Genomic instability induced in distant progeny of bystander cells depends on the connexins expressed in the irradiated cells

Abstract

Purpose: To examine the time window during which intercellular signaling though gap junctions mediates non-targeted (bystander) effects induced by moderate doses of ionizing radiation; and to investigate the impact of gap junction communication on genomic instability in distant progeny of bystander cells.

Materials and methods: A layered cell culture system was developed to investigate the propagation of harmful effects from irradiated normal or tumor cells that express specific connexins to contiguous bystander normal human fibroblasts. Irradiated cells were exposed to moderate mean absorbed doses from 3.7 MeV α particle, 1000 MeV/u iron ions, 600 MeV/u silicon ions, or ¹³⁷Cs γ rays. Following 5 h of co-culture, pure populations of bystander cells, unexposed to secondary radiation, were isolated and DNA damage and oxidative stress was assessed in them and in their distant progeny (20–25 population doublings).

Results: Increased frequency of micronucleus formation and enhanced oxidative changes were observed in bystander cells co-cultured with confluent cells exposed to either sparsely ionizing (¹³⁷Cs γ rays) or densely ionizing (α particles, energetic iron or silicon ions) radiations. The irradiated cells propagated signals leading to biological changes in bystander cells within 1 h of irradiation, and the effect required cellular coupling by gap junctions. Notably, the distant progeny of isolated bystander cells also exhibited increased levels of spontaneous micronuclei. This effect was dependent on the type of junctional channels that coupled the irradiated donor cells with the bystander cells. Previous work showed that gap junctions composed of connexin26 (Cx26) or connexin43 (Cx43) mediate toxic bystander effects within 5 h of co-culture, whereas gap junctions composed of connexin32 (Cx32) mediate protective effects. In contrast, the long-term progeny of bystander cells expressing Cx26 or Cx43 did not display elevated DNA damage, whereas those coupled by Cx32 had enhanced DNA damage.

Conclusions: In response to moderate doses from either sparsely or densely ionizing radiations, toxic and protective effects are rapidly communicated to bystander cells through gap junctions. We infer that bystander cells damaged by the initial co-culture (expressing Cx26 or Cx43) die or undergo proliferative arrest, but that the bystander cells that were initially protected (expressing Cx32) express DNA damage upon sequential passaging. Together, the results inform the roles that intercellular communication play under stress conditions, and aid assessment of the health risks of exposure to ionizing radiation. Identification of the communicated molecules may enhance the efficacy of radiotherapy and help attenuate its debilitating side-effects.

Keywords:

• Bystander effects
• genomic instability
• non-targeted effects
• gap junctions
• channel permeability
• radiation quality

Acknowledgements

We are ever grateful to Bill Morgan’s guidance through the years. We thank Drs Adam Rusek, Michael Sivertz, Peter Guida, and the late I-Hung Chang, and their colleagues, for their support during the experiments at the NASA Space Radiation Laboratory. We also thank Drs Roger W. Howell, Géraldine Gonon, Jie Zhang, Narongchai Autsavapromporn, Nicholas W. Colangelo and Jason D. Domogauer in the RUTGERS Division of Radiation Research for their support during experiments and for scientific exchanges.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Notes on contributors

Dr. Sonia M. de Toledo is an experimental radiobiologist. She has been involved in research and training of students examining the mechanisms underlying the biological effects of low doses/low fluences of ionizing radiations that differ in their biophysical characteristics.

Dr. Manuela Buonanno is currently an Associate Research Scientist at the Center for Radiological Research and the Radiological Research Accelerator Facility (RARAF) of Columbia University. Her doctoral studies at Rutgers University were focused at investigating the long-term consequences of radiation-induced bystander effects.

Dr. Andrew L. Harris is Professor of Pharmacology, Physiology and Neuroscience. His research investigates the nature of the chemical signals that can pass between cells through gap junction channels, and mechanisms of the modulation of that molecular permeability.

Dr. Edouard I. Azzam has been engaged for over two decades in studies of the roles and mechanisms of intercellular communication and oxidative metabolism in adaptive and bystander responses to different types of ionizing radiation.

Additional information

Funding

The authors are grateful for grant CA049062 from the National Institutes of Health, and grants NNJ06HD91G and NNX15AD62G from the National Aeronautics and Space Administration which supported this research.