From the Departments of Pharmacology,
Molecular Pharmacology, § Analytical/Physical
Chemistry, and ¶ Medicinal Chemistry, ISIS Pharmaceuticals,
Carlsbad, California 92008
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ABSTRACT |
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The use of antisense oligonucleotides to inhibit
the expression of targeted mRNA sequences is becoming increasingly
commonplace. Although effective, the most widely used oligonucleotide
modification (phosphorothioate) has some limitations. In previous
studies we have described a 20-mer phosphorothioate
oligodeoxynucleotide inhibitor of human protein kinase C- The protein kinase C
(PKC)1 family of isozymes is
composed of at least 11 different, but structurally related
serine/threonine kinases. These can be subdivided on the basis of
structural and biochemical similarities into three groups, the
conventional ( Considerable effort has been made over the last 10 years to develop
isozyme-specific inhibitors of PKC to allow the dissection of the
PKC-dependent signaling pathways (6). These efforts are
hampered by the similarities in protein structure of the many isozymes
of PKC, which make the identification of specific, small molecule
enzyme inhibitors difficult. To overcome this difficulty, we have used
antisense oligonucleotides to inhibit the expression of individual
isozymes of PKC (7, 8). Antisense oligonucleotides can be targeted to
mRNA sequences which are unique to a given PKC isozyme, leading to
the selective inhibition in expression of that isozyme (9, 10). The
long half-lives of some PKC proteins (and other proteins which have
been targeted with antisense oligonucleotides) have proven problematic,
as we have found that the most widely used oligonucleotide modification
available, the phosphorothioate (P=S) oligodeoxynucleotide, is
metabolized in cells over time. This leads to a loss of activity over a
48-72-h period, which can make inhibition of some PKC isozymes
difficult (7, 8, 11).
The factors which govern oligonucleotide activity are complex. Two
important parameters are the affinity with which an oligonucleotide hybridizes to a target mRNA, and the ability of the oligonucleotide to withstand degradation by intracellular nucleases (12-19). We have
therefore sought to improve these characteristics, with the anticipation that this would lead to the identification of antisense oligonucleotides with improved pharmacological activity compared with
those presently available. This should allow for the development not
just of improved antisense inhibitors of PKC, but of a more generalized
class of antisense effective against any mRNA target which encodes
a protein with a long half-life.
In the present study, the biophysical and pharmacological activity of
oligonucleotides containing the recently described
2'-O-(2-methoxy)ethyl (2'-MOE) modification (20), with both
2-O-methyl (2'-M) and 2'-deoxy containing oligonucleotides
are contrasted. Antisense oligonucleotides can inhibit the expression
of proteins by a number of potential mechanisms (21-24). One of the
most effective oligonucleotide-dependent mechanisms for
reducing protein expression is to cause an RNase H-mediated cleavage in
the hybridized target mRNA (7, 25, 26). Unfortunately, however,
2'-alkyl modifications (such as 2'-MOE) do not support RNase H-mediated
mRNA cleavage (12, 18, 27, 28). This can be overcome by the
inclusion of 2'-deoxy residues into an antisense oligonucleotide, in
combination with 2'-alkyl modifications, in a motif that will support
RNase H cleavage (chimeric oligonucleotides) (11, 15, 29-31). In our
studies reported here, an oligonucleotide containing 2-MOE modification in such a configuration was found to be at least 20-fold more active
than conventional P=S oligodeoxynucleotides at reducing the expression
of PKC- This has allowed us to examine the role played by PKC- Cell Culture--
Human A549 lung carcinoma cells were obtained
from the American Type Tissue Collection (ATCC). Cells were grown in
Dulbecco's modified Eagle's medium containing 1 g of
glucose/liter and 10% fetal calf serum and routinely passaged when
90-95% confluent.
Oligonucleotide Synthesis--
Phosphorothioate
oligodeoxynucleotides were synthesized as described previously (7).
2'-O-Methyl and 2'-MOE oligonucleotides were synthesized as
described (32).
Measurement of Oligonucleotide Tm--
Absorbance
versus temperature profiles were performed as described
previously (33). Briefly, antisense oligonucleotides were hybridized to
complementary RNA strands and Tm values and free
energies of duplex formation were obtained. Values are the averages of
three experiments.
