Journal of Chest Surgery

The Korean Society for Thoracic and Cardiovascular Surgery (대한심장혈관흉부외과학회)
권호 표지

Analysis of 5-aza-2'-deoxycytidine-induced Gene Expression in Lung Cancer Cell Lines

폐암 세포주에서 5-aza-2'-deoxycytidine 처치에 의해 발현되는 암항원 유전자 분석

  • 김창수 (고신대학교 의과대학 흉부외과학교실) ;
  • 이해영 (고신대학교 의과대학 흉부외과학교실) ;
  • 김종인 (고신대학교 의과대학 흉부외과학교실) ;
  • 장희경 (고신대학교 의과대학 병리학교실) ;
  • 박종욱 (계명대학교 의과대학 면역학교실) ;
  • 조성래 (고신대학교 의과대학 흉부외과학교실)
  • Published : 2004.12.01
Download PDF
⟨ Previous Next ⟩

Abstract

Background: DNA methylation is one of the important gene expression mechanisms of the cell. When cytosine of CpG dinucleotide in promotor is hypomethylated, expression of some genes that is controlled by this promoter is altered. In this study, the author investigated the effect of DNA demethylating agent, 5-aza-2'-deoxycytidine (ADC), on the expressions of cancer antigen genes, MHC and B7 in 4 lung cancer cell lines, NCIH1703, NCIH522, MRC-5, and A549. Material and Method: After treatment of cell lines, NCIH1703, NCIH522, MRC-5 and A549 with ADC (1 uM) for 48 hours, RT-PCR was performed by using the primers of MAGE, GAGE, NY-ESO-1, PSMA, CEA, and SCC antigen gene. In order to find the optimal ADC treatment condition for induction of cancer antigen, we studied the effect of ADC treatment time and dose on the cancer antigen gene expression. To know the effect of ADC on the expression of MHC or B7 and cell growth, cells were treated with 1 uM of ADC for 72 hours for FACS analysis or cells were treated with 0.2, 1 or 5 uM of ADC for 96 hours for cell counting. Result: After treatment of ADC (1 uM) for 48 hours, the expressions of MAGE, GAGE, NY-ESO-1, and PSMA genes increased in some cell lines. Among 6 MAGE isotypes tested, and gene expression of MAGE-1, -2, -3, -4 and -6 could be induced by ADC treatment. However, CEA gene expression did not change and SCC gene expression was decreased by ADC treatment. Gene expression was generally induced 24 - 28 hours after ADC treatment and expression of MAGE, GAGE, and NY-ESO-1 was maintained at least 14 days after ADC ADC teatment, and expression of MAGE, GAGE, and NY-ESO-1 was maintained at least 14 days after ADC teatment in ADC-Free medium. Most gene expression could be induced at 0.2 uM of ADC, but gene expression increased dependently on ADC treatment dose. The expression of MHC and B7 was not increased by ADC treatment in all four cell lines, and the growth rate of 4 cell lines decreased significantly with the increase of ADC concentrations. Conclusion: Treatment of lung cancer cell lines with ADC increases the gene expression MAGE, GAGE and NY-ESO-1 that are capable of induction of cytotoxic T lymphocyte response. We suggest that treatment with 1 uM of ADC for 48 hours and then culturing in ADC-free medium is optimal condition for induction of cancer antigen. However, ADC has no effect on MHC and B7 induction, additional modification for increase of expression of MHC, B7 and cytokine will be needed for production of efficient cancer cell vaccine.

