Journal: Journal of Cancer

Article Title: Functional Polymorphisms in BARD1 Association with Neuroblastoma in a regional Han Chinese Population

doi: 10.7150/jca.26719

Figure Lengend Snippet: (A) The BARD1 structure. The amino acid mutation caused by rs1048108, rs2229571 and rs3738888 in BARD1 were indicated. Rs1048108 (P24S) located near zinc finger ring region, which played a key role in the interaction of BARD1 and BRCA1. (B) Effect of the rs1048108 C or T allele on BARD1-BRCA1 interaction. Co-IP result showed no difference in BARD1-BRCA1 interaction between wild type and mutant type of rs1048108 C > T (P24S).

Article Snippet: To study the interaction between BARD1 and BRCA1, we obtained the human BRCA1 ORF expression plasmid (pcDNA3-HA-BRCA1) from Sino Biological lnc (Beijing, China).

Techniques: Mutagenesis, Co-Immunoprecipitation Assay

Journal: Molecular cancer research : MCR

Article Title: BRCA1 Loss Induces GADD153-Mediated Doxorubicin Resistance in Prostate Cancer

doi: 10.1158/1541-7786.MCR-11-0155

Figure Lengend Snippet: BRCA1 increases doxorubicin sensitivity in prostate cancer. A, BRCA1 expression was determined by WB in human prostate cancer cell lines. B, PC3 (pcDNA3, pcDNA3 BRCA1, shRNA scramble, and shRNA BRCA1) and LNCaP (shRNA scramble and shRNA BRCA1) stable cell lines were generated and BRCA1 expression was determined by WB. C, cells were exposed to doxorubicin or etoposide and viability was determined by MTS. Each sample was assayed in triplicate in 2 biological independent experiments. D, cells were stained with Annexin V–FITC and PI and analyzed by FACS after doxorubicin treatment (2 μmol/L, 24 hours). The average from 3 biological independent experiments (bottom) or 1 representative experiment (top) is shown. E, cells were treated with doxorubicin, fixed, and stained with PI. DNA content was determined by FACS. Histogram shows the percentage of cells in G1, S, and G2/M phases. The figure depicts the result of 3 biological independent experiments. F, cells were exposed to doxorubicin and WB analysis was carried out by using anti-p21Waf1/Cip1, -lamin A/C, or -cyclin D1, E, A, and B1 antibodies. In the Western blots, the numbers under the bands indicate BRCA1 quantitation normalized to actin B and PC3 cells (A) or control (B, F).*, P < 0.05.

Article Snippet: BRCA1 expression vector (pcDNA3 BRCA1) has also been previously described ( 23 ). shRNA scramble control and shRNA BRCA1 were from Upstate. siRNA control and siRNA GADD153 were from Santa Cruz Biotechnology.

Techniques: Expressing, shRNA, Stable Transfection, Generated, Staining, Western Blot, Quantitation Assay, Control

Journal: Molecular cancer research : MCR

Article Title: BRCA1 Loss Induces GADD153-Mediated Doxorubicin Resistance in Prostate Cancer

doi: 10.1158/1541-7786.MCR-11-0155

Figure Lengend Snippet: BRCA1 protein binds several genes involved in cell-cycle and DNA damage response. BRCA1-ChIP was conducted from PC3 cells exposed to doxorubicin. DNA-ChIP was analyzed by qPCR by using primers located at the proximal promoter region of BLM, FEN1, DDB2, H3F3B, BRCA2, CCNB2, GADD153, and MAD2L1 genes or β-Globin as negative control. Fold enrichment was calculated normalizing data to input and GAL4 antibody. *, P < 0.05; **, P < 0.01.

Article Snippet: BRCA1 expression vector (pcDNA3 BRCA1) has also been previously described ( 23 ). shRNA scramble control and shRNA BRCA1 were from Upstate. siRNA control and siRNA GADD153 were from Santa Cruz Biotechnology.

Techniques: Negative Control

Journal: Molecular cancer research : MCR

Article Title: BRCA1 Loss Induces GADD153-Mediated Doxorubicin Resistance in Prostate Cancer

doi: 10.1158/1541-7786.MCR-11-0155

Figure Lengend Snippet: BRCA1 protein regulates several genes involved in cell-cycle and DNA damage response. pcDNA3 and pcDNA3 BRCA1 (A) or shRNA scramble and shRNA BRCA1 (B) stable cell lines were exposed to doxorubicin (1 μmol/L; 24 hours). mRNA expression levels for the indicated genes were analyzed by RT-qPCR. Data were normalized to actin B. Media and SD from 3 biological independent experiments are shown. *, P < 0.05; **, P < 0.01.

