Bystander Effects in the Cellular Radiation Response
Authors: Diegeler Sebastian , Baumstark‐Khan Christa , Jordan Jens , Hellweg Christine Elisabeth 
Published: June 2021

The overall response to tumor radiation therapy results from direct radiation damage and indirect bystander effects (RIBE) mediated by secreted molecules and paracrine transfer of short‐lived mediators. RIBE can have both detrimental and protective actions on cancerous as well as healthy tissues. Nuclear Factor κB (NF‐κB), which governs immune responses, is a prime candidate for mediating RIBE. NF‐κB also modulates DNA repair while promoting cellular survival as part of the DNA damage response. After exposure to ionizing radiation NF‐κB is activated in directly targeted and non‐targeted bystander cells. We tested the hypothesis that the NF‐κB status is relevant for induction of RIBE, using a mouse embryonal fibroblasts (MEF) knock‐out variant that is unable to activate NF‐κB (NF‐κB essential modulator knock‐out (NEMO ko)).

To analyze influences of secreted soluble factors, cells were irradiated with X‐rays (200 kV) and incubated with fresh culture medium for 24 h. The conditioned medium was then transferred onto non‐irradiated cells and incubated for 24 h (bystander treatment). Bystander responses concerning cellular survival were determined with the colony forming ability test. NF‐κB activation and DNA double strand break (DSB) response were visualized by immunofluorescence staining (p65, γH2AX).

MEF wildtype (wt) bystander cells (conditioned medium from 4 Gy irradiated cells) showed a significantly stronger relative activation of NF‐κB (1.19 ± 0.02, p<0.001), indicating a role of the transcription factor in RIBE. In MEF wt and MEF NEMO ko, the number of DNA DSB was increased 6 h after bystander treatment. The number of γH2AX foci per cell after 4 Gy conditioned medium treatment was vastly increased in MEF NEMO ko cells (CtrlNEMO ko 0.01 ± 0.01 vs. 4 GyNEMO ko 1.48 ± 0.18, p < 0.01) compared to MEF wt cells with the same treatment (Ctrlwt 0.62 ± 0.05 vs. 4 Gywt 1.06 ± 0.06, p < 0.01). Cellular survival was most prominently affected by bystander treatment. MEF wt bystander cells showed significantly reduced survival above a threshold of 4 Gy conditioning dose (70 %). On the other hand, relative survival of MEF NEMO ko cells increased significantly (140 %) after bystander treatment of 1 to 8 Gy conditioning dose.

We conclude that, while MEF wt cells appear to be negatively affected by RIBE, NF‐κB (−) MEF cells seem to thrive on a treatment with conditioned medium despite an increased occurrence of DNA DSB. Mechanistically, the lack of NF‐κB activation in MEF NEMO ko cells may abolish an RIBE‐induced senescence phenotype and promote intracellular signaling cascades that regulate cellular growth. Therefore, NF‐κB is a promising target for immune‐assisted radiation therapy.

Support or Funding Information

Sebastian Diegeler was supported by a scholarship of the the Helmholtz Space Life Sciences Research School (SpaceLife), German Aerospace Center (DLR) Cologne, Germany, which was funded by the Helmholtz Association (Helmholtz‐Gemeinschaft) over a period of six years (Grant No. VH‐KO‐300) and received additional funds from the DLR, including the Aerospace Executive Board and the Institute of Aerospace Medicine. All authors were supported by the DLR grant Research & Development (FuE)‐Project “ISS LIFE” (Program RF‐FuW, partial program 475).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.