Cell and Tissue Journal

Abstract Aim: b-Catenin is a known proto-oncoprotein which its upregulation is involved in tumorigenesis of several human cancers including colorectal cancer, melanoma, pancreatic, ovarian and many other human malignancies. It is believed that this protein is a very potential target for cancer prevention and therapy. Although b-Catenin was originally identified as a component of the canonical Wnt signaling, there are many reports that this protein can also be regulated by several other signaling pathways including those through receptor tyrosine kinases, PI3-kinses and heterotrimeric G-proteins. It is quite likely that regulation of b-Catenin by multiple signaling pathways is a result of the cross-talk among these pathways. We and others have already provided evidence that activation of almost all members of the Ga subunits of heteritrimeric G-proteins can regulate b-Catenin via different mechanisms. For example, by using several cell systems, we have shown that activation of the Gaq class of Ga proteins leads to inhibition of GSK-3b and cellular accumulation of b-Catenin, suggesting that Gaq signaling may somehow positively regulate the canonical Wnt signaling. We have also confirmed the positive role of Gaq on b-Catenin-mediated signaling by using a couple of specific Gaq blocking peptides. Receptors for G-proteins (G protein-coupled receptors, GPCRs) are the most diverse family of proteins in mammals which are involved in many critical cellular processes. Around one thousand different GPCRs are encoded by human genome and more than 30% of the approved therapeutic drugs, target these receptors. Calcitonin receptor (CTR) is a member of the GPCR family (B1 subfamily) which is shown to couple to Gaq or Gas containing trimeric G proteins involved in primarily activation of phospholipase Cb1 and adenylate cyclase respectively. The known ligand for Calcitonin receptor is a 32 amino acid peptide called Calcitonin which is produced by several tissues in the body including thyroid, prostate, and central nervous system. CTR-mediated signaling has very important biological roles in many tissues including bones in which this signaling pathway is involved in maturation of osteoclasts and bone homeostasis. Calcitonin receptor is a GPCR family member which functions through activation of Gs and Gq trimeric G proteins and therefore, in this study we have examined whether activation of this receptor has any effect on b-Catenin transcriptional activity by measuring the expression of several b-Catenin-target genes including a reporter luciferase gene harboring several b-Catenin/T-cell factor recognition elements.
Material and Methods: HEK293T cell culture and transfection with appropriate genetic constructs plus gene expression assays at the level of transcription including quantitative RT-PCR and real-time PCR have been used in this study.
Results: The results showed that cellular activation of Calcitonin receptor increases the expression of several b-Catenin-responsive genes. Expression of the reporter luciferase, CCND1 (the Cyclin D1 encoding gene), c-MYC, and FGF-20 (Fibroblast growth factor-20) was increased upon the expression of Calcitonin receptor in HEK293T cells or treatment of these cells with Calcitonin.
Conclusion: The Calcitonin receptor-mediated signaling may activate b-Catenin and (or) the Wnt/b-Catenin pathway. Further investigations are required to find out the exact mechanism of regulation of the Wnt/b-Catenin pathway by Calcitonin receptor-mediated signaling. Since b-Catenin is a potential oncogene in human cancers, Calcitonin receptor and its signaling partners can be considered for clinical studies.
 

Abstract Aim: Different methods have been used for introducing DNA into mammalian cells. Calcium phosphate-based transfection protocols are inexpensive and easy to perform, however, the transfection efficiency in some cases is not satisfactory. In this study we try to increase the efficiency of calcium phosphate-mediated cell transfection.   Material and Methods: 2.0×10⁵ HEK293T cells were plated in each well of a 6-well plate containing a 10% poly L lysine-coated coverslip and grown to 60% confluency. The cells were then transfected with a plasmid encoding GFP (Green Fluorescence Protein), using a standard calcium phosphate protocol in the presence or absence of chloroquine phosphate. GFP protein expression was observed by immunofluorescence microscopy and quantified with the help of Image J software.  Results: We have shown that chloroquine phosphate can highly improve the efficiency of calcium phosphate-dependent transfection in the HEK293T cells, leading to an approximate 8-fold increase in the number of transfected cells. Chloroquine phosphate was not toxic to the cells, however, its positive effect on transfection efficiency was abolished in the presence of glycerol or DMSO (Dimethyl sulphoxide). Conclusion: we recommend chloroquine phosphate for increasing the efficiency of transfection by the inexpensive method of calcium phosphate. In addition, we recommend that chloroquine phosphate to be used alone and not along with other inducers of cell transfection.