-
BennettLesnik, Henri M. Sasmor and C. FrankCondon, Shin
Cheng-Flournoy, Elena A. Brenda F. Baker, Sidney S. Lot, Thomas
P.
Umbilical Vein Endothelial Cells Translation Initiation Complex
in Human Inhibit Formation of the ICAM-1Increase the ICAM-1 mRNA
Level and (ICAM-1) Oligonucleotides SelectivelyAnti-intercellular
Adhesion Molecule 1
-(2-Methoxy)ethyl-modifiedO-2GENETICS:SYNTHESIS, AND MOLECULAR
NUCLEIC ACIDS, PROTEIN
doi: 10.1074/jbc.272.18.119941997, 272:11994-12000.J. Biol.
Chem.
http://www.jbc.org/content/272/18/11994Access the most updated
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2*-O-(2-Methoxy)ethyl-modified Anti-intercellular Adhesion
Molecule1 (ICAM-1) Oligonucleotides Selectively Increase the ICAM-1
mRNALevel and Inhibit Formation of the ICAM-1 Translation
InitiationComplex in Human Umbilical Vein Endothelial Cells*
(Received for publication, November 19, 1996)
Brenda F. Baker, Sidney S. Lot, Thomas P. Condon, Shin
Cheng-Flournoy, Elena A. Lesnik,Henri M. Sasmor, and C. Frank
Bennett
From Isis Pharmaceuticals, Inc., Carlsbad, California 92008
Little is known about the mechanisms that accountfor inhibition
of gene expression by antisense oligonu-cleotides at the level of
molecular cell biology. For thispurpose, we have selected potent
2*-O-(2-methoxy)ethylantisense oligonucleotides (IC50 5 2 and 6 nM)
that tar-get the 5* cap region of the human intercellular adhe-sion
molecule 1 (ICAM-1) transcript to determine theireffects upon
individual processes of mRNA metabolismin HUVECs. Given the
functions of the 5* cap structurethroughout mRNA metabolism,
antisense oligonucleo-tides that target the 5* cap region of a
target transcripthave the potential to modulate one or more
metabolicstages of the message inside the cell. In this study
wefound that inhibition of protein expression by theseRNase H
independent antisense oligonucleotides wasnot due to effects on
splicing or transport of the ICAM-1transcript, but due instead to
selective interferencewith the formation of the 80 S translation
initiationcomplex. Interestingly, these antisense
oligonucleotidesalso caused an increase in ICAM-1 mRNA abundance
inthe cytoplasm. These results imply that ICAM-1 mRNAturnover is
coupled in part to translation.
Antisense oligonucleotides have been shown to be effectiveagents
for inhibition of gene expression at the mRNA level(13). They may
be described as exogenous regulators of mRNAmetabolism intended to
sterically interfere with one or moremetabolic processes upon
hybridization, such as initiation oftranslation, or to promote
enzyme-mediated mRNA degrada-tion by formation or exposure of a
region for nuclease activity,such as RNase H. The mode of action of
an antisense oligonu-cleotide in cells is dependent upon its
composition (sugar, back-bone, and base residues) and mRNA binding
site location (59-UTR, coding region, 39-UTR).1 Although several
types ofantisense oligonucleotides, which differ in composition and
tar-get site, have been found to be effective agents for
sequence-specific inhibition of gene expression in mammalian cells,
di-
rect or detailed evidence of their mode(s) of action
remainslimited (49).
Intercellular adhesion molecule 1 (ICAM-1) is one of severalcell
adhesion molecules expressed on the cell surface of vascu-lar
endothelium that participates in a broad range of immuneand
inflammatory responses (10). ICAM-1 is also expressed
onnonendothelial cells, such as keratinocytes, monocytes,
andfibroblasts in response to inflammatory mediators.
Elevatedlevels of ICAM-1 expression have been observed in a number
ofimmune-related human diseases (11, 12), e.g. rheumatoid
ar-thritis, psoriasis, and asthma. Thus, regulation of ICAM-1
geneexpression is of therapeutic interest (1315). The ICAM-1
genehas been sequenced, and the transcription initiation site
hasbeen characterized for several cell lines following induction
bya variety of cytokines (16, 17), including human umbilical
veinendothelial cells (HUVECs) with induction by TNF-a (18).
