American Society of Hematology

Figure 7.

$Figure 7. MSCV-Myc leukemias contain intact Arf-p53 pathways. (A) Germline DNA from spleens of leukemic mice was analyzed by Southern hybridization using an Ink4a exon 1β probe. Lanes 1 and 6 are Balb/c control mice and demonstrate the germline configuration of the locus. Arrowheads indicate size of germline fragments. Lanes 2 to 5 and 7 to 10 are MSCV-Myc spleen cells. Lanes 1 to 5 are spleen genomic DNA samples digested with XbaI and lanes 6 to 10 are digested with BglII. The band in lane 6 is due to inadvertent DNA overloading noted on ethidium-stained gel (data not shown). In all leukemias examined, the Ink4 locus was found to be in germline configuration. (B) Western blot analysis of p53 protein and the p53 target p21 in isolated leukemia cells before and after γ-irradiation. Leukemia cells were purified from spleens of moribund MSCV-Myc mice by FACS sorting on GFP and forward/side scatter profiles. Lysates were isolated at time zero (lanes 1-3) and from parallel cell aliquots 2 hours after irradiation with 500 Gy (lanes 4-6). All lanes have detectable p53 and the 2-hour samples reveal increased p21 levels, consistent with expected up-regulation by intact p53 response. β-actin levels indicated equivalent protein loading in all lanes. (C) Western analysis of proteins from tissues of leukemic and control animals that underwent transplantation. Myc protein is expressed in all MSCV-Myc and MSCV-Myc+Bcl2 mice. Bcl-2 is only expressed in MSCV-Myc+Bcl2 mice. Bcl-XL is expressed in normal bone marrow as well as unfractionated leukemia samples due to normal cell contamination (see panel E). (D) Western analysis of p19Arf and Bcl-2 proteins in animals that underwent transplantation. Arf is expressed in all tumors analyzed. Arf-null mouse embryonic fibroblasts (MEFs) are shown as negative control. Bcl-2 is expressed only in MSCV-Myc+Bcl2 samples. (E) Comparison of Bcl-XL protein levels in unfractionated and purified tumor-cell populations. Unfractionated tumor tissues show abundant Bcl-XL protein due to contamination with normal cells. Bcl-XL levels in purified leukemia cells are much lower (see panel B). (F) Comparison of c-Myc expression in Eμ-Myc lymphoma and MSCV-Myc leukemia. Whole-cell lysates were prepared from tumor-bearing lymph nodes from an Eμ-Myc mouse, and spleen cells from MSCV-Myc (Myc), MSCV-Myc+Bcl2 (Myc+Bcl2), and normal Balb/c control (BALB/c) mice. Expression levels appear equivalent in samples from retroviral and transgenic models.$

MSCV-Myc leukemias contain intact Arf-p53 pathways. (A) Germline DNA from spleens of leukemic mice was analyzed by Southern hybridization using an Ink4a exon 1β probe. Lanes 1 and 6 are Balb/c control mice and demonstrate the germline configuration of the locus. Arrowheads indicate size of germline fragments. Lanes 2 to 5 and 7 to 10 are MSCV-Myc spleen cells. Lanes 1 to 5 are spleen genomic DNA samples digested with XbaI and lanes 6 to 10 are digested with BglII. The band in lane 6 is due to inadvertent DNA overloading noted on ethidium-stained gel (data not shown). In all leukemias examined, the Ink4 locus was found to be in germline configuration. (B) Western blot analysis of p53 protein and the p53 target p21 in isolated leukemia cells before and after γ-irradiation. Leukemia cells were purified from spleens of moribund MSCV-Myc mice by FACS sorting on GFP and forward/side scatter profiles. Lysates were isolated at time zero (lanes 1-3) and from parallel cell aliquots 2 hours after irradiation with 500 Gy (lanes 4-6). All lanes have detectable p53 and the 2-hour samples reveal increased p21 levels, consistent with expected up-regulation by intact p53 response. β-actin levels indicated equivalent protein loading in all lanes. (C) Western analysis of proteins from tissues of leukemic and control animals that underwent transplantation. Myc protein is expressed in all MSCV-Myc and MSCV-Myc+Bcl2 mice. Bcl-2 is only expressed in MSCV-Myc+Bcl2 mice. Bcl-XL is expressed in normal bone marrow as well as unfractionated leukemia samples due to normal cell contamination (see panel E). (D) Western analysis of p19Arf and Bcl-2 proteins in animals that underwent transplantation. Arf is expressed in all tumors analyzed. Arf-null mouse embryonic fibroblasts (MEFs) are shown as negative control. Bcl-2 is expressed only in MSCV-Myc+Bcl2 samples. (E) Comparison of Bcl-XL protein levels in unfractionated and purified tumor-cell populations. Unfractionated tumor tissues show abundant Bcl-XL protein due to contamination with normal cells. Bcl-XL levels in purified leukemia cells are much lower (see panel B). (F) Comparison of c-Myc expression in Eμ-Myc lymphoma and MSCV-Myc leukemia. Whole-cell lysates were prepared from tumor-bearing lymph nodes from an Eμ-Myc mouse, and spleen cells from MSCV-Myc (Myc), MSCV-Myc+Bcl2 (Myc+Bcl2), and normal Balb/c control (BALB/c) mice. Expression levels appear equivalent in samples from retroviral and transgenic models.

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