One of the major barriers to efficient gene transfer and expression of nonviral vectors for gene therapy is passage across the nuclear envelope. We have previously shown that an oligolysine–RGD peptide that condenses plasmid DNA and binds to cell surface integrins can mediate increased internalisation of plasmid DNA into cells and synergistic enhancement of gene expression when complexed to a cationic lipid. In this report, we show that this enhancement is due to increased nuclear transfer of the plasmid DNA. We have applied the digitonin-permeabilised cell system that has been well established for the study of the nuclear transport of proteins to examine the nuclear transfer of plasmid DNA. Nuclear transfer of plasmid DNA complexed to an oligolysine–RGD peptide and lipofectamine appears to be an energy-dependent process involving the nuclear pore complex, since it is inhibited at 4°C and by treatment with wheat germ agglutinin or with an antibody to the nuclear pore complex which all block nuclear protein import. In accordance with active nuclear transport, we have shown that all these treatments inhibit expression of a luciferase reporter plasmid in permeabilised cells. Nuclear transfer of pDNA is enhanced in mitotic cells, but cell division is not a prerequisite for transfer. We propose that the oligolysine–RGD peptide acts as a nuclear localisation signal and that the cationic lipid is more important for cell entry and endosome destabilisation than nuclear transfer.
Nonviral vectors for gene therapy enter cells by endocytosis and traffic via endosomes before being released into the cytoplasm for nuclear transfer.1 One of the major barriers to efficient gene transfer is the nuclear envelope that selectively controls passage of macromolecules from the cytoplasm to the nucleus. Nuclear transport of oligonucleotides and plasmid DNA (pDNA) has been studied following: (1) transfection of whole cells;12345 (2) microinjection into the cytoplasm;678910111213 and (3) transfection of plasma membrane-permeabilised cells,1214151617 with the aim of determining the mechanism of transfer. Although the nuclear transport of pDNA is less well understood than nuclear protein transport a number of these studies571318 suggest that transfer occurs via the nuclear pore complex (NPC) similar to protein import and export. The NPC, a multiprotein complex, allows for active, energy-dependent, or passive transport of macromolecules (for review, see 1920). Active protein transport requires interaction of specific nuclear localisation signal (NLS) sequences on proteins with importins, that in turn interact with the protein RanGDP and carry cargo into the nucleus. These amino acid sequences characteristically bare a strong positive charge. An enhancement of nuclear transfer and/or expression of pDNA in the presence of a NLS, either covalently5101221 or noncovalently236162223242526 associated with the pDNA was found, which together with the observed interaction of NLS-pDNA constructs with the α-importin NLS nuclear import receptor321 supports the hypothesis of an involvement of the NPC machinery in the nuclear transfer of pDNA. However, it has also been observed that transfection is optimal in actively proliferating cells and thus it has been suggested that exogenous pDNA enters the nucleus during mitosis when breakdown of the nuclear envelope occurs.27282930
The majority of studies into nuclear transport of pDNA have been performed with either naked pDNA or a NLS-pDNA, while only a few studies have examined the mechanism of transfer of pDNA in the presence of a cationic polymer or liposome.1131 Yet the latter methodology is the most widely used for in vitro transfection.
We and others have previously demonstrated the principle of integrin-mediated gene transfer using a bifunctional oligolysine–RGD peptide which condenses pDNA and binds to RGD-recognising integrins on the cell surface.32333435 In addition, we have demonstrated that the presence of a liposome enhances substantially the level of gene expression of this construct.13236 It was, however, not clear whether this enhancement is entirely due to the targeting function of the oligo-peptide or if it may also be able to confer a nuclear targeting function by virtue of its strong positive charge. In this study, we set out to investigate the nuclear transport of the pDNA when complexed to an oligolysine–RGD peptide and/or liposome and to examine the role of the NPC in this transfer. We chose to use digitonin-permeabilised cells to examine nuclear transport since this cell system has proven to be invaluable in understanding nuclear protein import and export.37 The detergent digitonin selectively permeabilises membranes rich in cholesterol, such as the plasma membrane, while leaving intact the membranes of intracellular organelles with low levels of cholesterol, such as the nucleus37 and secretory granules.38 This system thereby allows direct access of various agents to the nucleus for the study of nuclear transfer and unlike microinjection a large population of cells can be examined. In addition, we have examined the role of the cell cycle in nuclear transfer with the aim of determining whether dismantling of the nuclear envelope during mitosis makes pDNA transfer more efficient.
