Departments of
Pharmacology (J.L., T.J.C., A.P., F.P.) and
Physiology (R.M.L.), University of Arizona Health Sciences Center,
Tucson, Arizona
Antisense oligodeoxynucleotides (ODN) have been used to inhibit the
function of a number of structurally defined neurotransmitter receptors
in vivo by transiently disrupting their expression in the
CNS. However, issues concerning the cellular and molecular mechanisms
of these ODN often raise questions about the specificity of such
ODN-mediated "knock-down" of target proteins. This study sought to
extend our in vivo "knock-down" of the delta
opioid receptor (DOR) by targeting this receptor in the NG 108-15 cells with an antisense ODN for the DOR and by using a polyclonal antibody raised against this receptor to determine the efficiency and
selectivity of the antisense ODN in inhibiting expression of the DOR.
By fluorescence tagging the ODN and immunofluorescence labeling the
DOR, we monitored the uptake efficiency of the ODN and the DOR density
in individual cells that had been treated with the antisense ODN or
with a mismatch control. Quantitative fluorescence image analysis
showed that the uptake of ODN by NG 108-15 cells was time- and
concentration-dependent and that it was not uniform within a
population. Treatment with the antisense ODN elicited an inverse
correlation between DOR immunoreactivity and the ODN fluorescence in
individual cells. No correlation was found in cells treated with the
mismatch control. These findings suggest that the antisense
ODN-mediated "knock-down" of the DOR is governed by the sequence
specificity of the ODN and the efficiency of its uptake by the target
cells in a time- and concentration-dependent manner. These data provide
further evidence in support of the selectivity of antisense ODN
targeting and the utility of these molecules as an effective tool in
neuropharmacological studies.
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Introduction |
Endogenous opioid peptides exert
their biological activity via the activation of at least
three types of opioid receptors, which have been classified
pharmacologically as delta, mu and kappa (Wood, 1982
). In addition, pharmacological evidence
primarily from in vivo studies of selective opioid ligands
has revealed the existence of subtypes for these receptors, further
confounding the molecular complexity of opioid action (Mattia et
al., 1991; Pick et al., 1991; Lai et al.,
1994a). The identification of three discrete genes that encode the
three opioid receptors not only confirms the molecular basis of the
heterogeneity of the opioid receptors but also provides the means to
extend the pharmacological analysis of opioid action at the molecular
level (Evans et al., 1992; Kieffer et al., 1992;
Thompson et al., 1993; Meng et al., 1993). The
deduced primary structure of the three opioid receptors, and their
putative secondary structures, as predicted, are closely related to the
superfamily of guanine nucleotide regulatory protein (G
protein)-coupled receptors. Furthermore, the three receptor polypeptides are highly homologous, with hydrophobic regions that are
structurally well conserved. On the other hand, discrete regions, including the amino and carboxyl terminal domains, contain sequences that are unique to each receptor. These discrete regions thus serve as
"markers" that distinguish the cloned receptor types, and they have
been particularly useful for anatomical localization analysis (Mansour
et al., 1994), the design of deletion and chimeric mutation
studies to examine ligand interaction with the receptors (Surratt
et al., 1994; Xue et al., 1994; Fukuda et
al., 1995) and the targeting of specific receptors by antisense
strategy (Lai et al., 1994b; Standifer et al.,
1994; Bilsky et al., 1996).
We previously explored the use of antisense ODN to transiently and
reversibly inhibit the expression of the cloned DOR in mouse brain (Lai
et al., 1994b; Bilsky et al., 1996). Antisense ODN are short, synthetic, single-stranded DNA whose mode of action is
through hybridization to complementary sequences in the target gene or
its messenger RNA; the latter results in the formation of an RNA/DNA
duplex that disrupts the normal translation of that gene and/or leads
to degradation of the messenger RNA by RNaseH (for review see Crooke,
1992 and Wahlestedt, 1994). Consequently, these molecular events result
in a reduction in the level, or "knock-down," of the protein
product. By virtue of the high affinity and specificity of the ODN for
their target sequences, the resultant "knock-down" of the target
protein could be orders of magnitude greater in specificity than
conventional antagonists and is directly correlated with the known
structural characteristics of that protein. In our studies, an
antisense ODN that is specific to the cloned DOR selectively inhibited
the supraspinal antinociceptive effect of
[D-Ala2,Glu4] deltorphin, a
delta-2 selective agonist, but had no effect on the
antinociceptive action of the delta-1 receptor agonist,
[D-Pen2,D-Pen5]enkephalin (Lai et
al., 1994b; Bilsky et al., 1996). The ability of this
antisense ODN to differentiate the antinociceptive effects of the two
pharmacologically defined, subtype-selective agonists implicates a
structural distinction between these putative receptor subtypes.
