Double-strand break induction and repair in V79-4 hamster cells: the role of core ionisations, as probed by ultrasoft X-rays.

PURPOSE: To compare the induction of double-strand breaks (DSB) in cells irradiated by 250 and 350 eV ultrasoft X-rays and assess the residual yield of breaks 2 hours post irradiation in order to unravel the correlation between the sharp increase in cell-killing efficiency of ultrasoft X-rays above versus below the carbon-K threshold (284 eV) and the induction of core events in DNA atoms. MATERIALS AND METHODS: V79-4 hamster cells were irradiated with synchrotron ultrasoft X-rays at isoattenuating energies of 250 eV and 350 eV. DSB were quantified using pulse field gel electrophoresis. RESULTS: A significant increase in DSB induction was observed for 350 eV ultrasoft X-rays above the carbon-K threshold, compared to 250 eV below the threshold, per unit dose to the cell. The DSB induced by the 350 eV ultrasoft X-rays were less repaired 2 h after irradiation. CONCLUSION: The increased DSB induction at 350 eV is attributed to the increase in the relative proportion of photon interactions in DNA resulting in significant dose inhomogeneity across the cell with a local increase in dose to DNA. It results from an increase in carbon-K shell interactions and the short range of the electrons produced. Core ionisations in DNA, through core-hole relaxation in conjunction with localised effects of spatially correlated low-energy photo- and Auger-electrons lead to an increase in number and the complexity of DSB.

Induction and repairability of DNA damage caused by ultrasoft X-rays: role of core events.

PURPOSE: To investigate the severity of damage induced in plasmid DNA by ultrasoft X-rays at different energies, in order to unravel the correlation between the sharp increase in cell-killing efficiency of ultrasoft X-rays above versus below the carbon K-threshold and the induction of core events in DNA atoms. MATERIALS AND METHODS: Bluescript (pBS, tight packing) and pSP189 (pSP, loose packing) plasmids were exposed to ultrasoft X-rays at 250, 380 and 760 eV energies, respectively, above phosphorus L-, carbon K- and oxygen K-thresholds. Complex DNA lesions were assayed by the repair protein Formamidopyrimidine DNA glycosylase (Fpg) and by in vitro repair assay using whole cell-free extracts. RESULTS: Clustered damage, as revealed by Fpg-induced double strand breaks, was observed at low level, but at similar rate at the three energies. Damage induced at 380 eV may be slightly less efficiently repaired by cell extracts than those produced at 250 eV. 760 eV photons which yield longer range electrons than 250 and 380 eV photons, induced more total damages which were more efficiently repaired, and thus likely more dispersed. CONCLUSION: It is demonstrated that ultrasoft X-rays induce complex damage, which do not exhibit the same ability to be repaired, depending on the energy and on DNA packing.

Targeted and nontargeted effects of ionizing radiation that impact genomic instability.

Radiation-induced genomic instability, in which the progeny of irradiated cells display a high frequency of nonclonal genomic damage, occurs at a frequency inconsistent with mutation. We investigated the mechanism of this nontargeted effect in human mammary epithelial cells (HMEC) exposed to low doses of radiation. We identified a centrosome-associated expression signature in irradiated HMEC and show here that centrosome deregulation occurs in the first cell cycle after irradiation, is dose dependent, and that viable daughters of these cells are genomically unstable as evidenced by spontaneous DNA damage, tetraploidy, and aneuploidy. Clonal analysis of genomic instability showed a threshold of >10 cGy. Treatment with transforming growth factor beta1 (TGFbeta), which is implicated in regulation of genomic stability and is activated by radiation, reduced both the centrosome expression signature and centrosome aberrations in irradiated HMEC. Furthermore, TGFbeta inhibition significantly increased centrosome aberration frequency, tetraploidy, and aneuploidy in nonirradiated HMEC. Rather than preventing radiation-induced or spontaneous centrosome aberrations, TGFbeta selectively deleted unstable cells via p53-dependent apoptosis. Together, these studies show that radiation deregulates centrosome stability, which underlies genomic instability in normal human epithelial cells, and that this can be opposed by radiation-induced TGFbeta signaling.

DNA core ionization and cell inactivation.

Whether inner-shell ionizations of DNA atoms, called core ionizations, are critical events for cell inactivation by ionizing radiations such as 100 keV electrons and gamma rays has been investigated. The number of core ionizations in DNA atoms per gray of the two types of radiations is calculated from various Monte Carlo track simulations. The probability that a core ionization leads to cell inactivation is deduced from experimental values of the RBEs of ultrasoft X rays. The contribution to V79 cell inactivation solely due to the core ionizations in DNA is found to be 75 +/- 27% for energetic electrons and gamma rays. This surprisingly large contribution strongly suggests the presence of new mechanisms associated with critical lesions for cell inactivation.

Imaging features that discriminate between foci induced by high- and low-LET radiation in human fibroblasts.

In this study, we investigated the formation of radiation-induced foci in normal human fibroblasts exposed to X rays or 130 keV/mum nitrogen ions using antibodies to phosphorylated protein kinase ataxia telangiectasia mutated (ATMp) and histone H2AX (gamma-H2AX). High-content automatic image analysis was used to quantify the immunofluorescence of radiation-induced foci. The size of radiation-induced foci increased for both proteins over a 2-h period after nitrogen-ion irradiation, while the size of radiation-induced foci did not change after exposure to low-LET radiation. The number of radiation-induced ATMp foci showed a more rapid rise and greater frequency after X-ray exposure and was resolved more rapidly such that the frequency of radiation-induced foci decreased by 90% compared to 60% after exposure to high-LET radiation 2 h after 30 cGy. In contrast, the kinetics of radiation-induced gamma-H2AX focus formation was similar for high- and low-LET radiation in that it reached a plateau early and remained constant for up to 2 h. High-resolution 3D images of radiation-induced gamma-H2AX foci and dosimetry computation suggest that multiple double-strand breaks from nitrogen ions are encompassed within large nuclear domains of 4.4 Mbp. Our work shows that the size and frequency of radiation-induced foci vary as a function of radiation quality, dose, time and protein target. Thus, even though double-strand breaks and radiation-induced foci are correlated, the dynamic nature of both contradicts their accepted equivalence for low doses of different radiation qualities.

Cellular inactivation and chromosomal aberrations: initial damage.

It has been proposed that unrepaired or misrepaired complex lesions of DNA are responsible for cell inactivation and chromosomal aberrations. The detailed features of the critical damage and the nature of initiating physical events are actively investigated. We studied the role of inner-shell (core) ionizations in DNA atoms is studied. Ultrasoft X-rays from LURE synchrotron radiation have been used to mimic core events induced by ionizing radiations. For biological matter, inner-shell photoionization is indeed the main interaction channel of these radiations. Moreover, by tuning the X-ray energy below and above the carbon K-threshold, it is possible to achieve a two-fold increase in the number of core-ionizations in DNA for a same dose. Cell survival and chromosome aberrations have thus been studied at three iso-attenuated energies: 250, 350, and 810 eV. Relative biological efficiencies (RBEs) for cell inactivation and chromosome aberrations were found to be strongly correlated with the yields of core events in DNA.