CERN Computer Newsletter: APRIL-JUNE 1994

List of 137 genes that were among the top 500 Robust-ProGENI-identified genes for at least 40 (over a quarter of 139 studied) treatments in the GDSC dataset (sheet 1). Also includes pathway and Gene Ontology enrichment analysis results of this set (sheet 2). (XLSX 126 kb

Bora prevents MDC1 and 53BP1 foci formation but not γH2AX.

<p>A. HupT3 cells were transfected with FLAG-tagged Bora, irradiated by 10 Gy and 0.5 h later stained with anti-FLAG antibody and γH2AX antibody, respectively. γH2AX foci were counted. At least 100 cells were counted in each experiment. Results are represented as number of foci per cell. Westernblot was performed to determine knockdown efficiency. B. MDC1 and 53BP1 foci formation in control HupT3 cells or cells with Bora overexpression. Cells were immunostained with anti- FLAG (red), MDC1 or 53BP1 (green) and DAPI (blue) antibodies. Quantification of MDC1 and 53BP1 foci. MDC1 and 53BP1 foci were counted and at least 100 cells were counted in each experiment. Results are represented as number of foci per cell. *** indicates p<0.01. Overexpression efficiency was indicated by Western blot analysis using anti-FLAG and anti-Bora antibody. C. Quantification of MDC1and 53BP1foci formation in control HupT3 cells or cells with Bora knockdown. Cells were immunostained with anti- FLAG (red), MDC1 or 53BP1 (green) and DAPI (blue) antibodies. Quantification of MDC1 and 53BP1 foci was performed in a similar fashion as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119208#pone.0119208.g003" target="_blank">Fig. 3B</a>, and *** indicates p<0.01. Knockdown efficiency was determined by Western blot analysis using anti-Bora antibody.</p

Bora inhibits MDC1 foci formation <i>via</i> interaction with MDC1 BRCT domain in a phosphorylation-dependent manner.

<p>A. Bora interacts with MDC1. IP was performed using anti-Bora antibody followed by blotting with anti-MDC1 antibody in 293T cells. B. Bora interacts with MDC1 <i>via</i> the MDC1 BRCT domain. Lysates from 293T cells overexpressing FLAG-tagged Bora were incubated with GST-BRCT or GST-FHA fusion protein immobilized on the glutathione agarose beads for 2 h before washing. The elution was subsequently analyzed by Western blot with anti-FLAG antibody. C. Effect of phosphorylation on Bora-MDC1 interaction. Lysates from 293T cells overexpressing FLAG-tagged Bora and HA-tagged MDC1 was either incubated with buffer alone or with lambda phosphatase for 15 min at 30°C. The mixture was then incubated with FLAG beads. There was a significant decrease in the binding between Bora and HA-tagged MDC1 in the presence of lambda phosphatase regardless of IR treatment. D. Bora C terminus fragment (313–559 aa), but not N terminus (1–312 aa) co-immunoprecipitates with HA-tagged MDC1. 293T cells were co-transfected with plasmids encoding FLAG-tagged Bora or various deletion constructs, and plasmids encoding HA-tagged MDC1. Lysates were incubated with FLAG beads, followed by Western blot analysis with anti-HA antibody.</p

Bora S325A phosphorylation mutant causes increased MDC1 and 53BP1 IRIF formation, DNA repair and resistance to IR treatment.

