Liposome-mediated transfection of fetal lung epithelial cells:
DNA degradation and enhanced superoxide toxicity
A. Keith
Tanswell,
Olivier
Staub,
Richard
Iles,
Rosetta
Belcastro,
Judy
Cabacungan,
Larisa
Sedlackova,
Brent
Steer,
Yanxia
Wen,
Jim
Hu, and
Hugh
O'Brodovich
The Medical Research Council Group in Lung Development and Lung
Biology Programme, Hospital for Sick Children Research Institute, and
Divisions of Neonatology and Respiratory Medicine, Department of
Paediatrics, University of Toronto, Toronto, Ontario, Canada M5G 1X8
 |
ABSTRACT |
Cationic liposomes, 1:1 (mol/mol)
1,2-dioleoyldimethylammonium
chloride-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine,
were used to transfect primary cultures of distal rat fetal lung
epithelial cells with pCMV4-based plasmids. A DNA-to-lipid ratio of
1:10 to 1:15 (wt/wt) optimized DNA uptake over a 24-h exposure. At a
fixed DNA-to-lipid ratio of 1:15, chloramphenicol acetyltransferase (CAT) reporter gene expression declined at lipid concentrations > 2.5 nmol/cm2 cell surface area,
whereas DNA uptake remained concentration dependent. CAT expression
peaked 48 h after removal of the liposome-DNA complex, declining
thereafter. Reporter gene expression was increased, and supercoiled
cDNA degradation was reduced by the addition of 0.2 mM nicotinamide and
10 µM chloroquine. Rat fetal lung epithelial cells
transfected with two different expression cassettes had an increased
susceptibility to superoxide-mediated cytotoxicity. This could be
attributed to a nonspecific delivery of exogenous DNA or some other
copurified factor. The DNA-dependent increase in superoxide-mediated
cytotoxicity, but not basal levels of cytotoxicity, was inhibited by
the addition of 0.2 mM nicotinamide and 10 µM chloroquine.
deoxyribonucleic acid; gene transfer; cytotoxicity; reactive oxygen
species
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INTRODUCTION |
THE LUNGS OF PREMATURE INFANTS are at particular risk
from pulmonary O2 toxicity. Not
only may their lung tissue be subjected to elevated
O2 for prolonged periods, which
will result in an increased production of superoxide and other
partially reduced oxygen species, but the immature lung also has
deficient antioxidant enzyme activities at the time of birth (30).
Tanswell and colleagues previously showed, both in vivo
(29) and in vitro (31), that treatment of fetal or neonatal rat lung
cells with antioxidant enzymes is protective against
O2-mediated cytotoxicity. Such
protein therapy is a useful experimental tool, but there are
significant practical limitations to this approach as a therapeutic
intervention (30). Theoretically, therefore, this population may
eventually become an appropriate candidate for the use of transient
antioxidant enzyme gene transfer administered via the airway route. The
feasibility of this approach has been demonstrated in adult animals
transfected with the human
1-antitrypsin gene (6), whereas
successful liposome-mediated transfection of the nasal epithelium of
patients suffering from cystic fibrosis has been recently reported (7). As the first step toward studies using liposome-mediated delivery of
antioxidant enzyme gene constructs in vivo, we have used reporter gene
constructs to establish an in vitro model system for the transfection
of primary epithelial cell cultures from the preterm lung.
Transfection of established cell lines with cationic liposomes is a
relatively simple and standard laboratory procedure. Primary cell
cultures have, however, been more difficult to successfully transfect
with standardized protocols. As stated above, the initial objective of
the experiments reported herein was simply to establish an in vitro
model system for the transfection of primary epithelial cell cultures
from the preterm lung. However, during the course of these studies, we
made several observations that may be of relevance to the design of in
vitro and in vivo gene transfer experiments. First, the degree of
reporter gene expression does not have a simple concentration-dependent
relationship with the amount of plasmid DNA delivered in that
efficiency declines with excess delivery. Second, effective
intracellular DNA delivery was much less of a limitation to successful
gene transfer than DNA damage occurring after cell uptake. Last,
delivery of exogenous DNA can render a cell more susceptible to
superoxide-mediated cytotoxicity by an as yet undefined mechanism.
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METHODS |
Materials.
35S-dATP was from ICN Biomedicals
Canada (Montreal, Quebec).
1,[2-14C]dioleoyl-sn-glycero-3-phosphoethanolamine
([14C]DOPE) was from
Amersham Canada (Oakville, Ontario).