Oligonucleotide Treatments of Cells--
A549 cells were grown to
60-70% confluence in T-75 flasks. The cells were then washed twice
with Dulbecco's modified Eagle's medium and then 5 ml of Dulbecco's
modified Eagle's medium containing 20 µg/ml N-[1-(2,
3-dioleyloxy)propyl]-n, n,n-trimethylammonium chloride/dioleoyl phosphatidylethanolamine (DOTMA/DOPE)
(Lipofectin®)(Life Technologies, Inc.) solution was added to the
flasks. Oligonucleotides were added to the required concentration from
a 10 µM stock solution and the flask swirled to mix the
solutions. The cells were then incubated at 37° C for 4 h and
then the DOTMA/DOPE/oligonucleotide solution was aspirated off and
replaced with medium for the indicated time.
In Vitro Nuclease Stability--
Oligonucleotide resistance to
snake venom 3'-phosphodiesterase was determined as described previously
(11). Briefly, the oligonucleotides were gel purified and
5'-end-labeled with high performance liquid chromatography-purified
[ Oligonucleotide Metabolism in A549 Cells--
A549 cells were
treated with 500 nM oligonucleotides as described above and
allowed to recover for 72 h. At this time, metabolites were
recovered and analyzed by capillary gel electrophoresis as described
previously (11). After digestion with proteinase K, oligonucleotide
metabolites were recovered by sequential passages through an anion
exchange column and a reverse phase column. Analysis of the samples by
capillary gel electrophoresis was performed on a Beckman 5010 P/ACE
capillary electrophoresis unit.
Measurement of PKC, jun, and fos mRNA Expression--
Total
mRNA was extracted from cells and resolved on agarose gels as
described previously (7). These were transferred to nylon membrane
(Bio-Rad) and probed with 32P-radiolabeled cDNA probes
for different PKC isozymes (7). Additionally, gels were probed with
[32P]cDNA probes for c-fos,
c-jun, junB (ATCC). Gels were routinely stripped
and reprobed with radiolabeled human glycerol-3-phosphate dehydrogenase
(GAPDH) probe to confirm equal loading. Radioactive bands were
quantitated using a PhosphorImager, and typically we measure only the
upper of the two PKC- Measurement of PKC Protein Expression--
PKC isozyme protein
expression was determined by Western blotting (7). The antibodies used
were obtained as indicated. PKC- Measurement of PKC Enzyme Activity--
A549 cells were treated
with oligonucleotides for 3 days. Cells were then washed in cold
phosphate-buffered saline, scraped, and pelleted into a sample
preparation buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 10 mM EGTA, 50 mM
2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 10 mM benzamidine). A cytosolic fraction was prepared by
centrifugation at 100,000 × g for 1 h at 4° C.
PKC enzyme activity was determined by measuring the ability of the
cytosolic protein extract to phosphorylate a synthetic peptide
substrate in the absence or presence of phosphatidylserine in an
enzyme-linked immunosorbent-based assay according to the manufacturers
instructions (MBL Co. Ltd., Nagoya, Japan). The final concentrations of
the reaction mixture used were 25 mM Tris-HCl, pH 7.0, 3 mM MgCl2, 0.1 mM ATP, 2 mM CaCl2, 0.5 mM EDTA, 1 mM EGTA, 5 mM 2-mercaptoethanol, ±50 µg/ml
phosphatidylserine. PKC activity is defined as
phosphatidylserine-dependent kinase activity.
Determination of Minimum Oligodeoxynucleotide Residues Required for
Oligonucleotide Activity--
The inhibition of PKC- Design and Hybridization Thermodynamics of 2'-Modified
Oligonucleotides--
A series of oligonucleotides were subsequently
synthesized with 8 contiguous central oligodeoxynucleotide residues
flanked with either 2'-O-methyl or 2'-MOE-modified sugar
residues. The 3' end base of each oligonucleotide was left
oligodeoxynucleotide for synthetic reasons. P=S backbone linkages were
always retained in the central oligodeoxynucleotide sequence of the
oligonucleotide to maintain resistance to endonucleases in this part of
the molecule (15). The flanking sequences were also prepared with
either phosphorothioate and phosphodiester backbones (Table
I). The incorporation of a total of 11 2'-O-methyl residues increased oligonucleotide affinity
toward a complementary mRNA from 52.1 to 61.9° C and 62.6° C
as either P=S or P=O backbone linkages. The 2'-MOE incorporations gave
a greater increase in Tm, to 64.8 and 69.6° C,
respectively.
