PI3K/ c-Myc/AFF4 axis promotes pancreatic tumorigenesis through fueling nucleotide metabolism.

MLL-AFF4 fusion gene has been discovered in acute leukemia, whether AFF4 alone plays a role in tumor, especially pancreatic tumorigenesis, is still elusive. Increasing evidence suggests that cancer cells altered nucleotide metabolism during tumorigenesis. In present study, we observed AFF4 overexpression promoted cell proliferation, colony formation and cell cycle progression while loss of AFF4 impairs above phenotypes of pancreatic ductal carcinoma (PDAC) cells. Using RNA-profiling, we revealed that HPRT1 and IMPDH2, two enzymes in the nucleotide metabolism pathway, were upregulated following AFF4 overexpression. Simultaneous expression of HPRT1 and IMPDH2 would mainly rescue the phenotypes of cells lacking AFF4. Additionally, xenograft study proved HPRT1 and IMPDH2 genetically function in the downstream of AFF4, which was recruited by PAX2 when CDK9 mediated AFF4 phosphorylation at S388 and drove HPRT1 and IMPDH2 expression. We further discovered PI3K/c-Myc axis is required for AFF4 expression in PDAC cells. Finally, we obtained the positive correlation between c-Myc and AFF4 or AFF4 and HPRT1/IMPDH2 in clinical PDAC samples. Otherwise, we conducted data-mining and found that the expression levels of AFF4 and HPRT1/IMPDH2 are correlated with patients' prognosis, establishing AFF4 as a potential biomarker and therapeutic target for PDAC.

Risk factors of positive resection margin differ in pancreaticoduodenectomy and distal pancreatosplenectomy for pancreatic ductal adenocarcinoma undergoing upfront surgery.

OBJECTIVE: Positive resection margin indicates worse prognosis. The present study identified the independent risk factors of R1 resection in pancreaticoduodenectomy (PD) and distal pancreatosplenectomy (DP) for patients with pancreatic ductal adenocarcinoma (PDAC). METHOD: Consecutive patients who were operated from 1st December 2017 to 30th December 2018 were analyzed retrospectively. A standardized pathological examination with digital whole-mount slide images (DWMSIs) was utilized for evaluation of resection margin status. R1 was defined as microscopic tumor infiltration within 1 mm to the resection margin. The potential risk factors of R1 resection for PD and DP were analyzed separately by univariate and multivariate logistic regression analyses. RESULTS: For the 192 patients who underwent PD, and the 87 patients who underwent DP, the R1 resection rates were 31.8% and 35.6%, respectively. Univariate analysis on risk factors of R1 resection for PD were tumor location, lymphovascular invasion, N staging, and TNM staging; while those for DP were T staging and TNM staging. Multivariate logistic regression analysis showed the location of tumor in the neck and uncinate process, and N1/2 staging were independent risk factors of R1 resection for PD; while those for DP were T3 staging. CONCLUSIONS: The clarification of the risk factors of R1 resection might clearly make surgeons take reasonable decisions on surgical strategies for different surgical procedures in patients with PDAC, so as to obtain the first attempt of R0 resection.

ACOT4 accumulation via AKT-mediated phosphorylation promotes pancreatic tumourigenesis.

The acyl-CoA thioesterase (ACOT) family catalyses the hydrolysis of acyl-CoA thioesters to their corresponding non-esterified fatty acid and coenzyme A (CoA). Increasing evidence suggests that cancer cells generally have altered lipid metabolism in different aspects. However, the roles of the ACOT family in cancer, especially in pancreatic ductal carcinoma (PDAC), are largely unknown. In the present study, we mined data to determine the clinical significance of all eleven ACOT genes among nine major solid tumour types from TCGA database and found that the expression of ACOT4 in PDAC was negatively correlated with patient survival, establishing ACOT4 as a potential biomarker of PDAC. Depletion of ACOT4 attenuated the proliferation and tumour formation of PDAC cells. Using mass spectrometry, HSPA1A was found to associate with ACOT4. Furthermore, we found that phosphorylation of ACOT4 at S392 by AKT decreased the binding of ACOT4 to HSPA1A, resulting in ACOT4 accumulation. The ACOT4 elevation promotes pancreatic tumourigenesis by producing excessive CoA to support tumour cell metabolism. Thus, our study expands the relationship between AKT signalling and lipid metabolism and establishes a functional role of ACOT4 in PDAC.