[8-14C]adenine was
from NEN (Boston, MA). Proteinase K and nick translation kits were from
Promega (Madison, WI). Sephadex G-50 and other chemicals were from
Sigma (St. Louis, MO). Porcine trypsin, heat-inactivated fetal bovine
serum (FBS), gentamicin, amphotericin B, topoisomerase I, DMEM, and
2,3-dioleoyloxyl-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate were from GIBCO
Canada (Burlington, Ontario). Collagenase and type I DNase (2,367 U/mg)
were from Worthington (Freehold, NJ). Restriction enzymes were from
Pharmacia (Baie d'Urfé, Quebec). 1,2-Dioleoyldimethylammonium
chloride (DODAC), 1,2-dioleoyl-3-N,N,N-trimethylaminopropane,
and
3
-[N-(N',-N'-dimethylaminoethane)-carbamoyl]-cholesterol hydrochloride were from Inex Pharmaceuticals (Vancouver, British Columbia).
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine
(DOPE), and fluorescein-labeled DOPE were from Avanti Polar Lipids
(Alabaster, AL). A 10-ml thermobarrel liposome extruder was from Lipex
Biomembranes (Vancouver, British Columbia), and extrusion filters were
from Nucleopore (Pleasanton, CA). The pCMV4-chloramphenicol
acetyltransferase (CAT) construct was a generous gift from Drs. K. Brigham and J. Conary (Vanderbilt University School of Medicine,
Nashville, TN). The pCMV4-secreted alkaline phosphatase (SEAP)
construct was purchased from Tropix (Bedford, MA).
Cell culture. Primary cultures of
19-day gestation rat distal fetal lung epithelial cells
(RFL19Ep) were prepared as
previously described (9, 16, 31). Briefly, the lungs of 19-day
gestation fetal rats were isolated and separated from major vessels and airways. The lungs were minced and gently vortexed to remove
erythrocytes. This step was repeated until the supernatant was clear.
The minced lung tissue was then subjected to proteolytic digestion with
trypsin and DNase, the process being arrested by the addition of FBS. The resultant cell suspension was eluted through 100-µm mesh nylon bolting cloth. The eluted cells were next subjected to a collagenase digestion, which was also arrested by the addition of FBS, and the
fibroblasts were removed by differential adherence. Epithelial cells of
>95% purity, as assessed by staining for cell cytoskeleton components, are obtained with this technique (16). For the purposes of
these experiments, cells were seeded in DMEM with 10% (vol/vol) FBS at
a sufficient cell density to allow near confluence 24-48 h after
seeding. Cells were maintained in a humidified gas mixture of 3%
O2-5%
CO2-92%
N2 to maintain the cells at a
normal fetal arterial oxygen tension of
20 mmHg. The culture medium
normally contained 50 µg/ml of gentamicin and 2.5 µg/ml of
amphotericin B, but these were omitted from the medium when cells were
exposed to the liposome-DNA complexes.
Cationic liposome preparation. An
equimolar stock solution of DODAC-DOPE in chloroform was dried down
under a stream of N2 until the
chloroform had completely evaporated. The dried lipids were then
maintained under vacuum for 24-48 h. The resulting lipid film was
rehydrated at 1 µg total lipid/µl PBS and sonicated for 2 × 30 s. For calculations of lipid recovery,
[14C]DOPE was included
in the initial lipid mixture. Liposomes were initially extruded through
sequentially smaller gauge syringe needles from 18- to 27-Fr and were
then subjected to five freeze-thaw cycles with liquid
N2 and extrusion through a 400-nm
filter (20). Liposomes were recovered by centrifugation at 165,000 g for 1 h. Plasmid DNA was added at
selected DNA-to-lipid ratios, and the mixture was vortexed gently, then
allowed to stand at room temperature for 20 min before use. This
preparation was diluted with culture medium as necessary.
Cytotoxicity index. Cytotoxicity was
assessed by the release of
[8-14C]adenine from
cells preincubated with 0.2 µCi/ml of
[8-14C]adenine (23).
As reported elsewhere, this assay has an ability to detect cell injury
equivalent to measurements of lactate dehydrogenase release (31) or
trypan blue exclusion (9). Exposures to liposomes ± DNA for various
intervals preceded the addition of
[8-14C]adenine, before
which the medium containing the liposomes or the liposome-free medium
for control conditions was removed and the cell monolayer was washed.
The medium containing
[8-14C]adenine was
added for 2 h before the monolayer was washed two times with fresh
medium. The percentage of preincorporated
[8-14C]adenine
released into the culture medium was assessed after 48 h and is
expressed relative to the basal release by cells not exposed to
liposomes, which is given an arbitrary value of 1.