Oligonucleotide Nuclease Resistance--
The nuclease resistance
of the chimeric oligonucleotides shown in Table I was determined using
a number of different strategies. An in vitro nuclease assay
was used to examine the ability to withstand digestion by a snake venom
phosphodiesterase (a 3'-exonuclease). Under conditions which resulted
in 50% digestion of the full-length P=S oligodeoxynucleotide (ISIS
3521) (60 min incubation time with the nuclease), approximately 75% of
a chimeric 2'-O-methyl/P=O compound (ISIS 8329) was degraded
(Fig. 2). In contrast, only approximately
40% of the 2'-MOE/P=O oligonucleotide (ISIS 9605) was degraded,
demonstrating that even combined with a P=O backbone, this latter
modification provides greater nuclease resistance than that obtained by
a P=S oligodeoxynucleotide substituent. When the two
2'-O-modified sugar residues were evaluated in the context
of a P=S backbone they provided considerable enhancement of stability
(Fig. 2). The 2'-MOE was superior, demonstrating no digestion for the
duration of the experiment.
Experiments were also performed to evaluate the effects of
incorporating the 2'-O-methyl and 2'-MOE modifications as
P=S on oligonucleotide stability in tissue culture cells. A549 cells were treated with oligonucleotides (500 nM in the presence
of cationic liposomes) and the oligonucleotide metabolites extracted from cells 72 h later and resolved by capillary gel
electrophoresis. At this time, extensive metabolism of ISIS 3521 had
occurred consistent with the successive removal of 3'-bases by
3'-exonucleases resulting in the appearance of n-1, -2 etc.
metabolites (Fig. 3). Some metabolism of
the 2'-O-methyl containing oligonucleotide (ISIS 5357) was also apparent (results not shown). In contrast, no metabolism of ISIS
9606 (the 2'-MOE modified oligonucleotide) was found (Fig. 3).
Effect of 2'-Modifications on Ability of Oligonucleotides to Reduce
Expression of PKC-
PKC- Effect of 2'-MOE Modified Oligonucleotide on PKC-
We next determined whether ISIS 9606 was able to deplete cells of PKC
enzyme activity. Using an enzyme-linked immunosorbent-based assay which
measures phosphatidylserine-dependent kinase activity we
were able to demonstrate an overall reduction in PKC enzyme activity of
approximately 70% (Fig. 8A).
Samples of protein extract used in the assay were analyzed by Western
blotting to confirm a specific reduction in PKC- Effect of PKC- Antisense oligonucleotides must possess certain characteristics in
order for them to demonstrate full biological activity. Oligonucleotides must be sufficiently resistant to intracellular nucleases, have sufficient affinity for targeted mRNA to bind with
a high degree of specificity and fidelity, and possess a mechanism for
inhibiting the expression of the protein encoded in the target
mRNA. The instability of conventional P=O DNA to nucleases largely
precludes their use as antisense oligonucleotides. This has lead to the
replacement by a sulfur atom for an equatorial oxygen atom in the
phosphate backbone of oligodeoxynucleotides, resulting in the widely
used P=S modification (17, 23, 42). The P=S modification provides
considerable stability to both exo- and endonucleases, and are widely
used as specific inhibitors of gene expression. The advantages in
nuclease stability obtained with this modification come with a price,
as each incorporation of a P=S generates a chiral center and reduces
the binding affinity for target mRNA by 1-0.5° C. Furthermore,
although P=S oligonucleotides are less sensitive to nucleases, they
will degrade in cells over time (11).
To overcome these drawbacks, considerable research has been undertaken
to identify oligonucleotide modifications which provide a more
attractive pharmacological profile than P=S oligodeoxynucleotides (27,
43, 44). These include modifications to the oligonucleotide backbone
(44-46), the oligonucleotide base (47, 48), and the C2' position of
the ribose (15, 49-52). To date, however, very few modifications have
been fully characterized with respect to their pharmacological activity
in tissue culture and animals. In the present study, we have
characterized the biophysical and pharmacological activities of
oligonucleotides containing 2'-MOE modifications and compared them to
both P=S oligodeoxynucleotides and 2'-O-methyl containing
oligonucleotides. As 2'-MOE and 2'-O-methyl modifications do
not support RNase H-mediated cleavage of hybridized mRNA, we have
generated "chimeric" oligonucleotides, composed of both
2'-modifications and 2'-deoxy residues.
