title: Properties of internalization factors contributing to the uptake of extracellular DNA into tumor-initiating stem cells of mouse Krebs-2 cell line.
abstract: BACKGROUND
  Previously, we demonstrated that poorly differentiated cells of various origins, including tumor-initiating stem cells present in the ascites form of mouse cancer cell line Krebs-2, are capable of naturally internalizing both linear double-stranded DNA and circular plasmid DNA.
  
  
  METHODS
  The method of co-incubating Krebs-2 cells with extracellular plasmid DNA (pUC19) or TAMRA-5'-dUTP-labeled polymerase chain reaction (PCR) product was used. It was found that internalized plasmid DNA isolated from Krebs-2 can be transformed into competent Escherichia coli cells. Thus, the internalization processes taking place in the Krebs-2 cell subpopulation have been analyzed and compared, as assayed by E. coli colony formation assay (plasmid DNA) and cytofluorescence (TAMRA-DNA).
  
  
  RESULTS
  We showed that extracellular DNA both in the form of plasmid DNA and a PCR product is internalized by the same subpopulation of Krebs-2 cells. We found that the saturation threshold for Krebs-2 ascites cells is 0.5 μg DNA/10(6) cells. Supercoiled plasmid DNA, human high-molecular weight DNA, and 500 bp PCR fragments are internalized into the Krebs-2 tumor-initiating stem cells via distinct, non-competing internalization pathways. Under our experimental conditions, each cell may harbor 340-2600 copies of intact plasmid material, or up to 3.097 ± 0.044×10(6) plasmid copies (intact or not), as detected by quantitative PCR.
  
  
  CONCLUSIONS
  The internalization dynamics of extracellular DNA, copy number of the plasmids taken up by the cells, and competition between different types of double-stranded DNA upon internalization into tumor-initiating stem cells of mouse ascites Krebs-2 have been comprehensively analyzed. Investigation of the extracellular DNA internalization into tumor-initiating stem cells is an important part of understanding their properties and possible destruction mechanisms. For example, a TAMRA-labeled DNA probe may serve as an instrument to develop a target for the therapy of cancer, aiming at elimination of tumor stem cells, as well as developing a straightforward test system for the quantification of poorly differentiated cells, including tumor-initiating stem cells, in the bulk tumor sample (biopsy or surgery specimen).
authors.affiliation: Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 10 Lavrentieva Ave., Novosibirsk, 630090, Russia. dolgova.ev@mail.ru.
authors.forename: Evgeniya V
authors.initials: EV
authors.lastname: Dolgova
authors.affiliation: Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 10 Lavrentieva Ave., Novosibirsk, 630090, Russia.
authors.forename: Ekaterina A
authors.initials: EA
authors.lastname: Potter
authors.affiliation: Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 10 Lavrentieva Ave., Novosibirsk, 630090, Russia.
authors.forename: Anastasiya S
authors.initials: AS
authors.lastname: Proskurina
authors.affiliation: Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 10 Lavrentieva Ave., Novosibirsk, 630090, Russia.
authors.forename: Alexandra M
authors.initials: AM
authors.lastname: Minkevich
authors.affiliation: Institute of Clinical Immunology, Siberian Branch of the Russian Academy of Medical Sciences, 14 Yadrintsevskaya Street, Novosibirsk, 630099, Russia.
authors.forename: Elena R
authors.initials: ER
authors.lastname: Chernych
authors.affiliation: Institute of Clinical Immunology, Siberian Branch of the Russian Academy of Medical Sciences, 14 Yadrintsevskaya Street, Novosibirsk, 630099, Russia.
authors.forename: Alexandr A
authors.initials: AA
authors.lastname: Ostanin
authors.affiliation: Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 10 Lavrentieva Ave., Novosibirsk, 630090, Russia.|Novosibirsk State University, 2 Pirogova Street, Novosibirsk, 630090, Russia.
authors.forename: Yaroslav R
authors.initials: YR
authors.lastname: Efremov
authors.affiliation: Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 10 Lavrentieva Ave., Novosibirsk, 630090, Russia.
authors.forename: Sergey I
authors.initials: SI
authors.lastname: Bayborodin
authors.affiliation: Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 10 Lavrentieva Ave., Novosibirsk, 630090, Russia.
authors.forename: Valeriy P
authors.initials: VP
authors.lastname: Nikolin
authors.affiliation: Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 10 Lavrentieva Ave., Novosibirsk, 630090, Russia.|Novosibirsk State University, 2 Pirogova Street, Novosibirsk, 630090, Russia.
authors.forename: Nelly A
authors.initials: NA
authors.lastname: Popova
authors.affiliation: Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 10 Lavrentieva Ave., Novosibirsk, 630090, Russia.
authors.forename: Nikolay A
authors.initials: NA
authors.lastname: Kolchanov
authors.affiliation: Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 10 Lavrentieva Ave., Novosibirsk, 630090, Russia.
authors.forename: Sergey S
authors.initials: SS
authors.lastname: Bogachev
mesh_terms: D000818:Animals; D001201:Ascites / Q000378:metabolism* / Q000473:pathology; D001692:Biological Transport; D045744:Cell Line, Tumor; D015169:Colony Count, Microbial; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D056915:DNA Copy Number Variations; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008808:Mice, Inbred CBA; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D014170:Transformation, Genetic; D014410:Tumor Stem Cell Assay
chemical_list: D004247:DNA
keywords: Ascites Krebs-2; DNA internalization factors; Extracellular DNA; Tumor-initiating stem cells