Multiple bystander effect of irradiated megacolonies of melanoma cells on non-irradiated neighbours

The multicellular megacolonies of human melanoma Me45 line growing on one part of the bottom of culture flasks were irradiated with 5 Gy (60Co), whereas megacolonies growing on the second part of the bottom were shielded. The bystander effect of radiation-traversed cells on non-traversed cells was studied during postradiation co-cultivation. Activity of superoxide dismutase (Mn and CuZn subunits), glutathione peroxidase (GSH-Pox) and malondialdehyde (MDA) concentration as a biochemical markers of bystander effect were monitored for a period of 72 h. The DNA damage was measured by the comet assay. Micronucleus induction, mitotic index and cellular death as apoptosis or necrosis were simultaneously estimated, based on morphologic criteria. The bystander effect of irradiated cells on their neighbours was observed as a slight increase of MDA concentration, comparable decrease of GSH-Pox activity, and some fluctuation of mitochondrial and cytoplasmic isoenzymes of SOD. DNA strand breaks and rejoining measured by comet assay as mean tail length, demonstrated clearly the bystander effect for nontraversed radiation cells, additionally verified by tail moment. There was also a significant increase of micronucleation and apoptosis generated by radiation traversed cells in shielded neighbours. Furthermore, significantly higher increase of necrosis in shielded neighbour cells compared to radiation traversed cells was observed. Proliferative activity showed a suppression in both, radiation traversed and shielded neighbour cells in all measured time points. The behaviour of used parameters points to the radical nature of modificators secreted by radiation traversed cells inducing bystander toxic damage in shielded neighbour cells.