OBJECTIVETo establish a syngeneic mouse model of liver tumor stably expressing hepatitis B virus (HBV) antigens.

METHODSMelanoma cell line B16 cells were transfected with pLXSN-2HBV. Cells (named B16/HBV) stably and persistently expressing HBV surface (HBsAg) and core (HBcAg) antigens were identified. The cells were injected into the hepatic subcapsular space of fifteen C57BL/6J mice. The mice were divided into 3 groups, receiving 100, 1000 or 5000 cells in a total volume of 5 µl per mouse, respectively, five mice in each group. Two weeks after the tumor cell inoculation, serum samples from the mice were collected weekly and the serum concentration of HBsAg and anti-HBs was quantified by ELISA. The tumor growth in the mouse liver was monitored by a high-resolution ultrasound system. Expression of HBsAg and HBcAg in the tumor tissues was determined by immunohistochemistry.

RESULTSLiver tumors were formed in all the mice receiving 1000 and 5000 B16/HBV cells per mouse, and in 80% of the mice receiving 100 B16/HBV cells. HBsAg and anti-HBs were detectable in their sera from 2 weeks after tumor cell inoculation. The mice receiving 100 cells per mouse began to die 4 weeks, those receiving 1000 cells per mouse began to die 3 - 4 weeks and those receiving 5000 cells began to die 2 - 3 weeks after the cell inoculation. All the tumor cells expressed HBsAg and HBcAg.

CONCLUSIONSThe B16/HBV cells stably and persistently express HBV antigens both in vitro and in vivo. A mouse model of transplanted liver tumor stably expressing HBV antigens has been successfully established by inoculation of those cells into the hepatic subcapsular space.

Animals ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; metabolism ; Liver Neoplasms, Experimental ; immunology ; virology ; Melanoma, Experimental ; metabolism ; pathology ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo investigate the antitumor immune response induced by dendritic cells vaccine coding AFPcDNA fragment with signal peptide (AFP(1)) and without signal peptide (AFP(2)), and to determine the inhibiting effect of the vaccine on the growth of hepatocarcinoma xenograft in Balb/c mice.

METHODSpcDNA3.1/AFP(1) and pcDNA3.1/AFP(2) were transfected into dendritic cells (DCs) by calcium phosphate nanoparticles and became DCs vaccine. Mouse spleen lymphocytes were stimulated by AFP(1)/DC and AFP(2)/DC. A Balb/c mouse model bearing mouse HCC xenograft was established on the day 14 after transplantation. Forty mice were divided equally into AFP(2)/DC group, AFP(1)/DC group and plasmid control group. The treated mice received DCs vaccine and the same amount of control plasmid.

RESULTSAFP(2)/DC stimulated T lymphocytel proliferation in vitro and improved CTL activity. The effects were better than AFP(1)/DC. The tumor-bearing mice injected intralesionally with AFP(1)/DC and AFP(2)/DC at a dose of 0.5 ml per mouse showed inhibition of tumor growth and prolongation of survival time. The tumor inhibition rate of the AFP(2)/DC group was 79.2% and the AFP(1)/DC group was 39.7% at 2 weeks after treatment. The tumor volume of AFP(2)/DC group was (726.7 +/- 298.2) mm(3), significantly smaller than the (1486.2 +/- 457.2) mm(3) of the AFP(1)/DC group and (2137.2 +/- 547.2) mm(3) of the plasmid control group (P < 0.05). The mean survival time of mice in the AFP(2)/DC group [(58.5 +/- 4.2) d] and AFP(1)/DC group [(45.2 +/- 4.8) d] were significantly longer than that of plasmid control group [(30.6 +/- 6.2) d, P < 0.05]. Bax-positive cell percentage was increased in the xenografts of AFP(2)/DC-treatment group compare with that of plasmid control group.

CONCLUSIONAFP(2)/DC and AFP(1)/DC vaccines show evident inhibiting effect on the growth of H22 xenograft in Balb/c mice through inducing efficient and specific immune response against the hepatocarcinoma cells.

