Bijsterbosch1 1 Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, the Netherlands; 2 Division of Molecular Toxicology, Leiden/Amsterdam Center for Drug Research, t
Trang 1Selection of effective antisense oligodeoxynucleotides with a green fluorescent protein-based assay
Discovery of selective and potent inhibitors of glutathione S -transferase Mu expression Peter A C.¢t Hoen1,2, Bram-Sieben Rosema1, Jan N M Commandeur2, Nico P E Vermeulen2,
Muthiah Manoharan3, Theo J C van Berkel1, Eric A L Biessen1and Martin K Bijsterbosch1
1 Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, the Netherlands; 2 Division of Molecular Toxicology, Leiden/Amsterdam Center for Drug Research, the Netherlands;3ISIS Pharmaceuticals, Carlsbad, California, USA
Antisense oligodeoxynucleotides (AS-ODNs) are frequently
used for the down-regulation of protein expression.Because
the majority of potential antisense sequences lacks
effect-iveness, fast screening methods for the selection of effective
AS-ODNs are needed.We describe a new cellular screening
assay for the evaluation of the potency and specificity of new
antisense sequences.Fusion constructs of the gene of interest
and the gene encoding the enhanced green fluorescent
pro-tein (EGFP) are cotransfected with AS-ODNs to COS-7
cells.Subsequently, cells are analysed for expression of the
EGFP fusion protein by flow cytometry.With the assay, we
tested the effectiveness of a set of 15 phosphorothioate
ODNs against rat glutathione S-transferase Mu1 (GSTM1)
and/or Mu2 (GSTM2).We found several AS-ODNs that
demonstrated potent, sequence-specific, and
concentration-dependent inhibition of fusion protein expression.At 0.5 lM,
AS-6 and AS-8 inhibited EGFP–GSTM1 expression by
95 ± 4% and 81 ± 6%, respectively.AS-5 and AS-10 were selective for GSTM2 (82 ± 4% and 85 ± 0.4% decrease, respectively).AS-2 and AS-3, targeted at homologous regions in GSTM1 and GSTM2, inhibited both isoforms (77–95% decrease).Other AS-ODNs were not effective or displayed non-target-specific inhibition of protein expres-sion.The observed decrease in EGFP expression was accompanied by a decrease in GSTM enzyme activity.As isoform-selective, chemical inhibitors of GSTM and GSTM knock-out mice are presently unavailable, the selected AS-ODNs constitute important tools for the study of the role of GSTM in detoxification of xenobiotics and protec-tion against chemical-induced carcinogenesis
Keywords: antisense oligodeoxynucleotide; carcinogenesis; genetic polymorphism; glutathione S-transferase; green fluorescent protein
Antisense oligodeoxynucleotides (AS-ODNs) are frequently
used for the down-regulation of gene expression, both
in vitro and in vivo [1–4].Due to the low stability of
phosphodiester ODNs (PO-ODNs) in biological systems,
more stable oligonucleotide analogues with a variety of
chemical modifications have been developed [5,6].ODNs
with a phosphorothioate-modified backbone (PS-ODNs)
are the most commonly used AS-ODNs.As AS-ODNs act
via Watson–Crick base pairing with their target mRNAs,
the nucleotide sequence of the target gene is in principle
sufficient information for the design of AS-ODNs.It appears, however, that not all AS-ODNs are potent inhibitors of protein expression.In studies where large sets
of PS-ODNs, directed against a single target gene, were tested for their ability to down-regulate their target mRNA and protein in cell culture [7,8], only 5–10% of the sequences tested appeared to be effective.Thus there is a need for rapid and accurate screening assays for the selection of effective and specifically acting AS-ODNs
Screening for effective antisense sequences is usually performed in free systems or in cell culture.Several cell-free assay systems have been described [9].These assays are fast, but not always reliable predictors for activity in biological systems.Use of differentiated cells generates more relevant information on the effectiveness of AS-ODNs in physiological systems.However, cellular assays are fre-quently hampered by low or irreproducible transfection of oligonucleotides.Furthermore, each new target requires the set-up and optimization of target-specific assays.Therefore,
we developed a new assay that uitilizes fusion constructs of a particular gene with the gene encoding enhanced green fluorescent protein (EGFP) as reporter.Because the screening is based on flow cytometric detection of EGFP expression, there is no need for development of target-specific assays.Reproducible transfections are achieved by using an easy transfectable cell line and the cotransfection of antisense PS-ODNs directed against the gene of interest and plasmids encoding the chimeric gene.In the present assay,