Pros and Cons: High Proportion of Stromal Component Indicates Better Prognosis in Patients With Pancreatic Ductal Adenocarcinoma-A Research Based on the Evaluation of Whole-Mount Histological Slides.

The study aimed to investigate the potential of tumor-stroma ratio (TSR) on digitalized whole-mount histopathology to predict prognosis in patients with pancreatic ductal adenocarcinoma (PDAC). The effectiveness were evaluated through internal validation. Data were retrospectively collected from consecutive patients who underwent primary pancreatic resection from December 2016 to August 2017 (developing cohort) and from September 2017 to April 2018 (validation cohort). Digitalized whole-mount slide images were used to evaluate TSR by both pathologists and a computerized model based on Conditional Generative Adversarial Model (cGAN), respectively. TSR>1 and ≤ 1 denoted low and high stromal component. Logistic regression analysis revealed intratumoral necrosis and R1 independently associated with low stromal component in the developing cohort. Cox regression analysis revealed tumor-node-metastasis (TNM) stage [II vs. I: hazard ratio (HR), 2.584; 95% CI, 1.386-4.819; P = 0.003; III vs. I: HR, 4.384; 95% CI, 2.285-8.411; P < 0.001], stromal component (low vs. high: HR, 1.876; 95% CI, 1.227-2.870; P = 0.004), tumor grade (G3 vs. G1/2: HR, 2.124; 95% CI, 1.419-3.179; P < 0.001), and perineural invasion (with vs. without: HR, 2.147; 95% CI, 1.187-3.883; P = 0.011) were independent prognostic factors in the developing cohort. Stromal component categories could classify patients into subgroups within TNM stages I, II, and III based on over survival. All results were validated in the validation cohort. The weighted kappa value for categorical assessments between pathologists' evaluation and computer-aided evaluation was 0.804 (95% CI, 0.573-0.951). TSR represents a simple and reliable metric for combining the prognostic value of TNM stage in patients with PDAC.

A decrease in miR-150 regulates the malignancy of pancreatic cancer by targeting c-Myb and MUC4.

OBJECTIVES: Pancreatic cancer is an aggressive cancer with high mortality. Conventional treatments have little impact on its progression. Limited research investigating the role of oncogene miR-150 specifically in pancreatic cancer has been published. The purpose of this study was to determine the tumorigenesis of miR-150 in pancreatic cancer. METHODS: One hundred six pancreatic ductal adenocarcinomas were analyzed together with their adjacent benign pancreatic tissues. The associations of miR-150, c-Myb, and MUC4 expression with survival rates were determined. Functional studies on miR-150 in pancreatic cancer were used to assess its effect on proliferation and malignancy in several pancreatic cell lines. RESULTS: miR-150 expression was significantly down-regulated in pancreatic ductal adenocarcinoma tissues compared with adjacent benign pancreatic tissues. Patients with low miR-150 expression had significantly higher mortality rates than those with high miR-150 expression. The in vitro and in vivo assays of pancreatic cancer cells showed that miR-150 overexpression leads to reduced cell growth, clonogenicity, migration, invasion, modular cell cycles, and induced apoptosis. Moreover, miR-150 expression was inversely correlated with c-Myb and MUC4 activities in pancreatic tissue, cell lines, and nude mouse model. CONCLUSIONS: miR-150 is an important suppressor of pancreatic ductal carcinoma and acts as a regulator of c-Myb and MUC4 in aggressive progress.

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.

Diagnostic and biological significance of microRNA-192 in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of human cancer. In the present study, we evaluated serum microRNA-192 (miR-192) as a potential biomarker in patients with PDAC and investigated its biological functions in this disease. miRNA expression profiling of human PDACs and adjacent normal pancreatic tissues identified 16 upregulated miRNAs including miR-192 and 8 downregulated miRNAs. Quantitative real-time polymerase chain reaction (PCR) revealed elevation of serum miR-192 levels in PDAC patients relative to these levels in duodenal adenocarcinoma patients and healthy controls. Receiver operating characteristic analysis demonstrated that serum miR-192 had a sensitivity of 76% and a specificity of 55% for detecting PDAC. Ectopic expression of miR-192 in PANC-1 pancreatic cancer cells enhanced cell proliferation and migration, reduced apoptosis and promoted cell cycle progression from the G0/G1 to the S phase. Western blot analysis showed that enforced expression of miR-192 decreased the expression of smad-interacting protein 1 (SIP1) and altered a set of cell cycle-related genes in the PANC-1 cells. miR-192 overexpression increased tumor volume in an orthotopic pancreatic cancer mouse model, coupled with suppression of SIP1 and elevation of collagen I. In conclusion, serum miR-192 may serve as a sensitive diagnostic biomarker for PDAC. Overexpression of miR-192 contributes to tumor growth and progression in PDAC, which is associated with repression of SIP1 and alteration of cell cycle regulatory genes.

