Conditional expression of Pim1 and c-Myc in murine B lineage cells and their functional consequences

This thesis was aimed at identifying proto-oncogenes that contribute to cell cycle entry, proliferation and survival of mouse B-lymphocytes at different stages of their development, i.e. in preB-I, preB-II, immature B-cells, mature resting and mature activated B-cells.
A central notion of tumour development of the B-lymphocyte cell lineages is that the activation of a single proto-oncogene, or the loss of a single tumour suppressor gene, is not sufficient to transform cells to full malignancy. One such example with which this cooperation of oncogenes has been studied is Eµ-myc induced lymphomagenesis. Transgenic Eµ-myc mice express the c-myc gene under the control of the IgH-chain gene enhancer in B-lineage cells. B-cell lymphomas are generated in these mice within several weeks. Retroviral insertional mutagenesis has identified pim1 as cooperating oncogene which accelerates lymphomagenesis in vivo.
Hence, I have chosen myc and pim1 to introduce them into B-cell progenitors by retroviral transduction, to test their effects alone or together on preB-I, preB-II, immature and mature B-cells in vitro and, upon transplantation into recipient mice, in vivo. As target cells for retroviral transduction, mouse fetal liver-derived preB-I-cells were chosen, since they proliferate long term in vitro on stromal cells in the presence of IL7.
I used inducible forms of these genes to turn on and off their expression in the transfected cells at different times for different time periods. After initial tests with the inducible estrogen-receptor system, the TetON inducible expression system was chosen which allows the expression of inducible transgenes upon addition of doxycycline.
In the here presented work show that overexpression of Myc alone in pre-BI cells in vitro enhances cell cycle entry without impairing apoptotis of pre-BI cells induced by the removal of IL-7 from the culture. Pim1 overexpression alone did not show any effect. However, in cooperation with Myc, Pim1 led to growth-factor-independent long term in vitro proliferation of pre-BI cells deprived of IL-7. This induction of proliferation could be reversed when doxycycline was removed again from the culture. During long term proliferation of these cells, differentiation to pre-BII and immature B-cells was slowed down but not completely blocked in vitro. The differentiated pre-BII and IgM+ immature B-cells also were induced to growth factor-independent proliferation by Pim1 plus Myc.
Transplantation of Myc-overexpressing pre-BI cells into sublethally irradiated Rag KO mice did not increase the numbers of B cells developing in the transplanted mice compared with mice transplanted with doxycycline-non-induced pre-BI cells. On the other hand, transplantation of pre-BI cells overexpressing Pim1 and Myc together expanded the immature IgM- (pre-B) cell compartment 100 fold and the immature IgM+ B-cell compartment 6 fold in the spleen within 1 month. Upon removal of doxycycline from the drinking water, the expanded numbers of B-cells reverted to almost normal levels, i.e. to the levels of B cell numbers of doxycycline-uninduced mice. Hence, overexpression of the proto-oncogenes Pim1 and Myc is able to induce growth-factor-independent proliferation of pre-B cells and immature IgM+ cells, in vitro and in vivo.
By contrast, mature B cells overexpressing Myc and Pim1 together, isolated from the spleens of pre-BI cell-transplanted mice, were not induced to cytokine-independent proliferation in vitro. Even the stimulation by LPS, IL-5, IL-4 + anti-CD40, and anti-Ig in vitro did not induce prolonged proliferation beyond the normal stimulation observed with non-transgenic, or with transgenic B cells in the absence of doxycycline. Likewise, in vivo antigenic stimulation with KLH of mice transplanted with Myc and Pim1-overexpressing pre-BI cells in the presence or absence of co-transplanted T-cells did not result in expanded, KLH-specific, class-switched antibody responses.
Hence, while pre-BI, pre-BII and immature B cells expand cytokine-independently in vitro and in vivo when Pim1 and Myc are overexpressed together, mature B cells generated from these transplanted pre-B cells in vivo do not manifest deregulated proliferation or survival in vitro or in vivo upon T-cell-independent or T-cell-dependent stimulation.