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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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Time-course of DNA-fragment transport across CaCo-2 cells.CaCo-2 cells on filters were incubated with a 633 bp long polymerase chain reaction (PCR) amplified fragment and Lucifer yellow (LY) at 37 °C and samples were collected at the time points indicated. A: The amount of DNA-fragments transported across the cells in the apical to basolateral (AB) direction and the BA direction was quantified by real-time PCR (qPCR) and normalized to the amount of DNA initially added to the cells and plotted against time. B: The amount of DNA-fragment left in the donor chambers after 90 min of incubation was quantified by qPCR and normalized to the amount of DNA initially added. C: All liquid in the basolateral donor chamber was collected from two wells and pooled before purification of DNA. PCR using the primers RRS SphI F and RRS SphI R was performed on the purified DNA before visualization on a 2% agarose gel to detect the full length DNA-fragment. D: After 90 min of incubation the amount of transcytosed LY was normalized to the amount of initially added LY in wells with or without addition of DNA-fragment. In A, B and D, the data shown are from one representative experiment with three replicates, showing mean +/−SD.
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pone-0056671-g001: Time-course of DNA-fragment transport across CaCo-2 cells.CaCo-2 cells on filters were incubated with a 633 bp long polymerase chain reaction (PCR) amplified fragment and Lucifer yellow (LY) at 37 °C and samples were collected at the time points indicated. A: The amount of DNA-fragments transported across the cells in the apical to basolateral (AB) direction and the BA direction was quantified by real-time PCR (qPCR) and normalized to the amount of DNA initially added to the cells and plotted against time. B: The amount of DNA-fragment left in the donor chambers after 90 min of incubation was quantified by qPCR and normalized to the amount of DNA initially added. C: All liquid in the basolateral donor chamber was collected from two wells and pooled before purification of DNA. PCR using the primers RRS SphI F and RRS SphI R was performed on the purified DNA before visualization on a 2% agarose gel to detect the full length DNA-fragment. D: After 90 min of incubation the amount of transcytosed LY was normalized to the amount of initially added LY in wells with or without addition of DNA-fragment. In A, B and D, the data shown are from one representative experiment with three replicates, showing mean +/−SD.

Mentions: The time-course of DNA uptake was studied by adding 5 nM of the PCR-fragment to polarized CaCo-2 cells, either apically or basolaterally. Samples were then collected at different time points as depicted in Figure 1A. The quantity of transcytosed PCR-fragment was measured using quantitative real time PCR (qPCR) as described in the Material and Methods section and plotted as % of initially added as a function of time (Figure 1A). The amount of DNA-fragments in the acceptor chambers increased over time and after 90 minutes 0.06% of initially added was transcytosed in the apical to basolateral direction while in the basolateral to apical direction 2×10−5% was transported. Thus a 3×103-fold difference in transport in the apical to basolateral direction was observed. Ten independent experiments were performed for the 90 minutes time point and analysed with a linear mixed-effects model, giving p = 0.003 (n = 10, number of observations  = 42). When taking samples from apical and basolateral compartments from control cells (without any additions), and adding PCR-fragment directly to these samples and incubating at 37 °C for 2 h, pronounced degradation of the fragment could be seen when running the DNA on an agarose gel (data not shown). The transcytosis of DNA-fragments as a function of time reached an apparent equilibrium after 60–90 min (Figure 1A). DNA degradation, reducing the available DNA in the donor chamber may explain this observation. We chose to use the 90 min time point as the standard time point in all the successive experiments because our interest is long term uptake and ultimately biological and health effects.


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)

Time-course of DNA-fragment transport across CaCo-2 cells.CaCo-2 cells on filters were incubated with a 633 bp long polymerase chain reaction (PCR) amplified fragment and Lucifer yellow (LY) at 37 °C and samples were collected at the time points indicated. A: The amount of DNA-fragments transported across the cells in the apical to basolateral (AB) direction and the BA direction was quantified by real-time PCR (qPCR) and normalized to the amount of DNA initially added to the cells and plotted against time. B: The amount of DNA-fragment left in the donor chambers after 90 min of incubation was quantified by qPCR and normalized to the amount of DNA initially added. C: All liquid in the basolateral donor chamber was collected from two wells and pooled before purification of DNA. PCR using the primers RRS SphI F and RRS SphI R was performed on the purified DNA before visualization on a 2% agarose gel to detect the full length DNA-fragment. D: After 90 min of incubation the amount of transcytosed LY was normalized to the amount of initially added LY in wells with or without addition of DNA-fragment. In A, B and D, the data shown are from one representative experiment with three replicates, showing mean +/−SD.
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Related In: Results  -  Collection

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pone-0056671-g001: Time-course of DNA-fragment transport across CaCo-2 cells.CaCo-2 cells on filters were incubated with a 633 bp long polymerase chain reaction (PCR) amplified fragment and Lucifer yellow (LY) at 37 °C and samples were collected at the time points indicated. A: The amount of DNA-fragments transported across the cells in the apical to basolateral (AB) direction and the BA direction was quantified by real-time PCR (qPCR) and normalized to the amount of DNA initially added to the cells and plotted against time. B: The amount of DNA-fragment left in the donor chambers after 90 min of incubation was quantified by qPCR and normalized to the amount of DNA initially added. C: All liquid in the basolateral donor chamber was collected from two wells and pooled before purification of DNA. PCR using the primers RRS SphI F and RRS SphI R was performed on the purified DNA before visualization on a 2% agarose gel to detect the full length DNA-fragment. D: After 90 min of incubation the amount of transcytosed LY was normalized to the amount of initially added LY in wells with or without addition of DNA-fragment. In A, B and D, the data shown are from one representative experiment with three replicates, showing mean +/−SD.
Mentions: The time-course of DNA uptake was studied by adding 5 nM of the PCR-fragment to polarized CaCo-2 cells, either apically or basolaterally. Samples were then collected at different time points as depicted in Figure 1A. The quantity of transcytosed PCR-fragment was measured using quantitative real time PCR (qPCR) as described in the Material and Methods section and plotted as % of initially added as a function of time (Figure 1A). The amount of DNA-fragments in the acceptor chambers increased over time and after 90 minutes 0.06% of initially added was transcytosed in the apical to basolateral direction while in the basolateral to apical direction 2×10−5% was transported. Thus a 3×103-fold difference in transport in the apical to basolateral direction was observed. Ten independent experiments were performed for the 90 minutes time point and analysed with a linear mixed-effects model, giving p = 0.003 (n = 10, number of observations  = 42). When taking samples from apical and basolateral compartments from control cells (without any additions), and adding PCR-fragment directly to these samples and incubating at 37 °C for 2 h, pronounced degradation of the fragment could be seen when running the DNA on an agarose gel (data not shown). The transcytosis of DNA-fragments as a function of time reached an apparent equilibrium after 60–90 min (Figure 1A). DNA degradation, reducing the available DNA in the donor chamber may explain this observation. We chose to use the 90 min time point as the standard time point in all the successive experiments because our interest is long term uptake and ultimately biological and health effects.

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