The E-box-binding zinc finger transcription factors Slug and ZEB1 are important repressors of E-cadherin, contributing to epithelial-mesenchymal transition (EMT) in primary epithelial cancers. Activator or repressor status of EMT transcription factors defines consequences for tumorigenesis. We show that changes in expression levels of Slug in melanoma cell lines lead to concomitant alterations of ZEB1 expression. Electrophoretic mobility shift, luciferase reporter, and chromatin immunoprecipitation assays identified Slug as a direct transcriptional activator at E-boxes of the ZEB1 promoter. Transcriptional activation of ZEB1 was demonstrated to be specific for Slug, as EMT regulators Snail and Twist failed to influence ZEB1 expression. Slug and ZEB1 cooperatively repressed E-cadherin expression resulting in decreased adhesion to human keratinocytes, but promoted migration of melanoma cells. Our results show that the transcriptional activity of ZEB1 is increased by Slug, suggesting a hierarchical organized expression of EMT transcription factors through directed activation, triggering an EMT-like process in melanoma.
Resistance to BRAF and MEK inhibition is a common phenomenon in melanoma. Cytokines and transcription factors have been attributed to contribute to the loss of sensitivity towards these inhibitors. Here, we show that transforming growth factor (TGF)-β1 if combined with PLX4032, a BRAF inhibitor, or GSK1120212, a MEK inhibitor, substantially increased cell death in BRAF-mutant melanoma cell lines. This increase was based on the combined regulatory decrease in Twist1, an antiapoptotic protein. Overexpression or silencing of Twist1 attenuated or aggravated induction of apoptosis through PLX4032 or GSK1120212, respectively. Exposure to tumour necrosis factor (TNF)-α, however, led to increased Twist1 levels and oppositional decrease in cell death if exposed to PLX4032 or GSK1120212. This increase in drug resistance again depended on Twist1 levels. Our studies suggest that Twist1 as a common downstream target of multiple signalling cascades plays a crucial role in mediating drug resistance to BRAF- and MEK-targeted molecular inhibitors.
US28, a constitutively active G-protein-coupled receptor encoded by the human cytomegalovirus, leads to mechanistically unknown programmed cell death. Here we show that expression of wild type US28 in human melanoma cells leads to apoptotic cell death via caspase 3 activation along with reduced cell proliferation. Reduced tumor growth upon US28 expression was observed in a xenograft mouse model. The signaling mute US28R129A showed a reduced anti-proliferative effect. On evaluating different G-proteins coupled to US28 for signal transduction, Gα13 was identified as the main G-protein executing the apoptotic effect. Silencing of Gα13 but not Gαq resulted in a substantial increase in cell survival. Over-expression of Gα13 but not Gαq and their GTPase deficient forms Gα13Q226L and GαqQ209L, respectively, confirmed the requirement of Gα13 for US28 mediated cell death. Increasing expression of Gα13 alone induced cell death underscoring its relay function for US28 mediated decreased cell viability. Further reduced expression of Gα13 in melanoma cell lines isolated from advanced lesions and melanoma tissue was observed. These findings identified Gα13 as crucial for US28 induced cell death, substantiating that the effect of US28 on cell fate depends on preferred G-protein binding.
The ability to grow a fully functional Hepatocyte in vitro would be a prodigious breakthrough for modern day drug testing and regenerative medicine. However there are many factors affecting Hepatocyte functionality in vitro that need to be studied before this is possible, like the effect of the underlying substrate that the Hepatocyte is grown on. To better understand this interaction, gene expression profiles of Hepatocytes were measured on four different substrates: Corning(r) CellBIND(r), Collagen, Corning Matrigel(r) and Locust Bean Gum (S906). Here, we present data analysis methods that enable data reduction, identification of differentially expressed genes, and grouping of genes with similar intensities by implementing data clustering. The optimum number of clusters in a given data set is estimated using the gap statistic method. We further employ Cytoscape to help visualize the data and incorporate GeneMania to help identify potentially interesting patterns across substrates. One of the interesting findings from our analysis suggests up-regulation of genes involved in vasculature development on all four substrates. Such findings can be used to design new experiments that can provide further insight on the interactions between hepatocytes and their substrates and help understand the mechanism behind responses of hepatocytes on these substrates.
This chapter outlines approaches for gene assembly and gene annotation of orphan crops (including sugarcane) that allow for sequence assembly even if no closely related sequence is available. It demonstrates the utility of full text mining for gene annotation and pathway discovery and shows that it can be substantially superior to the current 'best in class' annotation methodology, mapping to Gene Ontology terms. Using pathway mapping, significant advances have been achieved in the understanding of lignification both at the enzymatic and control levels. It is shown that text mining can be the key to understanding and extending existing pathways. Using Digitaria exilis as an exemplar, it has been shown that the systems designed for sugarcane are applicable to any orphan crop. Moreover, all the bioinformatics platforms and techniques discussed can be deployed on just a high-end laptop or desktop running Linux, putting the techniques within the reach of most research groups.
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