Cellular uptake and intracellular levels of the Bcl-2 antisense G3139 in cultured cells and treated patients with acute myeloid leukemia
Cellular uptake and intracellular levels of the Bcl-2 antisense G3139 in cultured cells and treated patients with acute myeloid leukemia
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DOI：
10.1158/1078-0432.ccr-04-1505
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发表时间：
2005-04-15
影响因子：
11.5
通讯作者：
Marcucci, G
中科院分区：
文献类型：
--
作者：
Dai, GW;Chan, KK;Marcucci, G
目的：目前正在临床研究的反义基因G3139下调Bcl-2可恢复耐药恶性细胞的化疗敏感性。迄今为止，体内给药后G3139在细胞内积累的机制仍有待阐明。本研究旨在评估体内是否可以检测到细胞内浓度的G3139，以及这些浓度与Bcl-2下调的关系。实验设计：使用一种新开发的、新颖的、高度敏感的基于elisa的检测方法，研究了从治疗患者收集的白血病骨髓细胞系和母细胞对G3139的细胞摄取。采用实时逆转录- pcr技术定量处理细胞中Bcl-2 mRNA的变化。结果：该方法得到充分验证，定量限为50 pmol/L。当暴露于0.33 ~ 10 μ mol/L G3139时，K562细胞内蛋白质浓度在2.1 ~ 11.4 pmol/mg之间。当G3139与阳离子脂质一起递送时，观察到细胞内浓度增加10至25倍。G3139在细胞核中积累，阳离子脂质使细胞核与细胞质之比增加了7倍。细胞内G3139浓度与Bcl-2 mRNA下调相关。在接受G3139治疗的急性髓系白血病患者的骨髓（3.4-40.6 pmol/mg蛋白）和外周血单个核细胞（0.47-19.4 pmol/mg蛋白）中，细胞内G3139的浓度稳定。结论：这是在急性髓性白血病患者体内可实现可测量的细胞内G3139水平的第一个证据，并且Bcl-2的下调可能依赖于可实现的细胞内浓度而不是血浆浓度。
Purpose: Down-regulation of Bcl-2 by the antisense G3139, currently under clinical evaluations, could restore chemosensitivity in otherwise resistant malignant cells. To date, the mechanism of intracellular accumulation of G3139 following in vivo administration remains to be elucidated. This study aimed to assess whether detectable intracellular concentrations of G3139 are achievable in vivo and how these relate to Bcl-2 down-regulation.Experimental Design: Cellular uptake of G3139 was studied in leukemia myeloid cell lines and blasts collected from treated patients using a newly developed, novel, and highly sensitive ELISA-based assay. Real-time reverse transcription-PCR was used to quantify Bcl-2 mRNA changes in treated cells.Results: The assay was fully validated and showed a limit of quantification of 50 pmol/L. When exposed to 0.33 to 10 mu mol/L G3139, K562 cells exhibited intracellular concentrations in the range of 2.1 to 11.4 pmol/mg protein. When G3139 was delivered with cationic lipids, a 10- to 25-fold increase of the intracellular concentrations was observed. There was an accumulation of G3139 in the nuclei, and the ratio of nucleus to Cytoplasm was increased 7-fold by cationic lipids. Intracellular concentrations of G3139 were correlated with Bcl-2 mRNA down-regulation. Robust intracellular concentrations of G3139 were achieved in vivo in bone marrow (range, 3.4-40.6 pmol/mg protein) and peripheral blood mononuclear cells (range, 0.47-19.4 pmol/mg protein) from acute myeloid leukemia patients treated with G3139.Conclusions: This is the first evidence that measurable intracellular levels of G3139 are achievable in vivo in acute myeloid leukemia patients and that Bcl-2 down-regulation is likely to depend on the achievable intracellular concentrations rather than on plasma concentrations.