Previous research has demonstrated that elevated expres-sion of
ICAM-1 may be controlled in cells by phosphorothioate-modified
antisense oligonucleotides (4, 5). At that time themost effective
oligonucleotides were those that were compatiblewith RNase H and
targeted the 39-UTR of the transcript. Sincethen advances in
chemical synthesis have brought forth a num-ber of oligonucleotide
modifications at the 29-sugar positionwhich give significant
increases in duplex stability and nucle-ase resistance but do not
support RNase H activity (19). Anti-sense oligonucleotides that
bind more tightly to the targetmRNA are expected to be more
effective at interfering with theprocesses of metabolism when bound
at suitable locations.Bulky substituents at this position also have
been shown toprovide a high degree of nuclease resistance.
Biophysical and biological analysis of a set of these
uniformly29-modified oligonucleotides (29-O-methyl (20), 29-O-allyl
(20),29-O-(2-methoxy)ethyl (21), and 29-fluoro (22)) that target
the 59terminus of the ICAM-1 transcript led to our selection of
theexceptionally active 29-O-(2-methoxy)ethyl-modified
oligonu-cleotides, ISIS 11158 and 11159, for an investigation of
theirintracellular mode of action in HUVECs (Fig. 1 and Table
I).The 59 cap of eukaryotic mRNA has been shown to be a struc-tural
element that functions throughout mRNA metabolism(2436). Therefore,
antisense oligonucleotides which target the59 cap region of a
designated transcript have the potential tomodulate one or more
metabolic stages of the message insidethe cell (37). In this study
the antisense mode of action wasdetermined by evaluation of the
target transcripts metabolicprocesses (splicing, transport,
translation, and stability) follow-ing antisense treatment and
induction of gene expression.
EXPERIMENTAL PROCEDURES
Cells and Cell CultureHUVECs were purchased from CloneticsCorp.
(San Diego, CA) and cultivated in the designated EBM medium
* The costs of publication of this article were defrayed in part
by thepayment of page charges. This article must therefore be
hereby markedadvertisement in accordance with 18 U.S.C. Section
1734 solely toindicate this fact.
To whom correspondence should be addressed: Isis
Pharmaceuti-cals, Inc., 2292 Faraday Ave., Carlsbad, CA 92008.
Tel.: 619-931-9200;Fax: 619-931-0209.
1 The abbreviations used are: UTR, untranslated region;
ICAM-1,intercellular adhesion molecule 1; HUVECs, human umbilical
veinendothelial cells; TNF-a, tumor necrosis factor a; PE,
phycoerythrin;DTT, dithiothreitol; PBS, phosphate-buffered saline;
G3PDH, glycerol-3-phosphate dehydrogenase.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 18, Issue of
May 2, pp. 1199412000, 1997 1997 by The American Society for
Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at
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supplemented with 10% fetal bovine serum from HyClone (Logan,
UT).Cells were used for experiments from passages two to ten at
8090%confluency.
Oligonucleotide SynthesisOligonucleotides were synthesized
utiliz-ing conventional solid-phase triester chemistry (38). The
29-deoxy (Per-septive Biosystems), 29-O-methyl (ChemGene), and
29-O-allyl (Boeh-ringer Mannheim) phosphoramidites were purchased
from commercialsources. 29-O-(2-Methoxy)ethyl and 29-fluoro
phosphoramidites weremanufactured either in house (Dr. Bruce Ross)
or under contract (R.I.Chemicals).
Oligonucleotide Treatment of HUVECsCells were washed threetimes
with Opti-MEM (Life Technologies, Inc.) pre-warmed to 37
C.Oligonucleotides were premixed with 10 mg/ml Lipofectin (Life
Tech-nologies, Inc.) in Opti-MEM, serially diluted to the desired
concentra-tions, and applied to washed cells. Basal and untreated
(no oligonucleo-tide) control cells were also treated with
Lipofectin. Cells wereincubated for 4 h at 37 C, at which time the
medium was removed andreplaced with standard growth medium with or
without 5 ng/ml TNF-a(R & D Systems). Incubation at 37 C was
continued until the indicatedtimes.
