Journal: Genes & Development

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand cross-links

doi: 10.1101/gad.336446.120

Figure Lengend Snippet: BRCA2 variants identified in individuals with atypical Fanconi anemia. ( A ) Family pedigree showing a sibling pair with Fanconi anemia (red circles) who are compound heterozygous for BRCA2 variants c.2330dupA (maternal inheritance) and c.8524C>T (paternal inheritance). Family history of breast cancer (purple, all diagnosed in 60s and 70s), skin cancer (gray), and colon cancer (green; diagnosed at 40 yr old). ( B ) Schematic of BRCA2 domain structure and key interacting proteins. ( C ) Alignment of exon 20 BRCA2 DBD peptide sequence demonstrating that it is evolutionary conserved across many species. In green are the amino acid residues modified by the patient variants, p.W2830_K2833del (c.8488-1G>A) and p.R2842C (c.8524C>T). Purple arrows indicate amino acid residues that contact DNA . ( D ) Immunoblot showing BRCA2 levels in WT (RA2985) control, FA-D1 (RA2525), and patient RA3105 and RA3106 LCLs. ( E ) Quantification of chromosome breaks following DEB treatment of WT (RA2985), FA-A (RA2939), and patient RA3105 and RA3106 LCLs. ( F , G ) Cell survival assays of patient-derived lymphoblast cell lines (LCLs) RA3105, FA-A (RA2939), WT (RA2985), and FA-D1 (RA2525) after MMC and PARP inhibitor olaparib (PARPi) treatment. Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate SD. ( H ) Quantification of chromosome breaks following MMC treatment of BJ wild-type fibroblasts, FA-A patient fibroblasts, and HSC62 fibroblasts. ( I ) Cell survival of HSC62 (c.8488-1G>A) fibroblasts compared with BJ WT fibroblast and complemented FA-A patient cells (RA3087) expressing wild-type FANCA (FA-A+A) or empty vector (FA-A+EV). Cells were treated with increasing concentrations of MMC. Relative cell survival was normalized to untreated controls to give the percent survival. Error bars indicate SD. ( J ) Cell survival of MMC-treated HSC62 uncorrected patient cell line (HSC62 mut ) compared with BJ WT fibroblast and CRISPR/Cas9 corrected wild-type HSC62 (HSC62 WT ) clones 1-3. ( K , L ) Cell survival of BJ WT fibroblasts, and CRISPR/Cas9-targeted BJ fibroblasts: BJ WT fibroblast clone ( BRCA2 WT ), c.8488-1G>A BJ clones ( BRCA2 8488-1G>A ), c.8524C>T BJ clones ( BRCA2 8524C>T ), and exon 20 BRCA2 frameshift mutant ( BRCA2 Trun. ). Cells were treated with increasing concentrations of MMC or PARPi. Error bars indicate SD. Kruskal-Wallis ANOVA, with Dunn's post-test. (***) P < 0.001; (****) P < 0.0001.

Article Snippet: Patient-derived fibroblast cell lines ( Supplemental Table S2 ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Sequencing, Modification, Western Blot, Control, Derivative Assay, Expressing, Plasmid Preparation, CRISPR, Clone Assay, Mutagenesis

