IMR90 bystander experiment 0.5 Gy alpha particle

The existence of a radiation bystander effect in which non-irradiated cells respond to signals from irradiated cells is well established. It raises concerns for the interpretation of risks from exposure to low doses of ionizing radiation. Sparse data exists about the bystander signaling mechanisms and the ability to transmit damaging effects both spatially and temporally. To understand early signaling and cellular changes in bystanders we have measured global gene expression 30 minutes after direct and bystander exposure to alpha particle in primary human lung fibroblasts. Gene ontology and pathway analyses suggested that the earliest measured changes at 30 minutes after treatment are in cell structure motility and adhesion categories and a significant number of genes belong to the category of inflammation and cell-to-cell communication. We investigated time course gene expression profiles of matrix metalloproteinases 1 and 3 (MMP1 and MMP3) chemokine ligands 2 3 and 5 (CXCL2 CXCL3 and CXCL5) interleukins 1a 1b 6 and 33 (IL1A IL1B IL6 and IL33) growth differentiation factor 15 (GDF15) and superoxide dismutase2 (SOD2) by real time quantitative PCR. These encode proteins involved in cellular signaling via the NFkappaB pathway and time course of mRNA levels revealed an increased response at 30 minutes after irradiation followed by another wave at 4 to 6 hours. We also investigated protein modifications in the AKT-GSK-3 signaling pathway and found that in irradiated cells AKT and GSK3beta are hyper-phosphorylated at 30 minutes and this effect is maintained until 4 hours after exposure. In bystanders there is a similar response with a delay of 30 minutes. In irradiated cells inactivated GSK3beta led to decreased phosphorylation of beta-catenin. Our results are the first to show that the radiation induced bystander signal can induce a widespread gene expression response as early as 30 minutes after exposure and that these changes are accompanied by protein modification of signaling modules such as AKT and GSK3beta. There are 12 total samples 4 corresponding biological replicates of IMR90 cells that were not irradiated (control=C) irradiated (alpha=A) and bystander (B) cells were harvested 0.5 hr after treatment