Article Figures & Data
Figures
- Figure 1.
Veliparib sensitizes GBM cell lines to TMZ in vitro. A, veliparib dose–response: T98G cells were pretreated with the indicated concentrations of veliparib and stimulated with 100 μmol/L H2O2 or incubated with 100 μmol/L TMZ. Immunoblotting for PAR and total PARP1 on the same membrane are shown. B, cytotoxicity assay: T98G cells were incubated with graded concentrations of veliparib and TMZ for 6 days and then analyzed in a CyQuant assay. Results shown are relative fluorescence for treated versus untreated cells. C, similar TMZ-sensitizing studies were performed for U251 and U251TMZ cells and results for all three lines at selected concentrations of drugs are shown. D, DNA-damage signaling: U251, U251TMZ, and T98G cells were treated as indicated for 24 or 72 hours and processed for immunoblotting with the indicated antibodies. Results for A and D are representative of three independent experiments, and in B and C, data plotted represent the mean ± SEM from a minimum of three independent experiments; *, P < 0.05; MW, molecular weight.
- Figure 2.
Veliparib promotes TMZ-induced γH2AX and RPA foci formation in GBM cell lines. U251 (A), U251TMZ (B), and T98G (C) cells cultured on glass cover slips were treated with DMSO, 10 μmol/L veliparib for 30 minutes and subsequently with or without 100 μmol/L TMZ for 24 or 72 hours and immunostained for RPA32 and γH2AX and counterstained with DAPI. Top, representative images (bar, 10 μm), and graphs depicted below present the mean ± SEM from three independent experiments for the percentage of cells with >20 foci/nuclei for each treatment; *, P ≤ 0.05.
- Figure 3.
Veliparib sensitizes GBM12 derivative TMZ-resistant xenograft lines in vitro. A and B, cytotoxicity assay: The sensitive GBM12 and derivative TMZ-resistant sublines, GBM12TMZ-mgmtLow and GBM12TMZ-mgmtHigh were analyzed for neurosphere growth following treatment with graded concentrations of veliparib and/or TMZ. A, the relative neurosphere counts for the indicated treatments are graphed as the mean ± SEM from three independent experiments. B, the calculated IC50 value for TMZ at different concentrations of veliparib is plotted as the mean ± SD from three independent experiments. C, DNA-damage signaling: GBM12 and derivative sublines were treated for 24 hours with the indicated doses of veliparib and/or TMZ and then processed for immunoblotting with the indicated antibodies. D, cells were analyzed for γH2AX foci as in Fig. 2 after drug treatment for 24 hours. Results are plotted as the mean ± SEM from a minimum of three independent experiments; *, P ≤ 0.05; NS, P > 0.05; MW, molecular weight.
- Figure 4.
Veliparib enhances the efficacy of TMZ in vivo in GBM12 but not in TMZ-resistant sublines. A, tumor regrowth analysis: Mice with established flank tumor xenografts were randomized into groups of 10 mice each and treated for three cycles with the indicated drugs. Time for reaching critical tumor volume of 1,500 mm3 is shown as endpoint for each group as Kaplan–Meier plots. B, pharmacodynamic analysis: Mice with GBM12 or GBM12TMZ-mgmtHigh xenografts were treated for 5 days and euthanized either 2 or 72 hours after the last dose of TMZ/veliparib. Three tumors were processed for each treatment/time point, and equal amounts of protein from these tumors were pooled for analysis in any given lane. All samples were run on the same gel, but two unloaded intervening lanes have been cropped from the images; MW, molecular weight. C, the pharmacokinetic profile of veliparib in tumor tissue: Groups of mice with flank xenografts were treated for 5 days with TMZ/veliparib and euthanized at time points ranging from 0 to 6 hours after the last dose of veliparib. The values plotted are the mean ± SD veliparib concentrations in five tumors at each time point.
- Figure 5.
PARP1 knockdown facilitates TMZ efficacy in U251TMZ in vitro and in vivo. A, PARP1 knockdown: U251TMZ cells and derivative stable transductants expressing PARP1-specific shRNA (D2, F6, and F10) or nontargeted shRNA (shRNA-NT) were treated with DMSO or veliparib and subsequently stimulated with H2O2 for 10 minutes. Samples were processed for immunoblotting with the indicated antibodies; MW, molecular weight. B, TMZ-sensitizing effects: Parental and shRNA-expressing U251TMZ cells were treated with TMZ and/or veliparib, and cell growth was compared using a CyQuant assay. Data plotted, mean ± SEM from a minimum of three independent experiments; *, P ≤0.05; NS, P > 0.05. C and D, the indicated cells were used to establish orthotopic tumors and groups of 10 mice with established tumors were randomized to therapy as indicated. Survival for mice in each treatment group is plotted in the Kaplan–Meier graphs and differences evaluated using the log-rank test.
Additional Files
Supplementary Material, Supplementary Figures S1-S6
Files in this Data Supplement:
- Data Supplement - Supplementary Material. Methods for analysis of apoptosis & pluripotency; and Figure Legends to the Supplementary Figures
- Data Supplement - Supplementary Figure S1. Sustained PARP inhibition by single Veliparib treatment and the effect of repeated drug treatment on cell growth in vitro
- Data Supplement - Supplementary Figure S2. Veliparib mediated enhancement of TMZ induced apoptosis in GBM cells
- Data Supplement - Supplementary Figure S3. Evidence of pluripotency in cells of PDX line GBM12 and derivative sublines
- Data Supplement - Supplementary Figure S4. Dose dependent increase in cytotoxic effects of veliparib, TMZ or their combination in multiple PDX GBM models of differential TMZ sensitivity
- Data Supplement - Supplementary Figure S5. Effects of Veliparib on TMZ induced DNA damage signaling in undifferentiated or differentiated GBM12 cells
- Data Supplement - Supplementary Figure S6. Measurements of tumor growth after treatment with veliparib, TMZ or their combination in TMZ-sensitive versus resistant GBM models in vivo.
Volume 20, Issue 14
