American Society of Hematology

Figure 1

$Figure 1. R115777 or PD184352 potentiates Chk1 inhibitor–induced γH2A.X expression and lethality in human MM cells in association with diminished Ras→ERK1/2 signaling. (A) U266 and RPMI8226 cells were exposed for 24 hours to UCN-01 (UCN, top panels) or Chk1i (bottom panels), after which Ras activation assays and Western blot analysis (WB) were performed to monitor Ras activation status and ERK1/2 phosphorylation, respectively. Ras activity was reflected by amount of Ras-GTP pulled down by Raf-1 RBD. Alternatively, membrane fractions were separated and subjected to WB. In parallel, the percentage of cell death (7AAD+) was assessed by flow cytometry to determine the toxicity of UCN-01 or Chk1i at the indicated concentrations in U266 (48h) or RPMI8226 cells (24h). (B,C) Cells were exposed to UCN-01 (RPMI8226, 100 nM for 24 hours; U266, 150 nM for 48 hours) or Chk1i (RPMI8226, 1 μM for 24 hours; U266, 2 μM for 48 hours) in the absence or the presence of either 5 μM R115777 (R115) or 5 μM PD184352 (PD184), after which cells were lysed and subjected to WB for phosphorylation of ERK1/2 and H2A.X (γH2A.X), as well as PARP degradation. For panels A through C, results of a representative experiment are shown; 2 additional studies yielded equivalent results. CF indicates cleavage fragment. (D) Alternatively, the percentage of apoptotic cells was determined by annexin V–FITC staining and flow cytometry. Veh indicates vehicle. Results represent the means plus or minus SD for 3 separate experiments performed in triplicate. ** indicates significantly greater than values for those treated with Chk1 inhibitors alone (P < .01). (E) U266 cells were treated with a range of R115777 and UCN-01 concentrations alone or in combination for 24 hours (for γH2A.X staining) or 48 hours (for 7AAD staining) at a fixed ratio (R115777/UCN-01, 50:1). At the end of this period, the percentage of cells with relative increases in γH2A.X expression (ie, drug treatment vs untreated controls, left panel) or 7AAD+ cells (right panel) was determined by flow cytometry, respectively. Median dose effect analysis was used to characterize the nature of the interaction. Two additional studies yielded equivalent results.$

R115777 or PD184352 potentiates Chk1 inhibitor–induced γH2A.X expression and lethality in human MM cells in association with diminished Ras→ERK1/2 signaling. (A) U266 and RPMI8226 cells were exposed for 24 hours to UCN-01 (UCN, top panels) or Chk1i (bottom panels), after which Ras activation assays and Western blot analysis (WB) were performed to monitor Ras activation status and ERK1/2 phosphorylation, respectively. Ras activity was reflected by amount of Ras-GTP pulled down by Raf-1 RBD. Alternatively, membrane fractions were separated and subjected to WB. In parallel, the percentage of cell death (7AAD⁺) was assessed by flow cytometry to determine the toxicity of UCN-01 or Chk1i at the indicated concentrations in U266 (48h) or RPMI8226 cells (24h). (B,C) Cells were exposed to UCN-01 (RPMI8226, 100 nM for 24 hours; U266, 150 nM for 48 hours) or Chk1i (RPMI8226, 1 μM for 24 hours; U266, 2 μM for 48 hours) in the absence or the presence of either 5 μM R115777 (R115) or 5 μM PD184352 (PD184), after which cells were lysed and subjected to WB for phosphorylation of ERK1/2 and H2A.X (γH2A.X), as well as PARP degradation. For panels A through C, results of a representative experiment are shown; 2 additional studies yielded equivalent results. CF indicates cleavage fragment. (D) Alternatively, the percentage of apoptotic cells was determined by annexin V–FITC staining and flow cytometry. Veh indicates vehicle. Results represent the means plus or minus SD for 3 separate experiments performed in triplicate. ** indicates significantly greater than values for those treated with Chk1 inhibitors alone (P < .01). (E) U266 cells were treated with a range of R115777 and UCN-01 concentrations alone or in combination for 24 hours (for γH2A.X staining) or 48 hours (for 7AAD staining) at a fixed ratio (R115777/UCN-01, 50:1). At the end of this period, the percentage of cells with relative increases in γH2A.X expression (ie, drug treatment vs untreated controls, left panel) or 7AAD⁺ cells (right panel) was determined by flow cytometry, respectively. Median dose effect analysis was used to characterize the nature of the interaction. Two additional studies yielded equivalent results.

This Feature Is Available To Subscribers Only