Figure 2 | Oncogene

Figure 2

From: BRCA1-mediated G2/M cell cycle arrest requires ERK1/2 kinase activation

Figure 2

Activation of ERK1/2 kinases by BRCA1 is required for BRCA1-induced G2/M cell cycle arrest. (a) MCF-7 cells were infected with Ad.BRCA1 at 50 PFU/cell and incubated in the presence of U0126 at the indicated doses for 24 h. For serum starvation experiment, log-phase growing cells were washed once with DMEM and then incubated in the medium containing DMEM and 0.1% serum for 24 h. The treated cells were collected, lysed, and the level of phosphorylated-ERK1/2 (pERK1/2) as well as the level of total ERK1/2 (ERK1/2) in each sample was determined by immunoblotting with relevant specific antibodies. (b) Cells were infected with Ad.Control (open bars) or Ad.BRCA1 (solid bars) at 50 PFU/cell and incubated for 24 h in the presence of U0126 at the indicated doses. The resulting cells were harvested, stained with PI and analysed for DNA contents by flow cytometry as described above. Percentage of cells with 4N-DNA content, indicative of G2/M phase of the cell cycle, is shown as the mean±s.d. of quadruplicate samples. (c) For treatment of PD98059, cells were infected with Ad.Control or Ad.BRCA1 (50 PFU/cell) and then incubated in medium containing 100 μ M PD98059 for 24 h prior to analysis (hatched bars). For experiments involving expression of dominant negative MEK1 (MEK1(dn)), cells were infected first with Ad.MEK1(dn) at 100 PFU/cell for 15 h, followed by infection with Ad.Control or Ad.BRCA1 at 50 PFU/cell for additional 24 h (open bars). As a control, a set of duplicate cell samples was infected with Ad.Control or Ad.BRCA1 without additional treatment (solid bars). The resulting cells were analysed for DNA content by FACS and for ERK1/2 phosphorylation by immunoblotting as described above. The percentage of G2 phase cells shown represents the average of two independent experiments. (d) MCF-7 cells were infected with Ad.Control or Ad.BRCA1 (50 PFU/cells) followed by incubation for 24 h in medium containing 0.1% DMSO (solid bars), 50 μ M U0126 (hatched bars), or 1 μ M wortmannin (open bars). Cell samples were analysed for DNA content and for ERK1/2 phosphorylation. The percentage of G2 phase cells shown represents the average of two independent experiments. (e) Cells were infected with or without Ad.BRCA1 at 30 PFU/cell for 24 h. Half of the Ad.BRCA1-infected and -uninfected MCF-7 cells were then either exposed to 10-Gy γ-irradiation or, as a control, remained untreated. The cells were then incubated for an additional 24 h and analysed by immunoblotting for levels of BRCA1 protein, total ERK1/2, and phosphorylated-ERK1/2. (f) Cells infected with or without Ad.BRCA1 at 30 PFU/cell for 24 h were treated with or without γ-irradiation at 10 Gy (solid bars). When U0126 was included in the experiment, U0126 (50 μ M) was either preincubated with uninfected MCF-7 cells for 2 h prior to irradiation treatment or added after 2 h following Ad.BRCA1 infection (open bars). Treated and control cells were analysed for DNA contents by flow cytometry. The results depict the percentage of cells with 4N-DNA content (G2 phase cells) and represent the average of two independent experiments

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