Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Cystic fibrosis (CF) is a frequent and severe genetic disease in which a progressive respiratory failure is the cause of early death in most cases. It is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a cyclic adenosine monophosphate (cAMP)-regulated chloride channel (1). Inasmuch as the only available treatments for CF so far are symptomatic, it is attractive to develop a treatment of this respiratory disease on the basis of the introduction of a normal copy of the gene into the airway epithelial cells. Viral and nonviral approaches have been used to deliver a functional CFTR gene to airway epithelium. Human trials have been initiated using recombinant adenoviruses and cationic lipids. However, several problems related to the antigenicity of the vectors and to a limited gene transfer efficiency arose. Further refinements of the vector systems and a better knowledge of the CF physiopathology are needed (2).

The in vitro studies and subsequently the in vivo protocols have focused on the efficiency of gene transfer into the surface airway epithelial cells. However, low levels of CFTR messenger RNA (mRNA) and protein were evidenced in cells of the normal surface epithelium (3). In contrast, high levels of CFTR mRNA and protein were demonstrated in epithelial cells of human submucosal glands and notably in a subpopulation of epithelial cells that comprise the serous tubules (4). Therefore, the airway gland serous cells may be important targets for gene therapy in CF. However, partly because of the difficulty in obtaining human healthy and CF tracheal tissues and in growing human gland serous cells, or partly because of the low availability of human gland serous cell lines, few studies so far have reported an efficient gene transfer into human submucosal glands, and in all of these the vector used was a recombinant adenovirus (5, 6).

Recently, we have described two human normal and CF tracheal gland serous cell lines named MM-39 and CF-KM4, respectively, that both retain serous secretory functions (6, 7). In the present study we examined the efficiency of various synthetic vectors to transfer genes into these human normal and CF tracheal gland serous cells. We used glycosylated polylysines (glycofectins) to take advantage of a receptor-mediated gene delivery via membrane lectins, i.e., cell surface receptors binding oligosaccharides (8, 9). We have previously shown that glycoplexes (glycofectin/DNA complexes) in conjunction with compounds helping to disrupt endosomal membranes are efficient vectors for transfection of airway surface epithelial cells in vitro (10, 11). We have also used another cationic polymer, polyethylenimine (PEI) (12) and commercially available cationic lipids. Both PEI and cationic lipids enter the cell through nonspecific endocytosis and disrupt the endosomal membrane, and both have been shown to be efficient vectors for gene transfer into airway surface epithelial cells. In the present study we have shown that normal and CF gland serous cells could be efficiently transfected using all these synthetic vectors.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials

Dulbecco's modified Eagle's medium/Ham's F12 mixture (DMEM/F12), L-glutamine, valine, and collagen type I were obtained from ICN (Costa Mesa, CA). Pyruvic acid, leucine, isoleucine, glutamic acid, cysteine, glucose, epinephrine, PEI 800 kD, PEI 25 kD, chloroquine, dithiothreitol (DTT), Triton X-100, glycerol, adenosine 5'-tri-phosphate (ATP), luciferin, and bicinchoninic acid (BCA) were from Sigma (St. Louis, MO). Ultroser G was from Biosepra (Villeneuve, La Garenne, France). Ethylenediaminetetraacetate (EDTA) and MgCl₂ were from Prolabo (Fontenay-sous-Bois, France). Lipofectin, a liposome containing an equimolar ratio of N-[1-(2,3-dioleyloxy)propyl]- N,N,N-trimethylammonium chloride and dioleoylphosphoethanolamine (DOPE), and lipofectAMINE, a lipid mixture of DOPE and 2,3-dioleyloxy-N-[2(sperminecarboxamido)- ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (1:3, by mass), were from GIBCO BRL (Gaithersburg, MD). The expression plasmids pCMVLuc (pUT 650, 5.15 kb; Cayla, Toulouse, France), pCMV GFP (pCMV GFP; Packard, Meriden, CT), and pCMV CFTR encoded genes for the firefly luciferase, the green fluorescent protein, and the human CFTR complementary DNA (pTG 5960; Transgène, Strasbourg, France), respectively, under the control of the human cytomegalovirus promoter. Supercoiled DNA plasmid was isolated by a standard alkaline lysis method followed by two successive CsCl gradient centrifugations in the presence of ethidium bromide, extensive extraction with n-butanol, and precipitation with ethanol. Poly-L-lysine (HBr 30,000-50,000; average molecular mass 40,000; average degree of polymerization 190; Bachem Feinchemikalien, Budendorf, Switzerland) dissolved in H₂O (1 g in 200 ml) was changed to poly-L-lysine p-toluene sulfonate as previously described (13).

