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.

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.

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.

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.

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.

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.

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.

[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.

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.

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.

[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.

[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.

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.

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.

[Significance of p33(ING1b) and p53 gene expression in hepatocellular carcinoma].

OBJECTIVE: To explore the significance of p33(ING1b) protein expression and p53 protein expression and investigate the mutation rate of ING1b gene in hepatocellular carcinoma (HCC). METHODS: Immunohistochemical method was employed to detect the expressions of p33(ING1b) and p53 proteins and the X antigen of hepatitis B virus (HBV) in 57 cases of HCC, 51 cases of surrounding non-tumor tissues surrounding HCC, and 12 cases of normal liver tissues; PCR-single strand conformation polymorphism (SSCP) was applied to reveal the mutation rates of two exons of ING1b gene in HCC. DNA fragments exhibiting abnormal shifting were cloned into PMD18-T vector and sequenced. RESULTS: The positive rates of p33(ING1b) protein and of p53 protein were 42.1% and 57.9% respectively among the 57 cases of HCC. Both p33(ING1b) protein and p53 protein were positive in 19 cases (33.3%) of HCC tissues, and they were distributed in the same areas, mostly in the nucleus. The expression of p33(ING1b) was significantly correlated with that of p53 protein (r = 0.783, P < 0.01). The matched surrounding non-tumor tissues and normal liver tissues all showed negative p53 staining, while weak positive expression of p33(ING1b) was observed in 13 cases out of the 51 non-tumor tissues and 2 cases out of 12 normal liver tissues. Thirty-five cases, all with hepatocellular carcinoma, out of the 57 HCC cases showed positive HBV X protein (76.4%). The correlation of HBV infection with the expression of p33(ING1b) and p53 proteins was significant (P < 0.01). PCR-SSCP showed that the mutation rate of ING1b gene in HCC was 7.1% (2/28). CONCLUSION: The expression of p33(ING1b) protein is significantly related to the expression of p53 protein in hepatocellular carcinoma; The existence of p33(ING1b) may have some other p53-independent pathway; Gene mutation was not the main reason that ING1b loses its tumor suppressing function in HCC. HBV infection has a close relationship with p33(ING1b) and p53 proteins expression.

[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.

[Expression of TTF-1 in thyroid tumors originating from follicular epithelium and its correlation with expression of RET, galectin-3 and mucin-1 genes].

OBJECTIVE: To explore the expression of thyroid transcription factor-1 (TTF-1) in thyroid tumors and in different parts of follicular epithelium and the correlation of expression of TTF-1 with the expression of RET, galectin-3 (Gal-3) and mucin-1 (MUC1) genes. METHODS: One hundred thirty-three samples of resected thyroid tumors were examined by streptavidin/peroxidase (S-P) immunohistochemical technique to detect the expression of TTF-1 and RET, Gal-3, and MUC1 genes. RESULTS: (1) TTF-1 was expressed in the nuclei and cytoplasm of different kinds of tumor cells and normal cells around the tumors. There was no significant difference between the expression of TTF-1 in nuclei of malignant thyroid tumor cells and that in nuclei of benign thyroid tumor cells (P > 0.05). The positive expression rate of TTF-1 was 27.40% in cytoplasm of benign tumor and was 66.10% in cytoplasm of malignant tumor was 66.10% (P < 0.05). (2) The expression rate of RET gene was 38.9% in thyroid adenoma, 88.4% in papillary carcinoma, and 87.5% in follicular carcinoma. The difference of expression rate of RET gene between adenoma and both of papillary and follicular carcinomas was statistically significant (P < 0.05). (3) The expression rate of MUC1 was 23.3% in benign tumor and 50.9% in malignant tumor. The expression rate of MUC1 was lower in benign thyroid diseases (nodular goiter and adenoma) than in thyroid papillary carcinoma and follicular carcinoma (P < 0.05). (4) The expression rate of Gal-3 gene was 38.4% in benign tumor and 88.1% in malignant tumor. (5) The expression rates of Gal-3 and MUC1 in benign lesions (nodular goiter and adenoma) were lower than those in thyroid papillary and follicular carcinomas (P < 0.05). The expression of Gal-3 and that of MUC1 in thyroid carcinomas were fundamentally parallel. However, the sensitivity of Gal-3 was higher than that of MUC1. In part of adenomas Gal-1 and MUC1 were positive locally simultaneously. The expression of TTF-1 in cytoplasm was associated with that of Gal-3 and MUC1. CONCLUSION: The expression of TTF-1 in nucleus is a phenomenon common to both normal and pathological thyroid follicles. Combined tests of RET, Gal-3 and MUC1 may act as the diagnostic markers to distinguish benign follicular tumor from malignant ones. Expression of TTF-1 in cytoplasm is probably a characteristic of malignant phenotypes of thyroid tumors.

[p33(ING1) gene expression and mutation in stomach cancer tissues and precarcinomatous tissues].

OBJECTIVE: To analyze the relationship of p33(ING1) gene expression and p33(ING1) exon-2 mutation to the pathogenesis, development and consequence of stomach cancer. METHODS: Envision immunohistochemical method was utilized to detect the p33(ING1) expression in 103 specimens of stomach cancer, 36 specimens of stomach mucosal atypical hyperplasia, and 32 specimens of normal stomach mucosa. PCR-SSCP was utilized to detect p33(ING1) exon-2 mutation in stomach cancer tissues. RESULTS: The p33(ING1) expression rate in stomach cancer was 54.4% (56/103), significantly lower than that in precarcinomatous tissues (94.4%, 34/36, P < 0.01) and that in normal tissues (100%, 32/32, P < 0.01). The p33(ING1) expression in stomach cancer was related to tumor growth, distant metastasis and tumor differentiation (all P < 0.05). p33(ING1) gene exon-2 mutation was detected in 3 cases of stomach cancer tissues (12%, 3/25), and not in other tissues by PCR-SSCP method. CONCLUSION: p33(ING1) low expression, and gene p33(ING1) exon-2 mutation may play an important role in the pathogenesis, development and consequence of stomach cancer.

[Expression of midkine and its relationship with HBV infection in hepatocellular carcinomas].

OBJECTIVE: To investigate expression of midkine (MK) in hepatocellular carcinoma (HCC) and its relationship with HBV infection. METHODS: Expression of MK mRNA, MK protein and HBV DNA were determined in 62 cases of human HCC and 10 cases of controls with in situ hybridization and immunohistochemistry. RESULTS: Positive rates of MK mRNA and MK protein in carcinoma tissue were 74.2% and 75.8% respectively, which were higher than in paratumorous liver tissue and normal controls (P < 0.01). Positive rate of HBV DNA in the nuclei of carcinoma tissue (62.9%) were higher than in paratumorous liver tissue (6.5%) (P < 0.01). There was a significant relationship between expression of MK mRNA and MK protein in the carcinoma tissue and HBV DNA in the nuclei of carcinoma tissue. CONCLUSIONS: HCC overexpresses MK at the mRNA and protein level. The overexpression of the MK protein might be correlated with the existence of HBV DNA in the nuclei of carcinoma tissue.