The molecular processes which control self-renewal and differentiation of hematopoietic stem cells (HSCs) are not completely understood. Elucidating these mechanisms and signaling pathways is important in possible ex vivo expansion and treatment of HSCs. It is likely that genes which are highly expressed in HSC populations have important roles in their biology. The stem cell database (http://stemcell.princeton.edu) identified many genes highly expressed in HSC populations using subtractive cDNA technology (Ivanova et al. Science, 2002. 298(5593): p. 601-04). We chose to study the novel gene, Ras homologue enriched in brain like-1 (RhebL1), identified in the stem cell database. Rheb and RhebL1, the two members of the Rheb GTPase family, have positive roles in activating the mammalian target of rapamycin (mTOR) pathway. The mTOR pathway positively affects proliferation, growth and survival of various cell types. Due to its expression pattern and its role in the mTOR pathway, we hypothesized that RhebL1 plays an important role in regulating hematopoietic cell biology. We cloned the full-length RhebL1 cDNA (BC016521) into the bicistronic MIEG3 retroviral vector, which contains the EGFP reporter. We transduced primary mouse bone marrow (mBM) cells with the MIEG3-RhebL1 vector and found positive effects of overexpression of RhebL1 on proliferation and colony-forming ability compared to cells transduced with empty vector MIEG3. Overexpression of RhebL1 increased the proliferation of mBM cells up to 2-fold over control cells in liquid cultures up to one week after transduction. In addition to the positive effect on proliferation, we found that overexpression of RhebL1 in mBM cells significantly increased the absolute number of hematopoietic progenitors, measured by methylcellulose colony assays, in liquid cultures of transduced cells up to one week after transduction. This positive effect on progenitor number was greatest for the CFU-granulocyte-macrophage (GM) progenitors (2.5 fold increase over MIEG3 cells). The number of CFU-GEMM progenitors was also significantly increased by RhebL1 overexpression, while the BFU-Erythroid progenitors were not significantly affected. The transduced cells remained GFP+ during the 6-day methylcellulose colony assay incubation period as determined by confocal microscopy. In addition to the overexpression studies with primary mBM cells, we have expanded our work to involve the BaF3 mouse pro-B cell line. BaF3 cells are dependent on the growth factor IL-3 for survival and proliferation. In addition, the mTOR pathway is involved in IL-3 signaling in these cells (Cruz et al. J. Leuk. Bio., 2005.78(6): p. 1378-85). We found that overexpression of RhebL1 in BaF3 cells increased proliferation after delayed IL-3 addition compared to cells expressing empty vector. Along with an increase in proliferation, BaF3 cells overexpressing RhebL1 had increased mTOR activation as measured by phosphorylated S6 protein levels within the cells during IL-3 starvation and after addition of IL-3. These results indicate that RhebL1 overexpression positively affects the proliferation and colony-formation of primary mouse BM cells. In the BaF3 cell line, RhebL1 overexpression increases mTOR signaling and positively affects the proliferation of the cells after delayed IL-3 addition. Our results suggest a role for RhebL1 in hematopoietic progenitor cell functions.

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