Treatment with a mismatch control ODN, on the other hand, had no effect
on the antinociceptive response to these drugs, which suggests that the
effect of the antisense ODN was specific to its sequence rather than
due to some nonspecific effect of the ODN. Antisense ODN against the
DOR also had no effect on mu or kappa selective
agonists (Bilsky et al., 1996). Furthermore, treatment of
mice with the ODN did not produce behavioral toxicity, changes in
feeding or alterations in base-line nociceptive threshold.
Thus the in vivo effect of the antisense ODN to the cloned
DOR is consistent with previous evidence for the existence of
delta opioid receptor subtypes. On the other hand, a number
of issues related to the mechanism of antisense targeting remain
unresolved. In particular, it is presumed that the effectiveness of the
antisense ODN-mediated "knock-down" depends on its stability, its
translocation into the cells that express the target protein and the
efficiency with which the antisense ODN suppresses the synthesis of
that protein. These issues have not been addressed in our studies using antisense ODN to target the delta opioid receptors in the
brain and spinal cord of mice. This communication describes a series of
experiments in which we extend our analysis by targeting the DOR in the
NG 108-15 cells with the DOR-specific antisense ODN and use a
polyclonal antibody that we raised against the DOR to determine the
efficiency and selectivity of the antisense ODN-mediated "knock-down" of the DOR. Our findings suggest that antisense
ODN-mediated "knock-down" of the target receptor is selective for
the target protein. The reduction in the level of the target receptor
is correlated with the sequence specificity and the efficiency of its
uptake by the target cells in a time- and concentration-dependent manner. These data provide further evidence of the utility of antisense
targeting as an effective tool in neuropharmacological studies.
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Materials and Methods |
Materials.
The cDNA for the mouse DOR was a gift from Dr.
Chris Evans (University of California, Los Angeles). The cDNA for the
rat KOR and the rat MOR were a gift from Dr. Huda Akil (University of Michigan); [3H]diprenorphine (39 Ci/mmol) and
[3H]naltrindole (34.7 Ci/mmol) were from DuPont NEN
(Wilmington, DE); tissue culture reagents were from Gibco BRL
(Gaithersburg, MD).
Production of polyclonal antibodies to a fusion protein
containing the C-terminal peptide of DOR.
A NotI/BalI fragment
(nucleotides 1040-1225), which contains the last 105 bp of the coding
region of the DOR, was subcloned into the fusion protein expression
vector, pGEX-4T-3 (Pharmacia, Piscataway, NJ) to form an in-frame,
contiguous coding sequence with that for GST. Transformation of
E. coli with this recombinant DNA resulted in clonal
transformants that expressed a high level of a 30-kDa polypeptide,
which is made up of a 26-kDa moiety of GST and a 4-kDa moiety of the
C-terminal peptide of the DOR. This fusion protein was affinity
purified, and 200 to 500 µg/ml was mixed with an equal volume of RIBI
adjuvant and injected s.c. into a New Zealand White rabbit. The first
boost was given 14 days later, followed by subsequent boosts every 4 weeks thereafter.
Stable expression of the DOR, MOR and KOR.
The cDNA for the
three receptors were subcloned into eukaryotic expression vectors (DOR
in LK-444; MOR and KOR in pCMV) and transfected into the mouse
hippocampal neuroblastoma cell line HN9.10 (Lee et al.,
1992) by the calcium phosphate precipitation method. Stable clonal cell
lines were selected by neomycin resistance (Geneticin, 1 mg/ml) and
maintained in selection medium for at least 10 passages (2 months).
Receptor density was monitored regularly by radioligand binding
analysis.
Immunocytochemical analysis.
Cells were maintained in
75-cm2 flasks in a humidified atmosphere with 95% air and
5% CO2. NG 108-15 cells were cultured in 5% fetal calf
serum/5% newborn calf serum/45% Hams F-12/45% DMEM/100 U
ml
1 penicillin/100 µg ml1 streptomycin.