The incorporation of 2'-MOE modifications into a 20-mer oligonucleotide
has a dramatic effect on the ability of the sequence to hybridize to a
target mRNA, approximately 1.5° C per base compared with
approximately 1° C per base for the 2'-O-methyl
modification. The increase in hybridizing affinity obtained by the
2'-MOE modification is thought to result from the conformation of the
sugar and the backbone. The sugar pucker is believed to change from
C2'-endo (associated with B-form DNA) to C2'-exo, which more closely
resembles RNA, and RNA/RNA duplexes are more stable than DNA/DNA
duplexes (20). In addition, this limited rotational freedom may produce an enhanced steric effect that limits nucleases from digesting the
3'-phosphodiester, resulting in the increased nuclease resistance seen
with this modification. Irrespective of the mechanism involved, the
2'-MOE modification enhances the nuclease resistance of a P=O
oligonucleotide to at least that of a P=S oligonucleotide. Combined
with a P=S modification, the 2'-MOE containing oligonucleotide exhibits
further resistance to intracellular nucleases.
Increases in oligonucleotide affinity for a targeted mRNA have been
reported to result in increased oligonucleotide potency, provided a
mechanism for preventing protein synthesis is maintained (15, 19, 28,
32, 48). The 2'-MOE modified oligonucleotides were about 5-fold more
potent than the parent P=S oligodeoxynucleotide sequence (at 24 h)
and >20-fold more potent after 72 h, both in the context of
either a P=S or a P=O backbone. The increase in potency for the 2'-MOE
modification versus the P=S oligodeoxynucleotide is the
result of a combination of enhanced nuclease resistance an increase in
hybridizing affinity. This modification is clearly superior to the
2'-O-methyl modification. The increased potency of the
oligonucleotide did not lead to any decrease in specificity. Control
oligonucleotides containing the same chemistry as the active sequence,
but with a randomized base composition were without effect on PKC- Relatively little is known about the specific signaling roles played by
individual members of the PKC family of isozymes, although recent
observations are beginning to shed light on this field. For example,
PKC-µ appears to associate with the B-cell antigen receptor complex
and is involved in regulating lymphocyte signaling (53); PKC- Our previous studies indicated that PKC- In summary, we have described the pharmacological characterization of a
potent and specific class of antisense oligonucleotide inhibitors of
gene expression. These 2'-MOE containing oligonucleotides are at least
20 times more active than the widely used P=S oligodeoxynucleotides. This increase in activity is derived from increases in both hybridizing affinity toward the targeted mRNA and in a substantial increase in
resistance toward intracellular nucleases. The oligonucleotide has been
used to demonstrate a role for PKC-
expression. In an effort to identify improved antisense inhibitors of
protein kinase C expression, a series of 2' modifications have been
incorporated into the protein kinase C-
targeting oligonucleotide,
and the effects on oligonucleotide biophysical characteristics and
pharmacology evaluated. The incorporation of 2'-O-(2-methoxy)ethyl
chemistry resulted in a number of significant improvements in
oligonucleotide characteristics. These include an increase in
hybridization affinity toward a complementary RNA (1.5° C per
modification) and an increase in resistance toward both 3'-exonuclease
and intracellular nucleases. These improvements result in a substantial
increase in oligonucleotide potency (>20-fold after 72 h). The
most active compound identified was used to examine the role played by
protein kinase C-
in mediating the phorbol ester-induced changes in
c-fos, c-jun, and junB
expression in A549 lung epithelial cells. Depletion of protein kinase
C-
protein expression by this oligonucleotide lead to a reduction in
c-jun expression but not c-fos or
junB. These results demonstrate that
2'-O-(2-methoxy)ethyl-modified antisense oligonucleotides are 1)
effective inhibitors of protein kinase C-
expression, and 2)
represent a class of antisense oligonucleotide which are much more
effective inhibitors of gene expression than the widely used
phosphorothioate antisense oligodeoxynucleotides.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
,
I,
II, and
), the novel (
,
,
,
and µ), and the atypical (
and
) (1-3). The classic PKCs and
the novel PKCs are activated by 1,2-diacylglycerol, which is generated
by phospholipase cleavage of membrane phospholipids. These
phospholipases are regulated by many growth factors and hormones, and
it is therefore widely thought that PKC isozymes play an important role
in regulating cell proliferation and differentiation, as well as
short-term cellular responses, such as secretion and ion flux (1). The identification of multiple members of the PKC family has lead to
speculation that individual isozymes play different roles in regulating
different cell functions (4). Much evidence is available to support
this hypothesis. For example, expression profiles of the individual
family members is extremely heterogeneous, both at the tissue and the
subcellular levels (4, 5). In addition, the substrate specificities of
purified proteins are very different, and the responses of isozymes to
stimuli differ not just between isotypes, but also between the same
isotype stimulated in different cell types.
