Expression of O<sup>6</sup>-Methylguanine-DNA Methyltransferase Examined by Alkyl-Transfer Assays, Methylation-Specific PCR and Western Blots in Tumors and Matched Normal Tissue — Oak Academic Publishing
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Expression of O<sup>6</sup>-Methylguanine-DNA Methyltransferase Examined by Alkyl-Transfer Assays, Methylation-Specific PCR and Western Blots in Tumors and Matched Normal Tissue
Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, USA
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Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, USA
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Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, USA
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Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, USA
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Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, USA
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Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, USA
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Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, USA
1 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, USA
2 Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, USA
3 Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, USA
4 Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, USA
5 Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, USA
6 Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, USA
7 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, USA
The tumor selectivity of alkylating agents that produce guanine O 6 -chloroethyl (laromustine and carmustine) and O 6 -methyl (temozolomide) lesions depends upon O 6 -methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included 1 ) alkyl-transfer assays using [benzene- 3 H] O 6 -benzylguanine as a substrate to assess functional MGMT activity, 2 ) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and 3 ) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tu mors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its mo lecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malig nancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT ex pression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumor selective reduction in MGMT activity exist in human tissue.
A. E. Pegg, “Multifaceted Roles of Alkyltransferase and Related Proteins in DNA Repair, DNA Damage, Resistance to Chemotherapy, and Research Tools,” Chemical Research in Toxicology, Vol. 24, No. 5, 2011, pp. 618-639. doi:10.1021/tx200031q
M. Christmann, B. Verbeek, W. P. Roos and B. Kaina, “O6-Methylguanine-DNA Methyltransferase (MGMT) in Normal Tissues and Tumors: Enzyme Activity, Promoter Methylation and Immunohistochemistry,” Biochimica et Biophysica Acta, Vol. 1816, No. 2, 2011, pp. 179-190.
B. Kaina, M. Christmann, S. Naumann and W. P. Roos, “MGMT: Key Node in the Battle against Genotoxicity, Carcinogenicity and Apoptosis Induced by Alkylating Agents,” DNA Repair, Vol. 6, No. 8, 2007, pp. 1079-1099. doi:10.1016/j.dnarep.2007.03.008
J. L. Tubbs, A. E. Pegg and J. A. Tainer, “DNA Binding, Nucleotide Flipping, and the Helix-Turn-Helix Motif in Base Repair by O6-Alkylguanine-DNA Alkyltransferase and Its Implications for Cancer Chemotherapy,” DNA Repair, Vol. 6, No. 8, 2007, pp. 1100-1115. doi:10.1016/j.dnarep.2007.03.011
S. L. Gerson, “MGMT: Its Role in Cancer Aetiology and Cancer Therapeutics,” Nature Reviews Cancer, Vol. 4, No. 4, 2004, pp. 296-307. doi:10.1038/nrc1319
K. Ishiguro, Y. L. Zhu, K. Shyam, P. G. Penketh, R. P. Baumann and A. C. Sartorelli, “Quantitative Relationship between Guanine O6-Alkyl Lesions Produced by Onrigin and Tumor Resistance by O6-Alkylguanine-DNA Alkyltransferase,” Biochemical Pharmacology, Vol. 80, No. 9, 2010, pp. 1317-1325. doi:10.1016/j.bcp.2010.07.022
T. P. Brent and J. S. Remack, “Formation of Covalent Complexes between Human O6-Alkylguanine-DNA Alkyltransferase and BCNU-Treated Defined Length Synthetic Oligodeoxynucleotides,” Nucleic Acids Research, Vol. 16, No. 14B, 1988, pp. 6779-6788. doi:10.1093/nar/16.14.6779
R. Stupp, W. P. Mason, M. J. van den Bent, M. Weller, B. Fisher, M. J. Taphoorn, K. Belanger, A. A. Brandes, C. Marosi, U. Bogdahn, J. Curschmann, R. C. Janzer, S. K. Ludwin, T. Gorlia, A. Allgeier, D. Lacombe, J. G. Cairncross, E. Eisenhauer and R. O. Mirimanoff, “Radiotherapy Plus Concomitant and Adjuvant Temozolomide for Glioblastoma,” The New England Journal of Medicine, Vol. 352, No. 10, 2005, pp. 987-996. doi:10.1056/NEJMoa043330
S. L. Gerson, J. E. Trey, K. Miller and N. A. Berger, “Comparison of O6-Alkylguanine-DNA Alkyltransferase Activity Based on Cellular DNA Content in Human, Rat and Mouse Tissues,” Carcinogenesis, Vol. 7, No. 5, 1986, pp. 745-749. doi:10.1093/carcin/7.5.745
-Alkylguanine-DNA Alkyltransferase
AGT)
[Benzene-<
sup>
3<
/sup>
H]O<
sup>
6<
/sup>
-Benzylguanine
Methylation-Specific PCR (MSP)
Laromustine (Onrigin
Cloretazine
VNP40101M
101M)
Temozolomide
R. C. Grafstrom, A. E. Pegg, B. F. Trump and C. C. Harris, “O6-Alkylguanine-DNA Alkyltransferase Activity in Normal Human Tissues and Cells,” Cancer Research, Vol. 44, No. 7, 1984, pp. 2855-2857.
