John B. Little, Edouard I. Azzam, Hatsumi Nagasawa, Sonia M. deToledo
Laboratory of Radiobiology, Harvard School of Public Health, Boston, MA
02115 USA
It has generally been considered that important biological effects of ionizing radiation arise as a consequence of DNA damage occurring in irradiated cells. We have examined this hypothesis by irradiating confluent cultures of fibroblasts and epithelial cells with very low fluences of alpha particles. The cultures are exposed such that as few as 1-2% of the cells in the population are traversed by an alpha particle and thus receive any radiation exposure. Genetic changes including an enhanced frequency of sister chromatid exchanges and HPRT mutations occurred in non-irradiated, "bystander" cells. These changes were associated with an increase in the frequency of micronuclei, indicating the induction of DNA damage in bystander cells. These results thus suggest that damage signals can be transmitted from irradiated cells to non-irradiated cells in the same population.
In order to gain information concerning molecular pathways, changes in gene expression were examined in confluent cultures of human diploid or mouse embryo-derived fibroblasts (MEFs) by western analysis and in-situ immunofluorescence. The expression levels of p53 and p21Waf1 were significantly enhanced in bystander cells. This was associated with an increase in the level of serine-15 phosphorylation of p53, suggesting that the activation of the p53 damage response pathway resulted from DNA damage induced in the bystander cells. The upregulation of p53 and p21Waf1 did not occur in cultures irradiated at low density, and was markedly reduced in the presence of gap junction inhibitors. The importance of gap-junction mediated intercellular communication was confirmed in connexin-43 mutant cells and knockout MEFs, providing direct evidence for the involvement of connexin-43 mediated intercellular communication in the transmission of damage signals to non-irradiated cells.
The bystander response was suppressed by incubation with superoxide dismutase and catalase, as well as an inhibitor of the generation of reactive oxygen species by NADP(H) oxidase. It was also associated with the induction of NFB, a transcription factor that is regulated by redox-dependent processes and is strongly induced by H202. Together, these findings suggest that the effect may be mediated by oxidative stress. This is of interest in light of the observation that most of the HPRT mutations induced in bystander cells were point mutations, as opposed to the high frequency of deletions observed in cells traversed by one or more alpha particles.
To further characterize the signaling pathways involved, we analyzed the activation of stress-related kinases and their downstream transcription factors by western blotting and electrophoretic mobility shift assays; a 2-4-fold increase in the phosphorylation levels of JNK, ERK1/2, p90RSK, Elk-1 and ATF2 was observed. These changes were detected by 15 min after exposure and persisted for at least 1 hour. These findings indicate that the activation of multiple signal transduction pathways occurs in bystander cells, involving signals arising from the plasma membrane as well as from DNA damage.
Supported by Research Grant FG02-98ER62685 from the U.S. Department of Energy.