HDGF-related protein-3 is required for anchorage-independent survival and chemoresistance in hepatocellular carcinomas.

OBJECTIVE: Hepatoma-derived growth factor (HDGF)-related proteins (HRPs) comprise a family of six members and are characterised by a conserved HATH domain. Among the family members, HDGF was the first to be identified as a mitogenic factor and shown to play an important role in hepatocellular carcinoma pathogenesis. The aim of the present study is to examine the relevance of HDGF-related protein-3 (HRP-3), another member of the HRP family in hepatocellular carcinoma (HCC). DESIGN: HRP-3 expression in HCC tissues was measured by quantitative reverse transcriptase PCR, western blot and immunohistochemistry analysis. The biological consequences of overexpression and knockdown of HRP-3 in HCC cell lines were studied in vitro and in vivo. RESULTS: Expression of HRP-3 mRNA and protein was shown to be highly upregulated in HCC tissues. While knockdown of HRP-3 by small interference RNAs failed to affect anchorage-dependent growth of HCC cells, it inhibited anchorage-independent growth of HCC cells in vitro and xenograft tumour growth in vivo. Further, knockdown of HRP-3 was shown to sensitise HCC cells to anoikis. Moreover, HRP-3 specifically activated the extracellular-signal-regulated kinase (ERK) pathway without affecting c-Jun N-terminal kinase (JNK), p38, AKT and signal transducer and activator of transcription 3 (STAT3). Importantly, inhibition of the ERK pathway diminished HRP-3-mediated protection of HCC cells from anoikis. Finally, knockdown of HRP-3 was shown to enhance apoptosis of HCC cells induced by multiple chemotherapeutic drugs. CONCLUSION: These findings indicate that HRP-3 plays an essential role in HCC pathogenesis and suggest that it may serve as a novel prognostic marker and molecular target for development of drugs for treatment of HCC.

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.

Reduced expression of EphB2 is significantly associated with nodal metastasis in Chinese patients with gastric cancer.

AIMS: EphB2 is a member of the Eph receptor tyrosine kinase family that has been involved in the regulation of cytoskeleton organization and cell migration in various cell types. Its role and regulation in carcinogenesis is controversial, especially in gastric cancer. We detected EphB2 expression and determined its clinical significance and explored its underlying molecular mechanism in gastric cancers. METHODS: Tissue microarray blocks containing primary gastric cancer, lymph node metastases, and adjacent normal mucosa specimens obtained from 337 Chinese patients were constructed. Expression of EphB2 in these specimens was analyzed using immunohistochemistry. Mutation analysis at the A9 tract in exon 17 and loss of heterozygosity analysis at the EphB2 gene locus were carried out in 13 sporadic EphB2-negative gastric cancers. RESULTS: Complete loss of EphB2 expression was observed in 177 (52.5%) of the 337 primary tumor and 41 (82%) of the 50 nodal metastases. Loss of EphB2 expression was significantly associated with advanced T stage, nodal metastasis, advanced disease stage, and poor histological differentiation. Loss of EphB2 expression correlated significantly with poor survival rates in both univariate and multivariate analysis. No frameshift mutation, but a higher frequency of allelic loss, was found in EphB2-negative primary and metastatic tumor samples. CONCLUSIONS: Frequent deletion and decreased expression of EphB2 protein suggested it as a negative biomarker for gastric carcinogenesis and a potential predictor of the outcome of patients with gastric cancer.

[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.

Preliminary comparison of tumor biologic factors in breast carcinomas from Australian and Chinese women.

OBJECTIVE: To compare the morphologic and immunohistochemical properties of breast carcinomas from Chinese and Australian women in order to define possible biologic differences between these carcinomas. STUDY DESIGN: Three hundred cases of breast carcinomas were assessed for histologic and immunophenotypic characteristics from the pathology archives of the Changhai and St. Vincent's Hospitals. RESULTS: The Chinese women had proportionally more grade 2 and 3 tumors, whereas Australian women had a higher proportion of grade 1 tumor. There was a higher proportion of younger patients with a larger tumor and patients with lymph node involvement in the Chinese group as compared with Australian women. There was no difference in rate of estrogen receptor positive tumors between the 2 groups. p53 Expression was statistically more common with less cyclin D1 expression in Chinese as compared with Australian women. CONCLUSION: This study indicates that both inherent tumor biology and stage at presentation influence adversely affect the outcome of breast carcinoma in Chinese compared as with Australian women.

