Hypomethylation of the MN/CA9 promoter and upregulated MN/CA9 expression in human renal cell carcinoma (original) (raw)

Abstract

MN/CA9 is a cancer-related gene, frequently activated in human renal cell carcinomas (RCCs). To reveal the activation mechanism, we investigated the relationship between methylation status of the MN/CA9 promoter region and gene expression using 13 human RCCs, and examined the effect of in vitro CpG methylation on the MN/CA9 promoter activity using a human RCC cell line (SK-RC-44), expressing MN/CA9. MN/CA9 expression was evaluated by RT-PCR and observed in 10 of 13 RCCs (77%). A total of 9 out of 10 _MN/CA9_-positive RCCs (90%) contained clear cell components. Methylation status of 6 CpGs in the MN/CA9 promoter region was decided by using the bisulfite genomic sequencing protocol. Out of 13 RCCs 9 (69%) showed partial hypomethylation of the CpG at −74 bp, while the other 4 RCCs and 3 normal kidney tissue samples showed complete methylation. Hypomethylation of the CpG at −74 bp was strongly correlated with MN/CA9 expression. Luciferase assay revealed that the MN/CA9 promoter activity was strongly suppressed by methylation of the CpG at −74 bp. These findings suggest that hypomethylation of the CpG at −74 bp in the MN/CA9 promoter region might play an important role in this gene activation of human RCC. © 2001 Cancer Research Campaign http://www.bjcancer.com

