model. NOG and NOD/SCID mice were obtained from the Central Institute for Experimental LY315920 sPLA2 inhibitor Animals and Clea Japan, respectively. Seven week old male NOD/SCID mice received 2Gy of total body irradiation from an X ray source, 24 h before administration of leukemic 2 107 cells. Everolimus, imatinib or vehicle were diluted with dH2O, and given daily at 10 ml/kg for 10 days by gavage. Bone marrow and spleen cells were stained with anti human CD19 PE and anti mouse CD45 PerCP, and acquired with FACS Aria to analyze chimerism. Cells were also stained with anti CD34 APC, anti CD38 PE Cy7 and anti CD45 APC Alexa Fluor 750, and were subsequently labeled with annexin V fluorescein isothiocyanate and PI as explained above. Protocols were approved by the Nagoya University Animal Ethics Committee.
Histopathology Livers, spleens and femurs were fixed in 15% buffered formalin. Hematoxylin and eosin, and CD34 staining were performed as previously described.20 22 Slides were examined at room temperature and images were captured by a fluorescence microscope with a charge coupled device camera fitted with 20 and 60 objective. Images were acquired by BZ analyzer software. Statistical PIK-90 c-kit inhibitor analysis Differences among more than two groups were analyzed with the Bonferroni test followed by one way analysis of variance. Statistical analyses were performed with STATA 9.2 software. Results Ex vivo treatment with imatinib for more residual quiescent CD34tCD38 population in Pht ALL cells We analyzed the cell cycle status of untreated spleen cells derived from the humanized Pht ALL leukemia murine model reported previously.
15 In the CD34tCD38 population, a higher percentage of Hoechstlow/PyroninYlow slow cycling quiescent cells was observed than in the CD34tCD38t population and CD34 population. A lower percentage of the StG2/M population was also observed among CD34tCD38 cells. We next treated these cells with imatinib for 48 h and analyzed the distribution of CD34/CD38 in residual viable cells. After treatment with imatinib at 0.3, 1 and 3 mM, more residual CD34tCD38 cells were observed than non treated cells. Significantly more slow cycling quiescent cells were observed within CD34t38 population after treated with 3 mM of imatinib, and less G0 cells in CD34 population. Treatment of CD34/CD38 sorted cells for 6 h with 3 mM imatinib caused equivalent inhibition of the phosphorylation of BCR ABL and direct substrate CrkL in each CD34/CD38 sub population.
Inhibition of phosphorylation with imatinib was also observed with intracellular staining of phospho CrkL. Expressions of BCR ABL and ABL were equivalent in each CD34/CD38 sub population. These results suggested that the slow cycling population derived from Pht leukemia NOG mice was insensitive to imatinib in spite of BCR ABL dephosphorylation. Ex vivo effects of everolimus on leukemic spleen cells, alone and in combination with imatinib Furthermore, we have introduced S 17 murine stromal cell lines to support the leukemic spleen cells in order to assess longerterm effect of treatment drugs,16 as the CD34t population of leukemic cells from the NOG mice eventually differentiated into CD34 cells and could not be maintained only with cytokines Everolimus overcomes resistance in quiescent Pht leukemia cells Y Kuwatsuka et al 2 for a longer period. If cultured with S17 cells, leukemic spleen cells were viable for more than 30 days.