Transfected and nontransfected HN9.10 cells were maintained in 5%
fetal calf serum/5% newborn calf serum/90% DMEM/1 mM
L-glutamine/100 U ml1 penicillin/100 µg
ml1 streptomycin. For experiments, cells were seeded onto
sterile cover slips contained in 60 mm petri dishes at 50,000 to
100,000 cells/plate 24 hr before the experiments. For experiments in
which cells were incubated with ODN, the cells were initially seeded onto cover slips at 100,000 cells/plate in normal media 24 hr before
treatment. On the day of the experiments, the cells were washed twice
with serum-free medium, and the culture media were replaced with
serum-free medium containing ODN at concentrations as specified. The
cells were maintained in serum-free conditions throughout the treatment
with ODN.
For immunostaining, the medium was aspirated and the cells washed twice
with serum-free medium. Immunolabeling was carried out as described
previously (Lynch et al., 1991). Briefly, the cells on the
cover slips were fixed with 4% paraformaldehyde and permeabilized with
0.1% Triton X-100. The cells were then incubated with a 1:10 dilution
of the antiserum in the presence of 0.1 mg/ml GST for 72 hr at 4°C.
The cells were then washed three times with 2 × SSC/0.05% Triton
X-100 and incubated with a FITC-conjugated goat anti-rabbit IgG
(Vector, Burlingame, CA) for 45 min at room temperature. The cover
slips were washed and mounted onto slides with 0.1% p-phenylene
diamine dissolved in 50% glycerol.
Oligodeoxynucleotide treatment of NG 108-15 cells.
Texas-red conjugated ODN. The ODN has the following
sequence: 5
-CTG TGG CCC CTT GCC GCT GC-3, which is complementary to the mismatch control sequence for the cloned DOR from NG 108-15 cells
(see below). The ODN was synthesized by solid-phase procedure, conjugated with Texas-red at the 5 end of the ODN and purified by
reverse-phase HPLC (Midland Certified Reagent Co., Midland, TX). This
Texas-red ODN was reconstituted in nuclease-free water and stored in
the dark at 4°C. Cover slips on which NG 108-15 cells were grown were
inverted on 40 µl of medium. The medium consisted of Texas-red ODN in
serum-free medium at a final concentration of 0.5 µM or 5 µM. The
cells on inverted cover slips were incubated in a humidified chamber
for 8, 16 and 24 hr, after which time the cells were washed with
phosphate buffered saline, fixed with 4% paraformaldehyde and mounted
with p-phenylene diamine.
Antisense and mismatch ODN. Antisense (5-GCA CGG GCA GAG
GGC ACC AG-3; complementary to nucleotide 7-26 of the DOR coding region) and mismatch (5-GCA GCG GCA AGG GGC CAC AG-3) ODN to the DOR
were used for these experiments. These two sequences, as well as that
of the Texas-red-tagged ODN, were screened through the Genbank Database
to ensure that these sequences were not likely to cross-react with
other known gene sequences. Cells were treated with the antisense or
the mismatch ODN at a final concentration of 5 µM for a total of 4 days, during which time the ODN-containing medium was refreshed daily.
The dosage was based on the uptake of the Texas-red ODN, and the
duration of treatment was based on our previous in vivo
antisense treatment, which showed both a maximal functional inhibition
and a reduction in the number of supraspinal delta opioid
receptors in the antisense ODN-treated, but not the mismatch
ODN-treated mice. On day 5, the cells were rinsed briefly and incubated
for 24 hr with 5 µM of the Texas-red-ODN as described above. At the
end of this incubation, the cells were processed for immunocytochemical
analysis as described above.
Uptake of eosin-labeled dextran by the NG 108-15 cells.
Cells were incubated overnight with 50 µg/ml of eosin-dextran (MW
70,000, fixable; Molecular Probes, Eugene, OR) after incubation with
the Texas-red ODN. Ability to incorporate the dextran into vesicles by
pinocytosis was taken as indicative of cell viability. These cells were
rinsed and then incubated with serum-free medium containing dextran.
Fluorescence microscopy.