Quantitation of ICAM-1 Protein Expression by
Fluorescence-activatedCell SorterCells were removed from plate
surfaces by brief trypsiniza-tion with 0.25% trypsin in PBS.
Trypsin activity was quenched with asolution of 2% bovine serum
albumin and 0.2% sodium azide in PBS
(1Mg/Ca). Cells were pelleted by centrifugation (1000 rpm,
BeckmanGPR centrifuge), resuspended in PBS, and stained with 3
ml/105 cells ofthe ICAM-1 specific antibody, CD54-PE (Becton
Dickinson) and 0.1 mgof the control antibody, IgG2b-PE
(Pharmingin). Antibodies were incu-bated with the cells for 30 min
at 4 C in the dark, under gentleagitation. Cells were washed by
centrifugation procedures and thenresuspended in 0.3 ml of FacsFlow
buffer (Becton Dickinson) with 0.5%formaldehyde (Polysciences).
Expression of cell surface ICAM-1 wasthen determined by flow
cytometry using a Becton Dickinson FACScan.Percentage of the
control ICAM-1 expression was calculated as
follows:[(oligonucleotide-treated ICAM-1 value) 2 (basal ICAM-1
value)/(non-treated ICAM-1 value) 2 (basal ICAM-1 value)].
Nuclear Runoff Transcription AnalysisCells were treated with
ol-igonucleotide at a concentration of 50 nM for 4 h. Cells were
harvested2 h post TNF-a induction. Preparation of nuclei and
measurement ofgene transcription were based upon published
procedures (39). Equalcounts/min of 32P-labeled RNA were hybridized
to slot-blot membranesloaded with cDNA fragments for ICAM-1 and
G3PDH. The fX 174 DNAHaeIII digest was included as a control.
Total RNA Isolation and Northern AnalysisTotal cellular RNA
wasisolated from HUVECs by lysis and precipitation with
Catrimox-14(Iowa Biotechnology Corp.), followed by extraction of
the DNA from theprecipitate with lithium chloride. Isolated RNA was
separated on a1.0% agarose gel containing 1.1% formaldehyde, then
transferred and
TABLE IBiological and biophysical profiles of modified antisense
oligonucleotides
Antisense activity (IC50) was determined by measurement of cell
surface expression of ICAM-1 protein following treatment with
eacholigonucleotide at six concentrations in the range of 1.650 nM,
as described under Experimental Procedures. IC50 values were
calculated fromthe average of two sets of dose-response data
points, where NA indicates that no IC50 was achieved or observed in
the given dose range. Thermalmelt analysis was performed as
described previously (23), where DTm (C) 5 DTm(parent) 2
DTm(modified), and DDG
037 5 DG
037(parent) 2
DG037(modified). Boldface highlights entry as most active 29
modification of the phosphodiester (P5O) and phosphorothioate
(P5S), respectively.
ISIS number 29-Sugar modificationAntisense activity Duplex
stability
Rank IC50 Tm DTm DDG0
37
nM C CPO
11158 -O-(2-Methoxy)ethyl 1 2.1 87.1 37.0 29.1412461 -O-Allyl NA
76.5 26.4 27.373214 -O-Methyl NA 83.7 33.6 27.853061 -H NA 58.4 8.3
22.8
PS11159 -O-(2-Methoxy)ethyl 2 6.5 79.2 29.1 26.9511665 -F 3 25
87.9 37.8 28.4712462 -O-Allyl 4 34 70.3 20.2 25.753067 -H 5 41 50.1
Parent Parent3224 -O-Methyl 6 .50 76.4 24.3 26.16
FIG. 1. A, 59-UTR sequence of theICAM-1 mRNA derived from normal
HU-VECs induced by TNF-a (18). The anti-sense binding region is
underlined. B, an-tisense oligonucleotide sequence andstructures.
Antisense oligonucleotides arecomplementary to nucleotides 120 of
theICAM-1 mRNA. The 39-terminal residueof each oligonucleotide was
a 29-deoxy.29-O-(2-Methoxy)ethyl-modified cytosineswere methylated
at the 5 position, e.g.5-methylcytosine. 29-O-Methyl,
29-O-allyl,and 29-fluoro modified oligonucleotidescontained uridine
in lieu of thymine.
