Important functions of IL3 are the regulation of growth and early differentiation of hae matopoietic progenitors as well as the control of the terminal differentiation of basophils, mast cells and dendritic cells. Recent publications suggest a strong link between RhoH e pression levels and B cell malignancies. We therefore used IL3 dependent BaF3 cells, a murine proB cell line, as a model system. These cells were shown to e press comparatively low levels of RhoH. Inhibitors,Modulators,Libraries We show that overe pression of RhoH decreases IL3 induced proliferation and the activ ity of STAT5. The surface e pression level of the IL3 receptor a chain is inversely correlated to the e pression levels of RhoH. In RhoH deficient cells, the STAT5 dependent gene interferon regulatory factor 1 is upregulated, eventually leading to an upregu lation of CD123.

Interestingly, only BaF3 cells that over e press RhoH are able to activate STAT1 after stimulation with IL3. This correlates with an upregula tion of the STAT1 dependent cell cycle inhibitors p21Cip1 and p27Kip1. Thus, our findings link the regula tory function of RhoH on proliferation to Inhibitors,Modulators,Libraries an interaction with the JAK STAT signalling pathway. Results RhoH regulates IL3 dependent cell proliferation In order to study the effects of RhoH on IL3 mediated signals, we used the IL3 dependent, murine proB cell line BaF3 to generate cell lines where the RhoH gene was silenced using the retroviral vector pSilen cer Retro 5. 1 U6 or Cilengitide that were retrovirally transduced with RhoH to overe press the protein. As a control, parental BaF3 Inhibitors,Modulators,Libraries cells were transduced with the empty vector.

Infected cells were selected with puromycin and the resulting stable Inhibitors,Modulators,Libraries cell lines were tested for RhoH e pression by quantitative real time PCR using GAPDH as a reference gene. Figure 1 A shows that in siRhoH cells the e pression was decreased to app. 10% com pared to control cells, while the RhoH cells showed a five fold overe pression of the gene. To corroborate successful retroviral transduction, we performed FACS analysis and compared CD4 e pression levels. It was shown previously that the level of e pression of the genes up and downstream of the IRES sequence are highly correlated in stably infected target cells. Figure 1B shows that control cells and RhoH transduced cells were app. 80% positive for CD4 e pression and e pressed the vector at comparable levels.

Parental cells that do not e press CD4 were used as a negative control. To investigate whether the overe pression of RhoH leads to changes in IL3 receptor mediated signalling events, proliferative responses were tested using control cells and BaF3 cells overe pressing RhoH. Cells were incubated with IL3 concentrations ranging from 0. 001 to 1 ng ml and factor dependent growth was determined after 48 hours through measurement of cel lular ATP.