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 lowdose irradiation and that, under these conditions, the whole cell should be considered a sensor of radiation exposure.

ATR-dependent radiation-induced γH2AX foci in bystander primary human astrocytes and glioma cells

“…NO-mediated signalling is probably not involved in early stages of bystander signalling causing DNA damage in NHA and T98G cells as inhibition of NO synthesis by aminoguanidine could not prevent bystander gH2AX foci formation (results not shown). Our previous studies have shown that in bystander T98G cells, micronuclei are produced (Shao et al, 2004), which are presumably a consequence of the induction of damage highlighted by the gH2AX signals reported here. These are NO dependent, suggesting that ROS interact with RNS at later times leading to longer term chromosomal damage.…”

“…Bystander effects induced by ionizing irradiation have been studied by various approaches including transfer of conditioned medium from irradiated cells (Lyng et al, 2002;Maguire et al, 2005), low particle fluence irradiation, where only a few percent of cells are irradiated (Azzam et al, 1998), and targeted irradiation of single cells and subcellular structures (Shao et al, 2004;Shao et al, 2003bShao et al, , 2005. They have been shown to occur in both tumour and normal cell types.…”

“…Most interestingly, several cytokines can be induced by ROS and NO/NOS (Ayache et al, 2002;Hsu and Wen, 2002;Kosmidou et al, 2002;Hwang et al, 2004;Ryan et al, 2004) providing a possible link between different factors potentially involved in bystander signalling. End points used for the study of bystander effects in vitro have included micronuclei formation (Azzam et al, 2002;Kashino et al, 2004;Shao et al, 2004), gene mutations and genomic instability (Zhou et al, 2000), gene expression changes (Azzam et al, 2002;Yang et al, 2005), transformation (Sawant et al, 2001), proliferation (Gerashchenko and Howell, 2003), cell survival, apoptosis (Belyakov et al, 2002;Lyng et al, 2002), cell cycle arrest (Azzam et al, 2000) and most recently the induction of gH2AX foci in bystander cells (Hu et al, 2005;Sokolov et al, 2005;Yang et al, 2005).…”

In vivo formation and repair of DNA double-strand breaks after computed tomography examinations

“…The assay's sensitivity and its demonstrated applicability to CT examinations allow the investigation of normal people without clinical symptoms who are exposed to well defined radiation doses. Thus, the assay has the potential to shed light on the in vivo significance of some radiobiological phenomena that have hitherto been restricted to in vitro measurements, such as the bystander effect (25) or low-dose hypersensitivity (26,27). Moreover, the assessment of ␥-H2AX foci can serve as a biologically relevant biomarker for exposure to low levels of IR.…”

Constitutive nitric oxide acting as a possible intercellular signaling molecule in the initiation of radiation-induced DNA double strand breaks in non-irradiated bystander cells

“…The release of NO from irradiated cells, measured to be in the micromolar level to the medium, induced an increase in radioresistance among the non-irradiated cells which were co-cultured with the irradiated ones (Matsumoto et al, 2001). Shao et al (2003aShao et al ( , 2004 presented evidence for a significant increase in the incidence of micronuclei in the non-irradiated bystander cells that were in the vicinity of ones irradiated through either the nuclei or the cytoplasm with a microbeam facility. Furthermore, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) (a scavenger of NO ) abolished excess micronuclei formation.…”