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. 2000 Nov;47(5):689–693. doi: 10.1136/gut.47.5.689

Evaluation of global DNA hypomethylation in human colon cancer tissues by immunohistochemistry and image analysis

F Hernandez-Blazque 1, M Habib 1, J Dumollard 1, C Barthelemy 1, M Benchaib 1, A de Capoa 1, A Niveleau 1
PMCID: PMC1728119  PMID: 11034586

Abstract

BACKGROUND—Global hypomethylation of DNA is frequently observed in human tumours. This alteration is detected in early adenomas in colorectal tumorigenesis. Information is currently acquired after extraction of DNA from tissues, digestion with nucleases, and analysis by reverse phase chromatography, or treatment with restriction enzymes followed by gel electrophoresis analysis and Southern hybridisation with radiolabelled probes.
AIMS—The purpose of our work was to evaluate the global methylation status of DNA in malignant lesions without loosing the histopathological features of the samples.
PATIENTS—The investigation was performed on paired normal-tumour tissues from 13 patients undergoing surgical resection of colorectal adenocarcinomas.
METHODS—Antibodies raised against 5-methylcytidine can be used to label methyl rich regions in interphase nuclei. This technique was adapted to the study of paraffin embedded tissues and an immunohistochemical method was developed to assess the global methylation status of individual nuclei while preserving cell morphology and tissue architecture. Computer assisted quantification of the staining intensity was performed on malignant and normal zones of human colon tissues to test the correlation between the immunolabelling signal and the respective histological patterns observed.
RESULTS—Qualitative and quantitative differences were observed and measured between the normal and malignant part of each sample. Morphologically altered nuclei displayed densely labelled spots within faintly labelled areas whereas normal nuclei were darker and uniformly stained. Image analysis allowed calculation of the average integrated optical density of the nuclei in both types of tissues, demonstrating a constant and significantly lower intensity for the former type of cells.


Keywords: colon adenocarcinoma; DNA hypomethylation; immunohistochemistry

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Figure 1  .

Figure 1  

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Indirect immunoperoxidase labelling. Example of dense nuclear labelling in normal colon tissue (A) and heterogeneous moderately stained nuclei in well differentiated colorectal adenocarcinoma (B). Before printing, colour slides were converted to black and white images without alteration of the range of density.

Figure 2  .

Figure 2  

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Dot blots of methylated DNA. Xp12 DNA was serially diluted with non-methylated λ phage DNA and 1 µl aliquots were dotted onto nitrocellulose, as described in materials and methods. Density levels were measured as described in materials and methods and plotted against percentage dilution.

Figure 3  .

Figure 3  

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DNA methylation assessment by Southwestern blotting. Blots: Example of results observed by Southwestern blotting with anti-5-MeCyd antibodies. Lane 1: non-digested human DNA; lane 2: human DNA digested with Eco R1; lane 3: human DNA digested with Mspl; and lane 4: human DNA digested with Hpall. Density profiles: DNA was extracted from paired normal-malignant tissues, digested with Mspl or Hpall, and submitted to gel electrophoresis, transferred onto nitrocellulose, and incubated with anti-5-MeCyd antibodies, as described in methods. Density profiles were registered and analysed as described in methods. The shaded area indicates the portion of the lane corresponding to satellite DNA. The ratios r between the surface of this portion and the surface of the entire lane are indicative of the sensitivity of each sample to digestion.

