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 Similar articles in PubMed 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 Qian, X. C. Articles by Brent, T. P. Search for Related Content PubMed PubMed Citation Articles by Qian, X. C. Articles by Brent, T. P.

Methylation Hot Spots in the 5' Flanking Region Denote Silencing of the O⁶-Methylguanine-DNA Methyltransferase Gene¹

Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105; and Department of Pharmacology, College of Medicine, University of Tennessee, Memphis, Tennessee 38163

The mechanism whereby the DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) is silenced in repair-deficient (Mer^-) human tumor cells is unknown. The role of methylation of the 5' CpG island in MGMT gene suppression is controversial. Althoug we previously showed by restriction enzyme analysis that CpG methylation in this region was associated with gene suppression, methylation at such sites was generally incomplete, suggesting heterogeneity. To clarify this issue, we have unequivocally defined the methylation status of every CpG by genomic sequencing of individual cloned copies of bisulfite-modified DNA. The region from -249 to +259 at the transcription start site was virtually methylation free in HT29 cells (Mer⁺), whereas in BE or HeLa S3 cells (Mer^-), this region was substantially methylated in every DNA copy, with "hot spots" from -249 to -103 and from +107 to +196. Up-regulation of MGMT in HeLa S3 cells induced by 5-azacytidine was accompanied by progressive demethylation and the appearance of totally unmethylated copies of DNA. We conclude that, in Mer^- cells, the MGMT promoter contains specific CpG methylation hot spots that are tightly linked to and are potential markers of gene silencing.

¹ This work was supported by NIH Grants CA14799 and Cancer Center Support (CORE) Grant P30 CA21765 and the American Lebanese and Syrian Associated Charities.

² To whom requests for reprints should be addressed, at Department of Molecular Pharmacology, St. Jude Children's Hospital, 332 North Lauderdale, Memphis, TN 38105-2794. Phone: (901) 495-3458; Fax: (901) 521-1668: E-mail: brent@stjude.org.

Received 5/23/97. Accepted 7/16/97.

This article has been cited by other articles:

				I. Vlassenbroeck, S. Califice, A.-C. Diserens, E. Migliavacca, J. Straub, I. Di Stefano, F. Moreau, M.-F. Hamou, I. Renard, M. Delorenzi, et al. Validation of Real-Time Methylation-Specific PCR to Determine O6-Methylguanine-DNA Methyltransferase Gene Promoter Methylation in Glioma J. Mol. Diagn., July 1, 2008; 10(4): 332 - 337. [Abstract] [Full Text] [PDF]

				W. Ji, L. Yang, L. Yu, J. Yuan, D. Hu, W. Zhang, J. Yang, Y. Pang, W. Li, J. Lu, et al. Epigenetic silencing of O6-methylguanine DNA methyltransferase gene in NiS-transformed cells Carcinogenesis, June 1, 2008; 29(6): 1267 - 1275. [Abstract] [Full Text] [PDF]

				J. N. Sarkaria, G. J. Kitange, C. D. James, R. Plummer, H. Calvert, M. Weller, and W. Wick Mechanisms of Chemoresistance to Alkylating Agents in Malignant Glioma Clin. Cancer Res., May 15, 2008; 14(10): 2900 - 2908. [Abstract] [Full Text] [PDF]

				R. Stupp, M. E. Hegi, M. R. Gilbert, and A. Chakravarti Chemoradiotherapy in Malignant Glioma: Standard of Care and Future Directions J. Clin. Oncol., September 10, 2007; 25(26): 4127 - 4136. [Abstract] [Full Text] [PDF]

				F. V. Jacinto and M. Esteller Mutator pathways unleashed by epigenetic silencing in human cancer Mutagenesis, July 1, 2007; 22(4): 247 - 253. [Abstract] [Full Text] [PDF]

				H. Ohgaki and P. Kleihues Genetic Pathways to Primary and Secondary Glioblastoma Am. J. Pathol., May 1, 2007; 170(5): 1445 - 1453. [Abstract] [Full Text] [PDF]

