Targeted cytoplasmic irradiation induces bystander responses

Abstract

The observation of radiation-induced bystander responses, in which cells respond to their neighbors being irradiated, has important implications for understanding mechanisms of radiation action particularly after low-dose exposure. Much of this questions the current dogma of direct DNA damage driving response in irradiated systems. In this study, we have used a charged-particle microbeam to target individual helium ions ((3)He(2+)) to individual cells within a population of radioresistant glioma cells cultured alone or in coculture with primary human fibroblasts. We found that even when a single cell within the glioma population was precisely traversed through its cytoplasm with one (3)He(2+) ion, bystander responses were induced in the neighboring nonirradiated glioma or fibroblasts so that the yield of micronuclei was increased by 36% for the glioma population and 78% for the bystander fibroblast population. Importantly, the yield of bystander-induced micronuclei was independent of whether the cytoplasm or nucleus of a cell was targeted. The bystander responses were fully eliminated when the populations were treated with 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide or filipin, which scavenge nitric oxide (NO) and disrupt membrane rafts, respectively. By using the probe 4-amino-5-methylamino-2',7'-difluorofluorescein, it was found that the NO level in the glioma population was increased by 15% after 1 or 10 cytoplasmic traversals, and this NO production was inhibited by filipin. This finding shows that direct DNA damage is not required for switching on of important cell-signaling mechanisms after low-dose irradiation and that, under these conditions, the whole cell should be considered a sensor of radiation exposure.

Fig. 1.

Cytoplasmic traversal-induced MN production in T98G population (A) and bystander AG0 population (B) with or without c-PTIO treatment. One or 10 cells in the T98G population were individually irradiated through the cytoplasm with one helium ion (*, P < 0.01 compared with nonirradiated control). Y_MN, yield of MN.

Fig. 3.

(A) Relative yield of MN (Y_MN) produced in T98G cells where either 10 or 100% of cells were irradiated with one helium ion through the nucleus or cytoplasm only. The MN yield of the nonirradiated T98G control was normalized as 1 (*, P < 0.01 compared with nonirradiated T98G control; **, P < 0.01 between 10-cell irradiation and 100% of cell irradiation). (B) Cytoplasmic traversal and nuclear traversal-induced MN produced in either the T98G or bystander AG0 populations. Ten cells in the T98G population were individually irradiated through cytoplasm or nucleus with one helium ion, respectively, (* and **, P < 0.01 compared with nonirradiated AG0 and T98G control, respectively).

Fig. 2.

Cytoplasmic traversal-induced MN production in the T98G population (A) and bystander AG0 population (B) with or without filipin treatment. One or 10 cells in the T98G population were individually irradiated through the cytoplasm with five helium ions (*, P < 0.01 compared with nonirradiated control).

Fig. 4.

Percentage of NO-positive cells (A) and relative NO-induced fluorescence intensity (B) in the cytoplasmic-traversed T98G population with or without treatment with c-PTIO or filipin. The fluorescence intensity of the nonirradiated T98G was considered as 1. A few cells within the T98G population were individually traversed through cytoplasm with one helium ion (*, P < 0.01 compared with nonirradiated control).