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1 KISEP Head and Neck Korean J Otolaryngol 2002;45: aza-2 -deoxycytidine처치에 의해 유도되는 B16F10 세포주의 유전자 발현 2 손진식 1 송달원 1 김태종 1 채한수 1 박종욱 Gene Expression of B16F10 Induced by Treatment with 5-aza-2 -deoxycytidine Jin-Sik Sohn, MD 1, Dal-Won Song, MD 1, Tae-Jong Kim, MD 1, Han-Soo Chae, MD 1 and Jong-Wook Park, MD 2 1 Department of Otolaryngology and 2 Immunology, School of Medicine, Keimyung University, Daegu, Korea ABSTRACT Background and ObjectivesThe goal of tumor vaccine is to activate immune system specifically against proteins expressed by a tumor, and many types of vaccines such as gene modified vaccines have been developed to increase immunogenicity of vaccine. We studied to determine whether or not 5-aza-2 -deoxycytidine ADC can increase the immunogenicity of B16F10 melanoma cell. Materials and MethodB16F10 cell was treated with ADC for the induction of DNA demethylation. An ADC treated B16F10 melanoma cell was analyzed first using the reverse transcriptase-polymerase chain reaction RT-PCR technique to evaluate the gene expression of tumor antigen MAGE-2, MAGE-5 and immunity-enhancing cytokines GM- CSF, IL-12, and then by flow cytometry to evaluate the expression of MHC and B7 that are responsible for antigen expression and T cell activation on B16F10 cell surface. In order to evaluate vaccination effect of ADC-treated B16F10 vaccine, each mouse group were injected with PBS, ADC, B16F10 vaccine or B16F10-ADC vaccine and they were also challenged with live B16F10 cell 7 days after vaccination. On the 20th day after live B16F10 cell challenge, the tumor mass size and the mouse survival period were determined. ResultsADC treatment for B16F10 melanoma cell increased expression of MHC and B7. ADC treatment also increased gene expression of MAGE-2, MAGE-5, GM-CSF and IL-12. The growth of tumor mass was decreased and the mouse survival period was elongated in B16F10-ADC vaccine immunized group. Conclusion ADC treatment may increase immunogenicity of B16F10 cell, and B16F10-ADC vaccine immunization can induce anticancer immunity in vivo. Korean J Otolaryngol 2002;45: KEY WORDS5-aza-2 -deoxycytidine Melanoma Cancer vaccine. 784

University, Daegu, Korea ABSTRACT Background and ObjectivesThe goal of tumor vaccine is to activate immune system specifically against proteins expressed by a tumor, and many types of vaccines such

3 Table 1. Primers used for RT-PCR Name Primer sequence sense/antisense GM-CSF 5 -GGATGTGGCTGCAGAATTTACTT-3 / 5 -TCATTTTTGGACTGGTTTTTTGCA-3 IL-12p40 5 -ACATGTGTCCTCAGAAGCTAACCATC-3 / 5 -ATCCTAGGATCGGACCCTGCAGGG-3 MAGE-2 5 -ATCGCTCTGCAGTGCCAGTT-3 / 5 -GTCCACCAAGTCATACACCT-3 MAGE-5 5 -GACATTGTGGACTCAGCACC-3 / 5 -GTCCACCAAGTCATACACCT-3 G3PDH 5 -GCCACCCAGAAGACTGTGGATGGC-3 / 5 -CATGTAGGCCATGAGGTCCACCAC Fig. 1. Effect of ADC on the proliferation of B16F10 cell. B16F10 cells were cultured in medium containing ADC for 24, 48 and 72 hours. Cells were detached by trypsin treatment from plate, and stained with trypan blue. The number of viable cells was counted in hemocytometer. 786 Korean J Otolaryngol 2002;45:784-90