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UV-crosslinked (Stratalinker 2400, Stratagene) to a Hybond N1
nylonmembrane (Amersham). Blots were hybridized with
Prime-a-GenecDNA probes (Promega) using RapidHyb (Amersham). Probes
weregenerated from the following human cDNA restriction or PCR
frag-ments: a 1.88-kilobase ICAM-1 fragment (BBG 58, R & D
Systems), a1.1-kilobase G3PDH fragment (pHcGAP, American Tissue
Culture Col-lection), and a 2-kilobase E-selectin fragment (4). A
Molecular Dynam-ics PhosphorImager was utilized to quantitate
Northern blot probesignals.
Nuclear and Cytoplasmic RNA FractionationHarvested cells
wereincubated in mild lysis buffer (0.5% Nonidet P-40, 10 mM
Tris-Cl (pH7.4), 140 mM KCl, 5 mM MgCl2, and 1 mM DTT) for 5 min at
4 C (40).Nuclei were separated from the cytosol by centrifugation
at 1000 3 g for5 min at 4 C. The cytosol was transferred to a
sterile tube containing3 volumes of a denaturing solution (5.3 M
guanidinium isothiocyanate,37.5 M sodium citrate (pH 7.0), 0.75%
Sarkosyl, and 0.15 M b-mercap-toethanol), phenol-extracted under
acidic conditions (pH 4.0), and iso-propyl alcohol-precipitated
(41). The cytosol precipitate was redissolvedin 300 ml of the
denaturing solution plus 30 ml of 2 M sodium acetate (pH4.0) and
reprecipitated with isopropyl alcohol. A second lysis step
wasperformed on the collected nuclei to ensure removal of the
cytosolfraction. Washed nuclei were lysed at room temperature by
the additionof 1 ml of Catrimox-14 surfactant. Nuclear RNA was
isolated by theLiCl extraction procedure.
Polysome Profile AnalysisApproximately 106
oligonucleotide-treated cells were pelleted, washed with PBS, then
mixed into 0.3 ml ofcold lysis buffer (0.5% Nonidet P-40, 10 mM
Tris-Cl (pH 7.4), 140 mMKCl, 5 mM MgCl2, 1 mM DTT, 100 mg/ml
cycloheximide, and RNaseinhibitor (5 Prime 3 Prime)) and incubated
for 5 min at 4 C. Nucleiwere pelleted at 1000 3 g, and the
resulting supernatant was layered ona 1035% (w/v) linear sucrose
gradient (4 ml), with a 50% cushion (0.75ml), in gradient buffer
(10 mM Tris (pH 8.0), 50 mM potassium acetate,1 mM magnesium
acetate, 1 mM DTT). Gradients were centrifuged at35,000 rpm for 3 h
at 5 C with a Beckman SW55 Ti rotor. 250-mlfractions were collected
with an Isco model 185 density gradient frac-tionator connected to
a Pharmacia UV monitor and fraction collector.Collected fractions
were treated with proteinase K (0.2 mg/ml) in 0.2%SDS at 42 C for
20 min, phenol-extracted, and ethanol-precipitated. 5to 10 mg of
tRNA was added to each fraction prior to precipitation.Precipitated
RNA was applied to a 1.0% denaturing agarose gel andanalyzed by
standard ethidium bromide staining and Northern blottingtechniques.
Fractions 1 and 2 and 3 and 4 were combined for gelanalysis.
RESULTS AND DISCUSSION
Scrambled control oligonucleotides were tested in a
dose-response analysis to verify that inhibition of ICAM-1
proteinexpression by the 29-O-(2-methoxy)ethyl-modified
oligonucleo-
tides, ISIS 11158 and 11159, was sequence-specific. The
respec-tive scrambled control oligonucleotides, ISIS 12344 and
12345,showed negligible effects on ICAM-1 protein expression
(Fig.2). As indicated in Table I, both the phosphodiester,
ISIS11158, and the phosphorothioate, ISIS 11159,
29-O-(2-methoxy)ethyl-modified oligonucleotides were more potent
inhibitors ofICAM-1 expression in HUVECs than the analogous
RNaseH-compatible phosphorothioate oligodeoxynucleotide,
ISIS3067.