Keywords: MN/CA9, hypomethylation, renal cell carcinoma

Full Text

The Full Text of this article is available as a PDF (143.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Busslinger M., Hurst J., Flavell R. A. DNA methylation and the regulation of globin gene expression. Cell. 1983 Aug;34(1):197–206. doi: 10.1016/0092-8674(83)90150-2. [DOI] [PubMed] [Google Scholar]
  2. Cho M., Grabmaier K., Kitahori Y., Hiasa Y., Nakagawa Y., Uemura H., Hirao Y., Ohnishi T., Yoshikawa K., Ooesterwijk E. Activation of the MN/CA9 gene is associated with hypomethylation in human renal cell carcinoma cell lines. Mol Carcinog. 2000 Mar;27(3):184–189. [PubMed] [Google Scholar]
  3. De Smet C., De Backer O., Faraoni I., Lurquin C., Brasseur F., Boon T. The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation. Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):7149–7153. doi: 10.1073/pnas.93.14.7149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hanada M., Delia D., Aiello A., Stadtmauer E., Reed J. C. bcl-2 gene hypomethylation and high-level expression in B-cell chronic lymphocytic leukemia. Blood. 1993 Sep 15;82(6):1820–1828. [PubMed] [Google Scholar]
  5. Henikoff S. Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 1987;155:156–165. doi: 10.1016/0076-6879(87)55014-5. [DOI] [PubMed] [Google Scholar]
  6. Herman J. G., Latif F., Weng Y., Lerman M. I., Zbar B., Liu S., Samid D., Duan D. S., Gnarra J. R., Linehan W. M. Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9700–9704. doi: 10.1073/pnas.91.21.9700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ivanov S. V., Kuzmin I., Wei M. H., Pack S., Geil L., Johnson B. E., Stanbridge E. J., Lerman M. I. Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes. Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12596–12601. doi: 10.1073/pnas.95.21.12596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kaluz S., Kaluzová M., Opavský R., Pastoreková S., Gibadulinová A., Dequiedt F., Kettmann R., Pastorek J. Transcriptional regulation of the MN/CA 9 gene coding for the tumor-associated carbonic anhydrase IX. Identification and characterization of a proximal silencer element. J Biol Chem. 1999 Nov 12;274(46):32588–32595. doi: 10.1074/jbc.274.46.32588. [DOI] [PubMed] [Google Scholar]
  9. Kaluzová M., Pastoreková S., Pastorek J., Kaluz S. P53 tumour suppressor modulates transcription of the TATA-less gene coding for the tumour-associated carbonic anhydrase MN/CA IX in MaTu cells. Biochim Biophys Acta. 2000 Apr 25;1491(1-3):20–26. doi: 10.1016/s0167-4781(00)00015-4. [DOI] [PubMed] [Google Scholar]
  10. Kuramasu A., Saito H., Suzuki S., Watanabe T., Ohtsu H. Mast cell-/basophil-specific transcriptional regulation of human L-histidine decarboxylase gene by CpG methylation in the promoter region. J Biol Chem. 1998 Nov 20;273(47):31607–31614. doi: 10.1074/jbc.273.47.31607. [DOI] [PubMed] [Google Scholar]
  11. Liao S. Y., Aurelio O. N., Jan K., Zavada J., Stanbridge E. J. Identification of the MN/CA9 protein as a reliable diagnostic biomarker of clear cell carcinoma of the kidney. Cancer Res. 1997 Jul 15;57(14):2827–2831. [PubMed] [Google Scholar]
  12. Lu S., Davies P. J. Regulation of the expression of the tissue transglutaminase gene by DNA methylation. Proc Natl Acad Sci U S A. 1997 Apr 29;94(9):4692–4697. doi: 10.1073/pnas.94.9.4692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. McKiernan J. M., Buttyan R., Bander N. H., de la Taille A., Stifelman M. D., Emanuel E. R., Bagiella E., Rubin M. A., Katz A. E., Olsson C. A. The detection of renal carcinoma cells in the peripheral blood with an enhanced reverse transcriptase-polymerase chain reaction assay for MN/CA9. Cancer. 1999 Aug 1;86(3):492–497. doi: 10.1002/(sici)1097-0142(19990801)86:3<492::aid-cncr18>3.0.co;2-r. [DOI] [PubMed] [Google Scholar]
  14. Merlo A., Herman J. G., Mao L., Lee D. J., Gabrielson E., Burger P. C., Baylin S. B., Sidransky D. 5' CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nat Med. 1995 Jul;1(7):686–692. doi: 10.1038/nm0795-686. [DOI] [PubMed] [Google Scholar]
  15. Nakayama M., Wada M., Harada T., Nagayama J., Kusaba H., Ohshima K., Kozuru M., Komatsu H., Ueda R., Kuwano M. Hypomethylation status of CpG sites at the promoter region and overexpression of the human MDR1 gene in acute myeloid leukemias. Blood. 1998 Dec 1;92(11):4296–4307. [PubMed] [Google Scholar]
  16. Oosterwijk E., Ruiter D. J., Hoedemaeker P. J., Pauwels E. K., Jonas U., Zwartendijk J., Warnaar S. O. Monoclonal antibody G 250 recognizes a determinant present in renal-cell carcinoma and absent from normal kidney. Int J Cancer. 1986 Oct 15;38(4):489–494. doi: 10.1002/ijc.2910380406. [DOI] [PubMed] [Google Scholar]
  17. Opavský R., Pastoreková S., Zelník V., Gibadulinová A., Stanbridge E. J., Závada J., Kettmann R., Pastorek J. Human MN/CA9 gene, a novel member of the carbonic anhydrase family: structure and exon to protein domain relationships. Genomics. 1996 May 1;33(3):480–487. doi: 10.1006/geno.1996.0223. [DOI] [PubMed] [Google Scholar]
  18. Pastorek J., Pastoreková S., Callebaut I., Mornon J. P., Zelník V., Opavský R., Zat'ovicová M., Liao S., Portetelle D., Stanbridge E. J. Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment. Oncogene. 1994 Oct;9(10):2877–2888. [PubMed] [Google Scholar]
  19. Saarnio J., Parkkila S., Parkkila A. K., Haukipuro K., Pastoreková S., Pastorek J., Kairaluoma M. I., Karttunen T. J. Immunohistochemical study of colorectal tumors for expression of a novel transmembrane carbonic anhydrase, MN/CA IX, with potential value as a marker of cell proliferation. Am J Pathol. 1998 Jul;153(1):279–285. doi: 10.1016/S0002-9440(10)65569-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schmutte C., Jones P. A. Involvement of DNA methylation in human carcinogenesis. Biol Chem. 1998 Apr-May;379(4-5):377–388. doi: 10.1515/bchm.1998.379.4-5.377. [DOI] [PubMed] [Google Scholar]
  21. Tsujiuchi T., Tsutsumi M., Sasaki Y., Takahama M., Konishi Y. Hypomethylation of CpG sites and c-myc gene overexpression in hepatocellular carcinomas, but not hyperplastic nodules, induced by a choline-deficient L-amino acid-defined diet in rats. Jpn J Cancer Res. 1999 Sep;90(9):909–913. doi: 10.1111/j.1349-7006.1999.tb00834.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Turner J. R., Odze R. D., Crum C. P., Resnick M. B. MN antigen expression in normal, preneoplastic, and neoplastic esophagus: a clinicopathological study of a new cancer-associated biomarker. Hum Pathol. 1997 Jun;28(6):740–744. doi: 10.1016/s0046-8177(97)90185-4. [DOI] [PubMed] [Google Scholar]
  23. Uemura H., Nakagawa Y., Yoshida K., Saga S., Yoshikawa K., Hirao Y., Oosterwijk E. MN/CA IX/G250 as a potential target for immunotherapy of renal cell carcinomas. Br J Cancer. 1999 Oct;81(4):741–746. doi: 10.1038/sj.bjc.6690757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yoshiura K., Kanai Y., Ochiai A., Shimoyama Y., Sugimura T., Hirohashi S. Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7416–7419. doi: 10.1073/pnas.92.16.7416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Zingg J. M., Jones P. A. Genetic and epigenetic aspects of DNA methylation on genome expression, evolution, mutation and carcinogenesis. Carcinogenesis. 1997 May;18(5):869–882. doi: 10.1093/carcin/18.5.869. [DOI] [PubMed] [Google Scholar]