DNA uptake. pCMV4-CAT plasmids were
nick translated with 35S-dATP, and
unincorporated nucleotides were removed by passing the reaction mixture
through a small column of Sephadex G-50. For uptake experiments,
35S-plasmid DNA was mixed with
unlabeled plasmid DNA to give a final specific activity of
6.5 × 106 dpm/µg DNA.
Integrity of the labeled DNA was confirmed by electrophoresis on 1%
(wt/vol) agarose gels with 0.2 µg/ml of ethidium bromide. For studies
of liposome-associated plasmid uptake, the cells were harvested at the
end of the incubation period by treatment with 0.1% (wt/vol)
trypsin and 0.001% (wt/vol) DNase and centrifugation. Trypsin
removes cell surface-associated liposomes (5), and DNase was included
to degrade any surface-associated DNA not associated with liposomes.
Total DNA uptake was calculated from the
35S content of the cell pellet.
For studies using fluorescence microscopy, plasmid DNA was
fluorescently labeled with ethidium monoazide (32).
DNA degradation. After a 24-h exposure
to DNA with cationic liposomes, the cells were washed two times with
culture medium to remove any free liposome-DNA complexes. Cells were
harvested immediately after being washed or after a further 24, 48, 72, or 120 h in culture with treatment with 0.1% (wt/vol) trypsin and 0.001% (wt/vol) DNase and centrifugation to remove cell
surface-associated liposomes and DNA (5). After resuspension in PBS, a
DNA extract was prepared from the cells with proteinase K (12) and was
subjected to electrophoresis on 1% (wt/vol) agarose gels with
0.2 µg/ml of ethidium bromide. Blots were probed with probes specific
to the CAT nucleotide sequence. In other studies, separation of certain bands by electrophoresis was enhanced by using 0.8% (wt/vol)
agarose gels without ethidium bromide.
Plasmid conformation. To relax
supercoiled pCMV4-CAT, 0.5 µg of the plasmid prepared with a cesium
chloride gradient was mixed in reaction buffer (50 mM
Tris · HCl, 50 mM KCl, 10 mM
MgCl2, 0.5 mM dithiothreitol, and
0.1 mM EDTA, pH 7.5) with 1 U of topoisomerase I in a final volume of
50 µl for 30 min at 37°C. The plasmid was linearized with 100 U
of the restriction enzyme Sma I with
100 µg of pCMV4-CAT in 100 µl of buffer (100 mM Tris acetate, 100 mM magnesium acetate, and 500 mM potassium acetate, pH 7.5) at 30°C
overnight. After extraction, the plasmids were subjected to
electrophoresis in 0.8% (wt/vol) agarose gels. For conformation studies, cell DNA extracts were similarly treated.
Reporter gene activity. The bacterial
CAT gene, which is not present in eukaryotic cells, was used as a
reporter gene to measure transgene expression in studies designed to
optimize the liposome delivery system. The gene product is an enzyme
that catalyzes the transfer of acetyl groups to the substrate
chloramphenicol from acetyl 1-CoA. Catalytic activity was determined by
a radiometric assay as described by Stribling et al. (28).
Superoxide cytotoxicity. Superoxide
was generated extracellularly by the addition of hypoxanthine and
xanthine oxidase essentially as described by Andreoli (2), except that
the release of preincorporated [14C]adenine was used
as a marker of cytotoxicity.
Poly(ADP-ribose) polymerase activity.
The activity of poly(ADP-ribose) polymerase was determined by the
measurement of acid-precipitable radioactivity after incubation of cell
lysates with [3H]NAD
as previously described (9).
Statistical analysis. All values are
means ± SE of each group. Where error bars are not evident, they
fall within the plot point. Where figures show data from representative
experiments, these experiments have been replicated on two to four
occasions. Statistical significance (P < 0.05) was generally determined by ANOVA for repeated measures
followed by assessment of differences with Duncan's multiple range
test, although paired t-tests were used for some experiments (24).
 |
RESULTS |
As the initial step in optimizing the transfection protocol for primary
cultures of RFL19Ep cells, we
assessed the cytotoxicity of the cationic liposome preparation (1:1
mol/mol DODAC-DOPE) to be used in our studies. As shown in Fig.
1A, the
cationic liposomes were cytotoxic (significantly increased release of
[8-14C]adenine) at
lipid concentrations
50 nmol/cm2 after a 2-h exposure.
