Specific molecular and cellular events induced by irradiated X-ray photoactivatable drugs raise the problem of co-toxicities: particular consequences for anti-cancer synchrotron therapy.

Synchrotrons are capable of producing intense low-energy X-rays that enable the photoactivation of high-Z elements. Photoactivation therapy (PAT) consists of loading tumors with photoactivatable drugs and thereafter irradiating them at an energy, generally close to the K-edge of the element, that enhances the photoelectric effect. To date, three major photoactivatable elements are used in PAT: platinum (cisplatin and carboplatin), iodine (iodinated contrast agents and iododeoxyuridine) and gadolinium (motexafin gadolinium). However, the molecular and cellular events specific to PAT and the radiobiological properties of these photoactivatable drugs are still misknown. Here, it is examined how standard and synchrotron X-rays combined with photoactivatable drugs impact on the cellular response of human endothelial cells. These findings suggest that the radiolysis products of the photoactivatable drugs may participate in the synergetic effects of PAT by increasing the severity of radiation-induced DNA double-strand breaks. Interestingly, subpopulation of highly damaged cells was found to be a cellular pattern specific to PAT. The data show that the efficiency of emerging anti-cancer modalities involving synchrotron photoactivation strongly depends on the choice of photoactivatable drugs, and important series of experiments are required to secure their clinical transfer before applying to humans.

Short-term effects of synchrotron irradiation on vasculature and tissue in healthy mouse brain.

The purpose of this study is to measure the effects of a tomographic synchrotron irradiation on healthy mouse brain. The cerebral cortexes of healthy nude mice were irradiated with a monochromatic synchrotron beam of 79 keV at a dose of 15 Gy in accordance with a protocol of photoactivation of cisplatin previously tested in our laboratory. Forty-eight hours, one week and one month after irradiation, the blood brain barrier (BBB) permeability was measured in the irradiated area with intravital multiphoton microscopy using fluorescent dyes with molecular weights of 4 and 70 kDa. Vascular parameters and gliosis were also assessed using quantitative immunohistochemistry. No extravasation of the fluorescent dyes was observed in the irradiated area at any measurement time (48 h, 1 week, 1 month). It appears that the BBB remains impermeable to molecules with a molecular weight of 4 kDa and above. The vascular density and vascular surface were unaffected by irradiation and no gliosis was induced. These findings suggest that a 15 Gy/79 keV synchrotron irradiation does not induce important damage on brain vasculature and tissue on the short term following irradiation.

Normoxic polyacrylamide gel doped with iodine: response versus X-ray energy.

The basis of Synchrotron Stereotactic Radio-Therapy (SSRT) is the incorporation of high atomic number atoms (iodine, for example) into the tumour mass followed by an irradiation with a monochromatic, low energy, X-ray beam from a synchrotron source. The purpose of the present study was to determine whether polymer gel dosimetry could be used to measure the enhancement of absorbed energy induced by the iodine in the media. We have used a standard nPAG formulation, loaded with NaI and the irradiations were performed either with monochromatic X-rays at the ESRF medical beamline or with a conventional 6 MV X-ray beam from a linear accelerator at the Grenoble University Hospital. We observed sensitivity increase with iodine loaded gels irradiated at low energies, in good agreement with the theoretical iodine dose-enhancement. As expected, the response of the iodine-doped polymer gel was not increased after irradiation with mega-voltage X-rays. We demonstrate in this study that polymer gel dosimeters can be used for measuring dose-enhancement due to iodine presence in SSR treatment.

Cadmium inhibits non-homologous end-joining and over-activates the MRE11-dependent repair pathway.

