Journal:

Article Title: A Rad50-dependent pathway of DNA repair is deficient in Fanconi anemia fibroblasts

doi: 10.1093/nar/gkh649

Figure Lengend Snippet: Effect of introduction of anti-Rad50, anti-Mre11 or anti-Nbs1 antibodies on DNA end-joining frequency in normal and FA fibroblasts. (A) The rejoining frequency of cohesive-ended DNA was determined in the HDFs in the absence of antibody (white bars) or in the presence of anti-Rad50 antibody (black bars). HDF, normal HDFs; FA-A, FA fibroblasts belonging to complementation group A; FA-C, FA fibroblasts belonging to complementation group C; FA-G, FA fibroblasts belonging to complementation group G. *P < 0.0001 compared to the same cell strain in the absence of antibody, χ2-test. (B) The rejoining frequency of blunt-ended DNA was determined in the same cells as in (A). *P < 0.0001 compared to the same cell type in the absence of antibody, χ2-test. (C) The rejoining frequency of cohesive- and blunt-ended DNA was determined in normal diploid fibroblasts co-electroporated with non-specific IgG (1), anti-Rad50 antibody (2), anti-Mre11 antibody (3), anti-Nbs1 antibody (4), both anti-Rad50 and anti-Mre11 antibodies (5), both anti-Rad50 and anti-Nbs1 antibodies (6), both anti-Mre11 and anti-Nbs1 antibodies (7), and anti-Rad50, anti-Mre11 and anti-Nbs1 antibodies (8). In all cases, antibody treatment significantly reduced plasmid end-joining levels compared to those observed in cells not treated with antibody, P < 0.0001, χ2-test.

Article Snippet: Mouse polyclonal anti-DNA ligase III, rabbit polyclonal anti-Fancd2, rabbit polyclonal anti-Rad50, rabbit polyclonal anti-Mre11 and rabbit polyclonal anti-Nbs1 antibodies were obtained from Novus Biologicals, Inc. (Littleton, CO).

Techniques: Plasmid Preparation

Journal:

Article Title: A Rad50-dependent pathway of DNA repair is deficient in Fanconi anemia fibroblasts

doi: 10.1093/nar/gkh649

Figure Lengend Snippet: Introduction of antibodies directed against proteins in the RMN complex sensitizes normal diploid fibroblasts to agents that damage chromosomal DNA. (A) Cell death was determined following treatment with 20 U of the restriction endonuclease PvuII in cells co-electroporated with non-specific IgG (1), anti-Rad50 antibody (2), anti-Mre11 antibody (3), anti-Nbs1 antibody (4), both anti-Rad50 and anti-Mre11 antibodies (5), both anti-Rad50 and anti-Nbs1 antibodies (6), both anti-Mre11 and anti-Nbs1 antibodies (7), and anti-Rad50, anti-Mre11 and anti-Nbs1 antibodies (8). (B) Cell death was determined after treatment with 200 ng/ml diepoxybutane of cells electroporated with the same antibodies as in (A).

Article Snippet: Mouse polyclonal anti-DNA ligase III, rabbit polyclonal anti-Fancd2, rabbit polyclonal anti-Rad50, rabbit polyclonal anti-Mre11 and rabbit polyclonal anti-Nbs1 antibodies were obtained from Novus Biologicals, Inc. (Littleton, CO).

Techniques:

Journal: Journal of Clinical Investigation

Article Title: Molecular disruption of RAD50 sensitizes human tumor cells to cisplatin-based chemotherapy

doi: 10.1172/jci33816

Figure Lengend Snippet: Figure 1 Native MRN expression in head and neck SCC cells. Native expression of MRE11, RAD50, and NBS1 was confirmed in both JHU012 and JHU029 SCC cells by Western blot. Lane 1, JHU029 cells; lane 2, JHU012 cells; lane 3, MagicMark Western Protein Standard with markers of 20, 30, 40, 50, 60, 80, 100, 120, and 220 kDa from bottom to top. Lanes were run on the same gel but were noncontiguous as indicated by the vertical white lines.

