The adhesion protein E-cadherin plays a central part in the process of epithelial morphogenesis. Expression of this protein is downregulated during the acquisition of metastatic potential at late stages of epithelial tumour progression. There is evidence for a transcriptional blockage of E-cadherin gene expression in this process. Here we show that the transcription factor Snail, which is expressed by fibroblasts and some E-cadherin-negative epithelial tumour cell lines, binds to three E-boxes present in the human E-cadherin promoter and represses transcription of E-cadherin. Inhibition of Snail function in epithelial cancer cell lines lacking E-cadherin protein restores the expression of the E-cadherin gene.

Activation of phosphatidylinositol 3-kinase (PI3K) and activation of the 70/85-kDa S6 protein kinases (␣II and ␣I isoforms, referred to collectively as pp70 S6k ) have been independently linked to the regulation of cell proliferation. We demonstrate that these kinases lie on the same signalling pathway and that PI3K mediates the activation of pp70 by the cytokine interleukin-2 (IL-2). We also show that the activation of pp70 S6k can be blocked at different points along the signalling pathway by using specific inhibitors of T-cell proliferation. Inhibition of PI3K activity with structurally unrelated but highly specific PI3K inhibitors (wortmannin or LY294002) results in inhibition of IL-2-dependent but not phorbol ester (conventional protein kinase C T cells are activated, representing G 0 -to-G 1 transition, by antigen presentation to the multimeric T-cell receptor. This results in the transcription, production, and secretion of the 15-kDa glycoprotein lymphokine, interleukin-2 (IL-2). Antigen stimulation also induces expression of the IL-2 receptor (IL-2R) ␣ subunit (p55) and increases the level of IL-2R ␤ subunit (p75). Together with the ␥ subunit, they form the high-affinity IL-2R (reviewed in references 75 and 103). IL-2 then stimulates activated T cells in an autocrine/paracrine fashion, driving G 1 -S transition and cell proliferation. The IL-2R has no intrinsic kinase activity, yet ligand binding increases tyrosine phosphorylation of many proteins, including the IL-2R ␤ chain. IL-2-dependent signalling also results in activation of c-Ras and phosphatidylinositol 3-kinase (PI3K) and increased serine/ threonine protein phosphorylation. Although much is known about Ras-regulated signal transduction (see references 12, 39, and 68 for reviews), the identities of the signalling proteins lying downstream of PI3K remain to be established.PI3K is a novel signal transducer composed of an 85-kDa SH2-and SH3-domain-containing regulatory subunit and a 110-kDa catalytic subunit with specificity toward the D3 hydroxyl in the inositol ring of phosphatidylinositol (37,54,81,98). Numerous studies provide evidence that PI3K, in association with various mitogenically active receptor and nonreceptor protein tyrosine kinases, mediates the transmission of growth-regulatory information within cells (reviewed in references 18, 21, and 82). These studies suggest that the activation of PI3K contributes a positive, but undefined, cell proliferation signal.The activity of the 70/85-kDa S6 protein kinases (␣I and ␣II isoforms, referred to collectively as pp70 S6k ) is also stimulated by IL-2 in IL-2-responsive cells (16,63,106) as well as in other cell types by many growth factors and oncogenes (reviewed in reference 36). However, the cytosolic mediators involved in its signal cascade have been previously unknown. pp70S6k was identified on the basis of its ability to phosphorylate the 40S ribosomal protein S6 in vitro. A number of other kinases, including the growth-regulated 90-kDa ribosomal S6 kinases (RSKs), can also phosphoryl...

The pp7O/85-kDa S6 kinases, collectively referred to as pp7OS6k, are thought to participate in transit through the GI phase of the cell cycle. pp7Os6k regulates the phosphorylation of the 40S ribosomal protein S6 and the transcription factor CREMT. pp7oS6k is regulated by serine/threonine phosphorylation, and although 1-phosphatidylinositol 3-kinase and phospholipase C have been implicated as upstream regulators, the mechanism of activation and identity of the upstream pp7QS6k kinases remain unknown. To improve our understanding of how this mitogenstimulated protein kinase is regulated by growth factors and the immunosuppressant rapamycin, we have initiated a structure/function analysis of pp79S6k. Our results indicate that both the N and C termini participate in the complex regulation of pp7OS6k activity.Two families of growth factor-regulated serine/threonine kinases that phosphorylate the 40S ribosomal protein S6 in vitro have been previously identified and partially characterized. One of these, the family of 85-92 kDa S6 kinases, referred to collectively as ppgorsk, has been shown to participate in Rasmediated signal transduction and is a direct target of erkencoded MAP kinases (MAPK) (1, 2). The other family, referred to as pp7os6k, consists of two enzymes termed p85a1-and p70aII-S6 kinase. The p85ac isoform is nuclear, while the p70aII isoform is both cytoplasmic and nuclear (ref. 3; M.M.C., unpublished data). Like ppgorsk, pp70S6k is regulated by serine/threonine phosphorylation (4, 5). In contrast to pp9orsk, a pp70S6k kinase has not been identified.