There was a small reduction in the cytotoxicity index at some of the
lesser lipid concentrations used, but this did not achieve
significance. Based on this finding and the desire to be well outside
the cytotoxic range of these lipids, we elected to use DODAC-DOPE at
5 nmol/cm2 in subsequent
experiments. As expected from the reported experience of others
(14a), the presence of serum in the culture medium caused a modest, although significant (P < 0.05), reduction in the uptake of
35S-DNA (data not shown), and
subsequent transfection experiments utilized serum-free medium. We next
examined the effect of DNA-to-lipid ratio on DNA uptake by
RFL19Ep cells, using a standard
concentration of 35S-DNA (0.1 µg/cm2) and varying the lipid
concentrations. As shown in Fig. 1B,
the optimal DNA-to-lipid ratio for DNA uptake by
RFL19Ep cells over a 6-h exposure
period under serum-free conditions was 1:10-15 (mg/mg). Over a
concentration range that bracketed the lipid concentrations selected
for study and with a fixed DNA-to-lipid ratio of 1:15 (mg/mg),
35S-DNA uptake over 6 h increased
in proportion to the lipid concentration applied to the cells (Fig.
1C). Optimal DNA uptake with
DODAC-DOPE at 5 nmol/cm2 and a
DNA-to-lipid ratio of 1:15 (mg/mg) was seen after a 24-h exposure (Fig.
1D). After a 24-h exposure to the
DNA-liposome complex, DNA that had been photolabeled with ethidium
monoazide was evident in the majority of cells (data not shown).
DODAC-DOPE liposomes, prepared with fluorescein-labeled DOPE, were
visualized in all cells after a 24-h exposure (data not shown).

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Fig. 1.
Subconfluent and serum-free primary cultures of
RFL19Ep cells were used for all
studies with cationic 1,2-dioleoyldimethylammonium chloride
(DODAC)-1,dioleoyl-sn-glycero-3-phosphoethanolamine
(DOPE; 1:1) liposomes. A: cells
exposed to liposomes (0-100
nmol/cm2) for 2 h. Cytotoxicity
was observed at liposome concentrations 50 nmol/cm2. Values are means ± SE; n = 3-5 cell
preparations. * P < 0.05 compared with liposome-free control value.
B: cells exposed to a fixed quantity
of 35S-DNA (0.1 µg/cm2) with increasing
concentrations of cationic liposome lipid for 6 h. Optimal DNA uptake
was seen at DNA-to-lipid ratios of 1:10-15 (mg/mg). Values are
means ± SE; n = 4 cell
preparations. * P < 0.05 compared with all other group values.
C: cells exposed to
35S-DNA with cationic liposomes at
a DNA-to-lipid ratio of 1:15 for 6 h. DNA uptake was linearly related
to liposome concentration
(r2 = 0.993).
Values are means ± SE; n = 4 cell preparations. D: cells
exposed to a fixed quantity of
35S-labeled DNA with cationic
liposomes at a DNA-to-lipid ratio of 1:15 for up to 48 h. Maximal
uptake was seen at 24 h. Values are means ± SE;
n = 3 cell preparations.
* P < 0.05 compared with
values seen before 24 h.
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CAT expression after a 24-h exposure to the liposome-DNA complex was
maximal 72 h after the start of the transfection period and 48 h after
the removal of the liposome-DNA complex, with a significant decline
thereafter (Fig.
2A). As
shown in Fig. 2B, the cationic
liposomes and plasmid were not cytotoxic at the concentrations used in
these experiments. With this more extended exposure protocol and lipid
component concentrations of 1-7.5
nmol/cm2, cationic liposome-DNA
complexes occasionally reduced the basal level of the cytotoxicity
index to below that seen under control conditions, as shown in this
particular experiment. Having defined an exposure protocol with
DODAC-DOPE that could reliably transfect primary
RFL19Ep cells, we wished to ensure
that other commercially available cationic liposome preparations would
not allow even better transfections with the same exposure protocol.
Under the conditions optimized for transfection with DODAC-DOPE, no
enhanced CAT activity was seen with any of the commercially available
liposome preparations tested (Table 1), and
DODAC-DOPE was retained for use in subsequent studies.

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Fig. 2.
A: transfection of subconfluent and
serum-free primary cultures of
RFL19Ep cells with
pCMV4-chloramphenicol acetyltransferase (CAT). Cells were exposed to 5 nmol/cm2 of liposomes at a
DNA-to-lipid ratio of 1:15 for 24 h. CAT activity, corrected for cell
protein and then expressed as percent maximum activity, peaked 72 h
after addition of liposomes and declined thereafter. Values are means ± SE; n = 4 cell preparations.