배경: DNA 메칠화란 유전자의 Promoter에 있는 CpG dinucleotide의 cytosine기에 메칠기가 붙는 현상을 말한다. CpG dinucleotide에 과메틸화가 일어나면 일부 유전자의 발현이 감소되며, 그 반대로 CpG dinucleotide의 메칠화가 억제되면 유전자 발현이 증가된다. DNA 메칠화 억제제인 5-aza-2'- deoxycytidine (ADC)을 폐암세포에 처치했을 때 암항원 유전자의 발현 유무와 이를 위한 최적 조건을 조사하고, 아울러 MHC와 B7의 발현과 세포 성장에 미치는 영향을 조사하여 암치료 백신에 ADC를 임상적으로 이용할 수 있는 지를 연구하였다. 대상 및 방법: 4개의 사람 폐암세포주 (NCIH1703, NCIH522, MRC-5 및 A549)에 ADC를 1 uM 농도로 처치한 후 48시간 뒤에 MAGE family, GAGE, NY-ESO-1, PSMA, CEA 및 SCC항원 유전자에 대한 RT-PCR을 실시하였고, 폐암세포에서 암항원의 발현을 증가시키는 최적의 ADC처치 조건을 규명하기 위하여 ADC농도와 처치 시간을 다양하게 하여 암세포를 자극한 후 암항원 유전자 발현성을 분석하였다. 또한 ADC 처리가 폐암 세포주의 MHC와 B7 발현을 증가시키는 가를 알아보기 위해 1 uM 농도의 ADC를 72시간 처치한 후 FACS 분석을 실시하였고, ADC가 세포성장에 미치는 영향을 알아보기 위하여, ADC를 0.2, 1 및 5 uM 농도로 96시간 처치 후 세포수를 측정하여 상대성장지수를 조사하였다. 결과: 세포주에 따라 차이는 있으나 MAGE, GAGE, NY-ESO-1 및 PSMA의 발현이 유도되었으며, MAGE아형 중에는 MAGE-1, -2, -3, -4, -6으로 나타났다. 그러나 비암항원인 CEA발현은 변화가 없었으며 SCC항원 유전자의 발현은 오히려 ADC처치에 의해 감소되었다. ADC 처치 후 24∼48 시간이 지난 뒤부터 암항원 유전자의 발현이 증가하였으며 ADC처리에 의해 유도된 유전자의 발현성은 ABC처치 후 최소 14일까지 유지되었다. 또 ADC를 0.2, 1, 5 uN 농도로 첨가하여 48시간 배양한 후 암항원 유전자 발현성을 측정한 결과 세포주에 따라 다소 차이는 있으나 대개 0.2 uM농도에서도 유전자 발현이 유도되었으며 1, 5 uM농도에서 매우 강하게 유도되었다. ADC 처리가 페암세포주의 MHC와 B7 발현을 증가시키는가를 알아보기 위해 1 uM 농도의 ADC를 72시간 처치한 후 FACS 분석을 실시한 결과 4개의 페암세포주에서 MHC 및 B7분자의 발현은 유도되지 않았다. 또 ADC농도가 세포성장에 미치는 영향을 알아보기 위하여 ADC를 0.2, 1, 5 uM농도로 96시간 처치 후 세포수를 측정하여 상대성장지수를 알아본 결과 ADC 처치 농도가 증가함에 따라 세포의 성장은 매우 감소하였다. 결론: 폐암세포주에서 ADC처치는 MAGE, GAGE 및 NY-ESO-1과 같은 세포독성 T 림프구 반응을 유도할 수 있는 암항원의 발현을 증가시킬 수 있으며, ADC의 세포독성과 항원 발현 유발시간을 분석할 때 1 uM 농도에서 48시간 처치한 후 ADC가 없는 배지에서 수일간 배양하는 것이 가장 효과적이라고 생각된다. 그러나, ADC를 처치하여도 MHC 및 B7의 발현의 변화는 없었으므로 ADC를 처치한 폐암세포를 암백신으로 사용하기 위해서는 MHC나 B7 및 cytokine의 발현을 증가시키는 추가적인 처치가 필요하다고 생각된다.