Correspondence to M.K.Bijsterbosch, Leiden/Amsterdam Center for
Drug Research, Division of Biopharmaceutics, PO Box 9502,
2300 RA Leiden, the Netherlands.
Fax: +31 71 5276032, Tel.: +31 71 5276038,
E-mail: bijsterb@lacdr.leidenuniv.nl
Abbreviations: ODN, oligodeoxynucleotide; AS, antisense; PO:
phosphodiester; PS, phosphorothioate; EGFP: enhanced green
fluorescent protein; GST, glutathione S-transferase; GSTM,
gluta-thione S-transferase Mu; CDNB, 1-chloro-2,4-dinitrobenzene; GSH,
glutathione; DOTAP,
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium; PMSF, phenylmethanesulfonyl fluoride;
TRITC, tetramethylrhodamine isothiocyanate; DMEM, Dulbecco’s
modified Eagle’s medium; FACS: fluorescence activated cell sorter.
Enzymes: glutathione S-transferase (EC 2.5.1.18).
(Received 13 December 2001, revised 4 April 2002,
accepted 9 April 2002)
Trang 2only transfected cells are analysed, thus eliminating the
background of expression in untransfected cells
With this assay, we determined the effectiveness of a set of
antisense PS-ODNs directed against the glutathione
S-transferase Mu1 (GSTM1) and Mu2 (GSTM2) isoforms
of the rat.GSTs play an important role in the detoxification
of DNA- and/or protein-reactive compounds by catalyzing
the conjugation of electrophilic groups with the tripeptide
glutathione [10].Approximately 50% of the Caucasian
population is deficient for GSTM1, the human orthologue
of rat GSTM1 and GSTM2 [11].Meta-analyses of
epide-miological studies reveal that this deficiency is associated
with an increased risk of lung and colorectal cancer,
especially when the GSTM-null genotype is combined with
high-inducibility of cytochrome P450 1A1 [12–18].It would,
however, be very important to demonstrate directly the
effect of differences in GSTM expression levels on the
prevalence of cancer biomarkers in in vitro and in animal
models.As GSTM knock-out mice are still unavailable,
temporal modulation of the expression of GSTM isoforms
by AS-ODNs in relevant in vitro and in vivo models is an
attractive possibility.In the current paper, several
target-specific AS-ODNs are selected from a set of 15 PS-ODNs
These ODNs selectively inhibit the expression of GSTM1
and/or GSTM2 and can be used to study the influence of
reduced GSTM expression on the detoxification of
xeno-biotics and protection against chemical-induced
carcino-genesis
M A T E R I A L S A N D M E T H O D S
Materials
PCR primers were from Eurogentec, Seraing, Belgium
PS-ODNs were synthesized according to standard
phos-phoramidite chemistry.The pEGFP-C1 plasmid and the
rabbit anti-EGFP Living Colors Peptide antibody were
from Clontech.Vent DNA Polymerase was from New
England Biolabs.1-chloro-2,4-dinitrobenzene (CDNB),
glutathione (GSH), dithiothreitol,
N-[1-(2,3-dioleoyl-oxy)propyl]-N,N,N-trimethylammonium salt (DOTAP),
and propidium iodide were from
Sigma.Phenyl-methanesulfonyl fluoride (PMSF) and Tween-20 were from
Merck.Tetramethylrhodamine isothiocyanate 5,6-mixed
isomers (TRITC) was from Molecular Probes.Cell culture
agents were from BioWhittaker.Milkpowder was from
Campina Melkunie (Eindhoven, the Netherlands).A
horseradish peroxidase-conjugated antirabbit IgG antibody
and an enhanced chemiluminescence assay were purchased
from Amersham Pharmacia Biotech.All other chemicals
were of analytical grade
Cloning of GSTM cDNAs into pEGFP-C1
Full-length cDNAs encoding GSTM1 (bases )21 to
+1039; GenBank accession no.X04229, cloned in the PstI
site of pBR322) and GSTM2 (bases)2 to +1036; GenBank
accession no.J03914, derived mRNA sequence, cloned in
the EcoRI site of pUC18) were kindly provided by D.Tu,
Pennsylvania State University, PA, USA.The cDNA
inserts were amplified and isolated by the sticky-end PCR
method [19].Briefly, the cDNAs were amplified with two
sets of PCR primers (forA + revA and forB + revB) for
each plasmid using Vent DNA polymerase (Table 1).The two PCR products were subjected to melting and cooling Four different double-stranded products were obtained, one