mRNA in A549 lung carcinoma cells. This inhibition
resulted in a time dependent and oligonucleotide-specific reduction in
expression of PKC-
protein.
in regulating
the expression of members of the AP-1 family of transcription factors
in A549 lung carcinoma cells. Activation of PKC by phorbol esters leads
to an increase in expression of the fos and jun
family members, by both increased transcription and increases in
mRNA stability. The PKC isoform responsible for this increase is
not clear, as A549 cells express multiple phorbol ester binding PKC isozymes, including PKC-
,
,
, and
. Depletion of PKC-
from A549 cells had a dramatic effect on the increase in
c-jun mRNA expression, reducing the up-regulation to
levels seen in control cells. In contrast, the phorbol ester dependent
increase in c-fos and junB mRNA expression
was not inhibited.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-32P]ATP (ICN). The oligonucleotides (100 nM) were then incubated with snake venom phosphodiesterase
(U. S. Biochemical Corp./Amersham) (5 × 10
3
units/ml) for the indicated times. The oligonucleotide metabolites were
then resolved on a 20% denaturing polyacrylamide gel followed by
quantitation by PhosphorImager (Molecular Dynamics) analysis.
transcripts, although both are reduced with
identical kinetics upon oligonucleotide treatment of
cells.2
, UBI; PKC-
, Santa Cruz
Biotechnology; PKC-
, a gift from Dr. Doriano Fabbro, Novartis
Pharmaceuticals; PKC-
, BioMol; PKC-µ, Santa Cruz Biotechnology;
and PKC-
(UBI).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
mRNA
expression by the uniform phosphorothioate oligodeoxynucleotide
sequence used here is believed to be mediated by RNase H (7). The
2'-modifications examined in the present study do not form substrates
for RNase H, and therefore need to be incorporated into the
oligonucleotide in combination with oligodeoxynucleotide residues to
effect this mechanism of mRNA degradation (18) (20, 34). The number
of contiguous oligodeoxynucleotide residues required in an
oligonucleotide to support RNase H cleavage of a hybridized RNA have
been proposed to range from 3 to 8 (15, 31, 35-39). To determine the
requirements for this oligonucleotide sequence, we have initially
incorporated 4, 6, or 8 contiguous oligodeoxynucleotide residues (deoxy
gap) into the center of a full phosphorothioated 2'-O-methyl
oligonucleotide, and determined the ability of these oligonucleotides
to reduce PKC-
mRNA expression in human lung A549 cells at 500 nM concentration. As shown previously, a fully
2'-O-methyl modified compound was unable to reduce PKC-
mRNA expression (Fig. 1, A
and B). However, increasing the number of
oligodeoxynucleotide residues (deoxy gap) present in the
oligonucleotide resulted in a progressive increase in the ability of
the sequence to reduce PKC-
mRNA expression. A contiguous
stretch of 8 oligodeoxy residues gave a greater than 90% reduction in
expression of PKC-
mRNA (Fig. 1, A and
B).
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Fig. 1.
Effect of number of oligodeoxy residues on
the ability of a phosphorothioate oligonucleotide to reduce PKC-
mRNA expression. A, A549 cells were treated with
oligonucleotides (500 nM) and DOTMA/DOPE as described under
"Experimental Procedures" for a period of 4 h. Cells were then
washed and allowed to recover for a further 20 h. At this time
PKC-
mRNA expression was determined by Northern blotting. The
gels were stripped and reprobed for expression of a housekeeping gene
(G3PDH) to confirm equal loading. The oligonucleotides used
were all full P=S. Oligodeoxy gap size refers to the number of
contiguous centrally placed 2'-oligodeoxy residues, with the remaining
residues being 2'-O-methyl. B, quantitation of
the above gel.