Article Snippet: BRCA1 expression vector (pcDNA3 BRCA1) has also been previously described ( 23 ). shRNA scramble control and shRNA BRCA1 were from Upstate. siRNA control and siRNA GADD153 were from Santa Cruz Biotechnology.

Techniques: shRNA, Stable Transfection, Expressing, Quantitative RT-PCR

Journal: Molecular cancer research : MCR

Article Title: BRCA1 Loss Induces GADD153-Mediated Doxorubicin Resistance in Prostate Cancer

doi: 10.1158/1541-7786.MCR-11-0155

Figure Lengend Snippet: BRCA1 induces several target genes expression in response to doxorubicin in prostate cancer xenografts. Nu/nu mice were inoculated with PC3 stable cells. Mice were injected i.p. with doxorubicin (8 mg doxorubicin/kg mouse) or vehicle (DMSO) on days 14 and 24. Mice were sacrificed 24 hours after the last injection and RT-qPCR analysis of candidate genes was carried out. Graph bar represents the average and SD from 5 tumors. Data were normalized to actin B. *, P < 0.05.

Article Snippet: BRCA1 expression vector (pcDNA3 BRCA1) has also been previously described ( 23 ). shRNA scramble control and shRNA BRCA1 were from Upstate. siRNA control and siRNA GADD153 were from Santa Cruz Biotechnology.

Techniques: Expressing, Injection, Quantitative RT-PCR

Journal: Molecular cancer research : MCR

Article Title: BRCA1 Loss Induces GADD153-Mediated Doxorubicin Resistance in Prostate Cancer

doi: 10.1158/1541-7786.MCR-11-0155

Figure Lengend Snippet: BRCA1 binds and regulates GADD153 promoter in prostate cancer cells. A, PC3 cells were exposed to different UV doses, incubated for 1 hour, and analyzed by WB by using anti-GADD153 and -actin antibodies. The numbers under the bands indicate GADD153 quantitation normalized to actin B and control. B, PC3 cells were exposed to different UV doses or doxorubicin and GADD153 mRNA expression levels were determined by RT-qPCR. Data were normalized to actin B (ACTB). One result from 3 biological independent experiments is shown. C, PC3 cells were transiently transfected with GADD153 luciferase plasmid, after 24 hours cells were treated as before, harvested, and luciferase activity was quantified. Data were normalized to total protein. All transfections were done in triplicate and each experiment was repeated 3 times. D, BRCA1-ChIP experiments were conducted from PC3 cells untreated or treated with doxorubicin as was described in Materials and Methods. qPCR was carried out with primers located at 1,900 or 200 bp upstream or 300 bp downstream from the TSS of the GADD153 gene. Fold enrichment was calculated normalizing data to input and IgG. E, binding assay was carried out from PC3 cells exposed or not to doxorubicin (1 μmol/L; 24 hours). F, PC3 stable cells were cotransfected with GADD153 luciferase plasmid and treated with doxorubicin (1 μmol/L; 24 hours) and harvested 48 hours posttransfection. Luciferase activity was measured. Transfections were done in triplicate in 3 biological independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: BRCA1 expression vector (pcDNA3 BRCA1) has also been previously described ( 23 ). shRNA scramble control and shRNA BRCA1 were from Upstate. siRNA control and siRNA GADD153 were from Santa Cruz Biotechnology.

Techniques: Incubation, Quantitation Assay, Control, Expressing, Quantitative RT-PCR, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Binding Assay

Journal: Molecular cancer research : MCR

Article Title: BRCA1 Loss Induces GADD153-Mediated Doxorubicin Resistance in Prostate Cancer

doi: 10.1158/1541-7786.MCR-11-0155

Figure Lengend Snippet: BRCA1 regulates doxorubicin-induced apoptosis and cell-cycle arrest through GADD153. A, PC3 cells were transfected with 50 pmol siRNA GADD153 and 72 hours posttransfection cells were harvest and GADD153 expression was determined by WB and RT-qPCR. The numbers under the bands indicate GADD153 quantitation normalized to actin B and control. B, PC3 stable cell lines (pcDNA3 or pcDNA3BRCA1) were transiently transfected with siRNA scramble or siRNA GADD153, exposed to doxorubicin (6 μmol/L) or vehicle (DMSO) during 24 hours, and double stained with Annexin-FITC and PI for FACS analysis. One representative experiment of 3 biological independent experiments is shown. C, alternatively, cells were exposed to doxorubicin (2 μmol/L; 24 hours) and then stained with PI for FACS analysis. Histogram shows the percentage of G1, S, and G2/M cells from 1 representative experiment from 3 biological independent experiments. *, P < 0.05.

