Furthermore, AnnexinV stainings of splenic B cells one day after setting up the in vitro cultures revealed that in contrast to pre-B cells, B cells did not respond to overexpression of Pim1 by increased survival (Fig. 5E). We conclude that overexpression of Pim1 and Myc does not induce ex vivo isolated splenic or peritoneal CD19+ sIgM+ immature or mature B cells to long-term polyclonal proliferation, or selective survival and extended proliferation in the

absence or presence of polyclonal B-cell stimulators. Our experiments presented in this paper describe the effect of the inducible learn more single or double overexpression of the proto-oncogenes Pim1 and Myc in mouse B-lymphocytes at different stages of development, starting at the DJH/DJH-rearranged pre-BI cell stage 1. Many experiments studying the effect of proto-oncogenes on hematopoietic cells have been done using transgenic mice, also in the case of Pim1 and Myc 18. These mice express the transgenes under the control of the μ enhancer of the immunoglobulin heavy chain (Eμ), which is expressed already at a very early stage of B-cell development. The limitation of such transgenic mice is that if the team play

of the transgenic proto-oncogenes leads to a block in differentiation at an early stage of cell differentiation (as it is the case in these Eμ Pim1/Myc transgenic mice), it is not possible to study effects of the proto-oncogenes on later differentiation stages of the cells using these mice. To circumvent this, we used Sirolimus cost an inducible system to overexpress the two proto-oncogenes, which allowed us to evaluate the effect of proto-oncogene overexpression at different stages of maturation. In the experiments presented here, we have used retroviral vectors to overexpress the proto-oncogenes in B-lymphocytes under the control of a doxycycline-inducible promoter. Retroviral vectors are known to induce transformations by themselves by activating surrounding host genes with their LTR promoters and enhancers. Hence,

we used self-inactivating vectors. It can be expected that three subsequent transductions, performed with the pre-BI cells, have generated a genetically heterogenous collection of transduced cells with differential inducibility of Pim1 and Myc. As one example, such transgenetic heterogeneity might well be the reason why only a fraction of the Pim1/Myc-double-transduced Cepharanthine pre-BI cells initiate proliferation upon proto-oncogene induction, probably either due to inactivation of a transgene or inappropriate overexpression levels of the transgene(s). In spite of these disadvantages, the results of our experiments show that retroviral vectors allow the rapid testing of different combinations of proto-oncogenes in our pre-BI cell lines and their differentiated descendants. Our cell cycle analyses with the Myc-single- and the Pim1/Myc-double-overexpressing pre-B cells show an increase of the frequency of cells in cell cycle.