Cell Culture

The immortalized human tracheal gland serous cell lines MM-39 and CF-KM4 were from a young non-CF adult who died of a head trauma and from a CF patient homozygous for the F508 mutation and undergoing bipulmonary transplantation, respectively (6, 7). In cell-attached patch-clamp experiments, MM39 cells but not CF-KM4 cells were shown to express a cAMP-regulated chloride channel. Both cell lines retain the features of the native gland serous cells, i.e., presence of cytokeratin and constitutive secretion of secretory leukocyte proteinase inhibitor (SLPI). In response to pharmacologic stimulations, an increase in SLPI expression was observed in MM-39 cells (14), whereas CF-KM4 cells have CF-specific defective secretory characteristics (6).

Cells were plated on tissue culture plasticware coated with collagen I and grown in DMEM/F12 supplemented with 1% Ultroser G, glucose (10 g/liter), sodium pyruvate (0.33 g/liter), and epinephrine (3 µM).

One day or 1 wk before transfection, MM-39 cells or CF-KM4 cells were seeded at 2 × 10⁵ cells per 4-cm² well in a 12-well plate and incubated at 37°C in a humidified atmosphere (5% CO₂). At the time of gene transfer procedure, and depending on the time of seeding, cell cultures were either 70% confluent or highly confluent. After 24 h or 1 wk of culture, MM-39 and CF-KM4 cells expressed cholinergic, adrenergic, and purinergic receptors. However, these receptors became functional only at confluency, i.e., MM-39 cells responded to cholinergic, adrenergic, and purinergic agonists by an increase in SLPI secretion (7). In contrast, CF-KM4 cells failed to respond to cholinergic and adrenergic agonists (6). Moreover, it was only at confluency that MM-39 and CF-KM4 cells became highly polarized as assessed by the formation of domes and the presence on electron microscopic analysis of tight junctions, desmosomes, and microvilli (M. Merten, personal communication).

Glycofectins

Glycosylated poly-L-lysines. Poly-L-lysine p-toluene sulfonate partially substituted with sugar residues (either -D-glucose [Glc], -Glc, -L-rhamnose [Rha], -L-fucose [Fuc], -D-mannose [Man], -D-N-acetyl-D-glucosamine [GlcNAc], -D-N-acetyl-D-galactosamine [GalNAc], -D-GalNAc, -D-lactose [Lac], -D-galactose [-Gal], or -D-galactose [-Gal]) was prepared as previously described (8, 15). Poly-L-lysine was substituted with 66 ± 5 lactosyl residues and with 76 ± 4 of the other monosaccharide residues. Fluorescein-labeled glycosylated polylysines, prepared as previously described (8), contained 4 ± 2 fluorescein residues.

Glycosylated partially gluconoylated polylysines. The poly-L-lysine, p-toluene sulfonate salt was partially substituted with gluconoyl (GlcA) residues as previously described (13, 16) and glycosylated as described above. Complexes made with glycosylated partially gluconoylated polylysines are smaller and more soluble than complexes made with glycosylated nongluconoylated polylysine. The average number of GlcA residues bound per poly-L-lysine molecule was 66 ± 3 residues; the average number of sugar residues was 24 ± 3.

Glycofection: Gene Transfer with Glycofectin/DNA Complexes

The glycoplexes were prepared as previously described (15). Glycosylated poly-L-lysine or glycosylated partially gluconoylated poly-L-lysine (10 to 15 µg in 0.3 ml of serum-free DMEM/F12) were mixed with the reporter gene plasmid (5 µg in 0.7 ml of serum-free DMEM/F12) and held for 30 min at 37°C. Using the lowest polymer-to-DNA ratio giving a complete retardation of the DNA in electrophoresis, no free polymer could be detected (15). Then 1 ml of DMEM/F12 containing the glycoplexes was supplemented with 1% Ultroser G and 100 µM chloroquine (unless otherwise specified) and added to each culture well after removal of the growth medium.