Fluorescence images were acquired
using a Photometrics liquid-cooled CCD camera attached to an Olympus
IMT-2 inverted microscope equipped with an Olympus 60X 1.4 NA objective
and 6.7X eyepiece. This camera has a linear response up to 5 × 105 counts/image element. An electronic shutter under
computer control was utilized to regulate exposure time. Standard
optics (Omega Optical, Brattleboro, VT) for FITC included a 10-nm band
pass excitation filter centered at 480 nm and a 20-nm band pass
emission filter centered at 520 nm. For Texas-red, the excitation and
emission were centered at 585 nm and 610 nm, respectively. For eosin,
excitation was centered at 525 nm and emission at 545 nm. The digitized
output of the camera was stored on a 386 based microcomputer. Image
analysis was performed using customized software on a Silicon Graphics IRIS 10/900. The fluorescence intensity within a single cell was quantified by acquiring images of all cells with a constant exposure time. This exposure time (for our studies, it was set at 2 sec) was
chosen to provide images with fluorescence intensities well above
background [>500 integrated optical density (IOD)] without causing
saturation of imaging elements. Thus all measurements were well within
the linear response range of the CCD camera.
Cells were selected for analysis on the basis of their Texas-red
fluorescence such that they represented the full range of Texas-red
intensities observed within that population. A second image was then
acquired of the FITC fluorescence in the same cells. The intensity of
fluorescence was determined for each cell by tracing around the cell
border of each cell and analyzing total IOD within the delineated area.
Fluorescence intensity of a cell was then corrected for noncell
background fluorescence. This was determined by using a standard
30 × 30 pixel square to sample fluorescence from at least four
different out-of-cell areas of the image; the average fluorescence
intensity then defined the noncell background. Specific labeling was
defined as a level of in-cell fluorescence above the noncell
background. NG 108-15 cells do not exhibit significant autofluorescence
at the Texas-red wavelength; however, the cells do express significant
autofluorescence over the fluorescence window. Specific labeling was
expressed as AUF per unit area (µm2). For
immunofluorescence of DOR, nonspecific labeling was defined as the
in-cell fluorescence (above the noncell background) of cells stained
with the secondary fluorophore-labeled antibody alone.
Estimated molecular density of Texas-red ODN.
Because ODN
and fluorophore have a stoichiometric ratio of 1:1, an estimate of
molecular density was calculated from the measured fluorescence
intensity through a sample calibration procedure as described
previously (Lynch et al., 1996). Briefly, droplets of
fluorophore approximately 0.2 µm in diameter were injected into a
layer of mineral oil on the microscope stage. Images of the droplets
were acquired, and the exact diameter of the droplet was measured.
Because the concentration of a specific molecule in the solution was
known, and the volume determined, a value for IOD/molecule could be
calculated from the measured fluorescence. For Texas-red ODN, the
conversion factor was 4 × 102 IOD/molecule, which
was used to estimate the molecular density of the ODN (in
atmol/µm2).
Confocal microscopy.
Confocal images for presentation were
also acquired using a Leica TCS-4D laser scanning confocal microscope
and SCANWARE software.
Radioligand binding analysis.
Cell membranes from
nontransfected or transfected HN9.10 cells, or with NG 108-15 cells,
were resuspended in 50 mM Tris/5 mM MgCl2, pH 7.2. Saturation binding analyses were carried out in the
Tris/MgCl2 buffer supplemented with 0.1 mM PMSF and 1 mg/ml bovine serum albumin in a final volume of 1 ml and 27 µg of membrane protein/assay tube. Nonspecific binding of the radioligand was defined
as that in the presence of 10 µM naloxone. Membranes were incubated
with [3H]naltrindole for 5 hr or with
[3H]diprenorphine for 3 hr at room temperature. The
reaction was terminated by rapid filtration through Whatman GF/B
filters, followed by five washes with ice-cold saline. The
radioactivity was determined by liquid scintillation counting.
Data analysis.
Radioligand binding data were analyzed by
nonlinear regression analysis using GraphPad Inplot (San Diego, CA).
Fluorescence data were analyzed by linear regression analysis and
repeated-measures ANOVA. Statistical significance is defined at the
95% confidence level (P < .05).
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Results |
Polyclonal antibody against the cloned DOR from mouse.
Immune
serum produced from the fusion protein displayed strong
immunoreactivity with the 30-kDa fusion protein in the presence of 0.1 mg/ml GST by Western analysis (data not shown). Immunocytochemical staining of the NG 108-15 cells with the antiserum showed a granular staining throughout the cytoplasm, whereas the nucleus exhibited little
staining (fig. 1). Furthermore, immunocytochemical
analysis of three transfected cell lines that expressed the cloned DOR (Evans et al., 1992), MOR (Thompson et al., 1993)
and KOR (Meng et al., 1993) showed that the antibody did not
cross-react with the MOR or KOR. These cell lines constitutively
express the opioid receptors at a density of 5.9 pmol/mg protein
(n = 2) for DOR (DORLKHN-8) based on
[3H]naltrindole binding, 1.7 pmol/mg protein
(n = 2) for MOR (MORCHN-1) and 2.0 pmol/mg protein
(n = 3) for KOR (KORCHN-8) based on
[3H]diprenorphine binding. Nontransfected HN9.10 cells
exhibited negligible levels of [3H]diprenorphine binding.
