2-o-(2-methoxy)ethyl-modified anti-intercellular adhesion molecule 1 (icam-1) oligonucleotides...

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.

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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 http://www-jbc.stanford.edu/jbc/11994


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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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FIG. 7. Inhibition of translation initiation proves to be key mode of action for 2*-O-(2-methoxy)ethyl-modified oligonucleotidesthat target the 5* cap region of the ICAM-1 transcript in HUVECs. Polysome distribution profiles for cells treated with ISIS 12344(phosphodiester scramble control), ISIS 11158 (phosphodiester antisense), ISIS 12345 (phosphorothioate scramble control), and 11159 (phosphor-othioate antisense), each at 50 nM. A, ethidium bromide-stained agarose gels; B, Northern blots. Oligonucleotide-treated cells were harvested 4 hpost TNF-a induction (1-h period) as described. A total RNA standard (fraction S) was included on each gel for blot to blot normalization.



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2-o-(2-methoxy)ethyl-modified anti-intercellular adhesion molecule 1 (icam-1) oligonucleotides...

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