Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Dinaciclib, a Cyclin-Dependent Kinase Inhibitor Promotes Proteasomal Degradation of Mcl-1 and Enhances ABT-737–Mediated Cell Death in Malignant Human Glioma Cell Lines

doi: 10.1124/jpet.115.230052

Figure Lengend Snippet: Combined treatment with dinaciclib and ABT-737 effectively kills glioma cells. (A) T98G cells were seeded at 60% confluence, allowed to attach overnight, and treated with dinaciclib (1.0 µM) or indicated signaling inhibitor (refer to Supplemental Table 2 for the concentrations used in this study) or the combination of both for 24 hours. Control cells received an equivalent amount of DMSO. Apoptosis was analyzed by flow cytometry as described in Materials and Methods. The results represent the mean of two independent experiments representing various stages of cell death. (B) U87, U87-EGFRviii, LNZ308, LN229, LN18, and T98G cells were treated with dinaciclib (100 nM (D), ABT-737 (100 nM) (A), or the combination of both (D and A). Control cells received DMSO (C). Apoptosis was analyzed by flow cytometry as described in Materials and Methods. The results represent the mean of three independent experiments. (C) In parallel, cell extracts (from U87, LNZ308, and T98G) were prepared, and equal amounts of protein were separated by SDS-PAGE and subjected to Western blot analysis with the indicated antibodies. β-actin served as loading control. The results of a representative study are shown; two additional experiments produced similar results.

Article Snippet: Dinaciclib, ribociclib, palbociclib, gefitinib, sorafenib, dasatinib, vorinostat, panobinostat, rapamycin, bortezomib, cucurbitacin-I, AZD-6244, YM-155, NVP-AUY922, AMG-925, ABT-737, ABT-263, MK-2206, XL-147, NVP-BKM120, and NVP-BEZ235 were purchased from Chemie Tek (Indianapolis, IN).

Techniques: Flow Cytometry, SDS Page, Western Blot, Produced

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Dinaciclib, a Cyclin-Dependent Kinase Inhibitor Promotes Proteasomal Degradation of Mcl-1 and Enhances ABT-737–Mediated Cell Death in Malignant Human Glioma Cell Lines

doi: 10.1124/jpet.115.230052

Figure Lengend Snippet: Effect of dinaciclib and ABT-737 on the cell-cycle profile and the expression levels of cell-cycle regulatory proteins. (A and B) Logarithmically growing U87, U87-EGFRviii, LNZ308 (upper panel), LN229, LN18, and T98G (lower panel) cells were treated with dinaciclib (indicated concentrations) or ABT-737 (100 nM) or the combination of both for 24 hours. Control cells received equivalent concentrations of vehicle, DMSO. Whole-cell extracts were prepared, and equal amounts of protein were separated by SDS-PAGE and subjected to Western blotting analysis with the indicated antibodies. β-actin served as loading control (A). Total Rb served as loading control (B). The results of a representative study are shown; two additional experiments produced similar results.

Article Snippet: Dinaciclib, ribociclib, palbociclib, gefitinib, sorafenib, dasatinib, vorinostat, panobinostat, rapamycin, bortezomib, cucurbitacin-I, AZD-6244, YM-155, NVP-AUY922, AMG-925, ABT-737, ABT-263, MK-2206, XL-147, NVP-BKM120, and NVP-BEZ235 were purchased from Chemie Tek (Indianapolis, IN).

Techniques: Expressing, SDS Page, Western Blot, Produced

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Dinaciclib, a Cyclin-Dependent Kinase Inhibitor Promotes Proteasomal Degradation of Mcl-1 and Enhances ABT-737–Mediated Cell Death in Malignant Human Glioma Cell Lines

