Bystander responses in three-dimensional cultures containing radiolabelled and unlabelled human cells

Research on the radiation-induced bystander effect has been carried out mainly in 2-D tissue culture systems. This study uses a 3-D model, wherein apparently normal human diploid fibroblasts (AG1522) are grown in a carbon scaffold, to investigate the induction of a G₁ checkpoint in bystander cells present alongside radiolabelled cells. Cultures were simultaneously pulse-labelled with ³H- deoxycytidine (³HdC) to selectively irradiate a minor fraction of cells, and bromodeoxyuridine (BrdU) to identify the radiolabelled cells. After thorough washing of cultures, iododeoxyuridine (IdU) was administered to detect proliferating bystander cells. The cultures were harvested at various times thereafter, and cells were reacted with two monoclonal antibodies specific to IdU/BrdU or BrdU, respectively, stained with propidium iodide, and subjected to multi-parameter flow cytometry. Cell-cycle progression was followed in radiolabelled cells (BrdU⁺) that were chronically irradiated by low energy beta particles emitted by DNA-incorporated ³H, and in unlabelled bystander cells (BrdU^-) by a flow cytometry based cumulative labelling index assay. As expected, radiolabelled cells were delayed, in a dose-dependent manner, in G₂ and subsequently G₁. No delay occurred in progression of bystander cells through G₁, when the labelled cells were irradiated at dose rates up to 0.32 Gy h^-1.