(A) Premalignant Bid-deficient bone marrow displays increased clonogenic potential in methylcellulose. For myeloid colonies, 10,000 cells were resuspended in Methylcult H4100 supplemented with cytokines: GCSF (2 ng/mL), GMCSF (10 ng/mL), Il3 (20 ng/mL), Il6 (4 ng/mL), Il11 (10 ng/mL), SCF (20 ng/mL). For erythroid colonies, cells were supplemented with cytokines SCF (20 ng/mL) and erythropoietin (4 U/mL). Colonies were counted on day 7. Appropriate assessment of colony morphology was verified by May Grunwald-Giemsa stained cytospins from representative colonies. (B,C) Bid-deficient myeloid precursor cells are resistant to anti-Fas Ab (Jo2)- and TNFα/actinomycin D-induced death. Lin-SCA1+ myeloid precursor cells from 8–12-wk-old mice were cultured in the presence of SCF, GCSF, and Il3. Cultures containing 80%–90% promyelocytes and myelocytes were treated with anti-Fas Ab (Jo2, μg/mL; B) or TNFα + actinomycin D (20 ng/mL; C), as TNFα alone was not effective. Viable cells were determined over a 36-h time course by trypan blue exclusion and annexin V staining. (D) Bid-deficient macrophages are resistant to TNF-α/actinomycin D-induced death. Primary bone marrow cells from 8–12-wk-old mice were cultured for 1 wk in MCSF and IL3. Cells were treated with TNFα (10 ng/mL) or TNFα+ actinomycin D (20 ng/mL). After 72 h, cells were stained with annexin V and analyzed by flow cytometry.

(A–D) Bid-deficient myeloid cells display a competitive advantage in repopulating assays. Eight thousand Ly5.2+ wild-type or Bid knockout bone marrow cells along with 1.2 × 10⁵ Ly5.1+ bone marrow cells (1:20 ratio) were injected intravenously into the tail vein of lethally irradiated Ly5.1+ wild-type congenic mice. Percentages of donor repopulation as assessed by percent Ly5.2+ cells (A), percent Ly5.2+CD11b+ cells (B), percent Ly5.2+B220+ cells (C), and percent Ly5.2+CD3+ cells (D) were determined by flow cytometric analysis of peripheral blood.

Bid-deficient mice display increased mortality and increased tumor incidence. (A) Kaplan-Meier survival plot for 38 wild-type and 74 Bid-deficient mice monitored over 24 mo. Bid−/− mice show increased mortality (p = 0.003), and a shortened time to death from tumor (p = 0.01). (B) Tumor incidence. Among mice that died of tumor, the presence of CMML between the Bid-deficient and wild type was highly significant (p = 0.002).

Massive hepatosplenomegaly typifies CMML of Bid-deficient mice. (A) Bid-deficient mouse with massive hepatosplenomegaly and lymphadenopathy. (B) Spleen of same Bid-deficient mouse (left) shown relative to an age-matched wild-type spleen (right).

(A) Leukemic Bid-deficient mice reveal increased mature monocytes and granulocytes. Peripheral blood May Grunwald-Giemsa stain 20× reveals leukocytosis comprised predominantly of maturing myeloid elements. (B) Bone marrow, May Grunwald-Giemsa stain 20× reveals features of a myeloproliferative disorder with marked myeloid hyperplasia consisting of predominantly mature granulocytes. (C) Peripheral blood, May Grunwald-Giemsa stain 100×. (D) Bone marrow, May Grunwald-Giemsa stain 100×. (E) Liver, hematoxylin & eosin (H&E) 20×. (F) Spleen, H&E 20×. Liver and spleen reveal a myeloid infiltrate and extramedullary hematopoiesis.

(A) Bid−/− bone marrow displays normal numbers of progenitor cells. FACS-staining profiles of progenitor populations from littermate Bid+/+ or Bid−/− bone marrow (14–16 mo of age). The lineage (Lin) cocktail consists of: CD3, CD4, CD8, B220, CD19, Ter119, Mac-1, Gr-1. Lin⁻, c-kit⁺, Sca-1⁻ cells were collected by flow sorting, and stained for CD34 and FcγII/III to delineate subpopulations of progenitor cells. HSCs were defined as Lin⁻, c-kit^high, Sca-1^high. Myeloid-erythroid progenitors were defined as Lin⁻ c-kit^highCD34⁻ FcγII/III^lo, GMPs were defined as Lin⁻ c-kit^highCD34⁺FcγII/III^high, and CMPs were defined as Lin⁻ c-kit^highCD34⁺FcγII/III^lo. (B) Transferred Bid−/− leukemic bone marrow demonstrates an expansion of GMP. FACS-staining profiles of progenitor populations of bone marrow from a secondary transplant of leukemic Bid−/− bone marrow reveals increased lin^neg, Sca1^pos, c-kit^pos cells consistent with expansion of GMP cells. Immunophenotype of bone marrow (C) and spleen (D) cells from a representative, primary leukemic Bid-deficient mouse. Flow cytometric analysis revealed an expansion of myeloid precursor cells and mature monocytes. There is a corresponding relative depletion of T cells (CD3+, CD4+, CD8+) and B cells (B220+). Plasma cells (CD138+B220−) and macrophages (F4/80+) were not expanded. Samples were gated on live cells based on forward and side scatter profiles.

(A) Leukemic cells from Bid-deficient animals display malignant potential. Microscopic analysis of secondary bone marrow transplant animals demonstrated aggressive tumor infiltrate in multiple organs. (A) Liver, H&E-stained paraffin section (20×) reveals a diffuse infiltrate of mature monocytic cells. (B) Lung, H&E-stained paraffin section (20×) reveals tumor infiltrate filling blood vessels. (C) Brain, H&E-stained paraffin section (20×) reveals tumor infiltrate obstructing blood vessels with associated hemorrhagic infarct. (D) May Grunwald-Giemsa stained peripheral blood (100×) shows mature myeloid elements.

Bid-deficient tumors are clonal, displaying chromosomal aberrations. A representative SKY of Bid-deficient leukemia. Pseudocolored chromosomes are on the left, inverted DAPI-banded chromosomes are in the middle, and classification-colored chromosomes are on the right. Tumor displays a t(11;6) (red box) and trisomy of multiple chromosomes (yellow arrows).