differentiation of pancreatic carcinoma.

Genetic alterations and reduced expression of tumor suppressor p33(ING1b) in human exocrine pancreatic carcinoma.

AIM: To detect the expression of p33(ING1b) protein and the change of p33(ING1b) gene in pancreatic carcinoma and to evaluate the significance of p33(ING1b) in pancreatic cell carcinogenesis. METHODS: Pathological specimens from pancreatic carcinoma and matched non-tumor pancreatic tissues were examined for p33(ING1b) expression and mutation by immunohistochemistry, polymerase chain reaction single-strand conformation polymorphisms (PCR-SSCP) and loss of heterozygosity (LOH). RESULTS: The rate of p33(ING1b) protein expression was 85% (34/40). A single germline missense mutation was detected in 1 of 40 tumors located at codon 215:TGC-TCC (Cys-Ser). Fourteen (60.9%) of 23 tumor samples showed LOH in all of the informative markers tested, but no mutation was detected in these tumors and only two of the informative tumors lacked expressions of p33(ING1b) protein. CONCLUSION: Mutation and loss of expression are not the main reasons for the disfunction of p33(ING1b) in pancreatic carcinoma, an abnormality at the level of chromosome and/or transcription may inhibit their normal functions, potentially contributing to pancreatic cell carcinogenesis.

[Interaction effect of p53 with HBV in hepatoma cell line SMMC-7721].

OBJECTIVES: To study the interaction of hepatitis virus B (HBV) and tumor suppressor p53. METHODS: 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. RESULTS: 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. CONCLUSION: p53 expression and effects could be enhanced by HBV and p53 had positive regulation effect on HBV replication.

[Effects of hepatitis B virus X gene on p21(WAF1) expression through p53-dependent and p53-independent pathways].

BACKGROUND & OBJECTIVE: It was reported that the hepatitis B virus X could inhibit the function of p53 and have contrary effects on p21(WAF1), a downstream gene of p53, but the mechanism is not clear up to now. So this study was designed to investigate HBx's effect on p21(WAF1) gene. METHODS: After co-transfection of sense or antisense wild type p53 gene (wtp53) with HBx into SMMC-7721 and HBx alone transfection into other hepatoma cell lines with different endogenous status of p53, we evaluated the luciferase expression level under the p21(WAF1) promoter by detecting the luciferase activity and alteration of cell cycle in these transfected cell lines by flow cytometry. Expressions of p53 and p21 (WAF1) in hepatoma cells after transfected with HBx were also estimated by Western blot analysis. RESULTS: The luciferase activity (1.007+/-0.098) in SMMC-7721 cells cotransfected with HBx and sense-wtp53 was higher than that in cells with wtp53 gene alone (0.490+/-0.012, P< 0.05), and also was depressed in the other experimental groups transfected with HBx (P< 0.05). Western blot analysis showed that after transfected with HBx, p53 expression was elevated in all hepatoma cell lines with different endogenous status of p53, and the expression of p21(WAF1) and luciferase activity under the p21(WAF1) promoter (0.053+/-0.010 vs. 0.094+/-0.013, P< 0.05) were both decreased in SMMC-7721 cells with low expression of p53, but relatively increased in HepG2 cell line with high expression of p53 (1.252+/-0.052 vs. 0.767+/-0.031, P< 0.05). Flow cytometry showed that fewer SMMC-7721 (42.31%) and Hep3B (36.96%) cells were arrested in G(0)/G(1) phase in transiently transfected HBx groups than in the control groups (47.10% and 42.90%), which was the opposite case in HepG2 cell line (63.62% vs. 57.42%). Moreover, after stably transfected with pcDNA3HBx, HepG2 cells reduced in G(0)/G(1) phase in compared with the control group (57.31% vs 61.49%). CONCLUSION: HBx may not only increase the expression of p21(WAF1) by introducing the accumulation of p53 in cytoplasm but also inhibit the transcriptional activity of p21(WAF1) promoter in a p53-independent manner.

[Interaction of p53 with p53 response element like binding sequence at upstream of hepatitis B virus enhancer I].

