BRCA2 affects the efficiency of DNA double-strand break repair in response to N-nitroso compounds with differing carcinogenic potentials

Abstract

The tumor suppressor gene breast cancer susceptibility gene 2 (BRCA2) is frequently mutated or epigenetically repressed in human cancer and has a significant role in the homologous recombination (HR) of DNA double-strand breaks (DSBs). Although N-nitrosodiethylamine (NDEA), N-nitrosodiethanolamine (NDELA) and N-nitrosodipropylamine (NDPA) have similar chemical structures and are able to induce DNA damage, they have varying carcinogenic risks. We hypothesized that the DNA damage repair pathways that are induced by these N-nitroso compounds (NOCs) may differ and that this may contribute to the genotoxic-carcinogenic effect of the NOCs. The present study aimed to characterize the formation of DSBs by NDEA, NDELA and NDPA and also to investigate whether BRCA2 is involved in the DNA damage response. The NOCs were observed to time-dependently induce DSBs and the expression of γ-H2AX in gastric cancer SGC7901 cells. It was observed that the DNA damage induced by NDEA, the most potent carcinogen, was not repaired as efficiently as that caused by NDELA or NDPA. The expression of BRCA2 and RAD51 was demonstrated to be inhibited by NDEA treatment but upregulated by NDELA or NDPA treatment. Furthermore, the knock down of BRCA2 expression impaired the DNA damage repair induced by NDELA or NDPA. The cells with this knock down exhibited an increased sensitivity to NDELA or NDPA treatment, but not to NDEA. These findings suggest that a BRCA2-mediated pathway contributes to differential DSB repair and sensitivity in response to NOC exposure and that it may be associated with the genotoxic-carcinogenic potential of NOCs.

Keywords: BRCA2; DNA damage repair; DNA double strand breaks; N-nitroso compounds.

Figure 1

NDEA, NDELA and NDPA-induced DNA double-strand breaks (DSBs). SGC-7901 cells were treated with 1X IC₅₀ concentrations of NDEA (0.76 mM), NDELA (1.09 mM) or NDPA (0.39 mM) for 15, 30 and 60 min. (A) DNA damage was determined using a neutral comet assay. Three compounds induced apparent DNA damage in SGC7901 cells. (B) The 50 cells per slide from (A) were analyzed to calculate the tail to head ratio of the SGC-7901 cells. Three compounds induced a time-dependent increase in extent of DNA damage. (C) The SGC-7901 cells were treated with 1X IC₅₀ concentrations of NDEA (0.76 mM), NDELA (1.09 mM) or NDPA (0.39 mM) for 1 h. Following treatment, the cells were stained with antibodies against γ-H2AX and counter-stained with DAPI. The cells treated with Dox (1 mM) for 1 h were used as positive controls. NOC treatment lead to formation of γ-H2AX. (D) The SGC-7901 cells were treated with 1X IC₅₀ concentrations of NDEA (0.76 mM), NDELA (1.09 mM) or NDPA (0.39 mM) for 15, 30 and 60 min. Following the treatment, the cells were collected and the cellular protein was prepared for western blotting using antibodies against γ-H2AX. Three compounds induced expression of γ-H2AX. NDEA, N-nitrosodiethylamine; NDELA, N-nitrosodiethanolamine; NDPA, N-nitrosodipropylamine; DAPI, 4′,6-diamidino-2-phenylindole; Dox, Doxorubicin.

Figure 2

Differential efficiency of DNA repair in response to NDEA, NDELA and NDPA treatment. The SGC7901 cells were treated with 1X IC₅₀ concentrations of NDEA (0.76 mM), NDELA (1.09 mM) or NDPA (0.39 mM) for 1 h. The cells were then incubated in a drug-free medium for 12 h to allow DNA repair. (A) DNA damage was detected by neutral comet assays. Dox (1 mM) treatment was used as a positive control. The tail to head ratio of comet DNA in cells treated with NDELA or NDPA was reduced. (B) The 50 cells per slide from (A) were analyzed by measuring the tail to head ratio. ^*P<0.05 and ^**P<0.01, T group vs. R group. The tail to head ratio of comet DNA in cells treated with NDELA or NDPA was significantly reduced. (C) The SGC7901 cells were treated with 1X IC₅₀ concentrations of NDEA (0.76 mM), NDELA (1.09 mM) or NDPA (0.39 mM) for 1 h. The cells were then incubated in drug-free medium for 12 h. The level of γ-H2AX was determined by western blotting. The expression of γ-H2AX in SGC7901 cells treated with NDELA or NDPA was significantly reduced. NDEA, N-nitrosodiethylamine; NDELA, N-nitrosodiethanolamine; NDPA, N-nitrosodipropylamine; Dox, Doxorubicin.

Figure 3

Involvement of the BRCA2-mediated pathway in the DNA repair of NOC-induced damage. (A) The SGC7901 cells were treated with 1X IC₅₀ concentrations of NDEA (0.76 mM), NDELA (1.09 mM) or NDPA (0.39 mM) for 15, and 60 min. Following an incubation in drug-free medium for 12 h, the cells were harvested and the expression of BRCA2 and RAD51 was determined by western blotting. The expression of BRCA2 and RAD51 was reduced in SGC7901 cells treated with NDEA, but were notably upregulated in cells treated with NDELA or NDPA. (B) Expression of BRCA2 was knocked down in siBRCA2-transfected SGC7901 cells. BRCA2-targeted siRNA and a control vector plasmid were stably transfected into the SGC7901 cells. (C–E) Differential expression levels of γ-H2AX induced by NOCs in vector- and siBRCA2-transfected cells following a 12-h drug-free incubation. The vector- and siBRCA2-transfected SGC7901 cells were treated with 1X IC₅₀ concentrations of (C) NDEA, (D) NDELA and (E) NDPA for 1 h, followed by a 12-h drug-free incubation. The expression levels of of γ-H2AX were determined. The DNA damage induced by NDELA or NDPA was not repaired as effectively as in the vector cells. DMSO (1 mM) treatment was used as a negative control. BRCA2, breast cancer susceptibity gene 2; NOC, N-nitroso compound; NDEA, N-nitrosodiethylamine; NDELA, N-nitrosodiethanolamine; NDPA, N-nitrosodipropylamine; siRNA, small interfering RNA.

Figure 4

BRCA2 confers sensitivity to NOC treatment. The sensitivity to (A) NDEA, (B) NDELA and (C) NDPA was determined using a clonogenic survival assay in vector- and siBRCA2-transfected SGC7901 cells. BRCA2, breast cancer susceptibity gene 2; NOC, N-nitroso compound; NDEA, N-nitrosodiethylamine; NDELA, N-nitrosodiethanolamine; NDPA, N-nitrosodipropylamine; siBRCA2, small interfering BRCA2 RNA.