Association between glutathione S-transferases M1 and T1 gene polymorphisms and esophageal cancer prognosi.

OBJECTIVE: To investigate the independent factors affecting the prognosis of patients after resection of esophageal cancer, and to inquire into the relationship between GSTM1, GSTT1 gene polymorphisms and esophageal cancer prognosis. METHODS: The clinical data of 273 patients with esophageal cancer were retrospectively analyzed. The patients were followed-up after their surgery, and the gene polymorphisms of GSTM1 and GSTT1 in each individual were detected by polymerase chain reaction (PCR). The clinical features along with the gene polymorphisms of GSTM1 and GSTT1 associated with the prognosis of patients were analyzed by using the method of univariate analysis and Cox proportional hazard model. The cumulative survival rate was estimated by Kaplan-Meier methods, and the survival curves were compared by using the log-rank test. RESULTS: The overall cumulative survival rate of first year, third year and fifth year is 94.6%, 58.5% and 17.8%, respectively. The median survival time (MST) is 38.7 months. The results of univariate analysis showed that: infiltration depth, length of tumor, the number of lymph node metastasis, the region of lymph node metastasis and the genetic polymorphism of GSTM1 and GSTT1 gene loci were associated with the survival of postoperative patients. Cox multivariate analysis further indicated that the length of tumor, the number of lymph node metastasis and the combined genotype (1) [GSTM1 (+/+) or (+/-) & GSTT1 (-/-)] were the independent prognostic factors. The length of tumor, the number of lymph node metastasis were the risk factors for the prognosis, and the combined genotype (1) had protective effect on survival when compared with reference [GSTM1 (+/+) or (+/-) & GSTT1 (+/+) or (+/-)]. CONCLUSION: The length of tumor, the number of lymph node metastasis were confirmed as the independent prognostic factors of esophageal carcinoma, and the null genotypes for GSTT1 (-/-) might be a protective factor for survival and GSTM1 (-/-) might be a potential negative prognostic factor in patients with esophageal cancer.

[Expression of RRM1 and ERCC1 genes in tumor tissues and peripheral blood lymphocytes of advanced non-small cell lung cancer].

OBJECTIVE: To investigate the expression of RRM1 and ERCC1 genes in tumor tissues and peripheral blood lymphocytes of advanced non-small cell lung cancer (NSCLC). METHODS: Tissue and peripheral blood samples were collected from 49 advanced NSCLC patients treated with gemcitabine plus carboplatin. The expressions of RRM1 and ERCC1 mRNA in tumor tissue and peripheral lymphocytes were detected by real-time fluorescent quantitative PCR. The relationship of gene expression with clinical characteristics,chemotherapy response and prognosis was analyzed. RESULTS: The RRM1 expression in tumor tissues was positively correlated with that in peripheral blood lymphocytes,while no significant correlation was observed between ERCC1 expression in tumor tissues and that in peripheral blood (rs=0.332 and 0.258; P=0.020 and 0.073, respectively). The expression of RRM1 and ERCC1 in tumor tissues peripheral lymphocytes was synchronous (rs=0.634 and 0.351; P<0.001 and 0.013, respectively). There was no significant correlation of gene expression with gender, age, smoking status, performance status, clinical stages and histological types of patients (P>0.05). Significant difference was found in response rate to chemotherapy (P<0.05,P<0.01,P<0.05),median survival time (P<0.05,P<0.01,P<0.05) and 1-year survival rate (P<0.01,<0.05,P<0.05) between patients with low RRM1 and ERCC1 expression levels in tumor tissues or low RRM1 expression levels in peripheral blood and those with high RRM1 and ERCC1 expression levels. The patients with low ERCC1 expression levels in tumor tissues gained higher 2-year survival rate (P<0.05). There was no correlation of the expression of ERCC1 in peripheral blood with the response to chemotherapy and prognosis (P>0.05). CONCLUSION: The expression of RRMI and ERCC1 genes in tumor tissues and RRM1 in peripheral blood lymphocytes is closely correlated with the response to chemotherapy and prognosis of patients with advanced NSCLC treated with gemcitabine plus carboplatin.

[Detection of RRM1, ERCC1 and BRCA1 gene expression in non-small cell lung cancer tissues and peripheral blood by SYBR real-time fluorescent quantitative PCR].

