Radiation-induced bystander effect (RIBE) is a poorly understood phenomenon wherein non-targeted cells exhibit effects of radiation. The researchers have reported that cell-free chromatin (cfCh) particles that are released from dying cells can integrate into genomes of surrounding healthy cells to induce DNA damage and inflammation.

This raised the possibility that RIBE might be induced by cfCh released from irradiated dying cells. When conditioned media from BrdU-labeled irradiated cells were passed through filters of pore size 0.22 µm and incubated with unexposed cells, BrdU-labeled cfCh particles could be seen to readily enter their nuclei to activate H2AX, active Caspase-3, NFκB, and IL-6.

A direct relationship was observed with respect to activation of RIBE biomarkers and radiation dose in the range of 0.1–50 Gy. We confirmed by FISH and cytogenetic analysis that cfCh had stably integrated into chromosomes of bystander cells and had led to extensive chromosomal instability.

The above RIBE effects could be abrogated when conditioned media were pre-treated with agents that inactivate cfCh, namely, anti-histone antibody complexed nanoparticles (CNPs), DNase I and a novel DNA degrading agent Resveratrol-copper (R-Cu). Lower hemi-body irradiation with γ-rays (0.1–50 Gy) led to activation of H2AX, active Caspase-3, NFκB, and IL-6 in brain cells in a dose-dependent manner.

RIBE biomarkers

Activation of these RIBE biomarkers could be abrogated by concurrent treatment with CNPs, DNase I and R-Cu indicating that activation of RIBE was not due to radiation scatter to the brain.

RIBE activation was seen even when mini-beam radiation was delivered to the umbilical region of mice wherein radiation scatter to brain was negligible and could be abrogated by cfCh inactivating agents.

These results indicate that cfCh released from radiation-induced dying cells are activators of RIBE and that it can be prevented by treatment with appropriate cfCh inactivating agents.

In order to investigate whether irradiated cells that survive, but carry damaged DNA extrude cfCh to induce RIBE? To address this issue, we irradiated MDA-MB-231 cells with a radiation dose of 0.1 Gy, which caused little cell death but had inflicted dsDNA breaks in 33.4% of cells.

Filtered conditioned medium from these cells, with extensive DNA damage, when applied to recipient NIH3T3 cells, however, did not lead to activation of RIBE (γH2AX) in the bystander cells. The level of H2AX activation was similar to that seen when un-irradiated conditioned medium had been applied to recipient NIH3T3 cells