Anisomycin Selectively Desensitizes Signalling Components Involved in Stress Kinase Activation and fos and jun Induction

Anisomycin, a translational inhibitor secreted by Streptomyces spp., strongly activates the stress-activated mitogen-activated protein (MAP) kinases JNK/SAPK (c-Jun NH₂-terminal kinase/stress-activated protein kinase) and p38/RK in mammalian cells, resulting in rapid induction of immediate-early (IE) genes in the nucleus. Here, we have characterized this response further with respect to homologous and heterologous desensitization of IE gene induction and stress kinase activation. We show that anisomycin acts exactly like a signalling agonist in eliciting highly specific and virtually complete homologous desensitization. Anisomycin desensitization of a panel of IE genes (c-fos, fosB, c-jun, junB, and junD), using epidermal growth factor (EGF), basic fibroblast growth factor, (bFGF), tumor necrosis factor alpha (TNF-α), anisomycin, tetradecanoyl phorbol acetate (TPA), and UV radiation as secondary stimuli, was found to be extremely specific both with respect to the secondary stimuli and at the level of individual genes. Further, we show that anisomycin-induced homologous desensitization is caused by the fact that anisomycin no longer activates the JNK/SAPK and p38/RK MAP kinase cascades in desensitized cells. In anisomycin-desensitized cells, activation of JNK/SAPKs by UV radiation and hyperosmolarity is almost completely lost, and that of the p38/RK cascade is reduced to about 50% of the normal response. However, all other stimuli produced normal or augmented activation of these two kinase cascades in anisomycin-desensitized cells. These data show that anisomycin behaves like a true signalling agonist and suggest that the anisomycin-desensitized signalling component(s) is not involved in JNK/SAPK or p38/RK activation by EGF, bFGF, TNF-α, or TPA but may play a significant role in UV- and hyperosmolarity-stimulated responses.     

Tubulin structure: insights into microtubule properties and functions

The structure of tubulin has recently been determined by electron crystallography, paving the way for a clearer understandin of the unique properties of tubulin that allow its varied functions within the cell. Some of the ongoing work on tubulin can be interpreted in terms of its structure, which can serve to guide future studies.     

Reactivity of new adhesion molecules on lymphocytes from patients with chronic graft versus host disease

Reaction patterns of the 7th Human Leukocyte Differentiation Antigen Workshop blind panel adhesion molecules were studied on CD3/CD4, CD3/CD8, CD3/TCR gamma delta double positive T cells from peripheral blood of patients with chronic graft versus host disease (n = 8) and healthy controls (n = 4). Reactivity of 14 adhesion antibodies was tested by three-colour immunophenotyping. The mean proportion of CD3+ T cells (69 +/- 19%). CD3/CD8++ (31 +/- 13%) and CD3/TCR gamma delta++ (4 +/- 2%) T sub-populations of patients were comparable with the healthy controls. However, the mean percentage of CD3/CD4++ T cell subset in patients (14 +/- 12%) proved to be significantly decreased in comparison with the normal control value (34 +/- 16%) presumably due to secondary immunodeficiency. The workshop antibodies proved to be reactive with three T cell subsets expressing the examined antigens. Based on the results of the adhesion molecule workshop new CD categories have been introduced: CD156b as a transmembrane protein, CD167a as an epithelial tyrosin kinase receptor, CD168 as a receptor for hyaluronan mediated motility (RHAMM) and CD171 as a co-stimulatory adhesion molecule. There were significant differences in the expression of the CD167a and CD156b antigens on the CD3/CD4++ subset between the samples of patients compared with the controls characterizing the CD4+ T lymphocyte subpopulation in chronic graft versus host disease.     

Behaviour of interleukin-2 serum levels in advanced non-small-cell lung cancer patients: relationship with response to therapy and survival

 Interleukin(IL)-2 is a T helper (Th) 1 type cytokine that has been shown to play an important role in antitumour immune responses. In this study, the prognostic significance of serum IL-2 levels was investigated in 60 advanced non-small-cell lung cancer (NSCLC) patients. IL-2 serum levels were determined before chemotherapy, at the end of chemotherapy and during follow-up, using a commercially available enzyme-linked immunoadsorbent assay kit. The results were analysed according to the response to therapy and were used to generate a model predicting overall survival and time to treatment failure. All 60 patients were shown to have higher IL-2 serum levels than controls (P < 0.0001). Stage IV patients had significantly lower IL-2 levels than stage III patients (P < 0.0001), although they were still significantly higher than controls (P < 0.0001). It is interesting that, when patients were divided into responders and non-responders according to the response to therapy, the former were shown to have significantly higher pre-chemotherapy levels than the latter (P < 0.0001). Moreover, a further significant increase in IL-2 serum levels (P=0.004) and a significant decrease (P < 0.0001) were shown in responders and non-responders, respectively at the end of the therapy. Using univariate and multivariate analyses, both overall survival and time to treatment failure were shown to be affected by the mean pathological levels of IL-2. Furthermore, the prognostic significance of the serum level of IL-2 was confirmed by the stepwise regression analysis. In conclusion, determination of pre-treatment IL-2 serum levels was shown to be of independent prognostic utility in patients with advanced NSCLC; therefore, its possible use for prediction of outcome is proposed. Received: 16 March 2000 / Accepted: 27 July 2000     

MDSCs Mediate Angiogenesis and Predispose Canine Mammary Tumor Cells for Metastasis via IL-28/IL-28RA (IFN-λ) Signaling

Background

Myeloid-derived suppressor cells (MDSCs) function in immunosuppression and tumor development by induction of angiogenesis in a STAT3-dependent manner. Knowledge of MDSC biology is mainly limited to mice studies, and more clinical investigations using spontaneous tumor models are required. Here we performed in vitro experiments and clinical data analysis obtained from canine patients.

