Enhanced DNA double-strand break repair of microbeam targeted A549 lung carcinoma cells by adjacent WI38 normal lung fibroblast cells via bidirectional signaling

Understanding the mechanisms underlying the radiation-induced bystander effect (RIBE) and bi-directional signaling between irradiated carcinoma cells and their surrounding non-irradiated normal cells is relevant to cancer radiotherapy. The present study investigated propagation of RIBE signals between human lung carcinoma A549 cells and normal lung fibroblast WI38 cells in bystander cells, either directly or indirectly contacting irradiated A549 cells. We prepared A549-GFP/WI38 co-cultures and A549-GFP/A549 co-cultures, in which A549-GFP cells stably expressing H2BGFP were co-cultured with either A549 cells or WI38 cells, respectively. Using the SPICE-NIRS microbeam, only the A549-GFP cells were irradiated with 500 protons per cell. The level of gamma-H2AX, a marker for DNA double-strand breaks (DSB), was subsequently measured for up to 24 h postirradiation in three categories of cells: (1) targeted/irradiated A549-GFP cells; (2) neighboring/non-irradiated cells directly contacting the targeted cells; and (3) distant/non-irradiated cells, which were not in direct contact with the targeted cells. We found that DSB repair in targeted A549-GFP cells was enhanced by co-cultured WI38 cells. The bystander response in A549-GFP/A549 cell co-cultures, as marked by gamma-H2AX levels at 8 h post-irradiation, showed a decrease to non-irradiated control level when approaching 24 h, while the neighboring/distant bystander WI38 cells in A549-GFP/WI38 co-cultures was maintained at a similar level until 24 h post-irradiation. Surprisingly, distant A549-GFP cells in A549-GFP/WI38 co-cultures showed time dependency similar to bystander WI38 cells, but not to distant cells in A549-GFP/A549 co-cultures. These observations indicate that gamma-H2AX was induced in WI38 cells as a result of RIBE. WI38 cells were not only involved in rescue of targeted A549, but also in the modification of RIBE against distant A549-GFP cells. The present results demonstrate that radiation-induced bi-directional signaling had extended a profound influence on cellular sensitivity to radiation as well as the sensitivity to RIBE.