Article Snippet: The primary antibodies used were a polyclonal rabbit anti-human RAD50 (zinc hook domain) (Novus Biologicals), which recognizes both wild-type and mutant RAD50, and the same polyclonal rabbit anti-human antibodies targeted against MRE11 and NBS1 that were used in the preceding screening experiment.

Techniques: Expressing, Western Blot

Journal: Journal of Clinical Investigation

Article Title: Molecular disruption of RAD50 sensitizes human tumor cells to cisplatin-based chemotherapy

doi: 10.1172/jci33816

Figure Lengend Snippet: Figure 2 Construction and function of the Ad-RAD50 vector. (A) A 326–base pair mutant RAD50 hook fragment from the wild-type RAD50 gene was cloned into a recombinant adenovirus vector to construct Ad-RAD50. The amino acid sequence of the integrated construct (residues 631–739) is highlighted in black, with the zinc hook region marked in yellow. PA, polyadenylation site. (B) Both the wild-type RAD50 protein and the mutant RAD50 protein were detected by Western blot in JHU012 tumor cells that were infected with Ad-RAD50 adenovirus. Lane 1, MagicMark Western Protein Standard; lane 2, JHU012 cells with no treatment; lane 3, JHU012 cells infected with Ad-RAD50.

Article Snippet: The primary antibodies used were a polyclonal rabbit anti-human RAD50 (zinc hook domain) (Novus Biologicals), which recognizes both wild-type and mutant RAD50, and the same polyclonal rabbit anti-human antibodies targeted against MRE11 and NBS1 that were used in the preceding screening experiment.

Techniques: Plasmid Preparation, Mutagenesis, Clone Assay, Recombinant, Construct, Sequencing, Western Blot, Infection

Journal: Journal of Clinical Investigation

Article Title: Molecular disruption of RAD50 sensitizes human tumor cells to cisplatin-based chemotherapy

doi: 10.1172/jci33816

Figure Lengend Snippet: Figure 3 Mutant RAD50 protein interacts with endogenous RAD50 and downregulates the MRN complex. (A) Anti-MRE11 antibody coimmunoprecipitated wild-type RAD50 in all cells, suggesting a direct interaction between these proteins. Mutant RAD50 was only coimmunoprecipitated in infected cells treated with zinc, indicating that the interaction between mutant RAD50 and the wild-type MRN complex is mediated by zinc-dependent dimerization of RAD50 hook domains and not direct interactions between mutant RAD50 and MRE11. All lanes shown were run simultaneously on a single gel as contiguous lanes. (B) Multiple MRN nuclear foci are seen at sites of DNA damage in JHU012 cells treated with Ad-RAD50 and cisplatin. Original magnification, ×63. There is spatial overlap of the signals corresponding to wild-type RAD50 (blue) and wild-type MRE11 (red), highly suggestive of a binding interaction between these proteins. (C) Nuclear foci from JHU012 cells treated with Ad-RAD50 and cisplatin (original magnification, ×63) demonstrate spatial overlap of the wild-type (red) and mutant RAD50 (blue) proteins, further suggesting direct bind- ing between these proteins. (D) Downregulation of wild-type MRE11 (P < 0.005), RAD50 (P < 0.001), and NBS1 proteins (P < 0.04) is seen after infection of JHU012 cells with Ad-RAD50 relative to noninfected controls. (Mean optical density of Western blot bands ± SEM is shown.) This is attributable to the demonstrated dimerization between mutant and wild-type RAD50 and inability of the mutant protein to directly bind MRE11. Consequently, only a single MRE112/RAD502 heterotetramer can form around the wild-type RAD50 with no complimentary heterotetramer assembling around mutant RAD50.

Article Snippet: The primary antibodies used were a polyclonal rabbit anti-human RAD50 (zinc hook domain) (Novus Biologicals), which recognizes both wild-type and mutant RAD50, and the same polyclonal rabbit anti-human antibodies targeted against MRE11 and NBS1 that were used in the preceding screening experiment.

