Interaction of amifostine and ionizing radiation on transcriptional patterns of apoptotic genes expressed in human microvascular endothelial cells (HMEC)
Interaction of amifostine and ionizing radiation on transcriptional patterns of apoptotic genes expressed in human microvascular endothelial cells (HMEC)
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DOI：
10.1016/j.ijrobp.2004.04.060
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发表时间：
2004-10-01
影响因子：
7
通讯作者：
Grdina, DJ
中科院分区：
文献类型：
--
作者：
Khodarev, NN;Kataoka, Y;Grdina, DJ
目的：氨磷汀是一种需要碱性磷酸酶去磷酸化才能激活的前药。这一过程在静脉注射后迅速在血液中发生。给正在接受选择的放射和化疗的癌症治疗的患者。因此，血管内皮细胞将代表一个正常的细胞系统，它是第一批体验到这种试剂的辐射防护效果的细胞系统之一。研究氨磷汀活性游离硫醇WR-1065对体外培养的人微血管内皮细胞(HMEC)辐射诱导的转录模式改变和随后的细胞凋亡的影响。方法与材料：体外培养人微血管内皮细胞，WR-1065浓度为4 mM，照射剂量为0~6Gy30min，WR-1065浓度为4 mM，照射前为30min。克隆形成实验检测细胞存活率，流式细胞仪分析细胞周期时相，流式细胞仪检测细胞凋亡，Anexin V染色和亚G1期分析，Clontech Atlas Human基因芯片分析基因表达，以确定阿米福汀和辐射条件对HMEC的协同和拮抗作用。结果：HMEC在照射前30min暴露于4mMWR-1065，其防护增强系数为2.0，即DO-IRR为1.25GY，DO-IRR+WR为2.56GY。表达谱显示29个基因在WR-1065和电离辐射的共同作用下被激活，另外12个基因被协同或相加抑制。特别是，包括caspase2、4、9和bcl家族不同成员在内的一组凋亡相关基因，以及与凋亡相关的受体，被确定为受到WR-1065和辐射联合治疗的显著影响。此外，一些表达细胞周期蛋白A、G1、G2和D3的细胞周期相关基因以及DNA损伤/检查点蛋白ATM、DNA-PK和RAD23B也受到显著影响。结论：WR-1065是氨磷汀的活性硫醇形式，通过克隆形成实验和细胞凋亡实验证实WR-1065是HMEC的有效辐射保护剂。表达谱成功地确定了HMEC对WR-1065和电离辐射暴露的转录反应，无论是单独还是联合，并展示了对不同细胞基因表达的协同和拮抗效应以及相应的功能反应。氨磷汀的辐射防护作用不仅限于其特有的物理化学性质，包括清除自由基、自氧化导致细胞内缺氧以及通过氢原子供体进行化学修复，还包括其调节参与细胞凋亡、细胞周期和DNA修复的基因的复杂转录调控的能力。(C)2004年爱思唯尔公司。
Purpose: Amifostine is a prodrug that requires dephosphorylation by alkaline phosphatase to become activated. This process occurs rapidly within the bloodstream after its i.v. administration to patients undergoing cancer treatment with selected radiation and chemotherapies. Vascular endothelial cells will, therefore, represent a normal cell system that is among the first to experience the radioprotective effects of this agent. Amifostine's active free thiol WR-1065 was investigated to determine its effect on radiation-induced changes in transcriptional patterns and subsequent apoptosis in human microvascular endothelial cells (HMEC) growing in vitro.Methods and Materials: Human microvascular endothelial cells were grown to confluency and then exposed to WR-1065 at a concentration of 4 mM for 30 min, radiation doses that ranged from 0 to 6 Gy, and WR-1065 at a concentration of 4 mM for 30 min before exposure to ionizing radiation. Cell survival was assessed by clonogenic assay, cell cycle phase was analyzed by flow cytometry, apoptosis was also assessed by flow cytometry in which Anexin V staining and sub-G1 fraction analysis were applied, and gene expression was analyzed by the Clontech Atlas Human cDNA array to identify synergistic and antagonistic effects as a function of amifostine and radiation exposure conditions with a focus on apoptotic-related factors.Results: Exposure of HMEC to 4 mM WR-1065 30 min before irradiation resulted in a protection enhancement factor of 2.0; that is, DO-IRR of 1.25 Gy and DO-IRR + WR of 2.56 Gy. Expression profiling revealed 29 genes that were synergistically activated by the combined action of WR-1065 and ionizing radiation, and an additional 12 genes were synergistically or additively suppressed. In particular, a subset of apoptosis-related genes that included caspases 2, 4, and 9 and different members of the bcl family, along with apoptosis-related receptors, were identified as being significantly affected by the combined treatment of WR-1065 and radiation exposure. In addition, a number of cell cycle-related genes that express cyclins A, G1, G2, and D3 and DNA damage/check point proteins ATM, DNA-PK and RAD23B were also found to be significantly affected. Functional assays of apoptosis were also performed that demonstrated the ability of WR-1065 to protect against radiation-induced apoptosis.Conclusions: WR-1065, the active thiol form of amifostine, is an effective radioprotector of HMEC as determined by use of clonogenic and apoptotic assays for cell survival. Expression profiling successfully defined the transcriptional response of HMEC to both WR-1065 and ionizing radiation exposure, either alone or in combination, and demonstrated both synergistic and antagonistic effects on the expression of different cellular genes, along with corresponding functional responses. The radioprotective effects of amifostine are not limited to its well-characterized physiochemical properties, which include free-radical scavenging, auto-oxidation leading to intracellular hypoxia, and chemical repair by hydrogen atom donation, but include its ability to modulate the complex transcriptional regulation of genes that are involved in apoptosis, cell cycle, and DNA repair. (C) 2004 Elsevier Inc.