BACKGROUND & OBJECTIVE: A p53 response element like binding sequence, 5'-TGCC(G)T-TGCCT-3' was found at upstream of hepatitis B virus (HBV) enhancer I from 1047 to 1059 nucleotides after analyzing the HBV genome by a computer program in our previous work. It indicated that the sequence could specifically bind P53 protein in vitro by electrophoretic mobility shift assay (EMSA) and electrophoretic mobility supershift assay (EMSSA). This study was designed to further investigate the interaction between P53 and p53 response element like binding sequence at upstream of HBV enhancer I. METHODS: HBV x gene enhancer and promoter which contains p53 response element like binding sequence TGCGT-TGCCT was in upstream of CAT enzyme in pX-CAT reporter plasmid. Firstly, we designed a point mutation in pX-CAT which change TGCGT TGCCT into TGTAT. TGTAT by polymerase chain reaction (PCR) in order to damage the p53 response element like sequence. After pX-CAT or mutation pX-CAT (mpX-CAT) transfected alone or cotransfected with pCMVp53 into HepG2 hepatoma cell, CAT activity was assayed to confirm the correlation of p53 protein with this DNA sequence. In addition, an antisense sequence corresponding to the p53 response element like sequence in HBV was reconstructed into the pZeoSV2 vector (alpha pZeoXP) and transfected into HepG2.2.15 cell line to block the binding of P53 with this sequence, the stable transfected HepG2.2.15 cell was observed about P53/P21 expression, cell cycle distribution and apoptosis rates. RESULTS: CAT enzyme of HepG2 had an higher expression in cotransfection with pCMVp53 and pX-CAT than alone pX-CAT (1.353 VS 0.738,P< 0.05), but it was lower in mpX-CAT(0.304) and pCMVp53 (0.402). Compared with the control group, P53/P21 expression and cell apoptosis rate decreased greatly in the stable transfected alpha pZeoXP HepG2.2.15 cell line (0.95% VS 7.84%), the cell number in S phase increased in the same cell line (16.37% VS 9.48%). CONCLUSION: Reporter gene expression of pX-CAT in intracellular could be promoted by P53, which further suggest that P53 could bind TGCC(G)T-TGCCT in upstream of HBV enhancer I and induce a prolonged P53 half life. Subsequently, P53 and p21 protein (downstream gene of P53) would have a higher expression and stronger activity.

[Expression of proteins in p53 (p14ARF-mdm2-p53-p21WAF/CIP1) pathway and their significance in exocrine pancreatic carcinoma].

OBJECTIVE: To investigate the expression of p14(ARF), p53, mdm2 and p21(WAF/CIP1) proteins and their relationship in exocrine pancreatic carcinoma. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Expression of ATM protein and its relationship with p53 in pancreatic carcinoma with tissue array.

ATM protein anticipates in the initiation of the DNA repair signal pathway and also mediates cell cycle arrest and repair. ATM deficiency predictably results in radiosensitivity, germ cell degeneration, chromosomal instability, immunodeficiency, and an extreme predisposition to tumors. Moreover, studies found that ATM is the upstream gene of the p53 pathway and would phosphorylate p53 directly after DNA damage, which would suppress tumorigenesis. Expression of ATM and p53 in 167 pancreatic cancer and 101 control specimens, benign lesions, and normal pancreata were detected by high-throughput tissue microarray and immunohistochemistry while seeking the role of ATM in the initiation and development of pancreatic carcinoma as well as its relationship with p53. We found that the positive rates of ATM and p53 expression in pancreatic carcinoma and its relative control specimen were 67.7% (113/167) and 82.2% (83/101) (P < 0.05) and 57.5% (96/167) and 5.0% (5/101) (P < 0.01), respectively. ATM positive staining is significantly relative to age and infiltration (P < 0.05), while the expression of p53 was significantly associated with tumor differentiation, lymph node metastasis, and nerve infiltration (P < 0.05). Expression of ATM and p53 was positively correlated. These findings suggest that expression of ATM deficiency may increase the transformative ability of pancreatic cancer cells. ATM may also cooperate with p53 in the repair of cell damage.

[Study of cooperation of tumor suppressor p33(ING1b) with p53 gene in hepatocellular carcinoma].

