Nicolas Foray

A subset of ATM- and ATR-dependent phosphorylation events requires the BRCA1 protein

BRCA1 is a central component of the DNA damage response mechanism and defects in BRCA1 confer sensitivity to a broad range of DNA damaging agents. BRCA1 is required for homologous recombination and DNA damage‐induced S and G2/M phase arrest. We show here that BRCA1 is required for ATM‐ and ATR‐dependent phosphorylation of p53, c‐Jun, Nbs1 and Chk2 following exposure to ionizing or ultraviolet radiation, respectively, and is also required for ATM phosphorylation of CtIP. In contrast, DNA damage‐induced phosphorylation of the histone variant H2AX is independent of BRCA1. We also show that the presence of BRCA1 is dispensable for DNA damage‐induced phosphorylation of Rad9, Hus1 and Rad17, and for the relocalization of Rad9 and Hus1. We propose that BRCA1 facilitates the ability of ATM and ATR to phosphorylate downstream substrates that directly influence cell cycle checkpoint arrest and apoptosis, but that BRCA1 is dispensable for the phosphorylation of DNA‐associated ATM and ATR substrates.

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.

Gamma-rays-induced death of human cells carrying mutations of BRCA1 or BRCA2

There is now evidence to suggest that BRCA1 and BRCA2 are involved in the response of cells to DNA damage and cell cycle checkpoint control. This report examines the death pathways of human cells with various BRCA1 and BRCA2 genotypes after exposure to gamma-rays. A lack of functional BRCA1 and BRCA2 led to defective repair of DNA double-strand breaks in irradiated cells. This impairment resulted in a relaxation of cell cycle checkpoints, production of micronuclei, and a loss of proliferative capacity. Heterozygous BRCA1 and BRCA2 mutations also led to enhanced radiosensitivity, with an impaired proliferative capacity after irradiation. The existence of a phenotype related to radiosensitivity in BRCA1+/− and BRCA2+/− cells raises the question of the response of heterozygous women to radiation.

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.

Constitutive Association of BRCA1 and c-Abl and Its ATM-Dependent Disruption after Irradiation

ABSTRACT BRCA1 plays an important role in mechanisms of response to double-strand breaks, participating in genome surveillance, DNA repair, and cell cycle checkpoint arrests. Here, we identify a constitutive BRCA1-c-Abl complex and provide evidence for a direct interaction between the PXXP motif in the C terminus of BRCA1 and the SH3 domain of c-Abl. Following exposure to ionizing radiation (IR), the BRCA1-c-Abl complex is disrupted in an ATM-dependent manner, which correlates temporally with ATM-dependent phosphorylation of BRCA1 and ATM-dependent enhancement of the tyrosine kinase activity of c-Abl. The BRCA1-c-Abl interaction is affected by radiation-induced modification to both BRCA1 and c-Abl. We show that the C terminus of BRCA1 is phosphorylated by c-Abl in vitro. In vivo, BRCA1 is phosphorylated at tyrosine residues in an ATM-dependent, radiation-dependent manner. Tyrosine phosphorylation of BRCA1, however, is not required for the disruption of the BRCA1-c-Abl complex. BRCA1-mutated cells exhibit constitutively high c-Abl kinase activity that is not further increased on exposure to IR. We suggest a model in which BRCA1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity.

Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts.

