The interferon-induced RNA-dependent protein kinase (PKR) is considered to play an important role in the r defense against viral infection and, in addition, has been suggested to be a tumor suppressor gene because of its growthsuppressive properties. Activation of PKR by double-stranded RNAs leads to the phosphorylation of the a subunit of eukaryotic initiation factor 2 (eIF-2a) and a resultant block to protein synthesis Initiation. To avoid the consequences of kinase activation, many viruses have developed strategies to down-regulate PKR. Recently, we reported on the purification and characterization of a cellular inhibitor of PKR (referred to as p58), which is activated during influenza virus infection. Subsequent cloning and sequencing has revealed that p58 is a member of the tetripeptide repeat (TPR) family of proteins. To further examine the physiological role of this PKR inhibitor, we stably ransfected NIH 3T3 cells with a eukaryotic expression plasmid contining p58 cDNA under control ofthe cytomegalovirus early promoter. By taking advantage of a recently characterized p58 species-specific monoclonal antibody, we isolated cell lines that overexpressed p58. These cells exhibited a transformed phenotype, owing at faster rates and higher saturation densities and exhibiting anchorage-independent growth. Most importantly, inoculation of nude mice with p58-overexpressing cells gave rise to the production of tumors. Finally, murine PKR activity and endogenous levels of eIF-2a phosphorylation were reduced in the p58-expressing cell lines compared with control cells. These data, taken together, suggest that p58 functions as an oncogene and that one meanism by which the protein induces malnt transformation is through the down-regulation of PKR and subsequent deregulation of protein synthesis. PKR (protein kinase, RNA dependent) is an interferoninduced protein kinase that plays a key role in the cellular defense against viral infection, the regulation of cellular gene expression, and control ofcell growth and proliferation (1-6). Activation by double-stranded RNAs (dsRNAs) or polyanions induces PKR autophosphorylation and, in turn, catalyzes phosphorylation of its natural substrate, the a subunit of the eukaryotic protein synthesis initiation factor, eIF-2a. These events lead to an often dramatic inhibition of protein synthesis initiation (1-3, 7). The activation of PKR can result from interactions with viral-specific RNAs (reviewed in refs.

Objective-Our objective was to determine a mechanism for the thrombocytopenia of murine Wiskott-Aldrich syndrome (WAS).Materials and Methods-Consumption rates of WAS protein (WASP)( −) and wild-type (WT) platelets were measured by injection of 5-chloromethylfluorescein diacetate (CMFDA)-labeled platelets into WT or WASP(−) recipients, and by in vivo biotinylation. Platelet and reticulated platelet counts were performed using quantitative flow cytometry. Bone marrow megakaryocyte number and ploidy was assessed by flow cytometry. Phagocytosis of CMFDA-labeled, opsonized platelets was assessed using bone marrow-derived macrophages. Serum antiplatelet antibodies were assayed via their binding to WT platelets.Results-CMFDA-labeled WASP(−) platelets are consumed more rapidly than WT platelets in either WT or WASP(−) recipients. In vivo biotinylation studies corroborate these findings and show a normal consumption rate for WASP(−) reticulated platelets. The number of reticulated platelets is reduced in WASP(−) mice, but a significant number of the mice show an increased proportion of reticulated platelets and more severe thrombocytopenia. Sera from some of the latter group contain antiplatelet antibodies. Compared to WT platelets, WASP(−) platelets opsonized with anti-CD61 or 6A6 antibody are taken up more rapidly by bone marrow-derived macrophages. In vivo consumption rates of WASP(−) platelets are more accelerated by opsonization than are those of WT platelets.Conclusion-Both rapid clearance and impaired production contribute to the thrombocytopenia of murine WAS. Increased susceptibility of opsonized WASP(−) platelets to phagocytosis leads to increased in vivo clearance. This correlates with a higher incidence of individuals with an elevated fraction of reticulated platelets, a more severe thrombocytopenia, and antiplatelet antibodies. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe Wiskott-Aldrich syndrome (WAS) is an X-linked recessive condition of variable penetrance, classically described as a triad of immunodeficiency, eczema, and thrombocytopenia [1,2]. It is caused by mutations that reduce the level of the WAS protein (WASP). WASP is a 54-kDa polypeptide that is expressed primarily, but not exclusively [3,4], in hematopoietic cells. WASP transmits and integrates signals arising at the cell membrane that result in actin polymerization. This can, in turn, have multiple effects, including but not limited to changes in cell shape and motility. While its function in T cells and macrophages has been studied in some detail [5,6], its biological role in platelets is not clear.WAS patients can have severe thrombocytopenia, with platelet counts ranging from 10,000 to 50,000 per uL in one series [7]. In some cases, the thrombocytopenia is the predominant clinical abnormality. These cases, formerly termed X-linked thrombocytopenia, are frequently due to missense mutations in the first four exons of the gene [8], and can show a fluctuating course resembling immune t...

Homeostasis of the extracellular matrix is a delicate balance between degradation and remodeling, the balance being maintained by the interaction of activated matrix metalloproteinases (MMPs) and specific tissue inhibitors of matrix metalloproteinases (TIMPs). Up-regulation of MMP activity, favoring proteolytic degradation of the basement membrane and extracellular matrix, has been linked to tumor growth and metastasis, as well as tumor-associated angiogenesis, whereas inhibition of MMP activity appears to restrict these processes. We have used retroviral-mediated gene delivery to effect sustained autocrine expression of TIMP-3 in murine neuroblas-