Intracellular distribution of the
29-O-(2-methoxy)ethyl-mod-ified oligonucleotides was determined by
fluorescence micros-copy, using fluorescein-labeled
oligonucleotides, to further com-pare and delineate the basis of
their antisense activity withrespect to the first generation
29-deoxyoligonucleotides (Fig. 3).As reported previously (42)
treatment of HUVECs with thefluorescein-labeled 29-deoxy
phosphorothioate oligonucleotide,in the presence of the cationic
lipid formulation (Lipofectin,Life Technologies, Inc.), resulted in
a heterogeneous accumu-lation of the oligonucleotide in the cell
nucleus as well as inpunctate cytoplasmic vesicles (Fig. 3A).
Treatment of HUVECswith the 29-deoxy phosphodiester analog showed a
diffuse dis-tribution of label in the cytoplasm (Fig. 3B),
attributed todegradation of the oligomer by nucleases (43, 44). In
compari-son, the fluorescein-labeled
29-O-(2-methoxy)ethyl-modifiedphosphorothioate oligonucleotide
yielded a distribution pattern(Fig. 3C) similar to the 29-deoxy
analog (Fig. 3A), with a highdegree of localization in the nucleus.
However in striking con-trast to the 29-deoxy analog (Fig. 3B), the
29-O-(methoxy)ethylphosphodiester showed a homogeneous nuclear
localization(Fig. 3D), attributed to its greater resistance to
nucleases, theabsence of nonspecific phosphorothioate-protein
interactions(45, 46), and possibly more compatible interactions
with thelipid formulation for uptake and delivery.
Total cellular RNA was isolated and analyzed to determinewhether
inhibition of ICAM-1 protein expression by ISIS 11158and 11159
resulted from antisense-promoted degradation ofthe target
transcript, an end point observed following treat-
FIG. 2. 2*-O-(2-Methoxy)ethyl-modified anti-ICAM-1
oligonu-cleotides are exceptionally effective at inhibiting ICAM-1
pro-tein expression in comparison to the RNase H-competent
ISIS3067. Dose-response analysis for ISIS 11158 and 11159, in
comparisonto ISIS 3067 and the controls ISIS 10588, 12344, and
12345. The controlsequence, 59-GATCGCGTCGGACTATGAAG-39, is a
scramble of theantisense sequence, i.e. identical in base
composition. ISIS 10588 is thephosphorothioate oligodeoxynucleotide
analog. ISIS 12344 is the 29-O-(2-methoxy)ethyl phosphodiester
analog and ISIS 12345 is the 29-O-(2-methoxy)ethyl phosphorothioate
analog.
FIG. 3. Modification-dependent differences in
intracellulardistribution of oligonucleotides observed 4 h post
delivery withLipofectin. Photographs from fluorescent microscopy
(Nikon Op-tiphot-2, 1003) of cells treated with
59-fluorescein-labeled anti-ICAM-1oligonucleotides. A, ISIS 3067
analog (29-deoxy phosphorothioate); B,ISIS 3061 analog (29-deoxy
phosphodiester); C, ISIS 11159 analog (29-O-(2-methoxy)ethyl
phosphorothioate); and D, ISIS 11158 analog (29-O-(2-methoxy)ethyl
phosphodiester). These data demonstrated thatboth the
phosphodiester and phosphorothioate 29-O-(2-methoxy)ethyl-modified
oligonucleotides were effectively delivered into the cell nucleusin
the presence of cationic lipids (42).
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ment with most active RNase H-compatible antisense
oligonu-cleotides (4, 5). Total RNA was harvested at 4 and 20 h
follow-ing a 1-h TNF-a induction period for untreated
andoligonucleotide-treated HUVECs. Interestingly, Northern
blotanalysis showed a significant increase in relative abundance
ofthe ICAM-1 transcript in those cells treated with the anti-ICAM-1
oligonucleotides at both time points (Fig. 4). Nuclearrunoff
experiments showed that this increase in transcriptabundance was
not due to an increase in the rate of transcrip-tion of the ICAM-1
gene (data not shown).
