In vitro and in vivo characterization of the transforming properties of FOP-FGFR1 kinase. (A) Proliferative effect of FOP-FGFR1 on murine Sca-1+ hematopoietic progenitors. Each point represents the mean number (SEM) of total colony-forming cells (CFCs) per 104 cells generated after sequential plating in methylcellulose from three independent transduction experiments. (B) Kaplan-Meier plots of recipient mice survival after transplantation of bone marrow Sca-1+ stem cells infected with FOP-FGFR1 and its mutants as indicated (n=number of mice). Arrows and asterisk indicate the FOP-FGFR1 Y511/F mice that developed a thymic lymphoma and that were killed for characterization (n=10), respectively. (C) Histopathology of mice transplanted with FOP-FGFR1 and derived mutants. Views of spleen (ii, iii), and liver (v, vi) of transplanted mice. (i, iv) Normal tissues in a negative control mouse transplanted with Sca-1+ bone marrow cells retrovirally transduced with MSCV. In the FOP-FGFR1 animals, spleen (ii) and liver (v) showed effacement of normal architecture due to marked extramedullary hematopoiesis with maturing myeloid and erythroid precursors, and numerous megakaryocytes (original magnification × 100, inserts × 400). A slight extramedullary hematopoiesis was also found in the FOP-FGFR1 Y511/F spleens (iii). The liver from FOP-FGFR1 Y511/F mouse (vi) was almost normal, with only a few foci of inflammation. The 5-μm deparaffinized sections were hematoxylin-eosinstained. (D) TCR gene rearrangement in FOP-FGFR1 (F-R1) and FOP-FGFR1Y511/F (Y511/F) transplanted mice. Representative results of blot hybridization with J2 probes of gels containing PCR products from indicated tissues and mice using D2 5′ and J2 3′ PCR primers19 . Purified thymocytes from wild-type (WT) and RAG-1-/- mice were used as positive and negative controls, respectively. Autoradiographs were exposed for 4 hours so that the faint bands corresponding to TCR rearrangements of some mature T cells in spleen samples are not visible. (E) FOP-FGFR1 and FOP-FGFR1 PLC-binding–defective (Y511) are expressed in tissues involved in the myeloproliferative disease. To evaluate expression of the introduced FOP-FGFR1 (left panel) and FOP-FGFR1 Y511/F (right panel) proteins, cell lysates prepared from different tissues as indicated were subjected to immunoprecipitation with anti–C-FGFR1 and analyzed by Western blot with anti–C-FGFR1 and/or antiphosphotyrosine antibodies. Note the strong expression of the mutant in spleen, and thymus (in the animals with thymus hypertrophy) and the faint one in bone marrow and liver, in agreement with the histopathological findings. (F-G) Southern blot detection of provirus integration in tissues of FOP-FGFR1 mice. Genomic DNAs from bone marrow (BM), spleen (S), liver (L), and thymus from several FOP-FGFR1 and FOP-FGFR1 Y511/F transplanted and diseased mice, were digested with either KpnI (F) or BamHI (G) and hybridized with a radioactive probe from the neomycin resistance gene. (F) KpnI cuts in both retroviral LTRs; the single band of expected size demonstrates the presence of provirus. GP+E86 packaging cell line FOP-FGFR1 stably transfected (GP+E FOP-FGFR1), and bone marrow cells from one of the control vector mice (MSCV) were used as controls. Asterisk indicates the thymus sample from a FOP-FGFR1Y511/F mouse that developed a malignant lymphoma 126 days after transplantation. (G) BamHI cuts only once in the provirus; this results in bands of different sizes corresponding to 2 to 4 genomic integration sites (arrowheads). Note the presence of the same clones in different tissues from the same mouse. Bone marrow cells from a non-infected mouse (WT) and from one of the vector control mice (MSCV) were used as controls.
