Molecular factors regulating E-cadherin expression in urothelial bladder cancer and their correlations with the clinicopathological features

This study aimed to assess the expression of S100A4, Twist and E-cadherin (mRNA and protein) in urothelial bladder cancer, investigate the correlation between them and evaluate their association with the clinicopathological features of the disease. The study included 54 patients diagnosed as urothelial bladder cancer of different stages and grades. The expression levels of S100A4, Twist and E-cadherin (mRNA and protein) in tissue samples were determined by quantitative RT-PCR and immunohistochemistry. The expression of S100A4 and Twist was significantly upregulated while E- cadherin was significantly downregulated in urothelial bladder cancer tissues compared to the adjacent surrounding normal bladder tissues at both mRNA and protein levels (p?<?0.001). Expression levels of S100A4 and Twist were significantly higher in recurrent tumor than in non-recurrent tumors (p?<?0.001) while the expression level of E-cadherin was significantly lower in recurrent tumors than in non-recurrent tumors at both mRNA and protein levels (p?<?0.001). There was a significant positive correlation between S100A4 and Twist expressions (r?=?0.875, p?<?0.001) while significant negative correlations were found between E- cadherin and S100A4 expressions(r=- 0.803, p?<?0.001) and between E-cadherin and Twist (r?=??0.809, p?<?0.001). Up-regulation of S100A4 and Twist and down-regulation of E-cadherin in urothelial bladder cancer tissues compared to adjacent normal tissues were observed. There was a significant negative correlation between S100A4 and E- cadherin and between E- cadherin and Twist expression. However, there was a significant positive correlation between S100A4 and Twist expressions. Furthermore, the alterations in the gene expression were associated with disease stage and grade.     

Cytotoxic and toxicogenomic effects of silibinin in bladder cancer cells with different <Emphasis Type="Italic">TP53</Emphasis> status

Silibinin is a natural phenol found in the seeds of the milk thistle plant. Recent data have shown its effectiveness for preventing/treating bladder tumours. Therefore, in this study we investigated the cytotoxic and toxicogenetic activity of silibinin in bladder cancer cells with different TP53 statuses. Two bladder urothelial carcinoma cell lines were used: RT4 (wild-type TP53 gene) and T24 (mutated TP53 gene). Cell proliferation, clonogenic survival, apoptosis rates, genotoxicity and relative expression profile of FRAP/mTOR, FGFR3, AKT2 and DNMT1 genes and of miR100 and miR203 were evaluated. Silibinin promoted decreased proliferation and increased late apoptosis in TP53 mutated cells. Increased early apoptosis rates, primary DNA damage, and decrease of cell colonies in the clonogenic survival assay were detected in both RT4 and T24 cell lines. Down-regulation of FRAP/mTOR, AKT2, FGFR3, DNMT1 and miR100 expression occurred in RT4 cells. Modulation of miR203 was observed in both cell lines. In conclusion, despite the reduction of clone formation in both cell lines, the toxicogenomic effect of silibinin on FRAP/mTOR, AKT2, FGFR3, DNMT1 and miR100 was dependent on the TP53 status. Taken together, the data confirmed the role of silibinin as an antiproliferative compound, whose mechanism of action was related to the TP53 status.     

The Differentiation of Skin Mesenchymal Stem Cells Towards a Schwann Cell Phenotype: Impact of Sigma-1 Receptor Activation

