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

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 3.7MeV α particle, 1000 MeV/u iron ions, 600MeV/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 (C×26) or connexin43 (C×43) mediate toxic bystander effects within 5 h of co-culture, whereas gap junctions composed of connexin32 (C×32) mediate protective effects. In contrast, the long-term progeny of bystander cells expressing C×26 or C×43 did not display elevated DNA damage, whereas those coupled by C×32 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 C×26 or C×43) die or undergo proliferative arrest, but that the bystander cells that were initially protected (expressing C×32) 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.