Incubation of DORLKHN-8 cells with the antibody resulted in a granular
cytoplasmic staining of these cells similar to that seen in the NG
108-15 cells. Concurrent staining of nontransfected HN9.10 cells,
MORCHN-1 and KORCHN-8 cells with the same antibody preparation showed a
small degree of staining above background. The result of the analysis
of the intensity of fluorescence in these cell lines is summarized in figure 2.
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Fig. 1.
Immunofluorescence image of NG 108-15 cells. The
cells were incubated with 1:10 dilution of the anti-DOR antiserum and
an FITC conjugated secondary antibody as described in "Materials and
Methods." Magnification: 400×. Scale bar corresponds to 10 µm.
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Fig. 2.
Immunocytochemical analysis of nontransfected HN9.10
(n = 9) and HN9.10 cell lines that express the KOR
(KORCHN-8) (n = 9), MOR (MORCHN-1) (n = 10) and DOR (DORLK-HN-8) (n = 12). Fluorescence of FITC
is expressed as AUF (in thousands)/µm2 above background.
The background fluorescence (secondary antibody only) was 8100 ± 125 (n = 6).
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Oligodeoxynucleotide (ODN) treatment of NG 108-15 cells.
Our
initial experiments showed that NG 108-15 cells remained viable in the
absence of serum over a period of several days if they were initially
established at relatively high density (50% confluency) in serum
containing medium and were subsequently maintained in serum-free medium
that was refreshed daily. These culturing conditions were subsequently
used for all our experiments in which the cells were treated with ODN
to circumvent the lability of ODN in serum (Akhtar et al.,
1991). Cells cultured under these conditions did not actively divide;
overnight loading of the cells with eosin-dextran showed that the cells
were metabolically viable and expressed substantial amounts of DOR
immunoreactivity (see below).
Texas-red ODN uptake by the cells. When the NG 108-15 cells
were incubated with the Texas-red ODN to monitor the uptake of ODN by
these cells, we observed that the cells accumulated the ODN in a time-
and concentration-dependent manner (fig. 3). On the
basis of the measured fluorescence intensities shown in figure 3, and a
conversion factor of 4 × 102 IOD/molecule of the
Texas-red ODN, we estimated the molecular density of the tagged ODN
that accumulated in these cells over time. Over a 24-hr incubation with
0.5 µM of tagged ODN, the estimated molecular density (EMD; mean ± S.E.M. in atmol/µm2) of ODN was 0.06 ± 0.003, 0.08 ± 0.005 and 0.41 ± 0.24 after 8, 16 and 24 hr of
incubation, respectively (fig. 3A). This time-dependent increase in the
EMD, however, did not reach statistical significance (P > .2;
repeated-measures ANOVA). Incubation with 5 µM of tagged ODN yielded
a significant increase in the EMD (mean ± S.E.M. in atmol/µm2) of ODN: 0.07 ± 0.02, 0.48 ± 0.24 and 2.33 ± 0.35 after 8, 16 and 24 hr, respectively (P < .0001; repeated-measures ANOVA) (fig. 3B). Furthermore, cells within a
population exhibited a highly variable level of accumulation of the
tagged ODN after 24 hr of incubation with 5 µM ODN (figs. 3B;
4). In three separate cultures, the EMD (in
atmol/µm2) ranged from 0.18 to 7.01 (n = 16), from 0.24 to 3.36 (n = 13) and from 0.17 to 2.97 (n = 27), which represents up to a 40-fold difference
in the EMD of ODN. The mean value of EMD was 1.33 ± 0.16 atmol/µm2 (n = 57), which corresponded to
a mean fluorescence intensity of 32,000 ± 3750 AUF/µm2.
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Fig. 3.
