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.
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.
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DOI：
10.1093/nar/gkl1156
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发表时间：
2007
影响因子：
14.9
通讯作者：
Marcucci G
中科院分区：
文献类型：
--
作者：
Liu Z;Liu S;Xie Z;Blum W;Perrotti D;Paschka P;Klisovic R;Byrd J;Chan KK;Marcucci G
DNA 高甲基化是恶性细胞中的常见现象，并已被探索作为低甲基化药物（例如地西他滨）的治疗靶点。 DNA 甲基化变化的检测可能作为药效学终点，以确定这些药物的生物活性并预测临床反应。我们开发并验证了一种快速、灵敏且特异的 LC-MS/MS 方法，用于体外和体内测定总体 DNA 甲基化 (GDM)。质量信号中 5-甲基-2'-脱氧胞苷 (5mdC) 与内标 2-脱氧鸟苷 (2dG) 的比率用于量化 GDM 水平。该测定在 40 fmol 至 200 pmol 5mdC 的线性范围内进行了验证。日内精度值范围为 2.8% 至 9.9%，日间精度值范围为 1.1% 至 15.0%。测定的准确度在 96.7% 至 109.5% 之间变化。该方法最初用于表征体外治疗的白血病细胞中地西他滨诱导的 GDM 变化。暴露于 2.5μM 地西他滨后，GDM 下降至基线值的约 50%。然后在接受地西他滨治疗的急性髓系白血病患者的骨髓样本中证明了该方法的临床适用性。我们的数据支持使用我们的 LC-MS/MS 方法来确定 GDM 体内变化的临床药效学。
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.