Clinical & Experimental Metastasis

, Volume 21, Issue 5, pp 389–397

Microarray analysis of gene expression in metastatic gastric cancer cells after incubation with the methylation inhibitor 5-aza-2′-deoxycytidine

  • Jie Chen
  • Christoph Röcken
  • Ludger Klein-Hitpass
  • Tobias Götze
  • Andreas Leodolter
  • Peter Malfertheiner
  • Matthias P.A. Ebert


While the exact mechanisms involved in cancer metastasis are not fully clarified, the altered expression of many different genes has been reported. Hypermethylation of the promoters of cancer-related genes is often associated with their inactivation during tumorigenesis and may also be involved in metastasis. Here we used cDNA microarrays to examine the different gene expression profiles of a primary gastric adenocarcinoma cell line RF1 and its derivative metastasis subline RF48. Compared with RF1, 49 genes were down-regulated and 8 genes were up-regulated in RF48. After treatment of RF48 cells with a DNA methylation inhibitor, 5-aza-2′-deoxycytidine, 101 genes were up-regulated and 1 gene was down-regulated in treated RF48 when compared with untreated RF48. Comparing gene expression patterns of untreated RF1, untreated RF48 and treated RF48 cells showed 5 genes expressed in RF1 but silenced in RF48, which were reactivated after 5-aza-2′-deoxycytidine treatment. Two of those 5 genes have CpG islands within their promoter regions, suggesting that those genes activated by 5-aza-2′-deoxycytidine may result from the direct inhibition of promoter methylation. In conclusion, using global gene expression analysis together with inhibition of DNA methylation, we demonstrate that hypermethylation of the promoters of certain cancer-related genes may play a role in cancer metastasis.

