Functional properties of the WNT11 new isoform, expressed in colon carcinoma cell line HT29

Colon carcinoma is a common type of neoplastic transformation. Mechanisms of its establishment and progression have been studying for several decades. Aberrant activation of the canonical Wnt signaling is frequently observed in colon carcinoma cells. Moreover, expression of the "noncanonical" Wnt ligands is also detected in this type of cancer. However, the implication of the noncanonical Wnt signaling in carcinogenesis and colorectal cancer (CRC) progression is still unclear. Here, to elucidate the characteristic features of the noncanonical Wnt signaling activation in CRC the expression of the "noncanonical" ligand hWnt11 has been studied. It was shown for the first time that expression of the hWnt11 in CRC is accompanied by the alternative splicing. The new hWnt11 isoform (hWnt11sp3) has been identified. Unlike to hWnt11, this isoform is not secreted and lacks the ability to inhibit the canonical Wnt signaling. Considering the canonical Wnt signaling inhibiting activity of hWnt11, different functional properties of the ligand and its isoform may reflect a special role of the alternative splicing in carcinogenesis and tumor progression. Thus, due to the difference in their functional properties an existence of several Wnt isoforms should be taken into account for the investigation of the role of Wnt ligands.     

Cloning and study of novel mammalian genes with structural homology with phage lysozyme

The mouse tag7 gene was cloned and characterized in our previous works. This work is devoted to identifying and cloning its homologs. The human homolog of the tag7 gene was cloned. Data of analysis of the primary structure of the human tag7 and results from the study of the production of the corresponding protein in human organs and tissues indicate that this gene plays a major role in the mammalian immune system. The mouse and human tagL gene carrying an extended region of structural homology with the tag7 gene was detected by computer analysis and was subsequently cloned. Sequence analysis suggested the possible membrane localization of the gene, whereas the specific expression pointed to the role of this gene in the immune system. Both genes, tag7 and tagL, were localized in the human chromosome 19.     

Classification of amino acids based on comparative analysis of contacts in DNA-protein complexes and specific DNA-protein interactions

We propose a classification of amino acid residues based on the events of contact formation between particular residues and DNA nucleotides, i.e., using the most integral properties that characterize interactions organizing DNA-protein complexes. We apply the Voronoi-Delaunay tessellation to draw statistics of contacts and of contact areas for a set of 1937 DNA-protein complexes. Similarity of amino acid residues is defined upon comparison of corresponding rows and matrices of contacts and areas of contacts. Nine measures of distance have been used to estimate the closeness of rows. Residues have been grouped by three hierarchical and two nonhierarchical clustering methods. In a total tree built using nine metrics with three hierarchical methods, we show that clustering centers (pairs of amino acids) in the main groups are always constant while other relationships between objects vary. Major classes of up to six amino acids correspond to certain local structures of the polypeptide chain. These data can be taken into account when designing DNA-protein ligands.     

Nuclear β-catenin localization is not sufficient for canonical Wnt signaling activation in human melanoma cell lines

In most cases, advanced stages of melanoma are practically incurable due to high metastatic potential of tumor cells. Multiple observations support the idea that aberrations in the Wnt signaling pathway play a significant role in melanoma development and progression. Canonical Wnt signaling activation results in stabilization and accumulation of the major effector molecule called & gb-catenin. Mutations promoting & gb-catenin stabilization and, thereby, activation of canonical Wnt signaling pathway are frequently found in different cancers but rarely observed in melanomas. Nevertheless, & gb-catenin nuclear and cytoplasmic accumulation is the feature of many human melanoma cell lines and original tumors. That is why the aim of the investigation was to elucidate the relation between & gb-catenin intracellular localization and activity status of Wnt signaling pathway in human melanoma cell lines. Ten human melanoma cell lines were characterized on the basis of the following parameters: canonical Wnt ligand expression, intracellular & gb-catenin localization and activity status of canonical Wnt signaling pathway. Here, it has been demonstrated that nuclear localization of & gb-catenin does not always correspond to active status of canonical Wnt signaling pathway. Moreover, in the majority of cell lines with nuclear & gb-catenin, canonical Wnt signaling cannot be activated by exogenous expression of an appropriate ligand. Human melanoma cell lines differ in activity of canonical Wnt signaling pathway as well as in mechanisms of its regulation. Therefore, pathway-targeted potential antineoplastic therapy requires the formation of a & ldmolecular pattern of cancer” for localization of the defect in Wnt signaling cascade in each case.     

Tag7 (PGLYRP1) in Complex with Hsp70 Induces Alternative Cytotoxic Processes in Tumor Cells via TNFR1 Receptor

Tag7 (also known as peptidoglycan recognition protein PGRP-S, PGLYRP1), an innate immunity protein, interacts with Hsp70 to form a stable Tag7-Hsp70 complex with cytotoxic activity against some tumor cell lines. In this study, we have analyzed the programmed cell death mechanisms that are induced when cells interact with the Tag7-Hsp70 complex, which was previously shown to be released by human lymphocytes and is cytotoxic to cancer cells. We show that this complex induces both apoptotic and necroptotic processes in the cells. Apoptosis follows the classic caspase-8 and caspase-3 activation pathway. Inhibition of apoptosis leads to a switch to the RIP1-dependent necroptosis. Both of these cytotoxic processes are initiated by the involvement of TNFR1, a receptor for TNF-α. Our results suggest that the Tag7-Hsp70 complex is a novel ligand for this receptor. One of its components, the innate immunity protein Tag7, can bind to the TNFR1 receptor, thereby inhibiting the cytotoxic actions of the Tag7-Hsp70 complex and TNF-α, an acquired immunity cytokine.     

Detection of a perforin-like protein from the soluble cytotoxic factor of natural killer cells

The protein composition of the cytolytic factor (NKCF) secreted by natural killer cells was analyzed. Natural killer cells were isolated from spleens of nude rats and purified in the Percole gradient. SDS-PAAG analysis of NKCF revealed the presence of major proteins with an approximate molecular weight of 62-67 kDa. Biochemical properties of these proteins such as fractionation on a TSK-3000 SW gel filtration column, electrophoretical mobility in reduced conditions, enhanced cytolytical activity in the presence of Ca2+ demonstrate the similarity with the pore-forming protein--perforin. Apart from the major component, proteins with m. w. 42 kDa, 31-28 kDa, 17 kDa and 12-14 kDa were also found. It is possible that the Ca2+-independent cytolytical activity is bound to one of these minor protein components of NKCF.