Relating Linear Energy Transfer to the Formation and Resolution of DNA Repair Foci After Irradiation with Equal Doses of X-ray Photons, Plateau, or Bragg-Peak Protons

Abstract

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.

Proton Irradiation Increases the Necessity for Homologous Recombination Repair Along with the Indispensability of Non-Homologous End Joining

“…These observations are consistent with the described action of particles, including protons, that induce a direct DNA damage within their defined track and therefore tend to induce accumulated DNA damage in closer proximity [13,14,64,65]. It has been previously suggested that the high energy of particles, e.g., protons, strongly correlates with clustered DNA damage in the form of coalesced DNA DSB due to denser ionizing events [6,13,14,64,66]. Moreover, a prediction analysis done by a new computational track structure model which simulates complexity of DNA damage after proton irradiation, endorses our assumption of a more complex DNA damage induced by proton irradiation [67].…”

“…Indeed, we observed that irradiation with SOBP protons induced γH2A.X DNA foci-clusters, whereas DNA damage foci induced by X-ray photons were smaller and more randomly distributed over the whole nucleus. These observations are consistent with the described action of particles, including protons, that induce a direct DNA damage within their defined track and therefore tend to induce accumulated DNA damage in closer proximity [13,14,64,65]. It has been previously suggested that the high energy of particles, e.g., protons, strongly correlates with clustered DNA damage in the form of coalesced DNA DSB due to denser ionizing events [6,13,14,64,66].…”

“…This maximized dose deposition at the end of the particle's range is represented as a Bragg peak of the depth-dose curve. In contrast, photons lose their energy exponentially, with higher values at the entry point and lower values at the deep-seated tissues [12][13][14]. Additionally, the rising number of particle therapy centers worldwide facilitates the use of protons or heavy ions in the clinical care of cancer patients and allows the validation of the suspected superiority of particle beams compared to photon beams in terms of normal tissue protection [15,16].…”

“…However, it is increasingly appreciated that not only the physical characteristics of the beam but also the microscopic pattern of energy deposition differs between photons and protons, particularly at the distal edge of the Bragg-peak, with a potential impact on the resulting biological effects [14,20]. In fact, depending on the tissue, the measured endpoint, dose and LET of the beam, the RBE values reported for protons vary between~1.1-1.7, with increasing RBE values for protons along the distal edge of the Bragg peak [18,21,[25][26][27].…”

“…Though an increasing number of reports point to different patterns of DNA damage induced by photon and proton irradiation with potential relevance for DNA repair [14,[28][29][30]46] there is a gap of knowledge about potential microscopic differences in the DNA damage pattern induced by protons from the entrance plateau (EP) versus protons from SOBP. Moreover, only limited data are available on variations in RBE caused by genetic deficiencies in components of the two major DNA DSB repair pathways, NHEJ and HRR, which are also observed in human cancer.…”