recombinant soluble mouse trail  (Biomol GmbH)

Journal:

Article Title: Treating Metastatic Solid Tumors With Bortezomib and a Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor Agonist Antibody

doi: 10.1093/jnci/djn113

Figure Lengend Snippet: Bortezomib (Bzb)-induced sensitization of renal and mammary mouse tumors to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL). A) Renca-FLAG, Renca-FLIP, and 4T1 tumor cells were untreated or pretreated with 20 nM Bzb for 3 hours. Cells were then treated with soluble recombinant mouse TRAIL (1 µg/mL) or medium. B) The cells were treated the same way except that instead of TRAIL they were treated with protein A–bound immobilized TRAIL receptor agonist monoclonal antibody MD5-1 (5 µg/mL). Cell viability was determined 18 hours later using 3-[4,5-dimethyiazol-2-yl-5]-[3-carboxymethyloxyphenyl]-2-[4-sulfophenyl]-2H tetrazolium assay. Percent decrease in cell number of treated cells compared with untreated cells is shown as means and 95% confidence intervals (n = 3) from one representative experiment of four (A) and three (B) independent experiments. *P < .001 (analysis of variance, two-sided) with respect to single agents.

Article Snippet: After 3–12 hours, cells were treated overnight with soluble recombinant mouse or human TRAIL (1000 ng/mL) (BioMol, Plymouth Meeting, PA) that had been cross-linked by prior treatment with mouse monoclonal anti-polyhistidine (R&D Systems, Minneapolis, MN; 2 μg antibody per μg TRAIL).

Techniques: Recombinant

Journal:

Article Title: Treating Metastatic Solid Tumors With Bortezomib and a Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor Agonist Antibody

doi: 10.1093/jnci/djn113

Figure Lengend Snippet: Bortezomib (Bzb)-induced sensitization to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–mediated apoptosis. Renca-FLAG and 4T1 tumor cells were untreated (Med) or treated with 20 nM Bzb for 3 hours before the addition of soluble recombinant mouse TRAIL (1 µg/mL) for a further 14–18 hours. The pan-caspase inhibitor zVAD-FMK (zVAD) or control analog zFA-FMK (zFA) (both at 40 µM final concentration) was added 2 hours before TRAIL treatment. Cell death was measured by annexin-V and propidium iodide staining using flow cytometry. Representative dot plots from one experiment of two independent experiments are shown. Numbers represent percentage of propidium iodide–stained and annexin-V–positive cells in the respective quadrants.

Article Snippet: After 3–12 hours, cells were treated overnight with soluble recombinant mouse or human TRAIL (1000 ng/mL) (BioMol, Plymouth Meeting, PA) that had been cross-linked by prior treatment with mouse monoclonal anti-polyhistidine (R&D Systems, Minneapolis, MN; 2 μg antibody per μg TRAIL).

Techniques: Recombinant, Control, Concentration Assay, Staining, Flow Cytometry

Journal:

Article Title: Treating Metastatic Solid Tumors With Bortezomib and a Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor Agonist Antibody

doi: 10.1093/jnci/djn113

Figure Lengend Snippet: Cytotoxicity of combination treatment with bortezomib (Bzb) and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL). Renca-FLAG, Renca-FLIP, and 4T1 cells were untreated (Med) or treated with 20 nM Bzb for 12 hours and washed with phosphate-buffered saline. Recombinant mouse TRAIL (1 µg/mL) was added to cells 12 or 24 hours later. Cell number was determined using the 3-[4,5-dimethyiazol-2-yl-5]-[3-carboxymethyloxyphenyl]-2-[4-sulfophenyl]-2H tetrazolium assay. A) Percent decrease in cell number following 18 hours of TRAIL treatment. B) Numbers of viable cells in the respective cell lines shown as absorbance (A) at 490 nm 6 days after Bzb treatment in the presence or absence of TRAIL, added at 12 hours after Bzb. Means and 95% confidence intervals from one representative experiment of four (A) and seven (B) independent experiments are shown. *P < .001 (analysis of variance, two-sided) with respect to single agents or medium. C) Renca-FLAG, Renca-FLIP, 4T1, and TBJ cells treated overnight with 0 or 10 nM of Bzb were analyzed by flow cytometry following 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolycarbocyanine iodide (JC-1) bivariate staining. A loss of J-aggregates (red fluorescence) was observed only in TBJ cells. Numbers represent percentage of JC-1-red–positive cells (upper quadrant, polarized mitochondria) and percentage of JC-1-red–negative cells (lower quadrant, depolarized). Representative dot plots from one experiment of five independent experiments are shown. D) Bcl-2–transduced (4T1-Bcl-2) or parental 4T1 cells were untreated or treated with 10 nM Bzb for 3 hours and then treated with mouse TRAIL (500 ng/mL). Percent decrease in cell number after 18 hours of treatment with TRAIL is shown (left). Percent decrease in cell number in 4T1-Bcl-2 or parental 4T1 cells following 18-hour exposure to staurosporine (0.2 µg/mL) is shown (right); *P < .001 (two-sided Student t test). One representative experiment out of three independent experiments is shown. For all graphs, means and 95% confidence intervals (error bars) are shown.

