Impact of Double-Stranded RNA Internalization on Hematopoietic Progenitors and Krebs-2 Cells and Mechanism.

It is well-established that double-stranded RNA (dsRNA) exhibits noticeable radioprotective and radiotherapeutic effects. The experiments conducted in this study directly demonstrated that dsRNA was delivered into the cell in its native form and that it induced hematopoietic progenitor proliferation. The 68 bp synthetic dsRNA labeled with 6-carboxyfluorescein (FAM) was internalized into mouse hematopoietic progenitors, c-Kit+ (a marker of long-term hematopoietic stem cells) cells and CD34+ (a marker of short-term hematopoietic stem cells and multipotent progenitors) cells. Treating bone marrow cells with dsRNA stimulated the growth of colonies, mainly cells of the granulocyte-macrophage lineage. A total of 0.8% of Krebs-2 cells internalized FAM-dsRNA and were simultaneously CD34+ cells. dsRNA in its native state was delivered into the cell, where it was present without any signs of processing. dsRNA binding to a cell was independent of cell charge. dsRNA internalization was related to the receptor-mediated process that requires energy from ATP. Synthetic dsRNA did not degrade in the bloodstream for at least 2 h. Hematopoietic precursors that had captured dsRNA reinfused into the bloodstream and populated the bone marrow and spleen. This study, for the first time, directly proved that synthetic dsRNA is internalized into a eukaryotic cell via a natural mechanism.

The New General Biological Property of Stem-like Tumor Cells (Part II: Surface Molecules, Which Belongs to Distinctive Groups with Particular Functions, Form a Unique Pattern Characteristic of a Certain Type of Tumor Stem-like Cells).

An ability of poorly differentiated cells of different genesis, including tumor stem-like cells (TSCs), to internalize extracellular double-stranded DNA (dsDNA) fragments was revealed in our studies. Using the models of Krebs-2 murine ascites carcinoma and EBV-induced human B-cell lymphoma culture, we demonstrated that dsDNA internalization into the cell consists of several mechanistically distinct phases. The primary contact with cell membrane factors is determined by electrostatic interactions. Firm contacts with cell envelope proteins are then formed, followed by internalization into the cell of the complex formed between the factor and the dsDNA probe bound to it. The key binding sites were found to be the heparin-binding domains, which are constituents of various cell surface proteins of TSCs-either the C1q domain, the collagen-binding domain, or domains of positively charged amino acids. These results imply that the interaction between extracellular dsDNA fragments and the cell, as well as their internalization, took place with the involvement of glycocalyx components (proteoglycans/glycoproteins (PGs/GPs) and glycosylphosphatidylinositol-anchored proteins (GPI-APs)) and the system of scavenger receptors (SRs), which are characteristic of TSCs and form functional clusters of cell surface proteins in TSCs. The key provisions of the concept characterizing the principle of organization of the "group-specific" cell surface factors of TSCs of various geneses were formulated. These factors belong to three protein clusters: GPs/PGs, GIP-APs, and SRs. For TSCs of different tumors, these clusters were found to be represented by different members with homotypic functions corresponding to the general function of the cluster to which they belong.

The new general biological property of stem-like tumor cells Part I. Peculiarities of the process of the double-stranded DNA fragments internalization into stem-like tumor cells.

Stem-like tumor cells of ascites carcinoma Krebs-2 and Epstein-Barr virus-induced B-lymphoma were shown to possess the innate capability of binding and internalizing the TAMRA-labeled double-stranded DNA (dsDNA) probe. The process of binding and internalizing is rather complicated and composed of the following successive stages: 1) initiating electrostatic interaction and contact of a negatively charged dsDNA molecule with a positively charged molecule(s) on the surface of a stem-like tumor cell; 2) binding of the dsDNA probe to a tumor stem cell surface protein(s) via the formation of a strong chemical/molecular bond; and 3) the very internalization of dsDNA into the cell. Binding of DNA to cell surface proteins is determined by the presence of heparin/polyanion-binding sites within the protein structure, which can be competitively blocked by heparin and/or dextran sulfate, wherein heparin blocks only the binding, while dextran sulfate abrogates both binding and internalization. The abrogation of internalization by dextran sulfate implies the role of scavenger receptors in this process. Cells were shown to uptake DNA in amounts constituting ∼0.008% of the haploid genome. Inhibitors of caveolae-dependent internalization abrogate the DNA uptake in Krebs-2 cells, and inhibitors of the clathrin/caveolar mechanism block the internalization in B-lymphoma cells. In the present report, it is shown for the first time that in contrast to the majority of committed tumor cells, stem-like tumor cells of Krebs-2 and B-lymphoma carry a general positive charge on their surface.

Identification of the xenograft and its ascendant sphere-forming cell line as belonging to EBV-induced lymphoma, and characterization of the status of sphere-forming cells.

BACKGROUND: We have characterized the human cell line arised from the Epstein-Barr virus (EBV) positive multiple myeloma aspirate subjected to the long-term cultivation. This cell line has acquired the ability to form free-floating spheres and to produce a xenograft upon transplantation into NOD/SCID mice. METHODS: Cells from both in vitro culture and developed xenografts were investigated with a number of analytical approaches, including pathomorphological analysis, FISH analysis, and analysis of the surface antigens and of the VDJ locus rearrangement. RESULTS: The obtained results, as well as the confirmed presence of EBV, testify that both biological systems are derived from B-cells, which, in turn, is a progeny of the EBV-transformed B-cellular clone that supplanted the primordial multiple myeloma cells. Next we assessed whether cells that (i) were constantly present in vitro in the investigated cell line, (ii) were among the sphere-forming cells, and (iii) were capable of internalizing a fluorescent TAMRA-labeled DNA probe (TAMRA+ cells) belonged to one of the three types of undifferentiated bone marrow cells of a multiple myeloma patient: CD34+ hematopoietic stem cells, CD90+ mesenchymal stem cells, and clonotypic multiple myeloma cell. CONCLUSION: TAMRA+ cells were shown to constitute the fourth independent subpopulation of undifferentiated bone marrow cells of the multiple myeloma patient. We have demonstrated the formation of ectopic contacts between TAMRA+ cells and cells of other types in culture, in particular with CD90+ mesenchymal stem cells, followed by the transfer of some TAMRA+ cell material into the contacted cell.