Techniques: Mutagenesis, Infection, Binding Assay, Western Blot

Journal: Journal of Clinical Investigation

Article Title: Molecular disruption of RAD50 sensitizes human tumor cells to cisplatin-based chemotherapy

doi: 10.1172/jci33816

Figure Lengend Snippet: Figure 4 Ad-RAD50 enhances cisplatin-induced suppression of SCC cell growth. (A) Combining Ad-RAD50 and cisplatin in JHU012 SCC cells produced a reduction in cell growth within 48 hours and a persistent suppression of cell proliferation relative to all other groups (P < 0.01). DL312 indicates DL312 control virus. (B) The addition of Ad-RAD50 to cisplatin treatment in JHU029 cells produced the greatest reduction in cell growth versus all other groups (P < 0.01). Cells were cloned from parental cell lines. Mean optical density ± SEM is shown.

Article Snippet: The primary antibodies used were a polyclonal rabbit anti-human RAD50 (zinc hook domain) (Novus Biologicals), which recognizes both wild-type and mutant RAD50, and the same polyclonal rabbit anti-human antibodies targeted against MRE11 and NBS1 that were used in the preceding screening experiment.

Techniques: Produced, Control, Virus, Clone Assay

Journal: Journal of Clinical Investigation

Article Title: Molecular disruption of RAD50 sensitizes human tumor cells to cisplatin-based chemotherapy

doi: 10.1172/jci33816

Figure Lengend Snippet: Figure 5 Ad-RAD50 enhances cisplatin-induced DNA damage. (A) Neutral comet assay was used to quantify the level of DNA DSB damage in JHU012 (shown) SCC cells. Damaged, fragmented DNA migrates toward the anode, producing a comet tail. Original magnification, ×20. (B) MTM as measured in the JHU012 cell line. The MTM is a correlate of DNA DSB damage. MTM ± SEM is shown. Ad-RAD50 combined with cisplatin increased the number of DNA DSBs by 437% relative to cisplatin monotherapy (P < 0.001) and by 173% compared with Ad-RAD50 alone (P < 0.02), suggesting that the enhanced cytotoxic effect of combined therapy is secondary to increased DNA DSB damage. (C) MTM as measured in the JHU029 cell line. MTM ± SEM is shown. Ad-RAD50 combined with cisplatin significantly increased DSB damage compared with all other groups (P < 0.003).

Article Snippet: The primary antibodies used were a polyclonal rabbit anti-human RAD50 (zinc hook domain) (Novus Biologicals), which recognizes both wild-type and mutant RAD50, and the same polyclonal rabbit anti-human antibodies targeted against MRE11 and NBS1 that were used in the preceding screening experiment.

Techniques: Neutral Comet Assay

Journal: Journal of Clinical Investigation

Article Title: Molecular disruption of RAD50 sensitizes human tumor cells to cisplatin-based chemotherapy

doi: 10.1172/jci33816

Figure Lengend Snippet: Figure 6 Ad-RAD50 and cisplatin combination treatment enhances telomere shortening in JHU012 SCC cells. (A) PNA FISH stains telomeres with a red signal, the intensity of which is proportional to telomere length. Original magnification, ×100. (B) Soft- ware-based quantification of telomere staining intensity indicated that combining Ad-RAD50 with cisplatin markedly attenuated telomere length by 73.9% versus cisplatin alone and by 65.2% versus Ad-RAD50 (P < 0.01). Telomere shortening beyond a critical threshold induces apoptosis, contributing to the cytotoxic effect of combination therapy in tumor cells. Mean ± SEM is shown.

Article Snippet: The primary antibodies used were a polyclonal rabbit anti-human RAD50 (zinc hook domain) (Novus Biologicals), which recognizes both wild-type and mutant RAD50, and the same polyclonal rabbit anti-human antibodies targeted against MRE11 and NBS1 that were used in the preceding screening experiment.

