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Nuclear accumulation of plasmid DNA can be enhanced by non-selective gating of the nuclear pore.

Vandenbroucke RE, Lucas B, Demeester J, De Smedt SC, Sanders NN - Nucleic Acids Res. (2007)

Bottom Line: Attempts to improve the transport of DNA to the nucleus through the use of nuclear localization signals or importin-beta have achieved limited success.Furthermore, in line with these observations, TCHD enhanced the transfection efficacy of both naked DNA and lipoplexes.In conclusion, based on the proposed structure of NPCs we succeeded to temporarily open the NPCs for macromolecules as large as pDNAs and demonstrated that this can significantly enhance non-viral gene delivery.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium.

ABSTRACT
One of the major obstacles in non-viral gene transfer is the nuclear membrane. Attempts to improve the transport of DNA to the nucleus through the use of nuclear localization signals or importin-beta have achieved limited success. It has been proposed that the nuclear pore complexes (NPCs) through which nucleocytoplasmic transport occurs are filled with a hydrophobic phase through which hydrophobic importins can dissolve. Therefore, considering the hydrophobic nature of the NPC channel, we evaluated whether a non-selective gating of nuclear pores by trans-cyclohexane-1,2-diol (TCHD), an amphipathic alcohol that reversibly collapses the permeability barrier of the NPCs, could be obtained and used as an alternative method to facilitate nuclear entry of plasmid DNA. Our data demonstrate for the first time that TCHD makes the nucleus permeable for both high molecular weight dextrans and plasmid DNA (pDNA) at non-toxic concentrations. Furthermore, in line with these observations, TCHD enhanced the transfection efficacy of both naked DNA and lipoplexes. In conclusion, based on the proposed structure of NPCs we succeeded to temporarily open the NPCs for macromolecules as large as pDNAs and demonstrated that this can significantly enhance non-viral gene delivery.

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Transfection efficiency of naked pDNA in Vero (A) and A549 (B) cells post-incubated for 1 h with different TCHD concentrations. The asterisk (*) represents data that significantly differ (P < 0.05) from the data point 0% (w/v) TCHD.
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Figure 4: Transfection efficiency of naked pDNA in Vero (A) and A549 (B) cells post-incubated for 1 h with different TCHD concentrations. The asterisk (*) represents data that significantly differ (P < 0.05) from the data point 0% (w/v) TCHD.

Mentions: The effect of TCHD on the transfection efficiency of naked pDNA was evaluated by incubating Vero and A549 cells with naked pDNA for 2 h. Subsequently, pDNA that was not incorporated into the cells was removed by washing, and the cells were exposed to increasing percentages (w/v) of TCHD for 1 h (Figure 4). The incubation with TCHD clearly increased the gene expression. This increase reached a maximal value at a TCHD percentage of 0.5 and 1.5% in Vero (Figure 4A) and A549 cells (Figure 4B), respectively. At these optimal concentrations, a 3- and 66-fold increase in gene expression was observed in Vero and A549 cells, respectively. At higher percentages no further increase and even a drop in gene expression was observed. Most likely this indicates that TCHD at these concentrations affects cellular processes that are not detected by the MTT assay. Indeed, it has been shown that the sensitivity of the MTT assay depends on the mechanism causing cytotoxicity (44). Between 0 and 1.5% (w/v) TCHD, a gradual increase in gene expression was observed in A549 cells (Figure 4B), which may indicate that the extent of NPC opening by TCHD is concentration dependent. Whether at 1.5% (w/v) TCHD a maximal opening of the NPCs is reached is not certain, since above this concentration also cytotoxic effects can play a role. This also explains the lower effects of TCHD in Vero cells. Indeed, in these cells the optimal concentration of TCHD to increase gene transfer is 0.5%. Based on the results in A549 cells, we can deduce that at such low TCHD concentration the opening of the NPC has not reached its maximum.Figure 4.