Ku86 has been shown to be involved in DNA double-strand break (DSB) repair and radiosensitivity in rodents, but its role in human cells is still under investigation. The purpose of this study was to evaluate the radiosensitivity and DSB repair after transfection of a Ku86-antisense in a human fibroblast cell line. Simian virus 40-transformed MRC5V1 human fibroblasts were transfected with a vector (pcDNA3) containing a Ku86-antisense cDNA. The main endpoints were Ku86 protein level, Ku DNA end-binding and DNA protein kinase activity, clonogenic survival, and DSB repair kinetics. After transfection of the Ku86-antisense, decreased Ku86 protein expression, Ku DNA end-binding activity, and DNA protein kinase activity were observed in the uncloned cellular population. The fibroblasts transfected with the Ku86-antisense showed also a radiosensitive phenotype, with a surviving fraction at 2 Gy of 0.29 compared with 0.75 for the control and 20% of unrepaired DSB observed at 24 hours after irradiation compared with 0% for the control. Several clones were also isolated with a decreased level of Ku86 protein, a surviving fraction at 2 Gy between 0.05 and 0.40, and 10–20% of unrepaired DSB at 24 hours. This study is the first to show the implication of Ku86 in DSB repair and in the radiosensitivity of human cells. This investigation strongly suggests that Ku86 could constitute an appealing target for combining gene therapy and radiation therapy.

100 Years of Individual Radiosensitivity: How We Have Forgotten the Evidence

The time has come to individualize radiation prescriptions on the basis of individual response rather than on population averages of organ tolerance.

BRCA1 carries tumor suppressor activity distinct from that of p53 and p21.

The loss of BRCA1 function appears as an essential step in breast and ovarian epithelial cells oncogenesis and is consistent with the concept that BRCA1 acts as a tumor suppressor gene. However, the mechanism underlying this activity is not understood. In 1996, a retroviral vector was used for BRCA1 delivery to demonstrate that the transfer of BRCA1 inhibits breast and ovarian cancer cell growth. Since this early observation, the tumor growth inhibitory activity of BRCA1 in vivo has not been further documented. Here we re-address this issue and report experiments designed to evaluate the potential of adenovirus-mediated BRCA1 delivery to suppress the growth of cells with various status of endogenous BRCA1 in comparison with p53 and p21. Delivery of wild-type BRCA1 by an adenovirus vector in breast and ovarian tumor cells, decreases in vitro proliferation and tumorigenicity. Similarly, in vivo administration of BRCA1 provokes tumor growth retardation or regression comparable to that obtained with p53 or p21. The antitumor effect of BRCA1 is not observed upon transfer of a mutant lacking the 542 C-terminal residues. The p53- or p21-mediated antiproliferative activities are likely to bear on their capacity to induce apoptosis and/or interfere with cell cycle checkpoint. By contrast, the data presented here show that neither of these mechanisms can account for the BRCA1-mediated antitumor activity and suggest the activation of an alternative route. Cancer Gene Therapy (2001) 8, 759–770

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.

Breast Cancer Induced by X-Ray Mammography Screening? A Review Based on Recent Understanding of Low-Dose Radiobiology

Screening mammography offers the possibility of discovering malignant diseases at an early stage, which is consequently treated early, thereby reducing the mortality rate. However, ionizing radiation as used in low-dose X-ray mammography may be associated with a risk of radiation-induced carcinogenesis. In the context of the harmful effects of ionizing radiation, this article reviewed novel radiobiological data and provided a simulation of the relative incidence of radiation-induced breast cancer due to screening against a background baseline incidence in a population of 100,000 individuals. The use of modern digital mammographic technology was assumed, giving rise to a glandular dose of 2.5 mGy from a 2-view per breast image. Assuming no latency time, this led to a ratio of induced incidence rate over baseline incidence rate of about 1.6‰ for biennial screening in women aged 50-74 years, although it cannot be excluded that the dose and dose rate effectiveness factor values relying on new radiobiological insights may lower this number to about 0.7‰. This carcinogenic risk is considered small in relation to the potential beneficial effects of screening, especially as latency time was not taken into consideration. However, individuals who are known to be carriers of risk-increasing genetic variations and/or have an inherited disposition of breast cancer should avoid ionizing radiation as much as possible and should be referred to ultrasound or magnetic resonance imaging. In addition, a significant, but difficult to quantify, risk of cancer is present for individuals who suffer from hypersusceptibility to ionizing radiation.