Gene Transfer with Polyethylenimine

To obtain various PEI/DNA charge ratios (net positive nitrogen charge from the PEI divided by the net negative phosphate charge of the DNA), a fixed amount of plasmid was complexed with different amounts of PEI, either 25 or 800 kD. Under physiologic pH conditions, PEI contains one positive nitrogen among six present in the molecule. Therefore a PEI:DNA charge ratio of 1.5 corresponds to nine nitrogen atoms per phosphate (12). The plasmid DNA (5 µg) and the desired amount of PEI (0.2 µl of 0.1 M PEI stock solution/µg of DNA for a charge ratio +/ of 1.5) were separately diluted into 50 µl of 150 mM NaCl, mixed together, and vortexed. PEI-DNA complexes were diluted into 1 ml of culture medium containing 1% Ultroser G.

Lipofection

Similarly, to obtain various lipid/DNA charge ratios (net positive charge from the cationic component of the lipid divided by the net negative charge of the DNA), a fixed amount of plasmid was complexed with different amounts of liposomes in polystyrene tubes. Lipofectin contains only one positive charge in the polar group and lipofectAMINE contains five positive charges per molecule. Lipofectin (1.9 µl of a 1 mg/ml solution per microgram of DNA for a charge ratio +/ of 0.5) or lipofectAMINE (1.9 µl of a 2 mg/ml solution per microgram of DNA for a charge ratio +/ of 5) were diluted in DMEM/F12 to a final volume of 50 µl, then gently mixed with 5 µg of plasmid previously prepared in 50 µl of DMEM/F12 and held 10 min at room temperature. Lipid-DNA complexes (lipoplexes) were diluted into 1 ml of culture medium containing 1% Ultroser G.

After incubation for 4 h, unless otherwise specified, at 37°C, the transfection medium was removed and the cells were further incubated at 37°C in 1 ml of complete growth medium.

Gene Expression Measurements

Luciferase gene expression. Luciferase gene expression was measured by luminescence according to De Wet and colleagues (17). After 48 h or at given intervals, the cells from each well were harvested by trypsinization and lysed with 200 µl of homogenization buffer (25 mM Tris-HCl [pH 7.8], 8 mM MgCl₂, 1 mM EDTA, 1 mM DTT, 15% glycerol, and 1% Triton X-100). The cell suspension was vortexed and kept for 10 min at room temperature. The solution was spun down (5 min, 800 × g). ATP (95 µl of a 2 mM solution in the homogenization buffer without Triton X-100) was added to 60 µl of supernatant. The luminescence was recorded for 4 s by means of a luminometer (Lumat LB 9501; Berthold, Wildbach, Germany) upon automatic addition of 150 µl of 167 µM luciferin in water. Measurements were done in duplicate. The luminescence was reported as relative light units (RLU).

Proteins were determined on each sample by using the BCA colorimetric method (18) modified according to Hill and Straka (19) to overcome the interference due to the presence of DTT in the homogenization buffer. After determination of the amount of proteins, the results were expressed as RLU per milligram of proteins.

Green fluorescent protein expression. Green fluorescent protein expression was analyzed by the cell fluorescence intensity using a FACSort (excitation wavelength: 488 nm; emission wavelength: 520 ± 20 nm).

Immunolocalization of CFTR. At 24 h after CFTR gene transfer into CF-KM4 cells using -glycosylated polylysine, cells were fixed for 20 min in 3% paraformaldehyde (pH 7.4) at room temperature, air-dried, and stored at -80°C until needed. Untransfected cells were similarly fixed and stored. The mouse anti-CFTR antibody mAb24-1 (Genzyme Corp., Cambridge, MA), dilution 1:100, raised against the amino acid sequence 1377-1480 of the carboxy-terminal domain of the CFTR protein was used. Secondary antibodies, goat biotinylated antimouse immunoglobulin G fractions (Boehringer Mannheim France S.A, Meylan, France) and streptavidin-fluorescein isothiocyanate (FITC) (Amersham International, Amersham, UK), were used at 1:100 dilution. Negative controls were obtained by omitting the primary antibody or replacing it with an isotype-matched antibody. The sections were mounted in vectashield-diamidino-phenylindole (DAPI) solution (Vector, Burlingame, CA) and examined with an MRC-1024 Bio-Rad confocal system (Bio-Rad, Hercules, CA) mounted on a Diaphot 300 inverted microscope. XY and XZ serial sections collected at steps of 0.5 µm were used to define the subcellular localization of CFTR, i.e., at the cell surface or in the cytoplasm.