<p>A. Bora S325 phosphorylation is required for its association with MDC1. Lysates from 293T cells overexpressing FLAG-tagged WT Bora, the S325E mutant and the S325A mutant with or without IR treatment were incubated with GST- MDC1 BRCT fusion protein immobilized on the glutathione agarose beads and subsequent analysis by Western blotting with anti-FLAG antibody. B. Effect of Bora deletion and mutant constructs on MDC1 and 53BP1 IRIF formation. Left Panel: Foci formation. Hela cells were transfected with wild type, Bora deletion, S501A or S325A mutant FLAG-tagged constructs. Forty-eight h after the transfection, cells were treated with 10 Gy IR and immunostained with indicated antibodies. Right Panel: Quantification of MDC1 and 53BP1-IRIF formation per cell is shown after 10 Gy IR. Error bars represent SEM calculated based on100 cells. C. Percentage of GFP positive cells observed in DR-GFP reporter assay in Hela cells that overexpressed different Bora mutant or deletion constructs. Data are presented as mean ± SEM from three independent experiments. Significance was calculated between WT Bora and S325A mutant. D. Effect of Bora deletion and S501mutant constructs on the Bora binding to MDC1 and IR sensitivity. Left Panel: Immunoprecipitation. Lysates from 293T cells overexpressing FLAG-tagged WT Bora and Bora mutants as well as Bora N or C terminal constructs were incubated with GST- MDC1 BRCT fusion protein immobilized on the glutathione agarose beads, with subsequent Western blot analysis with anti-FLAG antibody. Right Panel: Colony forming assays. Bora stably knockdown cell lines were transfected with WT Bora, S501A or C and N terminal constructs, and then treated with increasing dose of IR and cytotoxicity was determined by colony forming assays. E. Effect of S325 mutant construct on IR sensitivity. HupT3 and Hela cell lines with Bora stably knockdown were transfected with WT Bora and S325A mutant, and then treated with increasing dose of IR and cytotoxicity was determined by MTS assays and colony forming assays. P-values were calculated for the difference in AUC values between WT and S325A mutant.</p

Bora Ser325 can be phosphorylated by CDK9.

<p>A. CDK inhibitor blocks the interaction between MDC1-BRCT domain and Bora. FLAG-Bora from 293T cells was incubated <i>in vitro</i> with bacterially purified GST-BRCT domain of MDC1 in the presence of roscovitine. Interaction was determined by Western blot analysis. B. Roscovitine causes resistance to IR treatment in HupT3 and Hela cell lines overexpressing Bora. IR+20 μM roscovitine resulted in resistance phenotype when compared with treatment by IR alone in Bora overexpressed cells. All experiments were done in triplicate, and error bars represent SEM of three independent experiments. C. Depletion of CDK7 and CDK9 by RNAi impairs the association between Bora and MDC1. 293T cells overexpressing Bora were transfected with siRNA targeting different CDK family members. Binding assays were performed by incubating purified FLAG–Bora with bacterial purified GST–MDC1-BRCT fragment. qRT-PCR was performed to determine knockdown efficiency. D. <i>In vitro</i> phosphorylation assay shows that Bora is phosphorylated by CDK9, and CDK9 is activated upon IR treatment. Left panel: Bacterial purified full-length Bora or Bora S325A mutant was incubated in the presence of [γ-³²P] ATP with either CDK7/CyclinH1/MNAT1 or CDK9/CyclinK as indicated. Bora phosphorylation was determined by autoradiography. Bora protein levels were determined by Coomassie blue-staining. Right Panel: HupT3 cells were treated with 10 Gy IR and blotted with indicated antibodies. Data are representative of three independent experiments.</p

Effect of Bora on radiosensitivity and cell cycle in human tumor cell lines.

<p>A. Downregulation of Bora desensitizes cells to IR. Knockdown in HupT3 and Hela cells was performed using two Bora specific siRNAs, followed by MTS assay (upper panel) and colongenic assays (lower panel) to determine cell survival after treatment with increasing doses of IR. ** indicates p<0.05. Western blot was performed to determine the knockdown efficiency (left panel). B. Overexpression of Bora in HupT3 and Hela cells sensitizes cells to IR treatment as determined by MTS assay (upper panel), and clonogenic assays (lower panel). Western blot was performed to determine the overexpression efficiency (left panel). All experiments were performed in triplicate and error bars represent SEM of three independent experiments. Significance was defined by comparing the AUC values of radiation cytotoxicity curves between control and Bora knockdown cells. The x-axis indicates the radiation dose, and the y-axis indicates the surviving fraction after radiation exposure. ** indicates p<0.05. C. Bora affects radiosensitivity independent of its role in PLK1 pathway. HupT3 and Hela cells were transfected with indicated siRNAs, followed by MTS assay with increasing dosage of IR. Western blot was performed to determine the knockdown efficiency (left panel). All experiments were performed in triplicate and error bars represent SEM of three independent experiments. D. Down regulation of Bora slightly increased cells in S and G/M. HupT3 cells transfected with negative or Bora siRNAs were subjected to flow cytometry. Proportion of cells in each cell cycle was quantified. Western blot was performed to determine the knockdown efficiency (left panel). Error bar represents 3 independent experiments.</p

Mass spectrometry analysis of the affinity purified Bora immuno-complex in Her293-T cells overexpressing flag tagged Bora.

