Chinese Journal of Biochemistry and Molecular Biology PVAX-1 CD8 + T

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1 ISSN CN / Q Chinese Journal of Biochemistry and Molecular Biology ~ 477 PVAX-MAGE-1 H * PVAX-MAGE-1-1 melanoma antigen- 1MAGE-1 PVAX-MAGE-1 C57BL /6 ELISA PVAX-1 IL-2 IFN-γ P < CD8 + T P < 0 05 PVAX-MAGE-1 C57BL / 6 H22 PVAX-MAGE-1 T IL IFN -1 Q78 R735 Growth Inhibition of Mouse Ascites and Solid Hepatoma 22 With PVAX-MAGE-1 Gene Vaccine YANG Xiao-Ling 1 L Jing 1 WANG Jian-Hua 2 ZHANG Yue-Hong 1 1 NIU Bo 2 * 1 Department of Biochemistry and Molecular BiologyShanxi Medical UniversityTaiyuan 2 Biotechnology Research LaboratoryCapital Institute of PediatricsBeijing China China Abstract The melanoma antigen-1 MAGE-1 gene was inserted into a mammalian expression vector PVAX-1 and used to vaccinated C57BL / 6 mice using saline as the controls The splenocyte-secreted IL- 2 and IFN-γ were significantly higher than those of the controls P < 0 05 as well as the CTL activity of CD8 + T cells P < 0 05 In vivo experiments showed that the immunized C57BL /6 mice could inhibit the growth of the transplanted H22 ascites and solid tumor Our results indicated that PVAX- MAGE-1 gene vaccine was able to suppress the tumor growthand might involve the enhanced expression of IL and IFN Key words hepatocellular carcinoma melanoma antigen-1 MAGE-1 gene vaccine -1 melanoma antigen-1 MAGE CTL CTL HLA Ⅰ T CTL CTL MAGE * Tel Niub2004@ 126 com Received November Accepted January DNA DNA Niub2004@ 126 com * Corresponding author Tel

2 5 PVAX-MAGE-1 H PVAX-MAGE- Ⅰ A C57BL /6 60 A ~ 2 0 PVAX-1 Invitrogen H SMMC T4 EcoRⅠXhoⅠ 15 3 d TaKaRa RNA 5 ml / L 100 μl 3 QIAGEN IL-2 ELISA PVAX-MAGE-1 PVAX IFN-γ ELISA 100 μl 1 μg / μl 3 LDH Promega 10 d RPM I-1640 Sigma MAGE-1 A RNA RNA RT-PCR MAGE-1 Mouse T Cell CD8 Subset Column Kit R&D SYSTEMS Western 8 MAGE-1 Santacruz IL-2 IFNγ ELISA 5 SABC DAB PBS % GenBank MAGE-1 cdna M77481 CO 2 48 h ELISA 5 -GCCTCGAGTCCACCATGTCT CTTGAGCAGAGGAG-3 Xho Ⅰ 5 -CTGAATTCTCAGACTCCCTCTTCCTCCT-3 EcoRⅠ CD8 Subset Column Kit CD8 + T HCC RNA RT-PCR PCR LDH min94 60 s s s 35 CD8 + T SMMC s 0 8% Pvu Ⅱ EcoRⅠXhoⅠ 37 4 EcoRⅠXhoⅠ h LDH 50 PVAX-1 T4 DNA μl μl LDH PVAX-MAGE-1 30 min 50 μl JM109 EcoR Ⅰ Xho PVAX- MAGE-1 PVAX-1 TritonX-114 A 260 A 280 A 260 / l g / L C57BL /6 PVAX-MAGE-1 PVAX / ml % IL-2 IFN-γ CD8 + T Ficoll Mouse T Cell nm A % =

3 / - 100% mm 3 = / H22 MAGE H22 SPSS11 0 x 珋 ± S P < min 4 ABC d H H22 1 RT-PCR MAGE / ml % 750 bp 1000 bp ml 7 d bp / ml3 0 2 ml d 2 2 PVAX-MAGE d H ml 0 8% bp PVAX / ml bp PVAX-1 GenBank mm 3 = 2 /2 PVAX-MAGE-1 EcoR Ⅰ Xho Ⅰ MAGE C57BL /6 2 3 MAGE ml H22 RT-PCR Western 1 10 / ml PVAX-MAGE-1 MAGE-1 PVAX d Fig 1 Fig 1 Expressions of MAGE-1 gene in quadriceps muscle A RT-PCR for MAGE-1 mrna Total RNA was obtained from quadriceps muscle RT-PCR showed specific expression of MAGE-1 mrna in PVAX-MAGE-1 groupbut not in PVAX-1 and NaCl group M DNA marker 1 Normal group 2 NaCl group 3 PVAX-1 group 4 PVAX-MAGE-1 group B Expression of MAGE-1 gene products by Western blot analysis The molecular weight of MAGE-1 was 57 kd These proteins were exclusively observed in PVAX-MAGE-1 group 1 PVAX-MAGE-1 group 2 PVAX-1 group 3 NaCl group 4 Normal group 2 4 IL-2 IFN-γ P > 0 05 PVAX-1 IL-2 IFN-γ ELISA PVAX-1 A 0 0 pg / ml A t IL pg /

