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DNA-fragments are transcytosed across CaCo-2 cells by adsorptive endocytosis and vesicular mediated transport.

Johannessen LE, Spilsberg B, Wiik-Nielsen CR, Kristoffersen AB, Holst-Jensen A, Berdal KG - PLoS ONE (2013)

Bottom Line: The molecular mechanisms behind transport of DNA-fragments across the intestine and the effects of this transport on the organism are currently unknown.DNA-fragments were found to be transported across polarized CaCo-2 cells in the apical to basolateral direction (AB).DNA-transport across CaCo-2 cells was not competed out with oligodeoxynucleotides, fucoidan, heparin, heparan sulphate and dextrane sulphate, while linearized plasmid DNA, on the other hand, reduced transcytosis of DNA-fragments by a factor of approximately 2.

View Article: PubMed Central - PubMed

Affiliation: Norwegian Veterinary Institute, Oslo, Norway.

ABSTRACT
Dietary DNA is degraded into shorter DNA-fragments and single nucleosides in the gastrointestinal tract. Dietary DNA is mainly taken up as single nucleosides and bases, but even dietary DNA-fragments of up to a few hundred bp are able to cross the intestinal barrier and enter the blood stream. The molecular mechanisms behind transport of DNA-fragments across the intestine and the effects of this transport on the organism are currently unknown. Here we investigate the transport of DNA-fragments across the intestinal barrier, focusing on transport mechanisms and rates. The human intestinal epithelial cell line CaCo-2 was used as a model. As DNA material a PCR-fragment of 633 bp was used and quantitative real time PCR was used as detection method. DNA-fragments were found to be transported across polarized CaCo-2 cells in the apical to basolateral direction (AB). After 90 min the difference in directionality AB vs. BA was >10(3) fold. Even undegraded DNA-fragments of 633 bp could be detected in the basolateral receiver compartment at this time point. Transport of DNA-fragments was sensitive to low temperature and inhibition of endosomal acidification. DNA-transport across CaCo-2 cells was not competed out with oligodeoxynucleotides, fucoidan, heparin, heparan sulphate and dextrane sulphate, while linearized plasmid DNA, on the other hand, reduced transcytosis of DNA-fragments by a factor of approximately 2. Our findings therefore suggest that vesicular transport is mediating transcytosis of dietary DNA-fragments across intestinal cells and that DNA binding proteins are involved in this process. If we extrapolate our findings to in vivo conditions it could be hypothesized that this transport mechanism has a function in the immune system.

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Related in: MedlinePlus

Differentiation of CaCo-2 cells.A: Trans-epithelial electric resistance (TEER) was measured on CaCo-2 cells on filters during their differentiation. Measurements were performed before change of medium. TEER (Ω x cm2) was plotted against time. One representative experiment is shown with mean +/−SD from nine wells. B: Intestinal alkaline phosphatase (IAP) expression at mRNA level detected by reverse transcription followed by PCR in CaCo-2 cells, CaCo-2/HT29-MTX Mix (3∶1) and HT29-MTX cells. Control is HeLa total mRNA from the Superscript III cellsdirect cDNA synthesis kit (Invitrogen). One representative experiment out of two is shown.
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pone-0056671-g002: Differentiation of CaCo-2 cells.A: Trans-epithelial electric resistance (TEER) was measured on CaCo-2 cells on filters during their differentiation. Measurements were performed before change of medium. TEER (Ω x cm2) was plotted against time. One representative experiment is shown with mean +/−SD from nine wells. B: Intestinal alkaline phosphatase (IAP) expression at mRNA level detected by reverse transcription followed by PCR in CaCo-2 cells, CaCo-2/HT29-MTX Mix (3∶1) and HT29-MTX cells. Control is HeLa total mRNA from the Superscript III cellsdirect cDNA synthesis kit (Invitrogen). One representative experiment out of two is shown.

Mentions: Formation of a polarized cell layer and differentiation of CaCo-2 cells into enterocytes was measured by detecting trans-epithelial electric resistance (TEER) and intestinal alkaline phosphatase (IAP) expression (Figure 2). IAP expression was detected in CaCo-2 cells both when cultured alone and when co-cultured with HT29-MTX cells, while IAP was not expressed in HT29-MTX cells. IAP activity is indicative of brush border formation. Monolayer and tight junction formation could also be detected upon confocal analysis of CaCo-2 cells labelled with the nuclear stain Hoechst and the actin stain phalloidin (data not shown).