The results presented here indicate that both the oligolysine–RGD and –RGE peptides are able to mediate nuclear transfer of pDNA much more efficiently than oligolysine on its own, indicating that the charge is not the main factor involved in this function. We also found that the liposome lipofectamine promotes nuclear transfer in digitonin-permeabilised cells, but not in cells microinjected with the complex. Despite permeabilisation, lipofectamine may enhance cell entry and thus increase the amount of pDNA that comes in contact with the nucleus. In digitonin-permeabilised cells oligolysine–RGD and –RGE act synergistically with lipofectamine to enhance nuclear transfer of pDNA. In addition, the mechanism of [K]F16RGD/RGE nuclear transfer of pDNA appears to involve the nuclear pore complex machinery. Furthermore, this study also indicates that although nuclear transfer is more efficient in cells undergoing mitosis, substantial nuclear translocation also occurs in non-dividing cells.
The oligolysine–RGD peptide enhances transfer of pDNA to the nucleus
To examine nuclear transport of pDNA we adopted the well-established digitonin-permeabilised cell system which has been extensively used in the study of the nuclear transport of proteins.3739 To verify that the nuclear membrane of the human tracheal cells used in this study remains intact during permeabilisation with digitonin the nuclei of permeabilised cells were tested for their capacity to exclude FITC-labelled dextran of 70 kDa (Figure 1). No FITC label (green) could be detected by confocal microscopy in nuclei counterstained with the DNA binding dye propidium iodide (red), thus indicating that the nuclear membrane remains intact. This was confirmed by serial sectioning at the level of the nucleus using a confocal microscope.
When FITC-labelled [K]16RGD (Figure 2a) or [K]16RGE Figure 2bpeptides were incubated with permeabilised cells the label (green) was observed in the nucleus within 1 h by confocal sectioning of several cells at the nuclear level. The FITC-[K]16 peptide also transferred to the nucleus, but the label appeared to be less intense Figure 2cTo test if the oxidised/reduced state of the peptide affected nuclear transfer FITC-labelled oxidised (cyclic) or reduced (non-cyclic) peptides were examined for nuclear transfer (Figure 3). However, both peptides appeared to enter the nucleus within 30 min. When Cy5-labelled pDNA (pseudo-blue) alone was added to digitonin-permeabilised cells, no label was detected in nuclei counterstained with propidium iodide (red) (Figure 4a). When the Cy5-pDNA was condensed with the [K]16RGD peptide, only a low level of label was seen in nuclei Figure 4bWhen lipofectamine was complexed with Cy5-pDNA label was observed in the nuclei Figure 4cand addition of lipofectamine to the Cy5-pDNA:[K]16RGD complex clearly enhanced the amount of pDNA in the nucleus Figure 4dUnder these conditions colocalisation of Cy5-pDNA and FITC-[K]16RGD was observed in the nucleus as shown by the white signal resulting from the merger of red, blue and green (Figure 5a). Colocalisation of Cy5-pDNA and FITC-[K]16RGE Figure 5bor FITC-[K]16 Figure 5cwas also observed in the nucleus after complexing with lipofectamine. However, the proportion of the two fluorophores varied giving either a yellow (green and red) or mauve (blue and red) signal. Examination of the individual fluorophore images (ie non-merged) also allowed confirmation of colocalisation. No Cy5-pDNA and only a limited amount of peptide was observed in the nucleus when complexed with FITC-[K]16 without lipofectamine Figure 5dThese results correlate with the results for the luciferase activity levels in non-permeabilised cells, where the presence of both the [K]16RGD or [K]16RGE peptides with lipofectamine led to the highest expression and where the presence of the [K]16 peptide did not significantly modify the luciferase expression level mediated by lipofectamine Figure 5eIn the absence of lipofectamine, the [K]16RGD peptide enhanced gene transfer by specific interaction with cell surface integrins since the mutated peptide [K]16RGE which does not bind integrins did not increase the level of gene transfer above that of pDNA alone.3233 However, in the presence of lipofectamine a loss of specificity is observed since the [K]16RGE peptide gives equivalent levels of luciferase expression as does [K]16RGD.32 The observed synergistic effect on expression of both [K]16RGD and [K]16RGE in the presence of lipofectamine suggested to us that both peptides may be acting as an NLS and thereby enhancing gene expression by increasing nuclear transfer. Examination of the two peptide sequences on the protein data base PSORT II (prediction of protein sorting signals and localisation sites in amino acid sequences) predicts their nuclear localisation and thus supports this hypothesis. The basic amino acids [K]16 and R (arginine) in the peptides would together form a bipartite NLS. As lipofectamine, in contrast to the [K]16 RGD and [K]16 RGE peptides does not enter the nucleus in a detectable amount,1 we suggest that the mechanism by which it enhances nuclear transfer on its own is different from that of the two peptides.
The overall luciferase expression levels were higher in digitonin-permeabilised compared with non-permeabilised cells (Figure 6). This appears to be primarily due to an increase in the amount of pDNA, that enters cells and thus comes in contact with the nucleus irrespective of its complexing with a peptide. However, importantly the mean expression levels in permeabilised cells for the [K]16 RGD and [K]16 RGE peptides is higher than for [K]16 alone again suggesting that the RGD and RGE motifs enhance nuclear transfer.