Membrane Lectins Expressed by Gland Serous Cells and Uptake of Glycoplexes

Fluoresceinylated neoglycoproteins. Neoglycoproteins were prepared by coupling 4-isothiocyanatophenyl-glycosides to bovine serum albumin (BSA), fluoresceinylated by using FITC (FITC isomer I; Molecular Probes, Eugene, OR) and purified as previously described (20, 21). To remove any trace of free fluorescein, fluoresceinylated neoglycoproteins (1 mg/ml in phosphate-buffered saline [PBS]) were precipitated by adding 9 vol of ethanol, pelleted by centrifugation, and then dissolved in PBS. The average number of sugar moieties per BSA molecule was 25 ± 3, and the number of fluorescein molecules per neoglycoprotein was 3 ± 1. The fluorescence intensity of 1 µg/ml of fluoresceinylated neoglycoproteins was determined as previously described (22).

Uptake of neoglycoproteins and of glycoplexes by MM-39 and CF-KM4 cells. Cells (2 × 10⁵ cells in 2-cm² well) were incubated for 2 h at 37°C in 0.5 ml DMEM/F12 and 1% Ultroser G in the presence of 50 µg/ml fluoresceinylated neoglycoproteins or in the presence of plasmid DNA (2.5 µg/ml) complexed with fluoresceinylated glycofectins. Cells were washed with PBS containing 20 mg/ml BSA, 0.5 mM MgCl₂, and 1 mM CaCl₂; trypsinized; and suspended in sheath fluid. The cell-associated fluorescence intensity was analyzed using a FACSort flow cytometer (Becton Dickinson, Grenoble, France) before and after a final incubation for 15 min at 4°C in the presence of 50 µM monensin (21, 22). Monensin, which neutralizes acidic compartments, allows the recovery of the fluorescein fluorescence that is quenched in an acidic environment; thus, a higher fluorescence intensity of the monensin-treated cells demonstrates the presence of the fluorescein-labeled neoglycoproteins or glycoplexes in endocytic/lysosomal compartments. Data were analyzed using Cell Quest software (Becton Dickinson). The amounts of neoglycoproteins bound to MM-39 and CF-KM4 cells were determined after standardization of the flow cytometer by using calibrated, fluorescein-labeled beads as previously described (22).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Glycofection of 70% Confluent Normal (MM-39) and CF (CF-KM4) Gland Serous Cells

Glycofection was efficient for both cell lines and little variation in gene transfer efficiency was observed, depending on the sugar substitution of the polylysine (Figure 1). In both cell lines, all the glycosylated polylysines tested (-Glc, -GalNAc, -Gal, -Fuc, -Lac, -GlcNAc, -Glc, -Gal, -GalNAc, and -Rha) but one (-Man) gave a higher luciferase activity than that of the unsubstituted polylysine. But it is noteworthy that the luciferase activity was always much lower (10 times) in normal MM-39 cells than that observed in CF-KM4 cells.

View larger version (54K): [in this window] [in a new window]   Figure 1.   Gene transfer with glycosylated polylysines into (A) MM-39 and (B) CF-KM4 cells. At 24 h after seeding, cells were incubated for 4 h in the presence of glycosylated polylysines complexed with pCMVLuc (5 µg), 100 µM chloroquine, and 1% Ultroser G. After a subsequent culture in DMEM/F12 for 48 h, the cells were lysed, the luciferase activity was measured by chemiluminescence in a luminometer, and the proteins were determined using the BCA method. The number of RLU measured for 4 s are related to 1 mg protein. (None: complex obtained with unsubstituted polylysine; pCMVLuc: plasmid alone; results are means ± standard error of the mean.)

In contrast with the results obtained with glycosylated polylysines, the luciferase activity varied in both cell lines with the sugar substitution of the partially gluconoylated polylysine (Figure 2). In both cell lines, the highest luciferase activity was obtained with glycoplexes made of -GlcNAc-substituted gluconoylated polylysine as well as those made of -Lac- and -Fuc-substituted polylysine. The level of gene expression obtained in cells transfected with sugar-substituted gluconoylated polylysines was similar to that obtained with sugar-substituted nongluconoylated polylysines. As has been observed for glycosylated polylysines, the luciferase activity induced by glycosylated and gluconoylated polylysines was always 10 times lower in MM-39 cells than that observed in CF-KM4 cells.

View larger version (44K): [in this window] [in a new window]   Figure 2.   Gene transfer with partially glycosylated and gluconoylated polylysine into (A) MM-39 cells and (B) CF-KM4 cells. At 24 h after seeding, cells were incubated for 4 h in the presence of sugar-substituted partially gluconoylated polylysines complexed with pCMVLuc (5 µg), 100 µM chloroquine, and 1% Ultroser G. After a subsequent culture in DMEM/F12 for 48 h, the cells were lysed and the luciferase activity and the proteins were assayed as described in Figure 1 caption.

