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.

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.

Bleomycin is a cytotoxic antibiotic that generates DNA double-strand breaks (DSB) and DNA single-strand breaks (SSB). It is possible to introduce known quantities of bleomycin molecules into cells. Low amounts kill the cells by a slow process termed mitotic cell death, while high amounts produce a fast process that has been termed pseudoapoptosis. We previously showed that these types of cell death are a direct consequence of the DSB generated by bleomycin. Here, we use deglyco-bleomycin, a bleomycin derivative lacking the carbohydrate moiety. Although this molecule performs the same nucleophilic attacks on DNA as bleomycin, we show that deglyco-bleomycin is at least 100 times less toxic to Chinese hamster fibroblasts than bleomycin. In fact, deglyco-bleomycin treatment results in apoptosis induction. In contrast, however, deglyco-bleomycin was found to generate almost exclusively SSB. Our results suggest that more than 150 000 SSB per cell are required to trigger apoptosis in Chinese hamster fibroblasts and that SSB are 300 times less toxic than DSB. Taken together with previous studies on bleomycin, our data demonstrates that cells can die by apoptosis, mitotic cell death, or pseudoapoptosis, depending on the number of DNA breaks and on the ratio of SSB to DSB. © 2001 Cancer Research Campaign http://www.bjcancer.com

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.