BRCA2-dependent homologous recombination is required for repair of Arsenite-induced replication lesions in mammalian cells

Abstract

Arsenic exposure constitutes one of the most widespread environmental carcinogens, and is associated with increased risk of many different types of cancers. Here we report that arsenite (As[III]) can induce both replication-dependent DNA double-strand breaks (DSB) and homologous recombination (HR) at doses as low as 5 microM (0.65 mg/l), which are within the typical doses often found in drinking water in contaminated areas. We show that the production of DSBs is dependent on active replication and is likely to be the result of conversion of a DNA single-strand break (SSB) into a toxic DSB when encountered by a replication fork. We demonstrate that HR is required for the repair of these breaks and show that a functional HR pathway protects against As[III]-induced cytotoxicity. In addition, BRCA2-deficient cells are sensitive to As[III] and we suggest that As[III] could be exploited as a therapy for HR-deficient tumours such as BRCA1 and BRCA2 mutated breast and ovarian cancers.

Figure 1.

DNA double-strand breaks are induced by As[III] and repaired in AA8 Chinese hamster cells. (A) An example of γH2A.X foci (red) and DNA (blue) staining with and without As[III] treatment. (B) Quantification of γH2A.X foci after 24-h treatment with increasing doses of As[III]. Results are the average and standard deviation of three repeats, on each occasion at least 100 cells were counted for each condition. (C) Ethidium bromide staining (upper) and Southern analysis (lower) of DNA double-strand breaks visualized with pulsed-field gel electrophoresis after 24-h treatment with increasing doses of As[III]. Southern analysis used labelled fragmented genomic DNA as a probe. (D) The intensity of each band on the Southern blot. (E) DNA double-strand breaks following a 24 h 50 μM As[III] treatment and subsequent repair for times indicated.

Figure 2.

RAD51 foci and homologous recombination are induced by As[III]. (A) i. Examples of Rad51 foci staining. ii. The number of wild-type AA8 cells containing RAD51 foci following a 24-h treatment with As[III] at the doses indicated and remaining after 24-h treatment with 50 μM and repair for the time indicated. Foci after the repair times indicated. (B) Homologous recombination was determined in SPD8 cells. The reversion frequency from a non-functional to a functional hprt gene, giving resistance to HaST following a 24 h treatment with As[III] at various concentrations is shown. The average (symbol) and standard deviation (error bars) from three to five experiments are shown.

Figure 3.

DNA double-strand breaks are induced by As[III] but not repaired in V-C8 Chinese hamster cells. Ethidium bromide staining of DNA DSBs visualized with pulsed-field gel electrophoresis (A) with increasing doses of As[III] and (B) 24 h after removal of As[III], in the BRCA2 deficient (V-C8) and complemented (V-C8 B2) cells lines. (C and D) Apoptosis levels as indicated by FACS analysis of annexin V staining after 24 h treatment with As[III].

Figure 4.

Homologous recombination deficient cell lines are hypersensitive to As[III]. (A) Clonogenic survival of AA8 (wild type), irs1SF (XRCC3 deficient), PXR3 (XRCC3 complemented, V3-3 (DNA-PKcs deficient) and V3.3(hYAC) (DNA-PKcs complemented) cells with increasing doses of As[III]. (B) Clonogenic survival of V79-4 (wild-type), V-C8 (BRCA2 deficient), V-C8 B2 (V-C8 complimented with BRCA2), irs1 (XRCC2 deficient) and irsx2.2 (XRCC2 complemented) cells with increasing doses of As[III]. The average (symbol) and standard deviation (error bars) from three to four experiments are shown. (C) IC₅₀ As[III] values of each cell line.

Figure 5.

As[III] inhibits single-strand break repair, which causes replication forks to collapse and DSBs to form. (A) Ethidium bromide staining of DNA double-strand breaks visualized with pulsed-field gel electrophoresis after a 6-h treatment of AA8 cells with and without 3 μM aphidicolin (aph) and/or 100 μM As[III]. (B) Apoptosis measured by annexin-V staining after a 16-h treatment of AA8 cells with and without 3 μM aphidicolin (aph) and/or increasing doses of As[III]. (C) Clonogenic outgrowth of AA8 cells treated for 6 h with and without 3 μM aphidicolin (aph) and/or 200 μM As[III] then left to grow in normal media for 7 days. (D) Clonogenic survival of AA8 (wild type) and EM9 (XRCC1 deficient) cells with increasing doses of As[III]. (E) Clonogenic survival of AA8 (wild type) and EM9 (XRCC1 deficient) cells with and without 20 μM As[III] and increasing doses of methyl methanesulfonate (MMS).