Journal: Genes & Development

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand cross-links

doi: 10.1101/gad.336446.120

Figure Lengend Snippet: Defective ICL repair in BRCA2 DBD mutants results in increased ssDNA that is WRN and DNA2 dependent. ( A ) Immunofluorescence images of RAD51 foci, 8 h following 12 Gy ionizing radiation (IR) of BJ WT fibroblast and patient derived HSC62 fibroblast, detected with anti-RAD51 antibody. Third row images are individual cells enlarged to better demonstrate differences in RAD51 focus size. ( B ) Quantification of RAD51 foci 1 h, 8 h, and 24 h following 12 Gy IR of BJ WT fibroblast and HSC62 fibroblast. Error bars indicate SD of two independent experiments (≥200 cells per experiment). ( C ) Quantification of RAD51 foci 8 h after 12 Gy IR of BJ WT fibroblast, wild-type HSC62 (HSC62 WT ) clones 1–3, and HSC62 uncorrected patient cell line (HSC62 mut ). ( D ) Quantification of RAD51 foci 24 h following 1-h treatment with 3 µM MMC. Error bars indicate SD of three independent experiments (≥200 cells per experiment). ( E ) Quantification of RAD51 foci in isogenic BJ fibroblasts clones at 1 h, 8 h, and 24 h following 6 Gy IR of BJ WT fibroblasts, BJ WT fibroblast clone (BRCA2 WT ), BRCA2 8488-1G>A BJ clones 2–3, BRCA2 8524C>T BJ clones 1–2, and a BRCA2 homozygous truncation mutant, c.8531dupA ( BRCA2 Trun ). Error bars indicate SD of three independent experiments (≥200 cells per experiment). ( F ) Representative images of RAD51 foci in isogenic BJ fibroblasts clones, 8 h after 6 Gy IR, detected by immunofluorescence with anti-RAD51 antibody. Third row images are individual cells enlarged to better demonstrate differences in RAD51 focus size. ( G ) Quantification of RPA foci 24 h following 1-h treatment with 3 μM MMC of BJ WT fibroblast, CRISPR/Cas9 corrected wild-type HSC62 clones (HSC62 WT ), and HSC62 uncorrected patient cell line (HSC62 mut ). ( H ) Quantification of RPA foci 24 h following 1-h treatment with 3 μM MMC in HSC62 mut cells depleted of DNA2, MRE11, EXO1, CTIP, WRN, or BLM by siRNA compared with luciferase control (Luc). Error bars indicate SD of four independent experiments. ( I ) Quantification of RPA foci 24 h following 1 h treatment with 3 μM MMC in HSC62 mut cells depleted of DNA2, WRN, BLM, or codepleted of WRN and BLM by siRNA compared with luciferase control (Luc). Error bars indicate SD of three independent experiments. ( J ) Immunoblot analysis of RPA phosphorylation in isogenic BJ fibroblasts clones 24 h after 1-h treatment with 3 μM MMC. BRCA2 WT , BRCA2 8524C>T , and BRCA2 8488-1G>A BJ fibroblast cells were transfected with siRNA control luciferase (Luc) or siRNAs targeting DNA2 or WRN. ( K , L ) MMC cell survival of BJ BRCA2 WT , BRCA2 8488-1G>A , and BRCA2 8524C>T fibroblasts overexpressing (OE) WT RAD51 or empty vector (EV) control. Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate SD.

Article Snippet: Patient-derived fibroblast cell lines ( Supplemental Table S2 ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Immunofluorescence, Derivative Assay, Clone Assay, Mutagenesis, CRISPR, Luciferase, Control, Western Blot, Phospho-proteomics, Transfection, Plasmid Preparation

Journal: Genes & Development

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand cross-links

doi: 10.1101/gad.336446.120

Figure Lengend Snippet: Proper ICL repair is required to prevent aberrant nuclease processing. ( A ) Quantification of RPA foci 8 h, 24 h, and 48 h following 1-h treatment with 3 μM MMC of FA patient-derived fibroblasts compared with BJ wild-type fibroblasts. Patient cells lines from FA complementation group FA-R (RAD51/FANCR), FA-A (FANCA), FA-L (FANCL), FA-D2 (FANCD2), FA-I (FANCI), FA-J (FANCJ), and FA-P (SLX4/FANCP). FA-A patient complemented cell lines were generated by transducing WT FANCA cDNA or EV. Error bars indicate SD of two independent experiments. ( B ) FA-A patient cells expressing WT FANCA (FA-A+FANCA) or empty vector (FA-A+EV) were transfected with siRNA control luciferase (Luc) or siRNAs targeting DNA2 and WRN. Quantification of RPA foci 24 h following 1-h treatment with 3 μM MMC. Error bars indicate SD of two independent experiments. ( C ) FA-A+EV were transfected with siRNA Luc or siRNAs targeting DNA2 and BLM. Quantification of RPA foci 24 h following 1-h treatment with 3 μM MMC. Error bars indicate SD of two independent experiments. ( D ) FA-G patient cells expressing WT FANCG (FA-G+FANCG) or empty vector (FA-G+EV) were transfected with siRNA control luciferase (Luc) or siRNAs targeting DNA2, WRN, and BLM. Quantification of RPA foci 24 h following 1-h treatment with 3 μM MMC. Error bars indicate SD of three independent experiments.

