Communication between irradiated and un-irradiated (bystander) cells can cause damage in cells that are not directly targeted by ionizing radiation (IR); a process known as the bystander effect (BE). BE can also lead to genomic instability (GI) within the progeny of bystander cells, similar to the progeny of directly irradiated cells. The molecular factors that mediate this cellular communication can be transferred between cells via gap junctions or be released into the extracellular media/microenvironment of cells and tissue following irradiation. Although GI is thought to be a critical step in the onset and progression of cancer, BE response contributions in such processes are still not clear. Therefore, this study was designed to investigate the risks or benefits associated with the induction of non-targeted effects especially BE following exposure to low LET X-ray radiation using two different cell types. Additionally, the project aims to achieve an increased understanding of the mechanisms of non-targeted effects of ionizing radiation by examining the molecular signalling via exosomes within the irradiated, bystander and progeny of irradiated and bystander cell population. Different cell combinations were established between tumour (MCF7) and nontumour (HMT-3522S1) human breast epithelial cells using a 6-well plate co-culture system. The cells were irradiated with two doses of X-ray; 0.1 Gy (a iagnostic procedure relevant dose) and 2 Gy (therapeutic dose) and a sham-irradiation dose of 0 Gy (for control groups of experiment). The co-culturing time was 4 hours for all cell combination, whereupon a media transfer approach was used to induce BE within the cells in the exosome part of this study. The early and late cellular damage responses were evaluated by the following biological endpoints: cytogenetic/chromosomal analysis, apoptotic analysis, telomere length and telomerase activity measurements. In addition to these biological endpoints, the comet assay was utilised to estimate the initial and delayed DNA damage within the cells that had been treated with exosomes, previously extracted from the irradiated, bystander and control cell media. The results showed that 2 Gy direct irradated MCF7 and HMT cells were both able to induce early and late chromosomal damage in the bystander MCF7 and HMT cells. Furthermore, these bystander cells exhibited early and delayed telomeric instability, which could prompt further GI at later time-points. In comparison, 0.1 Gy direct ii irradiated MCF7 cells were only able to induce initial and delayed chromosomal damage within the bystander MCF7, which also demonstrated a high level of telomeric instability at early and late time-points. While, bystander HMT cells did not show chromosomal damage after 1, 12 and 24 generations/population doublings following co-culture with 0.1 Gy direct irradiated MCF7 or HMT cells. 0.1 Gy bystander HMT cells did reveal a high level of apoptosis at early and late time points, which might be due the removal of cells with a high level of chromosomal damage. Interestingly, the 0.1 Gy bystander HMT cells exhibited significant levels of telomeric instability at early and late time points, which could contribute to chromosomal instability at later time-points. The investigation in to the mechanisms of molecular signalling via exosomes showed that the exosomes of irradiated cell conditioned media (ICCM) from MCF7 cells had the ability to induce BE within MCF7 and HMT cells similar to the effects of ICCM following 2 Gy X-ray. The exosomes that were isolated from the MCF7 bystander cell media had a similar effect as the ICCM on the MCF7 and HMT bystander cells. These exosome-bystander cells also showed GI within their progeny after 24 generations and retained the ability to induce cellular damage to fresh un-irradiated MCF7 cells, demonstrating an underlying mechanism for propagating the delayed damage responses. The inhibition of the exosome’s cargo molecules by RNase treatment and protein denaturating (boiling of exosmes) significantly abrogated BE and GI in both MCF7 and HMT bystander cells following 2 Gy X-ray. Thus data demonstrated crucial roles for exosome RNA and protein molecules in the non-targeted effects of IR induction. In summary, our investigations demonstrate that BE has detrimental consequences within the tumour and non-tumour breast epithelial cells (MCF7 and HMT3522S1) following low and high doses of X-ray irradiation, and these detrimental consequences are frequently mediated by exosomes that contain RNA and protein molecules. Inhibition of these molecules can abrogate BE and GI following a radiotherapy dose, which can potentially have an application in clinical radiotherapy.
Al-Mayah, Ammar
Supervisors: Kadhim, Munira
Faculty of Health and Life SciencesDepartment of Biological and Medical Sciences
Year: 2012
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