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Efficient and Rapid Induction of a Chronic Myelogenous Leukemia-Like Myeloproliferative Disease in Mice Receiving P210 bcr/abl-Transduced Bone Marrow

Warren S. Pear, Juli P. Miller, Lanwei Xu, John C. Pui, Benny Soffer, Robert C. Quackenbush, Ann Marie Pendergast, Roderick Bronson, Jon C. Aster, Martin L. Scott and David Baltimore

Article Figures & Data

Figures

  • Fig. 1.

    (A) Structure of the retroviral vectors used to transduce murine bone marrow. The LTRs and vector backbone for all constructs was MSCV2.2. The EcoRI and Xba I restriction enzyme sites used for determining proviral integration are indicated. (B) Protocol for retroviral transduction of FU-treated bone marrow and subsequent reconstitution. During the 48-hour cocultivation, retroviral supernatants were added to the media (1 mL every 24 hours) or cells were transduced by spin infection without cocultivation (see Materials and Methods).

  • Fig. 2.

    Survival of mice receiving transduced bone marrow cells. The survival data are cumulative from two separate experiments for all retroviral constructs, except MSCV 210, which are from one experiment.

  • Fig. 3.

    Characteristics of the P210-induced myeloproliferative disease. (A) Peripheral blood from a mouse transduced with a control retrovirus (MigR1). Original magnification × 100. (B) Typical appearance of peripheral blood from mouse with myeloproliferative disease. Original magnification × 100. (C) Higher power view of (B). Original magnification × 1,000. (D) Splenomegaly associated with myeloproliferative disease. The diseased spleen is on top and the spleen from a MigR1 control animal is below. (E) Hematoxylin and eosin (H&E) section of spleen from (D). The red pulp is replaced by sheets of granulocytes. Original magnification × 400. (F) Hypercellular murine CML bone marrow. The great majority of cells are mature granulocytes. Original magnification × 400. (G) Liver in murine CML shows infiltration of mature myeloid cells and EMH in sinusoids. Original magnification × 100. This mouse did not have the macrophage expansion. (H) Murine CML liver with macrophage expansion (arrow). Note infiltrating hematopoietic cells in sinusoids. Original magnification × 400. (I) Pulmonary infiltrates of EMH in P210 mice. Original magnification × 100. (J) T-cell lymphoma associated with blast transformation (from an abdominal mass). Original magnification × 400. This tumor developed after 2 rounds of serial transplant of the myeloproliferative disease from mouse H2. (K) Wright-Giemsa staining of cells from peripheral blood of mouse receiving Mig210-transduced bone marrow cells. Original magnification × 400. (L) Abl expression in the cells from (K) as detected by the abl monoclonal antibody 24-11. Original magnification × 400. (M) Wright-Giemsa staining of cells from peripheral blood of mouse receiving Mig210-transduced bone marrow cells. Original magnification × 400. (N) Staining with an isotype control. Original magnification × 400.

  • Fig. 4.

    P210 bcr/abl is expressed in tissues involved in the myeloproliferative process. Cells (5 × 106) were loaded per lane and detected with the 8E9 abl-specific monoclonal antibody (Pharmingen). Many of the lower molecular weight bands are most likely the result of proteolysis due to the high percentage of neutrophils in the cell lysates.28 Lanes 1 through 4, bone marrow, peripheral blood, spleen, and liver cells from a Mig210 mouse. Lane 5, day-12 cells from bone marrow cultures transduced with Mig185. Mig185 is similar to Mig210, except Mig185 expresses P185 bcr/abl (Miller and Pear, unpublished data). Lane 6, Balb/c bone marrow cells. The band at approximately 140 kD in all lanes is c-abl. Arrows indicate the 210- and 185-kD proteins.

  • Fig. 5.

    Immunophenotypes of cells obtained from peripheral blood, spleen, bone marrow, and liver of mice receiving either P210 (Mig210) or control (MigR1) bone marrow cells. Mig210 expresses both P210 bcr/abl and GFP, whereas MigR1 expresses only GFP. GFP fluoresces in the FL1 channel. The lineage-specific antibodies (Gr1, Mac1, B220, Thy1.2, and Ter119) were directly or indirectly labeled with PE and fluoresce in the FL2 channel. Hematopoietic cells were not present in the livers of mice receiving MigR1. Ter119 staining was not performed on the cells derived from MigR1 peripheral blood. The Mig210 data were obtained from mouse BB12 and are a representative Mig210 phenotype. The MigR1 data were obtained from mouse FF4 and are a representative MigR1 phenotype.

  • Fig. 6.

    Proviral integration in P210 mice. (A through D) Tissues from mice receiving MSCV 210/grb2. (E) Tissues from mice receiving MSCV 210. (F) Tissues from mice receiving Mig210. All DNA preparations were digested with EcoRI except for lanes 1 and 3 in (F), which were digested with BamHI, which also cleaves once in the provirus. Digestion with Xba I showed the presence of intact proviral DNA for all samples (not shown). All samples are from primary mice and labeled with the tissue from which the DNA was derived, except for the following in (B): lane 2, G46A1 spleen-secondary recipient of spleen cells from G46, this mouse developed the myeloproliferative disease; lane 1, G46A1B2 spleen: recipient of spleen cells from G46A1, this mouse developed T-ALL; lanes 9 and 10, G41A1 spleen and peripheral blood, G41A2 spleen, and G41A3 peripheral blood: recipient of spleen cells from G41; these mice developed the myeloproliferative disease. Abbreviations: PB, peripheral blood; B, BamHI; R1,EcoRI. Sizes of the λ HindIII marker are shown.

