This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gonzalgo, M. L. Articles by Jones, P. A. Search for Related Content PubMed Articles by Gonzalgo, M. L. Articles by Jones, P. A.

Identification and Characterization of Differentially Methylated Regions of Genomic DNA by Methylation-sensitive Arbitrarily Primed PCR¹

Department of Biochemistry and Molecular Biology, Urological Cancer Research Laboratory, University of Southern California/Norris Comprehensive Cancer Center, University of Southern California, School of Medicine, Los Angeles, California 90033 [M. L. G., G. L., J-M. Z., P. A. J.]

We have developed a simple and reproducible fingerprinting method for screening the genome for regions of DNA that have altered patterns of DNA methylation associated with oncogenic transformation. Restriction enzymes with different sensitivities to cytosine methylation in their recognition sites were used to digest genomic DNAs from primary tumors, cell lines, and normal tissues prior to arbitrarily primed PCR amplification. Fragments that showed differential methylation were cloned and sequenced after resolving the PCR products on high-resolution polyacrylamide gels. The cloned fragments were then used as probes for Southern analysis to confirm differential methylation of these regions in colon tissues and cell lines. Forty-four DNA fragments associated with a total of five different regions of genomic DNA containing methylation sites were detected in 10 matched sets of normal and tumor colon DNAs and 7 colon cancer cell lines. A novel CpG island was also isolated that was found to be frequently hypermethylated in bladder and colon tumors. We have demonstrated that this technique is a rapid and efficient method that can be used to screen for altered methylation patterns in genomic DNA and to isolate specific sequences associated with these changes.

¹ Supported by United States Public Health Service Grant R35 CA49758 from the National Cancer Institute. W. M. R. is funded by a Burroughs-Wellcome Fund Hitchings-Elion Postdoctoral Fellowship.

² These authors contributed equally to this work.

³ Current address: The Scripps Research Institute, 10666 North Torrey Pines Road, MB-7, La Jolla, CA 92037.

⁴ Current address: Whitehead Institute, Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge, MA 02142.

⁵ To whom requests for reprints should be addressed. Phone: (213) 764-0816; Fax: (213) 764-0102.

Received 11/14/96. Accepted 1/ 2/97.

This article has been cited by other articles:

				J.-F. Coppin, W. Qu, and M. P. Waalkes Interplay between Cellular Methyl Metabolism and Adaptive Efflux during Oncogenic Transformation from Chronic Arsenic Exposure in Human Cells J. Biol. Chem., July 11, 2008; 283(28): 19342 - 19350. [Abstract] [Full Text] [PDF]

				J. M. Phillips and J. I. Goodman Identification of Genes that May Play Critical Roles in Phenobarbital (PB)-Induced Liver Tumorigenesis due to Altered DNA Methylation Toxicol. Sci., July 1, 2008; 104(1): 86 - 99. [Abstract] [Full Text] [PDF]

				M. R.H. Estecio, P. S. Yan, A. E.K. Ibrahim, C. S. Tellez, L. Shen, T. H.-M. Huang, and J.-P. J. Issa High-throughput methylation profiling by MCA coupled to CpG island microarray Genome Res., October 1, 2007; 17(10): 1529 - 1536. [Abstract] [Full Text] [PDF]

				M. Bettstetter, S. Dechant, P. Ruemmele, M. Grabowski, G. Keller, E. Holinski-Feder, A. Hartmann, F. Hofstaedter, and W. Dietmaier Distinction of Hereditary Nonpolyposis Colorectal Cancer and Sporadic Microsatellite-Unstable Colorectal Cancer through Quantification of MLH1 Methylation by Real-time PCR Clin. Cancer Res., June 1, 2007; 13(11): 3221 - 3228. [Abstract] [Full Text] [PDF]

				G. Wu, Z. Guo, X. Chang, M. S. Kim, J. K. Nagpal, J. Liu, J. M. Maki, K. I. Kivirikko, S. P. Ethier, B. Trink, et al. LOXL1 and LOXL4 Are Epigenetically Silenced and Can Inhibit Ras/Extracellular Signal-Regulated Kinase Signaling Pathway in Human Bladder Cancer Cancer Res., May 1, 2007; 67(9): 4123 - 4129. [Abstract] [Full Text] [PDF]

