American Society of Hematology

Figure 3

$Figure 3. The net function of p27 in BCR-ABL1–mediated leukemogenesis is tumor-suppressive. (A) BM harvested from 5-fluorouracil–treated p27+/+, p27+/−, and p27−/− mice (N = 3 per group) was analyzed by FACS to determine the proportions of Lin−/c-Kit+/Sca1+ (LKS) cells. The LKS population was gated for CD34 expression to distinguish between progenitor cells (LKS CD34+ for MPP) and the stem cell–enriched fraction (LKS CD34− for HSC). (B) Homing capacity was assessed in recipients of BM LKS cells from p27+/+, p27+/−, and p27−/− mice (N = 3 per group) using CFSE labeling. (C) Long-term engraftment was assessed in CD45.1+ recipients of CD45.2+ BM cells from p27+/+, p27+/− and p27−/− mice (N = 3 per group) after injecting all mice with MNCs (3 × 105 cells per mouse) and LKS cells (3000 cells per mouse). Starting 4 weeks after transplantation, the contribution of CD45.2+ cells in the peripheral blood was measured by weekly FACS. The data shown represent engraftment after 6 weeks. (D) BCR-ABL1–expressing p27+/+ and p27−/− BM cells were subjected to an in vivo competition experiment. To adjust for the 50% reduced LKS population in p27−/− mice, twice the number of BCR-ABL1–transduced BM cells from p27−/− CD45.2 mice (1.4 × 105 cells per mouse) mixed with BCR-ABL1–transduced BM cells from p27+/+ CD45.2/CD45.1 mice (0.7 × 105 cells per mouse) were injected into lethally irradiated wild-type congenic recipient mice (CD45.1). At the time of autopsy, BM cells were analyzed by FACS for the presence of GFP+ p27−/− CD45.2 or p27+/+ CD45.1 cells. (E-F) p27+/+ mice were transplanted with BCR-ABL1–transduced GFP+ BM LKS cells (5000 or 500 per mouse) from p27+/+, p27+/− and p27−/− mice and compared for survival using Kaplan-Meier statistics. (G-H) Spleen and liver weights of mice transplanted with BCR-ABL1–transduced LKS cells were compared according to genotype. (I) Representative histological sections of BM, liver, lung, and spleen from mice transplanted with BCR-ABL1–transduced LKS cells. Scale bars represent 100 μm *P < .050; **P < .010; **P < .001. CFSE, carboxyfluorescein succinimidyl ester; HSC, hematopoietic stem cells; MNC, mononuclear cell; MPP, multipotent progenitor.$

The net function of p27 in BCR-ABL1–mediated leukemogenesis is tumor-suppressive. (A) BM harvested from 5-fluorouracil–treated p27^+/+, p27^+/−, and p27^−/− mice (N = 3 per group) was analyzed by FACS to determine the proportions of Lin⁻/c-Kit⁺/Sca1⁺ (LKS) cells. The LKS population was gated for CD34 expression to distinguish between progenitor cells (LKS CD34⁺ for MPP) and the stem cell–enriched fraction (LKS CD34⁻ for HSC). (B) Homing capacity was assessed in recipients of BM LKS cells from p27^+/+, p27^+/−, and p27^−/− mice (N = 3 per group) using CFSE labeling. (C) Long-term engraftment was assessed in CD45.1⁺ recipients of CD45.2⁺ BM cells from p27^+/+, p27^+/− and p27^−/− mice (N = 3 per group) after injecting all mice with MNCs (3 × 10⁵ cells per mouse) and LKS cells (3000 cells per mouse). Starting 4 weeks after transplantation, the contribution of CD45.2⁺ cells in the peripheral blood was measured by weekly FACS. The data shown represent engraftment after 6 weeks. (D) BCR-ABL1–expressing p27^+/+ and p27^−/− BM cells were subjected to an in vivo competition experiment. To adjust for the 50% reduced LKS population in p27^−/− mice, twice the number of BCR-ABL1–transduced BM cells from p27^−/− CD45.2 mice (1.4 × 10⁵ cells per mouse) mixed with BCR-ABL1–transduced BM cells from p27^+/+ CD45.2/CD45.1 mice (0.7 × 10⁵ cells per mouse) were injected into lethally irradiated wild-type congenic recipient mice (CD45.1). At the time of autopsy, BM cells were analyzed by FACS for the presence of GFP⁺ p27^−/− CD45.2 or p27^+/+ CD45.1 cells. (E-F) p27^+/+ mice were transplanted with BCR-ABL1–transduced GFP⁺ BM LKS cells (5000 or 500 per mouse) from p27^+/+, p27^+/− and p27^−/− mice and compared for survival using Kaplan-Meier statistics. (G-H) Spleen and liver weights of mice transplanted with BCR-ABL1–transduced LKS cells were compared according to genotype. (I) Representative histological sections of BM, liver, lung, and spleen from mice transplanted with BCR-ABL1–transduced LKS cells. Scale bars represent 100 μm *P < .050; **P < .010; **P < .001. CFSE, carboxyfluorescein succinimidyl ester; HSC, hematopoietic stem cells; MNC, mononuclear cell; MPP, multipotent progenitor.

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