Sequence, chemistry, structure, and hybridizing affinity of the
oligonucleotides used in the present study
mRNA were synthesized with the
indicated 2'-sugar modifications (underlined sequences). The center of
the antisense oligonucleotides (not underlined) was
oligodeoxynucleotide and phosphorothioate. The 2'-modified regions were
synthesized with either phosphorothioate (lowercase s between bases) or
phosphodiester (lower case o between bases) backbone linkages. In
addition, the parent phosphorothioate oligodeoxynucleotide (ISIS 3521)
and a phosphodiester oligodeoxynucleotide (ISIS 11485) were
synthesized. Oligonucleotide sequence is shown 5'-3'. For indicated
Tm values, antisense oligonucleotides were
hybridized to complementary RNA strands and Tm
values and free energies of duplex formation were obtained as described
under "Experimental Procedures." Values are the averages of three
experiments.
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Fig. 2.
Digestion of oligonucleotides by snake venom
phosphodiesterases. Oligonucleotides were incubated with snake
venom phosphodiesterase for the indicated times as described under
"Experimental Procedures." The digested oligonucleotides were
resolved on 20% polyacrylamide gels and full-length 20-mer quantitated
using a PhosphorImager. , ISIS 9606;
, ISIS 5357;
, ISIS 9605;
, ISIS 3521;
, ISIS 8329;
, ISIS 11485.
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Fig. 3.
Oligonucleotide metabolism in A549
cells. A549 cells were treated with either ISIS 3521 (A
and B) or ISIS 9606 (C and D) for
either 0 h (A and C) or 72 h
(B and D) as described under "Experimental
Procedures." At these times metabolites were extracted and resolved
by capillary gel electrophoresis after spiking of the sample with a T27
oligonucleotide standard. Migration times of full-length (20-mer)
starting material was determined by comparison with this standard and
these peaks are indicated.
mRNA Expression--
Transfection of the P=S
oligodeoxynucleotide (ISIS 3521) into A549 cells results in a
concentration-dependent reduction in PKC-
mRNA
expression after 24 h (7). The IC50 for this reduction was approximately 100 nM. In the context of a P=S backbone,
the 2'-O-methyl (ISIS 5357) and 2'-MOE (ISIS 9606)
containing oligonucleotides demonstrate approximately a 2- and 5-fold
increase in potency, respectively (Fig.
4,A and B) as a
consequence of the enhanced hybridizing affinity of these two
compounds. In the context of a P=O backbone, the 2'-O-methyl
oligonucleotide (ISIS 8329) is inactive, even though this molecule has
a substantially higher Tm than the parent P=S
oligodeoxynucleotide (ISIS 3521). In contrast, the phosphodiester
containing 2'-MOE compound (ISIS 9605) is about 4-fold more active than
ISIS 3521 (Fig. 4, A and B).
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Fig. 4.
Dose response for the reduction in PKC-
mRNA expression in A549 cells by modified oligonucleotides 24 h after oligonucleotide addition to cells. A, A549
cells were treated with the indicated concentration of oligonucleotide
(0, 25, 50, or 100 nM) and DOTMA/DOPE as described under
"Experimental Procedures" for a period of 4 h. Cells were then
washed and allowed to recover for a further 20 h. At this time
PKC-
mRNA expression was determined by Northern blotting. The
gels were stripped and reprobed for expression of a housekeeping gene
(G3PDH) to confirm equal loading. The oligonucleotides used
were ISIS 9605, 9606, 8329, 5357, and 3521 (see Table I for
oligonucleotide chemistries). B, quantitation of the above
gels.
, ISIS 9606;
, ISIS 9605;
, ISIS 8329;
, ISIS 5357;
, ISIS 3521.
protein has a very long half-life (approximately 24 h)
(40) and therefore to substantially reduce expression of this protein
(by >80%) should require oligonucleotide activity for at least three
half-lives of the protein. The ability of the 2'-MOE modification to
withstand nuclease digestion prompted us to determine whether
oligonucleotides containing these modifications could reduce expression
of PKC-
mRNA for extended periods of time. Oligonucleotides ISIS
3521, ISIS 9605, and ISIS 9606 (as well as two scrambled control
oligonucleotides) were transfected into A549 cells and PKC-
mRNA
expression determined 72 h later. At this time ISIS 3521 is
inactive at concentrations up to 100 nM, whereas ISIS 9605 and ISIS 9606 are able to maintain reduced levels of PKC-
expression
with an oligonucleotide IC50 of approximately 100 nM (Fig. 5). The specificity
in oligonucleotide-dependent reduction in PKC-
mRNA
expression by 2'-MOE containing oligonucleotides was also examined.