Animals ; Calcium Phosphates ; pharmacology ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Cell Proliferation ; DNA, Complementary ; genetics ; immunology ; Dendritic Cells ; immunology ; Immunization ; Liver Neoplasms, Experimental ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Nanoparticles ; Neoplasm Transplantation ; Peptide Fragments ; Spleen ; cytology ; T-Lymphocytes ; pathology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; alpha-Fetoproteins ; genetics ; immunology

OBJECTIVETo investigate the effects of dendritic cells (DCs) infected with adenovirus vector encoding mTERT on induction of mTERT antigen specific immunity against H22 hepatoma in vivo.

METHODSForty Bal B/c mice were subcutaneously immunized with Ad-mTERT infected DC. Cytotoxicity of mTERT specific CTL was determined by 51Cr release assay. IL-2 and IFN-gamma were tested by ELISA. IFN-gamma ELISPOT assays were performed for measuring antigen specific IFN-gamma production by T cells. Tumor size and survival of the immunized mice were recorded and evaluated whether preexisting hepatoma metastases could be supressed after immunization with mTERT-expressing DCs.

RESULTSThe lytic activity of CTL, IL-2 (871.25 pg/ml), IFN-gamma (169.15 ng/ml) and IFN-gamma secreting cells (378/10(6) spleen cells) elicited by the Ad-mTERT infected DCs were much stronger and higher than that by Ad-GFP group (131.6 pg/ml, 15.4 ng/ml, 36/10(6) spleen cells, P<0.05), DC group (71.3 pg/ml, 10.5 ng/ml, 21/10(6) spleen cells, P<0.05), PBS group (65.8 pg/ml, 7.4 ng/ml, 18/10(6) spleen cells, P<0.05). In prophylaxis and treatment experiment the Ad-mTERT/DCs immunized mice lived significantly longer than other groups, demonstrating that primary DCs were genetically modified to express the mTERT antigen and could suppress the tumor growth.

CONCLUSIONAdenovirus vector mediated mTERT infected DCs can effectively induce mTERT antigen specific antitumor activity, and can induce protective and therapeutic antitumor immunity.

Adenoviridae ; genetics ; Animals ; Cell Line, Tumor ; Dendritic Cells ; immunology ; metabolism ; Female ; Genetic Vectors ; Immunization ; Interferon-gamma ; Interleukin-2 ; Liver Neoplasms, Experimental ; immunology ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Recombinant Proteins ; genetics ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Telomerase ; immunology ; metabolism ; Tumor Burden

OBJECTIVETo study the immunological suppressing effect of recombinant adenovirus vector rAD-mTERT promotor-m4-1BBL (rAD-mTERT) on mouse hepatoma cell line Hepa1-6 cells in co-culture with T lymphocytes.

METHODSAdding recombinant adenovirus rAD, rAD-CMV-m4-1BBL (rAD-CMV) and rAD-mTERT to Hepa1-6 and L929 cells, respectively, to observe the effect of these adenoviruses on growth and apoptosis of these cells in co-culture with T lymphocytes.

RESULTSAdding adenovirus significantly suppressed the growth and slightly increased apoptosis of the two types of cells (P < 0.05). rAD-mTERT promotor-m4-1BBL showed only pro-apoptotic effect on Hepa1-6 cells. When co-cultured with T lymphocytes, rAD-CMV-m4-1BBL showed promoting effect on apoptosis of the cells. Compared with that of T cells pre-co-culture, CD4(+) and CD8(+) T cells were proliferated, and the ratio of CD4/CD8 was significantly reduced (from 1.27 to 1.08).

CONCLUSIONAdding the recombinant adenoviruses only suppresses the cell growth, but not promotes their apoptosis. In co-culture with T lymphocytes, recombinant adenovirus vector rAD-mTERT promotor-m4-1BBL can targetingly suppress the growth and induce apoptosis of Hepa1-6 cells. The apoptosis is induced through the immunological killing effect of T lymphocytes.

4-1BB Ligand ; genetics ; physiology ; Adenoviridae ; genetics ; Animals ; Apoptosis ; CD4-CD8 Ratio ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; Coculture Techniques ; Fibroblasts ; cytology ; Genetic Vectors ; Liver Neoplasms, Experimental ; immunology ; pathology ; Mice ; Mice, Inbred C57BL ; Promoter Regions, Genetic ; Recombinant Proteins ; genetics ; T-Lymphocytes ; immunology ; Telomerase ; genetics ; Transfection

OBJECTIVETo 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.