Key words

array gastric cancer metastasis methylation inhibitor 5-aza-2′-deoxycytidine 





reverse transcription polymerase chain reaction


expressed sequence tags


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Pisani P, Parkin DM, Bray F et al. Estimates of the worldwide mortality from 25 cancers in 1990. Int J Cancer 1999; 83: 18–29.Google Scholar
  2. 2.
    Roukos DH. Current status and future perspectives in gastric cancer management. Cancer Treat Rev 2000; 26: 243–55.Google Scholar
  3. 3.
    Boussioutas A, Li H, Liu J et al. Distinctive patterns of gene expression in premalignant gastric mucosa and gastric cancer. Cancer Res 2003; 63: 2569–77.Google Scholar
  4. 4.
    Hippo Y, Taniguchi H, Tsutsumi S et al. Global gene expression analysis of gastric cancer by oligonucleotide microarrays. Cancer Res 2002; 62: 233–40.Google Scholar
  5. 5.
    Sakakura C, Hagiwara A, Nakanishi M et al. Differential gene expression profiles of gastric cancer cells established from primary tumour and malignant ascites. Br J Cancer 2002; 87: 1153–61.Google Scholar
  6. 6.
    Ji J, Chen X, Leung SY et al. Comprehensive analysis of the gene expression profiles in human gastric cancer cell lines. Oncogene 2002; 21: 6549–56.Google Scholar
  7. 7.
    Liang G, Gonzales FA, Jones PA et al. Analysis of gene induction in human fibroblasts and bladder cancer cells exposed to the methylation inhibitor 5-aza-2′-deoxycytidine. Cancer Res 2002; 62: 961–6.CrossRefGoogle Scholar
  8. 8.
    Hasegawa S, Furukawa Y, Li M et al. Genome-wide analysis of gene expression in intestinal-type gastric cancers using a complementary DNA microarray representing 23,040 genes. Cancer Res 2002; 62: 7012–7.Google Scholar
  9. 9.
    Shaver W. Establishment of a new human gastric adenocarcinoma cell line. Gastroenterology 1988; 94: A422.Google Scholar
  10. 10.
    Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet 1999; 21: 163–7.Google Scholar
  11. 11.
    Baylin SB, Herman JG, Graff JR et al. Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res 1998; 72: 141–96.CrossRefGoogle Scholar
  12. 12.
    Baylin SB, Herman JG. DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet 2000; 16: 168–74.Google Scholar
  13. 13.
    Esteller M, Herman JG. Cancer as an epigenetic disease: DNA methylation and chromatin alterations in human tumours. J Pathol 2002; 196: 1–7.Google Scholar
  14. 14.
    Jones PA, Taylor SM. Cellular differentiation, cytidine analogs and DNA methylation. Cell 1980; 20: 85–93.Google Scholar
  15. 15.
    Bender CM, Pao MM, Jones PA. Inhibition of DNA methylation by 5-aza-2′-deoxycytidine suppresses the growth of human tumor cell lines. Cancer Res 1998; 58: 95–101.Google Scholar
  16. 16.
    Santi DV, Garrett CE, Barr PJ. On the mechanism of inhibition of DNA-cytosine methyltransferases by cytosine analogs. Cell 1983; 33: 9–10.Google Scholar
  17. 17.
    Michalowsky LA, Jones PA. Differential nuclear protein binding to 5-azacytosine-containing DNA as a potential mechanism for 5-aza-2′-deoxycytidine resistance. Mol Cell Biol 1987; 7: 3076–83.Google Scholar
  18. 18.
    Baugh LR, Hill AA, Brown El et al. Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 2001; 29:E29.Google Scholar
  19. 19.
    Boon T, van der Bruggen P. Human tumor antigens recognized by T lymphocytes. J Exp Med 1996; 183: 725–9.Google Scholar
  20. 20.
    De Plaen E, Arden K, Traversari C et al. Structure, chromosomal localization, and expression of 12 genes of the MAGE family. Immunogenetics 1994; 40: 360–9.Google Scholar
  21. 21.
    Tureci O, Chen YT, Sahin U et al. Expression of SSX genes in human tumors. Int J Cancer 1998; 77: 19–23.Google Scholar
  22. 22.
    Gardiner-Garden M, Frommer M. CpG islands in vertebrate genomes. J Mol Biol 1987; 196: 261–82.Google Scholar
  23. 23.
    Hippo Y, Yashiro M, Ishii M et al. Differential gene expression profiles of scirrhous gastric cancer cells with high metastatic potential to peritoneum or lymph nodes. Cancer Res 2001; 61: 889–95.Google Scholar
  24. 24.
    Wang J, Chen S. Screening and identification of gastric adenocarcinoma metastasis-related genes using cDNA microarray coupled to FDD-PCR. J Cancer Res Clin Oncol 2002; 128: 547–53.Google Scholar
  25. 25.
    Mori M, Mimori K, Yoshikawa Y et al. Analysis of the gene-expression profile regarding the progression of human gastric carcinoma. Surgery 2002; 131: S39–47.Google Scholar
  26. 26.
    Virtanen I, Korhonen M, Kariniemi AL et al. Integrins in human cells and tumors. Cell Differ Dev 1990; 32: 15–27.Google Scholar
  27. 27.
    Cross GS, Speer A, Rosenthal A et al. Deletions of fetal and adult muscle cDNA in Duchenne and Becker muscular dystrophy patients. EMBO J 1987; 6: 3277–83.Google Scholar
  28. 28.
    Henry MD, Cohen MB, Campbell KP. Reduced expression of dystroglycan in breast and prostate cancer. Hum Pathol 2001; 32: 791–5.Google Scholar
  29. 29.
    Sgambato A, Migaldi M, Montanari M et al. Dystroglycan expression is frequently reduced in human breast and colon cancers and is associated with tumor progression. Am J Pathol 2003; 162: 849–60.Google Scholar
  30. 30.
    Shi Y, Ullrich SJ, Zhang J et al. A novel cytokine receptor-ligand pair. Identification, molecular characterization, and in vivo immunomodulatory activity. J Biol Chem 2000; 275: 19167–76.Google Scholar
  31. 31.
    Tangye SG, Phillips JH, Lanier LL. The CD2-subset of the Ig super-family of cell surface molecules: receptor-ligand pairs expressed by NK cells and other immune cells. Semin Immunol 2000; 12: 149–57.Google Scholar
  32. 32.
    Guenzi E, Topolt K, Cornali E et al. The helical domain of GBP-1 mediates the inhibition of endothelial cell proliferation by inflammatory cytokines. EMBO J 2001; 20: 5568–77.Google Scholar
  33. 33.
    Esteller M, Corn PG, Baylin SB et al. A gene hypermethylation profile of human cancer. Cancer Res 2001; 61: 3225–9.Google Scholar
  34. 34.
    Suzuki H, Gabrielson E, Chen W et al. A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer. Nat Genet 2002; 31: 141–9.Google Scholar
  35. 35.
    Karpf AR, Peterson PW, Rawlins JT et al. Inhibition of DNA methyl-transferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells. Proc Natl Acad Sci USA 1999; 96: 14007–12.Google Scholar
  36. 36.
    Stark GR, Kerr IM, Williams BR et al. How cells respond to interferons. Annu Rev Biochem 1998; 67: 227–64.Google Scholar
  37. 37.
    Ozaki K, Nagata M, Suzuki M et al. Isolation and characterization of a novel human lung-specific gene homologous to lysosomal membrane glycoproteins 1 and 2: significantly increased expression in cancers of various tissues. Cancer Res 1998; 58: 3499–503.Google Scholar
  38. 38.
    Hannigan BM, Barnett YA, Armstrong DB et al. Thymidine kinases: the enzymes and their clinical usefulness. Cancer Biother 1993; 8: 189–97.Google Scholar
  39. 39.
    Peifer M, Polakis P. Wnt signaling in oncogenesis and embryogenesis-a look outside the nucleus. Science 2000; 287: 1606–9.Google Scholar
  40. 40.
    Koliopanos A, Kleeff J, Xiao Y et al. Increased arylhydrocarbon receptor expression offers a potential therapeutic target for pancreatic cancer. Oncogene 2002; 21: 6059–70.Google Scholar
  41. 41.
    Andersson P, McGuire J, Rubio C et al. A constitutively active dioxin/aryl hydrocarbon receptor induces stomach tumors. Proc Natl Acad Sci USA 2002; 99: 9990–5.Google Scholar
  42. 42.
    Cameron EE, Bachman KE, Myohanen S et al. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet 1999; 21: 103–7.Google Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Jie Chen
    • 1
  • Christoph Röcken
    • 2
  • Ludger Klein-Hitpass
    • 3
  • Tobias Götze
    • 1
  • Andreas Leodolter
    • 1
  • Peter Malfertheiner
    • 1
  • Matthias P.A. Ebert
    • 1
  1. 1.Department of Gastroenterology, Hepatology and Infectious DiseasesOtto-von-Guericke UniversityMagdeburgGermany
  2. 2.Institute of PathologyOtto-von-Guericke UniversityMagdeburgGermany
  3. 3.Institute of Cell BiologyUniversity of EssenEssenGermany

Personalised recommendations