of which had the correct 5¢-EcoRI and 3¢-BamHI over-hanging ends.The PCR products were cloned into the EcoRI- and BamHI-digested pEGFP-C1 plasmid to gener-ate the C-terminal fusion constructs pEGFP-M1 and pEGFP-M2.Sequencing of the plasmids confirmed the in-frame ligation of the GSTM cDNAs and the absence of any PCR-induced mistakes in the inserts
TRITC-labelling of ODN
A 24-mer PO-ODN, provided with three PS-linkages at the 5¢-end and a 3¢-end primary amino group (sequence: T*A*A*GCTGTCCCGGGGTCTACGGCC), was label-led with TRITC by incubating 15 nmol ODN in 500 lL 0.1MNa-carbonate buffer (pH 9.0) with 10 molar equiv-alents of TRITC (dissolved in dimethylformamide at
2 mgÆmL)1).The mixture was incubated overnight with shaking at room temperature.The TRITC-labelled ODN was separated from unreacted TRITC by gel filtration on a Sephadex G-25 column (20· 0.4 cm), eluted with water The TRITC-ODN was precipitated from the eluent by adding 0.01 vols 1M MgCl2, 0 1 vols 3M NaAc pH 5.2, and 3 vols cold ethanol.The precipitate was formed by overnight incubation at )20 C and centrifugation for
30 min at 13000 g at 4C.The pellet was washed three times with 80% EtOH and subsequently dissolved in deionized water.The purity and identity of the TRITC-labelled ODN were checked by PAGE under denaturing conditions Cell culture and transfection
COS-7 cells (European Collection of Cell Cultures, Salis-bury, UK) were grown at 37C in a 5% CO2atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum, 2 mM L-glutamine,
100 UÆmL)1penicillin and 100 lgÆmL)1 streptomycin.At
24 h before transfection, cells were seeded in 12-wells plates
Table 1 Primers used for sticky-end PCR Separate PCR reactions were carried out with the following primer sets: GSTM1-forA and GSTM1-revA; GSTM1-forB and GSTM1-revB; GSTM2-forA and GSTM2-revA; GSTM2-forB and GSTM2-revB.Products from the first two PCR reactions were combined, melted and reannealed to give four GSTM1 cDNA products, of which one has the right EcoRI/ BamHI overhanging ends (underlined) to enable ligation in EcoRI and BamHI restricted pEGFP-C1.Combining the last two PCR reactions results in the formation of GSTM2 cDNA with EcoRI/BamHI over-hanging ends (underlined) which was cloned in a similar way into pEGFP-C1.
Primer Sequence
GSTM1-forA 5 ¢ -AATTCCATGCCTATGATACTGGGAT-3 ¢
GSTM1-forB 5 ¢ - C CATGCCTATGATACTGGGAT-3 ¢
GSTM1-revA 5 ¢ - C TAAAGATGAGACAGGCCTGG-3 ¢
GSTM1-revB 5 ¢ - GATCCTAAAGATGAGACAGGCCTGG-3 ¢
GSTM2-forA 5 ¢ -AATTCGATGCCTATGACACTGGGTTAC-3 ¢
GSTM2-forB 5 ¢ - C GATGCCTATGACACTGGGTTAC-3 ¢
GSTM2-revA 5 ¢ - C GTGGTTCACACTTTATTGCAAATC-3 ¢
GSTM2-revB 5 ¢ -GATCCGTGGTTCACACTTTATTGCAAATC-3 ¢
Trang 3at a density of 1· 105cells/well, which resulted in cultures
that were approximately 50% confluent at the day of
transfection.For cotransfections of plasmid and ODN, the
appropriate amount of plasmid [diluted to a concentration
of 0.2 lgÆlL)1 in HBS (0.15M NaCl in 20 mM Hepes,
pH 7.4)] was mixed with the appropriate amount of ODN
(diluted to a concentration of 0.2 lgÆlL)1in
HBS).Subse-quently, a transfection mixture was prepared by slowly
adding the plasmid/ODN mixture to a solution of DOTAP
in HBS (1 lgÆlL)1; charge ratio DNA : DOTAP¼ 1 : 5)
The DNA and DOTAP solutions were mixed by repeated
pipetting.The transfection mixture was diluted with HBS to
a total volume of 100 lL, and incubated for 15 min at room
temperature.Then, the culture medium was taken from the
cells and replaced by 400 lL of DMEM without serum or
antibiotics, and 100 lL of the transfection mixture was
slowly added.After 4 h, the transfection mixture was
removed from the cells, and serum-containing medium
was added.All analyses were performed after culture in the
serum-containing medium for a further 18 h
Flow cytometry
Cells were detached from the culture plates with trypsin,
centrifuged for 5 min at 400 g, washed once with 1 mL
NaCl/Pi, and dispersed in 1 mL NaCl/Pi.Immediately
before FACS analysis, 3 lL 1 lM propidium iodide was
added.Cellular fluorescence of approximately 3000 cells was