doi: 10.1124/jpet.115.230052

Figure Lengend Snippet: Cotreatment with dinaciclib and ABT-737 induces mitochondrial membrane potential dysfunction and conformational changes of the proapoptotic protein Bax. Logarithmically growing U87, U87-EGFRviii, LNZ308, LN229, LN18, and T98G cells were treated with dinaciclib (indicated concentrations) (A), ABT-737 (indicated concentrations (B), or the combination of both (indicated concentrations (C) for 24 hours. The integrity of the mitochondrial membranes of the cells was examined by DiOC6 staining and flow cytometry. (D) U87, U87-EGFRviii, LNZ308, LN229, LN18, and T98G cells were treated with dinaciclib (50 nM) or ABT-737 (50 nM) or the combination of both for indicated durations. Cytosolic extracts were prepared, and equal amounts of protein were separated by SDS-PAGE and subjected to Western blotting analysis with the indicated antibodies. (E), U87, U87-EGFRviii, LNZ308, LN229, LN18, and T98G cells were treated with dinaciclib (50 nM) or ABT-737 (50 nmol/L) or the combination of both for the indicated duration and lysed with 1% CHAPS buffer. An equal amount of protein (500 µg) was immunoprecipitated with monoclonal anti-Bax (6A7; Sigma-Aldrich) antibody and then subjected to Western blot analysis with polyclonal anti-Bax antibody (Cell Signaling Technology). (F) U87, U87-EGFRviii, LNZ308, LN229, LN18, and T98G cells were treated with dinaciclib (50 nM) or ABT-737 (50 nM) or the combination of both for 12 hours. Control cells received equivalent amounts of DMSO. Membrane fractions were obtained as described in Materials and Methods, and proportional amounts corresponding to total protein were analyzed for Bax oligomerization by Western blotting under nonreducing conditions. Slow-moving Bax oligomers in DSP cross-linked cells were derived from Bax monomers, and the molecular masses of oligomers containing Bax were calculated by plotting their migrations against migrations of molecular mass standards (left panel, mol. wt. marker). The results of a representative study are shown; two additional experiments produced similar results.

Article Snippet: Dinaciclib, ribociclib, palbociclib, gefitinib, sorafenib, dasatinib, vorinostat, panobinostat, rapamycin, bortezomib, cucurbitacin-I, AZD-6244, YM-155, NVP-AUY922, AMG-925, ABT-737, ABT-263, MK-2206, XL-147, NVP-BKM120, and NVP-BEZ235 were purchased from Chemie Tek (Indianapolis, IN).

Techniques: Staining, Flow Cytometry, SDS Page, Western Blot, Immunoprecipitation, Derivative Assay, Marker, Produced

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Dinaciclib, a Cyclin-Dependent Kinase Inhibitor Promotes Proteasomal Degradation of Mcl-1 and Enhances ABT-737–Mediated Cell Death in Malignant Human Glioma Cell Lines

doi: 10.1124/jpet.115.230052

Figure Lengend Snippet: Dinaciclib promotes proteasomal degradation of Mcl-1 and enhances ABT-737-mediated cell death in malignant human glioma cell lines. (A) Logarithmically growing T98G, U87, U87-EGFRviii, and LNZ308 cells were pretreated with 1.0 µM of MG-132 (proteasomal inhibitor) for 2 hours followed by dinaciclib (250 nM) for the indicated duration. Cell extracts were subjected to Western blot analysis with the indicated antibody. β-actin served as loading control. (B) U87, U87-EGFRviii, LNZ308, and T98G cells were transfected with nontarget (NT) or Mcl-1 shRNA as described in Materials and Methods. Forty-eight hours post-transfection, cells were treated with the indicated concentrations of ABT-737 for 24 hours, and viability was assessed by annexin V/PI apoptosis assay (lower panel). In parallel, cell lysates were collected and protein was subjected to Western blot analysis using Mcl-1 antibody. Immunoblots were stripped and reprobed with β-actin. (C) U87 and LNZ308 cells were transfected with vector (pCMV) or Mcl-1 expression vector as described in Materials and Methods. Forty-eight hours post-transfection, cells were treated with dinaciclib (dina, 100 nM) or ABT-737 (ABT, 100 nM) or the combination of both (dina + ABT) for 24 hours, and viability was assessed by annexin V/PI apoptosis assay (lower panel). In parallel, cell lysates were collected, and protein was subjected to Western blot analysis using Mcl-1 antibody. Immunoblots were stripped and reprobed with β-actin. Data are representative of triplicate studies from three independent experiments. *P < 0.005.

Article Snippet: Dinaciclib, ribociclib, palbociclib, gefitinib, sorafenib, dasatinib, vorinostat, panobinostat, rapamycin, bortezomib, cucurbitacin-I, AZD-6244, YM-155, NVP-AUY922, AMG-925, ABT-737, ABT-263, MK-2206, XL-147, NVP-BKM120, and NVP-BEZ235 were purchased from Chemie Tek (Indianapolis, IN).

