No abstract

Communication between irradiated and un-irradiated (bystander) cells can cause damage in cells that are not directly targeted by ionizing radiation, a process known as the bystander effect. Bystander effects can also lead to chromosomal/genomic instability within the progeny of bystander cells, similar to the progeny of directly irradiated cells. The factors that mediate this cellular communication can be transferred between cells via gap junctions or released into the extracellular media following irradiation, but their nature has not been fully characterized. In this study we tested the hypothesis that the bystander effect mediator contains an RNA molecule that may be carried by exosomes. MCF7 cells were irradiated with 2 Gy of X rays and the extracellular media was harvested. RNase treatment abrogated the ability of the media to induce early and late chromosomal damage in bystander cells. Furthermore, treatment of bystander cells with exosomes isolated from this media increased the levels of genomic damage. These results suggest that the bystander effect, and genomic instability, are at least in part mediated by exosomes and implicate a role for RNA.

In directly irradiating cells, telomere metabolism is altered and similar effects have been observed in nontargeted cells. Exosomes and their cargo play dominant roles in communicating radiation-induced bystander effects with end points related to DNA damage. Here we report novel evidence that exosomes are also responsible for inducing telomere-related bystander effects. Breast epithelial cancer cells were exposed to either 2 Gy X rays, or exposed to irradiated cell conditioned media (ICCM), or exosomes purified from ICCM. Compared to control cells, telomerase activity decreased in the 2 Gy irradiated cells and both bystander samples after one population doubling. At the first population doubling, telomere length was shorter in the 2 Gy irradiated sample but not in the bystander samples. By 24 population doublings telomerase activity recovered to control levels in all samples; however, the 2 Gy irradiated sample continued to demonstrate short telomeres and both bystander samples acquired shorter telomeres. RNase treatment of exosomes prevented the bystander effects on telomerase and telomere length that were observed at 1 population doubling and 24 population doublings, respectively. Thermal denaturation by boiling eliminated the reduction of telomere length in bystander samples, suggesting that the protein fraction of exosomes also contributes to the telomeric effect. RNase treatment plus boiling abrogated all telomere-related effects in directly irradiated and bystander cell populations. These findings suggest that both proteins and RNAs of exosomes can induce alterations in telomeric metabolism, which can instigate genomic instability in epithelial cancer cells after X-ray irradiation.

No effect of serotonin on the induction of either bystander effects of genomic instability was observed using this experimental system.