C. S. Brock, E. S. Newlands, S. R. Wedge, M. Bower, H. Evans, I. Colquhoun, M. Roddie, M. Glaser, M. H. Brampton and G. J. Rustin, “Phase I Trial of Temozolomide Using an Extended Continuous Oral Schedule,” Cancer Research, Vol. 58, No. 19, 1998, pp. 4363-4367.
K. Shyam, P. G. Penketh, R. H. Loomis, W. C. Rose and A. C. Sartorelli, “Antitumor 2-(Aminocarbonyl)-1,2-bis (methylsulfonyl)-1-(2-chloroethyl)-hydrazines,” Journal of Medicinal Chemistry, Vol. 39, No. 3, 1996, pp. 796-801. doi:10.1021/jm9505021
P. G. Penketh, K. Shyam and A. C. Sartorelli, “Comparison of DNA Lesions Produced by Tumor-Inhibitory 1,2-Bis(sulfonyl)hydrazines and Chloroethylnitrosoureas,” Biochemical Pharmacology, Vol. 59, No. 3, 2000, pp. 283-291. doi:10.1016/S0006-2952(99)00328-7
M. A. Badruddoja, S. T. Keir, I. King, J. Zeidner, J. J. Vredenburgh, L. H. Muhlbaier, D. D. Bigner and H. S. Friedman, “Activity of VNP40101M (Cloretazine) in the Treatment of CNS Tumor Xenografts in Athymic Mice,” Neuro-Oncology, Vol. 9, No. 3, 2007, pp. 240-244. doi:10.1215/15228517-2007-011
F. Giles, D. Rizzieri, J. Karp, N. Vey, F. Ravandi, S. Faderl, K. D. Khan, G. Verhoef, P. Wijermans, A. Advani, G. Roboz, H. Kantarjian, S. F. Bilgrami, A. Ferrant, S. M. Daenen, V. Karsten, A. Cahill, M. Albitar, G. Mufti and S. O’Brien, “Cloretazine (VNP40101M), a Novel Sulfonylhydrazine Alkylating Agent, in Patients Age 60 Years or Older with Previously Untreated Acute Myeloid Leukemia,” Journal of Clinical Oncology, Vol. 25, No. 1, 2007, pp. 25-31. doi:10.1200/JCO.2006.07.0961
O. Wiestler, P. Kleihues and A. E. Pegg, “O6-Alkylguanine-DNA Alkyltransferase Activity in Human Brain and Brain Tumors,” Carcinogenesis, Vol. 5, No. 1, 1984, pp. 121-124. doi:10.1093/carcin/5.1.121
K. Ishiguro, K. Shyam, P. G. Penketh and A. C. Sartorelli, “Development of an O6-Alkylguanine-DNA Alkyltransferase Assay Based on Covalent Transfer of the Benzyl Moiety from [Benzene-3H]O6-Benzylguanine to the Protein,” Analytical Biochemistry, Vol. 383, No. 1, 2008, pp. 44-51. doi:10.1016/j.ab.2008.08.009
M. E. Hegi, L. Liu, J. G. Herman, R. Stupp, W. Wick, M. Weller, M. P. Mehta and M. R. Gilbert, “Correlation of O6-Methylguanine Methyltransferase (MGMT) Promoter Methylation with Clinical Outcomes in Glioblastoma and Clinical Strategies to Modulate MGMT Activity,” Journal of Clinical Oncology, Vol. 26, No. 25, 2008, pp. 4189-4199. doi:10.1200/JCO.2007.11.5964
M. Weller, R. Stupp, G. Reifenberger, A. A. Brandes, M. J. van den Bent, W. Wick and M. E. Hegi, “MGMT Promoter Methylation in Malignant Gliomas: Ready for Personalized Medicine?” Nature Reviews Neurology, Vol. 6, No. 1, 2010, pp. 39-51. doi:10.1038/nrneurol.2009.197
