Prevention of radiation-induced bystander effects by agents that inactivate cell-free chromatin released from irradiated dying cells

Saurabh Kirolikar,Naveen Kumar Nair ,Naveen K Khare ,Sushma Shinde,Harshada Wani,Preeti Prasannan,Richa Pandey,G Raghuram ,Suhail Sarwar Siddiqui ,Sailee Parab,Pradip Chaudhari,Ratnam Prasad,Jenevieve D’souza ,Alfina Shaikh,Pritishkumar Tidke,Kavita Pal,Tannishtha Saha,Namrata Pancholi,Bhagyeshri Rane,Indraneel Mittra

doi:10.1038/s41419-018-1181-x

Abstract

Radiation-induced bystander effect (RIBE) is a poorly understood phenomenon wherein non-targeted cells exhibit effects of radiation. We 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. 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.

Highlights

Radiation-induced bystander effect (RIBE) is a phenomenon wherein cells not directly exposed to ionizing radiation show heritable changes that include DNA damage, mutations, chromosomal aberrations, chromosomal instability, senescence, apoptosis, and oncogenic transformations[1,2]
We have recently reported that cell-free chromatin (cfCh) from dying cancer cells can integrate into genomes of bystander healthy cells to induce DNA damage and inflammation[23]
Results of new experiments reported include; (1) demonstration that, following ionizing radiation, cfCh are released predominantly from apoptotic rather than necrotic cells; (2) EM demonstration of presence of cfCh in conditioned media of irradiated cells; (3) demonstration that fluorescently labeled cfCh released from irradiated dying cells are freely ingested by bystander cells to localize in the nuclei by 6 h; (4) demonstration that cfCh released from irradiated cells integrate into genomes of bystander cells leading to extensive genomic instability; (5) demonstration that cfCh released from irradiated dying cells activate RIBE in the form of activation of H2AX, active Caspase-3, NFκB, and IL-6 in bystander

Summary

Introduction

Radiation-induced bystander effect (RIBE) is a phenomenon wherein cells not directly exposed to ionizing radiation show heritable changes that include DNA damage, mutations, chromosomal aberrations, chromosomal instability, senescence, apoptosis, and oncogenic transformations[1,2]. Bystander effects have been reported using synchrotron—generated micro—beam irradiation[11,12], and targeted cytoplasmic irradiation has been shown to induce bystander responses[13], challenging the belief that direct damage to DNA is a prerequisite for RIBE. Serum from patients who have received focal radiation therapy have been shown to have RIBEinducing properties, and out-of-field RIBE has been reported in distant organs[16]. Evidence of RIBE was demonstrated in non-small cell lung cancer patients exposed to focal irradiation wherein DNA damage was observed in both irradiated and out-of-field normal cells[17]. Development of brain tumors in susceptible strains of mice exposed to trunk irradiation is another example of RIBE induced in distant organs[19]

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Conclusion