OBJECTIVE: To develop a method for the detection of RRM1, ERCC1 and BRCA1 gene expression by SYBR real-time fluorescent quantitative PCR in non-small cell lung cancer tissues and peripheral blood. METHODS: The plasmid standard of RRM1, ERCC1, BRCA1 and ß-actin genes was constructed. SYBR real-time PCR was performed, and the standard curve was established. The expressions of RRM1, ERCC1 and BRCA1 mRNA in non-small cell lung cancer tissues and peripheral blood were detected. RESULT: The standard curve presented linearity. The liquate curves of standard gene were all single apex, indicating that a good specificity was obtained. CONCLUSION: The developed SYBR real-time fluorescent quantitative PCR has advantage of convenient operation, low cost, good specificity and high veracity.

[Significance of K-ras detection in colorectal cancer].

OBJECTIVE: To determine the mutation status of K-ras gene in colorectal cancer and analyze the associations between its mutation status and clinicopathological characteristics in colorectal cancer so as to select the patients likely to benefit from a targeted therapy. METHODS: A total of 208 colorectal cancer tissue samples were collected from September 2008 to February 2009. DNA was extracted with a genomic DNA miniprep kit. Then PCR was performed with the designed primers and the product directly sequenced by the Sanger method. Then the associations between K-ras mutation status and clinicopathological characteristics in colorectal cancer were analyzed. RESULTS: Of 208 cases, 91 cases of K-ras gene mutation were detected. The 12 or 13 codon had a mutation rate of 43.8%. There were no significant differences in gender, tumor location, histopathological grading and Duke's stage between the wild and mutated groups. CONCLUSION: Detection of K-ras gene status in colorectal cancer will help to select the patients likely to benefit from the monoclonal antibody therapy of targeting epidermal growth factor receptor.

Construction and cellular immune response induction of HA-based alphavirus replicon vaccines against human-avian influenza (H5N1).

Several approaches are being taken worldwide to develop vaccines against H5N1 viruses; most of them, however, pose both practical and immunological challenges. One potential strategy for improving the immunogenicity of vaccines involves the use of alphavirus replicons and VP22, a herpes simplex type 1 (HSV-1) protein. In this study, we analysed the antigenic peptides and homogeneity of the HA sequences (human isolates of the H5N1 subtype, from 1997 to 2003) and explored a novel alphavirus replicon system of VP22 fused with HA, to assess whether the immunogenicity of an HA-based replicon vaccine could be induced and augmented via fusion with VP22. Further, replicon particles expressing VP22, and enhanced green fluorescent protein (EGFP) were individually used as controls. Cellular immune responses in mice immunised with replicons were evaluated by identifying specific intracellular cytokine production with flow cytometry (FCM). Animal-based experimentation indicated that both the IL-4 expression of CD4(+) T cells and the IFN-gamma expression of CD8(+) T cells were significantly increased in mice immunised with VPR-HA and VPR-VP22/HA. A dose titration effect vis-à-vis both IL-4 expression and IFN-gamma expression were observed in VPR-HA- and VPR-VP22/HA-vaccinated mice. Our results revealed that both VPR-VP22/HA and VPR-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza, and VP22 could enhance the immunogenicity of the HA antigens to which it is fused.

Effect of IFNalpha-2a on Fas expression and apoptosis rate of peripheral blood cytotoxic T cells in patients with hepatitis B.

BACKGROUND: Interferon(IFN) with antiviral and immunomodulatory activities is one of the most important therapeutic agents for the treatment of chronic hepatitis. The apoptotic effect of IFN is influenced by cell type and the types of IFN, which suppresses proliferation and induces apoptosis in some cell types while inhibiting apoptosis in others. The aim of this study was to explore the effect of IFNalpha-2a on Fas expression and the apoptosis rate of peripheral blood cytotoxic T cells(CTLs) in patients with hepatitis B. METHODS: Peripheral blood mononuclear cells were isolated from 26 patients with hepatitis B including 16 patients with chronic hepatitis B and 10 patients with chronic severe hepatitis B. Fas expression and apoptosis rate of CTLs were analyzed with flow cytometry before and after IFNalpha-2a treatment. RESULTS: Before IFNalpha-2a treatment, Fas expression and apoptosis rate of CTLs from patients with chronic hepatitis B were significantly higher than those from patients with chronic severe hepatitis B and healthy controls respectively. No significant difference was observed between Fas expression and apoptosis rate of CTLs from patients with chronic severe hepatitis B and healthy controls. After IFNalpha-2a treatment, Fas expression and apoptosis rate of CTLs from different groups were compared with those before IFNalpha-2a treatment, showing no significant difference despite alternation of different degree. CONCLUSIONS: Activation induced cell death (AICD) exists in peripheral blood CTLs from patients with hepatitis B. No effect of IFNalpha-2a exerts on Fas expression and apoptosis rate of Fas in patients with hepatitis B.

