Granulocyte colony-stimulating factor regulates neutrophil production by binding to a specific receptor, the granulocyte colony-stimulating factor receptor, expressed on cells of the granulocytic lineage. Recombinant forms of granulocyte colony-stimulating factor are used clinically to treat neutropenias. As part of an effort to develop granulocyte colony-stimulating factor mimics with the potential for oral bioavailability, we previously identified a nonpeptidyl small molecule (SB-247464) that selectively activates murine granulocyte colony-stimulating factor signal transduction pathways and promotes neutrophil formation in vivo. To elucidate the mechanism of action of SB-247464, a series of cell-based and biochemical assays were performed. The activity of SB-247464 is strictly dependent on the presence of zinc ions. Titration microcalorimetry experiments using a soluble murine granulocyte colony-stimulating factor receptor construct show that SB-247464 binds to the extracellular domain of the receptor in a zinc ion-dependent manner. Analytical ultracentrifugation studies demonstrate that SB-247464 induces self-association of the N-terminal three-domain fragment in a manner that is consistent with dimerization. SB-247464 induces internalization of granulocyte colony-stimulating factor receptor on intact cells, consistent with a mechanism involving receptor oligomerization. These data show that small nonpeptidyl compounds are capable of selectively binding and inducing productive oligomerization of cytokine receptors.

Harringtonine (HT), homoharringtonine (HHT) and isoharringtonine (IHT) are cephalotaxine alkaloids with anticancer activities isolated from Cephalotaxus hainanensis which is indigenous to China.Since the 1970s, HIT and HHT have been developed as effective anticancer drugs in China and have been used widely in the treatment of acute nonlymphoid leukemia and chronic granulocyte leukemia.Although IHT has the advantage of low toxicity, it has not been developed as an anticancer drug because of its very low content in the plant.The cell apoptosis induced by isoharringtonine was investigated in human acute promyelocytic leukemia HL-60 cells by agarose gel electrophoresis of DNA, flow cytometry and transmission electron microscopy.In these experiments IHT showed significant and rapid apoptotic inductive effect on HL-60 cells in both concentration-and time-dependent fashion.The characteristic apoptosis features could be seen in IHT treated HL-60 cells.Transmission electron microscopy of IHT treated HL-60 cells displayed chromatin condensation and aggregation under the nuclear membrane, nuclear fragmentation and apoptosis body formation.Typical DNA ladder in agarose gel electrophoresis and pre-G1 peak in flow cytometric analysis were also observed in the cells exposed to IHT.The apoptotic rate could reach 43.8% in the HL-60 cells treated for 120 minutes with IHT 10~7 mol/L.The cytotoxicity of IHT paralleled with cell apoptosis indicating that the anticancer activity of IHT results from the induction of apoptosis.These results are important impatus for further research and development of IHT as an anticancer agent.

Activation of members of the STAT (signal transducers and activators of transcription) family of latent transcription factors is an early event following the binding of many cytokines to their cognate receptors, Although the patterns of STATs activated by different cytokines are well described, the consequences of differential STAT activation are less well studied, We show by mutational analysis that STAT binding elements (SBEs) exist that discriminate between STAT complexes containing STAT1 alpha, STAT3 or both, and that these elements show altered cytokine responsiveness, We also show that in the context of a minimal promoter, single and multiple SBEs exhibit strikingly different patterns of transcriptional activation in response to IFN-gamma, IL-6, OSM or LIF. These differences in transcriptional activation are correlated with the differential ability of these cytokines to activate STAT1 alpha, STAT3 or both. Our results show that the pattern of STATs activated by a cytokine and the arrangement and sequence of the SBEs in the responding promoter have a profound effect on the ability of the cytokine to elicit a transcriptional response,

Induction of gene expression by interferon-gamma involves the activation of a latent cytoplasmic transcription factor, p91, by phosphorylation on a single tyrosyl residue. This phosphorylation triggers dimerization, nuclear translocation, and the binding of p91 to interferon-gamma response elements present in the promoters of induced genes. Phosphorylation of p91 requires the activation of two tyrosine kinases, JAK1 and JAK2, that themselves become phosphorylated on tyrosyl residues shortly after interferon-gamma binds to its receptor. The importance of tyrosine phosphorylation in this pathway prompted us to investigate the role of protein tyrosine phosphatases in the regulation of the pathway. We find that in the absence of interferon-gamma, treatment of cells with an inhibitor of tyrosine phosphatases causes a rapid and potent activation of the components of the interferon-gamma signal transduction pathway and induces an interferon-gamma-responsive gene. This suggests that tyrosine phosphatases act both to repress the interferon-gamma signal transduction pathway in the absence of interferon-gamma and to downregulate the pathway after interferon-gamma induction.