Journal: PLOS One

Article Title: Differential sensitivity of MCPH1- and BRCA2-deficient cancer cells to PARP-1 inhibition

doi: 10.1371/journal.pone.0345514

Figure Lengend Snippet: (A) . U2OS or Hela cells were transfected with Control (siCON), MCPH1 (siMCPH1) or BRCA2 (siBRCA2) siRNA for 48h or 120h and protein expression analysed via Western blotting with the indicated antibodies. MCPH1 expression in sgCON (control) and sgMCPH1 (MCPH1 knockout) cells was also investigated by Western blotting. (B) Quantification of MCPH1 or BRCA2 protein bands normalised to GAPDH and expressed as % change relative to siCON or sgCON. Data is the mean and standard error of three (48h) or two (120h and sgCON/sgMCPH1) independent experiments. (C) U2OS or Hela cells were transfected with MCPH1 siRNA for 72h before analysis of nuclear morphology (premature chromosome condensation, PCC) via DAPI staining and fluorescence microscopy. (D) The % of cells displaying PCC, with at least 100 cells analysed.

Article Snippet: Membranes were blocked with 5% dried skimmed milk/TBS-T (50mM Tris pH 7.6, 150mM NaCl and 0.2% v/v Tween-20) for a minimum of 1-hour prior to overnight incubation at 4°C with primary antibodies; MCPH1 (11962–1-AP, Proteintech), BRCA2 (29450–1-AP, Proteintech) and GAPDH (60004–1-Ig, Proteintech).

Techniques: Transfection, Control, Expressing, Western Blot, Knock-Out, Staining, Fluorescence, Microscopy

Journal: PLOS One

Article Title: Differential sensitivity of MCPH1- and BRCA2-deficient cancer cells to PARP-1 inhibition

doi: 10.1371/journal.pone.0345514

Figure Lengend Snippet: U2OS cells were transfected with control (siCON), BRCA2 (siBRCA2) or BRCA2 + MCPH1 (siBRCA2 + siMCPH1) siRNA for 48h before (A) Western blot analysis or (B) seeding into 96-well plates and treating with the indicated doses of AZD2461 or Talazoparib, with each treatment performed in triplicate. After 96-hours, cell viability was measured using an MTS assay. MTS data represents the mean and standard error from at least three independent experiments, with cell viability expressed as a % relative to the corresponding untreated sample. Statistical significance was measured using two-way ANOVA (p-values described in text, with significance set at p < 0.05).

Article Snippet: Membranes were blocked with 5% dried skimmed milk/TBS-T (50mM Tris pH 7.6, 150mM NaCl and 0.2% v/v Tween-20) for a minimum of 1-hour prior to overnight incubation at 4°C with primary antibodies; MCPH1 (11962–1-AP, Proteintech), BRCA2 (29450–1-AP, Proteintech) and GAPDH (60004–1-Ig, Proteintech).

Techniques: Transfection, Control, Western Blot, MTS Assay

Journal: American Journal of Cancer Research

Article Title: BRIT1 dysfunction confers synergistic inhibition of hepatocellular carcinoma by targeting poly (ADP-ribose) polymerases and PI3K

doi:

Figure Lengend Snippet: BRIT1 is aberrantly expressed and correlates with poor prognosis in HCC. A. Representative STS-based PCR analysis showed the presence of LOH in the BRIT1 locus in HCC sample 89-17 (n=3 technical repeats). STS3, STS5, STS6, and STS7 were 4 out of 13 sequence-tagged sites (STSs) used in this study. White star, lost band detected with STS5; N, non-cancerous liver tissue; T, HCC tumor tissue. LOH, loss of heterozygosity; STS, sequence-tagged site. B. Representative BRIT1 protein levels in 5 HCC samples (n=3 technical repeats). BRIT1 protein level was decreased in HCC samples as determined by Western blot analysis. C. BRIT1, but not BRCA1/2, was frequently altered in TCGA HCC samples (n=442). BRIT1 alterations include deep deletions, RNA downregulation, and mutations. For RNA expression level, z-score ≤-1.8. Data were retrieved from the cBioPortal as of June 27, 2017. D. BRIT1 deep deletion correlated with poor progression-free survival (PFS) of T1-stages TCGA HCC samples (n=186). HCC patients with diploid BRIT1, n=152 (with 62 relapsed), median =42.02; HCC patients with deep deletion of BRIT1, n=12 (with 7 relapsed), median =13.07. P=0.00836 with Log-rank test. E. BRIT1 low DNA copy number positively correlated with recurrence in the Guichard HCC cohort [34]. Normal, n=111; HCC samples without recurrence, n=27; HCC samples with recurrence, n=47. COPA=-7.344 with mCOPA analysis.