Gene Transfer with PEI into 70% Confluent MM-39 and CF-KM4 Cells

With both cell lines, PEI 25 kD and PEI 800 kD were quite efficient vectors for gene transfer. The most efficient gene transfer with PEI 25 kD used as a vector was obtained with the theoretical positive/negative charge ratios of 1.5 and 3 (Figure 3). The most efficient gene transfer with PEI 800 kD used as a vector was obtained with a theoretical positive/negative charge ratio of 1.5. For both PEI, the increase of the charge ratios up to 12 (data not shown) did not induce an increase in luciferase activity. With both cell lines, under optimal conditions, the gene transfer efficiency with PEI 25 kD was 4 times higher than with PEI 800 kD and close to that observed with glycofectins (Figures 1-3).

View larger version (34K): [in this window] [in a new window]   Figure 3.   Gene transfer with PEI 25 and 800 kD into (A) MM-39 and (B) CF-KM4 cells. At 24 h after seeding, cells were incubated for 4 h in the presence of PEI 25 or 800 kD complexed with pCMVLuc (5 µg) using the indicated charge ratios (+/) and 1% Ultroser G. After a subsequent culture in DMEM/F12 for 48 h, the cells were lysed and the luciferase activity and the proteins were assayed as described in Figure 1 caption.

Lipofection into 70% Confluent MM-39 and CF-KM4 Cells

With both cell lines, lipofectin and lipofectAMINE were quite efficient vectors for gene transfer. The most efficient gene transfer with lipofectin used as a vector was obtained with a theoretical positive/negative charge ratio of 1. The most efficient gene transfer with lipofectAMINE used as a vector was obtained when the positive/negative charge ratio was 5 or 10 in the case of the CF-KM4 cells and 10 in the case of the MM-39 cells (Figure 4). For both lipofectin and lipofectAMINE, increasing the charge ratios did not induce an increase in luciferase activity (data not shown). For both cell lines, gene transfer efficiency with lipofectAMINE was at least 4 times higher than the gene transfer efficiency observed with lipofectin and similar to that observed with glycofectins or PEI (Figures 1-4).

View larger version (38K): [in this window] [in a new window]   Figure 4.   Gene transfer with cationic lipids into (A) MM-39 and (B) CF-KM4 cells. Twenty-four hours after seeding, cells were incubated for 4 h in the presence of lipofectin or lipofectAMINE complexed with pCMVLuc (5 µg) using the indicated charge ratios (+/) and 1% Ultroser G. After a subsequent culture in DMEM/F12 for 48 h, the cells were lysed and the luciferase activity and the proteins were assayed as described in Figure 1 caption.

Effect of Confluency on Gene Transfer Efficiency

Gene transfer with glycofectins, PEI 25 and 800 kD, lipofectin, and lipofectAMINE was performed on CF-KM4 cells that had been cultivated 1 wk after seeding in order to study the efficiency of gene transfer on highly confluent gland serous cells. At that stage, cells stop growing and are more differentiated than growing cells.

For all vectors, the efficiency of gene transfer was reduced about 10 times in highly confluent cells as compared with 70% confluent cells (Figure 5). However, with all the vectors tested, the pattern of transfection efficiency was close to that observed in 70% confluent cells: most glycofectins were efficient (data not shown) and the most efficient gene transfer using PEI 25 and 800 kD, lipofectin, and lipofectAMINE as vectors was obtained with the same optimal theoretical charge ratios determined for 70% confluent cells. Similarly, PEI 25 kD and lipofectAMINE were more efficient than PEI 800 kD and lipofectin.

View larger version (53K): [in this window] [in a new window]   Figure 5.   Gene transfer into confluent CF-KM4 cells. At 1 wk after seeding, cells were incubated for 4 h in the presence of -D-Glc-substituted polylysine, PEI 25 kD, PEI 800 kD, lipofectin, or lipofectAMINE complexed with pCMVLuc (5 µg) using the indicated charge ratios (+/) and 1% Ultroser G. After a subsequent culture in DMEM/F12 for 48 h, the cells were lysed and the luciferase activity and the proteins were assayed as described in Figure 1 caption.