When the pDNA:[K]16 RGD complex was microinjected into the cytoplasm of cells, no label was observed in the nucleus at the control zero time (T0) but label was seen in the nucleus after 2 h (T 2h), whether or not the complex was prepared with lipofectamine (Figure 7). The label appeared to remain in a compact form within the nucleus. However, not all nuclei of microinjected cells took up the label (data not shown). No label was observed in the nuclei of cells microinjected with Cy5-labelled pDNA alone. These results suggest that the peptide is the major determining factor in nuclear transfer, but they appear to be in conflict with the data presented in Figure 4, which suggest that lipofectamine favours nuclear transfer. It may be that in digitonin-permeabilised cells lipofectamine enhances cell entry and protection of the pDNA and thus brings increased amounts in contact with the nucleus. It is, however, hazardous to draw quantitative conclusions from confocal images. It should also be noted that due to the small amount of sample injected into the cytoplasm of cells (about 1 pl) the amount of complex that comes in contact with the nucleus is potentially lower in microinjected cells than in permeabilised cells and the complex is presented in a microdrop form rather than as a diffuse solution enrobing the nucleus. In conclusion, we suggest that lipofectamine is more important for cell entry and protection of the pDNA, and possibly destabilisation of endosomes, than for nuclear transfer.
Nuclear transfer of complexed pDNA involves the nuclear pore complex
Nuclear transfer of NLS-containing proteins is inhibited at 4°C,37 and is blocked by agents which bind the nuclear pore complex such as the lectin wheat germ agglutinin (WGA),3739 or specific antibodies to a nuclear pore complex protein.40 To determine if nuclear transfer of oligolysine-RGD:lipofectamine complexed Cy5-pDNA was also inhibited by such treatments permeabilised cells were either maintained at 37°C (control) (Figure 8a) or at 4°C Figure 8b or they were pretreated with either WGA Figure 8c or a nuclear pore complex antibody Figure 8d before transfection and analysis by confocal microscopy. The 37°C control showed colocalisation of peptide and pDNA in the nucleus Figure 8a but no labelled pDNA or peptide could be seen in the nucleus when cells were maintained at 4°C Figure 8b. No peptide or pDNA was observed in nuclei when cells were pretreated with WGA Figure 8c. No pDNA but some peptide was observed in the nucleus when cells were treated with an antibody to the nuclear pore complex (yellow) Figure 8d. Since quantification of the fluorescence of confocal images is not very reliable, we attempted to confirm and quantify this inhibition by determining the luciferase activity expressed in digitonin-permeabilised cells. We hypothesised that since the protein luciferase localises to peroxisomes,41 it may not leak from permeabilised cells as do small macromolecules like ATP and cytoplasmic proteins such as lactate dehydrogenase (LDH) and thus synthesis of luciferase and detection of its activity could be possible. Permeabilised cells transfected at 4°C showed significantly reduced luciferase expression levels compared with 37°C (Figure 9a). Incubation of non-permeabilised control cells in the presence of WGA or an antibody to the nuclear pore complex resulted in a slight decrease in the level of expression Figure 9bHowever, permeabilisation also allows entry of the inhibitors into the cell and incubation of permeabilised cells in the presence of WGA or an antibody to the nuclear pore complex resulted in an approximate five-fold and three-fold decrease in the level of expression, respectively, compared with non-permeabilised treated cells. To verify that digitonin treatment did indeed result in permeabilisation of cells the release of ATP and LDH at different digitonin concentrations was determined. Digitonin at the concentration of 20 μg/ml resulted in the release of a substantial amount of ATP Figure 9cand LDH Figure 9dinto the extracellular medium. Release of LDH at high digitonin concentrations did not reach 100% probably due to the presence in cells of LDH in subcellular compartments, such as mitochondria.42 In addition, de novo protein synthesis is already detected 2 h after permeabilisation, although at a reduced level as determined by incorporation of [35S]-methionine into protein Figure 9eand 48 h later the number of non-viable cells for non-permeabilised and permeabilised cells was 9.0 ± 1.0 and 14.3 ± 1.5, respectively. This result and the measurements of luciferase expression suggest that digitonin momentarily forms pores in the plasma membrane allowing for entry of different agents, but that the cells recommence protein synthesis which reaches a normal level 48 h after the transfection period when the luciferase activity is determined. To further confirm the presence of the luciferase protein in permeabilised cells, fluorescence immunodetection of the protein was performed Figure 9fMicroscopic examination showed the presence of the luciferase protein in vesicles that were predominantly perinuclear. In addition, the luciferase protein was detected in the large majority of cells thus indicating that the detected luciferase activity was not due to just a few highly expressing cells that had not been permeabilised.
