Low dose, low-LET ionizing radiation-induced radioadaptation and associated early responses in unirradiated cells

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Abstract

Numerous investigators have reported that irradiation of cells with a low dose of ionizing radiation (IR) can induce a condition of enhanced radioresistance, i.e. a radioadaptive response. In this report, we investigated the hypothesis that a radioadaptive bystander effect may be induced in unirradiated cells by a transmissible factor(s) present in the supernatants of cells exposed to low dose γ-rays. Normal human lung fibroblasts (HFL-1) were irradiated with a 1 cGy dose of γ-rays and their supernatants were transferred to unirradiated HFL-1 as a bystander cell model. Compared with the directly irradiated cells, such treatment resulted in increased clonogenic survival following subsequent γ-irradiation with 2 and 4 Gy. This radioadaptive bystander effect was found to be preceded by early decreases in cellular levels of TP53 protein, increase in intracellular ROS, and increase in the redox and DNA repair protein AP-endonuclease (APE). The demonstration that radioadaptation can occur in unirradiated cells via a fluid-phase, transferable factor(s) adds to the complexity of the current understanding of mechanisms by which radioadaptive responses can be induced by low dose, low-LET IR.

Introduction

Several types of ionizing radiation (IR)-induced responses in unirradiated cells, or what are now called “bystander effects”, have been reported. Such effects, which can sometimes differ from those that occur in directly irradiated cells include [1]: the killing of unirradiated cells [2], increases in sister chromatid exchanges and chromosomal aberrations [3], [4], the induction of gene mutations and chromosomal instability [5], [6], [7], increases in intracellular reactive oxygen species (ROS) [8], and enhanced cell growth [1]. Although the mechanisms by which the bystander effects are induced remain poorly understood, recent evidence indicates that they can be mediated by soluble extracellular factors, including ROS, nitrogen oxide species and cytokines [1], [2], [3], [9], [10], direct cell–cell communications via gap junction communications [11], [12] and/or, perhaps, by exfoliated plasma membrane constituents [13].

Recently, Matsumoto et al. [10] reported reduced radiosensitivity in cells cultured in conditioned medium harvested from other cells that had been exposed to ≥2.5 Gy doses of X-rays. Such a finding suggests the possibility that at least relatively high dose, low-LET IR exposure may induce enhanced radioresistance in unirradiated cells via one or more soluble factors. Although this “bystander-like” response was observed under special conditions that likely lack generalization to other cell models, the investigation’s focus on enhanced radioresistance in unirradiated cells deviated from most prior investigations of radioadaptive responses in vitro, in which adaptive responses have been studied in cell populations that received direct exposure to a low “priming” dose of IR prior to being exposed to much higher dose IR (e.g. [14], [15], [16]). In the present study, we set out to extend upon the observation by Matsumoto et al. [10] by investigating the hypothesis that a radioadaptive response may be induced in unirradiated cells by a transmissible factor(s) present in the medium of cells exposed to a low dose of γ-rays like that used in more typical radioadaptive response experiments.

As described herein, we have indeed found evidence consistent with the induction of an adaptive response in bystander cells that is effectively akin to more conventional radioadaptive responses in which pre-exposure of cells to a low dose of IR results in increased radioresistance. While details about the mechanisms by which this effect is mediated remains to be determined, we have found that the adaptive bystander response is associated with decreases in basal levels of TP53 protein, increases in intracellular ROS, and increases in the redox and base excision repair protein AP-endonuclease (APE).

Section snippets

Cells and exposures to γ-rays

Normal human diploid lung fibroblasts (HFL-1) initially obtained from a human fetus (CCL 153, American Type Culture Collection, Rockville, MD) were routinely cultured in 175 cm2 tissue culture flasks in α-minimum essential medium (α-MEM, GIBCO-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone Laboratories Inc., Logan, UT). All cell cultures were incubated at 37 °C in humidified 5% CO2/95% air. Cells were harvested from the flasks by trypsinization and seeded in 60 

Radiosensitivity of directly irradiated and bystander cells

Surviving fractions from HFL-1 that were directly irradiated with 2 or 4 Gy doses of γ-rays in the absence of a prior lower dose exposure or any medium transfers were ∼0.5 and 0.2, respectively (Fig. 1). These values are virtually identical to those observed in an earlier study in which the survival of HFL-1 were assessed with these same doses [17]. Pretreatment of unirradiated cells with medium from γ-irradiated (1 cGy) cells before they were exposed to the higher doses of IR, on the other hand,

Discussion

The induction of enhanced radioresistance by “priming” low doses of IR (e.g. [14], [15], [16]) is a well-recognized yet poorly understood phenomenon [25]. Most prior investigations of such responses in vitro have focused on the radioadaptation of cell populations that received direct exposure to low dose IR prior to being exposed to higher dose IR. The adaptive-type response we have observed technically and conceptually deviates from this traditional approach for assessing for radioadaptive

Acknowledgments

This study was supported by the US Department of Energy and by the NIH/NCI (CA82598).

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