Microadenocarcinoma of the pancreas.

BACKGROUND: Microadenocarcinoma (MA) of the pancreas is a rare kind of neoplasm, whose status as an independent tumor entity is still a matter of controversy. METHODS AND RESULTS: In this article we investigated two patients with MA from the histological, immunohistochemical and genetic aspects. Morphologically, MA is composed of small, crowded microglandular structures, forming a cribriform pattern, sometimes solid sheets. Cells of MA were morphologically uniform and were less pleomorphic than those of the ductal adenocarcinoma. Immunohistochemistry revealed that MA, though with a certain extent of epithelial differentiation, possesses a different immunological phenotype from those of ductal carcinoma, acinar cell carcinoma, and endocrine tumors. Genetic analysis showed no abnormality of p53, K-ras, and beta-catenin, which were usually mutated in pancreatic ductal adenocarcinoma. CONCLUSION: Therefore, we suggest that MA should be taken as an independent tumor entity rather than a kind of growth pattern, but a final decision should be reached after cautious differential diagnosis of other kinds of pancreatic neoplasms.

[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.

HBV DNA can bind to P53 protein and influence p53 transactivation in hepatoma cells.

Hepatitis B virus (HBV) may contribute to hepatocarcinogenesis by blocking p53 function. A p53 response element-like binding sequences, TGCCT...TGCCT, was found in HBV genome. To clarify whether HBV DNA can, like some other DNA viruses, bind to P53 protein and form a DNA-protein complex, we used a series of plasmids encoding full-length or mutant HBV or p53 fragments to determine the binding ability of HBV DNA after cotransfected into cells by electrophoretic mobility shift (and supershift) assay. We found that HBV DNA could bind to P53 protein and form DNA-protein complexes in human hepatoma cell lines. Cotransfection with p53 and HBV DNA increased the replication of HBV, CAT activity, tumor cell apoptosis, and cytoplasmic P53 accumulation in the hepatoma cells. In conclusions, our observations suggest that the interaction of HBV and p53 at the levels of protein-protein and DNA-protein, which resulted in inactivation of p53 transactivation.

Human inhibitor of growth 1 inhibits hepatoma cell growth and influences p53 stability in a variant-dependent manner.

UNLABELLED: Inhibitor of growth 1 (ING1) is a type II tumor suppressor that affects cell function by altering chromatin structure and regulating transcription. Recently, three ING1 splice variants have been cloned, but their roles in apoptosis and p53 regulation in human hepatocellular carcinoma (HCC) have not been fully elucidated. The present study found that ING1, in a variant-dependent manner, inhibited hepatoma cell proliferation and colony formation, induced apoptosis and cell cycle arrest at G(0)/G(1) phase, and postponed tumor formation in nude mice. Expression of p33(ING1b) and p24(ING1c) variants, but not p47(ING1a), was markedly reduced in HCC samples. Reverse transcription polymerase chain reaction and western blotting analysis revealed that ectopic overexpression of p33(ING1b) or p24(ING1c) variant increased the expression of p53 downstream genes such as p21(waf1) and bax, and repressed bcl-2 expression (P < 0.01), whereas p47(ING1a) inactivated p21(waf1) promoter (P < 0.01). Furthermore, we found that p33(ING1b) and p24(ING1c) repressed Mdm2 expression (P < 0.01) and competed with Mdm2 for binding to p53. Interestingly, p33(ING1b)and p24(ING1c) did not directly bind to Mdm2 protein but strongly increased p14(arf) expression (P < 0.01) and interacted with p14(arf) protein to stimulate p53. Moreover, we found that ectopic overexpression of p33(ING1b) or p24(ING1c) significantly induced p53 protein acetylation at Lys-373/Lys-382 residue, but did not alter the phosphorylation status of p53. CONCLUSION: ING1 variants p33(ING1b) and p24(ING1c) may modulate p53 activity and subsequently inhibit hepatoma cell growth by at least two possible mechanisms: interacting with Mdm2 and p14(arf) to stabilize and activate p53, or increasing p53 acetylation.