Optimal expression in mitotic cells
To examine the role played by the cell cycle in nuclear transfer, cells were incubated in the presence of either demecolcine, which blocks cells in the G2/M phase, or hydroxyurea, which blocks cells in the G0/G1 phase. FACS analysis was used to determine the percentage of cells in the respective cycle phases. In the presence of demecolcine about 60% of the cells were in the G2/M phase (Figure 10a, left) and in the presence of hydroxyurea about 60% of the cells were in the G0/G1 phase (Figure 10a, right). In fact, half the control cells at 80% confluence were already in the G0/G1 phase. Treated cells were transfected in the presence of the respective drug that was then removed and the luciferase activity was determined after the 48-h post-transfection period. Cells in the G2/M phase showed an approximate four-fold increase in the luciferase expression level compared with non-synchronised control cells (Figure 10b, left), while cells in the G0/G1 phase showed a lower but nonetheless considerable level of expression similar to those of a non-synchronised control (Figure 10b, right). Confocal microscopy analysis showed the presence of labelled pDNA in the nuclei of cells treated either with demecolcine (Figure 10c, left) or hydroxyurea (Figure 10c, right) when transfected with the Cy5pDNA:[K]16RGD:lipofectamine complex. These results suggest that the dismantling of the nuclear membrane during mitosis favours nuclear transfer, but that transfer also occurs when the nuclear envelope is intact and that mitosis plays a relatively limited role in the entry of the [K]16 RGD/E pDNA complexes into the nucleus.
We tested the possibility that an increase in expression observed in cells in mitosis was due to an increase in the uptake of the complex by cells and not an increase in nuclear transfer. To this aim the uptake of [35S]-labelled pDNA complexed to [K]16RGD:lipofectamine was examined in control cells and cells treated with hydroxyurea or demecolcine. Since the cell-associated radioactivity for demecolcine-treated cells was lower than for either control or hydroxyurea-treated cells, we conclude that the increase in expression levels in mitotic cells was due to an increase in the nuclear transfer and not cell uptake (Figure 11). It should be pointed out that cell-associated radioactivity includes internalised complex, but also complex that may remain bound to the cell surface.
Inspired by the knowledge that protein transport into the nucleus requires specific amino acid sequences referred to as nuclear localisation signals (NLS), several attempts to enhance nuclear transfer of plasmid DNA (pDNA) by covalent5101221 or non-covalent association235162223242526 of pDNA with an NLS have been reported. The most frequently employed NLS is the classic monopartite sequence of the large T antigen of SV40 that is composed of a single stretch of basic amino acids; lysine and arginine. Sebestyén et al10 covalently attached this NLS to pDNA (up to 11 kb) and observed nuclear transfer of the pDNA in digitonin-permeabilised cells when the pDNA was conjugated with a high ratio of NLS peptide. Transfer was inhibited at 4°C and in the presence of wheat germ agglutinin (WGA) which is known to inhibit NLS-protein import through the nuclear pore complex, thus suggesting that nuclear transfer involves an energy-dependent active transport through the nuclear pore. The finding that NLS-pDNA constructs can interact with α-importin, an NLS nuclear import receptor321 supports this hypothesis. However, no nuclear transfer of this and another NLS–DNA conjugate could be observed after their microinjection into the cytoplasm of cells, possibly as a result of cytoplasmic sequestration.1221 Yet, other covalent NLS-pDNA constructs have been shown to be present in nuclear fractions of zebrafish embryos after microinjection into the cytoplasm,6 and to result in an increase in expression levels in cells transfected in the presence of either a cationic lipid or polymer.521
Non-covalent association of an NLS with the pDNA has also been reported to enhance expression levels in cells.3616222543 In addition, using a bifunctional peptide nucleic acid (PNA)-NLS, where PNA forms duplex hybrids with complementary DNA, Brandén et al2 demonstrated increased association of pDNA with the nucleus and expression in cells transfected in the presence of polyethylenimine (PEI). Thus the results concerning covalent and non-covalent association suggest that NLS binding to DNA and subsequent enhancement does not require a cross-linking agent and covalent binding may even be detrimental in abolishing transcription, in particular when many NLS molecules are present per pDNA.12
To determine whether the previously observed increase in expression and the increase in the amount of pDNA in isolated nuclear fractions in the presence of peptide and/or liposome133 is due in part to an increase in the transfer of the pDNA to the nucleus, we examined the nuclear transfer of the pDNA in digitonin-permeabilised cells with or without the oligolysine–RGD peptide and/or liposome. The results of this study demonstrate that pDNA (approximately 5 kb) alone does not transfer to the nucleus of digitonin-permeabilised or microinjected human tracheal cells, while transfer occurs when [K]16RGD or the liposome lipofectamine are complexed by non-covalent interaction with pDNA. In addition, the peptide and the liposome appear to act synergistically for nuclear transfer of the pDNA, as suggested previously by results of subcellular fractionation experiments.1 The observed synergistic increase in luciferase expression mediated by the [K]16RGD or [K]16RGE peptides, but not the [K]16, in the presence of an excess amount of liposome also suggested to us that the peptides may be acting as an NLS and thereby enhancing nuclear transfer. Increased expression in digitonin-permeabilised cells in the presence of the RGD/RGE peptides further supports this hypothesis. Examination of the peptide sequences on the protein data base PSORT II predict their nuclear localisation and suggest that they are bipartite NLS-like basic peptides.