Article Snippet: BRCA1 expression vector (pcDNA3 BRCA1) has also been previously described ( 23 ). shRNA scramble control and shRNA BRCA1 were from Upstate. siRNA control and siRNA GADD153 were from Santa Cruz Biotechnology.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Quantitation Assay, Control, Stable Transfection, Staining

Journal: Molecular cancer research : MCR

Article Title: BRCA1 Loss Induces GADD153-Mediated Doxorubicin Resistance in Prostate Cancer

doi: 10.1158/1541-7786.MCR-11-0155

Figure Lengend Snippet: BRCA1 protein binds several genes involved in cell-cycle and DNA damage response. BRCA1-ChIP was conducted from PC3 cells exposed to doxorubicin. DNA-ChIP was analyzed by qPCR by using primers located at the proximal promoter region of BLM, FEN1, DDB2, H3F3B, BRCA2, CCNB2, GADD153, and MAD2L1 genes or β-Globin as negative control. Fold enrichment was calculated normalizing data to input and GAL4 antibody. *, P < 0.05; **, P < 0.01.

Article Snippet: BRCA1 expression vector (pcDNA3 BRCA1) has also been previously described ( 23 ). shRNA scramble control and shRNA BRCA1 were from Upstate. siRNA control and siRNA GADD153 were from Santa Cruz Biotechnology.

Techniques: Negative Control

Journal: Molecular cancer research : MCR

Article Title: BRCA1 Loss Induces GADD153-Mediated Doxorubicin Resistance in Prostate Cancer

doi: 10.1158/1541-7786.MCR-11-0155

Figure Lengend Snippet: BRCA1 binds and regulates GADD153 promoter in prostate cancer cells. A, PC3 cells were exposed to different UV doses, incubated for 1 hour, and analyzed by WB by using anti-GADD153 and -actin antibodies. The numbers under the bands indicate GADD153 quantitation normalized to actin B and control. B, PC3 cells were exposed to different UV doses or doxorubicin and GADD153 mRNA expression levels were determined by RT-qPCR. Data were normalized to actin B (ACTB). One result from 3 biological independent experiments is shown. C, PC3 cells were transiently transfected with GADD153 luciferase plasmid, after 24 hours cells were treated as before, harvested, and luciferase activity was quantified. Data were normalized to total protein. All transfections were done in triplicate and each experiment was repeated 3 times. D, BRCA1-ChIP experiments were conducted from PC3 cells untreated or treated with doxorubicin as was described in Materials and Methods. qPCR was carried out with primers located at 1,900 or 200 bp upstream or 300 bp downstream from the TSS of the GADD153 gene. Fold enrichment was calculated normalizing data to input and IgG. E, binding assay was carried out from PC3 cells exposed or not to doxorubicin (1 μmol/L; 24 hours). F, PC3 stable cells were cotransfected with GADD153 luciferase plasmid and treated with doxorubicin (1 μmol/L; 24 hours) and harvested 48 hours posttransfection. Luciferase activity was measured. Transfections were done in triplicate in 3 biological independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: BRCA1 expression vector (pcDNA3 BRCA1) has also been previously described ( 23 ). shRNA scramble control and shRNA BRCA1 were from Upstate. siRNA control and siRNA GADD153 were from Santa Cruz Biotechnology.

Techniques: Incubation, Quantitation Assay, Control, Expressing, Quantitative RT-PCR, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Binding Assay

Journal: Molecular cancer research : MCR

Article Title: BRCA1 Loss Induces GADD153-Mediated Doxorubicin Resistance in Prostate Cancer

doi: 10.1158/1541-7786.MCR-11-0155

Figure Lengend Snippet: BRCA1 regulates doxorubicin-induced apoptosis and cell-cycle arrest through GADD153. A, PC3 cells were transfected with 50 pmol siRNA GADD153 and 72 hours posttransfection cells were harvest and GADD153 expression was determined by WB and RT-qPCR. The numbers under the bands indicate GADD153 quantitation normalized to actin B and control. B, PC3 stable cell lines (pcDNA3 or pcDNA3BRCA1) were transiently transfected with siRNA scramble or siRNA GADD153, exposed to doxorubicin (6 μmol/L) or vehicle (DMSO) during 24 hours, and double stained with Annexin-FITC and PI for FACS analysis. One representative experiment of 3 biological independent experiments is shown. C, alternatively, cells were exposed to doxorubicin (2 μmol/L; 24 hours) and then stained with PI for FACS analysis. Histogram shows the percentage of G1, S, and G2/M cells from 1 representative experiment from 3 biological independent experiments. *, P < 0.05.

