Figure 1.
Figure 1. RNAi leads to specific down-regulation of Bcr-Abl protein levels, resulting in a significant reduction of viability and sensitization to imatinib mesylate. (A) Western blot analysis of Bcr-Abl in 32Dp210-wt, 32Dp210-Thr315Ile, 32Dp210-His396Pro, M07p210, and primary CD34+/CML cells after siRNA treatment (800 nM). Lane 1, BAF7, siRNA homologous to bcr-abl b3a2 fusion site; lane 2, BAF8, mismatch siRNA; lane 3, EPC. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) level served as a loading control. The decrease in Bcr-Abl levels caused by BAF7 treatment was determined by densitometry—83% in 32Dp210-wt, 86.22% in 32Dp210-His396Pro, 71.83% in 32Dp210-Thr315Ile, 93.9% in M07p210, and 74.3% in primary CD34+/CML cells (because of the low expression of Bcr-Abl, a more sensitive technique was applied in primary CD34+ cells). (B) Western blot analysis of Bcr-Abl, c-Abl, Bcl-XL, and p27 in 32Dp210 cells after treatment with siRNA (800 nM), as indicated (lane1, BAF7; lane 2, BAF8; lane 3, EPC). GAPDH level served as a loading control. (C) BAF7 treatment led to a dose-dependent reduction of viability in 32Dp210 cells. Forty hours after the second treatment with 200 nM and 800 nM siRNA, respectively, viability of cells (relative to EPC) was determined by means of MTT (BAF7, white bars; BAF8, black bars). Values are means ± SD of 6 examinations. (D) Treatment with suboptimal amounts of breakpoint-specific siRNA (200 nM) results in sensitization to imatinib mesylate. After siRNA treatment, 32Dp210 cells were exposed to imatinib concentrations ranging from 0.03 μM to 1.0 μM. Cell viability was measured after 40 hours by means of MTT assay. BAF7-treated cells (▴) showed significantly higher sensitivity for imatinib than cells treated with BAF8 (•) or cells electroporated without any siRNA (EPC, □). The IC50 values for imatinib were 0.08 μM (BAF7), 0.27 μM (BAF8), and 0.27 μM (EPC), respectively. Values are means ± SD of triplicates. (E) After siRNA treatment, M07p210 cells were exposed to imatinib at a concentration of 0.05 μM for 21 hours. The percentage of apoptotic cells was then determined by Annexin V–FITC assay. Values are means ± SD of 3 independent experiments.

RNAi leads to specific down-regulation of Bcr-Abl protein levels, resulting in a significant reduction of viability and sensitization to imatinib mesylate. (A) Western blot analysis of Bcr-Abl in 32Dp210-wt, 32Dp210-Thr315Ile, 32Dp210-His396Pro, M07p210, and primary CD34+/CML cells after siRNA treatment (800 nM). Lane 1, BAF7, siRNA homologous to bcr-abl b3a2 fusion site; lane 2, BAF8, mismatch siRNA; lane 3, EPC. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) level served as a loading control. The decrease in Bcr-Abl levels caused by BAF7 treatment was determined by densitometry—83% in 32Dp210-wt, 86.22% in 32Dp210-His396Pro, 71.83% in 32Dp210-Thr315Ile, 93.9% in M07p210, and 74.3% in primary CD34+/CML cells (because of the low expression of Bcr-Abl, a more sensitive technique was applied in primary CD34+ cells). (B) Western blot analysis of Bcr-Abl, c-Abl, Bcl-XL, and p27 in 32Dp210 cells after treatment with siRNA (800 nM), as indicated (lane1, BAF7; lane 2, BAF8; lane 3, EPC). GAPDH level served as a loading control. (C) BAF7 treatment led to a dose-dependent reduction of viability in 32Dp210 cells. Forty hours after the second treatment with 200 nM and 800 nM siRNA, respectively, viability of cells (relative to EPC) was determined by means of MTT (BAF7, white bars; BAF8, black bars). Values are means ± SD of 6 examinations. (D) Treatment with suboptimal amounts of breakpoint-specific siRNA (200 nM) results in sensitization to imatinib mesylate. After siRNA treatment, 32Dp210 cells were exposed to imatinib concentrations ranging from 0.03 μM to 1.0 μM. Cell viability was measured after 40 hours by means of MTT assay. BAF7-treated cells (▴) showed significantly higher sensitivity for imatinib than cells treated with BAF8 (•) or cells electroporated without any siRNA (EPC, □). The IC50 values for imatinib were 0.08 μM (BAF7), 0.27 μM (BAF8), and 0.27 μM (EPC), respectively. Values are means ± SD of triplicates. (E) After siRNA treatment, M07p210 cells were exposed to imatinib at a concentration of 0.05 μM for 21 hours. The percentage of apoptotic cells was then determined by Annexin V–FITC assay. Values are means ± SD of 3 independent experiments.

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