G. Isowa, K. Ishizaki, T. Sadamoto, K. Tanaka, Y. Yamaoka, K. Ozawa and M. Ikenaga, “O6-MethylguanineDNA Methyltransferase Activity in Human Liver Tumors,” Carcinogenesis, Vol. 12, No. 7, 1991, pp. 1313-1317. doi:10.1093/carcin/12.7.1313
G. J. Kitange, B. L. Carlson, A. C. Mladek, P. A. Decker, M. A. Schroeder, W. Wu, P. T. Grogan, C. Giannini, K. V. Ballman, J. C. Buckner, C. D. James and J. N. Sarkaria, “Evaluation of MGMT Promoter Methylation Status and Correlation with Temozolomide Response in Orthotopic Glioblastoma Xenograft Model,” Journal of Neuro-Oncology, Vol. 92, No. 1, 2009, pp. 23-31. doi:10.1007/s11060-008-9737-8
L. Zhang, W. Lu, X. Miao, D. Xing, W. Tan and D. Lin, “Inactivation of DNA Repair Gene O6-MethylguanineDNA Methyltransferase by Promoter Hypermethylation and Its Relation to p53 Mutations in Esophageal Squamous Cell Carcinoma,” Carcinogenesis, Vol. 24, No. 6, 2003, pp. 1039-1044. doi:10.1093/carcin/bgg062
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 Research, Vol. 59, No. 4, 1999, pp. 793-797.
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,” The New England Journal of Medicine, Vol. 343, No. 19, 2000, pp. 1350-1354. doi:10.1056/NEJM200011093431901
U. K. Laemmli, “Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4,” Nature, Vol. 227, No. 5259, 1970, pp. 680-685. doi:10.1038/227680a0
R. S. Day III, C. H. J. Ziolkowski, D. A. Scudiero, S. A. Meyer and M. R. Mattern, “Human Tumor Cell Strains Defective in the Repair of Alkylation Damage,” Carcinogenesis, Vol. 1, No. 1, 1980, pp. 21-32. doi:10.1093/carcin/1.1.21
R. Sklar and B. Strauss, “Removal of O6-Methylguanine from DNA of Normal and Xeroderma Pigmentosum-Derived Lymphoblastoid Lines,” Nature, Vol. 289, No. 5796, 1981, pp. 417-420. doi:10.1038/289417a0
R. A. Kroes and L. C. Erickson, “The Role of mRNA Stability and Transcription in O6-Methylguanine DNA Methyltransferase (MGMT) Expression in Mer+ Human Tumor Cells,” Carcinogenesis, Vol. 16, No. 9, 1995, pp. 2255-2257. doi:10.1093/carcin/16.9.2255
L. C. Harris, P. M. Potter, K. Tano, S. Shiota, S. Mitra and T. P. Brent, “Characterization of the Promoter Region of the Human O6-Methylguanine-DNA Methyltransferase Gene,” Nucleic Acids Research, Vol. 19, No. 22, 1991, pp. 6163-6167. doi:10.1093/nar/19.22.6163
M. A. von Wronski, L. C. Harris, K. Tano, S. Mitra, D. D. Bigner and T. P. Brent, “Cytosine Methylation and Suppression of O6-Methylguanine-DNA Methyltransferase Expression in Human Rhabdomyosarcoma Cell Lines and Xenografts,” Oncology Research, Vol. 4, No. 4-5, 1992, pp. 167-174.
