Background:Aberrant activities of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathways have been implicated in the development and spread of various cancer entities, among them colorectal carcinoma (CRC). Transcription factors STAT3 and STAT1, both downstream effectors of interleukin (IL)-6 and its receptor, are involved in growth and developmental control of CRC cells. Constituents of the signalling network around IL-6 and STAT activation are discussed as potential biomarkers and therapeutic targets in CRC.Methods:By immunohistochemical analysis of a tissue microarray covering >400 CRC biopsies, the expression and activity status of STAT1, STAT3 as well as of IL-6 and the IL-6 receptor α-chain was determined. The outcome was correlated with clinical information and patients' survival data. Colorectal carcinoma biopsies were also analysed for specific DNA-binding activity of STATs.Results:Statistical analysis showed tendential associations between individual STATs, IL-6/IL-6 receptor-α and clinicopathological parameters. The study revealed a significant correlation of high STAT1 activity with longer patient overall survival. Surprisingly, strong STAT3 expression in surgical specimens was correlated with an increase in median overall survival by about 30 months. Statistical analysis revealed that high expression levels of STAT1 and STAT3 were associated. This finding was backed up by biochemical data that showed simultaneous STAT1 and STAT3 DNA-binding activity in randomly selected CRC biopsies.Conclusion:By multivariate data analysis, we could show that STAT3 expression and activity constitutes an independent favourable prognostic marker for CRC.
in this study, we present a straightforward approach for functional cell-based screening by coencapsulation of secretor yeast cells and reporter mammalian cells in millions of individual agarosecontaining microdroplets. our system is compatible with ultra-high-throughput selection utilizing standard fluorescence-activated cell sorters (FACS) without need of extensive adaptation and optimization. In a model study we co-encapsulated murine interleukin 3 (mIL-3)-secreting S. cerevisiae cells with murine Ba/F3 reporter cells, which express green fluorescent protein (GFP) upon stimulation with mIL-3, and could observe specific and robust induction of fluorescence signal compared to a control with yeast cells secreting a non-functional mIL-3 mutant. We demonstrate the successful enrichment of activating mIL-3 wt-secreting yeast cells from a 1:10,000 dilution in cells expressing the inactive cytokine variant by two consecutive cycles of co-encapsulation and fAcS. this indicates the suitability of the presented strategy for functional screening of high-diversity yeast-based libraries and demonstrates its potential for the efficient isolation of clones secreting bioactive recombinant proteins. The importance of biopharmaceuticals (biologics) in medicine is increasing at a fast pace and the biologics market is predicted to reach nearly 400 billion USD/year by 2025 1. Frequently applied biologics comprise substances such as cytokines, monoclonal antibodies, hormones, soluble receptors, recombinant DNAs, enzymes, and synthetic vaccines. While biologics in targeted therapies often demonstrate remarkable safety and specificity, especially in case of autoimmune diseases 2 and cancer 3 , the discovery of novel molecules and the necessary functional validation still represent a bottleneck in the development of novel biopharmaceuticals. To overcome these shortcomings, powerful display technologies such as phage display 4 and yeast surface display 5 have been developed which allow for the isolation of specific high-affinity molecules and respective genes from complex variant libraries. However, identified binders frequently show poor physiological activity in a biological context. Thus, extensive secondary functional screens are necessary for identification of hit molecules with the desired functional activity. Furthermore, those screens require elaborate subcloning of the surface-displayed hits into soluble expression formats and outcoming clones frequently demand further optimization of physicochemical and pharmacokinetic properties 6. Consequently, implementing functional assays and phenotypic screens in an earlier selection phase appears highly beneficial for the discovery of new potent biologic drugs or even first-in-class medicines with novel molecular mechanisms of action 7. In this context, a major limitation is represented by the relatively low throughput of classical phenotypic screens, falling far behind the performance of high-diversity library-based approaches resting on affinity-driven selection protocols 8. Complex p...
Small Kinetochore-Associated Protein (SKAP)/Kinastrin is a multifunctional protein with proposed roles in mitosis, apoptosis and cell migration. Exact mechanisms underlying its activities in these cellular processes are not completely understood. SKAP is predicted to have different isoforms, however, previous studies did not differentiate between them. Since distinct molecular architectures of protein isoforms often influence their localization and functions, this study aimed to examine the expression profile and functional differences between SKAP isoforms in human and mouse. Analyses of various human tissues and cells of different origin by RT-PCR, and by Western blotting and immunocytochemistry applying newly generated anti-SKAP monoclonal antibodies revealed that human SKAP exists in two protein isoforms: ubiquitously expressed SKAP16 and testis/sperm-specific SKAP1. In mouse, SKAP1 expression is detectable in testis at 4 weeks postnatally, when the first wave of spermatogenesis in mice is complete and the elongated spermatids are present in the testes. Furthermore, we identified Pontin as a new SKAP1 interaction partner. SKAP1 and Pontin co-localized in the flagellar region of human sperm suggesting a functional relevance for SKAP1-Pontin interaction in sperm motility. Since most previous studies on SKAP were performed with the testis-specific isoform SKAP1, our findings provide a new basis for future studies on the role of SKAP in both human somatic cells and male germ cells, including studies on male fertility.
The tumor microenvironment plays an important role in cancer progression. In particular, stromal fibroblasts have profound influence on migration and invasiveness of tumor cells. We employ bladder carcinoma cell lines of different de-differentiation grade to pursue the hypothesis that tumor cell malignancy correlates with the cells' potential to recruit support by tumor associated fibroblasts (TAFs). Bladder cancer cell lines RT112 and Cal29 were used as models for tumor cells of low or high de-differentiation grade, respectively. They were grown separately or in co-culture with tumor-associated fibroblast isolated from kidney carcinomas. Migratory and invasive cell properties were quantified by Boyden chamber (Matrigel) and wound healing assays. Cytokine production of cells was measured by intracellular staining followed by cytometry, by semi-quantitative PCR, or by subjecting conditioned medium to ELISA and Western blot analysis. Migration and invasiveness of bladder cancer cell lines Cal29 and RT112 was enhanced by co-cultivation with TAFs. The low grade cell line RT112, however, was much more dependent on TAF-derived signals to increase the malignancy parameters. Conditioned medium from tumor cells induced the secretion of Matrix Metalloproteinases (MMPs) and the release of GM-CSF, HGF, and Monocyte Chemotactic Protein (MCP-1, CCL2) by TAFs. We could identify tumor cell-derived IL-1α and β as well as TGF-β as stimuli responsible for the upregulation of GM-CSF and MCP-1, respectively, in fibroblasts. MCP-1 is implicated in inflammatory disease states characterized by monocytic infiltrates and was shown to promote cell migration of both bladder cancer cell lines under investigation. Employing pathway-specific reporter gene constructs we are currently addressing signal transduction reactions in both tumor cells and fibroblasts underlying this crosstalk. Supported by: IZKF Jena, European Regional Development Fund ERDF. Citation Format: Susanne Grimm, Susanne Jennek, Jens Bratsch, Andreas Wohlmann, Vrinda Mohta, Kerstin Junker, Karlheinz Friedrich. Malignancy of bladder cancer cells is enhanced by tumor associated fibroblasts through a cytokine-chemokine loop. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3597. doi:10.1158/1538-7445.AM2014-3597
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