Techniques: Western Blot, Transfection, shRNA, Apoptosis Assay, Plasmid Preparation, Expressing

Journal: Gut

Article Title: Senolytic elimination of Cox2-expressing senescent cells inhibits the growth of premalignant pancreatic lesions

doi: 10.1136/gutjnl-2020-321112

Figure Lengend Snippet: Bcl2-family protein inhibition reduces the numbers of senescent Cox2 + cells. (A) Timeline of treatments and analysis of Pdx1-Cre +/– ; LSL-Kras +/G12D mice, including the caerulein treatment point, followed by treatment with ABT-737 2 weeks subsequently, and analysis of mice 2 days later. (B) Pancreas sections from mice treated with ABT-737 or vehicle stained for cleaved caspase-3 (CC3, green), and for CK19 (red). (C) Percentage of CC3 + cells in PanINs from mice treated with ABT-737 or vehicle, quantified by image scoring. Values indicate mean across n=4 mice per group, ±SE. (D) Serial sections from same mice stained for SA-βGal (blue, top) and Cox2 (red, bottom). Arrowheads indicate representative regions of colocalisation. (E) Percentage of SA-βGal + cells out of CK19 + cells in pancreata from same mice, quantified by imaging cytometry. Values indicate mean across n=6 mice per group, ±SE. (F) Pancreas sections from ABT-737 and vehicle-treated mice stained for Cox2 (brown). (G) Percentage of Cox2 + cells in PanINs from same mice, quantified by image scoring. Values indicate mean across n=4 mice per group, ±SE. (H) Pancreas sections from ABT-737- and vehicle-treated mice stained for Bclxl (red). (I) Bclxl stain per section in PanINs from same mice, quantified by image analysis, scoring red signal per section area. Values indicate mean across n=4 mice per group, ±SE. *P<0.05, **P<0.01, t-test. a.u., arbitrary units; CC3, cleaved caspase 3; PanINs, pancreatic intraepithelial neoplasia.

Article Snippet: ABT-737 (WuXi AppTec) was dissolved in DMSO and prepared in a working solution of 30% propylene glycol, 5% Tween 80, 3.3% dextrose in water pH 4–5.

Techniques: Inhibition, Staining, Imaging, Cytometry

Journal: Gut

Article Title: Senolytic elimination of Cox2-expressing senescent cells inhibits the growth of premalignant pancreatic lesions

doi: 10.1136/gutjnl-2020-321112

Figure Lengend Snippet: Elimination of senescent cells blocks PanIN development and reduces PDAC incidence. (A) Timeline of caerulein and repeated ABT-737 treatments during PanIN and PDAC formation period. ABT-737 was administered for 2 days every month, for four consecutive months, and mice were either sacrificed after the last treatment (point 1), or maintained for a further 3.5 months (point 2). (B) Representative images of pancreatic sections from time point one stained for CK19, SA-βGal and CD45, labelling hematopoietic cells. (C) Percentage of tissue area containing ADM and PanIN lesions in mice treated with ABT-737 (n=6) or vehicle (n=3). Values indicate mean per group ±SE. (D) Levels of cytokines and chemokines in pancreata of Pdx1-Cre +/- ; LSL-Kras +/G12D mice treated with ABT-737, as measured by cytokine array. Values are shown relative to levels in vehicle-treated mice (pool of 3 mice per group). (E) Percentages of mice treated with ABT-737 or with vehicle that showed PDAC formation upon sacrifice at time point 2. Images show representative whole pancreas from mice in each group. Arrow indicates a large PDAC lesion. *P<0.05, χ 2 test between ABT-737 and vehicle treated groups. (F) Pancreas to body-weight ratio in same mice as in (E). Values indicate mean across mice ±SE, n numbers as in (E). (G) Representative images of pancreatic sections from time point two stained as indicated. (H) Percentage of tissue area occupied by ADM, PanINs or PDAC in the same mice. *P<0.05, t test. ADM, acinar to ductal metaplasias; ns, not significant; PanIN, pancreatic intraepithelial neoplasia; PDAC, pancreatic ductal adenocarcinoma.

Article Snippet: ABT-737 (WuXi AppTec) was dissolved in DMSO and prepared in a working solution of 30% propylene glycol, 5% Tween 80, 3.3% dextrose in water pH 4–5.

Techniques: Staining