First, two scrambled control oligonucleotides (ISIS 12963 and ISIS
13009) with the same base composition and chemistry as ISIS 9605 and
ISIS 9606, respectively, were found to be without effect on PKC-
mRNA expression at concentrations as high as 200 nM
(Fig. 5). Second, when the expression of multiple PKC-isozyme mRNA
transcripts from cells treated with ISIS 9606 was examined, only the
expression of PKC-
was found to be reduced (Fig.
6). Levels of PKC-
,
,
, and
mRNA were unaffected.
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Fig. 5.
Dose response for the reduction in PKC-
mRNA expression in A549 cells by 2'-MOE modified oligonucleotides
72 h after oligonucleotide addition to cells. A549 cells were
treated with the indicated concentration of oligonucleotide (0, 50, 100, or 200 nM) and DOTMA/DOPE as described under
"Experimental Procedures" for a period of 4 h. Cells were then
washed and allowed to recover for a further 68 h. At this time
PKC-
mRNA expression was determined by Northern blotting and
quantitated using a PhosphorImager. The oligonucleotides used were ISIS
9605 (
), ISIS 9606 (
), ISIS 3521 (
), ISIS 12963 (scrambled
ISIS 9605) (
), and ISIS 13009 (scrambled ISIS 9606) (
).
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Fig. 6.
Specificity of oligonucleotide mediated
reduction in PKC- mRNA expression. A549 cells were treated
with 100 nM ISIS 9606 and DOTMA/DOPE, or DOTMA/DOPE alone
as described under "Experimental Procedures" for a period of 4 h. Cells were then washed and allowed to recover for a further 20 h. At this time PKC-
,
,
,
, and
mRNA expression was
determined by Northern blotting.
Protein
Expression and PKC Enzyme Activity in A549 Cells--
The ability of
ISIS 9606 to maintain reduced levels of PKC-
mRNA expression for
up to 72 h suggested to us that this oligonucleotide would
effectively reduce PKC-
protein expression. A549 cells were treated
with 100 nM ISIS 9606 and PKC-
protein expression was
determined by Western blotting. PKC-
protein expression was reduced
in a time-dependent manner, consistent with a half-life of
the protein of approximately 24 h. The oligonucleotide-mediated reduction in PKC-
protein expression was again shown to be specific for this isozyme, as levels of PKC-
and -
were unaffected by ISIS
9606 (Fig. 7).
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Fig. 7.
Oligonucleotide mediated reduction in PKC-
protein expression in A549 cells. A, A549 cells were
treated with 100 nM of ISIS 9606 and DOTMA/DOPE as
described under "Experimental Procedures" for a period of 4 h.
Cells were then washed and allowed to recover for either a further 20, 44, or 68 h. At this time total cell protein was extracted and the
expression of PKC-
, -
, or -
determined by Western blotting.
B, quantitation of the gels in A: PKC-
(
),
-
(
), or -
(
).
protein expression
by ISIS 9606 (Fig. 8B). A scrambled control oligonucleotide
(ISIS 13009) was without effect on PKC-
protein expression or kinase
activity. The remaining kinase activity present after oligonucleotide
is likely to be due to a combination of residual PKC-
protein as well as other phospholipid-dependent kinases present in
A549 cells (PKC-
,
, and
).
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Fig. 8.
Oligonucleotide mediated reduction in PKC
enzyme activity in A549 cells. A, A549 cells were
treated with 100 nM of either ISIS 9606 or ISSI 13009 and
DOTMA/DOPE as described under "Experimental Procedures" for a
period of 4 h. Cells were then washed and allowed to recover for a
further 68 h. PKC enzyme activity was determined by measuring the
ability of a cytosolic protein extract to phosphorylate a synthetic
peptide substrate in the absence or presence of phosphatidylserine in
an enzyme-linked immunosorbent-based assay. Solid bars are
kinase activity in the presence of 50 µg/ml phosphatidylserine,
open bars in the absence of phosphatidylserine.