Methods

Using microarrays we examined changes in gene expression in canine mammary cancer cells due to their co-culture with MDSCs. Further, using Real-time rt-PCR, Western blot, IHC, siRNA, angiogenesis assay and migration/invasion tests we examined a role of the most important signaling pathway.

Results

In dogs with mammary cancer, the number of circulating MDSCs increases with tumor clinical stage. Microarray analysis revealed that MDSCs had significantly altered molecular pathways in tumor cells in vitro. Particularly important was the detected increased activation of IL-28/IL-28RA (IFN-λ) signaling. The highest expression of IL-28 was observed in stage III/IV mammary tumor-bearing dogs. IL-28 secreted by MDSCs stimulates STAT3 in tumor cells, which results in increased expression of angiogenic factors and subsequent induction of angiogenesis by endothelial cells, epithelial-mesenchymal transition (EMT) and increased migration of tumor cells in vitro. Knockdown of IL-28RA decreased angiogenesis, tumor cell invasion and migration.

Conclusions

We showed for the first time that MDSCs secrete IL-28 (IFN-λ), which promotes angiogenesis, EMT, invasion and migration of tumor cells. Thus, IL-28 may constitute an interesting target for further therapies. Moreover, the similarity in circulating MDSC levels at various tumor clinical stages between canine and human patients indicates canines as a good model for clinical trials of drugs targeting MDSCs.     

Mapping of nucleoside phosphorylase (Np-1) and esterase 10 (Es-10) on mouse chromosome 14

A method for detecting two alleles at Np-1 (nucleoside phosphorylase) and three alleles at Es-10 (esterase 10) from mouse blood by cellulose acetate electrophoresis is described. The allelic constitution at these loci for 44 inbred strains and stocks was determined. The location of Np-1 on chromosome 14 was established by backcross experiments in which alleles at Np-1 and Robertsonian translocations were segregating. Es-10 was shown to be linked to Np-1, and the following genetic map of Chr 14 was constructed: centromere-(8.9±4.0 cM)-[Np-1, Wc]-(10.2±1.9 cM)-Es-10-(15.5±3.7 cM)-s. The homologous human loci, NP and ES-D, are not linked.This work was supported by Contract E(11-1)-3267 with the Energy Research and Development Administration, by Contracts NO1-ES4-2156 and NO1-ES4-2159 with the National Institute of Environmental Health Sciences, and by Grants GM 19656 and GM 20919 from the National Institute of General Medical Sciences. D. A. K. was a participant in the 1975 Summer Program for College, Graduate, and Medical Students, which was supported, in part, by the Clark Foundation. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

La grotte de Santa Ana (Cáceres, Espagne) et l'évolution technologique au Pléistocène dans la Péninsule ibérique

The excavations carried out in the cave of Santa Ana (Cáceres, Spain), cave of the karstic network of the “Calerizo” of Cáceres, enabled us to know the existence, in stratigraphy, of the three lito-techniques modes which characterize the industrial development of lower and middle Pleistocene in the Iberian Peninsula. On standby of new research, the results obtained until now permit us two work out a diachronic assumption of technical evolution. In the Iberian Peninsula, there are only two karstic systems where we can fallow this technological development; one is the “Sierra de Atapuerca” (Burgos) and the other is the “Calerizo de Cáceres” (Cáceres).     

α‐Hemolysin enhances Staphylococcus aureus internalization and survival within mast cells by modulating the expression of β1 integrin

Mast cells (MCs) are important sentinels of the host defence against invading pathogens. We previously reported that Staphylococcus aureus evaded the extracellular antimicrobial activities of MCs by promoting its internalization within these cells via β1 integrins. Here, we investigated the molecular mechanisms governing this process. We found that S. aureus responded to the antimicrobial mediators released by MCs by up‐regulating the expression of α‐hemolysin (Hla), fibronectin‐binding protein A and several regulatory systems. We also found that S. aureus induced the up‐regulation of β1 integrin expression on MCs and that this effect was mediated by Hla‐ADAM10 (a disintegrin and metalloproteinase 10) interaction. Thus, deletion of Hla or inhibition of Hla‐ADAM10 interaction significantly impaired S. aureus internalization within MCs. Furthermore, purified Hla but not the inactive Hla_H35L induced up‐regulation of β1 integrin expression in MCs in a dose‐dependent manner. Our data support a model in which S. aureus counter‐reacts the extracellular microbicidal mechanisms of MCs by increasing expression of fibronectin‐binding proteins and by inducing Hla‐ADAM10‐mediated up‐regulation of β1 integrin in MCs. The up‐regulation of bacterial fibronectin‐binding proteins, concomitantly with the increased expression of its receptor β1 integrin on the MCs, resulted in enhanced S. aureus internalization through the binding of fibronectin‐binding proteins to integrin β1 via fibronectin.     

Location of phosphorylase kinase (Phk) in the mouse X chromosome

The phosphorylase kinase deficiency (Phk) locus has been located in the mouse X chromosome, the order of genes being centromere-Bn-Phk-Ta-jp. Since the Phk locus of the mouse may be identical to the locus responsible for the X-linked phosphorylase kinase deficiency trait of man, and there may be a high degree of gene-order homology in the X chromosome of all mammals, the location of Phk in the mouse reported here may aid in locating the phosphorylase kinase gene on the X chromosome of man.This research was supported by grants AM 13359 (to F.H.) and AM 14461 (to D.L.C.) from the National Institute of Arthritis and Metabolic Diseases, and by an allocation (to E.M.E.) from NIH General Research Support Grant RR-05545 from the Division of Research Resources to The Jackson Laboratory. F.H. is a recipient of a Research Career Development Award (AM 46 421) of the National Institute of Arthritis and Metabolic Diseases.