The mechanism by which [K]16RGD/E peptides permits nuclear transfer of pDNA appears to involve the nuclear pore and to be an active process, since transport is inhibited at 4°C and under conditions that block transport through the nuclear pore complex. Thus these results suggest that the peptide acts as an NLS and that the bipartite nature of the [K]16RGD peptide, ie the [K]16 motif and the arginine residue are necessary since the [K]16 peptide appears to have a lower affinity for the nucleus and does not enhance expression levels. Chan and Jans3 also found that polylysine in contrast to a polylysine/NLS conjugate does not increase nuclear import. Godbey et al28 showed that the polycation PEI which enhances gene expression undergoes nuclear localisation when added to cells with or without pDNA and Pollard et al11 showed that this polymer and polylysine promoted gene delivery from the cytoplasm to the nucleus in microinjected cells. An alternative explanation for the peptide-induced transfer observed in this study may be that the substantial amount of [K]16RGD/E peptides in the nucleus modifies the characteristics of the nucleus which in turn favours nuclear penetration of the pDNA.
The mechanism by which the liposome lipofectamine enhances nuclear transfer in this study is yet to be defined. It is possible that the hydrophobic and/or cationic characteristics of lipofectamine favour interaction with the nuclear membrane and/or that the condensation of the pDNA by a cation reduces its size and thus facilitates passage into the nucleus. It has been shown however that pDNA complexed to the cationic lipids when microinjected into the cell cytoplasm did not result in an increase in gene expression possibly as a result of cytoplasmic sequestration.1144 A recent report demonstrated that lactosylated poly-L-lysine resulted in nuclear translocation of pDNA by a NPC-dependent mechanism, but that unsubstituted poly-L-lysine or mannosylated poly-L-lysine were less efficient for transfer.45 The authors postulate that the lactose moiety provides for nuclear localisation by targeting a potential lectin-like protein of the NPC.
The nuclear pore complex acts as a gateway between the cytoplasm and the nucleus and is a multiprotein structure which forms a large central transporter channel (50–60 nm in diameter) surrounded by eight peripheral diffusion channels (approximately 10 nm in diameter).20 Diffusion of macromolecules appears to be limited to a size of approximately 50 kDa, thus peptides of about 3 kDa such as [K]16RGD can theoretically diffuse into the nucleus. However, the size of the [K]16RGD:pDNA complex, estimated to possess a diameter of about 20–100 nm,33 would exclude transport by diffusion. The addition of lipofectamine, estimated to have a mean diameter of about 90 nm46 would also increase the overall size, though it is possible that this component of the complex does not enter the nucleus. Our previous studies into the localisation of lipofectamine by electron microscopy in the cell did not reveal its presence in the nucleus.1 Thus the sizes of these components suggest that transfer occurs via the central energy-dependent channel unless the pDNA is capable of threading through the NPC in a worm-like manner as previously suggested.5
We were also interested in examining the relationship between the cell cycle and nuclear transfer since breakdown of the nuclear membrane during mitosis may open the way to nuclear transfer. Several studies have demonstrated that cells arrested in the G1 phase show reduced gene expression after transfection with either a cationic lipid or polycation,81128 while cells in mitosis show increased expression.2730 Nonetheless significant gene expression was observed in non-dividing cells8 and cell division was not required for expression of a pDNA complex to PEI when injected into the cell cytoplasm.5 Studies by Brunner et al27 and Escriou et al47 showed substantial increases in expression (30–500-fold) for mitotic cells compared with cells in the G1 phase when transfected with a cationic lipid or polymer. However, when an adenoviral system was used no substantial enhancement in expression (four-fold) was observed in mitotic cells. This suggests that when an efficient nuclear entry machinery is present as in the case of the adenovirus, the breakdown of the nuclear membrane does not influence the level of transfer.27 Our studies confirm the findings obtained with nonviral systems and suggest that the dismantling of the nuclear envelope favours nuclear transfer, but is not a prerequiste for transfer in particular if an efficient NLS-like mechanism which employs the nuclear entry machinery is present.
