by Evgeniya V. Dolgova | Institute of Cytology and Genetics SB RAS
Motivation and Aim: Previously, we have demonstrated poorly differentiated cells of various
origin and genesis, including tumor stem-like ones, to be capable of native, i.e. without
using any artificial compounds or procedures, internalizing extracellular double-stranded
DNA (dsDNA) fragments. However, the exact mechanism of extracellular dsDNA
internalization into cells has not yet been clarified.
Methods and Algorithms: In the current report, for murine Krebs-2 carcinoma and human
EBV+ B-lymphoma, there have been determined the mechanisms mediating the interaction
of dsDNA with the surface of tumor stem-like cells (TSCs) and its consequent internalization
into these cells. The following approaches were used: electrophoretic separation of cells in a
free volume or pre-exposure of cells to various proteases and/or compounds inhibiting pinoand endocytosis, followed by assessing the resulting efficiency of Alu-TAMRA DNA probe
internalization; analysis of the transcriptome of the indicated cell lines followed by the qPCR
to validate the differences in expression of some target genes identified as possible
participants in the process of dsDNA internalization.
Results: It was shown, that the process of binding and internalizing is rather complicated
and composed of the following successive stages: (i) initiating electrostatic interaction and
contact of a negatively charged dsDNA molecule with a positively charged molecule(s) on
the surface of a TSC; (ii) binding of the dsDNA probe to a tumor stem cell surface protein(s)
via the formation of a strong chemical/molecular bond; (iii) the very internalization of
dsDNA into the cell. Inhibitors of caveolae-dependent internalization abrogate the DNA
uptake in Krebs-2 cells, and inhibitors of clathrin/caveolar mechanism block the
internalization in EBV+ B-lymphoma cells.
Conclusion: The basic provisions of the concept characterizing the principle of the
organization of the stem-like tumor cell surface factors to be formulated as a concept of
“group-specific cell surface factors, which form the profile of tumor stem-like cells”.