				C. Plass Restriction Landmark Genomic Scanning Am. Assoc. Cancer Res. Educ. Book, April 14, 2007; 2007(1): 185 - 188. [Full Text] [PDF]

				J. M. Phillips, Y. Yamamoto, M. Negishi, R. R. Maronpot, and J. I. Goodman Orphan Nuclear Receptor Constitutive Active/Androstane Receptor-Mediated Alterations in DNA Methylation during Phenobarbital Promotion of Liver Tumorigenesis Toxicol. Sci., March 1, 2007; 96(1): 72 - 82. [Abstract] [Full Text] [PDF]

				V. F. Chekhun, N. Y. Lukyanova, O. Kovalchuk, V. P. Tryndyak, and I. P. Pogribny Epigenetic profiling of multidrug-resistant human MCF-7 breast adenocarcinoma cells reveals novel hyper- and hypomethylated targets Mol. Cancer Ther., March 1, 2007; 6(3): 1089 - 1098. [Abstract] [Full Text] [PDF]

				W E Farrell Pituitary tumours: findings from whole genome analyses. Endocr. Relat. Cancer, September 1, 2006; 13(3): 707 - 716. [Abstract] [Full Text] [PDF]

				A. N. Bachman, J. M. Phillips, and J. I. Goodman Phenobarbital Induces Progressive Patterns of GC-Rich and Gene-Specific Altered DNA Methylation in the Liver of Tumor-Prone B6C3F1 Mice Toxicol. Sci., June 1, 2006; 91(2): 393 - 405. [Abstract] [Full Text] [PDF]

				A. N. Bachman, G. M. Curtin, D. J. Doolittle, and J. I. Goodman Altered Methylation in Gene-Specific and GC-Rich Regions of DNA Is Progressive and Nonrandom during Promotion of Skin Tumorigenesis Toxicol. Sci., June 1, 2006; 91(2): 406 - 418. [Abstract] [Full Text] [PDF]

				T. K. WOJDACZ and L. L. HANSEN Techniques used in studies of age-related DNA methylation changes. Ann. N.Y. Acad. Sci., May 1, 2006; 1067: 479 - 487. [Abstract] [Full Text] [PDF]

				A. N. Bachman, L. M. Kamendulis, and J. I. Goodman Diethanolamine and Phenobarbital Produce an Altered Pattern of Methylation in GC-Rich Regions of DNA in B6C3F1 Mouse Hepatocytes Similar to That Resulting from Choline Deficiency Toxicol. Sci., April 1, 2006; 90(2): 317 - 325. [Abstract] [Full Text] [PDF]

				P. Schatz, J. Distler, K. Berlin, and M. Schuster Novel method for high throughput DNA methylation marker evaluation using PNA-probe library hybridization and MALDI-TOF detection. Nucleic Acids Res., January 1, 2006; 34(8): e59 - e59. [Abstract] [Full Text] [PDF]

				J.-i. Yasunaga, Y. Taniguchi, K. Nosaka, M. Yoshida, Y. Satou, T. Sakai, H. Mitsuya, and M. Matsuoka Identification of Aberrantly Methylated Genes in Association with Adult T-Cell Leukemia Cancer Res., September 1, 2004; 64(17): 6002 - 6009. [Abstract] [Full Text] [PDF]

				A. Bahar, D. J. Simpson, S. J. Cutty, J. E. Bicknell, P. R. Hoban, S. Holley, M. Mourtada-Maarabouni, G. T. Williams, R. N. Clayton, and W. E. Farrell Isolation and Characterization of a Novel Pituitary Tumor Apoptosis Gene Mol. Endocrinol., July 1, 2004; 18(7): 1827 - 1839. [Abstract] [Full Text] [PDF]

				R. E. Watson, J. M. McKim, G. L. Cockerell, and J. I. Goodman The Value of DNA Methylation Analysis in Basic, Initial Toxicity Assessments Toxicol. Sci., May 1, 2004; 79(1): 178 - 188. [Abstract] [Full Text] [PDF]