				K. Ishiguro, H. A. Seow, P. G. Penketh, K. Shyam, and A. C. Sartorelli Mode of action of the chloroethylating and carbamoylating moieties of the prodrug cloretazine. Mol. Cancer Ther., April 1, 2006; 5(4): 969 - 976. [Abstract] [Full Text] [PDF]

				D. A. Reardon, J. N. Rich, H. S. Friedman, and D. D. Bigner Recent Advances in the Treatment of Malignant Astrocytoma J. Clin. Oncol., March 10, 2006; 24(8): 1253 - 1265. [Abstract] [Full Text] [PDF]

				K. Ishiguro, K. Shyam, P. G. Penketh, and A. C. Sartorelli Role of O6-alkylguanine-DNA alkyltransferase in the cytotoxic activity of cloretazine Mol. Cancer Ther., November 1, 2005; 4(11): 1755 - 1763. [Abstract] [Full Text] [PDF]

				S Halford, A Rowan, E Sawyer, I Talbot, and I Tomlinson O6-methylguanine methyltransferase in colorectal cancers: detection of mutations, loss of expression, and weak association with G:C>A:T transitions Gut, June 1, 2005; 54(6): 797 - 802. [Abstract] [Full Text] [PDF]

				M. E. Hegi, A.-C. Diserens, T. Gorlia, M.-F. Hamou, N. de Tribolet, M. Weller, J. M. Kros, J. A. Hainfellner, W. Mason, L. Mariani, et al. MGMT Gene Silencing and Benefit from Temozolomide in Glioblastoma N. Engl. J. Med., March 10, 2005; 352(10): 997 - 1003. [Abstract] [Full Text] [PDF]

				R. P. Danam, S. R. Howell, T. P. Brent, and L. C. Harris Epigenetic regulation of O6-methylguanine-DNA methyltransferase gene expression by histone acetylation and methyl-CpG binding proteins Mol. Cancer Ther., January 1, 2005; 4(1): 61 - 69. [Abstract] [Full Text] [PDF]

				M. F. Paz, R. Yaya-Tur, I. Rojas-Marcos, G. Reynes, M. Pollan, L. Aguirre-Cruz, J. L. Garcia-Lopez, J. Piquer, M.-J. Safont, C. Balana, et al. CpG Island Hypermethylation of the DNA Repair Enzyme Methyltransferase Predicts Response to Temozolomide in Primary Gliomas Clin. Cancer Res., August 1, 2004; 10(15): 4933 - 4938. [Abstract] [Full Text] [PDF]

				B. R. Rood, H. Zhang, and P. H. Cogen Intercellular heterogeneity of expression of the MGMT DNA repair gene in pediatric medulloblastoma Neuro-oncol, July 1, 2004; 6(3): 200 - 207. [Abstract] [PDF]

				L. C. Pulling, B. R. Vuillemenot, J. A. Hutt, T. R. Devereux, and S. A. Belinsky Aberrant Promoter Hypermethylation of the Death-Associated Protein Kinase Gene Is Early and Frequent in Murine Lung Tumors Induced by Cigarette Smoke and Tobacco Carcinogens Cancer Res., June 1, 2004; 64(11): 3844 - 3848. [Abstract] [Full Text] [PDF]

				B. R. Vuillemenot, L. C. Pulling, W. A. Palmisano, J. A. Hutt, and S. A. Belinsky Carcinogen exposure differentially modulates RAR-{beta} promoter hypermethylation, an early and frequent event in mouse lung carcinogenesis Carcinogenesis, April 1, 2004; 25(4): 623 - 629. [Abstract] [Full Text] [PDF]

				D. Goldenberg, S. Harden, B. G. Masayesva, P. Ha, N. Benoit, W. H. Westra, W. M. Koch, D. Sidransky, and J. A. Califano Intraoperative Molecular Margin Analysis in Head and Neck Cancer Arch Otolaryngol Head Neck Surg, January 1, 2004; 130(1): 39 - 44. [Abstract] [Full Text] [PDF]

				K. K. Bhakat and S. Mitra CpG methylation-dependent repression of the human O6-methylguanine-DNA methyltransferase gene linked to chromatin structure alteration Carcinogenesis, August 1, 2003; 24(8): 1337 - 1345. [Abstract] [Full Text] [PDF]

