de Toledo Sonia M, Buonanno Manuela, Harris Andrew L, Azzam Edouard I
a Department of Radiology , RUTGERS New Jersey Medical School Cancer Center , Newark , NJ , USA.
b Pharmacology and Physiology and Neuroscience , RUTGERS New Jersey Medical School Cancer Center , Newark , NJ , USA.
Int J Radiat Biol. 2017 Oct;93(10):1182-1194. doi: 10.1080/09553002.2017.1334980. Epub 2017 Jun 15.
To examine the time window during which intercellular signaling though gap junctions mediates non-targeted (bystander) effects induced by moderate doses of ionizing radiation; and to investigate the impact of gap junction communication on genomic instability in distant progeny of bystander cells.
A layered cell culture system was developed to investigate the propagation of harmful effects from irradiated normal or tumor cells that express specific connexins to contiguous bystander normal human fibroblasts. Irradiated cells were exposed to moderate mean absorbed doses from 3.7 MeV α particle, 1000 MeV/u iron ions, 600 MeV/u silicon ions, or Cs γ rays. Following 5 h of co-culture, pure populations of bystander cells, unexposed to secondary radiation, were isolated and DNA damage and oxidative stress was assessed in them and in their distant progeny (20-25 population doublings).
Increased frequency of micronucleus formation and enhanced oxidative changes were observed in bystander cells co-cultured with confluent cells exposed to either sparsely ionizing (Cs γ rays) or densely ionizing (α particles, energetic iron or silicon ions) radiations. The irradiated cells propagated signals leading to biological changes in bystander cells within 1 h of irradiation, and the effect required cellular coupling by gap junctions. Notably, the distant progeny of isolated bystander cells also exhibited increased levels of spontaneous micronuclei. This effect was dependent on the type of junctional channels that coupled the irradiated donor cells with the bystander cells. Previous work showed that gap junctions composed of connexin26 (Cx26) or connexin43 (Cx43) mediate toxic bystander effects within 5 h of co-culture, whereas gap junctions composed of connexin32 (Cx32) mediate protective effects. In contrast, the long-term progeny of bystander cells expressing Cx26 or Cx43 did not display elevated DNA damage, whereas those coupled by Cx32 had enhanced DNA damage.
In response to moderate doses from either sparsely or densely ionizing radiations, toxic and protective effects are rapidly communicated to bystander cells through gap junctions. We infer that bystander cells damaged by the initial co-culture (expressing Cx26 or Cx43) die or undergo proliferative arrest, but that the bystander cells that were initially protected (expressing Cx32) express DNA damage upon sequential passaging. Together, the results inform the roles that intercellular communication play under stress conditions, and aid assessment of the health risks of exposure to ionizing radiation. Identification of the communicated molecules may enhance the efficacy of radiotherapy and help attenuate its debilitating side-effects.
研究通过间隙连接进行细胞间信号传导介导中等剂量电离辐射诱导的非靶向（旁观者）效应的时间窗；并研究间隙连接通讯对旁观者细胞远距离子代基因组不稳定性的影响。
开发了一种分层细胞培养系统，以研究从表达特定连接蛋白的受辐照正常细胞或肿瘤细胞向相邻旁观者正常人成纤维细胞传播有害效应。将受辐照细胞暴露于3.7 MeVα粒子、1000 MeV/u铁离子、600 MeV/u硅离子或铯γ射线的中等平均吸收剂量下。共培养5小时后，分离未暴露于二次辐射的旁观者细胞纯群体，并评估它们及其远距离子代（20 - 25次群体倍增）中的DNA损伤和氧化应激。
在与暴露于稀疏电离（铯γ射线）或密集电离（α粒子、高能铁或硅离子）辐射的汇合细胞共培养的旁观者细胞中，观察到微核形成频率增加和氧化变化增强。受辐照细胞在辐照后1小时内将信号传递给旁观者细胞，导致其发生生物学变化，且该效应需要间隙连接进行细胞偶联。值得注意的是，分离的旁观者细胞的远距离子代也表现出自发性微核水平升高。这种效应取决于将受辐照供体细胞与旁观者细胞偶联的连接通道类型。先前的研究表明，由连接蛋白26（Cx26）或连接蛋白43（Cx43）组成的间隙连接在共培养5小时内介导毒性旁观者效应，而由连接蛋白32（Cx32）组成的间隙连接介导保护效应。相比之下，表达Cx26或Cx43的旁观者细胞的长期子代未显示出DNA损伤增加，而由Cx32偶联的子代则有增强的DNA损伤。
响应稀疏或密集电离辐射的中等剂量，毒性和保护效应通过间隙连接迅速传递给旁观者细胞。我们推断，最初共培养受损的旁观者细胞（表达Cx26或Cx43）死亡或经历增殖停滞，但最初受到保护的旁观者细胞（表达Cx32）在连续传代时会表现出DNA损伤。总之，这些结果揭示了细胞间通讯在应激条件下所起的作用，并有助于评估电离辐射暴露的健康风险。鉴定通讯分子可能会提高放疗效果，并有助于减轻其使人衰弱的副作用。
