Biological responses in known bystander cells relative to known microbeam-irradiated cells

Normal human fibroblasts in plateau phase ( congruent with 95% G(1) phase) were stained with the vital nuclear dye Hoechst 33342 (blue fluorescence) or the vital cytoplasmic dye Cell Tracker Orange (orange fluorescence) and plated at a ratio of 1:1. Only the blue-fluorescing nuclei were microbeam-irradiated with a defined number of 90 keV/microm alpha particles. The orange-fluorescing cells were then "bystanders", i.e. not themselves hit but adjacent to cells that were. Hit cells showed a fluence-dependent induction of micronuclei as well as delays in progression from G(1) to S phase. Known bystander cells also showed enhanced frequencies of micronuclei (intermediate between those seen in irradiated and control cells) and transient cell cycle delays. However, the induction of micronuclei in bystander cells did not appear to be dependent on the fluence of the particles delivered to the neighboring hit cells. These are the first studies in which the bystander effect has been visualized directly rather than inferred. They indicate that the phenomenon has a quantitative basis and imply that the target for radiation effects cannot be considered to be the individual cell.