Time course and concentration dependence of uptake of
Texas-red ODN by the NG 108-15 cells in serum-free conditions. Each point represents the AUF (in thousands)/µm2 of a single
cell. A) 0.5 µM of ODN. Total number of cells sampled per time-point
is 11. B) 5 µM of ODN. Total number of cells sampled per time-point
is 21.
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Fig. 4.
Confocal fluorescence image of NG 108-15 cells that
had been incubated with 5 µM of Texas-red ODN for 24 hr in serum-free medium. Magnification: 100×.
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Irrespective of the levels of Texas-red staining, and thus of the level
of ODN accumulated in these cells, all the cells that were examined
also accumulated dextran (fig. 5). The ODN was found initially in endosome-like structures, and over time it was localized to both the cytoplasm and the nucleus. The uptake characteristics of
the tagged ODN, as well as dextran accumulation, were qualitatively similar in cells that had been preincubated with mismatch ODN (nontagged) for up to 4 days, which indicates that the cells were not
adversely affected by prolonged exposure to these ODN (data not shown).
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Fig. 5.
Uptake of Texas-red ODN and eosin-dextran by the NG
108-15 cells. The cells were incubated with 5 µM Texas-red ODN for 24 hr, followed by overnight incubation with 50 µg/ml eosin-dextran. A)
Eosin fluorescence. B) Texas-red fluorescence. Magnification: 400×.
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Antisense and mismatch ODN pretreatment of the cells.
Experiments were carried out to evaluate the relationship between the level of antisense ODN uptake and expression of DOR. Because longer periods of ODN treatment are required to block the expression (3-4
days) than to monitor uptake, cells were first incubated with 5 µM
ODN daily for 4 days and then labeled for uptake by incubation with 5 µM of Texas-red ODN for 24 hr. Under this treatment paradigm, we
found that cells that accumulated substantial amounts of Texas-red ODN
over 24 hr after DOR antisense ODN treatment consistently exhibited a
lower level of DOR antibody staining (fig. 6). Because
we could not determine directly the stoichiometric ratio of
FITC-conjugated second antibody to DOR molecules, we were not able to
correlate directly the density of DOR with tagged ODN uptake. Thus we
made a semiquantitative comparison between ODN accumulation and DOR
density by correlating the level of Texas-red and FITC fluorescence in
the same cell (fig. 7). We found that over the range of
4560 to 31,700 AUF/µm2 of Texas-red fluorescence, there
was a significant inverse correlation between the immunoreactivity of
DOR and the amount of Texas-red ODN accumulated in these cells (P < .01, linear regression, correlation coefficient = 
0.71) (fig.
7A). Furthermore, cells that accumulated over 25,000 AUF/µm2 of Texas-red all showed markedly reduced DOR
immunoreactivity averaging 2080 ± 159 AUF/µm2
(n = 11). In contrast, no correlation was observed in
cells that had been pretreated with the mismatch control ODN before
labeling with Texas-red ODN (P = .06; two-tailed t
test) (figs. 7B, 8). Among the cells that exhibited over
25,000 AUF/µm2 of Texas-red, the average DOR staining was
17,300 ± 2870 AUF/µm2 (n = 6). As
mentioned above, the mean fluorescence intensity of Texas-red ODN in
cells loaded with 5 µM of the ODN was 32,000 ± 3750 AUF/µm2. Thus it appears that the ability of a cell to
accumulate a large amount of ODN (within the upper 50% of the observed
range of ODN densities) correlates with up to 88% reduction in DOR
expression after antisense ODN treatment when compared with mismatch
ODN-treated cells. Qualitatively, the prevalence of cells that
accumulated within the upper range of Texas-red ODN was 50% to 80% of
a typical culture.
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Fig. 6.
Texas-red ODN and FITC immunofluorescence of DOR in
NG 108-15 cells pretreated with antisense ODN for the DOR. A) Texas-red fluorescence (in AUF/µm2) is 6250 (upper left) and 24,500 (lower right). B) FITC immunofluorescence (in AUF/µm2) is
13,900 (upper left) and 3620 (lower right). Magnification: 400×.
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Fig. 7.
Correlation between the fluorescence intensity of
Texas-red ODN and FITC immunofluorescence of DOR in individual NG
108-15 cells pretreated with antisense ODN for DOR (panel A), and
mismatch control ODN (panel B).
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Fig. 8.
