A Proteomic Study Suggests Stress Granules as New Potential Actors in Radiation-Induced Bystander Effects

Abstract

Besides the direct effects of radiations, indirect effects are observed within the surrounding non-irradiated area; irradiated cells relay stress signals in this close proximity, inducing the so-called radiation-induced bystander effect. These signals received by neighboring unirradiated cells induce specific responses similar with those of direct irradiated cells. To understand the cellular response of bystander cells, we performed a 2D gel-based proteomic study of the chondrocytes receiving the conditioned medium of low-dose irradiated chondrosarcoma cells. The conditioned medium was directly analyzed by mass spectrometry in order to identify candidate bystander factors involved in the signal transmission. The proteomic analysis of the bystander chondrocytes highlighted 20 proteins spots that were significantly modified at low dose, implicating several cellular mechanisms, such as oxidative stress responses, cellular motility, and exosomes pathways. In addition, the secretomic analysis revealed that the abundance of 40 proteins in the conditioned medium of 0.1 Gy irradiated chondrosarcoma cells was significantly modified, as compared with the conditioned medium of non-irradiated cells. A large cluster of proteins involved in stress granules and several proteins involved in the cellular response to DNA damage stimuli were increased in the 0.1 Gy condition. Several of these candidates and cellular mechanisms were confirmed by functional analysis, such as 8-oxodG quantification, western blot, and wound-healing migration tests. Taken together, these results shed new lights on the complexity of the radiation-induced bystander effects and the large variety of the cellular and molecular mechanisms involved, including the identification of a new potential actor, namely the stress granules.

Label-Free Direct Mass Spectrometry Analysis of the Bystander Effects Induced in Chondrocytes by Chondrosarcoma Cells Irradiated with X-rays and Carbon Ions

“…X-ray irradiations were performed with a PxiXradSmart 225cX irradiator using a tube tension of 225 kV, as previously described [21][22][23][24][25][26][27][28][29][30], and C-ion irradiations were performed in the GANIL facility (Caen, France) using the high-energy beam line IRABAT, as previously described [31,32]. For C-ions, a LET of 73 keV/µm was selected to reproduce clinical conditions.…”

“…Proteins were extracted from bystander chondrocytes (dry pellet) in lysis buffer (RIPA made without protease, in order not to interfere with proteases mass spectrometry) and left on ice for 20 min [29]. Mechanical grinding was carried out using a hand-held motorized pellet pestle for 20 s on ice, followed by 20 min at rest.…”

“…The experiments were performed in biological triplicates. The medium-transfer protocol was selected for consistency with our previous characterization of bystander cells and their secretome and proteome [21,29] (Supplementary Fig. 1).…”

“…In a previous analysis of the secretome of chondrosarcoma cells irradiated with low-dose X-rays, a set of forty modulated proteins was observed. These included a protein cluster related to the ribo-nucleosome compartment, cytoplasmic stress granules and proteins from exosomes involved in oxidative stress cell migration and motility [29]. In the same study, the biological response of nearby chondrocytes was assessed using a gel-based proteomic analysis.…”

“…In the same study, the biological response of nearby chondrocytes was assessed using a gel-based proteomic analysis. Observed bystander effects included changes in cytokinemediated signaling pathways, interleukin-12, cell junction and motility and extracellular exosome formation [29].…”