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The proto-oncogenes Myc and Pim1, which are deregulated in many types of cancers, are known to cooperate in B lymphoma development. Here we show that overexpression of retrovirally transduced, doxycycline-inducible Myc alone in IL-7-deprived, growtharrested pre-B cells enhanced cell cycle entry without impairing apoptosis. Overexpression of Pim1 decreased apoptosis, but had no effect on cell cycle entry. Coexpression of Pim1 and Myc inhibited apoptosis and led to IL-7-independent proliferation of the transduced pre-B cells in vitro, while blocking their differentiation to IgM 1 immature cells. Transplantation of Pim1/Myc overexpressing pre-BI cells into B-cell-deficient mice expanded the pre-B-cell compartments up to 100-fold within 4-8 weeks. Transformation remained dependent on the expression of both oncogenes, as removal of doxycycline in vitro and in vivo terminated proliferation and induced differentiation to IgM 1 B cells. In contrast, Pim1/Myc-transduced mature B cells that developed from the oncogene-transduced pre-BI cells in the absence of oncogene overexpression in vivo were not capable of long-term proliferation after induction of Pim and Myc overexpression, neither in vivo nor in vitro, neither with nor without stimulation by polyclonal activators.Key words: B-cell development . Inducible . Lymphoma . Myc . Pim1Supporting Information available online IntroductionDuring the development of B lymphocytes in the murine fetal liver, DJ H /DJ H -rearranged pre-BI cells develop shortly before birth. From these cells, long-term proliferating cell lines can be established in vitro on M-CSF-deficient BM stromal cell lines (OP9) in the presence of IL-7. Differentiation of these pre-BI cells can be induced in vitro by removal of IL-7, which culminates in the generation of sIgM 1 immature B cells [1].Transplantation of these fetal liver-derived pre-BI cell lines into B-cell-deficient recipient mice leads to one wave of B-cell development detectable in spleen and peritoneum, but not in the BM [1]. Using this adoptive transfer system, it is possible to study the effect of transgenes -introduced into the pre-BI cell lineson B-cell differentiation, survival and proliferation at different stages of B-cell maturation. We introduce doxycycline-inducible forms of oncogenes by retroviral vectors into such pre-BI cell lines and, therefore, are able to induce the expression of these oncogenes and study their effects at different stages of B-cell development in vitro and in vivo.The proto-oncogene Myc (c-Myc), a transcription factor of the basic helix-loop-helix/leucine zipper family, has been shown to be deregulated in different types of B-lymphoid tumors [2][3][4]. The Myc protein influences proliferation, differentiation and apoptosis of a variety of different cell types [5][6][7]. Deregulated Myc expression is known to facilitate transit from G1 into S-phase [8][9][10][11][12], and by decreasing levels and functions of P21cip1 and P27kip1, two inhibitors of cell cycle progression from G1-to S-phase [13,14]. Transgenic ...

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While c-myc often contributes to the generation of B cell transformation, its transgenic overexpression alone does not lead to full transformation of B-lineage cells. Synergistically acting second genes must cooperate. Here, we constructed doxycycline-inducible cDNA-libraries from pre-B cell mRNA. These libraries were retrovirally transduced as single copies into single cells and overexpressed in fetal-liver-derived c-myc-overexpressing pre-B cell lines. We scored transformation by survival and/or expansion of differentiating B-lineage cells in vitro and in vivo. Only one double c-myc/cDNA-library-expressing cell line was found in less than 5 × 10 library-transduced pre-B cells surviving and expressing a cDNA-library-derived transcript in vitro. This transcript was identified as a shortened form of the Exosc1 gene, encoding the RNA exosome complex component CSL4. Transplantations of double c-myc/Exosc1 short-form- or c-myc/Exosc1 full-length-transgenic cells into Rag1 mice resulted in survival, differentiation to CD19 CD93 sIgM CD5 CD11b mature B1 cells and, surprisingly, also vigorous expansion in vivo. Strikingly, after transplantations of c-myc/cDNA-library pre-BI cells the frequencies of double-transgenic pre-B cells and their differentiated progeny, expanding in vivo to heterogeneous phenotypes, was at least tenfold higher than in vitro. In a first analysis Ptprcap, Cacybp, Ndufs7, Rpl18a, and Rpl35a were identified. This suggests a strong influence of the host on B-cell transformation.

Deregulated expression of c-myc and bcl-xL is long known to generate transformed B cells in humans and mice. We overexpressed these genes to induce in vitro and in vivo differentiation of fetal liver-derived mouse pre-BI cells to B1-lineage pre-BII-like, immature and mature B-cell lines, and to Ig-secreting cells. In vitro, doxycycline-controlled c-myc/bcl-xL-overexpressing CD19 CD93 c-kikt IgM pre-BI cells differentiate to and survive as CD19 CD93 c-kit IgM immature B1 cells. Timed CpG stimulation of these oncogene-overexpressing pre-B or immature B1 cells generates either CD19 CD93 c-kit IgM SLC pre-BII-like or IgM MHCII CD73 CD80 CD40 mature B1-cell lines and IgM-secreting B1 cells in vitro and fixes their state of differentiation. All cell lines are clonable, but a majority of immature and mature B1-cell clones eventually reach a nonproliferating, surviving G -state. Transplanted in vivo, c-myc/bcl-xL-overexpressing pre-B cells expand to mature B1 cells, and to IgM- and IgA-secreting plasmablasts and plasma cells. Within 2 months, plasmablasts have expanded most prominently in BM and spleen, indicating that the host selectively expanded development of these transformed plasma cells. The sIgM B1-cell lines and clones offer the possibility to study their roles in the development of B1-Ab repertoires, of B1-cell-mediated autoimmune diseases and of B1-cell malignancies.

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