Pten suppresses leukemia stem cells. (A) BCR-ABL down-regulates Pten expression, and this down-regulation is abolished upon imatinib treatment. Bone marrow cells were transduced with GFP or BCR-ABL-GFP retrovirus, followed by transplantation into recipient mice. Some recipients of BCR-ABL-GFP–transduced bone marrow cells were treated with imatinib (100 mg/kg, twice a day by gavage), beginning at day 8 after BMT. At 24 hours later, GFP⁺Lin⁻c-Kit⁺Sca1⁺ cells in bone marrow were sorted from these mice by FACS, and total RNA was isolated for DNA microarray assay. (B) DNA microarray assay shows that the mRNA level of p53 was down-regulated by BCR-ABL in LSCs. (C) Bone marrow cells were isolated from mice with CML induced with BCR-ABL-GFP or BCR-ABL-PTEN-GFP. The percentage of GFP⁺Lin⁻c-Kit⁺Sca1⁺ cells in bone marrow was analyzed by FACS. (D) At day 20 after BMT, the percentages of GFP⁺Gr1⁺ leukemia cells in peripheral blood of recipients of bone marrow cells transduced with BCR-ABL-GFP or BCR-ABL-PTEN-GFP were analyzed by FACS. (E) Total numbers of leukemia cells in peripheral blood of recipients of bone marrow cells transduced with BCR-ABL-GFP or BCR-ABL-PTEN-GFP were analyzed by FACS. (F) Pten overexpression reduces the ability of leukemia stem cells to induce CML. Bone marrow cells from mice with CML induced with BCR-ABL-GFP or BCR-ABL-PTEN-GFP were sorted by lineage-depletion MACS columns (Miltenyi Biotec), followed by FACS analysis for the percentages of c-Kit⁺Sca1⁺ cells. After normalization, the same number (3 × 10⁴) of GFP⁺Lin⁻c-Kit⁺Sca1⁺ cells from each group was transferred into recipient mice (BCR-ABL-GFP, n = 7; BCR-ABL-PTEN-GFP, n = 5) to induce CML. (G) Rapamycin inhibits leukemia stem cells from CML mice in vitro. Bone marrow cells isolated from mice with CML induced by BCR-ABL-GFP were cultured (2 × 10⁶ cells/6-cm plate) under the stem cell conditions (see “Methods”) in the presence of DMSO or rapamycin (10μM) for 3 days, followed by FACS analysis of leukemia stem cells (GFP⁺Lin⁻c-Kit⁺Sca1⁺).