Notch signaling is a potential therapeutic target for various solid and hematopoietic malignancies. We have recently shown that downregulation of Notch-1 expression has significant anti-neoplastic activity in pre-clinical models. However, the mechanisms through which Notch modulation may affect cell fate in cancer remain poorly understood. We had previously shown that Notch-1 prevents apoptosis and is necessary for pharmacologically induced differentiation in murine erythroleukemia (MEL) cells. We investigated the mechanisms of these effects using three experimental strategies: (1) MEL cells stably transfected with antisense Notch-1 or constitutively active Notch-1, (2) activation of Notch-1 by a cell-associated ligand, and (d3) activation of Notch-1 by a soluble peptide ligand. We show that: (1) downregulation of Notch-1 sensitizes MEL cells to apoptosis induced by a Ca(2+) influx or anti-neoplastic drugs; (2) Notch-1 downregulation induces phosphorylation of c-Jun N-terminal kinase (JNK) while constitutive activation of Notch-1 or prolonged exposure to a soluble Notch ligand abolishes it; (3) Notch-1 has dose- and time-dependent effects on the levels of apoptotic inhibitor Bcl-x(L) and cell cycle regulators p21(cip1/waf1), p27(kip1), and Rb; and (4) Notch-1 activation by a cell-associated ligand is accompanied by rapid and transient induction of NF-kappaB DNA-binding activity. The relative effects of Notch-1 signaling on these pathways depend on the levels of Notch-1 expression, the mechanism of activation, and the timing of activation. The relevance of these findings to the role of Notch signaling in differentiation and cancer are discussed.
Recent advances in our understanding of highly conserved mechanisms that control cell fate determination are paving the way towards rationally designed biologics that modulate specific cell fate decisions. Cell fate decisions leading to proliferation, differentiation or apoptosis are crucial elements in the pathogenesis of countless human diseases. Biopharmaceuticals designed to regulate such processes in specific cell types in vivo or ex vivo have vast potential applications in oncology, stem cell technology, immunomodulation and neuropathology. One of the most conserved mechanisms controlling cell fate determination is based upon Notch-ligand interactions and subsequent signaling events. Recent studies have shown that this mechanism regulates cell differentiation, proliferation and apoptosis in a wide variety of cell maturation processes and in neoplastic cells. These observations identify the Notch signaling network as a promising drug target for numerous indications. In this review, we describe: 1) potential drug targets in the Notch signaling network; 2) the Notch agonists and antagonists developed so far, including recombinant proteins, antibody-based agents, synthetic peptides, antisense oligonucleotides and gene therapy approaches, as well as possible strategies to design novel Notch-targeting biopharmaceuticals; 3) the possible clinical applications of such biopharmaceuticals and 4) a model strategy for the selection and developement of a Notch-targeting biopharmaceutical.
Allograft rejection is induced by graft tissue infiltration of alloreactive T cells that are activated mainly in secondary lymphoid organs of the host. DOCK2 plays a critical role in lymphocyte homing and immunological synapse formation by regulating the actin cytoskeleton, yet its role in the in vivo immune response remains unknown. We show here that DOCK2 deficiency enables long-term survival of cardiac allografts across a complete mismatch of the major histocompatibility complex molecules. In DOCK2-deficient mice, alloreactivity and allocytotoxicity were suppressed significantly even after in vivo priming with alloantigens, which resulted in reduced intragraft expression of effector molecules, such as interferon-γ, granzyme B, and perforin. This is mediated, at least in part, by preventing potentially alloreactive T cells from recruiting into secondary lymphoid organs. In addition, we found that DOCK2 is critical for CD28-mediated Rac activation and is required for the full activation of alloreactive T cells. Although DOCK2-deficient, alloreactive T cells were activated in vitro in the presence of exogenous interleukin-2, these T cells, when transferred adoptively, failed to infiltrate into the allografts that were transplanted into RAG1-deficient mice. Thus, DOCK2 deficiency attenuates allograft rejection by simultaneously suppressing multiple and key processes. We propose that DOCK2 could be a novel molecular target for controlling transplant rejection.
This study investigated the effects of a blocking anti-CD28 antibody (Anti-CD28-PV1-IgG3) in vitro and in vivo. Anti-CD28-PV1-IgG3, a hamster-mouse chimeric antibody against murine CD28, which does not provide CD28-positive signaling during TCR-driven T cell activation, enabled long-term survival of heart allografts across a complete mismatch of the MHC in rats. Among the T cell signaling proteins tested in the spleens from recipients, we found that recipients treated with anti-CD28-PV1-IgG3 exhibited suppression of alloantigen-initiated proximal TCR signaling events, including Lck, Zap70, Vav, and PI3K expression, and their PKC theta- and JNK-regulated expression/activation. This leads to attenuation of intragraft T cell infiltration and expression of T cell effector molecules. These results indicate that targeting the CD28 receptor with a blocking antibody leads to long-term allograft survival by reducing activation of alloantigen-mediated key signaling events in T cells that might be crucial for full T cell activation.
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