X. Qian, M. A. von Wronski and T. P. Brent, “Localization of Methylation Sites in the Human O6-Methylguanine-DNA Methyltransferase Promoter: Correlation with Gene Suppression,” Carcinogenesis, Vol. 16, No. 6, 1995, pp. 1385-1390. doi:10.1093/carcin/16.6.1385
X. C. Qian and T. P. Brent, “Methylation Hot Spots in the 5' Flanking Region Denote Silencing of the O6-Methylguanine-DNA Methyltransferase Gene,” Cancer Research, Vol. 57, No. 17, 1997, pp. 3672-3677.
L. C. Harris, P. M. Potter, J. S. Remack and T. P. Brent, “A Comparison of Human O6-Methylguanine-DNA Methyltransferase Promoter Activity in Mer+ and Mer? Cells,” Cancer Research, Vol. 52, No. 22, 1992, pp. 6404-6406.
K. S. Srivenugopal, S. R. Mullapudi, J. Shou, T. K. Hazra and F. Ali-Osman, “Protein Phosphorylation Is a Regulatory Mechanism for O6-Alkylguanine-DNA Alkyltransferase in Human Brain Tumor Cells,” Cancer Research, Vol. 60, No. 2, 2000, pp. 282-287.
S. R. Mullapudi, F. Ali-Osman, J. Shou and K. S. Srivenugopal, “DNA Repair Protein O6-AlkylguanineDNA Alkyltransferase Is Phosphorylated by Two Distinct and Novel Protein Kinases in Human Brain Tumour Cells,” Biochemical Journal, Vol. 351, No. 2, 2000, pp. 393-402. doi:10.1042/0264-6021:3510393
K. S. Srivenugopal, S. R. Mullapudi and F. Ali-Osman, “Phosphorylation of O6-Alkylguanine-DNA Alkyltransferase: Experience with a GST-Fusion Protein and a New Pull-Down Assay,” Cancer Letters, Vol. 181, No. 1, 2002, pp. 87-93. doi:10.1016/S0304-3835(01)00823-0
I. K. Lim, T. J. Park, J. W. Jee, M. S. Lee and W. K. Paik, “Differential Expression of O6-Methylguanine-DNA Methyltransferase during Diethylnitrosamine-Induced Carcinogenesis and Liver Regeneration in Sprague-Dawley Male Rats,” Journal of Cancer Research and Clinical Oncology, Vol. 125, No. 8-9, 1999, pp. 493-499. doi:10.1007/s004320050307
I. K. Lim, T. J. Park and W. K. Paik, “Phosphorylation of Methylated-DNA-Protein-Cysteine S-Methyltransferase at Serine-204 Significantly Increases Its Resistance to Proteolytic Digestion,” Biochemical Journal, Vol. 352, No. 3, 2000, pp. 801-808. doi:10.1042/0264-6021:3520801
G. N. Major, E. J. Gardner, A. F. Carne and P. D. Lawley, “Purification to Homogeneity and Partial Amino Acid Sequence of a Fragment Which Includes the Methyl Acceptor Site of the Human DNA Repair Protein for O6Methylguanine,” Nucleic Acids Research, Vol. 18, No. 6, 1990, pp. 1351-1359. doi:10.1093/nar/18.6.1351
G. N. Major, M. Brady, G. B. Notarianni, J. D. Collier and M. S. Douglas, “Evidence for Ubiquitin-Mediated Degradation of the DNA Repair Enzyme for O6-Methylguanine in Non-Tumour Derived Human Cell and Tissue Extracts,” Biochemical Society Transactions, Vol. 25, No. 2, 1997, p. 359S.