Although cadmium still represents a public health problem and despite the fact that it has been classified as an IARC Group-I carcinogen, the molecular and cellular mechanisms responsible for the toxicity and the carcinogenicity of cadmium compounds are poorly known. Since unrepaired DNA double-strand breaks (DSBs) are considered to be key-lesions in cell lethality, and because misrepaired DSBs are a source of genomic instability leading to cancer proneness, the activity of the major DSB-repair pathways, i.e. non-homologous end-joining (NHEJ) and recombination, has been evaluated in human endothelial cells exposed to cadmium chloride and cadmium diacetate. Exposure to cadmium results in the production of DSBs a few hours after incubation. These breaks trigger the phosphorylation of H2AX proteins, which was used as an indirect measure of DSB in this study. The presence of cadmium in cells decreases the repair rate of X-ray-induced DSBs, suggesting an impact of cadmium upon the reparability of DSBs. Such an interpretation was consolidated by the finding that the DNA-PK kinase activity, essential for NHEJ, is affected by the presence of cadmium. These results suggest that the toxicity of cadmium compounds may be explained by the propagation of persistent DSBs. In parallel, the presence of cadmium was also associated with an over-activation of the MRE11-dependent repair pathway that may favour genomic instability. Altogether, our data provide a first example of the impact of cadmium upon DSB repair and signalling.

DNA double-strand break repair defects in syndromes associated with acute radiation response: at least two different assays to predict intrinsic radiosensitivity?

PURPOSE: Human diseases associated with acute radiation responses are rare genetic disorders with common clinical and biological features including radiosensitivity, genomic instability, chromosomal aberrations, and frequently immunodeficiency. To determine what molecular assays are predictive of cellular radiosensitivity whatever the genes mutations, the existence of a quantitative correlation between cellular radiosensitivity and unrepaired DNA double-strand breaks (DSB) repair defects was examined in a collection of 40 human fibroblasts representing 8 different syndromes. MATERIALS AND METHODS: A number of techniques such as pulsed-field gel electrophoresis, plasmid assay and immunofluorescence with antibodies against MRE11, MDC1, 53BP1 and phosphorylated forms of H2AX, DNA-PK were applied systematically. RESULTS AND CONCLUSIONS: Survival fraction at 2 Gy was found to be inversely proportional to the amount of unrepaired DSB, whatever the genes mutations and the assay applied. However, no single assay discriminates the full range of human radiosensitivity. Particularly, nuclear foci formed by the phosphorylation of H2AX do not predict well moderate radiosensitivities. Our findings suggest the existence of an ATM-dependent interplay between the activation of DNA-PK and MRE11. A classification of diseases according their cellular radiosensitivity, their molecular response to radiation and the functional assays permitting their evaluation is proposed.

Induction and repair rate of DNA damage: a unified model for describing effects of external and internal irradiation and contamination with heavy metals.

DNA is a key-target for genotoxic stress. Hence, the knowledge of induction and repair rate of DNA damage are crucial to describe and predict the impact of stress situations. Unfortunately, DNA damage induction and repair rates are generally assessed separately whereas they act either concomitantly or transiently in living organisms. Furthermore, the interplay of induction and repair raises the question whether DNA repair adapts to respond to different amounts of DNA damage. In a previous report, we proposed a stochastic interpretation of the repair rate of the major radiation-induced DNA damage. We provided evidence that the repair rate of individual DNA damage is time-independent whereas that of a population of DNA damage is time-dependent (Foray, N., Charvet, A.-M., Duchemin, D., Favaudon, V., Lavalette, D., 2005. The repair rate of radiation-induced DNA damage: a stochastic interpretation based on the gamma function. J. Theor. Biol. 236, 448-458). Here, to better describe situations in which DNA damage induction and repair occur together, our biostatistical model was modified by the introduction of a DNA damage induction parameter. Theoretical and experimental data were compared and discussed by taking concrete experimental situations: X-rays irradiation at different dose-rates, internal irradiation with radioactive compound, contamination with heavy metal and detection of DNA damage by immunofluorescence. By assuming that DNA repair rate is invariant whatever the amount of DNA damage, our model provides good prediction of experimental data suggesting its relevance for the description of complex situations of co-toxicities.

Molecular and cellular response of the most extensively used rodent glioma models to radiation and/or cisplatin.