Article Snippet: Patient-derived fibroblast cell lines ( Supplemental Table S2 ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Derivative Assay, Generated, Expressing, Plasmid Preparation, Transfection, Control, Luciferase

Journal: Genes & Development

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand cross-links

doi: 10.1101/gad.336446.120

Figure Lengend Snippet: BRCA2 DBD and C-terminal domain variants confer a moderate defect in HR and disrupt replication fork protection function. ( A ) Levels of mClover-positive cells were normalized to WT HEK293T (siLuc). Error bars indicate SD of three independent experiments performed in triplicate. P -values were determined by ANOVA and Tukey's multiple comparison. (****) P < 0.0001. ( B ) Sister chromatid exchange (SCE) assay in BJ WT fibroblast and HSC62 patient derived fibroblast following treatment with 0.1 μg/mL or 0.2 μg/mL MMC. ( C ) Isogenic BJ fibroblast BRCA2 mutants, BRCA2 Trun . , BRCA2 8524C>T , and BRCA2 8488-1G>A were analyzed for replication fork resection. Cells were labeled with DNA analogs, IdU for 20 min, and then CldU for 20 min. Cells were then incubated in 6 mM HU with and without MRE11 inhibitor mirin (50 µM) for 4 h before being harvested. DNA fibers were prepared and visualized by immunofluorescence detection of IdU and CldU and measured. Error bars indicate SD. ( D ) Isogenic BJ fibroblast BRCA2 mutants, BRCA2 Trun. , BRCA2 8524C>T , BRCA2 8488-1G>A , and BRCA2 S3291A were transfected with siRNA control luciferase (Luc) or siRNAs targeting DNA2 or MRE11. Cells were treated and labeled with DNA analogs as above. Error bars indicate SD. ( E ) BJ fibroblast with BRCA2 variants, BRCA2 8524C>T and BRCA2 8488-1G>A , were analyzed for replication fork resection when depleted of RADX by shRNA or transduced with shRNA control (shCONT.). Cells were treated and labeled with DNA analogs as above. Data of two replicates plotted. Error bars indicate SD. ( F ) Quantification of chromosome breaks in isogenic BJ fibroblast BRCA2 mutants following 5 h of 6 mM HU and released into colcemid. Breakage was not significantly increased in BRCA2 8524C>T and BRCA2 8488-1G>A compared with BRCA2 WT . Kruskal-Wallis ANOVA, with Dunn's post-test. (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001.

Article Snippet: Patient-derived fibroblast cell lines ( Supplemental Table S2 ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Comparison, Derivative Assay, Labeling, Incubation, Immunofluorescence, Transfection, Control, Luciferase, shRNA, Transduction

Journal: Genes & Development

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand cross-links

doi: 10.1101/gad.336446.120

Figure Lengend Snippet: SNF2 translocases are not required for ICL repair. ( A ) BJ fibroblast mutants BRCA2 8524C>T and BRCA2 8488-1G>A were analyzed for replication fork resection when depleted of either SMARCAL1 or ZRANB3 by shRNA or transduced with control shRNA (shLuc). Cells were labeled with DNA analogs, IdU for 20 min and then CldU for 20 min. Cells were then incubated in 6 mM HU for 4 h before being harvested. DNA fibers were prepared and visualized by immunofluorescence detection of IdU and CldU and measured. Error bars indicate SD ( B ) Quantification of RPA foci in isogenic BJ fibroblasts clones 24 h following 1-h treatment with 3 µM MMC in cells depleted of SMARCAL1 or ZRANB3. Error bars indicate SD of two independent experiments. ( C ) Quantification of RPA foci in BJ fibroblasts clones 24 h following 1-h treatment with 3 µM MMC in cells depleted of HLTF. Error bars indicate SD of two independent experiments. ( D ) MMC cell survival of isogenic BJ BRCA2 WT fibroblasts depleted of SMARCAL1 or ZRANB3 by shRNA or transduced with shRNA luciferase control (shLuc). Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate SD. ( E , F ) MMC and CPT cell survival assay of isogenic BJ BRCA2 8488-1G>A or BRCA2 8524C>T clones depleted of either SMARCAL1 or ZRANB3 by shRNA or transduced with shRNA luciferase control (shLuc). Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate SD. Kruskal-Wallis ANOVA, with Dunn's post-test. (***) P < 0.001; (****) P < 0.0001.

Article Snippet: Patient-derived fibroblast cell lines ( Supplemental Table S2 ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: shRNA, Transduction, Control, Labeling, Incubation, Immunofluorescence, Clone Assay, Luciferase, Clonogenic Cell Survival Assay