Optimized Conditions for Glycofection into 70% Confluent CF-KM4 Cells Using -Glc-Substituted Polylysine As a Vector

A high luciferase activity was detected as early as 4 h after the end of a 4-h incubation of the cells in the presence of glycoplexes, was maximal 24 h after the end of the glycofection step, and lasted up to 72 h (Figure 6A). A low luciferase activity was still detected 6 d after the glycofection step. With MM-39 cells, the time course of the luciferase expression was quite similar, the level being, however, always 5 to 10 times lower than in the case of CF-KM4 cells (data not shown).

View larger version (51K): [in this window] [in a new window]   Figure 6.   Gene transfer with -D-Glc substituted polylysine into CF-KM4 cells. (A) Duration of gene expression and (B) duration of glycofection. At 24 h after seeding, cells were incubated in the presence of -Glc-substituted polylysine complexed with pCMVLuc (5 µg), 100 µM chloroquine, and 1% Ultroser G. (A) After 4   h of transfection, CF-KM4 cells were cultured in DMEM/F12 and at the indicated times, the cells were lysed. (B) The duration of the glycofection was varied as indicated. Cells were then incubated in DMEM/F12 for 48 h. The cells were lysed and the luciferase activity ( filled bars) was assayed as described in the caption to Figure 1. Protein contents (linked filled squares) were determined with the BCA colorimetric method.

The duration of glycofection into CF-KM4 cells in the presence of 5 µg of plasmid and 100 µM of chloroquine led to a high luciferase activity after a 4-h glycofection step and to a maximal activity after an 8-h glycofection step (Figure 6B). However, after 24 h of incubation, the protein content of the well dramatically decreased. This reflects a decrease in the total number of the cells per well that might be related to the duration of incubation in the presence of 100 µM chloroquine. When the effect of the duration of glycofection was studied in MM-39 cells, a similar pattern of efficiency was observed, the luciferase activity being as usual 5 to 10 times lower at any time (data not shown).

Effect of Repeated Glycofections into 70% Confluent CF-KM4 Cells on Gene Transfer Efficiency

The effect of repeated transfections of 4 h in the presence of 5 µg of plasmid complexed to -Glc-substituted polylysine was studied. One, two, or three transfections of 4 h were applied daily on 70% confluent CF-KM4 cells on 3 consecutive days and the number of transfected cells was evaluated on the fourth day. Luciferase activity increased with the number of transfections applied (Figure 7A). The number of transfected cells was determined by using a plasmid encoding the gene for the green fluorescent protein to transfect the cells and a flow cytometer to count the labeled cells. After a single transfection of 4 h the first day, 6% of the cells were labeled. After two daily transfections of 4 h, 15% of the cells were labeled. Finally, after three daily transfections of 4 h, 20% of the cells were labeled (Figure 7B).

View larger version (38K): [in this window] [in a new window]   Figure 7.   Influence of repeated glycofections and evaluation of the number of CF-KM4 transfected cells. At 24 h after seeding, cells were incubated in the presence -Glc-substituted polylysine complexed with (A) pCMVLuc (5 µg) or (B) pCMVGFP (5 µg), chloroquine (100 µM), and 1% Ultroser G. Incubations of 4 h with CF-KM4 cells were applied daily as indicated (D1: a 4-h glycofection on the first day; D1+D2: a 4-h glycofection on the first and the second days; D1+D2+D3: a 4-h glycofection on the first, second, and third days). Cells were subsequently cultured in DMEM/F12. (A) Luciferase activity ( filled bars) and proteins (linked filled squares) and (B) the percentage of fluorescent cells by flow cytometry were determined on the fourth day.

Immunolocalization of CFTR in CF-KM4 Cells

In untransfected CF-KM4 cells, no CFTR labeling was observed or a very light labeling exclusively localized to the cytoplasm was identified (Figure 8A). At 24 h after CFTR gene transfer into CF-KM4 cells using -glycosylated polylysine, an intense fluorescent signal was observed. The sublocalization of the transgenic CFTR was analyzed by laser scanning confocal microscopy and the plasma membrane was identified as being the predominant localization of CFTR expression (Figure 8B). A diffuse cytoplasmic CFTR staining was sometimes associated.

View larger version (27K): [in this window] [in a new window]   Figure 8.   Immunolocalization of CFTR in (A) untransfected or (B) transfected CF-KM4 cells. An indirect immunofluorescence with antibody mAb 24-1 was performed (A) on untransfected cells or (B) 24 h after CFTR gene transfer into CF-KM4 cells using -glycosylated polylysine. The sections were counterstained with DAPI and analyzed under a confocal microscope. An intense labeling of the plasma membrane (M) is present in transfected cells. Bar: 10 µm.