Texas-red ODN and FITC immunofluorescence of DOR in
NG 108-15 cells pretreated with mismatch control ODN for DOR. A)
Texas-red fluorescence (in AUF/µm2) is (clockwise from
top right): 17,000, 71,000, 5800 and 63,000. B) FITC immunofluorescence
(in AUF/µm2) is (clockwise from top right): 21,900, 25,300, 19,300 and 24,700. Magnification: 400×.
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We also assessed the change in DOR density by radioligand binding
analysis. Membranes prepared from antisense ODN-treated cells showed a
40% reduction in specific [3H]diprenorphine binding when
compared with membranes from untreated or mismatch control-treated
membranes. The specific binding values (in fmol/106 cells)
of [3H]diprenorphine at a saturating concentration of 4 nM for untreated, mismatch control-treated and antisense ODN-treated NG
108-15 cell membranes were 450, 500 and 270, respectively
(n = 2).
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Discussion |
This study examines the selectivity of antisense ODN in mediating
a "knock-down" of its target protein. By employing fluorescence imaging and computer-assisted image analysis, we monitored the level of
the antibody/DOR complex and that of a fluorescence-labeled ODN
simultaneously in individual cells. These results suggest a direct
relationship between the uptake of antisense ODN for the DOR and a
reduction of its target, the endogenous DOR in the NG 108-15 cells. The
effects of the antisense ODN are sequence-dependent, because a mismatch
control ODN did not bring about any change in the level of DOR in these
cells.
Fusion proteins are now well established as a means of efficiently
producing antigens for the generation of polyclonal, as well as
monoclonal, antibodies against defined epitopes from specific proteins.
A number of other antibodies also have been developed recently for the
opioid receptors and applied in a variety of immunohistochemical
analyses (Arvidsson et al., 1995a,b,c). The polyclonal
antibodies generated from the fusion protein containing the C-terminal
epitope of the DOR display reactivity toward both the fusion protein
and NG 108-15 cells (fig. 1). The polyclonal antibody also does not
exhibit significant cross-reactivity to the other opioid receptor
types. As shown in figure 2, the transfected cells expressing the DOR
exhibited an intensity of fluorescence/µm2 over 6-fold
that of nontransfected or transfected cells that express the MOR or the
KOR. Both the NG 108-15 cells and transfected cells expressing the DOR
exhibited significant cytoplasmic immunoreactivity. Interestingly, this
cytoplasmic localization of the DOR resembles the distribution of the
receptor in spinal cord and brainstem neurons using a DOR-specific
antibody (Arvidsson et al., 1995a). The cellular and
molecular basis for the subcellular distribution of the DOR currently
remains speculative. Our antibody to the DOR enabled us to monitor the
level and distribution of the DOR in experiments in order to examine
the effect of transient targeting of this receptor by antisense ODN in
individual NG 108-15 cells.
Initial experiments using fluorescence-tagged ODN showed that viable NG
108-15 cells accumulated the ODN in a time- and concentration-dependent manner (fig. 3). These observations are consistent with a number of
previous studies in which phosphodiester ODN (Loke et al., 1989; Yakubov et al., 1989) or phosphorothioate ODN (Gao
et al., 1993; Beltinger et al., 1995) were shown
to be taken up by a number of different cell lines, and this uptake was
thought to be mediated by both adsorptive endocytosis and fluid-phase
pinocytosis. On the basis of the purity of the tagged ODN preparation
(HPLC purified) and its stability in serum-free conditions (thin-layer
chromatography of the incubation medium containing 5 µM Texas-red ODN
after 24 hr of incubation with cells did not detect any free dye in the medium), our data indicate that the fluorescence detected in the NG
108-15 cells corresponded directly to the level of intact ODN that had
been taken up. Independently of the concentration of the ODN or the
duration of incubation, however, the uptake of the ODN was not uniform
throughout the cell population (fig. 3). This result argues against a
diffusion mechanism for uptake, which again is consistent with previous
findings (Loke et al., 1989). The heterogeneity of ODN
uptake by the cells was not due to their viability, based on dextran
uptake. Moreover, cells that showed intense Texas-red ODN uptake were
comparable morphologically under phase-contrast microscopy to those
that were less fluorescent throughout all time-points and different
concentrations. These data suggest that the cultured cells may
accumulate the ODN at a different rate and/or to a different degree;
because dead cells do not accumulate ODN (Loke et al.,
1989), such rate and degree of accumulation of the ODN may depend on
cell metabolism. It should be noted that fluorescence does not
necessarily indicate the localization of full-length ODN, because
degradation of ODN readily occurs intracellularly (Yakubov et
al., 1989; Crooke et al., 1995), but it does directly
reflect the amount of tagged ODN that has been accumulated by the
cells. Furthermore, fluorescence intensity increased both in the
cytoplasm and in the nucleus, which suggests that the ODN had access to
both cell compartments
a result similar to observations made with
other cell types exposed to ODN (Loke et al., 1989; Gao
et al., 1993; Beltinger et al., 1995; Crooke et al., 1995).