Selected References

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

  1. Ahuja N., Mohan A. L., Li Q., Stolker J. M., Herman J. G., Hamilton S. R., Baylin S. B., Issa J. P. Association between CpG island methylation and microsatellite instability in colorectal cancer. Cancer Res. 1997 Aug 15;57(16):3370–3374. [PubMed] [Google Scholar]
  2. Bernardino J., Roux C., Almeida A., Vogt N., Gibaud A., Gerbault-Seureau M., Magdelenat H., Bourgeois C. A., Malfoy B., Dutrillaux B. DNA hypomethylation in breast cancer: an independent parameter of tumor progression? Cancer Genet Cytogenet. 1997 Sep;97(2):83–89. doi: 10.1016/s0165-4608(96)00385-8. [DOI] [PubMed] [Google Scholar]
  3. Cheah M. S., Wallace C. D., Hoffman R. M. Hypomethylation of DNA in human cancer cells: a site-specific change in the c-myc oncogene. J Natl Cancer Inst. 1984 Nov;73(5):1057–1065. [PubMed] [Google Scholar]
  4. Counts J. L., Goodman J. I. Hypomethylation of DNA: an epigenetic mechanism involved in tumor promotion. Mol Carcinog. 1994 Dec;11(4):185–188. doi: 10.1002/mc.2940110402. [DOI] [PubMed] [Google Scholar]
  5. Cravo M., Pinto R., Fidalgo P., Chaves P., Glória L., Nobre-Leitão C., Costa Mira F. Global DNA hypomethylation occurs in the early stages of intestinal type gastric carcinoma. Gut. 1996 Sep;39(3):434–438. doi: 10.1136/gut.39.3.434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cunningham J. M., Christensen E. R., Tester D. J., Kim C. Y., Roche P. C., Burgart L. J., Thibodeau S. N. Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability. Cancer Res. 1998 Aug 1;58(15):3455–3460. [PubMed] [Google Scholar]
  7. Fearon E. R., Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990 Jun 1;61(5):759–767. doi: 10.1016/0092-8674(90)90186-i. [DOI] [PubMed] [Google Scholar]
  8. Feinberg A. P., Gehrke C. W., Kuo K. C., Ehrlich M. Reduced genomic 5-methylcytosine content in human colonic neoplasia. Cancer Res. 1988 Mar 1;48(5):1159–1161. [PubMed] [Google Scholar]
  9. Feinberg A. P., Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature. 1983 Jan 6;301(5895):89–92. doi: 10.1038/301089a0. [DOI] [PubMed] [Google Scholar]
  10. Feinberg A. P., Vogelstein B. Hypomethylation of ras oncogenes in primary human cancers. Biochem Biophys Res Commun. 1983 Feb 28;111(1):47–54. doi: 10.1016/s0006-291x(83)80115-6. [DOI] [PubMed] [Google Scholar]
  11. Gama-Sosa M. A., Slagel V. A., Trewyn R. W., Oxenhandler R., Kuo K. C., Gehrke C. W., Ehrlich M. The 5-methylcytosine content of DNA from human tumors. Nucleic Acids Res. 1983 Oct 11;11(19):6883–6894. doi: 10.1093/nar/11.19.6883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goelz S. E., Vogelstein B., Hamilton S. R., Feinberg A. P. Hypomethylation of DNA from benign and malignant human colon neoplasms. Science. 1985 Apr 12;228(4696):187–190. doi: 10.1126/science.2579435. [DOI] [PubMed] [Google Scholar]
  13. Goelz S. E., Vogelstein B., Hamilton S. R., Feinberg A. P. Hypomethylation of DNA from benign and malignant human colon neoplasms. Science. 1985 Apr 12;228(4696):187–190. doi: 10.1126/science.2579435. [DOI] [PubMed] [Google Scholar]
  14. Gonzalez-Zulueta M., Bender C. M., Yang A. S., Nguyen T., Beart R. W., Van Tornout J. M., Jones P. A. Methylation of the 5' CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing. Cancer Res. 1995 Oct 15;55(20):4531–4535. [PubMed] [Google Scholar]
  15. Greenblatt M. S., Bennett W. P., Hollstein M., Harris C. C. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 1994 Sep 15;54(18):4855–4878. [PubMed] [Google Scholar]
  16. Greenblatt M. S., Bennett W. P., Hollstein M., Harris C. C. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 1994 Sep 15;54(18):4855–4878. [PubMed] [Google Scholar]
  17. Greger V., Debus N., Lohmann D., Höpping W., Passarge E., Horsthemke B. Frequency and parental origin of hypermethylated RB1 alleles in retinoblastoma. Hum Genet. 1994 Nov;94(5):491–496. doi: 10.1007/BF00211013. [DOI] [PubMed] [Google Scholar]
  18. Habib M., Fares F., Bourgeois C. A., Bella C., Bernardino J., Hernandez-Blazquez F., de Capoa A., Niveleau A. DNA global hypomethylation in EBV-transformed interphase nuclei. Exp Cell Res. 1999 May 25;249(1):46–53. doi: 10.1006/excr.1999.4434. [DOI] [PubMed] [Google Scholar]
  19. Hakkarainen M., Wahlfors J., Myöhänen S., Hiltunen M. O., Eskelinen M., Johansson R., Jänne J. Hypermethylation of calcitonin gene regulatory sequences in human breast cancer as revealed by genomic sequencing. Int J Cancer. 1996 Dec 20;69(6):471–474. doi: 10.1002/(SICI)1097-0215(19961220)69:6<471::AID-IJC9>3.0.CO;2-1. [DOI] [PubMed] [Google Scholar]
