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Expression of Activated Mutants of the Human Interleukin-3/Interleukin-5/Granulocyte-Macrophage Colony-Stimulating Factor Receptor Common β Subunit in Primary Hematopoietic Cells Induces Factor-Independent Proliferation and Differentiation

Matthew P. McCormack and Thomas J. Gonda

Article Figures & Data

Figures

  • Fig. 1.

    Time-course of proliferation of infected fetal liver cells in liquid culture. Cells were infected with RufNeo retroviral constructs containing the indicated hβc subunits as described in the Materials and Methods. Cells were then washed and 2 × 105 cells were cultured with (A) and without (B) 500 U/mL mIL-3 and mGM-CSF and 2 U/mL hEpo. Cell counts were performed at weekly intervals. (□) hβc; (▴) FIΔ; (▾) V449E; (⋄) I374N.

  • Fig. 2.

    In situ morphology of fetal liver liquid cultures. (A) Cells infected with wild-type hβc retroviruses grown in the presence of growth factors (mIL-3, mGM-CSF, and hEpo). (B) Cells infected with V449E retroviruses grown in the absence of growth factors. (C) Cells infected with FIΔ retroviruses grown in the absence of growth factors. (D) Cells infected with I374N retroviruses grown in the absence of growth factors. Photographs are at 300× original magnification and were taken at day 14 of culture.

  • Fig. 3.

    Expression and confirmation of identity of hβc mutants in factor-independent fetal liver cells. (A) Flow cytometric analyses of mutant hβc expression. Dashed lines represent staining with an irrelevant isotype control antibody. Solid lines represent staining with an anti-hβc MoAb. (B) Map of hβc cDNA showing Nco I (N), BstYI (Bs), and Bgl II (Bg) restriction sites used to authenticate each form of hβc, as well as the region duplicated in FIΔ (indicated by boxes). The restriction sites affected by the point mutations are indicated as Bg+ (gained in V449E) and Bs (lost in I374N). Arrows indicate the positions of PCR primers used to amplify hβc fragments from genomic DNA. (C) Electrophoretic analysis of PCR products generated from genomic DNA of factor-independent fetal liver cells infected with constructs containing the indicated hβc mutants. As a negative control, a reaction was performed containing no DNA (−). Lanes M contain DNA size standards (SPP-1 phage DNA digested with EcoRI [Bresatec Ltd, Adelaide, South Australia]). For comparison, PCR products were generated from RufNeo-hβc plasmids (labeled wild-type). PCR products were either undigested (lanes 1), digested with BglII (lanes 2), or digested with BstYI (lanes 3). Bands in each digest that differ between the mutants and the wild-type are indicated by asterisks.

  • Fig. 4.

    Morphology of factor-dependent and -independent fetal liver cells at day 21 of liquid suspension culture. (A) Cells infected with RufNeo-hβc grown in IL-3, GM-CSF, and Epo. (B) Cells infected with RufNeo-FIΔ grown in the absence of growth factors. (C) Cells infected with RufNeo-V449E grown in the absence of growth factors. (D) Cells infected with RufNeo-I374N grown in the absence of growth factors. Photographs are at 780× original magnification.

  • Fig. 5.

    Expression of lineage-specific cell surface antigens on G418-resistant (G418R) and factor-independent (FI) fetal liver cells. Flow cytometric analyses were performed as described in the Materials and Methods. Dashed lines represent staining with an irrelevant isotype control antibody. Solid lines represent staining with MoAbs directed to the indicated cell surface markers.

  • Fig. 6.

    Colony formation by mock-infected and infected fetal liver cells in methyl-cellulose. Mock-infected and infected fetal liver cells were plated in the indicated conditions in semisolid methyl-cellulose medium and the resultant colonies were scored after 7 days. The data shown are a combination of two separate experiments. In each experiment, duplicate dishes were counted in the case of those containing growth factors, whereas in the case of dishes containing Epo alone or without added growth factors, eight dishes were counted. GFs, growth factors (IL-3, GM-CSF, and Epo).

  • Fig. 7.

    Morphology of factor-independent fetal liver cell lines. (A) For comparison, mast cells obtained from fetal liver cultures infected with RufNeo-hβc and cultured for 6 weeks in the presence of growth factors (IL-3, GM-CSF, and Epo). (B and C) RTVE1 and RTVE2 cell lines, respectively, obtained after infection of fetal liver cells with RufNeo-V449E. Photographs are at 780× original magnification.

Tables

  • Table 1.

    Differential Cell Counts of Factor-Dependent and Factor-Independent Fetal Liver Cells

    RufNeo ConstructAdditionsCell Type (%)
    EosMastNeutMKMErythMyelBlast
    Day 7
    hβcIL-3, GM, Epo0.62.932.80.627.04.626.45.2
    IL3, GM, Epo, G4181.045.50.318.39.320.65.0
    FIΔEpo67.328.72.71.3
    69.730.3
    V449EEpo0.31.347.80.34.310.032.63.3
    2.00.748.80.70.72.339.95.0
    I374NEpo4.092.93.2
    11.288.8
    Day 21
    hβcIL-3, GM, Epo9.041.041.09.0
    IL-3, GM, Epo, G4181.00.379.313.36.0
    FIΔEpo99.30.7
    100.0
    V449EEpo8.36.01.372.012.3
    0.72.793.33.3
    I374NEpo100.0
    100.0
    • Data are representative of three separate experiments.

    • Abbreviations: Eos, eosinophils; Mast, mast cells; Neut, neutrophils; MK, megakaryocytes; M, monocytes/macrophages; Eryth, erythroid cells; Myel, promyelocytes/myelocytes; Blast, blast cells; GM, GM-CSF.

  • Table 2.

    Factor-Dependent and -Independent Colonies Formed by Fetal Liver Cells Infected With Wild-Type and Mutant hβc Subunits

    RufNeo ConstructAdditionsColony types (%)Colonies Examined
    GEoMastMGMMixBFU-EBlast
    hβcIL-3, GM, Epo1214393384103
    IL-3, GM, Epo, G4181836382655109
    FIΔEpo342436680
    17453884
    V449EEpo91122617293369
    1387302018561
    I374NEpo47915173
    1891093
    • Data are a combination of two separate experiments. Colony types were determined at day 7 of culture.

    • Abbreviations: G, neutrophilic granulocyte; Eo, eosinophil; Mast, mast cell; M, macrophage; GM, granulocyte-macrophage; Mix, mixed myeloid/erythroid; BFU-E, erythroid burst-forming unit; Blast, blast cell; GM, GM-CSF.