To examine the potential of everolimus to overcome resistance due to quiescence in Pht leukemia cells, everolimus treatment was investigated ex vivo alone and in combination with imatinib on S17 stromal cells. Everolimus treatment at 100 nM for 5 days increased the sub MGCD0103 HDAC inhibitor G1 population, and combination of everolimus and imatinib further increased the sub G1 population. Cell cycle status was also investigated after 250K CD34CD38 CD34CD38 CD34 SG2/M 58.6 29.8 43.7 15.7 65.9 16.1 7.66 SG2/M SG2/M 25.9 5.06 G0 G0 G0 G1 G1 G1 250K 200K 200K 150K 150K Hoechst 100K 100K Pyronin Y 50K 50K 1500 105 1.49 12.8 77.3 73.8 0.06 3.03 40.5 27.2 49.2 7.28 CD34CD38 CD34CD38 CD34 2.5 1.4 1.2 1 0.8 0.6 0.4 0.2 0 1.2 1 0.8 0.6 0.4 0.2 0 2 1.5 Go cells number 1 0.
5 0 0 1 CD34 CD38 pY245 BCR ABL BCR ABL ABL Tubulliin pY207 CrkL Imatinib CD34 CD38 CD34 CD38 Imatinib 3 0 1 3 0 1 3 95 65 0.088 7.71 CD38 4.61 9.74 20.1 BMS-708163 1146699-66-2 104 92.4 103 PI CD34 102 0 105 104 103 PI 102 0 105 104 103 102 0 CD34 105 104 103 102 0 No treat Imatinib 1000 # Cells 500 0 1500 1000 # Cells 500 0 0 102 103 104 huCD45 Annexin V 105 0 102 103 104 huCD45 105 0 102 103 104 105 Annexin V 0 102 103 104 105 0 102 103 104 105 CD38 0 102 103 104 105 0 250K 200K 150K 100K Pyronin Y 50K 0 250K 200K 150K 100K Pyronin Y 50K 0 0 150K 200K 250K Hoechst 0 50K 100K 150K 200K 250K Hoechst 0 50K 100K a b c d Figure 1 Ex vivo analysis of humanized mouse positive acute lymphoblastic leukemia cells. Leukemic spleen cells were CD34 positively selected with MACS column. CD34t cells were stained with Hoechst, PyroninY and CD38 allophycocyanin.
Cells including CD34 population that had flowed through the column were stained with Hoechst, PyroninY and CD34 APC. Leukemic spleen cells were ex vivo cultured with cytokines and treated with or without imatinib for 48 h. Human CD45t propidium iodide Annexin V viable population was analyzed for CD34 and CD38 distribution. Panels show a representative experiment. After treated with IM for 48 h, CD34t cells were positively selected with MACS column, and stained with Hoechst, PyroninY and CD38 APC. Cells including CD34 population that had flowed through the column were stained with Hoechst, PyroninY and CD34 APC. Graphs show the number of forward scatter/side scatter gated G0 cells in each CD34/CD38 sub population, each relative to the untreated control. Bars indicate means.d. values of three independent experiments.
CD34/CD38 sorted populations were treated with or without IM 3 mM for 6 h. Expression of BCRABL and phosphorylation of BCR ABL and CrkL in each population was examined by western blotting analysis. Everolimus overcomes resistance in quiescent Pht leukemia cells Y Kuwatsuka et al 3 treatment with S 17 for 5 days. Although the untreated control and imatinib treated cells showed 35% of Hoechstlow/PyroninYlow cells in total acquired cells, combination of imatinib and everolimus decreased these quiescent cells to 13% of total acquired cells. Significant difference was found between imatinib alone and combination of imatinib plus everolimus. Treatment with everolimus and imatinib for 5 days induced substantial cell death in CD34t38 population relative to dimethylsulfoxide control. These results indicated that ex vivo combination treatment with imatinib and everolimus was also effective for the quiescent CD34t38 cells. Evaluation of molecular biomarkers during cell death i