Journal: Genes

Article Title: Up-Regulation of Non-Homologous End-Joining by MUC1

doi: 10.3390/genes15060808

Figure Lengend Snippet: MUC1 stimulates non-homologous end-joining (NHEJ), while partially suppressing homologous recombination (HR). ( A ) Western blotting analysis of the expression of MUC1 in U2OS-DR and U2OS-EJ5. MUC1 was stably expressed in U2OS-DR cells and U2OS-EJ5 cells, which specifically detect homologous recombination (HR) and non-homologous end-joining (NHEJ), respectively. Tubulin was used as a loading control. ( B ) MUC1-overexpression stimulates NHEJ and partially suppresses HR. Cells were transfected by a plasmid with I-SceI to introduce DSBs. Successful repair of DSBs by HR or NHEJ results in the expression of GPF. DSB repair activity was expressed as %GPF positive cells by FACS. Gray and black columns indicate the DSB repair activity without and with the MUC1-overexpression, respectively. Bars indicate standard deviations obtained from at least three independent experiments. Student’s t -tests showed ** p < 0.01 and * p < 0.05. ( C ) The addition of deoxyribonucleosides (dNs) in the medium stimulates NHEJ but not HR. After pre-incubation of DR or EJ5 cells in the presence of 50 µM dNs for 16 h, DSB repair activities were measured by the cell-based DSB repair assay. At least three independent experiments were performed. Bars represent standard deviations. * p < 0.05, and ** p < 0.01 were obtained by Student’s t -test.

Article Snippet: Pancreatic cancer cell line S2.013 with MUC1 overexpression (S2.013 MUC1) and MUC1-knockdown cell lines CAPAN2-shMUC1 and CFPAC1-shMUC1 [ , , ] are a generous gift from Dr. Pankaj Singh (OU Health Stephenson Cancer Center, University of Oklahoma Health Sciences Center).

Techniques: Non-Homologous End Joining, Homologous Recombination, Western Blot, Expressing, Stable Transfection, Control, Over Expression, Transfection, Plasmid Preparation, Introduce, Activity Assay, Incubation

Journal: Genes

Article Title: Up-Regulation of Non-Homologous End-Joining by MUC1

doi: 10.3390/genes15060808

Figure Lengend Snippet: Pharmacological inhibitions of NHEJ preferentially kill MUC1-expressed pancreatic cancer cell lines. The expression of MUC1 in each cell line was confirmed by Western blotting, shown on the left. ( A ) S2.013 with and without MUC1 overexpression, ( B ) CAPAN2 with and without suppression of endogenous MUC1 by shRNA, and ( C ) CFPAC1 with and without suppression of endogenous MUC1 by shRNA. The MUC1-expressed cells showed a higher sensitivity to NU7441 (DNA-PK inhibitor), HDAC inhibitor LBH589 (pan-HDAC inhibitor), and FK228 (selective HDAC1 and 2 inhibitor), compared to the cells with low/little MUC1 expression. The cytotoxicity of each chemical was examined by the colony-forming assay. Cells were treated with indicated concentrations of chemicals until colonies formed. The error bars represent standard deviations from three independent experiments. * p < 0.05 and ** p < 0.01 determined by Student’s t -test.

Article Snippet: Pancreatic cancer cell line S2.013 with MUC1 overexpression (S2.013 MUC1) and MUC1-knockdown cell lines CAPAN2-shMUC1 and CFPAC1-shMUC1 [ , , ] are a generous gift from Dr. Pankaj Singh (OU Health Stephenson Cancer Center, University of Oklahoma Health Sciences Center).

Techniques: Expressing, Western Blot, Over Expression, shRNA

Journal: Genes & Development

Article Title: ATDC binds to KEAP1 to drive NRF2-mediated tumorigenesis and chemoresistance in pancreatic cancer

doi: 10.1101/gad.344184.120

Figure Lengend Snippet: ATDC regulates oxidative stress and chemoresistance in pancreatic cancer cells. Dose response curves for gemcitabine (GEM) ( A ) and 5-fluorouracil (5-FU) ( B ) treatment in S2-013 or HPAC cells with either ATDC overexpression (S2-013) or ATDC shRNA expression (HPAC). ( C ) Steady state ROS levels in control (Scramble, Scr) and sh ATDC Capan-2 and HPAC cells, and control (vector [Vec]) and ATDC-overexpressing S2-013 and MIA Paca-2 cells. ( D ) Ratio of reduced (GSH) to oxidized (GSSG) glutathione in the PDA cell lines listed in C . ( E ) Lipid peroxidation levels in the same isogenic PDA cell lines listed in C . Data are representative of three independent experiments. (*) P < 0.05, (**) P < 0.01, (***) P < 0.005. Mean ± SEM.

Article Snippet: Isogenic pancreatic cancer cells (5 × 10 5 ; S2-013 or HPAC) were orthotopically implanted in the pancreas of 8-wk-old female immunocompromised NSG mice (NOD.Cg-Prkdc scid ;Il2rg tm1Wjl/SzJ ; The Jackson Laboratory 005557) ( n = 10 animals/group).

Techniques: Over Expression, shRNA, Expressing, Control, Plasmid Preparation