METHODSDendritic 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.

RESULTSA 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).

CONCLUSIONmAFP transgenic DC tumor vaccine inoculation may induce strong immunoprotection against liver carcinogenesis and tumor development and reduce PLC incidence induced by DEN/CCl4/ethanol.

Adenoviridae ; genetics ; Animals ; B7-1 Antigen ; metabolism ; Cancer Vaccines ; Carbon Tetrachloride ; Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; metabolism ; Diethylnitrosamine ; Ethanol ; Genetic Vectors ; Histocompatibility Antigens Class I ; metabolism ; Histocompatibility Antigens Class II ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Liver Neoplasms, Experimental ; chemically induced ; immunology ; prevention & control ; Male ; Mice ; Mice, Inbred C57BL ; Random Allocation ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; alpha-Fetoproteins ; genetics ; metabolism

OBJECTIVETo study the relationship between the change of regulatory T cell number in CD4+ T subset and the growth of tumor in H22 hepatocellular carcinoma-bearing mice.

METHODSTumor-bearing mice were established by subcutaneous inoculation of H22 hepatocelluler carcinoma cells. Flow cytometry was used to detect the expression of CD4 and CD25 molecules of the T cells which came from the tumor-bearing mice. The Foxp3 gene expression was detected by RT-PCR and flow cytometry. CD4+ CD25+ T cells and CD4+ CD25- T cells were separated and purified by immuno-magnetic beads. The proliferation and suppressive function of the CD4+ CD25+ T cells coming from tumor-bearing mice was measured by [3H]-thymidines incorporation experiment in vitro, and then effect of CD4+ CD25+ T cells originated from hepatocellular carcinoma-bearing mice on tumor growth was observed in vivo.

RESULTS(1) Compared with mice of the control group, the percentage of CD4+ CD25+ T cells of CD4+ T cells in tumor-bearing mice is not only higher in draining lymph nodes (18.80% < or = 0.06%) vs. (9.50% +/- 0.03%), (P < 0.01), but also higher in non-draining lymph nodes (LN) and spleen (SP), LN: (16.28% +/- 0.02%) vs. (9.50% +/- 0.03%), P < 0.01; SP: (17.28% +/- 0.06%) vs. (11.08% +/- 0.04%), (P < 0.05). The expression of regulatory T cell specific marker Foxp3 gene was also increased. In the same tumor-bearing mice, the number of CD4+ CD25+ T cells in draining lymph node was relatively higher than the contralateral nondraining lymph node, but the difference was statistically not significant (18.8% +/- 0.06%) vs. (16.28% +/- 0.02%), (P > 0.05). (2) The CD4+ CD25+ T cells purified from tumor-bearing mice--like naturally occurring regulatory T cells--were anergic to anti-CD3 monoclonal antibody stimulation in vitro, but it could suppress CD4+ CD25- T cells proliferation. (3) The percentage of CD4+ CD25+ T cells was positively related to tumor size. It could also suppress the anti-tumor effect of CD4+ CD25- T cells in vivo. Conclusion The growth of hepatocellular carcinoma in mice can boost the amount of regulatory T cells. The amount of regulatory T cells is positively related to tumor size, indicating that attack on regulatory T cells could be used as one of modalities in cancer treatment in the future.

Animals ; CD4 Antigens ; immunology ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Female ; Flow Cytometry ; Forkhead Transcription Factors ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Interleukin-2 Receptor alpha Subunit ; immunology ; Liver Neoplasms ; immunology ; metabolism ; pathology ; Liver Neoplasms, Experimental ; immunology ; metabolism ; pathology ; Lymph Nodes ; immunology ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Reverse Transcriptase Polymerase Chain Reaction ; Spleen ; immunology ; metabolism ; T-Lymphocytes, Regulatory ; cytology ; immunology ; metabolism

OBJECTIVETo understand the influence of co-expression of CD80, CD86 and CD137L genes on tumor immunogenicity in hepatocellular carcinoma H22-BAL B/c mouse models.