To determine whether the antisense effect on transcriptabundance
was specific to ICAM-1, blots were probed for theE-selectin
transcript whose expression is also transiently in-duced by TNF-a
in HUVECs. As with ICAM-1 an increase inabundance of the E-selectin
transcript was observed only inthose cells treated with the
29-O-(2-methoxy)ethyl-modified ol-igonucleotide, ISIS 11929, that
targets the 59-terminal regionof the E-selectin transcript (Fig.
5). The increase in targettranscript abundance following antisense
treatment may be anattribute of these transiently expressed
transcripts in combi-
nation with the antisense mode of action.The 59 cap structure of
mRNA has been shown to facilitate
splicing of the first intron of pre-mRNA constructs in
severaldifferent systems (48, 49). The ICAM-1 gene consists of
sevenexons separated by six introns, with intron 1
approximately4000 nucleotides in length (16). Evaluation of the
Northernblots showed that this set of modified antisense
oligonucleo-tides had no effect on splicing of the ICAM-1 pre-mRNA
to themature transcript, as evidenced by lack of ICAM-1 mRNAspecies
or intermediates of longer lengths (data not shown).
Nuclear and cytosolic fractionation was utilized to determineif
antisense inhibition of ICAM-1 protein expression resultedfrom
inhibition of transport of the mature transcript out of thenucleus
to the cytoplasm. Fractionated mRNA was evaluatedby Northern
analysis 2 h post TNF-a induction for 29-O-(2-methoxy)ethyl
oligonucleotide-treated (phosphorothioate andphosphodiester;
antisense and control each at 50 nM) and un-treated cells (Fig. 6).
At this time point no substantial alter-ation in the abundance of
the ICAM-1 transcript was observedin the nuclear fractions of
antisense treated cells (110114%)versus scrambled control treated
(113118%) and untreated(100%). In contrast, a significant increase
in the abundance ofthe ICAM-1 transcript was observed in the
cytosolic fractionfrom the antisense treated cells, ISIS 11158
(230%) and ISIS
FIG. 4. Increase in target transcript abundance occurs in
cellstreated with 2*-O-(2-methoxy)ethyl-modified antisense
oligonu-cleotides which complement the 5* cap region of ICAM-1.
North-ern analysis of total cellular RNA. A, Northern blots for
ICAM-1 andG3PDH. B, bar graph showing relative abundance of ICAM-1
tran-script, normalized to the glyceraldehyde-3-phosphate
dehydrogenasemRNA (G3PDH). Each oligonucleotide was tested at a
concentration of50 nM. Cells were harvested at 4 and 20 h post 1-h
induction by TNF-a.ISIS 11929 is a phosphodiester
29-O-(2-methoxy)ethyl-modified anti-E-selectin oligonucleotide used
as a control in this experiment.
FIG. 5. Increase in target transcript abundance also occurs
incells treated with 2*-O-(2-methoxy)ethyl-modified antisense
oli-gonucleotides which target the 5* cap region of
E-selectin.Northern analysis of total cellular RNA. A, Northern
blots for E-selectinand G3PDH. B, bar graph showing relative
abundance of E-selectintranscript, normalized to the G3PDH
transcript. Each oligonucleotidewas tested at a concentration of 50
nM. Cells were harvested at 4 and20 h post full-time induction by
TNF-a. ISIS 11929, 59-GAAGTCAGC-CAAGAACAGCT-39, is complementary to
nucleotides 120 of the E-selectin transcript (47).
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11159 (181%), in comparison to the scrambled control
treated,ISIS 12344 (133%) and ISIS 12345 (108%), and untreated
cells(100%). Relative abundance of the ICAM-1 transcript in
eachcompartment was also determined 4 h post 1-h TNF-a induc-tion.