-ATCCTAGGATCGGACCCTGCAGGG-3 MAGE-2 5 -ATCGCTCTGCAGTGCCAGTT-3 / 5 -GTCCACCAAGTCATACACCT-3 MAGE-5 5 -GACATTGTGGACTCAGCACC-3 / 5 -GTCCACCAAGTCATACACCT-3 G3PDH 5

4 Fig. 3. Effect of ADC on the MHC and B7 gene expression of B16F10 cell. B16F10 cells were cultured in medium containing 1 mol of ADC for 48 hours, and stained with FITC-conjugated anti-mhc or anti-b7 antibody for FACS analysis. Black color space represent control group, and white color space represent ADC-treated group. G3PDHGlyceraldehyde-3-phosphate dehydrogenase. Fig. 4. Effect of ADC on the MHC and B7 expression of B16F10 cell. B16F10 cells were cultured in medium containing 1 mol of ADC for 48 hours, and stained with FITC-conjugated anti-mhc H-2k b or anti-b7 CD80 antibody for FACS analysis. Black color space represent control group, and white color space represent ADC-treated group. Fig. 2. Effect of ADC on the cell cycle of B16F10 cell. B16F10 cells were cultured in medium containing ADC for 24 hours. Cells were detached by trypsin treatment from plate, and stained with propidium iodide, and cell cycle was analyzed by FACS scan. M1, M2 and M3 represent G1, S and G2/M phase, respectively. Fig. 5. Effect of ADC on the GM-CSF and IL-12 gene expression of B16F10 cell. B16F10 cells were cultured in medium containing ADC for 1, 6, 24 and 48 hours, and total RNA was isolated. The gene expression of GM-CSF and IL-12 was measured by RT-PCR. G3PDHGlyceraldehyde-3-phosphate dehydrogenase. 787

Black color space represent control group, and white color space represent ADC-treated group. G3PDHGlyceraldehyde-3-phosphate dehydrogenase. Fig. 4.

5 Fig. 6. Effect of vaccination with B16F10-ADC on the tumor growth. Mice immunized with PBS, ADC, B16F10 or B16F10- ADC were challenged with live B16F10 cell. Twenty days after tumor challenge, the size of tumor mass of each group was measured. PBSPhosphate buffered saline, ADC5-aza -2- deoxycytidine Table 2. Delay of mice death by immunization with B16F10-ADC 788 Day Number of survived mice PBS ADC B16F10 B16F10-ADC PBSPhosphate buffered saline ADC5-aza -2-deoxycytidine - Korean J Otolaryngol 2002;45:784-90

Twenty days after tumor challenge, the size of tumor mass of each group was measured.

6 REFERENCES 1) Chang AE, Li Q, Bishop DK, Normolle DP, Redman BD, Nickoloff BJ. Immunogenetic therapy of human melanoma utilizing autologous tumor cells transduced to secrete granulocyte-macrophage colony-stimulating factor. Hum Gene Ther 2000;11: ) Schmitt WE, Stassar MJ, Schmitt W, Little M, Cochlovius B. In vitro induction of a bladder cancer-specific T-cell response by mrna-transfected dendritic cells. J Cancer Res Clin Oncol 2001; 127: ) Monzavi-Karbassi B, Kieber-Emmons T. Current concepts in cancer vaccine strategies. Biotechniques 2001;30: ) Van der Meijden AP. Non-specific immunotherapy with bacille 789

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infiltration, Fas/Fas ligand gene expression, and mammary epithelial apoptosis. J Immunol 2001;166:1156-68. 6) Finke J, Ferrone S, Frey A, Mufson A, Ochoa A. Where have all the T cells gone?

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( )Jkstro011.hwp 비인강암의방사선치료결과및생존율에관한예후인자분석 2005 2 1 2005 3 28. :, Tel: 053)250-7665, Fax: 053)250-7984 E-mail: jhkim@dsmc.or.kr 정영연외 2 인 : 비인강암의예후인자분석 정영연외 2 인 : 비인강암의예후인자분석 Carcinoma of the nasopharynx treated by radiotherapy