Munetoshi Maeda

Bystander Cell Killing in Normal Human Fibroblasts is Induced by Synchrotron X-Ray Microbeams

Abstract The radiation-induced bystander response is defined as a response in cells that have not been directly targeted by radiation but that are in the neighborhood of cells that have been directly exposed. In the work described here, it is shown that bystander cell killing of normal human fibroblast WI-38 cells was induced by synchrotron microbeam X radiation. Cell nuclei in confluent WI-38 cells were irradiated with the microbeam. All of the cells on the dish were harvested and plated 24 h after irradiation. It was found that the bystander cell killing effect showed a parabolic relationship to the radiation dose when five cells were irradiated. At doses above 1.9 Gy, the surviving fraction increased to approximately 1.0. This suggests that induction of bystander cell killing may require some type of activity in the targeted cells, because the dose resulting in 37% cell survival was about 2.0 Gy. Bystander cell killing was suppressed by a pretreatment with aminoguanidine [an inhibitor of inducible nitric oxide (NO) synthase] or carboxy-PTIO (a scavenger of NO). These results suggest that NO is the chief initiator/mediator of bystander cell killing induced by X-ray microbeams.

Bystander Cell Death is Modified by Sites of Energy Deposition within Cells Irradiated with a Synchrotron X-Ray Microbeam

Abstract Radiation-induced bystander effects are the biological responses exhibited by cells adjacent to cells that have been traversed by charged particles. Using a synchrotron X-ray microbeam irradiation system, we irradiated five cells in two different ways: by targeting the nuclei with 10 µm × 10-µm 5.35 keV X-ray beams and by irradiating the whole cells with 50 µm × 50-µm 5.35 keV X-ray beams. Then we measured the clonogenic survival of the bystander cells. When only the nuclei were irradiated, a parabolic enhancement of bystander cell death was observed in a dose-dependent manner in the low-dose region around 1 Gy. In contrast, the surviving fraction of bystander cells decreased monotonically when whole cells were irradiated. Addition of carboxy-PTIO, a specific scavenger of nitric oxide (NO), suppressed bystander cell death in both cases. These results indicate that NO is a mediator in the induction of the parabolic and monotonic types of bystander cell death. Moreover, from the spatial analysis, we found that the parabolic type of bystander cell death was induced primarily within 1 mm of irradiated cells. Our findings demonstrate that the induction of bystander cell death depends on the sites of energy deposition in irradiated cells.

Dose Response of Soft X-Ray-Induced Bystander Cell Killing Affected by p53 Status

A radiation-induced bystander response, which is generally defined as a cellular response that is induced in nonirradiated cells that received bystander signals from directly irradiated cells within an irradiated cell population. In our earlier X-ray microbeam studies, bystander cell killing in normal human fibroblasts had a parabolic relationship to the irradiation dose. To elucidate the role of p53 in the bystander cell killing, the effects were assessed using human non-small cell lung cancer cells expressing wild-type or temperature-sensitive mutated p53. The surviving fraction of bystander wild-type p53 cells showed a parabolic relationship to the irradiation dose; survival was steeply reduced up to 0.45 Gy, recovered toward to 2 Gy, and remained at control levels up to 5 Gy. In contrast, in the mutated p53 cells at a nonpermissive temperature, the surviving fraction was steeply reduced up to 1 Gy and remained at the reduced level up to 5 Gy. When the mutated p53 cells were incubated at a permissive temperature, the decrease in the surviving fraction at 2 Gy was suppressed. The wild-type p53 cells were not only restrained in releasing bystander signals at 2 Gy, but were also resistant to the signals released by the mutated p53 cells. These results suggest that the X-ray-induced bystander cell killing depends on both the irradiation dose and the p53 status of the targeted cells and the bystander cells.