Bystander-mediated genomic instability after high LET radiation in murine primary haemopoietic stem cells

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Abstract

Communication between irradiated and unirradiated (bystander) cells can result in responses in unirradiated cells that are similar to responses in their irradiated counterparts. The purpose of the current experiment was to test the hypothesis that bystander responses will be similarly induced in primary murine stem cells under different cell culture conditions. The experimental systems used here, co-culture and media transfer, are similar in that they both restrict communication between irradiated and bystander cells to media borne factors, but are distinct in that with the media transfer technique, cells can only communicate after irradiation, and with co-culture, cells can communication before, during and after irradiation. In this set of parallel experiments, cell type, biological endpoint, and radiation quality and dose, were kept constant. In both experimental systems, clonogenic survival was significantly decreased in all groups, whether irradiated or bystander, suggesting a substantial contribution of bystander effects (BE) to cell killing. Genomic instability (GI) was induced under all radiation and bystander conditions in both experiments, including a situation where unirradiated cells were incubated with media that had been conditioned for 24 h with irradiated cells. The appearance of delayed aberrations (genomic instability) 10–13 population doublings after irradiation was similar to the level of initial chromosomal damage, suggesting that the bystander factor is able to induce chromosomal alterations soon after irradiation. Whether these early alterations are related to those observed at later timepoints remains unknown. These results suggest that genomic instability may be significantly induced in a bystander cell population whether or not cells communicate during irradiation.

Introduction

The deterministic ‘hit-effect’ relationship between radiation and cellular response has been reconsidered in recent years due to evidence of radiation-like effects in bystander cells that have not themselves been irradiated, but have been in direct physical contact with or shared media with irradiated cells [1]. Bystander effects (BE) may be induced by very low doses of radiation, and the effect appears to plateau at higher doses [2], [3], [4]. BE require that hit and non-hit cells communicate, which can occur via gap junction intercellular communication [5], [6] and/or communication via the release of various factors into cell culture medium [7], [8], [9]. However, whether BE are induced via GJIC or media-borne factors largely depends on the experimental design of the study, e.g. the type of cells selected for study or whether cells are allowed to physically touch. BE have been observed with many cell types, and have been reported very soon after irradiation as increased micronucleus formation [10], [11] and chromatid aberrations [12] and at delayed times after irradiation as increased mutation frequency [13], [14], [15], delayed reproductive cell death [4], transformation [16], and delayed chromosomal instability (defined herein as genomic instability, GI) [17], [18]. The observation of BE using such a wide range of biological endpoints suggests that a variety of mechanisms may govern these responses at the cellular level. Alternatively, most labs exploit their own expertise with a particular cell type, irradiation and bystander condition and biological endpoint, which may frustrate the identification of a single ‘bystander factor’, should it exist. An example is the current debate as to whether the genetics of the cell sending [19] or receiving [20] the bystander signal is responsible for the bystander response.

Many innovative systems have been developed to study bystander effects, such as grid shielding techniques [18], microbeam techniques [10], [17], [21], media transfer techniques (e.g. ref. [19]) and co-culture techniques [11], [12], [22], [23]. Some of the first experiments demonstrating radiation-induced BE after exposure to broad beam α-particle irradiation relied on mathematical extrapolation to determine the contribution of BE to the observed biological endpoint [24]. Lorimore et al. [18] interposed a grid between the α-source and the cells, thereby irradiating a fraction of the cells, while others remained unirradiated. Even when half of the cells were shielded the level of GI was equivalent to the situation where all cells were irradiated, and both were significantly elevated compared to controls. Hill et al. [22], using the same shielding approach as Lorimore et al. [18], reported a similar pattern in the bystander cells when they assessed induction of DNA-bound PCNA, suggesting that initiation of early (visualized with PCNA staining; [22]) and delayed damage (visualized as GI; [18]) in bystander cells may involve overlapping pathways. Under shielding conditions, irradiated and bystander cells may communicate before, during and after irradiation. In Hill et al. [22], cells were cultured for 6 h post-irradiation; however, in Lorimore et al. [18], irradiated and bystander populations were cultured and expanded together 7 days (10–13 population doublings) for analysis of GI, so the precise contribution of the bystander population to GI could not be examined independently.

In order to examine the irradiated and bystander populations separately, we selected for this study a media transfer approach and co-culture system that has recently been developed at MRC Harwell [22]. This co-culture system is similar in principle to a system described at Columbia University [11], [12], [23]; however, these studies (and ref. [22]) have primarily have focused on relatively early biological events in bystander cells. In both experimental designs here, each population, irradiated and bystander, can be expanded and analyzed separately. The media transfer technique restricts communication between irradiated and bystander cells to media-borne factors at some time after irradiation (e.g. [19]); however, included in the irradiation media are factors that are produced immediately after irradiation, until the irradiated media is removed. The co-culture system allows communication via media-borne factors before, during and after irradiation, until the populations are separated for expansion.

Using these two related, but distinct bystander systems, we examined clonogenic survival and chromosomal aberration at 2 and 7 days post-irradiation in primary murine haemopoietic stem cells were irradiated with 1 Gy 238Pu α-particles. Examination of early (Day 2) as well as delayed (Day 7) chromosomal damage allowed us to compare early bystander-mediated chromosomal damage and that chromosomal damage associated with bystander-mediated genomic instability.

Section snippets

Cell culture

For both experiments, whole bone marrow was extracted from 9-week-old male CBA mice (n = 16 femurs) and used to prepare cell suspension in MEM alpha medium supplemented by 10% horse serum, l-glutamine and antibiotics [18]. The culture vessels used for irradiation have been described [18], [22]. Briefly, Hostaphan dishes are 30 mm internal diameter glass walled dishes with bases of 2.5 μm Hostaphan film. A small amount of cell suspension (10 μl) was plated at 2 × 106 per hostaphan base and formed a

Survival

Survival was measured on Days 7 and 11 post-irradiation by the CFU-A colony forming assay as described (ref. [18] and references therein).

Discussion

In the current set of experiments, we used two distinct systems, co-culture and media transfer, to assess radiation-induced bystander responses in murine primary haemopoietic stem cells. In both designs, bystander and irradiated cells are restricted to communication via media-borne factors and irradiated and bystander cells can be expanded separately. Both systems differ in that the co-culture system allows communication between irradiated and bystander cells before, during and after

Acknowledgements

The authors thank Mr. Steward Townsend for FACS and confocal analysis, Mr. David Stevens and Richard Doull for irradiations, Mr. David Papworth for statistical analysis, and Dr. Gwyneth Watson, Ms. Kim Chapman, Ms. Catherine Gibbons and Mr. James Kelley for valuable discussions. This work was supported by the Medical Research Council and a UK Department of Health Grant to MAK.

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    1

    These authors contributed equally to this paper.

    2

    Present address: Unilever Centre for Molecular Science Informatics, Department of Chemistry; Lensfield Road, Cambridge, CB2 1EW, UK.

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