Analysis of bystander signaling between targeted cancer cells and neighboring normal cells. (S14-TK01: 2015)

Radiation induced bystander effect (RIBE) is generally known to be the phenomena that mediated from irradiated cells to non-irradiated cells. This RIBE may become one of the major concern not only in the field of radiation protection, but also in the radiation cancer therapy. In the recent studies reported inverse direction in the RIBE pathway that showed non-irradiated cells rescues the irradiated cells. Therefore, mechanistic study of radiation induced bi-directional signaling between normal cells and cancer cells will give important information for radiation risk assessment. In this study, A549, human lung cancer cells, A549GFP cells that stably express H2B-GFP, and WI38, human lung normal cells were used. As shown in Figure 1, A549GFP cells were co-cultured with either A549 cells or WI38 cells and only the A549GFP cells in the cell population were targeted with 500 protons to the nucleus by SPICE-NIRS microbeam [1]. After irradiation, cells were fluorescently immuno-stained against γ-H2AX, which is a well know maker for DNA double strand breaks (DSBs) and total fluorescence per nucleus were quantified from the microscopic images.In the previous fiscal year, we reported our preliminary results that neighboring un-irradiated WI38 cells inhibited radiation induced DSBs repair of irradiated A549GFP, and DSBs of the WI38 cells increased due to RIBE. Also, consequently, both the gap-junction intercellular communication (GJIC) pathway and the cell to cell non-contact Media-Transfer (MT) pathway may be strongly involved in the bi-directional cellular responses. During this fiscal year, we repeated these experiments of irradiating only A549-GFP cells within the mixed population to clearly show the bi-directional cellular response between targeted A549-GFP cells and non-targeted WI-38 cells. We also started an experiment to answer whether this bi-directional signaling would be affected by the irradiation of WI-38 cells. In this setup, we targeted both cells, A549-GFP and WI-38 cells with 500 protons to see if there is any difference when the neighboring WI-38 cells were targeted equally with A549GFP cells (Figure 2 A). Figure 2B and C are the representative result obtained from a single beam time. As a result, in all the time points after irradiation, the cells in the non-targeted area showed higher γ-H2AX signals compared to the controls. This indicates the bystander signals from the targeted cells produced DNA double strand breaks in the cells of non-targeted area. This was seen similarly with and without WI38 cells in the population. We also compared the results of γ-H2AX in targeted A549GFP cells in A549GFP-A549 samples, and A549GFP-WI-38 samples. The samples with only A549 cells showed faster DNA dsb repair compared to those samples with WI38. Further analysis is still in progress and will be presented