Character of HBV (hepatitis B virus) polymerase gene rtM204V/I and rtL180M mutation in patients with lamivudine resistance.

OBJECTIVES: To investigate the relationship between HBV (hepatitis B virus) polymerase gene 180 and 204 sites mutation and lamivudine resistance. METHODS: One hundred forty-one patients with lamivudine resistance after lamivudine treatment and 60 chronic hepatitis B patients without lamivudine treatment were enrolled in this study. The serum HBV DNA mutation was analyzed by sequence detection via polymerase chain reaction (PCR). The sequences of the same patient were analyzed before and after lamivudine treatment. RESULTS: One hundred and nine lamivudine resistance patients had HBV YMDD (tyrosine-methionine-aspartate-aspartate) mutation. Among them, 45 patients had rtL180M/M204V mutation (41.28%), 28 patients had rtL180M/M204I mutation (25.70%) and 36 patients had rtM204I mutation (33.02%). There were 6 patients with rtL180M mutation in 32 lamivudine resistance patients. Sixty chronic hepatitis patients without lamivudine treatment had no mutations. CONCLUSIONS: HBV mutations, which play an important role in lamivudine resistance usually locate at polymerase gene 204 site; 180 site mutation was also observed in these patients. Evaluation of the anti-virus therapy by surveillance of the two sites mutations is of importance.

[Involvement of TRAIL up-regulation of CD4+, CD8+ T cells in liver injury in chronic hepatitis B].

OBJECTIVE: To describe a novel mechanism for TRAIL up-regulation of CD4+, CD8+ T cells to participate in the pathophysiological process in patients with chronic hepatitis B (CHB). METHODS: The serum levels of soluble TRAIL (sTRAIL), IFN-gamma and membrane bound TRAIL expression on peripheral leucocytes from 58 CHB patients were examined by ELISA and flow cytometry respectively. The levels of TRAIL were compared with the baseline levels of 15 healthy controls, and correlation analysis were performed between ALT, TBil and PT, morphological change in hepatic tissues. RESULTS: The results showed that TRAIL levels on membranes of CD4+, CD8+ T cells in CHB patients were much higher than the healthy controls (P < 0.001), which of CD4+ T cells positively correlated with serum TBil (r=0.354, P = 0.008), Serum IFN-gamma level (r=0.302, P = 0.011) and which of CD8+ T cells positively correlated with serum TBil (r=0.522, P = 0.000), ALT (r=0.393, P = 0.003), PT (r=0.385, P = 0.004), serum IFN-gamma level (r=0.307, P = 0.009). The serum levels of soluble TRAIL only correlated with serum HBeAg expression (r=0.695, P = 0.001). CONCLUSION: These findings suggest that the expression of TRAIL on the membranes of lymphocytes was up-regulated, which may take part in the immunopathogenesis in CHB patients. TRAIL expression can be induced either by virus-specific protein expression or by inflammation cytokine IFN-gamma

[Effect of fragile histidine triad gene on apoptosis of MGC-803 gastric cancer cells].

OBJECTIVE: To investigate the effect of fragile histidine triad (FHIT) gene on the apoptosis of gastric cancer cells. METHODS: FHIT gene was transfected into gastric cancer cell line MGC-803 by liposome. Western blot and immunohistochemical assay were employed to detect the expression of FHIT. Apoptosis and cell cycle were analyzed by flow cytometry (FCM). Morphological changes were observed by light and electron microscope. RESULTS: Stable expression of FHIT protein in transfected MGC-803 cells was obtained. The rates of G0/G1 phase and apoptosis cells were significantly higher in FHIT-transfected MGC-803 cells than those in control cells (64.4%compared with 49.5%and 32.0%compared with 12.2%, respectively). CONCLUSION: The results indicate that FHIT gene might be involved in the apoptosis of gastric cancer cells.

Expression of co-stimulator 4-1BB molecule in hepatocellular carcinoma and adjacent non-tumor liver tissue, and its possible role in tumor immunity.