* P < 0.05 compared with
maximum activity group. B: this
decline in intensity could not be attributed to cytotoxicity of
cationic liposome-DNA complex. With a fixed DNA-to-lipid ratio of 1:15
and 0-20 nmol/cm2 of lipid
for a 24-h exposure, cytotoxicity was only evident at a lipid
concentration of 20 nmol/cm2.
Values are means ± SE; n = 4 cell preparations. * P < 0.05 compared with values for liposome-free cells.
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In pursuing the relationship between DNA delivery and transgene
expression, we observed that transgene expression was only dependent on
the amount of DNA delivered, up to a phospholipid concentration of 2.5 nmol/cm2, with transgene
expression declining with further increases in DNA delivery (Fig.
3). When activity and delivery were
compared as a ratio, the reduced efficiency of transfection per
microgram of DNA was even more evident. This observation led us to
speculate that excess transfected plasmid might adversely affect
translational efficiency through an excessive activation of
intracellular DNA repair processes after DNA injury after endosomal
uptake. A number of interventions that might alter DNA degradation were
studied (Table 2), including
diphenylphenyldiamine to stabilize lysosomes (22), aurintricarboxylic
acid to inhibit DNase activity (3), nicotinamide to inhibit
poly(ADP-ribose) polymerase activity (18), and chloroquine to inhibit
endosomal acidification (15). The concentrations used were defined in a
series of preliminary concentration curves to define their cytotoxicity
and optimal effects, if any, on gene expression. Diphenylphenyldiamine
was found to have no consistent effect on reporter gene expression. A
trend toward enhanced reporter gene expression was observed with both
aurintricarboxylic acid and nicotinamide, although significance was not
achieved. Enhanced reporter gene expression with chloroquine was
significant (P < 0.05). A series of
studies was performed with various combinations of these agents to
detect the presence of additive effects (data not shown). Although a
significant additive effect was not observed, the combination of
chloroquine and nicotinamide tended to enhance reported gene expression
over that seen with chloroquine alone (Table
3), and this combination was used in
subsequent studies.

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Fig. 3.
With a standard DNA-to-lipid ratio of 1:15 (mg/mg) and a transfection
time of 24 h, uptake of
35S-plasmid DNA ( )
increased in a linear fashion over a DODAC-DOPE (1:1) concentration
range of 0-10 nmol/cm2. In
contrast, CAT activity ( ), corrected for cell protein and then
expressed as percent maximum activity, was maximal at a lipid
concentration of 2.5 nmol/cm2 and
was reduced when increased concentrations of lipid-DNA complex were
used. Values are means ± SE; n = 4 cell preparations. Plot points with similar superscripts (a or b) are
not significantly different. Relative decline in efficiency of
transfection at increased lipid-DNA complex concentrations is most
evident when corresponding values are shown as a ratio of CAT activity
to DNA delivery ( ).
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Using a CAT cDNA probe and electrophoretic separation of cell DNA
extracts on 1% agarose (wt/vol) gels with ethidium bromide (Fig.
4A),
we were able to confirm that cells treated with a combination of
chloroquine and nicotinamide had an overall reduction in degradation of
delivered DNA. After transfection, cells had three
high-molecular-weight bands labeled by the CAT cDNA probe seen at the
48- and 72-h time points. The cellular DNA band that comigrated with
linearized plasmid declined in intensity with time after transfection,
and there was no obvious effect of interventions on this band.
Interventions did have an effect on the band that comigrated with
relaxed plasmid. This band represented 6 ± 0.2% (SE) of the total
band intensity in untreated samples compared with 13 ± 9% in
treated samples (n = 3) at 48 h and 9 ± 3 vs. 42 ± 11% at 72 h. The band that comigrated with
supercoiled plasmid was only clearly distinguishable in treated samples
at the 48- and 72-h time points, being 2 ± 1% of the total at 48 h
and 6 ± 2% at 72 h. These values are derived from vertical scans
of each lane, and because the amount of linearized DNA may vary from
lane to lane, direct horizontal comparisons cannot be made. Preparation
of liposome-DNA complexes had no effect on the proportion of DNA in the
supercoiled configuration (Fig. 4B),
but the extraction procedure used to isolate cell DNA, when applied to
plasmid alone, did convert a portion (
10-12%) of the supercoiled DNA to a relaxed DNA configuration as reported by others
(10). Enhanced separation of supercoiled from linearized DNA was
achieved by using 0.8% (wt/vol) gels without ethidium bromide
for electrophoretic separation, although separation of relaxed from
linearized DNA became less clear (Fig.
5A).
