© 1991 Oxford University Press
research-article |
Use of antibodies to human O6-alkylguanine-DNA alkyltransferase to study the content of this protein in cells treated with O6-benzylguanine or N-methyl-N'-nitro-N-nitrosoguanidine
Departments of Cellular and Molecular Physiology and of Phamamlogy, Pennsylvania State University College of Medicine, The Milton S.Hershey Medical Center PO Box 850, Hershey, PA 17033
1Carcinogen-Modified Nucleic Acid Chemistry, Advanced BioScience Laboratories, Inc. -Basic Research Program, NCI-Frederick Cancer Research and Developement Center PO Box B, Frederick , MD 21701
2Division of Hematology-Oncology, The University of Chicago Medical Center 5841 S. Maryland Avenue, Box 420 Chicago, IL 60637, USA
Antisera raised in rabbits to three peptides corresponding to amino acid sequences found in human O6 -alkylguanine-DNA alkyltransferase were used to study the fate of the alkyltransferase protein in human colon tumor cells after exposure to N-methyl-N'-nitro-N-ntrosoguanidine or to O6 -benzylguanidine. Under these conditions, the alkyltransferase protein becomes inactivated, presumably by the conversion of its cysteine acceptor site to S-methylcysteine or S-benzylcysteine rrspectively. It was found that the protein was rapidly degraded after such inactivation both in intact cells and in cell-free extracts. It is probable that a conformational change in the protein is brought about by conversion of the alkyltransferase to the Inactive form by alkylation of the cysteine acceptor site. This change may render the protein very sensitive to proteolytic degradation. The rapid degradation of the inactive form of the protein may serve as a signal for its resynthesis but in the short term ensures that its reactivation by regeneration of the cysteine acceptor site is unlikely to occur to any sigificant extent. The short halflife of the inactivated alkyltranferase protein makes it probable that measurement of the content of the alkyltransferase protein by immunohistochemistry, which is likely to measure the sum of the active and inactivated forms of the protein, will nevertheless yield an accurate estimation of the cellular capacity to repair O6-methylguanine provided that procedures with sufficient specificity and affinity can be developed.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Philip, S. Swaminathan, S. G. Kuznetsov, S. Kanugula, K. Biswas, S. Chang, N. A. Loktionova, D. C. Haines, P. Kaldis, A. E. Pegg, et al. Degradation of BRCA2 in Alkyltransferase-Mediated DNA Repair and Its Clinical Implications Cancer Res., December 1, 2008; 68(23): 9973 - 9981. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-Y. Tserng, S. T. Ingalls, E. M. Boczko, T. P. Spiro, X. Li, S. Majka, S. L. Gerson, J. K. V. Willson, and C. L. Hoppel Pharmacokinetics of O6-Benzylguanine (NSC637037) and Its Metabolite, 8-Oxo-O6-Benzylguanine J. Clin. Pharmacol., August 1, 2003; 43(8): 881 - 893. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. Rasimas, A. E. Pegg, and M. G. Fried DNA-binding Mechanism of O6-Alkylguanine-DNA Alkyltransferase. EFFECTS OF PROTEIN AND DNA ALKYLATION ON COMPLEX STABILITY J. Biol. Chem., February 28, 2003; 278(10): 7973 - 7980. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Dolan, M. Posner, T. Karrison, J. Radosta, G. Steinberg, D. Bertucci, L. Vujasin, and M. J. Ratain Determination of the Optimal Modulatory Dose of O6-Benzylguanine in Patients with Surgically Resectable Tumors Clin. Cancer Res., August 1, 2002; 8(8): 2519 - 2523. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Liu, M. Xu-Welliver, S. Kanugula, and A. E. Pegg Inactivation and Degradation of O6-Alkylguanine-DNA Alkyltransferase after Reaction with Nitric Oxide Cancer Res., June 1, 2002; 62(11): 3037 - 3043. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Xu-Welliver and A. E. Pegg Degradation of the alkylated form of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase Carcinogenesis, May 1, 2002; 23(5): 823 - 830. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. M. Noll and N. D. Clarke Covalent capture of a human O6-alkylguanine alkyltransferase-DNA complex using N1,O6-ethanoxanthosine, a mechanism-based crosslinker Nucleic Acids Res., October 1, 2001; 29(19): 4025 - 4034. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Dowlati, J. Haaga, S. C. Remick, T. P. Spiro, S. L. Gerson, L. Liu, S. J. Berger, N. A. Berger, and J. K. V. Willson Sequential Tumor Biopsies in Early Phase Clinical Trials of Anticancer Agents for Pharmacodynamic Evaluation Clin. Cancer Res., October 1, 2001; 7(10): 2971 - 2976. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Liu, T. P. Spiro, X. Qin, S. Majka, J. Haaga, J. Schupp, J. K. V. Willson, and S. L. Gerson Differential Degradation Rates of Inactivated Alkyltransferase in Blood Mononuclear Cells and Tumors of Patients after Treatment with O6-Benzylguanine Clin. Cancer Res., August 1, 2001; 7(8): 2318 - 2324. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. L. Kreklau, N. Liu, Z. Li, K. Cornetta, and L. C. Erickson Comparison of Single- Versus Double-Bolus Treatments of O6-Benzylguanine for Depletion of O6-Methylguanine DNA Methyltransferase (MGMT) Activity in Vivo: Development of a Novel Fluorometric Oligonucleotide Assay for Measurement of Mgmt Activity J. Pharmacol. Exp. Ther., April 12, 2001; 297(2): 524 - 530. [Abstract] [Full Text]
|
|
|
|
|
|
L. P. Encell and L. A. Loeb Enhanced in vivo repair of O4-methylthymine by a mutant human DNA alkyltransferase Carcinogenesis, July 1, 2000; 21(7): 1397 - 1402. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Maze, C. Kurpad, A. E. Pegg, L. C. Erickson, and D. A. Williams Retroviral-Mediated Expression of the P140A, but not P140A/G156A, Mutant Form of O6-Methylguanine DNA Methyltransferase Protects Hematopoietic Cells against O6-Benzylguanine Sensitization to Chloroethylnitrosourea Treatment J. Pharmacol. Exp. Ther., September 1, 1999; 290(3): 1467 - 1474. [Abstract] [Full Text]
|
|
|
|
|
|
T. P. Spiro, S. L. Gerson, L. Liu, S. Majka, J. Haaga, C. L. Hoppel, S. T. Ingalls, J. M. Pluda, and J. K. V. Willson O6-Benzylguanine: A Clinical Trial Establishing the Biochemical Modulatory Dose in Tumor Tissue for Alkyltransferase-directed DNA Repair Cancer Res., May 1, 1999; 59(10): 2402 - 2410. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Edara, S. Kanugula, and A. E. Pegg Expression of the inactive C145A mutant human O6-alkylguanine-DNA alkyltransferase in E.coli increases cell killing and mutations by N-methyl-N'-nitro-N-nitrosoguanidine Carcinogenesis, January 1, 1999; 20(1): 103 - 108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. H. Wu, K. E. Lohrbach, O. I. Olopade, D. M. Kokkinakis, H. S. Friedman, and M. E. Dolan Lack of Evidence for a Polymorphism at Codon 160 of Human O6-Alkylguanine-DNA Alkyltransferase Gene in Normal Tissue and Cancer Clin. Cancer Res., January 1, 1999; 5(1): 209 - 213. [Abstract] [Full Text] [PDF]
|
|