Bcl-2 is a critical determinant of the sensitivity of early myeloid progenitors to cytokine withdrawal.

Chimeric mice with a bcl-2^−/− or wt hematopoietic system were generated by reconstitution of lethally irradiated C57BL/6-Ly5.1 mice with 2 × 10⁶ fetal liver cells from E14 bcl-2^−/− or wt embryos (C57BL/6-Ly5.2) produced from intercrosses ofbcl-2^+/− animals. Absolute numbers of T cells, B cells, macrophages, and granulocytes in blood (A,C) or spleen (B,D) of reconstituted mice were determined by cell counting and flow cytometric analysis staining cells with surface marker–specific mAbs (anti-B220, anti–Thy-1, anti–Gr-1, or anti–Mac-1). Host and donor-derived cells were distinguished by staining with an FITC-conjugated mAb to Ly5.2. Data shown for the peripheral blood represent arithmetic means ± SEs of 9bcl-2^−/− reconstituted and 5 wt reconstituted animals that were analyzed 10 weeks after fetal liver stem cell transplantation. Data shown for the spleen represent arithmetic means ± SEs of 6 bcl-2^−/− and 4 wt reconstituted animals analyzed 10 to 16 weeks after reconstitution. (E-F) Survival analysis of bone marrow–derived myeloid progenitors. Cells (2.5 × 10⁴) from bone marrow of (E) wt mice, lethally irradiated wt mice reconstituted with either wt or bcl-2^−/− fetal liver stem cells, and from bone marrow of (F) wt, bim^−/−, andbcl-2 transgenic mice were cultured in 0.3% agar in triplicate or quadruplicate and stimulated with mSCF (100 ng/mL) and mIL-3 (2500 U/mL). Cytokines were added at the time of plating or after a delay of 12, 18, 24, 48, or 72 hours. After 7 days of stimulation with cytokines at 37°C, colonies were counted using a dissection microscope. Data shown represent arithmetic means ± SDs of 2 to 3 independent experiments performed using 4 to 6 animals of each genotype.