Membrane Lectins Expressed by Gland Serous Cells and Uptake of Glycoplexes

To determine the sugar specificity of putative membrane lectins mediating endocytosis of their ligands, CF-KM4 and MM-39 cells were incubated in the presence of various fluorescein-labeled neoglycoproteins. After a 2-h incubation, CF-KM4 cells were strongly labeled with BSA bearing -Man, less with BSA bearing -Lac, -Rha, -6-phospho-Man (Man-6P), -Glc, -Glc, and -Gal residues and weakly with BSA bearing various other sugar residues (Figure 9A). The normal MM-39 cells were also strongly labeled with BSA bearing -Man residues; less with BSA bearing -Rha, -Man-6P, -Glc, and -Lac residues; and weakly with BSA bearing various other sugar residues (Figure 9A). Because receptor-mediated endocytosis conveys the ligand into acidic vesicles in which the fluorescein fluorescence is quenched, a postlabeling incubation with the proton/sodium ionophore monensin was performed to neutralize the acidic compartments and restore the fluorescence of internalized fluorescein-labeled neoglycoproteins. The fluorescence intensity of CF-KM4 and MM-39 cells incubated with any neoglycoprotein tested increased 1.5 times after incubation with monensin (Figure 9A), indicating that fluorescein-labeled neoglycoproteins were internalized in acidic compartments of both CF-KM4 and MM-39 cells.

View larger version (33K): [in this window] [in a new window]   Figure 9.   Flow cytometric analysis of the uptake of (A) neoglycoproteins and (B) glycoplexes by CF-KM4 and MM-39 cells. At 24 h after seeding, CF-KM4 and MM-39 cells were incubated for 2 h in the presence of (A) 50 µg/ml fluoresceinylated neoglycoproteins and (B) 5 µg/ml pCMVLuc complexed with fluoresceinylated glycosylated polylysines. The fluorescence intensity of cells was measured before (open bars) and after ( filled bars) a postlabeling incubation at 4°C in the presence of 50 µM monensin. To take into account the specific fluorescence intensity of each neoglycoprotein, the cell fluorescence intensity was corrected with the IBSA/Ineo ratio, where IBSA and Ineo represent the fluorescence intensity of 1 µg/ml fluorescein-labeled BSA and of a given fluorescein-labeled neoglycoprotein, respectively. To take into account the specific fluorescence intensity of each glycoplex, the cell fluorescence intensities were corrected with the Icomp/Iglycoplex ratio, where Icomp and Iglycoplex represent the fluorescence intensity of 1 µg/ml of fluorescein-labeled polylysine/ DNA complexes and of a given fluorescein-labeled glycoplex, respectively.

Knowing that the endocytosis mediated by lectins may depend on the size of the glycoconjugate, we used the same approach with glycoplexes instead of neoglycoproteins. CF-KM4 cells were incubated in the presence of fluorescein-labeled glycoplexes for 2 h. The fluorescence intensity of CF-KM4 cells incubated with several glycoplexes was much higher than the fluorescence intensity of cells incubated with polyplexes made with unsubstituted polylysine (Figure 9B). In the presence or absence of monensin, the highest fluorescence intensity was observed when CF-KM4 and MM-39 cells were incubated with glycoplexes made of polylysine substituted with -Man and to a lower extent when cells were incubated with glycoplexes made of polylysine substituted with -GalNAc, -Gal, and -Lac. Glycoplexes bearing -Fuc and -GalNAc led to a very low labeling. After the postincubation with monensin, the fluorescence intensity of CF-KM4 and MM-39 cells incubated with these glycoplexes increased 2.5 times, indicating that glycoplexes were sequestered in compartments more acidic than those in which the neoglycoproteins were localized (Figure 9B).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We have shown that an efficient gene transfer can be achieved in human airway gland serous cells using various nonviral vectors. To our knowledge, this is the first report on gene transfer into airway gland serous cells using synthetic vectors. Gene transfer into airway gland serous cells may be of importance in CF because several lines of evidence suggest that these cells contribute to the primary pathogenesis of lung disease in CF: high levels of CFTR mRNA and protein were indeed demonstrated in human airway gland serous cells (4). Moreover, accumulation of airway secretions with abnormal viscoelastic properties is a common feature in CF lung disease and submucosal glands are thought to be the primary source of mucous secretion (23). Tracheal submucosal gland cultures from patients with CF also appear to have altered ion transport properties (24, 25) and abnormalities in their protein secretion (26). Finally, postmortem analysis of tissue from patients with CF who die in the neonatal period have shown that the only abnormalities were localized in the submucosal glands that demonstrated glandular hyperplasia and ductular obstruction (27).

