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Properties of internalization factors contributing to the uptake of extracellular DNA into tumor-initiating stem cells of mouse Krebs-2 cell line.

ABSTRACT: 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.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).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 ?g 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.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).

SUBMITTER: Dolgova EV

PROVIDER: S-EPMC4881173 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Properties of internalization factors contributing to the uptake of extracellular DNA into tumor-initiating stem cells of mouse Krebs-2 cell line.

Dolgova Evgeniya V EV Potter Ekaterina A EA Proskurina Anastasiya S AS Minkevich Alexandra M AM Chernych Elena R ER Ostanin Alexandr A AA Efremov Yaroslav R YR Bayborodin Sergey I SI Nikolin Valeriy P VP Popova Nelly A NA Kolchanov Nikolay A NA Bogachev Sergey S SS

Stem cell research & therapy 20160525 1

<h4>Background</h4>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.<h4>Methods</h4>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 ...[more]

PMID: 27225522

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Project description:BackgroundAs in many Sub-Saharan African countries, the health system in Somalia is not operating at the capacity needed to lift childhood vaccination coverage to ninety percent or above, as recommended by United Nations Children's Fund. Current national estimates of coverage for the six major vaccine preventable childhood diseases range from thirty to sixty percent. Infectious disease outbreaks continue to pose significant challenges for the country's health authorities.ObjectiveThis important qualitative study, conducted in Galkayo District, Somalia, investigates limiting factors associated with childhood vaccination uptake from the perspective of both communities and health care workers.MethodsQualitative information was collected through six focus group discussions with parents (n = 48) and five one-to-one interviews with health workers (n = 15) between March and May 2017, in three settings in the Galkayo District - Galkayo city, Bayra and Bacadwayn.ResultsFrom a health system perspective, the factors are: awareness raising, hard to reach areas, negative attitudes and perceived knowledge of health workers, inadequate supplies and infrastructure, and missed vaccination opportunities. From the perspective of individuals and communities the factors are: low trust in vaccines, misinterpretation of religious beliefs, vaccine refusals, Somalia's patriarchal system and rumours and misinformation. Parents mostly received immunization information from social mobilizers and health facilities. Fathers, who are typically family decision-makers, were poorly informed. The findings highlight the need for in-service training to enable health workers to improve communication with parents, particularly fathers, peripheral communities and local religious leaders.ConclusionsEnhancing knowledge and awareness of vaccination among parents is crucial. Fathers' involvement is lacking. This may be boosted by highlighting fathers' obligation to protect their children's health through vaccination. It is also important that men engage with the wider community in decision-making and advance towards the global vaccination targets.

Dataset Information

Properties of internalization factors contributing to the uptake of extracellular DNA into tumor-initiating stem cells of mouse Krebs-2 cell line.

Publications

Properties of internalization factors contributing to the uptake of extracellular DNA into tumor-initiating stem cells of mouse Krebs-2 cell line.

Similar Datasets

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