Neural crest stem cells (NCSCs) are the source of mature Schwann cells in the peripheral nervous system (PNS). The NCSC population resides in the bulge of hair follicles and in the dermis. Recently, it was shown that 2–3% of the human dermis mesenchymal stem cell (MSC) population expresses the NCSC marker CD271, thus enabling the use of skin MSCs for studying Schwann cell differentiation in vitro. The aims of this study were to establish a protocol for human skin MSC differentiation towards Schwann cell-like cells (SC-lcs) and to analyse the expression of sigma-1 receptor (S1R) in SC-lcs. The impact of S1R ligands, namely the selective agonist PRE-084, the positive allosteric modulator E1R and the selective antagonist NE-100, on Schwann cell differentiation was assessed. The expression of the neuron-specific genes Tubulin-βIII and Integrin-6α, the Schwann cell-specific gene S100b, MBP and the NCSC-specific genes p75NTR, Sox10, Notch1, Integrin-4α, Ap2α and Pax6 was analysed in MSCs and SC-lcs by real-time RT-PCR. BDNF secretion was evaluated by ELISA. The effect of S1R ligands on SC-lc differentiation was measured using BDNF ELISA and MBP flow cytometry. After MSC differentiation, NCSC markers p75NTR and Integrin-4α were downregulated 3.5-fold and 2-fold, respectively. To the contrary, MBP and S100b were significantly upregulated in SC-lcs. S1R ligands showed a tendency to increase the secretion of BDNF by the SC-lc population. PRE-084 and E1R increased MBP expression in the SC-lc population, whereas 3 μM NE-100 inhibited MBP expression in SC-lcs. In conclusion, our data demonstrate that S1R plays an important role in skin MSC differentiation towards myelinating Schwann cells.     

Sorbitol-6-phosphate dehydrogenase gene (<Emphasis Type="Italic">S6PDH</Emphasis>) polymorphism in tribe Pyreae (Rosaceae) species

The sorbitol-6-phosphate dehydrogenase gene (S6PDH) sequences of eight tribe Pyreae species (Rosaceae) are studied for the first time. The exon–intron structure and polymorphism of the nucleotide and amino acid sequences of this gene are characterized. The interspecific polymorphism of the S6PDH coding sequences in the studied Pyreae species is 8.36%. Sorbitol-6-phosphate dehydrogenase gene expression in S. aucuparia, A. melanocarpa, and M. domestica (cv. Skala) leaves is studied. The highest level of S6PDH expression is detected in mature leaves.     

Dependence of expression of regulatory master genes of embryonic development in pancreatic cancer cells on the intracellular concentration of the master regulator PDX1

Exogenous expression of the gene encoding the pancreatic master regulator PDX1 in cell lines with different degrees of differentiation of pancreatic cancer cells is accompanied by changes in the expression of known master genes involved in cancer progression. In BxPC3^PDX+ cells, as compared to BxPC3^PDX–, we detected an increased expression of the following genes: NKX6.1 (2 times), NR5A2 (2.5 times), KLF5 (1.8 times), ZEB1 (3 times), and ONECUT1 (1.3 times), as well as a decreased expression of MUC1 and SLUG genes (3 and 2 times, respectively). In PANC1^PDX+ cells, as compared to the control PANC1^PDX– cells, we detected a decreased expression of ISL1 (2 times) and an increased expressed of KRT8 (2 times) and MUC1 (by 30%). In the high-grade cell lines (including the BxPC3 line studied), the total content of sites containing the marks of active enhancers was higher than that in the low-grade cell lines (PANC1).     

New <Emphasis Type="Italic">slbo</Emphasis>-Gal4 driver lines for the analysis of border cell migration during <Emphasis Type="Italic">Drosophila</Emphasis> oogenesis

Border cell (BC) migration during Drosophila oogenesis is an excellent model for the analysis of the migratory and invasive cell behavior. Most studies on BC migration have exploited a slbo-Gal4 driver to regulate gene expression in these cells or to mark them. Here, we report that the slbo-Gal4 transgene present in the line #6458 from the Bloomington Stock Center is inserted within chickadee (chic), a gene encoding the actin-binding protein Profilin, which promotes actin polymerization and is known to be involved in cell migration. The chic⁶⁴⁵⁸ mutation caused by the transgene insertion behaves as a null chic allele and is homozygous lethal. To evaluate possible effects of chic⁶⁴⁵⁸ on the assessment of BC behavior, we generated new lines bearing the slbo-Gal4 transgene inserted into different second chromosome loci that do not appear to be involved in cell migration. Using these new lines and the slbo-Gal4-chic⁶⁴⁵⁸ line, we defined the functional relationships between the twinfilin (twf) and chic in BC migration. Migration of BCs is substantially reduced by mutations in twf, which encodes an actin-binding protein that inhibits actin filament assembly. The defects caused by twf mutations are significantly suppressed when the slbo-Gal4-chic⁶⁴⁵⁸, but not the new slbo-Gal4 drivers were used. These findings indicate twf and chic interact and function antagonistically during BC migration in Drosophila oogenesis.     