B, samples of protein extract used in the above assay were
analyzed by Western blotting to confirm a specific reduction in PKC-
protein expression by ISIS 9606.
Reduction on Phorbol Ester-mediated AP-1 Gene
Expression in A549 Cells--
AP-1 is a sequence-specific
transcriptional complex composed of members of the fos and
jun families (41). Activation of the AP-1 family of
transcription factors is complex, involving both transcriptional and
post-translational regulation. The phorbol ester class of tumor
promoters are known to be strong inducers of AP-1 via activation of
PKC. In A549 cells, 12-O-tetradecanoylphorbol-13-acetate (TPA) causes a time-dependent accumulation of mRNA
transcripts of multiple members of the AP-1, including
c-jun, junB, and c-fos. A maximum
increase in expression of these transcripts occurs at between 30 and 60 min after TPA addition (data not shown). In order to determine whether
PKC-
is involved in regulating this response, A549 cells were
treated with ISIS 9606 prior to treatment with TPA. The induction of
both c-fos and junB mRNA were unaffected by
PKC-
depletion, however, the up-regulation of c-jun was
almost completely inhibited (Fig. 9). A
scrambled control oligonucleotide was without effect on the
up-regulation of any of these AP-1 family members (Fig. 9).
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Fig. 9.
Effect of PKC- depletion on the phorbol
ester-mediated increase in transcription factor mRNA
expression. A, A549 cells were treated with 100 nM of either ISIS 9606 or ISSI 13009 and DOTMA/DOPE as
described under "Experimental Procedures" for a period of 4 h.
Cells were then washed and allowed to recover for a further 68 h.
Cells were then treated with 100 nM TPA for 30 min and the
expression of either c-fos, junB, c-jun, or GAPDH mRNA
expression determined by Northern blotting. B, quantitation
of the expression of c-fos, junB, and c-jun,
mRNA shown in A.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
mRNA and protein expression. In addition, only expression of
PKC-
was reduced by oligonucleotide treatment of the cells.
is
thought to mediate transcriptional activation of the human
transglutaminase 1 gene (54); and PKC-
has been shown to selectively
stimulate the transcription factor complex AP-1 in T-lymphocytes (55).
Our strategy has been to use oligonucleotides to help define functions
for PKC isozymes, this has proven successful for PKC-
, -
, and
-
(7) (56, 57).
expression was required for
the phorbol ester-mediated up-regulation of the cell adhesion molecule
ICAM-1 expression in A549 cells (7). The 5'-regulatory domain of the
ICAM-1 gene has been shown to contain multiple promoter sites (58-60).
Of these, the most important regulators of ICAM-1 gene expression
appear to be NF
B and TPA-responsive elements (61). In A549 cells the
key regulators of AP-1 that are expressed are c-jun, junB
and c-fos (Fig. 9).2 The effectiveness of the
2'-MOE modified oligonucleotides at reducing PKC-
protein expression
allowed us to examine whether the phorbol ester-mediated increase in
expression of these three transcription factors could be regulated by
this PKC isozyme. Of the three, only the expression of c-jun
was inhibited, suggesting that PKC-
increases ICAM-1 expression
through a mechanism which requires c-jun expression.
in regulating c-jun expression in A549 epithelial cells, suggesting that this
TPA-responsive element binding transcription factor may be required for
complete up-regulation of ICAM-1 mRNA expression in response to
phorbol esters.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom reprint requests should be addressed. Tel.: 760-603-2364; Fax: 760-603-2600; E-mail: nick_dean{at}isisph.com.
The abbreviations used are: PKC, protein kinase C; AP-1, activator protein-1; CGE, capillary gel electrophoresis; DOTMA/DOPE, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride/dioleoyl phosphatidylethanolamine; GAPDH, glycerol-3-phosphate dehydrogenase; 2'-MOE, 2'-O-(2-methoxy)ethyl; P=O, phosphodiester; P=S, phosphorothioate; TPA, 12-O-tetradecanoylphorbol-13-acetate; ICAM, intracellular adhesion molecule.
2 R. A. McKay, L. J. Miraglia, L. L. Cummins, S. R. Owens, H. Sasmor, and N. M. Dean, unpublished observation.
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