Overexpression of Pten delays CML development. (A) Structure of BCR-ABL-PTEN-GFP retroviral construct. (B) Western blot analysis shows expression of BCR-ABL, PTEN, and GFP from BCR-ABL-PTEN-GFP retrovirus. NIH3T3 cells were transduced with BCR-ABL-GFP or BCR-ABL-PTEN-GFP retrovirus for 3 hours. Then, 2 days later, protein lysates were analyzed be Western blotting by the use of the antibodies indicated. (C) Overexpression of Pten alone or in combination imatinib treatment prolongs survival of CML mice. Mice with CML induced with BCR-ABL-GFP (n = 20) or BCR-ABL-PTEN-GFP (n = 20) were treated with a placebo (n = 7) or imatinib (n = 7, 100 mg/kg, twice a day by gavage), beginning at day 8 after transplantation. (D) Flow cytometry analysis showed a slower accumulation of GFP⁺Gr1⁺ leukemia cells in peripheral blood of recipients of BCR-ABL-PTEN-GFP–transduced bone marrow cells than that in recipients of BCR-ABL-GFP–transduced bone marrow cells. (E) CML was induced with BCR-ABL-GFP or BCR-ABL-PTEN-GFP, and the difference in peripheral blood leukemia cell counts (white blood cell count × the percentage of GFP⁺Gr1⁺ cells) in CML mice treated with a placebo or imatinib was determined at day 20 after BMT. (F) Spleen weight of CML mice induced with BCR-ABL-GFP or BCR-ABL-PTEN-GFP. (G) Photomicrographs of hematoxylin and eosin–stained lung sections from mice with CML induced with BCR-ABL-GFP or BCR-ABL-PTEN-GFP at day 20 after transplantation. (H) At day 20 after BMT, peripheral blood cells were stained with Gr1 and Hoechst blue. The S + G₂M phase of leukemia cells (GFP⁺Gr1⁺) was represented by the percentage of Hoechst blue–positive cells. Mean percentage for each cell population (n = 3) was shown. (I) At day 20 after BMT, peripheral blood cells were stained with Gr1, annexin V, and propidium iodide (PI). Apoptotic leukemia cells were represented by the GFP⁺Gr1⁺annexinV⁺PI⁺ population. Mean percentage for each cell population (n = 3) was shown.