1. GLUT-4 glucose-transporter protein and mRNA levels were assessed in heart, red muscle and white muscle, as well as in brown and white adipose tissue from 7-day streptozotocin-induced diabetic and 48 h-fasted rats. 2. In agreement with previous data, white adipose tissue showed a substantial decrease in GLUT-4 mRNA and protein levels in response to both diabetes and fasting. Similarly, GLUT-4 mRNA and protein markedly decreased in brown adipose tissue in both insulinopenic conditions. 3. Under control conditions, the level of expression of GLUT-4 protein content differed substantially in heart, red and white skeletal muscle. Thus GLUT-4 protein was maximal in heart, and red muscle had a greater GLUT-4 content compared with white muscle. In spite of the large differences in GLUT-4 protein content, GLUT-4 mRNA levels were equivalent in heart and red skeletal muscle. 4. In heart, GLUT-4 mRNA decreased to a greater extent than GLUT-4 protein in response to diabetes and fasting. In contrast, red muscle showed a greater decrease in GLUT-4 protein than in mRNA in response to diabetes or fasting, and in fact no decrease in GLUT-4 mRNA content was detectable in fasting. On the other hand, preparations of white skeletal muscle showed a substantial increase in GLUT-4 mRNA under both insulinopenic conditions, and that was concomitant to either a modest decrease in GLUT-4 protein in diabetes or to no change in fasting. 5. These results indicate that (a) the effects of diabetes and fasting are almost identical and lead to changes in GLUT-4 expression that are tissue-specific, (b) white adipose tissue, brown adipose tissue and heart respond similarly to insulin deficiency by decreasing GLUT-4 mRNA to a larger extent than GLUT-4 protein, and (c) red and white skeletal muscle respond to insulinopenic conditions in a heterogeneous manner which is characterized by enhanced GLUT-4 mRNA/protein ratios.

No abstract

Recombinant adeno-associated viruses (rAAVs) are promising vectors in preclinical and clinical assays for the treatment of diseases with gene therapy strategies. Recent technological advances in amplification and purification have allowed the production of highly purified rAAV vector preparations. Although quantitative polymerase chain reaction (qPCR) is the current method of choice for titrating rAAV genomes, it shows high variability. In this work, we report a rapid and robust rAAV titration method based on the quantitation of encapsidated DNA with the fluorescent dye PicoGreen®. This method allows detection from 3×10(10) viral genome/ml up to 2.4×10(13) viral genome/ml in a linear range. Contrasted with dot blot or qPCR, the PicoGreen-based assay has less intra- and interassay variability. Moreover, quantitation is rapid, does not require specific primers or probes, and is independent of the rAAV pseudotype analyzed. In summary, development of this universal rAAV-titering method may have substantive implications in rAAV technology.

Denervation induces insulin resistance of the glucose transport process in skeletal muscle. To determine whether this is due to alterations in the expression of muscle glucose transporters (GLUT) in different fiber types, we evaluated the amount of GLUT-1 and GLUT-4 protein and messenger RNA (mRNA) in extensor digitorum longus (EDL) and soleus at 1, 2, and 3 days after sciatotomy. Denervation elevated the basal rate of 2-[1,2-3H]deoxy-D-glucose (2-DOG) uptake in the EDL and decreased the insulin-stimulated DOG uptake in both muscles. Denervation after 1 day did not modify the GLUT-1 or the GLUT-4 protein level in either muscle. However, it increased GLUT-1 mRNA by 66% and decreased GLUT-4 mRNA by 70% in the EDL, but not in the soleus (P < 0.05). After 2 days of denervation, by which time GLUT-1 mRNA was increased 2-fold and GLUT-4 mRNA was reduced by 70%, we observed a 2-fold increase in GLUT-1 protein (P < 0.01) in the EDL and a 40-45% decrease in GLUT-4 protein in both muscles (P < 0.01). These results indicate that modifications in the expression of GLUT-1 and GLUT-4 protein cannot explain the insulin resistance of the glucose transport process in the EDL or soleus 1 day after denervation. After 2 days of denervation, however, alterations in GLUT-1 and GLUT-4 protein levels may contribute to the change in basal and insulin-stimulated DOG uptake in both the EDL and the soleus muscles.

Canine adenovirus vectors (CAV2) are currently being evaluated for gene therapy, oncolytic virotherapy, and as vectors for recombinant vaccines. Despite the need for increasing volumes of purified CAV2 preparations for preclinical and clinical testing, their purification still relies on the use of conventional, scale-limited CsCl ultracentrifugation techniques. A complete downstream processing strategy for CAV2 vectors based on membrane filtration and chromatography is reported here. Microfiltration and ultra/diafiltration are selected for clarification and concentration of crude viral stocks containing both intracellular and extracellular CAV2 particles. A DNase digestion step is introduced between ultrafiltration and diafiltration operations. At these early stages, concentration of vector stocks with good recovery of viral particles (above 80%) and removal of a substantial amount of protein and nucleic acid contaminants is achieved. The ability of various chromatography techniques to isolate CAV2 particles was evaluated. Hydrophobic interaction chromatography using a Fractogel propyl tentacle resin was selected as a first chromatography step, because it allows removal of the bulk of contaminating proteins with high CAV2 yields (88%). An anion-exchange chromatography step using monolithic supports is further introduced to remove the remaining contaminants with good recovery of CAV2 particles (58-69%). The main CAV2 viral structural components are visualized in purified preparations by electrophoresis analyses. Purified vector stocks contained intact icosahedral viral particles, low contamination with empty viral capsids (10%), and an acceptable total-to-infectious particle ratio (below 30). The downstream processing strategy that was developed allows preparation of large volumes of high-quality CAV2 stocks.