Using this approach, we were able to show that the band that comigrated with supercoiled plasmid was present in DNA extracts of cells treated
with nicotinamide and chloroquine at the 24-h time point (Fig.
5B). That the lower band, assumed to
be supercoiled cDNA, was indeed supercoiled was confirmed by
demonstrating its sensitivity to topoisomerase (Fig.
5B). Treatment with topoisomerase
reduced the amount of DNA in the supercoiled configuration from 6% of the total, as assessed by densitometry, to <2% of the total in the
example shown. Contrary to our expectations, liposome-DNA complexes
containing largely supercoiled cDNA or cDNA that was largely relaxed
after treatment with topoisomerase I had equivalent transfection
efficiencies, in contrast to linearized plasmid that had no
transfection capacity (Fig.
6).

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Fig. 4.
A:
RFL19Ep cells were exposed to
DODAC-DOPE (1:1) at a lipid concentration of 2.5 nmol/cm2 with pCMV4-CAT at a
DNA-to-lipid ratio of 1:15 (mg/mg) for 24 h. Cell DNA was extracted
from cell monolayers 24, 48, or 72 h from addition of liposome-DNA
complex. DNA was separated by gel electrophoresis in 1% agarose
(wt/vol) with ethidium bromide for application of probes
specific for CAT insert. Cells treated with nicotinamide and
chloroquine (T), but not control (C) cells, had a band consistent with
supercoiled cDNA (S) evident at 48 and 72 h after addition of
liposome-DNA complex. Band comigrating with relaxed cDNA (R), but not
band comigrating with linearized plasmid (L), was better preserved in
cells treated with nicotinamide and chloroquine. Freshly prepared
unlinearized (P) and linearized plasmids were subjected to gel
electrophoresis for comparison. B:
relaxed (R) and supercoiled (S) pCMV4-CAT were separated by gel
electrophoresis (lane 1). After
pCMV4-CAT was mixed with liposomes and DNA was extracted
(lane 2), there was some increase in
proportion of relaxed cDNA. This could be attributed to extraction
procedure and not to mixing with liposomes because plasmid alone
subjected to the same extraction procedure had a similar degree of
increase in relaxed cDNA (lane 3).
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Fig. 5.
A: electrophoretic mobility of
pCMV4-CAT plasmid DNA (lane 1) on
0.8% (wt/vol) agarose gels without ethidium bromide for application of
probes specific for CAT insert and after linearization
(lanes
2-4) and
relaxation with topoisomerase I (lane
5) enhanced separation of supercoiled cDNA from
linear DNA but reduced separation of relaxed and linear cDNA (R/L).
B: with this approach, it was possible
to confirm that a band comigrating with plasmid (lane
1) supercoiled cDNA was present in extracts of cells
treated with chloroquine and nicotinamide (lane
2) after a 24-h transfection and was sensitive to
relaxation by topoisomerase I (lane
3).
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Fig. 6.
Control (C) plasmid that was largely in a supercoiled (S)
configuration, plasmid that had been largely converted to a relaxed (R)
form by treatment with topoisomerase I (T), as assessed by gel
electrophoresis (inset), and linearized (L) plasmid were
assessed for their effect on CAT activity assayed 72 h after addition
of liposome-DNA complexes to
RFL19Ep cells. Values are means ± SE; n = 4 cell
preparations. * P < 0.05 compared with values for cells transfected with control plasmid.
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Superoxide was generated in the culture medium to establish a
RFL19Ep cell cytotoxicity assay
(Fig.
7A).
RFL19Ep cell cultures were
transfected with the CAT expression cassette to determine whether the
presence of transfected DNA rendered these cells more susceptible to
oxidant injury. The sensitivity of the cells in primary culture to
exogenously generated superoxide was significantly increased by prior
exposure to the lipid-DNA complex (P < 0.05; Fig. 7B). To confirm that
this effect was not plasmid specific, the experiment was repeated with
different constructs, including one containing the SEAP reporter gene
(Fig. 7C), with the same result. To
put the cytotoxicity index in perspective, the mean increased release
of [14C]adenine with
exogenously generated superoxide in this latter experiment represented
the total intracellular content of 58% of the cells in the monolayer,
which increased to a mean of 74% of the monolayer after transfection
with the SEAP construct. The enhanced sensitivity to
superoxide-mediated cytotoxicity could not be accounted for by the
lipid component alone and could only be attributed to the delivery of
plasmid DNA. The addition of nicotinamide and chloroquine (Fig.