METHODSThe mice were randomly divided into five groups, named A, B, C, D and E, and control groups A and B, 20 mice in each group. Hepatocellular carcinoma H22-BAL B/c mouse model was established by subcutaneous injection of cells H22-Wt, H22-neo, H22-CD80/CD86+, H22-CD137L+ and H22-CD80/CD86/CD137L+, respectively. The rate and incubation period of tumor development, survival rate, and the tumor growth in vivo were observed and recorded. The effects of gene transduction on immunogenicity of the tumor and antitumor immunity of the animals were assessed by re-innoculation of wild type H22 cells.

RESULTSThe rate of tumor development in group E was only 50%, much lower than that in other four groups (P < 0. 01). The tumor growth in group C was reduced with complete tumor regression in two hosts (20%, 2/10). In group E, there was more pronounced reduction of tumor size. The maximal tumor sizes were remarkably smaller than those of group C, and there was complete tumor regression in three mice (60%, 3/5). No tumor regression was found in the other three groups. Survival rates of group C and E were significantly higher than that of animals in groups A, B and D (P < 0. 01), but no significant difference was seen between group C and E. The results of re-inoculation test showed that tumor formation rate was 40% (4/10) in group C, 100% (8/8) in group D, and 0 (0/5) in group E. There were significant differences between groups C and E and control group, between group E and C, but not between C and D. After the third time of re-inoculation with H22-Wt cells at the 56th day, tumor occurred in 6/6 mice (100%) of group C, but 0 (0/5) in group E. The difference was very significant. Five animals without tumor formation after the first inoculation in group E, were re-inoculated with H22-Wt cells on the 21st day and the third re-inoculation on the 56th day, no tumor was found (0/5).

CONCLUSIONBoth co-expression and solo-expression of CD80+ CD86 and CD137L genes reduce the tumorigenicity of wild type H22 cells, but co-expression of CD80, CD86 and CD137L genes can more significantly improve the immunogenicity of H22-CD80/CD86/CD137L+ cells.

4-1BB Ligand ; genetics ; immunology ; metabolism ; Animals ; B7-1 Antigen ; genetics ; immunology ; metabolism ; B7-2 Antigen ; genetics ; immunology ; metabolism ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; Liver Neoplasms, Experimental ; genetics ; immunology ; pathology ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Random Allocation ; Survival Analysis

PTEN, a dual-specificity phosphatase, exerts its tumor-suppressive effects through the inhibition of cell cycle progression and cell immigration, therefore could be an important candidate for tumor-suppression. As study on prokaryotic expression of PTEN and its anti-tumor functions has not been reported, the present study aims at an efficient expression of functional PTEN in Escherichia coli and the investigation of its tumor-suppression activity. PTEN cDNA cloned in our lab previously was recombined into prokaryotic expression vector pET-44a(+) to construct pET-PTEN (pEP) and pET-Nus-PTEN (pENP). PTEN was fused with 6 x His tag in pEP, and with Nus in pENP, which could be useful for a stable and soluble expression. The recombinant vectors were transformed into both BL21 (DE3) (BL) and Rosetta-gami (DE3) pLysS (RG). The former is a normal expression host while the latter is optimized for expression of eukaryotic genes and folding of target proteins. On the induction of 0.5mmol/L IPTG, 55kD and 118kD specific protein bands were observed, corresponding to His-PTEN and Nus-PTEN fusion proteins, respectively. Western blot analysis showed the recombinant fusion proteins could react with PTEN polyclonal antibody. The recombinant HTEN was expressed both in soluble fraction and inclusion body. Higher expression levels of recombinant PTEN were obtained in BL (His-PTEN: 10.3%; NusA-PTEN: 18.7%), whereas the higher percentages of soluble recombinant proteins were observed in RG (His-PTEN: 4.7%; Nus-PTEN: 6.6%). The obtained recombinant fusion proteins were purified by affinity chromatography and were showed to be homogeneous in SDS-PAGE. In tumor-suppression experiments, His-PTEN was proved to have significant inhibition on growth of mice solid tumor with an inhibitory ratio of 58.76%, and on the proliferation of DU-145 tumor cells with an inhibitory ratio of 46.16%. The cell cycle progression of DU-145 tumor cells was also arrested from G0/G1 to S phase. His-PTEN from RG was proved to have significantly higher tumor-suppression activity than that from BL, indicating that there may be some advantages for eukaryotic genes to be expressed in the former host. This is the report of functional recombinant PTEN expressed in Escherichia coli. Purified His-PTEN was used for immunizing Kunming mice, and ascitic polyclonal antibodies raised against His-PTEN were generated using sarcoma 180 cells. At 1:2000 dilution, the antibodies could interact with PTEN by western blot. The present study has laid a foundation for application of PTEN in cancer therapy.
Animals ; Antibodies, Neoplasm ; biosynthesis ; immunology ; Antineoplastic Agents ; pharmacology ; Escherichia coli ; genetics ; metabolism ; Humans ; Liver Neoplasms, Experimental ; therapy ; Mice ; PTEN Phosphohydrolase ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