Techniques: Staining

Journal: Journal of Clinical Investigation

Article Title: Molecular disruption of RAD50 sensitizes human tumor cells to cisplatin-based chemotherapy

doi: 10.1172/jci33816

Figure Lengend Snippet: Figure 7 Ad-RAD50 chemosensitizes JHU012 human head and neck cancer to cisplatin in vivo. (A) Externally measured tumor volume ± SEM from the time of initial tumor palpability on day 4, to treatment initiation on day 13, and through the treatment period to day 20. (B) Mean internally mea- sured tumor volume ± SEM immediately prior to treatment on day 13 and at the time of animal sacrifice on day 24. (C) Mean internally measured change in tumor volume ± SEM. During the 11 day period between treatment initiation and animal sacrifice, a single administration of Ad-RAD50 combined with a single intraperitoneal dose of cisplatin, at either 3 mg/kg or 5 mg/kg, was able to significantly enhance the therapeutic efficacy of cisplatin (3 mg/kg or 5 mg/kg) monotherapy. For Ad-RAD50 with cisplatin (3 mg/kg), P < 0.001 vs. control; P < 0.05 vs. cisplatin (3 mg/kg) and cisplatin (5 mg/kg) with DL312; and P < 0.05 vs. Ad-RAD50 alone. For Ad-RAD50 with cisplatin (5 mg/kg), P < 0.001 vs. control, cisplatin (3 mg/kg), cisplatin (5 mg/kg) with DL312, and Ad-RAD50 alone.

Article Snippet: The primary antibodies used were a polyclonal rabbit anti-human RAD50 (zinc hook domain) (Novus Biologicals), which recognizes both wild-type and mutant RAD50, and the same polyclonal rabbit anti-human antibodies targeted against MRE11 and NBS1 that were used in the preceding screening experiment.

Techniques: In Vivo, Drug discovery, Control

Journal: Journal of Clinical Investigation

Article Title: Molecular disruption of RAD50 sensitizes human tumor cells to cisplatin-based chemotherapy

doi: 10.1172/jci33816

Figure Lengend Snippet: Figure 8 Quantification of apoptosis in human head and neck cancer tumors. (A) TUNEL staining was performed on tumor SCC samples after tumor harvest. A selection of 1 high-powered field (original magnification, ×40) from each treatment group is shown. (B) Mean percentage apoptosis per high-powered field ± SEM is shown. The combination of Ad-RAD50 and cisplatin enhanced induction of apoptosis relative to all other groups (P < 0.001). This is consistent with the tumor volume reduction seen in the combination therapy groups.

Article Snippet: The primary antibodies used were a polyclonal rabbit anti-human RAD50 (zinc hook domain) (Novus Biologicals), which recognizes both wild-type and mutant RAD50, and the same polyclonal rabbit anti-human antibodies targeted against MRE11 and NBS1 that were used in the preceding screening experiment.

Techniques: TUNEL Assay, Staining, Selection

Journal: Oncogene

Article Title: The p53-inducible gene 3 (PIG3) contributes to early cellular response to DNA damage.

doi: 10.1038/onc.2009.438

Figure Lengend Snippet: Figure 9 p53-inducible gene 3 (PIG3) affects the recruitment of DNA damage sensors and mediators at the sites of DNA damage. The control- and PIG3-depleted HeLa cells were treated with 200 ng/ml neocarzinostatin (NCS). At 1 h after NCS treatment, cells were fixed and immunostained with anti-PIG3, anti-53BP1, anti-NBS1, anti-Mre11, anti-RAD50 or anti-phospho-ataxia telangiectasia mutated (ATM). The nuclei were visualized by 40-6-diamidino-2-phenylindole (DAPI) staining.

Article Snippet: The following antibodies were used for immunofluorescence staining: anti-53BP1 polyclonal antibody (Santa Cruz, 1:50), antiMre11 monoclonal antibody (BD Pharmingen, San Jose, CA, USA, 1:200), anti-Nbs1 polyclonal antibody (Cell Signaling Technology, Boston, MA, USA, 1:200), anti-Rad50 monoclonal antibody (BD Pharmingen, 1:200) and anti-ATM protein kinase pS1981 monoclonal antibody (Rockland, Immunochemicals Ins., 1:500, Philadelphia, PA, USA).

Techniques: Control, Staining