Bystander response in human lymphoblastoid TK6 cells.

The mechanisms of the medium-mediated bystander response induced by gamma-rays in non-irradiated TK6 cells were investigated. Cell cultures were irradiated and the culture medium discarded immediately after irradiation and replaced with a fresh one. In cells incubated with conditioned medium from irradiated cells (CM), a significant decrease in cell viability and cloning efficiency was observed, together with a significant increase in apoptosis, also in directly irradiated cells. To examine whether bystander apoptosis involved the extrinsic pathway, an inhibitor of caspase-8 was added to CM cultures, which significantly decreased apoptosis to control levels. The addition to CM of ROS scavengers, Cu-Zn superoxide dismutase and N-acetylcysteine did not affect the induction of apoptosis. To assess whether CM treatment activates a DNA damage response, also the formation of gamma-H2AX foci, as markers of double-strand breaks and their colocalisation with 53-binding protein 1 (53BP1) and the protein mutated in the Nijmegen breakage syndrome 1 (NBS1) was analysed. In cultures treated for 2h with CM, 9-11% of cells showed gamma-H2AX foci, which partially or totally lacked colocalisation with 53BP1 and NBS1 foci. About 85% of irradiated cells were positive for gamma-H2AX foci, which colocalised with 53BP1 and NBS1 proteins. At 24h from irradiation, very few irradiated cells retained foci, fitting DNA repair kinetics. The number of foci-positive bystander cells also decreased to background values 24h after CM incubation. Our results suggest that irradiated TK6 cells release into the medium some soluble factors, not ROS, which are responsible for the cytotoxic effects induced in bystander cells. In our experimental system, the role of ROS appeared to be of minor importance in inducing cell mortality, but probably critical in activating the DNA damage response in the responsive fraction of bystander cells.

"Modeled microgravity" affects cell response to ionizing radiation and increases genomic damage.

The aim of this work was to assess whether "modeled microgravity" affects cell response to ionizing radiation, increasing the risk associated with radiation exposure. Lymphoblastoid TK6 cells were irradiated with various doses of gamma rays and incubated for 24 h in a modeled microgravity environment obtained by the Rotating Wall Vessel bioreactor. Cell survival, induction of apoptosis and cell cycle alteration were compared in cells irradiated and then incubated in 1g or modeled microgravity conditions. Modulation of genomic damage induced by ionizing radiation was evaluated on the basis of HPRT mutant frequency and the micronucleus assay. A significant reduction in apoptotic cells was observed in cells incubated in modeled microgravity after gamma irradiation compared with cells maintained in 1g. Moreover, in irradiated cells, fewer G2-phase cells were found in modeled microgravity than in 1g, whereas more G1-phase cells were observed in modeled microgravity than in 1g. Genomic damage induced by ionizing radiation, i.e. frequency of HPRT mutants and micronucleated cells, increased more in cultures incubated in modeled microgravity than in 1g. Our results indicate that modeled microgravity incubation after irradiation affects cell response to ionizing radiation, reducing the level of radiation-induced apoptosis. As a consequence, modeled microgravity increases the frequency of damaged cells that survive after irradiation.