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Direct visualization of different types of DNA lesions in monolayer and three dimensional cell culture models
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Dr. David Jen Chi Chen
Professor, Division of Molecular Radiation Biology, Department of Radiation Oncology,
University of Texas Southwestern Medical Center, Dallas, Texas, USA |
| Ionizing radiation (IR) induces a variety of DNA lesions among which simple and/or complex DNA double-strand breaks are the biologically most significant. Recruitment and retention of DNA repair and response proteins at DSBs can be conveniently visualized by fluorescence imaging of repair foci, however, some of the challenging questions cannot be answered. Therefore, use of live cell imaging technology to directly monitor single, double-strand breaks and base damages in single living cell has many advantages. Using 53BP1 fused to yellow fluorescent protein (YFP-53BP1) as a DNA DSB surrogate marker, we noticed that the number of DSBs formed, as measured by the number of YFP-53BP1 formed, was linear with dose from 5 mGy to 1 Gy in monolayer culture (2D). The DSBs induced by very low radiation doses (5 mGy) were repaired with efficiency similar to repair of DSBs induced at higher doses (>100 mGy) of low-Let IR. On the other hand, currently, it is unclear why clustered DNA damages are difficult to repair and what is the fundamental mechanism by which these lesions trigger carcinogenic events? To answer these questions, we used 53BP1 (double-strand), XRCC1 (single-strand) and OGG1 (base damage) foci analysis to monitor complex DNA lesions repair in situ. We show, consistent with biophysical analysis, that the kinetics of complex DNA lesions loss in human fibroblasts is substantially compromised. Further, we provide evidence that the difficulties associated with the repair of these lesions is due to the spatial distribution of DSBs, SSBs and base damages within the clustered DNA lesions and is not because of the physical location of these damages in the sub-nuclear domains. Further, the yield and the type of aberrations are dictated by the nature of the unrepaired clustered DNA damage in response to HZE particles radiation. Although 2D cell culture systems provide useful and powerful information, these systems do not recapitulate the three-dimensional (3D) structural organization or functional differentiation of the cells in vivo. Therefore, we have developed a novel human lung 3D model and have assessed cellular responses to DNA damage induced by IR. Though the number of DSBs induced after low- and high-LET IR was comparable between 2D and 3D structures, DSBs were repaired with slower kinetics in 3D structures as compared to 2D. Thus, cells in 2D and 3D culture responded to radiation differently. This data will have important implications for evaluating risks due to radiation exposure. Use of our newly developed system to directly follow the induction and repair of different types of DNA damages in situ opens up new experimental approaches to study low and high-LET IR induced DNA damages in 2D, 3D and animal models. |
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