Nuclear accumulation of plasmid DNA can be enhanced by non-selective gating of the nuclear pore.

Vandenbroucke RE, Lucas B, Demeester J, De Smedt SC, Sanders NN - Nucleic Acids Res. (2007)

Transfection efficiency of naked pDNA in Vero (A) and A549 (B) cells post-incubated for 1 h with different TCHD concentrations. The asterisk (*) represents data that significantly differ (P < 0.05) from the data point 0% (w/v) TCHD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919477&req=5

Figure 4: Transfection efficiency of naked pDNA in Vero (A) and A549 (B) cells post-incubated for 1 h with different TCHD concentrations. The asterisk (*) represents data that significantly differ (P < 0.05) from the data point 0% (w/v) TCHD.
Mentions: The effect of TCHD on the transfection efficiency of naked pDNA was evaluated by incubating Vero and A549 cells with naked pDNA for 2 h. Subsequently, pDNA that was not incorporated into the cells was removed by washing, and the cells were exposed to increasing percentages (w/v) of TCHD for 1 h (Figure 4). The incubation with TCHD clearly increased the gene expression. This increase reached a maximal value at a TCHD percentage of 0.5 and 1.5% in Vero (Figure 4A) and A549 cells (Figure 4B), respectively. At these optimal concentrations, a 3- and 66-fold increase in gene expression was observed in Vero and A549 cells, respectively. At higher percentages no further increase and even a drop in gene expression was observed. Most likely this indicates that TCHD at these concentrations affects cellular processes that are not detected by the MTT assay. Indeed, it has been shown that the sensitivity of the MTT assay depends on the mechanism causing cytotoxicity (44). Between 0 and 1.5% (w/v) TCHD, a gradual increase in gene expression was observed in A549 cells (Figure 4B), which may indicate that the extent of NPC opening by TCHD is concentration dependent. Whether at 1.5% (w/v) TCHD a maximal opening of the NPCs is reached is not certain, since above this concentration also cytotoxic effects can play a role. This also explains the lower effects of TCHD in Vero cells. Indeed, in these cells the optimal concentration of TCHD to increase gene transfer is 0.5%. Based on the results in A549 cells, we can deduce that at such low TCHD concentration the opening of the NPC has not reached its maximum.Figure 4.

Bottom Line: Attempts to improve the transport of DNA to the nucleus through the use of nuclear localization signals or importin-beta have achieved limited success.Furthermore, in line with these observations, TCHD enhanced the transfection efficacy of both naked DNA and lipoplexes.In conclusion, based on the proposed structure of NPCs we succeeded to temporarily open the NPCs for macromolecules as large as pDNAs and demonstrated that this can significantly enhance non-viral gene delivery.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium.

ABSTRACT
One of the major obstacles in non-viral gene transfer is the nuclear membrane. Attempts to improve the transport of DNA to the nucleus through the use of nuclear localization signals or importin-beta have achieved limited success. It has been proposed that the nuclear pore complexes (NPCs) through which nucleocytoplasmic transport occurs are filled with a hydrophobic phase through which hydrophobic importins can dissolve. Therefore, considering the hydrophobic nature of the NPC channel, we evaluated whether a non-selective gating of nuclear pores by trans-cyclohexane-1,2-diol (TCHD), an amphipathic alcohol that reversibly collapses the permeability barrier of the NPCs, could be obtained and used as an alternative method to facilitate nuclear entry of plasmid DNA. Our data demonstrate for the first time that TCHD makes the nucleus permeable for both high molecular weight dextrans and plasmid DNA (pDNA) at non-toxic concentrations. Furthermore, in line with these observations, TCHD enhanced the transfection efficacy of both naked DNA and lipoplexes. In conclusion, based on the proposed structure of NPCs we succeeded to temporarily open the NPCs for macromolecules as large as pDNAs and demonstrated that this can significantly enhance non-viral gene delivery.

Show MeSH
Related in: MedlinePlus