				L. Zhang, W. Lu, X. Miao, D. Xing, W. Tan, and D. Lin Inactivation of DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation and its relation to p53 mutations in esophageal squamous cell carcinoma Carcinogenesis, June 1, 2003; 24(6): 1039 - 1044. [Abstract] [Full Text] [PDF]

				R. Danesi, F. De Braud, S. Fogli, T. M. De Pas, A. Di Paolo, G. Curigliano, and M. Del Tacca Pharmacogenetics of Anticancer Drug Sensitivity in Non-Small Cell Lung Cancer Pharmacol. Rev., March 1, 2003; 55(1): 57 - 103. [Abstract] [Full Text] [PDF]

				J. Brabender, H. Usadel, R. Metzger, P. M. Schneider, J. Park, D. Salonga, D. D. Tsao-Wei, S. Groshen, R. V. Lord, N. Takebe, et al. Quantitative O6-Methylguanine DNA Methyltransferase Methylation Analysis in Curatively Resected Non-Small Cell Lung Cancer: Associations with Clinical Outcome Clin. Cancer Res., January 1, 2003; 9(1): 223 - 227. [Abstract] [Full Text] [PDF]

				L. Liu, S. Schwartz, B. M. Davis, and S. L. Gerson Chemotherapy-induced O6-Benzylguanine-resistant Alkyltransferase Mutations in Mismatch-deficient Colon Cancer Cancer Res., June 1, 2002; 62(11): 3070 - 3076. [Abstract] [Full Text] [PDF]

				N. Kohya, K. Miyazaki, S. Matsukura, H. Yakushiji, Y. Kitajima, K. Kitahara, M. Fukuhara, Y. Nakabeppu, and M. Sekiguchi Deficient Expression of O6-Methylguanine-DNA Methyltransferase Combined With Mismatch-Repair Proteins hMLH1 and hMSH2 Is Related to Poor Prognosis in Human Biliary Tract Carcinoma Ann. Surg. Oncol., May 1, 2002; 9(4): 371 - 379. [Abstract] [Full Text] [PDF]

				M. Esteller, G. Gaidano, S. N. Goodman, V. Zagonel, D. Capello, B. Botto, D. Rossi, A. Gloghini, U. Vitolo, A. Carbone, et al. Hypermethylation of the DNA Repair Gene O6-Methylguanine DNA Methyltransferase and Survival of Patients With Diffuse Large B-Cell Lymphoma J Natl Cancer Inst, January 2, 2002; 94(1): 26 - 32. [Abstract] [Full Text] [PDF]

				M. Nakamura, T. Watanabe, Y. Yonekawa, P. Kleihues, and H. Ohgaki Promoter methylation of the DNA repair gene MGMT in astrocytomas is frequently associated with G:C {->} A:T mutations of the TP53 tumor suppressor gene Carcinogenesis, October 1, 2001; 22(10): 1715 - 1719. [Abstract] [Full Text] [PDF]

				M. Esteller, J. Garcia-Foncillas, E. Andion, S. N. Goodman, O. F. Hidalgo, V. Vanaclocha, S. B. Baylin, and J. G. Herman Inactivation of the DNA-Repair Gene MGMT and the Clinical Response of Gliomas to Alkylating Agents N. Engl. J. Med., November 9, 2000; 343(19): 1350 - 1354. [Abstract] [Full Text] [PDF]

				H. Kinoshita, Y. Shi, C. Sandefur, L. F. Meisner, C. Chang, A. Choon, C. R. Reznikoff, G. S. Bova, A. Friedl, and D. F. Jarrard Methylation of the Androgen Receptor Minimal Promoter Silences Transcription in Human Prostate Cancer Cancer Res., July 1, 2000; 60(13): 3623 - 3630. [Abstract] [Full Text]

				A. Bearzatto, M. Szadkowski, P. Macpherson, J. Jiricny, and P. Karran Epigenetic Regulation of the MGMT and hMSH6 DNA Repair Genes in Cells Resistant to Methylating Agents Cancer Res., June 1, 2000; 60(12): 3262 - 3270. [Abstract] [Full Text]

