Supplementary Figures 2-8 from Quantitative, Noninvasive Imaging of Radiation-Induced DNA Double-Strand Breaks In Vivo
ARTICLE ABSTRACT
DNA double-strand breaks (DSB) are a major form of DNA damage and a key mechanism through which radiotherapy and some chemotherapeutic agents kill cancer cells. Despite its importance, measuring DNA DSBs is still a tedious task that is normally carried out by gel electrophoresis or immunofluorescence staining. Here, we report a novel approach to image and quantify DSBs in live mammalian cells through bifragment luciferase reconstitution. N- and C-terminal fragments of firefly luciferase genes were fused with H2AX and MDC1 genes, respectively. Our strategy was based on the established fact that at the sites of DSBs, H2AX protein is phosphoryated and physically associates with the MDC1 protein, thus bringing together N- and C-luciferase fragments and reconstituting luciferase activity. Our strategy allowed serial, noninvasive quantification of DSBs in cells irradiated with X-rays and 56Fe ions. Furthermore, it allowed for the evaluation of DSBs noninvasively in vivo in irradiated tumors over 2 weeks. Surprisingly, we detected a second wave of DSB induction in irradiated tumor cells days after radiation exposure in addition to the initial rapid induction of DSBs. We conclude that our new split-luciferase–based method for imaging γ-H2AX–MDC1 interaction is a powerful new tool to study DSB repair kinetics in vivo with considerable advantage for experiments requiring observations over an extended period of time. Cancer Res; 71(12); 4130–7. ©2011 AACR.