8) restored
superoxide-mediated cytotoxicity to the same level as seen in
untransfected cells (P > 0.05). A
possible explanation for these findings was the activation of nuclear
poly(ADP-ribose) polymerase by short nucleotide strands entering the
nucleus after cytosolic DNA degradation, which could amplify any
activation initiated by superoxide alone. However, direct measurement
of poly(ADP-ribose) polymerase activity (Table
4) showed no activation of the enzyme by
delivery of either linearized or circular plasmid.

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Fig. 7.
A: to establish an assay to assess
sensitivity of cells to superoxide, cells were incubated with either 25 ( ) or 50 ( ) mU/ml of xanthine oxidase and 5 mM hypoxanthine for
30, 60, or 90 min. Release of preincorporated
[14C]adenine was
compared with control cells not exposed to xanthine oxidase ( ).
Values are means ± SE; n = 4 cell
preparations. Significantly different
(P < 0.05 by ANOVA) compared with:
* control cells;
# cells exposed to 25 mU/ml
of xanthine oxidase. B: cells that had
been exposed to 2.5 nmol/cm2 of
liposomes (Lip) for 24 h showed no independent increase in cytotoxicity
in presence or absence of 25 mU of xanthine oxidase and 5 mM
hypoxanthine (H/X) for 60 min. However, when plasmid DNA was added to
liposomes at a DNA-to-lipid ratio of 1:15 (H/X+Lip+CAT), there was a
significant increase in sensitivity to superoxide-mediated
cytotoxicity. C: enhanced sensitivity
to superoxide-mediated cytotoxicity was also seen with a different
reporter gene construct [H/X+Lip+secreted alkaline phosphatase
(SEAP)]. In this instance, only increases ( ) in cytotoxicity
index above control value with liposomes alone are shown. Values are
means ± SE; n = 3 cell
preparations. For both B and C, significantly
different (P < 0.05 by paired
t-test) compared with: * cells
not exposed to H/X; # all
other groups.
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Fig. 8.
Cytotoxicity indexes for untransfected control and transfected cells.
Some cells were treated with 25 mU of xanthine oxidase and 5 mM
hypoxanthine to generate superoxide (SO) for 60 min in absence or
presence of nicotinamide (0.2 mM) and chloroquine (10 µM; Ch/N).
Control cells had no additives. Values are means ± SE for 3 separate cell preparations. Exposure to superoxide resulted in a
significant increase in cytotoxicity.
* P < 0.05 compared with all
values for cells not exposed to superoxide. N.S., not significant.
Relative to untransfected cells, cells transfected with DODAC-DOPE
(1:1) at a lipid concentration of 2.5 nmol/cm2 with pCMV4-CAT at a
DNA-to-lipid ratio of 1:15 for 24 h had a significantly increased
sensitivity to superoxide. This increase over basal injury of
untransfected cells was attenuated in presence of nicotinamide (0.2 mM)
and chloroquine (10 µM).
|
|
 |
DISCUSSION |
There is increasing evidence that the degree of epithelial integrity
maintained after lung injury is a critical determinant of pulmonary
fibrosis (1). If true, increased antioxidant gene expression or
manipulation of other critical determinant genes in epithelial cells
may be a future approach to therapeutic intervention. Adenoviral
approaches to gene therapy have, to date, been disappointing due to
viral- and reporter gene protein-mediated immune responses that limit
the duration of any initial response as well as the efficacy of
subsequently administered adenoviral vectors (17). This has led to a
renewed interest in the use of cationic liposome-DNA complexes for
transfection despite their relative inefficiency compared with viral
vectors. Expression cassettes delivered with liposomes via the airway
will be mostly taken up by lung epithelium, which also eliminates the
problem of minimal lung delivery of cationic liposomes after parenteral
injection (29). As a first step toward an in vivo delivery system, we
undertook these in vitro studies to define an effective liposome-DNA
preparation. Because our primary interest is in neonatal lung injury
and lung maturity has a direct effect on the sensitivity of lung tissue to oxidant injury (14), we used primary cultures of immature lung
epithelium rather than cell lines that adapt to their culture environment during serial passages.
Our approach to the development of an effective transfection protocol
for primary cultures was similar to that taken by Caplen et al. (8)
with permanent epithelial cell lines, recognizing that the efficacy of
liposome-mediated DNA transfection is markedly dependent on several
parameters including the toxicity of the lipids used, the cell type
transfected, the amounts of DNA and lipid used, and their ratio. The
liposome preparation used in our studies was cytotoxic when sufficient
concentrations of lipid were applied to the cells. The amount of DNA
taken up by the cells was dependent on the concentrations of lipid and
DNA used as well as on their ratio. If a lung cell DNA content of 7 pg/cell (27) and an average cell density of 7.5 × 105
cells/cm2 are assumed, the
observed DNA delivered with a 24-h exposure increased total cell DNA by
0.2%. Reporter gene expression decreased over time in culture. This
temporally related to a progressive loss of the delivered plasmid DNA.
