Bim deficiency in Eμ-Myc mice results in excess B lymphoid cells. (A) White blood cell counts in healthy 3-wk-old mice of indicated Bim genotype. Open bars, nontransgenic mice; black bars, Eμ-Myc-transgenic mice. Differences between all Bim genotypes were statistically significant for Eμ-Myc-transgenic mice (P < 0.0005), as were the differences between nontransgenic and transgenic mice for each Bim genotype (P = 0.03, P < 0.0001 and P < 0.0001 for Bim^+/+, Bim^+/-, and Bim^-/- mice, respectively). (B) Frequency of pre-B cells (B220⁺ sIgM^-) and B cells (B220⁺ sIgM⁺) per ml of peripheral blood of healthy 3-wk-old wild-type C57BL/6 mice and Eμ-Myc mice of the indicated genotype, determined by flow cytometric analysis. B cells were more numerous in Bim^-/- Eμ-Myc mice (P = 0.03) than in wild-type C57BL/6, whereas in Bim^+/+ Eμ-Myc and Bim^+/- Eμ-Myc mice, the difference from Bim^+/+ Eμ-Myc mice was not significant (P = 0.44 and 0.24, respectively). The number of pre-B cells was significantly different between Bim^+/+ Eμ-Myc mice and wild-type C57BL/6 (P < 0.003), but not between wild-type C57BL/6 and either Bim^+/- Eμ-Myc (P = 0.26) or Bim^-/- Eμ-Myc (P = 0.29) mice. Bim^+/+ Eμ-Myc mice had significantly more pre-B cells than did Bim^+/- Eμ-Myc or Bim^-/- Eμ-Myc mice (P < 0.02 for each comparison).

Bim and p19Arf expression in Eμ-Myc tumors. (A) Western blot analysis of Bim protein in individual tumors from Eμ-Myc-transgenic mice of the indicated Bim genotype. BimEL and BimL are the two most abundant Bim isoforms. Heat shock protein 70 protein served as loading control. (B) Western blot analysis of p19Arf in the same tumors. Heat shock protein 70 and a 60-kDa protein detected by nonspecific staining with the anti-p19 antibody served as loading control, and p53-null mouse embryo fibroblasts (MEF) provided a positive control for p19Arf expression. p19Arf was detected with rabbit antibody. The diagram depicts Myc regulation of the p19Arf/Mdm2/p53 axis (modified from ref. 22).

Loss of Bim accelerates lymphoma development initiated by Eμ-Myc transgene. (A) Cumulative incidence of all tumors in mice of the indicated genotype. Tumors occurred earlier in Bim^+/- mice (P < 0.0001) and Bim^-/- mice (P < 0.0001) than in Bim^+/+ mice. (B) B cell lymphoma development was accelerated in Eμ-Myc mice by the loss of one allele (P = 0.0008) or two alleles of Bim (P < 0.0001). (C) Pre-B lymphoma development was not altered in Bim^+/- mice (P = 1), and a trend toward earlier onset in Bim^-/- mice was not statistically significant (P = 0.1). (D) Frequency of total leukocytes in the blood of sick Eμ-Myc mice of indicated Bim genotype. Bim^+/- mice had higher leukocyte counts than Bim^+/+ mice (P < 0.0001), and Bim^-/- mice had significantly higher counts than in either Bim^+/- (P < 0.0001) or in Bim^+/+ mice (P < 0.0001). The Bim^-/- and Bim^+/- mice had much higher numbers of B cells than did Bim^+/+ mice (P < 0.0001 and P = 0.003, respectively). Pre-B cell numbers were somewhat higher in the Bim^-/- mice than in the Bim^+/+ mice (P = 0.01), but were comparable in the Bim^+/- mice (P = 0.9). Leukocyte counts were 7.9 ± 0.3 × 10⁶ per ml in 45 healthy wild-type C57BL/6 mice and 16 ± 3 × 10⁶ per ml in 39 healthy 3-wk-old Eμ-Myc Bim^+/+ mice.

Bim levels are elevated in preleukemic Eμ-Myc–expressing B lymphoid cells, and loss of Bim enhances their survival. (A) Loss of Bim enhances survival of B lymphoid cells deprived of cytokines. Sorted splenic B cells (B220⁺ IgM⁺; Left) and bone marrow pre-B cells (B220⁺ IgM^-; Right) from healthy young Eμ-Myc mice were cultured and their viability was analyzed by flow cytometry at the indicated intervals, as described in Materials and Methods. The data are pooled for three Bim^-/- Eμ-Myc-transgenic mice and four mice of each of the other genotypes. Error bars, SEM. (B) Loss of Bim protects against apoptosis induced by crosslinking surface IgM. B cells were sorted from the spleen of nontransgenic C57BL/6 animals of the indicated genotype and were incubated in the presence of 0.5 μg/ml anti-IgM Fab fragment. The survival of treated cells is shown, corrected for the spontaneous apoptosis that occurred in culture (see A). Loss of one or both alleles of Bim protected B cells significantly (Fisher's protected least significant difference for 40-h values, P = 0.0025 and P < 0.0001, respectively). (C)Eμ-Myc transgene modulates Bcl2 and Bim expression. Western blot analysis of Bim and Bcl2 protein in sorted pre-B and B cells from healthy 3-wk-old control and Eμ-Myc-transgenic mice. Samples were adjusted for total protein content, and actin staining was used as a loading control. Extracts from two independent sorts are shown in each group and are representative of the results from five experiments. BimEL and BimL are the two most abundant Bim isoforms.

Model for Myc regulation of apoptosis. Data presented in this paper suggest that Bim is a p53-independent target of Myc, either directly or indirectly. Conceivably, as indicated by the broken line, p53-independent induction of apoptosis by p19Arf may also involve activation of Bim (see Discussion).