				M. Esteller, M. Toyota, M. Sanchez-Cespedes, G. Capella, M. A. Peinado, D. N. Watkins, J.-P. J. Issa, D. Sidransky, S. B. Baylin, and J. G. Herman Inactivation of the DNA Repair Gene O6-Methylguanine-DNA Methyltransferase by Promoter Hypermethylation Is Associated with G to A Mutations in K-ras in Colorectal Tumorigenesis Cancer Res., May 1, 2000; 60(9): 2368 - 2371. [Abstract] [Full Text]

				E. E. Cameron, S. B. Baylin, and J. G. Herman p15INK4B CpG Island Methylation in Primary Acute Leukemia Is Heterogeneous and Suggests Density as a Critical Factor for Transcriptional Silencing Blood, October 1, 1999; 94(7): 2445 - 2451. [Abstract] [Full Text] [PDF]

				T. Rein, T. Kobayashi, M. Malott, M. Leffak, and M. L. DePamphilis DNA Methylation at Mammalian Replication Origins J. Biol. Chem., September 3, 1999; 274(36): 25792 - 25800. [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]

				D. J. Wong, S. A. Foster, D. A. Galloway, and B. J. Reid Progressive Region-Specific De Novo Methylation of the p16 CpG Island in Primary Human Mammary Epithelial Cell Strains during Escape from M0 Growth Arrest Mol. Cell. Biol., August 1, 1999; 19(8): 5642 - 5651. [Abstract] [Full Text] [PDF]

				M. Eileen Dolan, B. L. McRae, E. Ferries-Rowe, M. Belanich, G. A. van Seventer, J. Guitart, D. Pezen, T. M. Kuzel, and D. B. Yarosh O6-Alkylguanine-DNA Alkyltransferase in Cutaneous T-Cell Lymphoma: Implications for Treatment with Alkylating Agents Clin. Cancer Res., August 1, 1999; 5(8): 2059 - 2064. [Abstract] [Full Text] [PDF]

				G. M. Venkataraman, M. Yatin, R. Marcinek, and K. B. Ain Restoration of Iodide Uptake in Dedifferentiated Thyroid Carcinoma: Relationship to Human Na+/I- Symporter Gene Methylation Status J. Clin. Endocrinol. Metab., July 1, 1999; 84(7): 2449 - 2457. [Abstract] [Full Text]

				P. G. Corn, S. J. Kuerbitz, M. M. van Noesel, M. Esteller, N. Compitello, S. B. Baylin, and J. G. Herman Transcriptional Silencing of the p73 Gene in Acute Lymphoblastic Leukemia and Burkitt's Lymphoma Is Associated with 5' CpG Island Methylation Cancer Res., July 1, 1999; 59(14): 3352 - 3356. [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]

				M. Esteller, S. R. Hamilton, P. C. Burger, S. B. Baylin, and J. G. Herman Inactivation of the DNA Repair Gene O6-Methylguanine-DNA Methyltransferase by Promoter Hypermethylation is a Common Event in Primary Human Neoplasia Cancer Res., February 1, 1999; 59(4): 793 - 797. [Abstract] [Full Text] [PDF]

				H. Kawate, K. Sakumi, T. Tsuzuki, Y. Nakatsuru, T. Ishikawa, S. Takahashi, H. Takano, T. Noda, and M. Sekiguchi Separation of killing and tumorigenic effects of an alkylating agent in mice defective in two of the DNA repair genes PNAS, April 28, 1998; 95(9): 5116 - 5120. [Abstract] [Full Text] [PDF]

				K. K. Bhakat and S. Mitra Regulation of the Human O6-Methylguanine-DNA Methyltransferase Gene by Transcriptional Coactivators cAMP Response Element-binding Protein-binding Protein and p300 J. Biol. Chem., October 27, 2000; 275(44): 34197 - 34204. [Abstract] [Full Text] [PDF]

				H. Yamada, K. Vijayachandra, C. Penner, and A. Glick Increased Sensitivity of Transforming Growth Factor (TGF) beta 1 Null Cells to Alkylating Agents Reveals a Novel Link between TGFbeta Signaling and O6-Methylguanine Methyltransferase Promoter Hypermethylation J. Biol. Chem., May 25, 2001; 276(22): 19052 - 19058. [Abstract] [Full Text] [PDF]