Qin Taichun, Youssef Emile M, Jelinek Jaroslav, Chen Rong, Yang Allen S, Garcia-Manero Guillermo, Issa Jean-Pierre J
Department of Leukemia, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Clin Cancer Res. 2007 Jul 15;13(14):4225-32. doi: 10.1158/1078-0432.CCR-06-2762.
1-beta-D-Arabinofuranosylcytosine (cytarabine; ara-C) is the most active agent in myeloid leukemia. 5-Aza-2'-deoxycytidine (DAC) is a cytosine analogue that inhibits DNA methylation and also has activity in myeloid leukemia. Therefore, we investigated combining these two drugs in human leukemia cell lines in vitro.
We initially examined the effects of ara-C and DAC on human leukemia cell lines HL60, ML-1, RAji, and Jurkat. We measured IC(50) of DAC and ara-C in these cell lines and calculated a combination index of these two drugs given either simultaneously or sequentially. In searching for mechanisms relative to epigenetic regulation for this effect, we examined DNA methylation of LINE and Alu repetitive elements as a surrogate for global genomic DNA methylation. In addition, we sorted Annexin V positive and negative cells and measured differences in LINE methylation between them.
The combination of DAC and ara-C showed additive induction of cell death in ML-1 and synergistic induction in HL60, Raji, and Jurkat. Sequentially, DAC followed by ara-C was a synergistic combination in all cell lines. Low-dose DAC induced more hypomethylation than high doses of the drug, whereas ara-C had no effects on methylation. The combination of ara-C with DAC either together or DAC followed by ara-C resulted in inhibition of LINE demethylation in HL60. The RIL gene, which is silenced by DNA hypermethylation, was activated by DAC, but the addition of ara-C to DAC reduced RIL gene activation. DAC treatment increased H3 Lys(9) acetylation of Alu elements, whereas ara-C had no effect, and the addition of ara-C to DAC inhibited this effect. Finally, we showed that after DAC exposure, Annexin V positive cells were more hypomethylated than Annexin V negative cells.
The combination of DAC and ara-C showed additive or synergistic effects on cell death in four human leukemia cell lines in vitro, but antagonism in terms of epigenetic effects. One possible explanation for these paradoxical observations is that hypomethylated cells are sensitized to cell killing by ara-C. These data suggest that DAC used in combination with ara-C has clinical potential in the treatment of acute myeloid leukemia.
1-β-D-阿拉伯呋喃糖基胞嘧啶（阿糖胞苷；ara-C）是髓系白血病中最具活性的药物。5-氮杂-2'-脱氧胞苷（DAC）是一种胞嘧啶类似物，可抑制DNA甲基化，在髓系白血病中也具有活性。因此，我们在体外研究了将这两种药物联合应用于人类白血病细胞系。
我们首先检测了阿糖胞苷和DAC对人类白血病细胞系HL60、ML-1、Raji和Jurkat的作用。我们测定了这些细胞系中DAC和阿糖胞苷的半数抑制浓度（IC50），并计算了这两种药物同时或序贯给药时的联合指数。为了寻找这种作用的表观遗传调控相关机制，我们检测了长散在核元件（LINE）和短散在核元件（Alu）重复元件的DNA甲基化，作为全基因组DNA甲基化的替代指标。此外，我们对膜联蛋白V阳性和阴性细胞进行了分选，并检测了它们之间LINE甲基化的差异。
DAC与阿糖胞苷联合应用在ML-1细胞中显示出相加性细胞死亡诱导作用，在HL60、Raji和Jurkat细胞中显示出协同诱导作用。序贯给药时，先给予DAC再给予阿糖胞苷在所有细胞系中都是一种协同组合。低剂量DAC比高剂量药物诱导更多的低甲基化，而阿糖胞苷对甲基化无影响。阿糖胞苷与DAC联合应用或先给予DAC再给予阿糖胞苷均可导致HL60细胞中LINE去甲基化受到抑制。因DNA高甲基化而沉默的RIL基因被DAC激活，但在DAC中加入阿糖胞苷会降低RIL基因的激活。DAC处理增加了Alu元件的组蛋白H3赖氨酸9（H3 Lys(9)）乙酰化，而阿糖胞苷无此作用，在DAC中加入阿糖胞苷会抑制这种作用。最后我们发现，在DAC处理后，膜联蛋白V阳性细胞比膜联蛋白V阴性细胞甲基化程度更低。
DAC与阿糖胞苷联合应用在体外对四种人类白血病细胞系的细胞死亡显示出相加或协同作用，但在表观遗传效应方面存在拮抗作用。对这些矛盾观察结果的一种可能解释是，低甲基化细胞对阿糖胞苷诱导的细胞杀伤更敏感。这些数据表明，DAC与阿糖胞苷联合应用在急性髓系白血病治疗中具有临床潜力。