Article Snippet: BRCA1 expression vector (pcDNA3 BRCA1) has also been previously described ( 23 ). shRNA scramble control and shRNA BRCA1 were from Upstate. siRNA control and siRNA GADD153 were from Santa Cruz Biotechnology.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Quantitation Assay, Control, Stable Transfection, Staining

Journal: Cancer Research

Article Title: Transcriptional Autoregulation by BRCA1

doi: 10.1158/0008-5472.can-09-1477

Figure Lengend Snippet: Figure 3. BRCA1 and BARD1 negatively regulate transcription from the BRCA1 promoter. A, Jurkat T cells were cotransfected with luciferase reporter constructs (see schematic above) spanning the indicated lengths of regulatory sequences upstream of the BRCA1 TSS (pGL12 or pGL35) in combination with either of the BRCA1 WT (pcDNA3 BRCA1), BRCA1 COOH-terminal mutant (pcDNA3 ΔBRCT), or empty (pcDNA3) expression vectors. B, Jurkat T cells were cotransfected with the indicated BRCA1 promoter reporters and either shRNA BRCA1 or control shRNA with scrambled sequence expression vectors. C, Jurkat T cells were cotransfected with the indicated BRCA1 promoter reporters and either WT BARD1 (BARD1) or empty expression vectors, or BARD1 siRNA or control duplex siRNA oligonucleotides. Columns, mean from at least three independent transfections; bars, SEM. D, ChIP analysis of BARD1 enrichment at the BRCA1 promoter with and without pretreatment with 1 μmol/L doxorubicin. Columns, mean derived from two independent biological replicates; bars, SEM.

Article Snippet: BARD1 expression vector (pcDNA3 BARD1) was purchased from Origene Technologies. shRNA control and shRNA BRCA1 were from Upstate. siRNA control and siRNA BARD1 (AACAGUAACAUGUCCGAUGAAdTdT) were from Dharmacon, Inc. shRNA E2F1 A, shRNA E2F1 B, and pGIPZ control plasmids were obtained from OpenBiosystems.

Techniques: Luciferase, Construct, Mutagenesis, Expressing, shRNA, Control, Sequencing, Transfection, Derivative Assay

Journal: Cancer Research

Article Title: Transcriptional Autoregulation by BRCA1

doi: 10.1158/0008-5472.can-09-1477

Figure Lengend Snippet: Figure 4. Reciprocal regulation of BRCA1 promoter activation and transcription in PC3 prostate cell lines by stable overexpression or depletion of BRCA1 protein. A, PC3 cells stably transfected with control empty vector (Control) or BRCA1 expression vector (BRCA1) were transiently transfected with BRCA1-luc reporter constructs (pGL12 or pGL35). B, PC3 cells stably expressing shRNA BRCA1 or control shRNA with scrambled sequence shRNA expression vectors were transfected with BRCA1-luc reporter (pGL12). Luciferase activity was normalized to protein. Columns, mean from three independent transfections; bars, SEM. C, PC3 prostate cancer cells that stably overexpress BRCA1 as in A were analyzed for nascent BRCA1 RNA levels (unspliced transcript) by qRT-PCR and compared with cells stably expressing empty vector (Ctrl). Similarly, cells stably depleted of BRCA1 as in B were analyzed for nascent BRCA1 RNA levels compared with cells stably producing shRNA containing a scrambled sequence (Ctrl).

Article Snippet: BARD1 expression vector (pcDNA3 BARD1) was purchased from Origene Technologies. shRNA control and shRNA BRCA1 were from Upstate. siRNA control and siRNA BARD1 (AACAGUAACAUGUCCGAUGAAdTdT) were from Dharmacon, Inc. shRNA E2F1 A, shRNA E2F1 B, and pGIPZ control plasmids were obtained from OpenBiosystems.

Techniques: Activation Assay, Over Expression, Stable Transfection, Transfection, Control, Plasmid Preparation, Expressing, Construct, shRNA, Sequencing, Luciferase, Activity Assay, Quantitative RT-PCR