Article Snippet: Total cell extracts were separated with SDS-PAGE, transferred to nitrocellulose membrane and probed with antibodies against human BRIT1 [ 8 ], PAR (IBL-America, Minneapolis, MN; catalog number: 10407), PARP1 (BD Biosciences, San Jose, CA; catalog number: 51-6639GR), phospho-AKT (T308) (Cell Signaling Technology, Danvers, MA; catalog number: 9275), β-actin (Sigma-Aldrich, St. Louis, MO; catalog number: A5044), and α-Tubulin (Sigma-Aldrich, St. Louis, MO; catalog number: T5168).

Techniques: Sequencing, Western Blot, RNA Expression

Journal: American Journal of Cancer Research

Article Title: BRIT1 dysfunction confers synergistic inhibition of hepatocellular carcinoma by targeting poly (ADP-ribose) polymerases and PI3K

doi:

Figure Lengend Snippet: A splicing-acceptor mutant of BRIT1 is identified in HCC. A. BRIT1 mutant K659fsX10 identified in HCC sample 89-17 by PCR-based sequencing (n=3 technical repeats). Red star, position of K659fsX10; blue arrow, overlapping nucleotides including the normal and the mutant. WT, wild-type; MT, mutant. ag refers to the common acceptor site, and AG refers to the cryptic acceptor site. Italic AAAGCAG indicates the lost 7 nucleotides. B. Representative immunofluorescent staining showed that BRIT1 mutant K659fsX10 failed to localize onto the damaged DNA induced by IR. Bar graph illustrates the average percentage of cells with BRIT1 foci formation. At least 100 cells were counted for each condition from individual experiment (n=4 biological repeats). FLAG, FLAG-BRIT1. Red arrow, co-localization of BRIT1 and γ-H2AX; white arrow, no co-localization; n.s., not significant. *, P<0.05.

Article Snippet: Total cell extracts were separated with SDS-PAGE, transferred to nitrocellulose membrane and probed with antibodies against human BRIT1 [ 8 ], PAR (IBL-America, Minneapolis, MN; catalog number: 10407), PARP1 (BD Biosciences, San Jose, CA; catalog number: 51-6639GR), phospho-AKT (T308) (Cell Signaling Technology, Danvers, MA; catalog number: 9275), β-actin (Sigma-Aldrich, St. Louis, MO; catalog number: A5044), and α-Tubulin (Sigma-Aldrich, St. Louis, MO; catalog number: T5168).

Techniques: Mutagenesis, Sequencing, Staining

Journal: American Journal of Cancer Research

Article Title: BRIT1 dysfunction confers synergistic inhibition of hepatocellular carcinoma by targeting poly (ADP-ribose) polymerases and PI3K

doi:

Figure Lengend Snippet: BRIT1-deficient cells are hypersensitive to PARP inhibitor. A. BRIT1 was proficient in SNU449 cells, but deficient in Hep3B and HepG2. DNA copy number retrieved from the Cancer Cell Line Encyclopedia (CCLE). The mRNA and protein levels determined by RT-PCR with the primers mentioned in the Methods section and Western blot analysis, respectively (n=3 biological repeats). B. Generation of BRIT1-knockout HCC cells (449.KO) by CRISPR/Cas9. The BRIT1 null mutations induced by two sets of guide RNA were validated by PCR-based sequencing, showing a 1-bp deletion on both alleles in 449.KO1, and a 10-bp deletion on both alleles in 449.KO2, and by Western blot analysis of BRIT1 protein. C. HR activity was reduced in BRIT1-deficient HCC cells. The HR activity was determined by using DR-GFP/I-SceI-based HR repair assay and represented by the relative percentage of GFP+cells in each cell type. It was calculated by subtracting the percentage of GFP+cells in I-SceI-transfected cells with that in control plasmid pCAGGS-transfected cells, followed by normalizing with transfection efficiency determined by pEGFP-C1 plasmid. Each value in the bar graph was relative to the percentage of GFP+cells in SNU449, which was set at 1, and the data were obtained from repeated experiments (n=3 biological repeats). *, P<0.05 compared to SNU449 cells. D. Colony formation assay (n=3 biological repeats) showed BRIT1-deficient Hep3B and HepG2 cells were sensitive to olaparib. *, P<0.05 compared to SNU449. E. Colony formation assay (n=3 biological repeats) showed BRIT1 knockout 449.KO cells exhibited the hypersensitivity to olaparib. *, P<0.05 compared to SNU449. F. Colony formation assay (n=3 biological repeats) showed ectopic BRIT1 induced the resistance of Hep3B and HepG2 cells to PARPi olaparib. Survival rate was also determined by colony formation assay. *, P<0.05 when compared to respective parental cells.

