Gong B, Almasan A
Department of Cancer Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Ohio 44195, USA.
Cancer Res. 2000 Oct 15;60(20):5754-60.
Ionizing radiation is a major tool for cancer treatment. The response of eukaryotic cells to ionizing radiation includes apoptosis, a process which requires activation of multiple genes. We sought to determine whether radiation-induced gene expression plays a role in radiation-induced apoptosis. We found Apo2 ligand (Apo2L, also called TRAIL) mRNA induction following gamma-irradiation of Jurkat, MOLT-4, CEM, and PBMC, all human T lineage-derived cells. Increased Apo2L protein levels were found in MOLT-4 and Jurkat cells. Radiation also activated the Apo2L death receptor (DR)5 (also called Apo2, TRAIL-R2, or KILLER) in MOLT-4 cells, which harbor a wild-type p53. We isolated 1152 bp of 5' flanking region of the Apo2L gene and a shorter fragment of 716 bp, both of which showed promoter activity driving the expression of a luciferase reporter gene; however, the response to radiation in MOLT-4 cells was lost when only 430 bp of 5' proximal flanking sequence was maintained. Exogenous Apo2L induced phosphatidylserine exposure on cell membranes, caspase 8 and caspase 3 activation, key markers of apoptosis, confirming that the Apo2L/DR5 pathway is functional in these cells. Bid, a Bcl-2 family protein also known to contribute to receptor-mediated apoptosis, was also activated. To determine whether Apo2L and DR5 were critical for radiation signaling to apoptosis, we stably expressed a dominant negative DR5delta-receptor in Jurkat cells. Cell survival was significantly augmented, indicating that increased Apo2L expression contributed to radiation-induced apoptosis. Clonogenic assays demonstrated that purified, recombinant soluble Apo2L enhanced the lethality of low, therapeutic doses (1-2 Gy) of gamma-irradiation. These data suggest that production of Apo2L may cooperate synergistically with the cytotoxic effect of radiation, and that combinations of Apo2L and radiation may become a powerful tool in clinical therapy.
电离辐射是癌症治疗的主要手段。真核细胞对电离辐射的反应包括凋亡，这一过程需要多个基因的激活。我们试图确定辐射诱导的基因表达是否在辐射诱导的凋亡中发挥作用。我们发现，在对Jurkat、MOLT-4、CEM和PBMC（所有这些都是人T淋巴细胞系来源的细胞）进行γ射线照射后，Apo2配体（Apo2L，也称为TRAIL）的mRNA被诱导。在MOLT-4和Jurkat细胞中发现Apo2L蛋白水平升高。辐射还激活了MOLT-4细胞中的Apo2L死亡受体（DR）5（也称为Apo2、TRAIL-R2或KILLER），该细胞具有野生型p53。我们分离出Apo2L基因5'侧翼区域的1152 bp片段和一个716 bp的较短片段，这两个片段均显示出驱动荧光素酶报告基因表达的启动子活性；然而，当仅保留5'近端侧翼序列的430 bp时，MOLT-4细胞对辐射的反应消失。外源性Apo2L诱导细胞膜上磷脂酰丝氨酸暴露、半胱天冬酶8和半胱天冬酶3激活，这些是凋亡的关键标志物，证实Apo2L/DR5途径在这些细胞中具有功能。Bid，一种也已知有助于受体介导的凋亡的Bcl-2家族蛋白，也被激活。为了确定Apo2L和DR5对于辐射信号传导至凋亡是否至关重要，我们在Jurkat细胞中稳定表达了显性负性DR5δ受体。细胞存活率显著提高，表明Apo2L表达增加有助于辐射诱导的凋亡。克隆形成试验表明，纯化的重组可溶性Apo2L增强了低治疗剂量（1 - 2 Gy）γ射线照射的致死性。这些数据表明，Apo2L的产生可能与辐射的细胞毒性作用协同发挥作用，并且Apo2L与辐射的联合应用可能成为临床治疗中的有力工具。