  20. Issa J. P., Vertino P. M., Wu J., Sazawal S., Celano P., Nelkin B. D., Hamilton S. R., Baylin S. B. Increased cytosine DNA-methyltransferase activity during colon cancer progression. J Natl Cancer Inst. 1993 Aug 4;85(15):1235–1240. doi: 10.1093/jnci/85.15.1235. [DOI] [PubMed] [Google Scholar]
  21. Jakob C. A., Guldenschuh I., Hürlimann R., Müllhaupt B., Müller A., Ammann R., Fried M., Roth J. 5'-Cytosine DNA-methyltransferase mRNA levels in hereditary colon carcinoma. Virchows Arch. 1999 Jan;434(1):57–62. doi: 10.1007/s004280050305. [DOI] [PubMed] [Google Scholar]
  22. Jones P. A. DNA methylation errors and cancer. Cancer Res. 1996 Jun 1;56(11):2463–2467. [PubMed] [Google Scholar]
  23. Laird P. W., Jaenisch R. The role of DNA methylation in cancer genetic and epigenetics. Annu Rev Genet. 1996;30:441–464. doi: 10.1146/annurev.genet.30.1.441. [DOI] [PubMed] [Google Scholar]
  24. Lengauer C., Kinzler K. W., Vogelstein B. DNA methylation and genetic instability in colorectal cancer cells. Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2545–2550. doi: 10.1073/pnas.94.6.2545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Little M., Wainwright B. Methylation and p16: suppressing the suppressor. Nat Med. 1995 Jul;1(7):633–634. doi: 10.1038/nm0795-633. [DOI] [PubMed] [Google Scholar]
  26. Mayer W., Niveleau A., Walter J., Fundele R., Haaf T. Demethylation of the zygotic paternal genome. Nature. 2000 Feb 3;403(6769):501–502. doi: 10.1038/35000656. [DOI] [PubMed] [Google Scholar]
  27. Qu G., Dubeau L., Narayan A., Yu M. C., Ehrlich M. Satellite DNA hypomethylation vs. overall genomic hypomethylation in ovarian epithelial tumors of different malignant potential. Mutat Res. 1999 Jan 25;423(1-2):91–101. doi: 10.1016/s0027-5107(98)00229-2. [DOI] [PubMed] [Google Scholar]
  28. Razin A., Riggs A. D. DNA methylation and gene function. Science. 1980 Nov 7;210(4470):604–610. doi: 10.1126/science.6254144. [DOI] [PubMed] [Google Scholar]
  29. Reynaud C., Bruno C., Boullanger P., Grange J., Barbesti S., Niveleau A. Monitoring of urinary excretion of modified nucleosides in cancer patients using a set of six monoclonal antibodies. Cancer Lett. 1992 Jan 31;61(3):255–262. doi: 10.1016/0304-3835(92)90296-8. [DOI] [PubMed] [Google Scholar]
  30. Schmutte C., Yang A. S., Nguyen T. T., Beart R. W., Jones P. A. Mechanisms for the involvement of DNA methylation in colon carcinogenesis. Cancer Res. 1996 May 15;56(10):2375–2381. [PubMed] [Google Scholar]
  31. Sharrard R. M., Royds J. A., Rogers S., Shorthouse A. J. Patterns of methylation of the c-myc gene in human colorectal cancer progression. Br J Cancer. 1992 May;65(5):667–672. doi: 10.1038/bjc.1992.142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Steinbeck R. G. Chromosome division figures reveal genomic instability in tumorigenesis of human colon mucosa. Br J Cancer. 1998 Apr;77(7):1027–1033. doi: 10.1038/bjc.1998.171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tran R., Kashmiri S. V., Kantor J., Greiner J. W., Pestka S., Shively J. E., Schlom J. Correlation of DNA hypomethylation with expression of carcinoembryonic antigen in human colon carcinoma cells. Cancer Res. 1988 Oct 15;48(20):5674–5679. [PubMed] [Google Scholar]
  34. Wahlfors J., Hiltunen H., Heinonen K., Hämäläinen E., Alhonen L., Jänne J. Genomic hypomethylation in human chronic lymphocytic leukemia. Blood. 1992 Oct 15;80(8):2074–2080. [PubMed] [Google Scholar]
  35. de Capoa A., Di Leandro M., Grappelli C., Menendez F., Poggesi I., Giancotti P., Marotta M. R., Spanò A., Rocchi M., Archidiacono N. Computer-assisted analysis of methylation status of individual interphase nuclei in human cultured cells. Cytometry. 1998 Feb 1;31(2):85–92. doi: 10.1002/(sici)1097-0320(19980201)31:2<85::aid-cyto3>3.3.co;2-8. [DOI] [PubMed] [Google Scholar]
  36. de Capoa A., Febbo F. R., Giovannelli F., Niveleau A., Zardo G., Marenzi S., Caiafa P. Reduced levels of poly(ADP-ribosyl)ation result in chromatin compaction and hypermethylation as shown by cell-by-cell computer-assisted quantitative analysis. FASEB J. 1999 Jan;13(1):89–93. doi: 10.1096/fasebj.13.1.89. [DOI] [PubMed] [Google Scholar]
  37. de Capoa A., Grappelli C., Febbo F. R., Spanò A., Niveleau A., Cafolla A., Cordone I., Foa R. Methylation levels of normal and chronic lymphocytic leukemia B lymphocytes: computer-assisted quantitative analysis of anti-5-methylcytosine antibody binding to individual nuclei. Cytometry. 1999 Jun 1;36(2):157–159. doi: 10.1002/(sici)1097-0320(19990601)36:2<157::aid-cyto10>3.3.co;2-b. [DOI] [PubMed] [Google Scholar]

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