[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.

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.

[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.

[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.

[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.

[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.

[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.

[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.

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

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

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

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

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

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

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

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

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

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