<p>Mass spectrometry analysis of the affinity purified Bora immuno-complex in Her293-T cells overexpressing flag tagged Bora.</p

Effect of Bora on DNA repair.

<p>A. Bora knockdown results in a faster repair of DSBs. HupT3 cells were transfected with negative or Bora siRNA and treated with 10 Gy IR for indicated period of time, followed by fixation and staining with anti-γH2AX antibody and DAPI at each time point. B. Quantification of the γH2AX foci in Bora knockdown and control HupT3 cells at different time points post IR (error bars indicate SEM.; n = 100 cells per time point per experiment). The graph indicates the percentage of positive γH2AX foci at indicated time points after IR. There was a significant difference in the number of γH2AX foci between Bora knockdown cells and control cells over time after IR treatment. C. Downregulation of Bora increases IR-induced DNA repair. Control or Bora knocked down Hela cells were transfected with an I-SceI expression vector. Flow cytometric analysis was used to determine the percentage of GFP positive cells as an indication of DNA repair capability of I-SceI-generated DSBs. The data are presented as mean ± SEM from three independent experiments. *** indicates p<0.01. Western blot was performed to determine knockdown efficiency. D. Downregulation of Aurora A decreases IR-induced DNA repair. Control or Aurora A knocked down Hela cells were transfected with an I-SceI expression vector. Percentage of GFP positive cells was determined by flow cytometric analysis. The data are presented as mean ± SEM from three independent experiments. *** indicates p<0.01. qRT-PCR was performed to determine knockdown efficiency.</p

SNPs near the cysteine proteinase cathepsin O gene <i>(CTSO)</i> determine tamoxifen sensitivity in ERα-positive breast cancer through regulation of BRCA1

<div><p>Tamoxifen is one of the most commonly employed endocrine therapies for patients with estrogen receptor α (ERα)-positive breast cancer. Unfortunately the clinical benefit is limited due to intrinsic and acquired drug resistance. We previously reported a genome-wide association study that identified common SNPs near the <i>CTSO</i> gene and in <i>ZNF423</i> associated with development of breast cancer during tamoxifen therapy in the NSABP P-1 and P-2 breast cancer prevention trials. Here, we have investigated their roles in ERα-positive breast cancer growth and tamoxifen response, focusing on the mechanism of CTSO. We performed <i>in vitro</i> studies including luciferase assays, cell proliferation, and mass spectrometry-based assays using ERα-positive breast cancer cells and a panel of genomic data-rich lymphoblastoid cell lines. We report that CTSO reduces the protein levels of BRCA1 and ZNF423 through cysteine proteinase-mediated degradation. We also have identified a series of transcription factors of BRCA1 that are regulated by CTSO at the protein level. Importantly, the variant CTSO SNP genotypes are associated with increased CTSO and decreased BRCA1 protein levels that confer resistance to tamoxifen. Characterization of the effect of both <i>CTSO</i> SNPs and <i>ZNF423</i> SNPs on tamoxifen response revealed that cells with different combinations of <i>CTSO</i> and <i>ZNF423</i> genotypes respond differently to Tamoxifen, PARP inhibitors or the combination of the two drugs due to SNP dependent differential regulation of BRCA1 levels. Therefore, these genotypes might be biomarkers for selection of individual drug to achieve the best efficacy.</p></div

CTSO regulates ER+ breast cancer cell proliferation and tamoxifen response.

<p>(A) Knock down of CTSO decreased proliferation in CAMA1 and ZR75-1 cell lines through BRCA1. Error bars represent SEM; ** P< 0.01 compared to baseline (negative control). The knock down efficiency was determined by qRT-PCR and western blot. (B) Knock down of CTSO conferred sensitivity to tamoxifen through BRCA1. Knock down of BRCA1 abrogated CTSO effects on proliferation in CAMA1 and ZR75-1 cells in the presence of 4-OH TAM. Error bars represent SEM. * = p < 0.05; ** = p < 0.01.</p