Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

A second-generation profiling system for quantitative methylation analysis of multiple gene promoters: application to lung cancer

Oncogene volume 26, pages 6518–6525 (2007)Cite this article

Abstract

Cancer-specific gene promoter methylation has been described in many types of cancers, and various semi-quantified results have shown their usefulness. Here, we show a more sensitive and specific second-generation system for profiling the DNA methylation status. This method is based on bisulfite reaction of DNA and real-time PCR using two TaqMan MGB probes labeled with different fluorescence, followed by clustering analysis. Primers were designed with CpG-less sequences, and TaqMan MGB probes were designed to contain three or four CpG sites and to be shorter than conventional TaqMan probes. We have added new criteria for primer and probe design for further specificity. We confirmed the reliability of this system and applied it to analysis of lung cancers. Using 10 promoters, 90 primary lung cancers were clustered into six groups consisting of cases having similar smoking status and pathological findings. EGFR mutation and p16 promoter DNA methylation were exclusive, as previously reported; however, DNA methylation in other genes was unrelated to EGFR mutation. This system was also useful to distinguish double primary lung cancers from a single cancer with intrapulmonary metastasis. As above, our system has widespread availability in clinical use and biological research.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  • Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A et al. (2000). Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403: 503–511.

    Article  CAS  Google Scholar 

  • Brabender J, Usadel H, Danenberg KD, Metzger R, Schneider PM, Lord RV et al. (2001). Adenomatous polyposis coli gene promoter hypermethylation in non-small cell lung cancer is associated with survival. Oncogene 20: 3528–3532.

    Article  CAS  Google Scholar 

  • Chen G, Gharib TG, Wang H, Huang CC, Kuick R, Thomas DG et al. (2003). Protein profiles associated with survival in lung adenocarcinoma. Proc Natl Acad Sci USA 100: 13537–13542.

    Article  CAS  Google Scholar 

  • Clark SJ, Harrison J, Paul CL, Frommer M . (1994). High sensitivity mapping of methylated cytosines. Nucleic Acids Res 22: 2990–2997.

    Article  CAS  Google Scholar 

  • de Hoon MJ, Imoto S, Nolan J, Miyano S . (2004). Open source clustering software. Bioinformatics 20: 1453–1454.

    Article  CAS  Google Scholar 

  • Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, Shibata D et al. (2000). MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 28: E32.

    Article  CAS  Google Scholar 

  • Eisen MB, Spellman PT, Brown PO, Botstein D . (1998). Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 95: 14863–14868.

    Article  CAS  Google Scholar 

  • Feil R, Khosla S . (1999). Genomic imprinting in mammals: an interplay between chromatin and DNA methylation? Trends Genet 15: 431–435.

    Article  CAS  Google Scholar 

  • Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW et al. (1992). A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA 89: 1827–1831.

    Article  CAS  Google Scholar 

  • Fukami T, Fukuhara H, Kuramochi M, Maruyama T, Isogai K, Sakamoto M et al. (2003). Promoter methylation of the TSLC1 gene in advanced lung tumors and various cancer cell lines. Int J Cancer 107: 53–59.

    Article  CAS  Google Scholar 

  • Gu J, Berman D, Lu C, Wistuba II, Roth JA, Frazier M et al. (2006). Aberrant promoter methylation profile and association with survival in patients with non-small cell lung cancer. Clin Cancer Res 12: 7329–7338.

    Article  CAS  Google Scholar 

  • Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB . (1996). Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 93: 9821–9826.

    Article  CAS  Google Scholar 

  • Izumi H, Inoue J, Yokoi S, Hosoda H, Shibata T, Sunamori M et al. (2005). Frequent silencing of DBC1 is by genetic or epigenetic mechanisms in non-small cell lung cancers. Hum Mol Genet 14: 997–1007.

    Article  CAS  Google Scholar 

  • Jones PA, Baylin SB . (2002). The fundamental role of epigenetic events in cancer. Nat Rev Genet 3: 415–428.

    Article  CAS  Google Scholar 

  • Kim YT, Park SJ, Lee SH, Kang HJ, Hahn S, Kang CH et al. (2005). Prognostic implication of aberrant promoter hypermethylation of CpG islands in adenocarcinoma of the lung. J Thorac Cardiovasc Surg 130: 1378.

    Article  CAS  Google Scholar 

  • Kutyavin IV, Afonina IA, Mills A, Gorn VV, Lukhtanov EA, Belousov ES et al. (2000). 3′-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res 28: 655–661.

    Article  CAS  Google Scholar 

  • Lehmann U, Hasemeier B, Lilischkis R, Kreipe H . (2001). Quantitative analysis of promoter hypermethylation in laser-microdissected archival specimens. Lab Invest 81: 635–638.

    Article  CAS  Google Scholar 

  • Martini N, Melamed MR . (1975). Multiple primary lung cancers. J Thorac Cardiovasc Surg 70: 606–612.

    CAS  PubMed  Google Scholar 

  • Mitsudomi T, Yatabe Y, Koshikawa T, Hatooka S, Shinoda M, Suyama M et al. (1997). Mutations of the P53 tumor suppressor gene as clonal marker for multiple primary lung cancers. J Thorac Cardiovasc Surg 114: 354–360.