Article Snippet: Total cell extracts were separated with SDS-PAGE, transferred to nitrocellulose membrane and probed with antibodies against human BRIT1 [ 8 ], PAR (IBL-America, Minneapolis, MN; catalog number: 10407), PARP1 (BD Biosciences, San Jose, CA; catalog number: 51-6639GR), phospho-AKT (T308) (Cell Signaling Technology, Danvers, MA; catalog number: 9275), β-actin (Sigma-Aldrich, St. Louis, MO; catalog number: A5044), and α-Tubulin (Sigma-Aldrich, St. Louis, MO; catalog number: T5168).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Knock-Out, CRISPR, Sequencing, Activity Assay, Transfection, Control, Plasmid Preparation, Colony Assay

Journal: American Journal of Cancer Research

Article Title: BRIT1 dysfunction confers synergistic inhibition of hepatocellular carcinoma by targeting poly (ADP-ribose) polymerases and PI3K

doi:

Figure Lengend Snippet: PARP inhibitor synergizes with PI3K inhibitor to suppress BRIT1-deficient HCC in vitro. A. Colony formation assay showed BRIT1-deficient HCC cells Hep3B and HepG2 exhibited the synergy of PARPi and PI3Ki. Cells were treated with PARPi olaparib (2.5 µM) and PI3Ki BEZ235 (2.5 nM) alone or in combination for 2 weeks, and the inhibitory effects were determined by using colony formation assay (n=3 biological repeats). *, P<0.05 compared to control. B. Colony formation assay showed BRIT1 ablation by CRISPR/Cas9 enhanced the synergy of olaparib and BEZ235 in HCC cells (n=3 biological repeats). *, P<0.05 compared to control. C. Colony formation assay showed ectopic BRIT1 attenuated the synergistic effect of olaparib and BEZ235 (n=3 biological repeats). *, P<0.05. D. Apoptotic rate was increased in BRIT1-deficient cells treated with olaparib or BEZ235 alone, and further enhanced by the combination. Ctrl, control; OLA, olaparib; BEZ, BEZ235; OLA (or O)+BEZ, combination of olaparib and BEZ235. *, P<0.05 compared to SNU449 cells.

Article Snippet: Total cell extracts were separated with SDS-PAGE, transferred to nitrocellulose membrane and probed with antibodies against human BRIT1 [ 8 ], PAR (IBL-America, Minneapolis, MN; catalog number: 10407), PARP1 (BD Biosciences, San Jose, CA; catalog number: 51-6639GR), phospho-AKT (T308) (Cell Signaling Technology, Danvers, MA; catalog number: 9275), β-actin (Sigma-Aldrich, St. Louis, MO; catalog number: A5044), and α-Tubulin (Sigma-Aldrich, St. Louis, MO; catalog number: T5168).

Techniques: In Vitro, Colony Assay, Control, CRISPR

Journal: American Journal of Cancer Research

Article Title: BRIT1 dysfunction confers synergistic inhibition of hepatocellular carcinoma by targeting poly (ADP-ribose) polymerases and PI3K

doi:

Figure Lengend Snippet: DNA damage is significantly induced by PARP inhibitor and PI3K inhibitor. A and B. DSBs were induced by olaparib and BEZ235 alone and exaggerated by combination treatment in BRIT1-deficient 449.KO2 and Hep3B cells. A. Representative neutral-pH comet assay of DNA damage. B. Quantitative analysis of ~100 cells for each condition (n=3 biological repeats). More than 97% untreated SNU449 cells contained tail moments <2, which was set as the parameter for cells with intact DNA. Cells with the parameter >2 were regarded as the one with DSBs. Ctrl, control; OLA, olaparib. BEZ, BEZ235; OLA+BEZ, olaparib+BEZ235. *, P<0.05. C and D. γ-H2AX foci were significantly elevated by treatment of olaparib or BEZ235 alone and aggravated by combination treatment in BRIT1-deficient 449.KO2 and Hep3B cells. C. Representative γ-H2AX immunofluorescent staining of γ-H2AX foci. D. Quantitative analysis of ~100 cells for each condition (n=3 biological repeats). *, P<0.05. E. Western blot analysis showed that olaparib induced a high level of phosphor-AKT in BRIT1-deficient cells (n=5 biological repeats). OLA, olaparib; PAR, poly (ADP-ribose); p-AKT, phosphorylated AKT. *, P<0.05. F. Western blot analysis demonstrated that BEZ235 induced a high level of PAR formation in BRIT1-deficient cells (n=5 biological repeats). PAR, poly (ADP-ribose); p-AKT, phosphorylated AKT. *, P<0.05.