				L. Luo, B. Li, and T. P. Pretlow DNA Alterations in Human Aberrant Crypt Foci and Colon Cancers by Random Primed Polymerase Chain Reaction Cancer Res., October 1, 2003; 63(19): 6166 - 6169. [Abstract] [Full Text] [PDF]

				R. E. Watson, G. M. Curtin, D. J. Doolittle, and J. I. Goodman Progressive Alterations in Global and GC-Rich DNA Methylation during Tumorigenesis Toxicol. Sci., October 1, 2003; 75(2): 289 - 299. [Abstract] [Full Text] [PDF]

				M. F. Paz, S. Wei, J. C. Cigudosa, S. Rodriguez-Perales, M. A. Peinado, T. H.-M. Huang, and M. Esteller Genetic unmasking of epigenetically silenced tumor suppressor genes in colon cancer cells deficient in DNA methyltransferases Hum. Mol. Genet., September 1, 2003; 12(17): 2209 - 2219. [Abstract] [Full Text] [PDF]

				T. Matsuyama, M. T. Kimura, K. Koike, T. Abe, T. Nakano, T. Asami, T. Ebisuzaki, W. A. Held, S. Yoshida, and H. Nagase Global methylation screening in the Arabidopsis thaliana and Mus musculus genome: applications of virtual image restriction landmark genomic scanning (Vi-RLGS) Nucleic Acids Res., August 1, 2003; 31(15): 4490 - 4496. [Abstract] [Full Text] [PDF]

				W. A. Palmisano, K. P. Crume, M. J. Grimes, S. A. Winters, M. Toyota, M. Esteller, N. Joste, S. B. Baylin, and S. A. Belinsky Aberrant Promoter Methylation of the Transcription Factor Genes PAX5 {alpha} and {beta} in Human Cancers Cancer Res., August 1, 2003; 63(15): 4620 - 4625. [Abstract] [Full Text] [PDF]

				J. Tost, P. Schatz, M. Schuster, K. Berlin, and I. G. Gut Analysis and accurate quantification of CpG methylation by MALDI mass spectrometry Nucleic Acids Res., May 1, 2003; 31(9): e50 - e50. [Abstract] [Full Text] [PDF]

				S. E. COTTRELL and P. W. LAIRD Sensitive Detection of DNA Methylation Ann. N.Y. Acad. Sci., March 1, 2003; 983(1): 120 - 130. [Abstract] [Full Text] [PDF]

				A. KANEDA, D. TAKAI, M. KAMINISHI, E. OKOCHI, and T. USHIJIMA Methylation-Sensitive Representational Difference Analysis and Its Application to Cancer Research Ann. N.Y. Acad. Sci., March 1, 2003; 983(1): 131 - 141. [Abstract] [Full Text] [PDF]

				A. PATEL, J. D. GROOPMAN, and A. UMAR DNA Methylation as a Cancer-Specific Biomarker: From Molecules to Populations Ann. N.Y. Acad. Sci., March 1, 2003; 983(1): 286 - 297. [Abstract] [Full Text] [PDF]

				C. Plass Cancer epigenomics Hum. Mol. Genet., October 1, 2002; 11(20): 2479 - 2488. [Abstract] [Full Text] [PDF]

				R. E. Watson and J. I. Goodman Effects of Phenobarbital on DNA Methylation in GC-Rich Regions of Hepatic DNA from Mice That Exhibit Different Levels of Susceptibility to Liver Tumorigenesis Toxicol. Sci., July 1, 2002; 68(1): 51 - 58. [Abstract] [Full Text] [PDF]

				K. W. Choy, C. P. Pang, K. F. To, C. B. O. Yu, J. S. K. Ng, and D. S. C. Lam Impaired Expression and Promotor Hypermethylation of O6-Methylguanine-DNA Methyltransferase in Retinoblastoma Tissues Invest. Ophthalmol. Vis. Sci., May 1, 2002; 43(5): 1344 - 1349. [Abstract] [Full Text] [PDF]