J. D. Collier, M. F. Bassendine, A. D. Burt and G. N. Major, “Characterisation of the DNA Repair Enzyme for O6-Methylguanine in Cirrhosis,” Journal of Hepatology, Vol. 25, No. 2, 1996, pp. 158-165. doi:10.1016/S0168-8278(96)80068-7
K. S. Srivenugopal, X. H. Yuan, H. S. Friedman and F. Ali-Osman, “Ubiquitination-Dependent Proteolysis of O6Methylguanine-DNA Methyltransferase in Human and Murine Tumor Cells Following Inactivation with O6Benzylguanine or 1,3-Bis(2-chloroethyl)-1-nitrosourea,” Biochemistry, Vol. 35, No. 4, 1996, pp. 1328-1334. doi:10.1021/bi9518205
M. Xu-Welliver and A. E. Pegg, “Degradation of the Alkylated Form of the DNA Repair Protein, O6-Alkylguanine-DNA Alkyltransferase,” Carcinogenesis, Vol. 23, No. 5, 2002, pp. 823-830. doi:10.1093/carcin/23.5.823
L. Shen, Y. Kondo, G. L. Rosner, L. Xiao, N. S. Hernandez, J. Vilaythong, P. S. Houlihan, R. S. Krouse, A. R. Prasad, J. G. Einspahr, J. Buckmeier, D. S. Alberts, S. R. Hamilton and J. P. Issa, “MGMT Promoter Methylation and Field Defect in Sporadic Colorectal Cancer,” Journal of the National Cancer Institute, Vol. 97, No. 18, 2005, pp. 1330-1338. doi:10.1093/jnci/dji275
T. Kawasaki, M. Ohnishi, K. Nosho, Y. Suemoto, G. J. Kirkner, J. A. Meyerhardt, C. S. Fuchs and S. Ogino, “CpG Island Methylator Phenotype-Low (CIMP-Low) Colorectal Cancer Shows Not Only Few Methylated CIMP-High-Specific CpG Islands, but Also Low-Level Methylation at Individual Loci,” Modern Pathology, Vol. 21, No. 3, 2008, pp. 245-255. doi:10.1038/modpathol.3800982
J. A. Maxwell, S. P. Johnson, J. A. Quinn, R. E. McLendon, F. Ali-Osman, A. H. Friedman, J. E. Herndon II, K. Bierau, J. Bigley, D. D. Bigner and H. S. Friedman, “Quantitative Analysis of O6-Alkylguanine-DNA Alkyltransferase in Malignant Glioma,” Molecular Cancer Therapeutics, Vol. 5, No. 10, 2006, pp. 2531-2539. doi:10.1158/1535-7163.MCT-06-0106
M. Christmann, G. Nagel, S. Horn, U. Krahn, D. Wiewrodt, C. Sommer and B. Kaina, “MGMT Activity, Promoter Methylation and Immunohistochemistry of Pretreatment and Recurrent Malignant Gliomas: A Comparative Study on Astrocytoma and Glioblastoma,” International Journal of Cancer, Vol. 127, No. 9, 2010, pp. 2106-2118. doi:10.1002/ijc.25229
S. Hongeng, T. P. Brent, R. A. Sanford, H. Li, L. E. Kun and R. L. Heideman, “O6-Methylguanine-DNA Methyltransferase Protein Levels in Pediatric Brain Tumors,” Clinical Cancer Research, Vol. 3, No. 12, 1997, pp. 2459-2463.
M. Nagane, K. Kobayashi, A. Ohnishi, S. Shimizu and Y. Shiokawa, “Prognostic Significance of O6-Methylguanine-DNA Methyltransferase Protein Expression in Patients with Recurrent Glioblastoma Treated with Temozolomide,” Japanese Journal of Clinical Oncology, Vol. 37, No. 12, 2007, pp. 897-906. doi:10.1093/jjco/hym132
Y. Kishida, A. Natsume, H. Toda, Y. Toi, K. Motomura, H. Koyama, K. Matsuda, O. Nakayama, M. Sato, M. Suzuki, Y. Kondo and T. Wakabayashi, “Correlation between Quantified Promoter Methylation and Enzymatic Activity of O6-Methylguanine-DNA Methyltransferase in Glioblastomas,” Tumour Biology, Vol. 33, No. 2, 2012, pp. 373-381. doi:10.1007/s13277-012-0319-1
S. L. Gerson, K. Miller and N. A. Berger, “O6 Alkylguanine-DNA Alkyltransferase Activity in Human Myeloid Cells,” The Journal of Clinical Investigation, Vol. 76, No. 6, 1985, pp. 2106-2114. doi:10.1172/JCI112215
J. A. Allay, O. N. Koc, B. M. Davis and S. L. Gerson, “Retroviral-Mediated Gene Transduction of Human Alkyltransferase Complementary DNA Confers Nitrosourea Resistance to Human Hematopoietic Progenitors,” Clinical Cancer Research, Vol. 2, No. 8, 1996, pp. 1353-1359.