Overall, these findings show that uptake of the ODN was prevalent over
24 hr when the NG 108-15 cells were exposed to 5 µM ODN. Repeated
dosing of the cells with the antisense ODN at 5 µM for 4 days, a
treatment paradigm that was intended to mimic that employed to
successfully "knock-down" DOR in mice, showed that the antisense
ODN significantly reduced the overall level of DOR in the NG 108-15 cells when compared with cells that had been treated with the mismatch
ODN at the same concentration. DOR antisense ODN, but not the mismatch
control, resulted in a 40% reduction in
[3H]diprenorphine binding sites, which is consistent with
that observed previously (Standifer et al., 1994; Zhou
et al., 1994). At the single-cell level, a reduction in the
DOR, based on its immunoreactivity, was observed only in cells that had
subsequently accumulated a substantial amount of the Texas-red ODN.
This outcome establishes a strong physical correlation between the
accumulation of the antisense and the Texas-red ODNs and a reduction in
the level of DOR. It is interesting that there is a statistically
significant inverse correlation between the level of DOR and ODN
fluorescence in the antisense ODN-treated cells. However, because of
the relatively small sample size, and because the intracellular
disposition of ODN over the treatment period is complex, the precise
concentration relationship between the antisense ODN and DOR remains to
be determined. Nevertheless, the reduction of the DOR was not due to
uptake of the Texas-red ODN itself, because its sequence was not
specific to the DOR. Furthermore, cells that had been pretreated with
antisense ODN or mismatch control ODN were subsequently loaded with the same Texas-red ODN. The reduction in the DOR level also could not be
due to toxicity of the ODN, because preliminary studies (discussed
above) had ruled out possible changes in DOR expression due to cell
viability alone, and, moreover, cells that had been pretreated with the
mismatch control ODN exhibited, on average, a much higher level of DOR
irrespective of Texas-red ODN accumulation (fig. 7B). These
observations suggest, therefore, that the cells were more susceptible
to the antisense pretreatment when they exhibited efficient ODN uptake.
Indeed, the most obvious observation from cells that had been treated
with the antisense ODN was the marked reduction in DOR immunoreactivity
when compared with the mismatch ODN-treated cells over the upper range
of ODN uptake.
This clear-cut correlation between Texas-red ODN uptake and the DOR
immunoreactivity is consistent with the assumption that the efficiency
of the Texas-red ODN uptake in the last 24 hr of incubation paralleled
the extent to which the cells had continually accumulated the antisense
ODN during the 4-day treatment. Further, the rate and/or extent of
antisense ODN accumulation was sufficient to abolish completely the
expression of the DOR in some cells. The reduction in the level of DOR
was specifically due to the antisense sequence for the DOR, because
pretreatment with the mismatch ODN did not alter the level of DOR,
irrespective of the level of ODN uptake by cells within the population.
The selectivity of the DOR antisense ODN for the DOR has also been
observed in vivo; treatment with this antisense ODN had no
effect on the antinociception of either kappa or
mu opioid receptor selective agonists (Bilsky et
al., 1996). It should be pointed out that Texas-red-labeled antisense ODN, or its mismatch control, was not used for the 4-day treatment paradigm because of concern that the 5-end labeling with the
fluorophore might alter the affinity of the ODN for its RNA target, and
also because of the prohibitive cost of the procedure. Overall, these
findings provide direct evidence at the single-cell level that the
antisense ODN, but not the mismatch ODN, mediated "knock-down" of
the DOR in a highly selective manner. This action of the antisense ODN
is governed by the sequence of the ODN and depends critically on the
translocation of the ODN into the NG 108-15 cells.
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Abstract
Introduction
opioid receptor: Isolation of a cDNA by expression cloning and pharmacological characterization.
Proc. Natl. Acad. Sci. U.S.A.
89: 12048-12052, 1992
opioid receptor.
Proc. Natl. Acad. Sci. U.S.A.
90: 9954-9958, 1993