Article Snippet: After 3–12 hours, cells were treated overnight with soluble recombinant mouse or human TRAIL (1000 ng/mL) (BioMol, Plymouth Meeting, PA) that had been cross-linked by prior treatment with mouse monoclonal anti-polyhistidine (R&D Systems, Minneapolis, MN; 2 μg antibody per μg TRAIL).

Techniques: Saline, Recombinant, Flow Cytometry, Staining, Fluorescence

Journal:

Article Title: Treating Metastatic Solid Tumors With Bortezomib and a Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor Agonist Antibody

doi: 10.1093/jnci/djn113

Figure Lengend Snippet: Bortezomib (Bzb)-induced sensitization to the extrinsic tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptor–mediated pathway of apoptosis in renal and mammary carcinomas. Renca-FLAG, Renca-FLIP, and 4T1 cells were treated overnight with Bzb (10 or 20 nM) or medium. A) Surface expression of death receptor DR5 by MD5-1 staining on tumor cells sensitized with 20 nM Bzb. Representative histograms from four independent experiments with similar results are shown (thin line, isotype control; filled histogram, untreated; heavy line, Bzb treated). B) Caspase-8 and caspase-3 activation in cells that were treated with a combination of Bzb (10 nM, overnight) and TRAIL (TR, 1000 ng/mL, 2 hours), with either agent alone, or left untreated (Med). Caspase activation was assayed by the luciferase-based enzymatic reaction. Mean luminescence and 95% confidence intervals (error bars) from one representative experiment of four independent experiments are shown. *P < .001 (analysis of variance, twosided) compared with single agents and untreated cells. C) Immunoblots of caspase-8 expression in whole-cell lysates of Renca-FLAG, Renca-FLIP, and 4T1 cells that were treated with media (Med) or treated overnight with 20 nM Bzb and then treated with or without recombinant mouse TRAIL (TR, 1000 ng/mL, 5 hours). Representative blots from three independent experiments are shown. D) Immunoblots of cFLIP, cIAP-1, and XIAP protein expression in whole-cell lysates of Renca-FLAG, Renca-FLIP, and 4T1 cells treated overnight with 0 or 20 nM Bzb. Representative blots from three independent experiments are shown.

Article Snippet: After 3–12 hours, cells were treated overnight with soluble recombinant mouse or human TRAIL (1000 ng/mL) (BioMol, Plymouth Meeting, PA) that had been cross-linked by prior treatment with mouse monoclonal anti-polyhistidine (R&D Systems, Minneapolis, MN; 2 μg antibody per μg TRAIL).

Techniques: Expressing, Staining, Control, Activation Assay, Luciferase, Western Blot, Recombinant

Journal:

Article Title: Treating Metastatic Solid Tumors With Bortezomib and a Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor Agonist Antibody

doi: 10.1093/jnci/djn113

Figure Lengend Snippet: Bortezomib (Bzb)-induced sensitization of human tumors to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL). Cell viability of human cancer cell lines treated with or without 20 nM of Bzb for 3 hours was determined 18 hours after treatment with soluble recombinant human TRAIL in the continued presence of Bzb. A) Renal cell carcinomas ACHN and A498. B) Breast carcinomas MDA-MB-231 and BT-549. Percent decrease in cell number is shown as means and 95% confidence intervals (n = 3) from one representative experiment of five independent experiments. *P < .001 (analysis of variance, two-sided) with respect to single agents.

Article Snippet: After 3–12 hours, cells were treated overnight with soluble recombinant mouse or human TRAIL (1000 ng/mL) (BioMol, Plymouth Meeting, PA) that had been cross-linked by prior treatment with mouse monoclonal anti-polyhistidine (R&D Systems, Minneapolis, MN; 2 μg antibody per μg TRAIL).

Techniques: Recombinant