Multigene targeting with antisense oligodeoxynucleotides: An exploratory study using primary human leukemia cells
Multigene targeting with antisense oligodeoxynucleotides: An exploratory study using primary human leukemia cells
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DOI：
10.1158/1078-0432.ccr-05-0106
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发表时间：
2005-07-01
影响因子：
11.5
通讯作者：
Gewirtz, AM
中科院分区：
文献类型：
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作者：
Opalinska, JB;Machalinski, B;Gewirtz, AM
目的：我们之前报道过 c-myb 和 Vav 原癌基因可以用反义寡脱氧核苷酸沉默，并且抑制其中任何一个都会损害白血病细胞的生长。由于尚不清楚这些基因的表达是否存在关联，因此我们试图确定在 K562 和原发性患者 (n = 9) 慢性粒性白血病细胞中同时沉默这两个基因的治疗价值。 实验设计：将 K562 和原发性慢性粒性白血病细胞暴露于反义寡脱氧核苷酸（单独或组合）24 或 72 小时，然后在甲基纤维素培养物中克隆。确定对 K562 簇和母细胞形成单位（红系集落和粒细胞-巨噬细胞集落形成单位）的影响，并将其与下调目标 mRNA 的能力相关联。结果：暴露 24 小时后，c-myb 或 Vav 反义寡脱氧核苷酸以序列特异性、剂量响应的方式抑制 K562 细胞生长。同时暴露于两种寡脱氧核苷酸显着增强了生长抑制并加速了细胞凋亡。原代细胞结果更为复杂。暴露于抗vav或抗myb反义寡脱氧核苷酸24小时和72小时后，观察到同等的集落形成单位抑制。将细胞同时暴露于两种反义寡脱氧核苷酸24小时不会导致集落形成的额外抑制。然而，与两种寡脱氧核苷酸一起孵育 72 小时后，与单独使用任一寡脱氧核苷酸相比，集落形成显着减少（粒细胞-巨噬细胞集落形成单位从约 30% 到约 78%；母细胞形成单位 - 红系从约 50% 到约 80%）。结论：我们假设将原代白血病细胞暴露于针对两个或可能更多的基因可能会显着增强这些分子的治疗效用。
Purpose: We previously reported that the c-myb and Vav proto-oncogenes are amenable to silencing with antisense oligodeoxynucleotides and that inhibition of either impairs leukemic cell growth. Because the expression of these genes is not known to be linked, we sought to determine the therapeutic value of silencing both genes simultaneously in K562 and primary patient (n = 9) chronic myelogenous leukemia cells.Experimental Design: K562 and primary chronic myelogenous leukemia cells were exposed to antisense oligodeoxynucleotides (alone or in combination) for 24 or 72 hours and then cloned in methylcellulose cultures. Effects on K562 cluster, and blast-forming unit - erythroid colonies and granulocyte-macrophage colony-forming units were determined and correlated with the ability to down-regulate the targeted m RNA.Results: After 24-hour exposure, K562 cell growth was inhibited in a sequence specific, dose-responsive manner with either c-myb or Vav antisense oligodeoxynucleotides. Exposure to both oligodeoxynucleotides simultaneously considerably enhanced growth inhibition and accelerated apoptosis. Primary cell results were more complex. After 24- and 72-hour exposures to either anti-vav or anti-myb antisense oligodeoxynucleotides, equivalent colony-forming unit inhibition was observed. Exposing cells to both antisense oligodeoxynucleotides simultaneously for 24 hours did not result in additional inhibition of colony formation. However, after 72-hour incubation with both oligodeoxynucleotides, colony formation was diminished significantly when compared with either oligodeoxynucleotides alone (from similar to 30% to similar to 78% for granulocyte-macrophage colony-forming unit; similar to 50% to similar to 80% for blast-forming unit - erythroid).Conclusions: We hypothesize that exposing primary leukemic cells to antisense oligodeoxynucleotides targeted to two, or possibly more, genes might significantly augment the therapeutic utility of these molecules.