4 5 PVAX-MAGE-1 H mlifn-γ pg / ml 2 5 CD8 + T 50 1 PVAX-MAGE-1 CTL 40 27% PVAX % 14 5% Fig 2 P < 0 05 Fig 2 Cytotoxicity T-lymphocyte CTL assay Groups of mice n = 15 were immunized with PVAX- MAGE-1 filled diamonds PVAX-1 filled squares or NaCl filled triangles Cytotoxicity was measured by LDH release assay CD8 + T cells were used as effector cells and SMMC-7721 cells were used as target cells CD8 + T cell were incubated with SMMC cells at the indicated effecter target cell ratio E / T for 4 hours at 37 Specific lysis was observed in PVAX-MAGE-1 immunization groupwhich was statistically significant compared with the PVAX-1 and NaCl P < H22 MAGE-1 Fig 3A Fig 3B H22 Fig 3 Detection for expression of MAGE-1 in H22 cells by immunohistochemistry 400 About 5 μm thick sections were prepared from H22 cell Immunohistochemical analysis using an ABC kit was performed with MAGE-1 antibody A Positive staining The result indicated that the MAGE-1 stains H22 cell in H-E staining B Negative staining 2 7 PVAX-1 H22 3 d 9 d 9 d 13 d 11 ± 1 34 d PVAX-1 10 d 15 d d 18 d 24 d d H22 P < 0 05 C57BL /6 H22 40 d Table 1 Table 1 Tumor volumetumor mass and tumor forming rate in mice after immunotherapy n = 5 珔 x ± s Group Tumor volume cm 3 Tumor mass g Tumor forming rate % NaCl group ± ± PVAX-1 group ± ± PVAX-MAGE-1 group ± ± 0 67 * 40 * P < 0 05 compared with NaCl group and PVAX-1 group 2 8 PVAX-1 C57BL /6 H22 10 d PVAX- MAGE-1 Fig 100% 10 mm dpvax-1 11 d 21 d 1 06 ± cm 3

5 ± cm 3 PVAX-MAGE-1 P < ± cm MAGE MAGE-1 1 MAGE-1 MHC HLA-I HLA-CW* MAGE-1 MHC T MHCⅠ TCR 3 -MHC CTL MAGE-1 DNA PVAX-MAGE-1 PVAX-1 MAGE-1 FDA Igκ V-J2-C 9-11 PVAX-MAGE-1 DNA H22 T Fig 4 Inhibitory effect of three groups on tumor growth in mouse in vivo C57BL /6 mice five per group were first challenged by subcutaneous injection with 0 2 ml H22 tumor cells / ml Ten days after tumor challengethe tumor forming rate was 100% The mice of three groups were vaccinated with PVAX-MAGE- 1PVAX-1and NaCl three times with 10-day intervals The mice were monitored for evidence of tumor growth by tumor volume per 8 days Tumor volume was measured starting from day 10 after tumor challenge Each curve denoted the growth curve of a tumor mass of a mouse as measured at different time points during the experiment five mice per group The growth velocity of tumor was markedly reduced in PVAX-MAGE-1 group PVAX-MAGE-1 IL INF DNA PVAX-MAGE- References 1 INF-γ IL-2 T Th1 1 Lupo LRanieri ERotelli M Tet al Gene expression of the MAGE CD8 + T J P < 0 05 PVAX-MAGE-1 MHCⅠ CTL T 14 MAGE-1-3and -6 antigens in hepatic carcinomaj Tumori -1 Guan Xiang-HongXu He- FeiLuo-Bing The expression of MAGE-1 gene in human gastric

6 5 PVAX-MAGE-1 H carcinomaj Acta Acad Med Qingdao Univ Shichijo STsunosue RMasuoka Ket al Expression of MAGE gene family on human lymphocytic leukemiaj Cancer Immunol Immunother Lee K DLee H HJoo H Bet al Expression of MAGE A1-6 mrna in sputa of head and neck cancer patients a preliminary reportj Anticancer Res B Kim HKim TChung Jet al The detection of peripheral lung cancer by MAGEA1-6 RT-nested PCR in bronchial washing specimensj Lung Cancer Hara IHara SMiyake Het al Expression of MAGE gene in testicular germ cell tumorsj Urology Grau EOltra SMaritinez Fet al MAGE-A1 expression is associated with good prognosis in neuroblastoma tumorsj J Cancer Res Clin Oncol J E F T and the induction of immunityj J Leukocyte Bio M Sambrook JFritsch E FManiatis T Molecular Cloning A Laboratory Manual 2 nd edm New York Cold Spring Harbor Laboratory Press Ottaviani SZhang YBoon Tet al A MAGE-1 antigenic peptide recognized by human cytolytic T lymphocytes on HLA-A2 tumor cellsj Cancer immunol immunother MAGE-1 T J Lv Jian-FengLeng Xi-ShengPeng Ji-Runet al Induction of cytotoxic T lymphocytes from the peripheral blood of a hepatocellular carcinoma patient using a MAGE-1 peptide NYKCRFPEI in vitroj Chin J Gen Surg Toh H CWang W WChia W Ket al Clinical benefit of allogeneic melanoma cell lysate-pulsed autologous dendritic cell vaccine in MAGE-positive colorectal cancer patientsj Clin Cancer Res M Jin Bo-Quan Medical Immunology5 th edm Beijing People s Medical Publishing House Shedlock DJWeiner DB DNA vaccination antigen presentation 14 J Jia-Rui Yan Jin- Qi Liu Guo-Dong et al Construction and eukaryotic expression of cancer vaccine encoding multitarget complex antigenj Lett Biotechnol