Nucleic Acids Research Advance Access originally published online on January 30, 2007
Nucleic Acids Research 2007 35(5):e31; doi:10.1093/nar/gkl1156
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Nucleic Acids Research, 2007, Vol. 35, No. 5 e31
© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Characterization of in vitro and in vivo hypomethylating effects of decitabine in acute myeloid leukemia by a rapid, specific and sensitive LC-MS/MS method
1College of Pharmacy 2Division of Hematology-Oncology 3Department of Molecular Virology, Immunology and Medical Genetics and 4The Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210
*To whom correspondence should be addressed. Tel: +1 614 293 9273; Fax: +1 614 293 2695; Email: guido.marcucci{at}osumc.edu
Received July 11, 2006. Revised November 3, 2006. Accepted December 14, 2006.
DNA hypermethylation is a common finding in malignant cells and has been explored as a therapeutic target for hypomethylating agents (e.g., decitabine). Detection of changes in DNA methylation might serve as a pharmacodynamic endpoint to establish the biological activity of these agents and predict clinical response. We developed and validated a rapid, sensitive and specific LC-MS/MS method for determination of global DNA methylation (GDM) in vitro and in vivo. Ratios of 5-methyl-2'-deoxycytidine (5mdC) to the internal standard 2-deoxyguanosine (2dG) in mass signal were used to quantify GDM levels. The assay was validated in a linear range from 40 fmol to 200 pmol 5mdC. The intra-day precision values ranged from 2.8 to 9.9% and the inter-day values from 1.1 to 15.0%. The accuracy of the assay varied between 96.7 and 109.5%. This method was initially applied for characterization of decitabine-induced GDM changes in in-vitro-treated leukemia cells. Following exposure to 2.5 µM decitabine, GDM decreased to 
50% of the baseline value. The clinical applicability of this method was then demonstrated in bone marrow samples from patients with acute myeloid leukemia treated with decitabine. Our data support the use of our LC-MS/MS method for clinical pharmacodynamic determination of changes in GDM in vivo.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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