Interleukin 6 (IL-6) is the major survival factor for myeloma tumor cells and induces signaling through the STAT proteins. We report that one STAT family member, Stat3, is constitutively activated in bone marrow mononuclear cells from patients with multiple myeloma and in the IL-6-dependent human myeloma cell line U266. Moreover, U266 cells are inherently resistant to Fas-mediated apoptosis and express high levels of the antiapoptotic protein Bcl-xL. Blocking IL-6 receptor signaling from Janus kinases to the Stat3 protein inhibits Bcl-xL expression and induces apoptosis, demonstrating that Stat3 signaling is essential for the survival of myeloma tumor cells. These findings provide evidence that constitutively activated Stat3 signaling contributes to the pathogenesis of multiple myeloma by preventing apoptosis.

We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 g of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.Genes can be transferred into skeletal muscle cells of rodents and primates by intramuscular injection of plasmid DNA, and the resulting gene expression has been reported to last as long as several months (1, 2). Similarly, various viral vectors such as adenoviral, retroviral, and AAV-based vectors (3), have been used to transduce myofibers in vivo. The i.m. injection of plasmid DNA, however, has several advantages over viral vectors. First, plasmid DNA vectors are easier to construct and can be prepared as pharmaceutical-grade solutions (4) without the risk of contamination with wild-type infectious particles. Second, previous infection by wild-type adenovirus or AAV may induce a neutralizing antibody response that could preclude administration of the recombinant virus. In contrast, anti-DNA antibodies have never been detected in experiments of muscle DNA injection (2), therefore it is possible to readminister plasmid DNA by i.m. injection if repeated therapy or escalation is required.Despite the promise of i.m. injection of plasmid vectors for treating serum protein deficiencies, several important issues remain to be addressed before this approach becomes feasible for human gene therapy. The potential clinical usefulness of direct gene transfer to muscle of plasmid DNA is in fact limited by the low and highly variable level of gene expression (1, 2, 5, 6). Therefore, although DNA injection is potentially very powerful as a vaccination method because a low level of gene expression is sufficient to trigger immunoresponses, it is necessary to increase the efficiency of DNA uptake after i.m. injection of plasmid vectors before using this technique as a standard gene correction procedure.One of the most efficient methods implemented to achieve gene transfer and expression in mammalian cells is based on electric pulses (7). Electroporation has been used to introduce foreign DNA in different cell types (7), but it has also recently met with some success in in vivo applications. Gene transfer by electrical permeabilization has been obtained in skin (8, 9), corneal endothelium (10), melanoma (11), brain (12), liver, (13) and muscle (14) of experimental animals.We have shown previously that electropermeabilization can increase severalfold the uptake by rat muscle of a plasmid encoding the Escherichia coli lacZ gene (15). In this study...

Helper-dependent (HD) adenoviral vectors devoid of all viral coding sequences provide for safe and highly efficient gene transfer with long-lasting transgene expression. High titer stocks of HD vectors can be generated by using the cre-recombinase system. However, we have encountered difficulties with this system, including rearranged HD vectors and variable efficiency of HD vector rescue. These problems represent a major hindrance, particularly with regard to large-scale production. To overcome these limitations, we have modified the system in two ways: We constructed a new helper virus with a modified packaging signal and enhanced growth characteristics. We also redesigned the vector backbones by including noncoding adenovirus sequences adjacent to the right inverted terminal repeat and by incorporated a number of different segments of noncoding DNA of human origin as ''stuffer.'' Comparison of these vectors showed that the nature of the stuffer sequence affects replication of the HD vector. Optimization of the system resulted in a more robust and consistent production of HD vectors with low helper contamination and high in vivo potency.

The helical cytokine interleukin (IL) 6 and its specific binding subunit IL‐6R alpha form a 1:1 complex which, by promoting homodimerization of the signalling subunit gp130 on the surface of target cells, triggers intracellular responses. We expressed differently tagged forms of gp130 and used them in solution‐phase binding assays to show that the soluble extracellular domains of gp130 undergo dimerization in the absence of membranes. In vitro receptor assembly reactions were also performed in the presence of two sets of IL‐6 variants carrying amino acid substitutions in two distinct areas of the cytokine surface (site 2, comprising exposed residues in the A and C helices, and site 3, in the terminal part of the CD loop). The binding affinity to IL‐6R alpha of these variants is normal but their biological activity is poor or absent. We demonstrate here that both the site 2 and site 3 IL‐6 variants complexed with IL‐6R alpha bind a single gp130 molecule but are unable to dimerize it, whereas the combined site 2/3 variants lose the ability to interact with gp130. The binding properties of these variants in vitro, and the result of using a neutralizing monoclonal antibody directed against site 3, lead to the conclusion that gp130 dimer is formed through direct binding at two independent and differently oriented sites on IL‐6. Immunoprecipitation experiments further reveal that the fully assembled receptor complex is composed of two IL‐6, two IL‐6R alpha and two gp130 molecules. We propose here a model representing the IL‐6 receptor complex as hexameric, which might be common to other helical cytokines.

DNA oligonucleotide complementary to sequences in the 5′ third of U3 snRNA were injected into Xenopus oocyte nuclei to disrupt endogenous U3 snRNA. The effect of this treatment on rRNA processing was examined. We found that some toads have a single rRNA processing pathway, whereas in other toads, two rRNA processing pathways can coexist in a single oocyte. U3 snRNA disruption in toads with the single rRNA processing pathway caused a reduction in 20S and ‘32S’ pre‐rRNA. In addition, in toads with two rRNA processing pathways, an increase in ‘36S’ pre‐rRNA of the second pathway is observed. This is the first in vivo demonstration that U3 snRNA plays a role in rRNA processing. Cleavage site #3 is at the boundary of ITS 1 and 5.8S and links all of the affected rRNA intermediates: 20S and ‘32S’ are the products of site #3 cleavage in the first pathway and ‘36S’ is the substrate for cleavage at site #3 in the second pathway. We postulate that U3 snRNP folds pre‐rRNA into a conformation dictating correct cleavage at processing site #3.