OBJECTIVE: To investigate the cooperation effect of p33(ING1b), coded protein of the novel tumor-suppressor gene ING1b, with p53 gene on the cell growth, cell cycle arrest, apoptosis and expression of the p53 downstream gene p21(WAF1/CIP1) in hepatocellular carcinoma. METHODS: Recombinant plasmids pcDNA3-INGIb containing sense p33(ING1b) cDNA, and pCMV-wtp53 containing sense p53 gene, and were transfected into HepG2, PLC/PRF/5 and Hep3B hepatoma cell lines with different p53 endogenous expression status using lipofecTAMINE method. Recombinant plasmids pcDNA3-alphaING1b containing antisense p33(ING1b) cDNA, pCMV-alphawtp53 containing antisense p53 gene, and plain vector as control were transfected into the cells. Forty-eight hours after transfection, the apoptosis rates were detected and cell cycle arrest were analyzed by flow cytometry. The expression of p33(ING1b) and wtp53 proteins were detected by Western blot. The luciferase gene reporter plasmid drived by p21(WAF1/CIP1) promoter was also transfected into the cells to analyze the activation of p21(WAF1/CIP1) gene in hepatoma cells. Then 6% ethanol was added into the in DMEM culture medium and all of the above experiments were repeated. RESULTS: After the p33(ING1b) gene was transfected, the apoptosis rate of HepG2 cells which express endogenous wtp53 was enhanced (22.53%), the number of cells arrested in G(0)/G(1) phase was increased (67.45%), and the activation of p21(WAF1/CIP1) promoter reached the highest level (13.08) (all P < 0.01). In the PLC/PRF/5 cells which express endogenous mutant p53, after combined p33(ING1b) and wtp53 gene transfection the percentage of cells arrested in G(0)/G(1) phase (78.16%) and the activation of p21(WAF1/CIP1) promoter (12.99) were higher than those in the experiment groups transfected with p33(ING1b) or wtp53 gene alone (both P < 0.01), however, the difference in apoptosis rate was not statistically significant (P > 0.05). After 6% ethanol treatment, apoptosis rate of PLC/PRF/5 cells transfected with combined p33(ING1b) and wtp53 gene was increased significantly (42.8%). The apoptosis rate and cell cycle arrest were not significantly different between the Hep3B cells with deletion of wtp53 which were transfected with p33(ING1b) and wtp53 gene and those of the control group (P > 0.05), however, the activity of the p21(WAF1/CIP1) promoter was activated in the combined transfectlion group (10.32, P < 0.01). CONCLUSION: p33(ING1b) cooperate with wtp53 in the process of inhibiting hepatoma cell growth, inducing apoptosis, and activating p53 downstream gene p21(WAF1/CIP1), and after chemical inducing reagent treatment the cooperation between p33(ING1b) and wtp53 gene is enhanced.

[Immunohistochemical demonstration of cyclins A, B, D1, D3 and E in hepatocellular carcinomas using tissue microarrays].

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

[Determination of expression of eight p53-related genes in hepatocellular carcinoma with tissue microarrays].

BACKGROUND & OBJECTIVE: Hepatocarcinogenesis was a multistage process involving a number of genes. Molecular biological studies indicated that abnormal expression of p53 gene family is correlated with the development of hepatocellular carcinoma (HCC). This study was designed to investigate the expression difference and its clinicopathologic significance of eight kinds of p53-related oncogenes and tumor-suppressor genes in HCC and adjacent-tumor liver tissues using tissue microarray technique. METHODS: The HCC tissue microarrays comprised 273 cases of HCC tissues, 144 adjacent-tumor liver tissues, and 10 normal liver tissues obtained from autopsy. The diameter of each specimen on tissue microarrays was 2.0 mm. Immunohistochemistry was employed to detect the expression of p16(INK4a), p21(WAF1), p33(ING1b), p53, p57(KIP2), p73, mdm2, and ATM genes in tumor and adjacent tumor liver tissue, respectively. The relationship between HBV infection rate and the expression of these genes was also analyzed. RESULTS: Three paraffin-embedded HCC tissue microarrays were successfully constructed, composed of 136, 143, and 148 tissue dots, respectively. The positive staining rates of these eight genes in HCC and surrounding-tumor tissues were: (p16(INK4a)) 35.9% and 9.0%, (p21(WAF1)) 41.8% and 11.1%, (p33(ING1b)) 43.65% and 14.6%, (p53) 46.2% and 12.5%, (p57(KIP2)) 39.2% and 16.7%, (p73) 9.5% and 2.8%, (mdm2) 41.4% and 9.0%, (ATM) 6.6% and 1.4%, respectively. The expression of all these genes in HCC were stronger than that in the surrounding-tumor liver tissues (P< 0.05). The expression differences of these genes in HCC tissues with varied differentiated grades were not significant(P >0.05). Except p16(INK4a), p21(WAF1), and p73 genes(P >0.05), the expression of p33(ING1b), p53, mdm2, ATM, and p57(KIP2) genes in HCC with portal vein invasion were higher than those in HCC without portal vein invasion (p33(ING1b), p53, mdm2, ATM, P< 0.01; p57(KIP2), P< 0.05). The infection rate of HBV did not have a significant correlation to the expression of these eight genes (P >0.05). CONCLUSION: Tissue microarray technique had the advantage of high-throughput in the detection of HCC-related oncogenes and tumor-suppressor genes, which can analyze larger amounts of specimens, more target genes, and cost less working time.

[The role of hepatitis B virus X gene and p53 on hepatocellular carcinoma cell growth].

OBJECTIVE: To explore the effects of hepatitis B virus X gene and p53 on hepatocellular growth. METHODS: 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. RESULTS: 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. CONCLUSION: 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.