Aurélie Joubert

Cure of Fisher Rats Bearing Radioresistant F98 Glioma Treated with cis-Platinum and Irradiated with Monochromatic Synchrotron X-Rays

High-grade gliomas are usually of poor prognosis, and conventional radiotherapy, even combined with chemotherapy, still fails to improve the survival of patients. Here, we propose an innovative therapeutic approach combining synchrotron radiation with cis-diamminedichloroplatinum (II) (CDDP). As suggested previously, monochromatic synchrotron irradiation of CDDP at 78.8 keV, just above the 78.4 keV platinum absorption K-edge, leads to an enhanced photoelectric effect and an increased local toxicity. To select a particular radiation energy that could provide supra-additive effect, we used pulsed-field gel electrophoresis to assess yields of DNA double-strand breaks induced in rat F98 glioma cells after CDDP treatment combined with synchrotron X-rays. Thereafter, intracerebral CDDP injection combined with synchrotron X-rays was applied to Fisher rats bearing F98 glioma. CDDP concentrations were mapped by synchrotron X-ray microfluorescence. An extra number of more slowly repaired double strand breaks were observed when irradiating CDDP-treated F98 cells at 78.8 keV. In vivo treatments were then performed with different radiation doses and CDDP concentrations. All cell inoculations in rat brain resulted in tumor development, and tumor presence was controlled by computed tomography. Among all of the conditions tested, the combination of 3 μg of CDDP with 15 Gy resulted in the largest median survival time (206 days). After 1 year, about 34% of treated rats were still alive. This preclinical finding, validated by molecular analysis, represents the most protracted survival reported with this radioresistant glioma model and demonstrates the interest in powerful monochromatic X-ray sources as new tools for cancer treatments.

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.

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 etxa0al., 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.

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.

Biologie des radiations : avancées majeures et perspectives pour la radiothérapie

At the beginning of the 21st century, radiation biology is at a major turning point in its history. It must meet the expectations of the radiation oncologists, radiologists and the general public, but its purpose remains the same: to understand the molecular, cellular and tissue levels of lethal and carcinogenic effects of ionizing radiation in order to better protect healthy tissues and to develop treatments more effective against tumours. Four major aspects of radiobiology that marked this decade will be discussed: technological developments, the importance of signalling and repair of radiation-induced deoxyribonucleic acid (DNA) damage, the impact of individual factor in the response to radiation and the contribution of radiobiology to better choose innovative therapies such as protontherapy or stereotactic body radiation therapy (SBRT). A translational radiobiology should emerge with the help of radiotherapists and radiation physicists and by facilitating access to the new radio and/or chemotherapy modalities.

In Vitro and In Vivo Optimization of an Anti-glioma Modality Based on Synchrotron X-Ray Photoactivation of Platinated Drugs

Abstract For the past 5 years, a radio-chemotherapy approach based on the photoactivation of platinum atoms (PAT-Plat) consisting of treating tumors with platinated compounds and irradiating them above the platinum K edge (78.4 keV) has been developed at the European Synchrotron Radiation Facility (Grenoble, France). Compared to other preclinical modalities, PAT-Plat provides the highest survivals of rats bearing the rodent F98 glioma. However, further investigations are required to optimize its efficiency and to allow its clinical application. Here we examined in vitro and in vivo whether monochromatic X rays are more efficient than high-energy photons in producing the PAT-Plat effect by measuring DNA double-strand breaks (DSBs) and survival of glioma-bearing rats and whether an increase in the platinum concentration in the tumor results in increased rat survival. DSBs were assessed by pulsed-field gel electrophoresis with different DNA fragment migration programs and with γ-H2AX immunofluorescence. In vivo, F98 glioma cells were injected intracerebrally, treated with a single intracranial injection of cisplatin or carboplatin 13 days after tumor implantation, and irradiated the day after with 78.8 keV X rays or 6 MV photons. Our results indicate that 78.8 keV X rays are more efficient than high-energy photons at producing the PAT-Plat effect. At low concentrations, cisplatin is more efficient than carboplatin; this is likely due to more efficient DNA binding and DSB repair inhibition. High concentrations of carboplatin inside tumors do not necessarily lead to protracted survival of rats. The therapeutic benefit of anti-glioma synchrotron strategies appears to be correlated with the percentage of unrepaired DSBs but not with the number of DSBs induced.

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.

DNA Double-Strand Breaks Repair and Signaling of Human Gliomas and Normal Brain Cells in Response to Radiation: Potential Impact of the ATM-and BRCA1- Dependent Pathways

Deriving from glial, astrocyte, or dendrocyte cells, gliomas are the most frequent tumors of central nervous system. Unfortunately, most of gliomas are refractory to standard radiotherapy treatments. The median survival for patients bearing grade IV gliomas (glioblastomas) does not exceed one year even after both aggressive surgery and radiotherapy treatment (Behin et al., 2003). A standard of 60 Gy delivered in 30 fractions during six weeks remains the best radiotherapy modality against gliomas (Behin et al., 2003). This last conclusion raises the possibility that human gliomas might be generally more radioresistant than other tumor tissues. However, there is no consensus in literature about a specific radioresistance of human gliomas. Besides, the complexity of the molecular and cellular features of radiation response and the difficulty to define reliable endpoints to account for radiosensitivity whatever the tissue type may have limited the extent of some reports. In addition, three specific features of radiobiology of gliomas can be also evoked: cellular in vitro endpoints like clonogenic survival seem to be less appropriate to predict gliomas radiocurability than that of other tumour types (Taghian et al., 1992, 1993); animal models extensively used in glioma research may not reflect specificities of human gliomas and may bias in anti-glioma strategies (Holland, 2001); DNA repair capacity of gliomas is poorly documented and most of the investigations about genes mutations concern actors of proliferation rather than upstream DNA repair proteins (Zhu & Parada, 2002). In 2004, our group obtained the most protracted survival of rats bearing radioresistant rodent gliomas by using synchrotron X-rays combined with intracerebral cisplatin injection. Such so-called PAT-plat treatment triggers the photoactivation of platinum atoms and produces some additional DNA double-strand breaks (DSBs) at the vicinity of cisplatininduced DNA adducts (Biston et al., 2004). The severity of PAT-Plat-induced DSBs was shown to be due to the inhibition of the major DSB repair pathway in mammalians, namely