[Effects of hMIP-1beta gene modification on in vivo tumorigenicity and vaccine efficacy of tumor cells].

UNLABELLED: OBJECTIVE To explore the effects of human macrophage inflammatory protein-1 beta (hMIP-1beta) modification on the in vivo tumorigenicity and vaccine efficacy of tumor cells. METHODS: Murine colorectal adenocarcinoma CT26 cells were transfected with a recombinant adenovirus carring the hMIP-1beta gene (AdhMIP-1beta). The efficacy of gene transfection was tested by X-gal staining. The hMIP-1beta level in the supernatant of hMIP-1beta gene-modified CT26 cells was assayed by ELISA, and the chemotactic activity for CD4+ T cells, CD8+ T cells, NK cells and immature dendritic cells (imDCs) were assayed by a transwell chamber. The changes of growth characteristics and in vivo tumorigenicity of hMIP-1beta gene-modified CT26 cells were also assessed. BALB/c mice were immunized with hMIP-1beta gene-modified CT26 tumor vaccine and the antitumor effect was evaluated. RESULTS: hMIP-1beta gene could be transfected into CT26 cells by AdhMIP-1beta with an efficiency over 95%. The level of hMIP-1beta in the culture supernatant of hMIP-1beta gene-modified CT26 cells was 980 pg/ml and the supernatant displayed ramarkable chemotactic activity to CD4+ T cells, CD8+ T cells, NK cells and imDCs compared with LacZ gene-modified CT26 cells and control. When the hMIP-1beta gene-modifited CT26 cells were subcutaneously inoculated in BALB/c mice, the tumorigencity was delayed and suppressed, and overt necrosis and lymphocyte infiltration were observed in the tumor tissue, but not in those inoculated with LacZ gene-modified CT26 cells or parental CT26 cells. The mice immunized with hMIP-1beta gene-modified CT26 tumor vaccine could induce tumor specific CTL activity and nonspecific NK activity, and exhibited resistance to later challenge with wild-type CT26 cells. CONCLUSION: hMIP-1beta gene-modified CT26 cells exhibit decreased tumorigenicity, and hMIP-1beta gene-modified tumor vaccine may induce a powerful specific and nonspecific antitumor response. The data suggested that hMIP-1beta gene-modified tumor vaccine may play a potent role in prevention of metastasis and recurrence of malignant tumors.

[Dendritic cell vaccine modified by murine mAFP gene enhances immunoprotective effect on liver carcinogenesis and tumor development in mice].

OBJECTIVE: To construct a dendritic cell vaccine transduced by murine alpha-fetoprotein (mAFP) gene, and evaluate its immunoprotective effect on C57BL/6J mice during the induction of hepatocellular carcinoma by diethylnitrosamines, carbon tetrachloride and ethanol. METHODS: Dendritic cells (DCs) were induced and augmented by murine IL-4 and GM-CSF, and transfected by recombinant adenovirus engineered with mAFP gene. Major MHC class I and II, B7.1 (CD80), B7.2 (CD86), CD18a, and CD54 molecules on DC were analyzed by FACS. 80 C57BL/6J male mice were randomly divided into 4 groups (20 mice per group): Simple DC inoculated group, pAdBM5-mAFP-DC inoculated group, pAdBM5-mAFP plasmid inoculated group, and PBS control group. They were immunized once with 5 x 10(5) DCs (0.1 ml)/mouse administered s. c. in the left flank or 100 mg pAdBMS-mAFP plasmid/mouse administered i. m. in the left tibialis anterior muscle. Inoculation was conducted once a week for 4 weeks after 3 times consecutive immunization initially. At the same time of immunization, DEN/CCl4/ethanol were given to induce hepatocellular carcinoma. Tumor incidence was assessed after 20 weeks. RESULTS: A transgenic DC vaccine was successfully constructed and the mAFP transgenic DCs expressed high level molecules of major MHC class I and II , B7.1, B7.2, CD18a, and CD54. After the 20-week induction, the incidence of primary hepatocellular carcinoma (PLC) was 70.0% in simple DC inoculated group, 25.0% in pAdBMS-mAFP-DC inoculated group, 65.0% in pAdBM5-mAFP plasmid inoculated group, and 75.0% in PBS control group. There was a significant difference between group B and other groups (P < 0.05). CONCLUSION: mAFP transgenic DC tumor vaccine inoculation may induce strong immunoprotection against liver carcinogenesis and tumor development and reduce PLC incidence induced by DEN/CCl4/ethanol.

A reversed-phase high performance liquid chromatography method for quantification of methotrexate in cancer patients serum.

A simple, rapid and sensitive reversed-phase high performance liquid chromatography (HPLC) method has been developed for the determination of methotrexate in human serum. After deproteinization of the serum with 40% silver nitrate solution, methotrexate and internal standard (IS) were separated on a reversed-phase column with a mobile phase consisting of 10mM sodium phosphate buffer (pH6.40)-methanol (78:22%, v/v) and ultraviolet detection at 310nm. The linearity is evaluated by a calibration curve in the concentration range of 0.05-10.0µg/mL and presented a correlation coefficient of 0.9995. The absolute recoveries were 97.52±3.9% and 96.87±3.7% for methotrexate and ferulic acid (internal standard), respectively. The intra- and inter-day precision were less 6.19 and 5.89%, respectively (n=6). The limit of quantitation was 0.02µg/mL and the limit of detection was 0.006µg/mL. The complete analysis was achieved less than 10min with no interference from endogenous components or 22 examined drugs. This method was validated by using serum samples from high-dose methotrexate treated patients with osteosarcoma, breast cancer, acute leukemia and lymphoma. The method was demonstrated to be a simple, rapid and reliable approach in quantification of methotrexate in serum samples from patients with high-dose methotrexate therapy.

Two-stage model of chemically induced hepatocellular carcinoma in mouse.

The aim of this study was to develop an efficient and reproducible mouse model for hepatocellular carcinoma (HCC) research and assess the expression of two proto-oncogenes (c-myc and N-ras) and tumor suppressor gene p53 in the carcinogenic process. In this study, we found that diethylnitrosamine initiation with CCl4 and ethanol promotion could induce a short-term, two-stage liver carcinogenesis model in male BALB/c mice, the process of hepatocarcinogenesis including liver damage, liver necrosis/cell death, liver inflammation, liver proliferation, liver hyperplasia, liver steatosis, and liver cirrhosis and hepatocellular nodules, which mimicked the usual sequence of events observed in human HCC. We also identified that the increase in expression of the p53 gene is related to the proliferation of hepatocytes, whereas overexpression of the c-myc and N-ras genes is associated with hepatocarcinogenesis. This animal model may serve as a basis for recapitulating the molecular pathogenesis of HCC seen in humans.