AIM: To investigate the expression of 4-1BB molecule in hepatocellular carcinoma (HCC) and its adjacent tissues. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the gene expression of 4-1BB in hepatocarcinoma and its adjacent tissues, and peripheral blood mononuclear cells (PBMCs) from both HCC and health control groups. Flow cytometry was used to analyse the phenotypes of T cell subsets from the blood of HCC patients and healthy volunteers, and further to determine whether 4-1BB molecules were also expressed on the surface of CD4+ and CD8+ T cells. The localization of 4-1BB proteins on tumor infiltrating T cells was determined by direct immunofluorescence cytochemical staining and detected by confocal microscopy. RESULTS: 4-1BB mRNA, which was not detectable in normal liver, was found in 19 liver tissues adjacent to tumor edge (<1.0 cm). Low expression of 4-1BB mRNA was shown in 8 tumor tissues and 6 liver tissues located within 1 to 5 cm away from tumor edge. In PBMCs, 4-1BB mRNA was almost not detected. Percentage of CD4+, CD8+ and CD3+/CD25+ T cells, as well as ratio of CD4 to CD8 revealed no difference between groups (P>0.05, respectively), while a significant lower percentage of CD3+ T cell was found in HCC group as compared to healthy control group (P<0.05). However, 4-1BB molecules were almost not found on the surface of CD4+ and CD8+ T cells in HCC and healthy control group. Double-staining of 4-1BB+/CD4+ and 4-1BB+/CD8+ immunofluorescence on tumor infiltrating T cells was detected in 13 liver tissues adjacent to tumor edge (<1.0 cm) by confocal microscopy. CONCLUSION: Although HCC may escape from immune attack by weak immunogenicity or downregulated expression of MHC-1 molecules on the tumor cell surface, tumor infiltrating T cells can be activated via other costimulatory signal pathways to exert a limited antitumor effect on local microenvironment. The present study also implicates that modulating 4-1BB/4-1BBL costimulatory pathway may be an effective immunotherapy strategy to augment the host response.

Expression of 4-1BB molecule on peripheral blood T cells in liver transplanted patients and its clinical implication.

OBJECTIVE: To investigate the gene expression of 4-1BB in peripheral blood mononuclear cells (PBMCs) and its possible significance in clinical liver transplantation. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the gene expression of 4-1BB in PBMCs from 22 patients receiving liver transplantation, 13 patients with primary liver carcinoma (PLC), and 12 healthy controls. To determine whether 4-1BB molecule is also expressed on the surface of CD4+ and CD8+ T cell, flow cytometry was used to analyse the phenotype of T cell subsets from the blood of liver transplantation patients. RESULTS: 4-1BB mRNA was detected in PBMCs from stable survivors after liver transplantation, but almost not detected in PBMCs from PLC patients and healthy controls. Meanwhile, 4-1BB was almost not expressed on the surface of CD4+ and CD8+T cells in healthy controls and PLC patients. A low level of 4-1BB expression, however, was found on the surface of CD4+ and CD8+ T cells from the stable survivors after liver transplantation. CONCLUSIONS: This study demonstrates that although patients are stable after liver transplantation, effector T-cells can also be activated through the signal of 4-1BB molecule and persistent immune response to grafts. Blockage of 4-1BB/4-1BBL pathway may benefitially reduce the clinical dosage of immunosuppressive agents and prolong the survival of grafts.

[Influence of combined cyclosporine A and tacrolimus with 5-fluorouracil on hepatocellular carcinoma rats].

OBJECTIVE: To investigate the effect of combined CsA and FK506 with 5-FU on hepatocellular carcinoma rats. METHODS: A syngeneic rat model of hepatocellular carcinoma was used. Control group (A) underwent 4 ml 5% GS. Treatment group was divided into 3 groups namely, group B: only 5-FU and 5% GS; group C: 5-FU, CsA and 5% GS; group D: 5-FU, FK506 and 5%GS. Cell cycle, apoptosis, necrosis and mitochondrial transmembrane potential were measured by flow cytometry, laser scanning confocal microscopy, and electron transmission microscopy. Statistical analysis was performed by SPSS 10.0 for Windows software. Statistical comparisons were made with ANOVA followed by Dunnett's T3 or LSD test. RESULTS: Compared to the control group, the percentage of apoptotic cells including trifle necrotic cells was significantly higher, and among the treatment group, group D was the highest, and group C was higher than group B. In the treatment group, cell cycle of hepatoma cells was mainly arrested at S phase, but in group D, G0/G1 phase cells were significantly decreased and S phase cells significantly increased. Compared to the control group, mitochondrial transmembrane potential was significantly decreased in the treatment group, among with, group B was the lowest, group C was higher than group D. Morphological changes demonstrated by electron microscopy included dispersed nuclear chromatin, loss of nucleoli, membrane bleeding, cell shrinkage, typical apoptotic bodies and marked swelling of mitochondria in the treatment group. In the control group, however, they were characterized by normal cell ultrastructure. CONCLUSIONS: The present study reveals that 5-FU combined with CsA or FK506 demonstrated a synergistic effect on hepatocellular carcinoma rats. For FK506, the powerful mutual effect is related to the increase of tumor cell's quantity in S phase. Both CsA and FK506 can provide protection on mitochondrial transmembrane potential reduction against hepatoma cells damage from 5-FU.