DNA-fragments are transcytosed across CaCo-2 cells by adsorptive endocytosis and vesicular mediated transport.

Johannessen LE, Spilsberg B, Wiik-Nielsen CR, Kristoffersen AB, Holst-Jensen A, Berdal KG - PLoS ONE (2013)

Differentiation of CaCo-2 cells.A: Trans-epithelial electric resistance (TEER) was measured on CaCo-2 cells on filters during their differentiation. Measurements were performed before change of medium. TEER (Ω x cm2) was plotted against time. One representative experiment is shown with mean +/−SD from nine wells. B: Intestinal alkaline phosphatase (IAP) expression at mRNA level detected by reverse transcription followed by PCR in CaCo-2 cells, CaCo-2/HT29-MTX Mix (3∶1) and HT29-MTX cells. Control is HeLa total mRNA from the Superscript III cellsdirect cDNA synthesis kit (Invitrogen). One representative experiment out of two is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569430&req=5

pone-0056671-g002: Differentiation of CaCo-2 cells.A: Trans-epithelial electric resistance (TEER) was measured on CaCo-2 cells on filters during their differentiation. Measurements were performed before change of medium. TEER (Ω x cm2) was plotted against time. One representative experiment is shown with mean +/−SD from nine wells. B: Intestinal alkaline phosphatase (IAP) expression at mRNA level detected by reverse transcription followed by PCR in CaCo-2 cells, CaCo-2/HT29-MTX Mix (3∶1) and HT29-MTX cells. Control is HeLa total mRNA from the Superscript III cellsdirect cDNA synthesis kit (Invitrogen). One representative experiment out of two is shown.
Mentions: Formation of a polarized cell layer and differentiation of CaCo-2 cells into enterocytes was measured by detecting trans-epithelial electric resistance (TEER) and intestinal alkaline phosphatase (IAP) expression (Figure 2). IAP expression was detected in CaCo-2 cells both when cultured alone and when co-cultured with HT29-MTX cells, while IAP was not expressed in HT29-MTX cells. IAP activity is indicative of brush border formation. Monolayer and tight junction formation could also be detected upon confocal analysis of CaCo-2 cells labelled with the nuclear stain Hoechst and the actin stain phalloidin (data not shown).

Bottom Line: The molecular mechanisms behind transport of DNA-fragments across the intestine and the effects of this transport on the organism are currently unknown.DNA-fragments were found to be transported across polarized CaCo-2 cells in the apical to basolateral direction (AB).DNA-transport across CaCo-2 cells was not competed out with oligodeoxynucleotides, fucoidan, heparin, heparan sulphate and dextrane sulphate, while linearized plasmid DNA, on the other hand, reduced transcytosis of DNA-fragments by a factor of approximately 2.

View Article: PubMed Central - PubMed

Affiliation: Norwegian Veterinary Institute, Oslo, Norway.

ABSTRACT
Dietary DNA is degraded into shorter DNA-fragments and single nucleosides in the gastrointestinal tract. Dietary DNA is mainly taken up as single nucleosides and bases, but even dietary DNA-fragments of up to a few hundred bp are able to cross the intestinal barrier and enter the blood stream. The molecular mechanisms behind transport of DNA-fragments across the intestine and the effects of this transport on the organism are currently unknown. Here we investigate the transport of DNA-fragments across the intestinal barrier, focusing on transport mechanisms and rates. The human intestinal epithelial cell line CaCo-2 was used as a model. As DNA material a PCR-fragment of 633 bp was used and quantitative real time PCR was used as detection method. DNA-fragments were found to be transported across polarized CaCo-2 cells in the apical to basolateral direction (AB). After 90 min the difference in directionality AB vs. BA was >10(3) fold. Even undegraded DNA-fragments of 633 bp could be detected in the basolateral receiver compartment at this time point. Transport of DNA-fragments was sensitive to low temperature and inhibition of endosomal acidification. DNA-transport across CaCo-2 cells was not competed out with oligodeoxynucleotides, fucoidan, heparin, heparan sulphate and dextrane sulphate, while linearized plasmid DNA, on the other hand, reduced transcytosis of DNA-fragments by a factor of approximately 2. Our findings therefore suggest that vesicular transport is mediating transcytosis of dietary DNA-fragments across intestinal cells and that DNA binding proteins are involved in this process. If we extrapolate our findings to in vivo conditions it could be hypothesized that this transport mechanism has a function in the immune system.

Show MeSH
Related in: MedlinePlus