Article Snippet: Total cell extracts were separated with SDS-PAGE, transferred to nitrocellulose membrane and probed with antibodies against human BRIT1 [ 8 ], PAR (IBL-America, Minneapolis, MN; catalog number: 10407), PARP1 (BD Biosciences, San Jose, CA; catalog number: 51-6639GR), phospho-AKT (T308) (Cell Signaling Technology, Danvers, MA; catalog number: 9275), β-actin (Sigma-Aldrich, St. Louis, MO; catalog number: A5044), and α-Tubulin (Sigma-Aldrich, St. Louis, MO; catalog number: T5168).

Techniques: Single Cell Gel Electrophoresis, Control, Staining, Western Blot

Journal: American Journal of Cancer Research

Article Title: BRIT1 dysfunction confers synergistic inhibition of hepatocellular carcinoma by targeting poly (ADP-ribose) polymerases and PI3K

doi:

Figure Lengend Snippet: BRIT1-deficient xenograft tumor growth is remarkably inhibited by PARP inhibitor in combination with PI3K inhibitor in vivo. A. BRIT1-deficient Hep3B tumor growth was deterred by olaparib (n=6 for each group) as compared to control. *, P<0.05. B. Macroscopical images showed that Hep3B xenograft tumor growth was attenuated by olaparib (n=6 mice each group). Bar graph, the average tumor weight after sacrifice. *, P<0.05 when compared to control. C. Western blot analysis demonstrated that PAR formation was abolished by olaparib in Hep3B xenograft tumors (n=3 biological repeats). D. Macroscopical images (left) and bar graph of tumor volume (right) demonstrated that tumor growth of Hep3B xenografts was remarkably inhibited by olaparib and BEZ235 in combination (n=10 mice each group). *, P<0.05 compared to control. E. Representative γ-H2AX staining and apoptotic cells in Hep3B xenografts tumors showed that combination treatment of olaparib and BEZ235 remarkably triggered γ-H2AX foci intensity and apoptosis. γ-H2AX foci formation was determined by immunohistochemistry, and apoptotic cells were stained by using TUNEL assay (n=3 mice for each condition). *, P<0.05 compared with control or single drug treatment.

Article Snippet: Total cell extracts were separated with SDS-PAGE, transferred to nitrocellulose membrane and probed with antibodies against human BRIT1 [ 8 ], PAR (IBL-America, Minneapolis, MN; catalog number: 10407), PARP1 (BD Biosciences, San Jose, CA; catalog number: 51-6639GR), phospho-AKT (T308) (Cell Signaling Technology, Danvers, MA; catalog number: 9275), β-actin (Sigma-Aldrich, St. Louis, MO; catalog number: A5044), and α-Tubulin (Sigma-Aldrich, St. Louis, MO; catalog number: T5168).

Techniques: In Vivo, Control, Western Blot, Staining, Immunohistochemistry, TUNEL Assay

Journal: American Journal of Cancer Research

Article Title: BRIT1 dysfunction confers synergistic inhibition of hepatocellular carcinoma by targeting poly (ADP-ribose) polymerases and PI3K

doi:

Figure Lengend Snippet: Schematic diagram of the proposed model illustrates the enhanced synthetic lethality achieved by co-targeting of PARPi and PI3Ki in BRIT1-deficient HCC. In BRIT1-deficient HCC, BRIT1 deficiency causes DNA damage. Under this condition, PARP inhibition by PARPi leads to the increase of DNA damage, accompanied with a high PI3K/AKT activity for compensatory DNA repair. Meanwhile, PI3K inhibition by PI3Ki leads to the accumulation of DNA damage as well as an increased PARP activity. As such, concurrent inhibition of PI3K and PARP remarkably enhances the synthetic lethality under BRIT1 deficiency, leading to much more severe DNA damages than in the context of BRIT1 proficiency.

Article Snippet: Total cell extracts were separated with SDS-PAGE, transferred to nitrocellulose membrane and probed with antibodies against human BRIT1 [ 8 ], PAR (IBL-America, Minneapolis, MN; catalog number: 10407), PARP1 (BD Biosciences, San Jose, CA; catalog number: 51-6639GR), phospho-AKT (T308) (Cell Signaling Technology, Danvers, MA; catalog number: 9275), β-actin (Sigma-Aldrich, St. Louis, MO; catalog number: A5044), and α-Tubulin (Sigma-Aldrich, St. Louis, MO; catalog number: T5168).

Techniques: Inhibition, Activity Assay