  • Fig. 7.

    Development of blast transformation after serial passage. (A) Cartoon showing disease development in recipients of cells from serial passage. Secondary recipients (A1 through 5) received a 1:1 mixture of spleen and bone marrow cells from mouse H2. Spleen or bone marrow cells were transferred to tertiary recipients. Only mice receiving the spleen cells developed the myeloproliferative disease. All mice receiving bone marrow cells developed T-cell lymphomas with characteristics similar to H2A4B1. Cells derived from the spleen of tertiary recipients that developed the myeloproliferative disease were transferred to quaternary recipients. All of these mice developed T-cell lymphomas. (B) Proviral integration pattern shows a common single proviral integration site in all H2 mice. Lanes 1 and 2 are from primary mice. Lanes 3 through 8 are from secondary mice. (C) Both the myeloproliferative disease and T-cell lymphomas show an identical proviral integration pattern. Lanes 2 through 4 are derived from T-ALL. Lanes 5 through 7 are secondary and tertiary recipients. (D) Tissues from the H2 mice show the presence of an intact integrated provirus. For (B) and (C), genomic DNA was digested with EcoRI and genomic DNA shown in (D) was digested with Xba I. The blots were hybridized with the cDNA expressing the neomycin resistance gene.

Tables

  • Table 1.

    Summary of murine P210 bcr/abl bone marrow transfer experiments

    ConstructNo. of Independent, ExperimentsNo. of Evaluable MiceNo. of Mice With Murine CMLLatency (d)WBC Range (× 106 cells/mL)Spleen Weight Range in Grams% With Pulmonary Involvement% With Macrophage Foci
    MSCV 2101111121-3136-2650.6-0.910091
    MSCV 210/grb22191919-2650-1600.6-0.910047
    Mig 2102101016-1960-3900.6-0.7510020
    • The results summarize five independent experiments. For MSCV 210/grb2, 23 mice were reconstituted; however, 4 mice died before analysis and tissue degradation precluded diagnosis. Latency is defined as either time to death or time to onset of terminal symptoms consisting of cachexia, decreased activity, and hunched posture with tachypnea. For control mice transduced with either MigR1 or MSCV grb2, the WBC ranged from 5 to 15 × 106/mL cells during this period and spleen weights were 0.1 to 0.25 grams.

  • Table 2.

    Disease Characteristics of Murine CML

    MouseLatency (wks)WBCDifferential CountSpleen Weight (g)
    Grb21545P/50L/5M0.2
    H2047584P/5L/3M/6NR0.6
    H21410073P/11L/1M/11NR0.8
    H2237769P/7L/3M/9NR0.6
    H2347780P/7L/2M/1NR0.6
    H2433651P/9L/1M/36NR/3Blasts0.7
    H25426586P/2L/1M/11NR0.9
    H26425090P/3L/2M/1NR0.75
    H27353ND0.6
    H28422079P/13L/3M/5NR0.8
    H29320076P/4L/20NR0.85
    • Characteristics of the P210-induced myeloproliferative disease in one transplant experiment. Disease was induced by transduction with MSCV 210 (see Table 1). Grb2 is a mouse that received MSCV Grb2, which only expresses grb2. This mouse (as did all MSCV Grb2 mice) remained healthy for the entire 9-month observation period. WBC count is 106 cells/mL. For the differential counts, P indicates polymorphonuclear granulocytes, L indicates lymphocytes, M indicates monocytes, NR indicates nucleated red blood cells, and ND indicates not determined.

  • Table 3.

    Summary of Serial Transplants

    MouseConstructPrimarySecondaryTertiaryQuaternaryIdentical Proviral Integration
    H2210CMLCMLCML, T-ALLT-ALLYes
    G4210/grb2CMLT-ALLNDYes
    G28210/grb2CMLCML, ALLND 3-150
    G41210/grb2CMLCMLALL 3-150
    G46210/grb2CMLCMLALLYes
    G57210/grb2CMLCMLNDYes
    SI2210/grb2CMLCMLND 3-150
    • Summary of serial transplant experiments. The different constructs are MSCV 210 and MSCV 210/grb2. The diseases are listed under each round of transplant. Where there is more than one disease listed in a column, mice in the cohort developed either one or the other disease, but not both diseases. Proviral integration was determined by digestion of genomic DNA with EcoRI and hybridization with either an IRES or neo probe as described in Materials and Methods. All four tumors analyzed showed an identical proviral integration pattern in secondary, tertiary, and quaternary serial transplant recipients.

    • Abbreviation: ND, not done.

    • F3-150 Mice died before workup. For these mice, diagnosis was made by morphology. Integrated proviral DNA was present by Xba digestion and PCR (not shown); however, the proviral integration pattern could not be assessed due to degradation.