    Article  CAS  Google Scholar 

  • Panning B, Jaenisch R . (1998). RNA and the epigenetic regulation of X chromosome inactivation. Cell 93: 305–308.

    Article  CAS  Google Scholar 

  • Safar AM, Spencer H, Su X, Coffey M, Cooney CA, Ratnasinghe LD et al. (2005). Methylation profiling of archived non-small cell lung cancer: a promising prognostic system. Clin Cancer Res 11: 4400–4405.

    Article  CAS  Google Scholar 

  • Sambrook J, Russel DW . (2001). Molecular Cloning 3rd edn., vol 2, chapter 9. CSHL Press: New York.

    Google Scholar 

  • Suzuki M, Shigematsu H, Iizasa T, Hiroshima K, Nakatani Y, Minna JD et al. (2006). Exclusive mutation in epidermal growth factor receptor gene, HER-2, and KRAS, and synchronous methylation of nonsmall cell lung cancer. Cancer 106: 2200–2207.

    Article  CAS  Google Scholar 

  • Takai D, Jones PA . (2002). Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc Natl Acad Sci USA 99: 3740–3745.

    Article  CAS  Google Scholar 

  • Takai D, Yagi Y, Wakazono K, Ohishi N, Morita Y, Sugimura T et al. (2001). Silencing of HTR1B and reduced expression of EDN1 in human lung cancers, revealed by methylation-sensitive representational difference analysis. Oncogene 20: 7505–7513.

    Article  CAS  Google Scholar 

  • Toyooka S, Tokumo M, Shigematsu H, Matsuo K, Asano H, Tomii K et al. (2006). Mutational and epigenetic evidence for independent pathways for lung adenocarcinomas arising in smokers and never smokers. Cancer Res 66: 1371–1375.

    Article  CAS  Google Scholar 

  • Tsou JA, Shen LY, Siegmund KD, Long TI, Laird PW, Seneviratne CK et al. (2005). Distinct DNA methylation profiles in malignant mesothelioma, lung adenocarcinoma, and non-tumor lung. Lung Cancer 47: 193–204.

    Article  Google Scholar 

  • Ushijima T . (2005). Detection and interpretation of altered methylation patterns in cancer cells. Nat Rev Cancer 5: 223–231.

    Article  CAS  Google Scholar 

  • Usuda K, Saito Y, Takahashi S, Okaniwa G, Handa M, Sakuma T et al. (1996). Diagnosis and surgical treatment of lung cancer with synchronous multiple lesions. Japanese J Lung Cancer 36: 359–364.

    Article  Google Scholar 

  • Virmani AK, Tsou JA, Siegmund KD, Shen LY, Long TI, Laird PW et al. (2002). Hierarchical clustering of lung cancer cell lines using DNA methylation markers. Cancer Epidemiol Biomarkers Prev 11: 291–297.

    CAS  PubMed  Google Scholar 

  • Wang J, Lee JJ, Wang L, Liu DD, Lu C, Fan YH et al. (2004). Value of p16INK4a and RASSF1A promoter hypermethylation in prognosis of patients with resectable non-small cell lung cancer. Clin Cancer Res 10: 6119–6125.

    Article  CAS  Google Scholar 

  • Zeschnigk M, Bohringer S, Price EA, Onadim Z, Masshofer L, Lohmann DR . (2004). A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA): analysis of the retinoblastoma locus. Nucleic Acids Res 32: e125.

    Article  Google Scholar 

Download references

Acknowledgements

We thank Peter W Laird for his excellent advice. DT is supported by a grant from the Smoking Research Foundation, a grant from the Ministry of Education, Culture, Sports, Science and Technology, a grant from Japan Society for the Promotion of Science, a grant from the Takeda Science Foundation, and a grant from the Life Science Foundation of Japan. H Kitagawa is supported by a grant from the Smoking Research Foundation, and a grant from the Japan Society for the Promotion of Science.

Author information

Author notes

  1. A Sano, H Kage and K Sugimoto: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of General Thoracic Surgery, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan

    A Sano, J Nakajima & S Takamoto

  2. Department of Respiratory Medicine, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan

    H Kage, K Sugimoto, H Kitagawa, N Aki, T Nagase & N Ohishi

  3. Department of Pathology, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan

    A Goto & M Fukayama

  4. Department of Clinical Laboratory, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan

    Y Yatomi & D Takai

Authors
  1. A Sano
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. H Kage
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K Sugimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. H Kitagawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. N Aki
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A Goto
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. M Fukayama
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. J Nakajima
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. S Takamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. T Nagase
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Y Yatomi
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. N Ohishi
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. D Takai
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to D Takai.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sano, A., Kage, H., Sugimoto, K. et al. A second-generation profiling system for quantitative methylation analysis of multiple gene promoters: application to lung cancer. Oncogene 26, 6518–6525 (2007). https://doi.org/10.1038/sj.onc.1210483

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.onc.1210483

Keywords

This article is cited by

Search

Advanced search

Quick links