A WD40 protein,AtGHS40, negatively modulates abscisic acid degrading and signaling genes during seedling growth under high glucose conditions

The Arabidopsis thaliana T-DNA insertion mutant glucose hypersensitive (ghs) 40-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The ghs40-1 mutant displayed severely impaired cotyledon greening and expansion and showed enhanced reduction in hypocotyl elongation of dark-grown seedlings when grown in Glc concentrations higher than 3 %. The Glc-hypersensitivity of ghs40-1 was correlated with the hyposensitive phenotype of 35S::AtGHS40 seedlings. The phenotypes of ghs40-1 were recovered by complementation with 35S::AtGHS40. The AtGHS40 (At5g11240) gene encodes a WD40 protein localized primarily in the nucleus and nucleolus using transient expression of AtGHS40-mRFP in onion cells and of AtGHS40-EGFP and EGFP-AtGHS40 in Arabidopsis protoplasts. The ABA biosynthesis inhibitor fluridone extensively rescued Glc-mediated growth arrest. Quantitative real time-PCR analysis showed that AtGHS40 was involved in the control of Glc-responsive genes. AtGHS40 acts downstream of HXK1 and is activated by ABI4 while ABI4 expression is negatively modulated by AtGHS40 in the Glc signaling network. However, AtGHS40 may not affect ABI1 and SnRK2.6 gene expression. Given that AtGHS40 inhibited ABA degrading and signaling gene expression levels under high Glc conditions, a new circuit of fine-tuning modulation by which ABA and ABA signaling gene expression are modulated in balance, occurred in plants. Thus, AtGHS40 may play a role in ABA-mediated Glc signaling during early seedling development. The biochemical function of AtGHS40 is also discussed.     

Localization of gene expression,tissue specificity of <Emphasis Type="Italic">Populus</Emphasis> xylosyltransferase genes by isolation and functional characterization of their promoters

Efficient bioconversion of cellulose into glucose using lignocellulose as the feedstock requires improved cell wall traits. Xylan is part of the matrix covering cellulose and linkages of xylan and lignin inhibit cellulases access. Xylose, an uncommon sugar in the yeast fermentation process, represents 20% of the lignocellulose mass fraction, thus xylan related genes are important targets for biotechnology. Initially, the isolation of candidate genes and their functional characterization is a prerequisite. A common strategy is to perform exhaustive promoter characterizations for candidate genes. Here, we report on the characterization of two xylosyltransferases-related gene promoters that were isolated upstream of the respective candidate genes in poplar, a promising feedstock for bioenergy that is characterized by its short-rotation. The species is known to have undergone recent whole genome duplication, thus finding duplicated gene sequences for xylosyltransferases based on phylogeny reconstruction but with distinct expression patterns is expected. To investigate these two genes closely related to the single, well-characterised Arabidopsis thaliana (At) irx10 gene, we constructed two binary vectors, each studying the full-length promoter of the respective gene. For the poplar (Ptr) gene, most likely the functionally closest to the At irx10 gene, localization of gene expression was studied using the green fluorescence protein (GFP). Poplar’s glucuronoxylan glucuronosyltransferase protein, PtrGUT2B, expression was localized in the inflorescence, and specifically in the xylem and along the fibres, suggesting that PtrGUT2B gene expression is associated with fibre production similar to the At irx10 gene. Conversely, the second, duplicated, poplar gene termed PtrGUT2A showed expression in the cambium/phloem, vascular bundles, in the primary xylem and the central cylinder that actively undergo cell divisions and differentiation as evidenced by driving ß-glucuronidase (GUS) expression in these specific tissues. There was no staining detected in the mature secondary xylem or interfascicular fibres. Our study demonstrated that the PtrGUT2B is the true ortholog of irx10 and the two isolated promoters can be used to target tissue specific expression in plant biotechnology assays.