Fig 1.
ENZ induces telomere damage, and inhibiting repair of damage with an ATMi leads to cell death, in multiple CRPC cell lines.
(A-C) ENZ induces telomere damage in CRPC cells. Based on the dose-response data shown in S1A Fig, CRPC cells were treated for 24 hr with 5 μM ENZ (22Rv1) or 10 μM ENZ (C4-2B and LNCaP/AR), then labeled with antibodies to DNA damage marker γ-H2AX and the telomere marker TIN2. Colocalization of γ-H2AX and TIN2 indicate DNA damage at telomeres. Cells with a TIF response to ENZ (>5 dual-labeled foci) were counted in enlarged (1000X) photomicrographs of representative fields. Data are expressed as mean ± SD of 3 independent experiments. (D-E) Combining ENZ with ATMi KU60019 leads to cell death. 22Rv1 (D), C4-2B (E), and LNCaP/AR (F) cells were treated with 5 μM ENZ in the presence or absence of 10 μM KU60019 for 24 hr, then washed to remove drugs and allowed to grow for 14 days (colony formation assay). The survival fraction is plotted relative to vehicle-treated controls; mean ± SD of 3 independent experiments.
Fig 2.
Telomere-associated AR in prostate cancer cells.
(A-D) Actinomycin D-resistant AR is preferentially associated with telomeres. (A) Actinomycin D treatment facilitates immunofluorescent identification of AR at telomeres. LNCaP cells were treated with or without 0.5 μg/ml actinomycin D (Act D) for 4 hr, then fixed, permeabilized, and equilibrated in cytoskeleton buffer, and then subjected to dual labeling with antibodies against AR (AR-441, red) and TIN2 (green). Actinomycin D treatment greatly decreases the amount of nuclear AR without affecting TIN2. Dual-labeled foci (yellow in the merge panels) represent AR at telomeres, and are more evident in actinomycin D-treated cells. (B) Schematic of location of PCR primers used to amplify a region spanning the sub-telomere and telomere region of chromosome 17p. (C) Actinomycin D treatment decreases AR association with AREs but not with telomeres. Chromatin was isolated from LNCaP cells that had been treated with or without actinomycin D and then fixed; chromatin immunoprecipitates (ChIPs) were prepared using antibodies against AR (N-20), GR, and PR, and analyzed by PCR for the presence of androgen response elements (AREs) of PSA and NDRG1 gene promoters, or chromosome 17p telomere DNA. The data shown is representative of 3 experiments. (D) Full length AR, but not splice variant AR-V7, is associated with telomeres in 22Rv1 cells. Immunofluorescent images of 22Rv1 cells that were treated with or without 0.5 μg/ml Act D for 4 hr, then labeled with TIN2 antibody and either antibody AR-441 (which recognizes both full-length AR and splice variant AR-V7) or an AR-V7-specific antibody are shown. The merge panels show colocalization of antibody AR441 with TIN2, but not of AR-V7 with TIN2 (both in vehicle-treated and actinomycin D-treated cells); this suggests that only full-length AR is associated with telomeres. (E) Quantitation of labeled foci in actinomycin D-treated cells in D. Labeled foci were counted in twenty Act D-treated cells/group in each of three separate experiments, and expressed as the percentage of TIN2 foci colocalized with AR. Error bar represents mean ± SD, n = 3.
Fig 3.
Effect of knockdown of full-length AR or AR-V7 on telomere DNA damage in 22Rv1 cells.
22Rv1 cells were transfected with siRNA targeting AR exon 1 to knockdown both full-lengh AR (f-AR) and AR-V7, AR exon 7 to knockdown only f-AR, or AR exon CE3 to knockdown only AR-V7. Scrambled siRNA was used as control. (A) Cell extracts were prepared, and western blot analysis was performed using an AR antibody (N-20) that recognizes both f-AR and AR-V7, an AR-V7-specific antibody, or a GAPDH antibody. (B) The effect of knockdown of f-AR and AR-V7, or of only AR-V7, on telomere DNA damage was assessed by dual immunofluorescent staining of the DNA damage marker γ-H2AX (red) and the telomere marker TIN2 (green). Colocalization of γ-H2AX and TIN2 (indicated by yellow arrows) is shown in the ‘merge’ panels. Higher magnification inserts of representative cells in the merge images facilitate the visualization of the presence or absence of colocalization. (C) Bar chart of the percentage of cells with a TIF response, a measure of DNA damage at telomeres. Eighty cells/treatment were counted in each of three separate experiments; mean ± SD, n = 3. *, p = 0.04; ***, p = 0.0001.
Fig 4.
Effect of f-AR or AR-V7 knockdown in the presence of ATMi in 22Rv1 cells.
A) 22Rv1 cells were transfected with siRNAs targeting AR exon 1 to knockdown both f-AR and AR-V7, AR exon 7 to knockdown only f-AR, or AR exon CE3 to knockdown only AR-V7. Scrambled siRNA was used as control. Transfected cells were then treated for 24 hr with or without 10 μM ATMi KU60019, then washed to remove drugs and subjected to a colony formation assay (14 day growth). B) Bar chart shows the survival fraction of cells from the colony formation assay, expressed relative to the survival of control cells transfected with scrambled siRNA and treated with vehicle; mean ± SD of 3 independent experiments.
Fig 5.
Combined treatment with AR antagonist ENZ plus ATMi inhibits growth of CRPC 22Rv1 xenograft tumors in mice that are resistant to each drug alone.
22Rv1 tumor-bearing athymic nude mice were randomly assigned to vehicle control (Cont), ENZ, ATMi KU59403 (KU), or combined ENZ+KU treatment for 4 weeks. A) Tumor size over time is presented as mean tumor volume (mm3) of each treatment group (n = 6 or 7 mice/group). Error bars represent standard deviation. Statistical analysis was performed for comparison between ENZ alone or KU alone and KU+ENZ treatments: *, p<0.05; **, p<0.001; ***, p<0.0001. B) The doubling time of each tumor was calculated from a plot of log tumor volume vs. time. Doubling times of each treatment group are presented as Box-Whisker plots; horizontal lines represent mean, first and third quartiles, and whiskers represent the minimum and maximum doubling time of each group. The doubling time (days, mean ± SEM) of each group was: Control, 4.94 ± 1.0; KU, 5.50 ± 1.36; ENZ, 5.46 ± 1.36; ENZ+KU, 12.71 ± 4.38. P values are shown in the chart. C) Body weight of mice during the treatment period, relative to day 0 of treatment of each group. D) ENZ induces ATM activation in 22Rv1 xenograft tumors. Immunostaining of pATM is shown in a representative 22Rv1 xenograft tumor section from each treatment group. E) Evaluation of cell death in serial sections of 22Rv1 tumors. TUNEL assay to detect cell death was performed as described in the manufacturer’s protocol (InVitrogen). Images show ~1, 000 cells (blue) in a representative tumor tissue section from each treatment group. Cell death was analyzed by counting the percentage of cells that were dead (red) in each image.