Studies on the Biological Effects of Radiation-Induced Bystander Responses and Rescue Effects


Student thesis: Doctoral Thesis

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Awarding Institution
Award date23 May 2017


Radiation-induced non-targeted/bystander response refers to a phenomenon that tissue/cells that are not directly irradiated with ionizing radiation but are in close proximity to those that are, or have received signals from such tissue/cells, can participate in the cellular damage processes caused by radiation. The presence of non-targeted effects, so-called radiation-induced bystander effects (RIBE), has revolutionized our conceptual thinking on the relevant target of ionizing radiation-induced DNA damage. That is, both extranuclear and extracellular targets can contribute to the signaling event that may lead to DNA damage in the targeted and non-targeted cells. RIBE has been shown by several biological endpoints in mammalian cell cultures previously, and recently in 3 dimensional (3D) human tissues as well as animal models. However, the underlying mechanism and the effect of bystander response on human health are not well-established. In recent years, a new phenomenon which is closely associated with RIBE has been reported, that of "rescue effect" or the "reciprocal bystander effect". By co-culturing with the non-irradiated bystander cells, the biological effects in directly irradiated cells found to be rescued from receiving feedback signals from non-irradiated bystander cells.

The overall aims of the current study were to (1) verify the role of Cycloxygenase-2 (COX-2) in radiation-induced bystander mutagenic response under both in vivo and in vitro conditions and (2) investigate the existence of rescue effects in cells using different approaches. COX-2 knockout (KO) mice and mouse embryonic fibroblasts (MEFs) with the gpt delta gene in the C57BL/6J background were generated to examine the contribution of COX-2 in radiation-induced non-targeted effects. In the study of rescue effects, Chinese hamster ovary (CHO) K1 cells and its mutant cell line xrs5 cells, and the mouse embryonic fibroblasts NIH/3T3 cells were used.

Chapter 1 gives the introduction and literature review.

Chapter 2 reports the in vivo bystander experiment where 2 Gy X-rays were delivered to a 1 cm2 area of the lower abdomen of the gpt COX-2 WT and KO mice. The COX-2 level in non-irradiated bystander lung tissues among irradiated WT mice showed a 1.9 fold increase when compared with non-irradiated control. Similarly, the induction of γ-H2AX foci and spi mutant yield in non-irradiated bystander lung tissues among WT mice also showed ~2 and ~3 fold increases, respectively, when compared with non-irradiated control tissues. However, in similarly irradiated COX-2 KO mice, there was no bystander response in out of field lung tissues when compared with non-irradiated control KO mice at 24 h post-irradiation.

Chapter 3 reports the study of in vitro bystander experiment in gpt COX-2 WT and KO MEFs using α-particles as an irradiation source. Our results showed that the COX-2 level, the MN induction and the spi MF in non-irradiated bystander WT MEFs after 2 Gy of α-particles irradiation using the Track Segment Charged-Particle Irradiator significantly increased by 2.5 fold, 2.5 fold and 1.6 fold, respectively, compared with non-irradiated control cells. Similar to the in vivo studies described above, COX-2 KO MEFs showed little or no bystander response when compared with the corresponding untreated KO controls. Using a microbeam irradiator to selectively irradiate a random proportion of cells with a lethal dose of 30 α-particles, we found that both the expression level of 8-OHdG and number of γ-H2AX foci/cell in non- irradiated bystander WT MEFs peaked at 60 min post-irradiation. When compared with sham-irradiated controls at the same time frame, the fold change of 8-OHdG and γ-H2AX in bystander WT MEFs intermixed with irradiated cells were 2.6 and 1.6, respectively. In contrast, similarly treated COX-2 KO MEFs showed little or no increase.

Chapter 4 illustrates the dependence of rescue effect on cell lines and radiation sources. In this study, CHO cells and xrs5 cells were both exposed to (1) 5 and 20 cGy of alpha-particles and (2) 20 cGy and 2 Gy of x-rays and were followed for up to two weeks. Two endpoints were assessed, (i) micronucleus (MN) formation, and (ii) cell number of the irradiated cells. Our results indicated that both CHO cells and xrs5 cells induced rescue effect post α-particles irradiation; however, only CHO cells showed similar behavior with X-rays irradiation. When compared to uniformly irradiated cells, less damaging effects were induced in the irradiated cells co-cultured with non-irradiated bystander cells. In addition, the differences in effects between the two irradiated cells decreased over time.

Chapter 5 describes the involvement of NF-κB pathway in the rescue effect using NIH/3T3 cell line. Our results showed that a stronger rescue effect was induced in cell population containing a larger ratio of the bystander (97.5%) to irradiated cells (2.5%) after 5 cGy α-particles irradiation, using 53BP1 assay (~2.2 folds) and NF-κB expression (~1.1 folds) as endpoints. The effect of rescue signals (secreted by the bystander cells) on the irradiated cells was studied. The induction of nuclear 53BP1 foci showed a decrease in conditioned-medium (CM) treated irradiated cells, when compared to the non-treated irradiated cells (~1.24 folds). However, the rescue effect was not seen when NF-κB activation inhibitor BAY-11-7082 was added to the CM.

Chapter 6 are conclusions and future directions.