Disruption of cell cycle kinetics by benzo[a]pyrene: inverse expression patterns of BRCA-1 and p53 in MCF-7 cells arrested in S and G2

Abstract

The effects of a ligand of the aromatic hydrocarbon receptor (AhR), benzo[a] pyrene (B[ a]P), and its metabolite, BPDE (7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene), on BRCA-1 levels and cell cycle kinetics were determined in MCF-7 breast cancer cells. Exposure of asynchronous MCF-7 cells for 72 hours to a non-cytotoxic dose of 0.5 microM B[a]P triggered a three-fold reduction in BRCA-1 protein. In MCF-7 cells resistant (20% to 30%) to genotoxic concentrations of B[a]P (1 to 5 microM), the loss of BRCA-1 protein was coupled with pausing in S-phase and G2/M, and accumulation of p53, mdm2 and p21. Treatment of MCF-7 cells synchronized in S-phase (72%) with B[a]P prolonged the arrest in S-phase, although this checkpoint was transient since cells resumed to G2/M after 12 hours with reduced levels of BRCA-1. In these cells, levels of p53 were increased, whereas the cellular content of p21 remained unaltered. In contrast, the co-treatment with the AhR antagonist, alpha-naphthoflavone (ANF), abrogated the deleterious effects of B[a]P on BRCA-1 expression, while preventing the accumulation of p53 and disruption of cell cycle profile. These findings suggest that the AhR mediated the inverse expression patterns of BRCA-1 and p53 upon exposure to B[a]P. The treatment with BPDE induced S-phase arrest and reduced BRCA-1 mRNA levels. The negative effects of BPDE on BRCA-1 expression were not transient since removal of BPDE did not allow complete reversal of the repression. These cumulative data suggest that the B[a]P metabolite, BPDE, may play a key role in disruption of BRCA-1 expression and cell cycle kinetics in breast epithelial cells.

Figure 1

Dose-response effects of B[a]P on cell viability and BRCA-1. (A) MCF-7 cells were cultured in the presence of increasing concentrations of B[a]P for 72 hours. Bars represent average cell number±SD from three independent wells counted in triplicate (n = 9). (B) Western blotting of BRCA-1 protein. Cell extracts were obtained from MCF-7 cells cultured for 72 hours in control DMEM/F12 containing 10% FCS, basal medium plus vehicle (DMSO), or increasing concentrations of B[a]P. The bands in panel B are immunocomplexes visualized by incubating Western blots with BRCA-1 (Ab-2) or β-actin (Ab-1) antibodies.

Figure 2

Time-dependent effects of B[a]P on BRCA-1 and cell cycle checkpoints. Asynchronous MCF-7 cells were cultured for various periods of time in basal DMEM/F12 plus 10% FCS or basal medium containing 5 µM B[a]P. At the end of the incubation periods, cell extracts were analyzed for their content in BRCA-1, p53, p21, and mdm2 protein. The control bands are β-actin immunocomplexes.

Figure 3

B[a]P induces S-phase and G₂/M accumulation. Asynchronous MCF-7 cells were cultured for 24, 48, and 72 hours in basal DMEM/F12 plus 10% FCS, basal medium containing the vehicle (DMSO) or vehicle plus 5 µM B[a]P. Cell cycle profiles were analyzed by flow cytometry as described in Materials and Methods section. Bars represent percentages of cells in G₀/G₁, S, and G₂/M representative of three separate experiments with standard deviations lower than 3%.

Figure 4

B[a]P delays escape from S-phase and extends transit through G₂/M. MCF-7 cells were synchronized in S-phase with aphidicolin (1 µg/ml) for 24 hours, after which cells were released into (C and D) basal DMEM/F12 plus 10% FCS (aphidicolin/DMEM) or (E and F) basal medium plus 5 µM B[a]P (aphidicolin/B[a]P). Cells were harvested at 12 and 24 hours after release. Cell cycle distribution was examined by flow cytometry after propidium-iodide staining. Arrowheads below the DNA histograms represent channels with most events for specific phases of the cell cycle. The profiles are representative of three independent experiments.

Figure 5

B[a]P-dependent accumulation in G₂/M coincides with loss of BRCA-1. (A) MCF-7 cells were synchronized in S-phase with aphidicolin (1 µg/ml) for 24 hours, after which cells were released into basal DMEM/F12 plus 10% FCS, or basal medium plus 5 µM B[a]P, colchicine (0.25 µM), or colchicine plus B[a]P. Cells were harvested at 12 and 24 hours after release. Cell cycle distribution was examined by flow cytometry after propidium-iodide staining. The profiles are representative of three independent experiments. (B) Western blotting of BRCA-1, p53 and p21. After synchronization in S-phase with aphidicolin (1 µg/ml) for 24 hours, cells were released into DMEM/F12 plus 10% FCS or basal medium containing 5 µM B[a]P. Cell extracts were collected at 12 and 24 hours after release. The control bands are β-actin immunocomplexes.

Figure 6

ANF restores normal cell cycle distribution, BRCA-1, and inhibits the accumulation p53. Asynchronous MCF-7 cells were cultured for 72 hours in basal DMEM/F12, basal medium containing vehicle (DMSO), or 5 µM B[a]P plus increasing concentrations of ANF. Bands represent immunocomplexes of BRCA-1 and p53. The control bands are β-actin immunocomplexes.

Figure 7

BPDE induces S-phase arrest and reduces the potential for BRCA-1 expression. (A) Flow cytometry profile of MCF-7 cells cultured for 24 hours in basal DMEM/F12 plus 10% FCS, or basal medium plus increasing concentrations of BPDE. Bars represent average percentage of cells in G₀/G₁, S, and G₂/M from two separate experiments. (B) RT-PCR analysis of BRCA-1 mRNA from MCF-7 cells treated with nanomolar concentrations of BPDE for 24 hours. (C) MCF-7 cells were treated for 72 hours in basal medium, or pre-treated in BPDE (500 nM)-containing medium for 0.5 1, 6 and 12 hours. At the end of the pre-incubation periods, cells were washed and cultured up to 72 hours in fresh DMEM/F12 plus 10% FCS free of BPDE. In (B) and (C), changes in BRCA-1 mRNA were assessed by RT-PCR analysis of total RNA. The PCR products represent BRCA-1 (712 bp) and control ribosomal 18S RNA (488 bp) from input cDNA corresponding to 400 ng of total RNA. Experimental conditions for semi-quantitative RT-PCR analysis of BRCA-1 were those described in Material and Methods section [22]. (D) Western blot analysis of BRCA-1 and p53. MCF-7 cells were pre-treated with BPDE (500 nM) for various periods of time and then cultured up to 72 hours in basal DMEM/F12 plus 10% FCS medium. Bands represent immunocomplexes of BRCA-1, p53 and p21. The control bands are β-actin immunocomplexes.