In conclusion, results presented here indicate that: (1) the peptide [K]16RGD/RGE enhances nuclear transfer of pDNA, probably as a result of its bipartite NLS-like sequence and interaction with the NPC; (2) the liposome lipofectamine is probably more important for cell entry and possibly endosome escape than nuclear translocation; and (3) cells in mitosis possess enhanced nuclear transfer, but cell division is not a prerequisite for nuclear transfer.
Materials and methods
The fetal human tracheal epithelial cell line 56FHTE8o- was kindly provided by Dr D Gruenert.48 Cells were grown in a 1:1 mixture of Dulbecco's minimum essential medium and Ham's F-12 nutrient medium (DMEM/F-12) and supplemented with foetal calf serum (10% v/v) (Biowest, Nuaillé, France). Cells were maintained at 37°C in 5% CO2–95% air and the media changed every second day. Cells were seeded on either six- or 12-well plates, 25 cm2 or 75 cm2 cultured flasks or Labtek eight-well plates (Gibco BRL, Cergy Pontoise, France) until approximately 80% confluence.
Peptides and plasmid DNA
The peptides N-[K]16GGCRGDMFGCA ([K]16RGD) and N-[K]16GGCRGEMFGCA ([K]16RGE) were synthesized and cyclised as previously described33 and [K]16, [K]16RGD and [K]16RGE peptides were labelled with FITC as previously described.32 An American firefly (Photinus pyralis) luciferase pDNA (5256 bp) (pGL3) under the control of an SV40 promoter and enhancer was used as a reporter gene. pDNA was purified and labelled with Cy5 by a modified nick-translation technique giving predominantly an open circle form as previously described.3249 The pDNA (1 μg) was labelled in the presence of 10-6 units of DNase I and the integrity of the pDNA confirmed by electrophoresis on an agarose (0.8%) gel and staining with ethidium bromide (data not shown). The Cy5-label was detected using a Molecular Dynamics fluorescence scanning system (Storm 860) with excitation at 649 and emission at 670 nm (data not shown).
The [K]16RGD, [K]16RGE peptides (FITC labelled or non-labelled) were mixed with the pDNA at a ratio of five to one (w:w), respectively, and incubated 15 min at room temperature in HEPES buffered saline. Peptide at this ratio condenses the DNA as shown by electron microscopy and renders it resistant to Dnase.33 Lipofectamine (2 mg/ml) at 3:1 (w:w) mixture of a polycationic lipid (DOSPA) and a neutral lipid (DOPE), was then added (final ratio DNA:peptide:lipofectamine; 1:5:18 or 1:5:24 (w:w:w) and the mixture incubated a further 15 min at room temperature.
Cells were synchronised in the G2/M phase by pretreatment with demecolcine (0.06 μg/ml), for 16 h at 37°C or in the G0/G1 phase with hydroxyurea (2.5 mM) for 18 h at 37°C. Cells were then harvested in trypsin (0.05%)/EDTA (0.02%) and fixed in cold 70% ethanol overnight. After washing with PBS, cells were resuspended in PBS containing RNase A at 1 mg/ml and propidium iodide at 50 μg/ml. After 30 min at room temperature, the red fluorescence of the propidium iodide was detected and analysed on a FACScalibur (Becton Dickinson, Le Pont de Claix, France) equipped with an argon laser emitting at 488 nm.
Transfection and luciferase detection
The complex (pDNA:[K]16RGD:lipofectamine, 0.08 μg:0.4 μg:1.4 μg, per well) was added to permeabilised or non-permeabilised cells in a final volume of 300 μl of Opti-MEM (Gibco BRL). Cells were then incubated at 37°C for 4 h after which the complex was removed. The cells were then washed in PBS, the normal supplemented media added and incubated for a further 48-h post-transfection period. Forty-eight hours was found to give optimal luciferase activity (data not shown). Luciferase activity was assayed as previously described32 and the protein concentration of cell lysates was determined using the Bradford method.50 For inhibition of nuclear pore transport, permeabilised cells were pretreated with 50 μg/ml WGA (Sigma, St-Quentin Fallavier, France) for 30 min at room temperature before the addition of the complex and subsequent detection of luciferase activity. This lectin binds N-acetylglucosamine residues present on a class of nuclear pore complex (NPC) proteins. In addition, inhibition of nuclear pore transport in permeabilised cells was performed by pretreatment with the Mab 414 antibody which recognizes a related family of NPC proteins (Babco, Berkley, USA)40 for 30 min at room temperature at a working dilution of 1/10 000. Cells synchronised in the G2/M phase by pretreatment with demecolcine (0.06 μg/ml), for 16 h at 37°C or in the G0/G1 phase with hydroxyurea (2.5 mM) for 18 h at 37°C were transfected in the presence of drug as described above and the 48-h post-transfection was performed in the absence of drug.