Purpose Anti-glioma strategies are generally based on trials involving rodent models whose choice remains based on proliferative capacity and availability. Recently, our group obtained the most protracted survival of rats bearing F98 gliomas by combining synchrotron X-rays and intracerebral cisplatin injection (Biston et al., Cancer Res, 64:2317-2323, 2004). The response to such treatment was suggested to be dependent on BRCA1, a tumour suppressor known to be involved in the response to radiation and cisplatin. In order to verify the impact of BRCA1 functionality upon success of anti-glioma trials, radiobiological features and BRCA1-dependent stress signalling were investigated in the most extensively used rodent glioma models. Methods Cell death pathways, cell cycle arrests, DNA repair and stress signalling were evaluated in response to radiation and cisplatin in C6, 9L and F98 models. Results Rodent glioma models showed a large spectrum of cellular radiation response. Surprisingly, BRCA1 was found to be functionally impaired in C6 and F98 favouring genomic instability, tumour heterogeneity and tolerance of unrepaired DNA damage. Significance Our findings strengthened the importance of the choice of the glioma model on genetic and radiobiological bases, inasmuch as all these rat glioma models are induced by nitrosourea-mediated mutagenesis that may favour specific gene mutations. Particularly, BRCA1 status may condition the response to anti-glioma treatments. Furthermore, since BRCA1 acts as a tumour suppressor in a number of malignancies, our findings raise also the question of the implication of BRCA1 in brain tumours formation.

Lead contamination results in late and slowly repairable DNA double-strand breaks and impacts upon the ATM-dependent signaling pathways.

Despite a considerable amount of data, evaluation of the potential genotoxicity and cancer proneness of lead compounds remains unclear, probably due to the plethora of experimental procedures, biological endpoints and cellular models used. In parallel, the understanding in DNA damage formation, repair and signaling has considerably progressed all along these last years, notably for DNA double-strand breaks (DSBs). Here, were examined DNA damage formation and repair in human cells exposed to lead nitrate (Pb(NO(3))(2)) and their consequences upon the ATM-dependent stress signaling, cell cycle progression and cell death. As observed with anti-pH2AX immunofluorescence, exposure to Pb(NO(3))(2) results in formation of late DSBs, that would not originate from conversion of nucleotide damage but likely by a direct production of single-strand breaks. Lead contamination inhibits non-homologous end-joining repair process by preventing the DNA-PK kinase activity whereas the MRE11-dependent repair pathway is exacerbated. Lead contamination triggers successive synchronization of cells in G2/M phase in which the RAD51-dependent homologous recombination was found to be activated. Altogether, our findings support that lead contamination generates late unrepairable DSBs that impact upon the ATM-dependent stress signaling pathway by favoring propagation of errors. Such findings should help to consider more carefully the biological action of lead compounds in the frame of public and occupational exposures.

Consequences of the bleed-through phenomenon in immunofluorescence of proteins forming radiation-induced nuclear foci.

PURPOSE: By allowing the visualization of the proteins inside cells, the immunofluorescence technique has revolutionized our view of events that follow radiation response. Particularly, the formation of nuclear foci, their kinetic of appearance and disappearance, and the association-dissociation of protein partners are useful endpoints to better understand the effects of ionizing radiation. Recently, the technique based on the phosphorylation of the histone 2A family, member X (H2AX) has generated a plethora of reports concerning the interaction between the major proteins involved in DNA repair and stress signaling pathways. However, some unavoidable overlaps of excitation and emission wavelength spectra (the so-called bleed-through phenomenon) of the available fluorescent markers are still generating discrepancies and misinterpretations in the choreography of DNA damage response. Biases are particularly strong with the fluorescein isothiocyanate (FITC)-rhodamine couple, tetramethyl rhodamine iso-thiocyanate (TRITC), the most extensively used markers. METHOD AND RESULTS: Here, two representative examples of biased co-immunofluorescence with pH2AX proteins that form radiation-induced nuclear foci or not are presented. A brief review of literature points out differences in kinetic of appearance and association-dissociation of radiation-induced pH2AX and MRE11 foci. CONCLUSION: Through this report, we would like authors to consider more carefully protein co-localizations by performing systematically, before any co-immunofluorescence, immunofluorescence of each protein separately to avoid bleed-through artifacts.