Lyng F M, Maguire P, McClean B, Seymour C, Mothersill C
Radiation and Environmental Science Centre, Focas Institute, Dublin Institute of Technology, Dublin 8, Ireland.
Radiat Res. 2006 Apr;165(4):400-9. doi: 10.1667/rr3527.1.
Much evidence now exists regarding radiation-induced bystander effects, but the mechanisms involved in the transduction of the signal are still unclear. The mitogen-activated protein kinase (MAPK) pathways have been linked to growth factor-mediated regulation of cellular events such as proliferation, senescence, differentiation and apoptosis. Activation of multiple MAPK pathways such as the ERK, JNK and p38 pathways have been shown to occur after exposure of cells to radiation and a variety of other toxic stresses. Previous studies have shown oxidative stress and calcium signaling to be important in radiation-induced bystander effects. The aim of the present study was to investigate MAPK signaling pathways in bystander cells exposed to irradiated cell conditioned medium (ICCM) and the role of oxidative metabolism and calcium signaling in the induction of bystander responses. Human keratinocytes (HPV-G cell line) were irradiated (0.005-5 Gy) using a cobalt-60 teletherapy unit. The medium was harvested 1 h postirradiation and transferred to recipient HPV-G cells. Phosphorylated forms of p38, JNK and ERK were studied by immunofluorescence 30 min-24 h after exposure to ICCM. Inhibitors of the ERK pathway (PD98059 and U0126), the JNK pathway (SP600125), and the p38 pathway (SB203580) were used to investigate whether bystander-induced cell death could be blocked. Cells were also incubated with ICCM in the presence of superoxide dismutase, catalase, EGTA, verapamil, nifedipine and thapsigargin to investigate whether bystander effects could be inhibited because of the known effects on calcium homeostasis. Activated forms of JNK and ERK proteins were observed after exposure to ICCM. Inhibition of the ERK pathway appeared to increase bystander-induced apoptosis, while inhibition of the JNK pathway appeared to decrease apoptosis. In addition, reactive oxygen species, such as superoxide and hydrogen peroxide, and calcium signaling were found to be important modulators of bystander responses. Further investigations of these signaling pathways may aid in the identification of novel therapeutic targets.
目前已有大量关于辐射诱导旁观者效应的证据，但信号转导所涉及的机制仍不清楚。丝裂原活化蛋白激酶（MAPK）通路与生长因子介导的细胞增殖、衰老、分化和凋亡等细胞事件的调节有关。细胞暴露于辐射及其他多种毒性应激后，已显示出多种MAPK通路如ERK、JNK和p38通路会被激活。先前的研究表明氧化应激和钙信号在辐射诱导的旁观者效应中很重要。本研究的目的是调查暴露于受辐照细胞条件培养基（ICCM）的旁观者细胞中的MAPK信号通路，以及氧化代谢和钙信号在诱导旁观者反应中的作用。使用钴-60远距离治疗装置对人角质形成细胞（HPV-G细胞系）进行辐照（0.005 - 5 Gy）。辐照后1小时收集培养基，并转移至受体HPV-G细胞。在暴露于ICCM后30分钟至24小时，通过免疫荧光研究p38、JNK和ERK的磷酸化形式。使用ERK通路抑制剂（PD98059和U0126）、JNK通路抑制剂（SP600125）和p38通路抑制剂（SB203580）来研究旁观者诱导的细胞死亡是否可以被阻断。细胞还在超氧化物歧化酶、过氧化氢酶、EGTA、维拉帕米、硝苯地平和毒胡萝卜素存在的情况下与ICCM一起孵育，以研究由于对钙稳态的已知影响，旁观者效应是否可以被抑制。暴露于ICCM后观察到JNK和ERK蛋白的活化形式。抑制ERK通路似乎会增加旁观者诱导的凋亡，而抑制JNK通路似乎会减少凋亡。此外，发现活性氧如超氧化物和过氧化氢以及钙信号是旁观者反应的重要调节因子。对这些信号通路的进一步研究可能有助于识别新的治疗靶点。