In both the normal and CF cell lines, gene transfer was efficient with all the vectors tested. However, gene transfer efficiency was 10-fold higher in the CF cells than in the normal cells. We have already reported such differences in airway surface epithelial cells (11). Inasmuch as difference in gene transfer efficiency was observed with all the vectors tested, this difference might be unrelated to the vector and due to the difference in the CFTR protein expressed by MM-39 and CF-KM4 cells (6, 7). It has been suggested that CFTR played a role in acidification of intracellular organelles (28) and in the regulation of endosome fusion (29) and of plasma membrane recycling (30). All of these characteristics are likely to influence the intracellular trafficking of the complexes. Therefore, according to the type of CFTR protein expressed by the cells, a common step in the intracellular trafficking of synthetic vectors complexed to DNA might be modified and result in a difference of gene transfer efficiency.

We have tested the efficiency of gene transfer into gland serous cells using various nonviral vectors: glycofectins that form with plasmid complexes taken up by cells through a receptor-mediated mechanism via membrane lectins (8); PEI, a cationic polymer (12); and lipofectin and lipofectAMINE, cationic lipids that induce a nonspecific endocytosis of the complexes and spontaneously disrupt the endosomal membrane. We have shown that all these vectors were efficient in transfecting normal and CF gland serous cells and the efficiency was close to the one we have previously described for normal and CF airway surface epithelial cells (11). However, a receptor-mediated gene delivery is likely to be the most efficient way for uptaking plasmid-containing complexes in vivo. Indeed, such complexes are roughly neutral and therefore avoid nonspecific binding to mucus proteins (31). In many cases the receptor-directed ligand is a proteinaceous material; unfortunately, this may lead to immunogenic particles, and further, the preparation of reproducible and stable particles is difficult. In the present study we used simple sugar residues to target lectins present at the membrane surface of airway gland serous cells. As we have previously described for airway surface epithelial cells (11), mannosylated neoglycoproteins and mannosylated glycoplexes were very efficiently taken up. However, mannosylated glycoplexes were dramatically inefficient in gene transfer; glycosylated, lactosylated, or N-acetyl-glucosaminylated glycoplexes that were moderately taken up were found to be the most efficient devices in gene transfer. This highlights the finding that gene transfer efficiency is determined partly by the uptake of the complexes and also by their subsequent intracellular trafficking. In addition, our results show that polylysines bearing the same sugar residues could mediate an efficient gene transfer into both airway surface epithelial cells (11) and airway gland serous cells (this report). This might be of interest for future in vivo applications if, together with airway surface epithelial cells, airway gland serous cells are important targets for CF gene therapy.

Although a high transfection efficiency was achieved in 70% confluent cells, transfection efficiency was quite low in highly confluent gland serous cells. This decrease in gene transfer efficiency was observed for all the vectors tested and is likely to be related to the status of the cells. The level of endocytosis or a common step of the intracellular trafficking of the complexes might be impaired in nondividing and differentiated cells. In the case of cationic-lipid gene transfer to airway surface epithelial cells, such a decreased gene transfer efficiency in nondividing and polarized cells has been attributed in part to a low cellular binding and uptake of the complexes (32). Moreover, the breakdown of the nuclear membrane that occurs during mitosis is thought to facilitate the nuclear translocation of the plasmid DNA from the cytoplasm. Therefore, the decrease in gene transfer efficiency in a differentiated epithelium may also be due to its low mitotic activity (33). However, epithelial cell proliferation rates are elevated in highly inflamed CF lungs (34), suggesting that perhaps CF airways may be more amenable than normal airways to transfection.

We have shown in the present study that in vitro gene transfer into human airway gland serous cells is successfully achieved by using the vectors described to efficiently transfect human airway surface epithelial cells. If, together with airway surface epithelial cells, airway gland serous cells are a necessary target for gene therapy in CF, the possibility of using the same vector efficiently for both cell types may make the task a little easier. However, it does not withdraw the need to improve the vectors for efficient gene transfer in vivo, the low accessibility of gland serous cells in vivo, and our ignorance about the number of CF gland serous cells that need a correction of the chloride ion transport defect.