After two rinses in chilled PBS, cells were incubated in the presence or absence of digitonin (20 μg/ml) (Calbiochem, Fontenay-sous-Bois, France) for 5 min on ice and washed twice in chilled PBS. Cells were then incubated in the presence of [35S]-methionine (15 μCi/well, six-well plates) for 2 h at 37°C. In order to determine free label associated with the cells, control cells were incubated in the same conditions at 4°C. After three rinses in chilled PBS cells were incubated with 5% TCA for 30 min on ice and rinsed twice with 80% ethanol. Lysis was performed in the presence of 20% KOH for 15 min on ice. The protein concentration was determined by the Bradford method50 and the radioactivity measured by scintillation counting with Ecolite in an LKB Rackbeta 1209 counter.
ATP and lactate dehydrogenase release
Cell ATP and lactate dehydrogenase (LDH) release was measured after permeabilisation of cells with digitonin (10–150 μg/ml). For ATP release,cells were rinsed three times with DMEM/F-12 medium and incubated with ATP degradation inhibitors: P1,P5-di (adenosine-5′) pentaphosphate (Sigma) and adenosine 5′-diphosphate (Sigma) respectively at 0.5 mM and 1 mM in DMEM/F-12 medium, 3 min at 37°C. Cells were then prechilled and permeabilised with digitonin (10–150 μg/ml) containing inhibitors, for 5 min on ice (600 μl per well). The medium was collected and the ATP measured using an adenosine 5′-triphosphate bioluminescence assay kit (Sigma) and detected as relative light units (RLU) emitted. Results are expressed as extracellular ATP. For detection of the lactate dehydrogenase (LDH) activity cells were prechilled and incubated 5 min on ice with the indicated digitonin concentration. After two rinses with PBS, cells were scrapped into 1 ml of PBS and centrifuged for 10 min at 4°C at 800 r.p.m. The pellet was resuspended in hypotonic lysis buffer (500 mM Tris HCl pH 7.4, 100 mM NaCl, 3 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 0.5% deoxycholate, 0.1% SDS, Nonidet P40) and the cytoplasmic marker lactate dehydrogenase was assayed with a Synchron CX4 CE (Beckman Gagny, France) analyser using pyruvate and NADH as substrates. Results are expressed as a percentage of the LDH in non-permeabilised cells. Cell viability was determined by the trypan blue exclusion assay and triplicate samples were counted in a haemocytometer.
Cell uptake of [35S]-labelled pDNA
Cells were synchronised in the G2/M or G0/G1 phase by pretreatment with demecolcine (0.06 μg/ml), for 16 h at 37°C or with hydroxyurea (2.5 mM) for 18 h at 37°C, respectively. The plasmid pGL3 was labelled with [35S]dATP and the complex composed of [35S]pGL3:[K]16RGD:lipofectamine (1:5:18) was prepared as previously described.32 Cells were then incubated with the complex for the indicated times. After rinsing with PBS, cells were harverested with trypsin/EDTA, the trypsin was blocked with of 1% BSA in PBS and cells recovered by centrifugation (10 min, 1100 g). The cell pellet was rinsed once in PBS by centrifugation before lysis in 1 N NaOH. The radioactivity was measured by scintillation counting in a 1600TR analyser (Packard, Rungis, France) and the protein content determined by the method of Bradford. Results are expressed as c.p.m/mg of protein.
Disulphide bridge peptide reduction
To determine the impact on nuclear transport of disulphide bond formation between the cysteine residues of the peptide, a sample of the peptide was reduced with dithiotreitol (DTT). DTT was added in a five-fold excess relative to thiol groups (ie 0.3 mM) to a 1 mg/ml solution of peptide in 0.1 M ammonium bicarbonate pH 8.0 and incubated for 6 h at room temperature in a nitrogen atmosphere. The mixture was then acidified to pH 7.4 with acetic acid. To verify reduction of the disulphide bridge in the peptide an Ellman's assay was performed. Fifty μl of 3 mM DNTB (5,5′-dithio-bis(2-nitrobenzoicacid) was added to 1 ml of 0.1 mg/ml peptide solution. The absorbance at 412 nm was measured on a spectrophotometer after incubation for 15 min. The concentration of sulphydryl groups was calculated from the molar extinction coefficient of 14150/cm using the following equation, [SH] = [A412(sample) - A412(reference)]/14 150.
Cells (1.5×105/ml) were grown on Esco glass slides (22×22 mm) (Erie Scientific, Portsmouth, USA) in 35 mm Petri dishes for 48 h. Either pDNA (pGL3) labelled with Cy5 (20 μg/μl) or Cy5-pGL3 complexed to FITC- [K]16RGD in the presence or absence of lipofectamine at a ratio of 1:5:18 (pGL3: [K]16RGD:lipofectamine) were injected into the cytoplasm of cells with an Eppendorf 5242 microinjector using glass micropipettes (Clark, Reading, UK). About 1 pl was injected per cell. Cells were then rinsed twice with OpiMEM and incubated at 37°C for the indicated times.