Keywords

References

  1. Knuth A, Jager D, Jager E. Cancer immunotherapy in clinical oncology. Cancer Chemother Pharmacol 2000;46: Suppl: S46-51 https://doi.org/10.1007/PL00014050
  2. Ockert D, Schmitz M, Hampl M, Rieber EP. Advances in cancer immunotherapy. Immunol Today 2000;20:63-5. https://doi.org/10.1016/S0167-5699(98)01388-7
  3. Ockert D, Schmitz M, Hampl M, Rieber EP. Advances in cancer immunotherapy. Immunol Today 2000;20:63-5. https://doi.org/10.1016/S0167-5699(98)01388-7
  4. Schadendorf D, Paschen A, Sun Y. Autologous, allogeneic tumor cells or genetically engineered cells as cancer vaccine against melanoma. Immunol Lett 2000;74:67-74 https://doi.org/10.1016/S0165-2478(00)00251-0
  5. Timmerman JM, Levy R. Dendritic cell vaccines for cancer immunotherapy. Annu Rev Med 1999;50:507-29. https://doi.org/10.1146/annurev.med.50.1.507
  6. Gong J, Nikrui N, Chen D, et al. Fusions of human ovarian carcinoma cells with autologous or allogeneic dendritic cells induce antitumor immunity. J Immunol 2000;165:1705-11 https://doi.org/10.4049/jimmunol.165.3.1705
  7. Mendiratta SK, Quezada A, Matar M, et al. Intratumoral delivery of IL-12 gene by polyvinyl polymeric vector system to murine renal and colon carcinoma results in potent antitumor immunity. Gene Ther 1999;6:833-9. https://doi.org/10.1038/sj.gt.3300891
  8. Lee SG, Heo DS, Yoon SJ, et al. Effect of GM-CSF and IL-2 co-expression on the anti-tumor immune response. Anticancer Res 2000;20:2681-6.
  9. Levitsky HI, Lazenby A, Hayashi RJ, Pardoll DM. In vivo priming of two distinct antitumor effector populations: the role of MHC class I expression. J Exp Med 1994;179: 1215-24. https://doi.org/10.1084/jem.179.4.1215
  10. Wachsman JT. DNA methylation and the association between genetic and epigenetic changes: relation to carcinogenesis. Mutat Res 1997;375:1-8. https://doi.org/10.1016/S0027-5107(97)00003-1
  11. Gonzalgo ML, Jones PA. Mutagenic and epigenetic effects of DNA methylation. Mutat Res 1997;386:107-18. https://doi.org/10.1016/S1383-5742(96)00047-6
  12. Momparler RL, Bovenzi V. DNA methylation and cancer. J Cell Physiol 2000;183:145-54. https://doi.org/10.1002/(SICI)1097-4652(200005)183:2<145::AID-JCP1>3.0.CO;2-V
  13. Tureci O, Sahin U, Zwick C, Koslowski M, Seitz G, Pfreundschuh M. Identification of a meiosis-specific protein as a member of the class of cancer/testis antigens. Proc Natl Acad Sci USA 1998;95:5211-6 https://doi.org/10.1073/pnas.95.9.5211
  14. Celis E, Fikes J, Wentworth P, et al. Identification of potential CTL epitopes of tumor-associated antigen MAGE-1 for five common HLA-A alleles. Mol Immunol 1994;31:1423-30. https://doi.org/10.1016/0161-5890(94)90158-9
  15. van der Bruggen P, Bastin J, Gajewski T, et al. A peptide encoded by human gene MAGE-3 and presented by HLA-A2 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE-3. Eur J Immunol 1994;24:3038-43. https://doi.org/10.1002/eji.1830241218
  16. Zeuthen J, Dzhandzhugazyan K, Hansen MR, Kirkin AF. The immunogenic properties of human melanomas and melanoma-associated antigens recognized by cytotoxic T lymphocytes. Bratisl Lek Listy 1998;99:426-34
  17. Chen JL, Dunbar PR, Gileadi U, et al. Identification of NY- ESO-1 peptide analogues capable of improved stimulation of tumor-reactive CTL. J Immunol 2000;165:948-55. https://doi.org/10.4049/jimmunol.165.2.948
  18. Chen ME, Lin SH, Chung LW, Sikes RA. Isolation and characterization of PAGE-1 and GAGE-7. New genes expressed in the LNCaP prostate cancer progression model that share homology with melanoma-associated antigens. J Biol Chem 1998;273:17618-25. https://doi.org/10.1074/jbc.273.28.17618
  19. Russo V, Traversari C, Verrecchia A, Mottolese M, Natali PG, Bordignon C. Expression of MAGE gene family in primary and metastatic human breast cancer: implications for tumor-specific immunotherapy. Int J Cancer 1995;64:216-21 https://doi.org/10.1002/ijc.2910640313
  20. Mori M, Inoue H, Mimori K, et al. Expression of MAGE genes in human colorectal carcinoma. Ann Surg 1996;224: 183-8. https://doi.org/10.1097/00000658-199608000-00011
  21. Russo V, Dalerba P, Ricci A, et al. MAGE BAGE and GAGE genes expression in fresh epithelial ovarian carcinomas. Int J Cancel 1996;67:457-60. https://doi.org/10.1002/(SICI)1097-0215(19960729)67:3<457::AID-IJC24>3.0.CO;2-3
  22. Fujie T, Mori M, Ueo H, Sugimachi K, Akiyoshi T. Expression of MAGE and BAGE genes in Japanese breast cancers. Ann Oncol 1997;8:369-72. https://doi.org/10.1023/A:1008255630202
  23. Li J, Yang Y, Fujie T, et al. Expression of the MAGE gene family in human gastric carcinoma. Anticancer Res 1997; 17:3559-63
  24. Picardo AL, Torres AJ, Maestro M, et al. Quantitative analysis of carcinoembryonic antigen, squamous cell carcinoma antigen, CA 125, and CA 50 cytosolic content in non-small cell lung cancer. Cancer 1994;73:2305-11. https://doi.org/10.1002/1097-0142(19940501)73:9<2305::AID-CNCR2820730911>3.0.CO;2-D
  25. Lee YC, Yang PC, Kuo SH, Luh KT. Tissue polypeptide antigen and carcinoembryonic antigen as tumor markers in lung cancer. J Formos Med Assoc 1991;90:631-6.
  26. Coral S, Sigalotti L, Gasparollo A, et al. Prolonged upregulation of the expression of HLA class I antigens and costimulatory molecules on melanoma cells treated with 5-aza- 2'-deoxycytidine (5-AZA-CdR). J Immunother 1999;22:16-24. https://doi.org/10.1097/00002371-199901000-00003
  27. De Backer O, Arden KC, Boretti M, et al. Characterization of the GAGE genes that are expressed in various human cancers and in normal testis. Cancer Res 1999;59:3157-65.
  28. De Backer O, Arden KC, Boretti M, et al. Characterization of the GAGE genes that are expressed in various human cancers and in normal testis. Cancer Res 1999;59:3157-65.
  29. Coral S, Sigalotti L, Gasparollo A, et al. Prolonged upregulation of the expression of HLA class I antigens and costimulatory molecules on melanoma cells treated with 5-aza-2'- deoxycytidine (5-AZA-CdR). J Immunother 1999;22:16-24. https://doi.org/10.1097/00002371-199901000-00003
  30. Redondo M, Ruiz-Cabello F, Concha A, et al. Differential expression of MHC class II genes in lung tumour cell lines. Eur J Immunogenet 25:385-91.