OBJECTIVESTo investigate immune responses and the anti-tumor effect of a constructed Chimeric AFP-Mt.HSP70 DNA vaccine in mice.

METHODSChimeric AFP-Mt.HSP70 was constructed by molecular clone techniques. Spleen cells derived from mice immunized twice were induced to secrete IFN gamma and were assayed using ELISA. The activity of the cytotoxic lymphocytes (CTL) derived from spleen cells was assayed using lactate dehydrogenase (LDH). 4 x 10(6) Hepa1-6 cells/200 microl were injected subcutaneously into the right axilla of each mouse bearing the tumor. The anti-tumor effect of the recombinant DNA vaccine was evaluated by measuring tumor sizes of the mice.

RESULTSAFP-specific CTL reaction was induced by our chimeric DNA vaccine and Mt.HSP70 enhanced this effect (P < 0.05). The CTL activity was about 32% at E/T=50:1. The IFN gamma secreted by spleen cells of mice immunized with chimeric plasmids was about 200 pg/ml. It was higher than those in the other groups; Tumor sizes of mice immunized with fused plasmids were smaller than those in the other groups. Survival times of mice immunized with the fused plasmids were prolonged.

CONCLUSIONChimeric DNA vaccine can induce AFP-specific CTL reaction and has an anti-tumor effect on transplanted tumors in our murine experiment.

Animals ; Cancer Vaccines ; immunology ; Carcinoma, Hepatocellular ; drug therapy ; Cell Line, Tumor ; Female ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Liver Neoplasms, Experimental ; drug therapy ; Mice ; Mice, Inbred C57BL ; Plasmids ; Spleen ; cytology ; T-Lymphocytes, Cytotoxic ; immunology ; Vaccines, DNA ; immunology ; alpha-Fetoproteins ; genetics ; immunology

OBJECTIVETo study the cytotoxic activity against tumor cells and cytokines production of spleen cells induced in vitro by murine 4-1BBL gene transfected Hepa1-6.

METHODSThe eukaryotic expression vector pCDNA3.1(+)-m4-1BBL was transfected into murine hepatocellular carcinoma cell line Hepa1-6 by Liposomes. Then the transfected cells were selected in medium containing G418 (400 - 800 micro g/ml) and termed as Hepa1-6-m4-1BBL. The TCV-m4-1BBL was obtained by treating them with mitomycin (MMC). Cocultivation TCV with syngeneic murine spleen cells, then the lymphocytes were tested for cytotoxic activity against Hepa1-6-wt cells and the supernatants were harvested for detecting the cytokines (IL-2, TNF-alpha and GM-CSF).

RESULTSHepa1-6-m4-1BBL cells expressed 4-1BBL protein with highest cell surface level. The 4-1BBL mRNA could still be detected in the cells when cultured 48 h after treated with MMC (80 mg/L). Comparing with TCV-Hepa1-6, the tumor cell vaccine derived from Hepa1-6-m4-1BBL (TCV-m4-1BBL) could induce a more efficient cytotoxic activity of syngeneic murine lymphocyte against its parental tumor cell Hepa1-6 (P < 0.05), but not against non-parental tumor cell H22 and NIH3T3. Higher levels of IL-2, TNF-alpha and GM-CSF were released by the splencytes after stimulated by TCV-m4-1BBL.

CONCLUSIONSThese results suggest the expression of m4-1BBL by tumor cells is effective in inducing antitumor immune responses.

4-1BB Ligand ; Animals ; Cancer Vaccines ; immunology ; Female ; In Vitro Techniques ; Liver Neoplasms, Experimental ; immunology ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Tumor Necrosis Factors ; genetics ; physiology