				R. E. Watson and J. I. Goodman Epigenetics and DNA Methylation Come of Age in Toxicology Toxicol. Sci., May 1, 2002; 67(1): 11 - 16. [Abstract] [Full Text] [PDF]

				G. Liang, M. F. Chan, Y. Tomigahara, Y. C. Tsai, F. A. Gonzales, E. Li, P. W. Laird, and P. A. Jones Cooperativity between DNA Methyltransferases in the Maintenance Methylation of Repetitive Elements Mol. Cell. Biol., January 15, 2002; 22(2): 480 - 491. [Abstract] [Full Text] [PDF]

				G. J. R. Brock, T. H.-M. Huang, C.-M. Chen, and K. J. Johnson A novel technique for the identification of CpG islands exhibiting altered methylation patterns (ICEAMP) Nucleic Acids Res., December 15, 2001; 29(24): e123 - e123. [Abstract] [Full Text] [PDF]

				I. D.C. Markl, J. Cheng, G. Liang, D. Shibata, P. W. Laird, and P. A. Jones Global and Gene-specific Epigenetic Patterns in Human Bladder Cancer Genomes Are Relatively Stable in Vivo and in Vitro over Time Cancer Res., August 1, 2001; 61(15): 5875 - 5884. [Abstract] [Full Text] [PDF]

				J. F Costello and C. Plass Methylation matters J. Med. Genet., May 1, 2001; 38(5): 285 - 303. [Abstract] [Full Text]

				M. Esteller, P. G. Corn, S. B. Baylin, and J. G. Herman A Gene Hypermethylation Profile of Human Cancer Cancer Res., April 1, 2001; 61(8): 3225 - 3229. [Abstract] [Full Text]

				G. Liang, K. D. Robertson, C. Talmadge, J. Sumegi, and P. A. Jones The Gene for a Novel Transmembrane Protein Containing Epidermal Growth Factor and Follistatin Domains Is Frequently Hypermethylated in Human Tumor Cells Cancer Res., September 1, 2000; 60(17): 4907 - 4912. [Abstract] [Full Text]

				C. Salem, G. Liang, Y. C. Tsai, J. Coulter, M. A. Knowles, A.-C. Feng, S. Groshen, P. W. Nichols, and P. A. Jones Progressive Increases in de Novo Methylation of CpG Islands in Bladder Cancer Cancer Res., May 1, 2000; 60(9): 2473 - 2476. [Abstract] [Full Text]

				A. R. Karpf, P. W. Peterson, J. T. Rawlins, B. K. Dalley, Q. Yang, H. Albertsen, and D. A. Jones Inhibition of DNA methyltransferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells PNAS, November 23, 1999; 96(24): 14007 - 14012. [Abstract] [Full Text] [PDF]

				M. Toyota, C. Ho, M. Ohe-Toyota, S. B. Baylin, and J.-P. J. Issa Inactivation of CACNA1G, a T-Type Calcium Channel Gene, by Aberrant Methylation of Its 5' CpG Island in Human Tumors Cancer Res., September 1, 1999; 59(18): 4535 - 4541. [Abstract] [Full Text] [PDF]

				R. Singal and G. D. Ginder DNA Methylation Blood, June 15, 1999; 93(12): 4059 - 4070. [Full Text] [PDF]

				M. Toyota, C. Ho, N. Ahuja, K.-W. Jair, Q. Li, M. Ohe-Toyota, S. B. Baylin, and J.-P. J. Issa Identification of Differentially Methylated Sequences in Colorectal Cancer by Methylated CpG Island Amplification Cancer Res., May 1, 1999; 59(10): 2307 - 2312. [Abstract] [Full Text] [PDF]

				M. Shiraishi, Y. H. Chuu, and T. Sekiya Isolation of DNA fragments associated with methylated CpG islands in human adenocarcinomas of the lung using a methylated DNA binding column and denaturing gradient gel electrophoresis PNAS, March 16, 1999; 96(6): 2913 - 2918. [Abstract] [Full Text] [PDF]

				P. A. Jones and M. L. Gonzalgo Altered DNA methylation and genome instability: A new pathway to cancer? PNAS, March 18, 1997; 94(6): 2103 - 2105. [Full Text] [PDF]