Confocal laser scanning microscopy
Cells were permeabilised for 5 min on ice with digitonin (20 μg/ml). The wells were rinsed twice with PBS and then incubated at 37°C or 4°C with different components: Cy5-labelled pDNA:FITC-labelled [K]16RGD/E:lipofectamine at the above indicated optimal ratio 1:5:18 (w/w/w, 0.3 μg of pDNA per well) or Cy5-pDNA:FITC-[K]16RGD (1:5, 0.3 μg pDNA per well) or Cy5-pDNA:lipofectamine (1:18, 0.3 μg pDNA per well), or FITC-[K]16RGD/E (5 μg per well) or FITC-polylysine [K]16 (5 μg per well) or Cy5-pDNA alone (0.3 μg pDNA per well) in OptiMEM for 1 h. Pretreatment with the lectin wheat germ agglutinin (WGA, 50 μM) or with the Mab 414 antibody (1/10 000) was performed for 30 min at room temperature. To verify the integrity of the nuclear membrane of permeabilised cells, they were incubated with 500 μg/ml FITC-dextran (MW 70 000, Sigma) for 30 min at 37°C. Cells synchronised in the G0/G1 or G2/M phase were permeabilised and the nuclear transfer of the Cy5-pDNA:[K]16RGD:lipofectamine complex at the optimal ratio of 1:5:18 (w/w/w) for 1 h at 37°C examined. To test the nuclear transfer of cyclised and non-cylised peptide, cells were permeabilised and rinsed twice with PBS and incubated with 0.1 mg/ml of cyclised or non- cyclised [K]16RGD, 30 min at 37°C. Control cells pretreated with DTT were incubated a further 30 min at room temperature with 500 μg/ml FITC-dextran to determine nuclear membrane integrity. Cells were washed twice in PBS, fixed for 20 min at room temperature in 4% paraformaldehyde (PFA), rinsed in 50 mM NH4Cl for 5 min at room temperature. Cells were treated with 1 mg/ml of RNase A 10 min at room temperature and nuclei were then stained with propidium iodide (3 μg/ml) for 3 min at room temperature. Cells were then mounted in DABCO/glycergel (Dako, Trappes, France). For detection of the luciferase protein, cells were transfected as described above. After a 48-h post-transfection period, cells were incubated in the presence or absence of digitonin (20 μg/ml) for 5 min on ice, washed twice in PBS, fixed for 20 min at room temperature in 4% PFA and rinsed four times in 50 mM NH4Cl for 5 min at room temperature. Cells were then incubated with a fluorescein-conjugated IgG anti-luciferase (firefly, Photinus pyralis) antibody (Rockland, Gilbetsville, USA) for 1 h at room temperature at a working dilution of 1/7500 in the presence of 0.% saponin. Cells were then washed four times with 50 mM NH4Cl for 5 min at room temperature and treated as described above. Confocal microscopy was performed using a TCS SP Leica (Lasertechnichk) microscope, equipped with a ×40 objective (plan apo; NA = 1.25). For FITC excitation, an argon-krypton ion laser adjusted at 488 nm was used, 568 nm for propidium iodide and 647 nm for Cy5 excitation. For each optical section, triple fluorescence images were obtained in the sequential mode (ie FITC first, TRITC second and Cy5 third). The signal was treated by line averaging to integrate the signal collected over four lines in order to reduce noise. The confocal pinhole was adjusted to allow for a minimum field depth. A focal series was collected for each specimen. The focal step between each section was 0.9 μm. Selected sections were then processed to produce a single composite overlay image (colour merged). Images were printed on a colour ink jet printer (Epson Stylus color 850) using Photoshop 5.0 software.
Data are presented as the mean ± s.e.m of triplicate determinations and are representative of results obtained in three or four independent experiments that produced similar relative results. Note that the luciferase mean values varied for different batches of cells and for cells of distant passage number. Confocal images show representative results of at least two individual experiments using different batches of labelled pDNA. Statistical analysis was performed using the nonparametric rank test of Wilcoxon. Probability values <0.05 were considered statistically significant.
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This work was supported by the Association Française de Lutte contre la Mucoviscidose and MC was supported by a grant from this association. We are very grateful to Dr Andrew D Miller of the Department of Chemistry Imperial College, London, UK, for synthesising the [K]16RGD and [K]16RGE peptides. We thank Dr Elizabeth Lasnier for the assay of LDH.
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Colin, M., Moritz, S., Fontanges, P. et al. The nuclear pore complex is involved in nuclear transfer of plasmid DNA condensed with an oligolysine–RGD peptide containing nuclear localisation properties. Gene Ther 8, 1643–1653 (2001). https://doi.org/10.1038/sj.gt.3301572
- cationic lipid
- gene transfer
- in vitro transfection
- nuclear transfer
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