After uptake across the cell membrane, as much as 99.9% of the
delivered plasmid DNA may fail to escape the endosome to be available
for transgene delivery (11). Thereafter, most of the DNA that escapes
the endosome will not cross the nuclear membrane (25). Any free
cytosolic DNA, unprotected by a coating of cationic lipid, will be
subject to degradation by a recently recognized cytosolic DNase (19). These factors would account for the rapid and extensive DNA degradation observed in control cell monolayers such that only linearized cDNA was
evident 24 h after completion of exposure to the liposome-DNA complex,
indicative of endonuclease activity. Treatment of cell monolayers with
chloroquine and nicotinamide was partially protective against DNA
degradation, preserving some of the DNA in relaxed and supercoiled cDNA
configurations up to 72 h from the onset of transfection. The most
probable explanation for this finding is a chloroquine-mediated
limitation of liposome-DNA complex degradation in the endosome that, on
endosomal escape, allowed some cDNA to resist the action of cytosolic
DNase. Although simply extending the survival of intracellular plasmid
would not necessarily have any beneficial effect on reporter gene
expression because any preserved DNA would not automatically increase
the traffic of DNA across the nuclear membrane, increased reporter gene
expression was seen with cells exposed to chloroquine and nicotinamide.
Neither relaxed nor linearized cDNA is believed to be capable of
reporter gene expression (21). Although this was true for linearized cDNA, supercoiled cDNA that had been extensively relaxed with topoisomerase I gave equivalent reporter gene expression to that seen
with largely supercoiled cDNA. This suggests that either the small
amount of supercoiled cDNA that was resistant to topoisomerase I was
sufficient to generate the amount of reporter gene activity observed or
fetal distal lung epithelial cells have repair mechanisms that allow
repair of nicked cDNA to reconstitute a supercoiled configuration (26).
The observation that reporter gene expression declined with excessive
DNA delivery was intriguing and suggested to us that the large
percentage of delivered DNA that becomes linearized may have adverse
effects on the processing of uninjured and functional forms. If this
observation is confirmed in vivo, it has significant implications for
current approaches to gene therapy, for which it is generally assumed
that increased gene delivery will equate to increased gene expression.
Of even greater concern for gene therapy initiatives with liposome-DNA
complexes is the potential for enhancing the sensitivity of transfected
cells to oxidant injury. Cystic fibrosis has been the major focus for
lung gene therapy to date. When limited to studies of nasal
electrophysiology, adverse effects secondary to enhanced oxidant injury
would not be anticipated. When delivered into an inflammed airway, the
risk of enhancing oxidant injury could be considerably increased.
Superoxide causes single-strand scissions in plasmid DNA (13), and
excessive DNA injury, resulting in poly(ADP-ribose) polymerase
activation with secondary adenine nucleotide and ATP depletion, is a
well-recognized pathway for lethal oxidant injury (4). A reasonable
explanation for the observed effect of DNA delivery on superoxide
sensitivity would be DNA degradation, resulting in the release of
nucleotide strands, which could cross the nuclear membrane to cause a
sublethal activation of poly(ADP-ribose) polymerase. This would allow
an additive effect on exposure to superoxide. However, direct
measurement of poly(ADP-ribose) polymerase activity showed no
activation with delivery of either circular or linearized DNA, and the
mechanism of the enhanced sensitivity to superoxide by transfected DNA
or some other copurified component remains unclear.
 |
ACKNOWLEDGEMENTS |
This work was supported by a Group Grant from the Medical Research
Council of Canada, a Development Grant from the Hospital for Sick
Children (HSC), a Research and Development Programme Grant from the
Canadian Cystic Fibrosis Foundation, and an Equipment Grant from the
Ontario Thoracic Society.
 |
FOOTNOTES |
A. K. Tanswell is the HSC Women's Auxiliary Chair in Neonatology. H. M. O'Brodovich was a career investigator of the Heart and Stroke
Foundation of Ontario during the tenure of these studies. R. Iles and
O. Staub were Fellows of the Canadian Cystic Fibrosis Foundation.
Address for reprint requests: K. Tanswell, Division of Neonatology,
Hospital for Sick Children, 555 University Ave., Toronto, Ontario,
Canada M5G 1X8.
Received 28 March 1997; accepted in final form 15 May 1998.
 |
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