Under these conditions, the relative abundance of theICAM-1
transcript was 451 and 425% in the cytosolic fractionand 128 and
126% in the nuclear fractions of the antisensetreated cells, ISIS
11158 and 11159, respectively, relative tothe untreated cells. The
significant increase in abundance ofthe transcript in the cytoplasm
of the antisense treated cellssuggested a decrease in the rate at
which the transcript isnormally degraded. The lack of a substantial
change in ICAM-1mRNA abundance in the nuclear fraction indicated
that theantisense oligonucleotides did not significantly affect the
nu-cleocytoplasmic transport rate of the mature
ICAM-1transcript.
Polysome profiles were utilized to determine the effect
ofantisense oligonucleotide treatment upon the translation proc-ess
of the target ICAM-1 transcript (Fig. 7). ICAM-1 proteinand mRNA
were evaluated 4 h after a 1-h TNF-a inductionfrom cells treated
with antisense oligonucleotides ISIS 11158and 11159, and their
respective scrambled controls ISIS 12344and 12345. Cytosolic
extracts were sedimented by linear su-crose gradient centrifugation
(1035%). The ethidium bromide-stained gel of the fractionated RNA
showed a respectable sep-aration of the subpolysomal and polysomal
pools (Fig. 7A).Assignment of the fractions were verified by UV
absorbanceplots obtained during fractionation. Northern blots
showed asignificant difference in the polysomal distribution of
theICAM-1 transcript in cells treated with ISIS 11158 and 11159,in
comparison to those of the controls, ISIS 12344 and 12345(Fig. 7B).
The polysome profiles for the ISIS 11158 and 11159
treated cells showed the majority (71 and 65%, respectively)
ofthe full-length target transcript localized in the
subpolysomefractions, e.g. 40 s and 60 s, whereas the ISIS 12344
and 12345polysome profiles showed the majority (77 and 86%,
respec-tively) of the target transcript in the monosome and
polysomefractions.
The polysome profile data for ISIS 11158 and 11159 indicatethat
inhibition of ICAM-1 protein expression occurs throughinterference
with translation initiation and specifically riboso-mal assembly,
as indicated by the dramatic redistribution oftranscript into the
subpolysome fractions. The formation of astable antisense-mRNA
duplex (or secondary structure) in the59 cap region is likely the
basis of this effect (see Table I). Theincrease in abundance of the
ICAM-1 mRNA in the cytosolicfraction of the antisense treated cells
in conjunction with thechange in the polysome distribution patterns
indicates that oneof the target transcripts decay pathways is
coupled to transla-tion. These data are consistent with
observations of transcriptsthat contain stability determinants in
the coding region, e.g.c-fos and c-myc (50, 51).
Regulation of gene expression may occur at one or morestages of
mRNA metabolism. The most well known examples ofregulation through
mRNA metabolism have been found at thestages of translation (52)
and degradation (53) of the maturetranscript, where certain mRNA
sequences and structural ele-ments have been found to be key
regulatory determinants. Ofparticular relevance, it has been shown
that stable secondaryand tertiary structures located in the
59-terminal regionmay regulate initiation of translation (5456).
The 29-O-(2-me-thoxy)ethyl-modified antisense oligonucleotides,
complemen-tary to the 59-terminal region of the target transcript
(nucleo-tides 120), mimic this endogenous mode of regulation in
cells
FIG. 6. 2*-O-(2-Methoxy)ethyl-modi-fied antisense
oligonucleotides haveno effect on nuclear-cytosolic distri-bution
of the ICAM-1 transcript. Theincrease in ICAM-1 mRNA abundance
oc-curs primarily in the cytoplasm. Northernanalysis of nuclear and
cytoplasmic RNAfractions 2 h post TNF-a induction. A,Northern blots
for ICAM-1 and G3PDH.B, bar graphs showing relative abun-dance of
ICAM-1 transcript, normalizedto G3PDH. Each oligonucleotide
wastested at a concentration of 50 nM.
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by inhibiting formation of the 80 S translation initiation
com-plex. We believe that this event in turn affects the
turnoverrate of the transiently expressed ICAM-1 transcript.
AcknowledgmentsWe thank John Brugger and Pierre Villiet
foroligonucleotide synthesis, Tracy Reigle for graphic
illustrations, LexCowsert for technical advice, and Stan and
Rosanne Crooke for theircomments on the manuscript.
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(1993) Antisense Research and Applications
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