Novel strong tissue specific promoter for gene expression in human germ cells

<p>Abstract</p> <p>Background</p> <p>Tissue specific promoters may be utilized for a variety of applications, including programmed gene expression in cell types, tissues and organs of interest, for developing different cell culture models or for use in gene therapy. We report a novel, tissue-specific promoter that was identified and engineered from the native upstream regulatory region of the human gene <it>NDUFV1 </it>containing an endogenous retroviral sequence.</p> <p>Results</p> <p>Among seven established human cell lines and five primary cultures, this modified <it>NDUFV1 </it>upstream sequence (mNUS) was active only in human undifferentiated germ-derived cells (lines Tera-1 and EP2102), where it demonstrated high promoter activity (~twice greater than that of the SV40 early promoter, and comparable to the routinely used cytomegaloviral promoter). To investigate the potential applicability of the mNUS promoter for biotechnological needs, a construct carrying a recombinant cytosine deaminase (RCD) suicide gene under the control of mNUS was tested in cell lines of different tissue origin. High cytotoxic effect of RCD with a cell-death rate ~60% was observed only in germ-derived cells (Tera-1), whereas no effect was seen in a somatic, kidney-derived control cell line (HEK293). In further experiments, we tested mNUS-driven expression of a hyperactive <it>Sleeping Beauty </it>transposase (SB100X). The mNUS-SB100X construct mediated stable transgene insertions exclusively in germ-derived cells, thereby providing further evidence of tissue-specificity of the mNUS promoter.</p> <p>Conclusions</p> <p>We conclude that mNUS may be used as an efficient promoter for tissue-specific gene expression in human germ-derived cells in many applications. Our data also suggest that the 91 bp-long sequence located exactly upstream <it>NDUFV1 </it>transcriptional start site plays a crucial role in the activity of this gene promoter <it>in vitro </it>in the majority of tested cell types (10/12), and an important role - in the rest two cell lines.</p

Pancreatic Lineage Specifier PDX1 Increases Adhesion and Decreases Motility of Cancer Cells

Intercellular interactions involving adhesion factors are key operators in cancer progression. In particular, these factors are responsible for facilitating cell migration and metastasis. Strengthening of adhesion between tumor cells and surrounding cells or extracellular matrix (ECM), may provide a way to inhibit tumor cell migration. Recently, we demonstrated that PDX1 ectopic expression results in the reduction of pancreatic cancer line PANC-1 cell motility in vitro and in vivo, and we now provide experimental data confirming the hypothesis that suppression of migration may be related to the effect of PDX1 on cell adhesion. Cell migration analyses demonstrated decreased motility of pancreatic Colo357 and PANC-1 cell lines expressing PDX1. We observed decreased expression levels of genes associated with promoting cell migration and increased expression of genes negatively affecting cell motility. Expression of the EMT regulator genes was only mildly induced in cells expressing PDX1 during the simulation of the epithelial-mesenchymal transition (EMT) by the addition of TGFβ1 to the medium. PDX1-expressing cancer cell lines showed increased cell adhesion to collagen type I, fibronectin, and poly-lysine. We conclude that ectopic expression of PDX1 reduces the migration potential of cancer cells, by increasing the adhesive properties of cells and reducing the sensitivity to TGFβ1-induced EMT

Activity of the upstream component of tandem TERT/survivin promoters depends on features of the downstream component.

We spliced the promoters of the human telomerase and human survivin genes (PhTERT and PhSurv, respectively) widely used for gene therapy and known to have the broadest cancer type spectrum of activity. Two head-to-tail constructs were obtained: the PhTERT-PhSurv and PhSurv-PhTERT tandems. The splicing caused quantitative and qualitative changes in the promoter features. In both constructs, only the promoter proximal to the transcribed gene retained its ability to initiate transcription, whereas the distal promoter was silent, the phenomenon never reported before. However, the distal promoter modulated the activity of the proximal one by increasing its strength and causing an appearance of additional transcription start sites. We suggested that this suppression might be due to the presence of Sp1 transcription factor binding sites in both promoters and Sp1-bridges between these sites. Such Sp1-bridges might convert the tandem promoter linear DNA into a stem-loop structure. If localized inside the formed loop, the distal promoter could lose its ability to initiate transcription. To test this hypothesis, we constructed two modified double promoters, where the proximal PhSurv promoter was replaced either by a shortened variant of the survivin promoter (PhSurv269) or by the mouse survivin promoter. Both PhSurv substitutes were considerably shorter than PhSurv and had different numbers and/or positions of Sp1 sites. In modified tandems, transcription was initiated from both promoters. We also prepared two mutant forms of the PhSurv-PhTERT tandem with two or four Sp1 sites removed from the distal "long" PhSurv promoter. In the first case, the distal PhSurv promoter remained silent, whereas the removal of four Sp1 binding sites restored its activity. In the majority of studied cancer cell lines the efficiency of transcription from the hTERT-(shortened hSurv269) promoter tandem was markedly higher than from each constituent promoter. In normal lung fibroblast cells, the tandem promoter activity was considerably lower

SOX9 Protein in Pancreatic Cancer Regulates Multiple Cellular Networks in a Cell-Specific Manner

SOX9 is upregulated in the majority of pancreatic ductal adenocarcinoma cases. It is hypothesized that the increased expression of SOX9 is necessary for the formation and maintenance of tumor phenotypes in pancreatic cancer cells. In our research, we studied six pancreatic cancer cell lines, which displayed varying levels of differentiation and a range of oncogenic mutations. We chose the method of downregulation of SOX9 expression via siRNA transfection as the main method for investigating the functional role of the SOX9 factor in pancreatic cancer cells. We discovered that the downregulation of SOX9 expression in the cell lines leads to cell-line-specific changes in the expression levels of epithelial and mesenchymal protein markers. Additionally, the downregulation of SOX9 expression had a specific effect on the expression of pancreatic developmental master genes. SOX9 downregulation had the greatest effect on the expression levels of the protein regulators of cell proliferation. In three of the four cell lines studied, the transfection of siSOX9 led to a significant decrease in proliferative activity and to the activation of proapoptotic caspases in transfected cells. The acquired results demonstrate that the SOX9 protein exerts its multiple functions as a pleiotropic regulator of differentiation and a potential promoter of tumor growth in a cell-specific manner in pancreatic cancer cells

Primers used in the experiments.

<p>Primers used in the experiments.</p

Relative activity of the studied single and double promoters in different cell lines.

<p>The values represent relative promoter activities as ratios of the luciferase activity expressed by plasmids containing promoters under study to the luciferase activity expressed by a pGL3-PV plasmid containing only the SV40 promoter. Mean values (± SEM) of relative luciferase activity were calculated from three independent experiments.</p><p>Sign “<b>+</b>” in the first column means that data are calculated as an arithmetic sum of activities of single constituent promoters. The average value of promoter activity over five cancer cell lines (Calu-1, A375, A549, PANC-1, and HT1080) was calculated as a sum of the activities in each line divided by 5. The rightmost column corresponds to normal lung fibroblasts IVL-11NS.</p