TERT promoter mutations and polymorphisms as prognostic factors in primary glioblastoma TERT promoter mutations and polymorphisms as prognostic factors in primary glioblastoma

Technical improvements in clinical radiotherapy for maximizing cytotoxicity to the tumor while limiting negative impact on co-irradiated healthy tissues include the increasing use of particle therapy (e.g., proton therapy) worldwide. Yet potential differences in the biology of DNA damage induction and repair between irradiation with X-ray photons and protons remain elusive. We compared the differences in DNA double strand break (DSB) repair and survival of cells compromised in non-homologous end joining (NHEJ), homologous recombination repair (HRR) or both, after irradiation with an equal dose of X-ray photons, entrance plateau (EP) protons, and mid spread-out Bragg peak (SOBP) protons. We used super-resolution microscopy to investigate potential differences in spatial distribution of DNA damage foci upon irradiation. While DNA damage foci were equally distributed throughout the nucleus after X-ray photon irradiation, we observed more clustered DNA damage foci upon proton irradiation. Furthermore, deficiency in essential NHEJ proteins delayed DNA repair kinetics and sensitized cells to both, X-ray photon and proton irradiation, whereas deficiency in HRR proteins sensitized cells only to proton irradiation. We assume that NHEJ is indispensable for processing DNA DSB independent of the irradiation source, whereas the importance of HRR rises with increasing energy of applied irradiation.

Proton beam therapy is increasingly applied for the treatment of human cancer, as it promises to reduce normal tissue damage. However, little is known about the relationship between linear energy transfer (LET), the type of DNA damage, and cellular repair mechanisms, particularly for cells irradiated with protons. We irradiated cultured cells delivering equal doses of X-ray photons, Bragg-peak protons, or plateau protons and used this set-up to quantitate initial DNA damage (mainly DNA double strand breaks (DSBs)), and to analyze kinetics of repair by detecting γH2A.X or 53BP1 using immunofluorescence. The results obtained validate the reliability of our set-up in delivering equal radiation doses under all conditions employed. Although the initial numbers of γH2A.X and 53BP1 foci scored were similar under the different irradiation conditions, it was notable that the maximum foci level was reached at 60 min after irradiation with Bragg-peak protons, as compared to 30 min for plateau protons and photons. Interestingly, Bragg-peak protons induced larger and irregularly shaped γH2A.X and 53BP1 foci. Additionally, the resolution of these foci was delayed. These results suggest that Bragg-peak protons induce DNA damage of increased complexity which is difficult to process by the cellular repair apparatus.

The quantitative analysis of foci plays an important role in various cell biological methods. In the fields of radiation biology and experimental oncology, the effect of ionizing radiation, chemotherapy or molecularly targeted drugs on DNA damage induction and repair is frequently performed by the analysis of protein clusters or phosphorylated proteins recruited to so called repair foci at DNA damage sites, involving for example γ-H2A.X, 53BP1 or RAD51. We recently developed “The Focinator” as a reliable and fast tool for automated quantitative and qualitative analysis of nuclei and DNA damage foci. The refined software is now even more user-friendly due to a graphical interface and further features. Thus, we included an R-script-based mode for automated image opening, file naming, progress monitoring and an error report. Consequently, the evaluation no longer required the attendance of the operator after initial parameter definition. Moreover, the Focinator v2-0 is now able to perform multi-channel analy...

Cancer bioenergetics fuel processes necessary to maintain viability and growth under stress conditions. We hypothesized that cancer metabolism supports the repair of radiation-induced DNA double-stranded breaks (DSBs). We combined the systematic collection of metabolic and radiobiological data from a panel of irradiated cancer cell lines with mathematical modeling and identified a common metabolic response with impact on the DSB repair kinetics, including a mitochondrial shutdown followed by compensatory glycolysis and resumption of mitochondrial function. Combining ionizing radiation (IR) with inhibitors of the compensatory glycolysis or mitochondrial respiratory chain slowed mitochondrial recovery and DNA repair kinetics, offering an opportunity for therapeutic intervention. Mathematical modeling allowed us to generate new hypotheses on general and individual mechanisms of the radiation response with relevance to DNA repair and on metabolic vulnerabilities induced by cancer radiotherapy. These discoveries will guide future mechanistic studies for the discovery of metabolic targets for overcoming intrinsic or therapy-induced radioresistance.