Reactive oxygen species-induced release of signalling factors in irradiated cells triggers membrane signalling and calcium influx in bystander cells

Purpose: The aim of this study was to elucidate the sequence of very early bystander signalling events and to determine the role of the different signalling molecules in both the production of the bystander signal and the response to this signal. Materials and methods: Human keratinocytes (HaCaT cell line) were irradiated (0.005, 0.05 and 0.5 Gy) using a cobalt 60 teletherapy unit, the medium was harvested one hour post irradiation and transferred to recipient HaCaT cells. Membrane permeability and levels of calcium, reactive oxygen species and nitric oxide were measured in the recipient cells immediately after the addition of irradiated cell conditioned medium (ICCM). Inhibitors of reactive oxygen species (ROS), nitric oxide (NO), calcium and membrane signalling were used in both donor and recipient cells to investigate if bystander effects could be blocked. Results: It was found that membrane signalling followed by calcium influx was the first response in the recipient cells to addition of ICCM. ROS, NO and calcium were all found to be important signalling molecules involved in bystander responses, while ROS and calcium were found to be involved in the production of the bystander signal. Conclusions: The data suggest that calcium and/or ROS induce irradiated cells to release long-lived signalling factors which can trigger membrane signalling and an influx of calcium further inducing ROS in unirradiated cells.