determined in a Becton Dickinson FACS Calibur flow
cytometer.The EGFP signal was detected in the FL-1
channel; TRITC and propidium iodide signals were detected
in the FL-3 channel.Only single cells were gated in forward/
sideward scatter plots; dead cells were excluded from the
analysis by gating of propidium iodide-positive cells
GST activity assay
COS-7 cells were transfected with either pEGFP,
pEGFP-M1 or pEGFP-M2 as described above.Then, the cells were
washed twice with NaCl/Pi and lysed in 300 lL 10 mM
sodium phosphate buffer (pH 7.4) containing 2 mM
dithio-threitol, 1 mMEDTA, and 50 lMPMSF.The lysates were
homogenized by short sonication.Total GST activity was
analysed in a CDNB conjugation assay, essentially as
described before [20].The assay makes use of the
GST-catalyzed addition of GSH to CDNB.The CDNB–GSH
conjugate formed can be measured spectrophotometrically
To this end, 50 lL of protein lysate ( 10 lg protein,
concentration determined with the Bradford protein assay
[21]) was incubated with 150 lL of a solution of 1.67 mM
CDNB in 0.1Mpotassium phosphate buffer pH 6.5 Lysis
buffer, instead of lysate, was used as a blank.The reaction
was started by the addition of 50 lL 5 mMGSH dissolved
in potassium phosphate buffer pH 6.5 CDNB–GSH
con-jugate formation was monitored over time with a
Perkin-Elmer HTS7000 bioassay plate reader at 340 nm and 37C
The rate of conjugate formation was constant 15–45 min
after the addition of GSH
Western blotting
Lysates of COS-7 cells transfected with pEGFP,
pEGFP-M1 or pEGFP-M2, prepared as described above, were
analysed for GFP expression by Western blotting.Five lg total cellular protein, dissolved in denaturing loading buffer (62 mM Tris/HCl pH 6.8, 12.5% v/v glycerol, 1.25% w/v SDS, 2.5% v/v 2-mercaptoethanol, and 0.25% w/v Bromo-phenol blue) were heated for 4 min at 96C, and subjected
to gel electrophoresis in an SDS/15% polyacrylamide gel Proteins were blotted overnight at 4C onto a nitrocellulose membrane at a current of 76 mA.Thereafter, the nitrocel-lulose membrane was incubated for 1 h in blocking buffer, consisting of 10 mM Tris/HCl pH 8.0, 150 mM NaCl, 0.5 mMCaCl2, 5% w/v milkpowder, 1% w/v BSA, 0.25% v/v Tween-20.Then, the membrane was incubated for 1 h at room temperature with the primary anti-EGFP antibody (100· diluted in blocking buffer without milk powder, containing 0.5% v/v Tween-20) The membrane was washed 10 times with NaCl/Pi containing 0.02% v/v Tween-20, and incubated with a horseradish peroxidase-conjugated donkey antirabbit IgG (10· diluted in 10 · diluted blocking buffer).EGFP was detected by an enhanced chemiluminescence assay, according to the manufacturer’s protocol
Statistical analysis Data were analysed statistically for significance with a one
or two sample student t-test.GRAPHPAD INSTAT Software version 3.00, GraphPad Software Inc (San Diego, CA, USA), was used for this purpose
R E S U L T S Cloning of EGFP–GSTM fusion constructs Cellular screening of antisense sequences for their potential
to inhibit gene expression is often complicated by irrepro-ducible transfection procedures and lack of good quantita-tive assays for monitoring of gene expression.To circumvent these problems, we developed a screening assay, based on fusion proteins of the target protein with EGFP, that enables accurate determination of the effects of AS-ODNs by flow cytometry.C-terminal fusion constructs
of GSTM1 and GSTM2 with EGFP (named pEGFP-M1 and pEGFP-M2, respectively) were made by ligating PCR-amplified cDNAs, coding for GSTM1 and GSTM2 (PCR primers in Table 1), into the multiple-cloning site of the pEGFP-C1 vector.Sequence analysis confirmed a correct in-frame ligation of the two cDNAs and the absence of any sequence errors in the inserts.By flow cytometric analysis, it was shown that transfection of COS-7 cells with pEGFP, pEGFP-M1, or pEGFP-M2 proceeded with equal efficien-cies (30 ± 2%, 31 ± 1%, and 29 ± 1%, respectively) The average intensity of the fluorescent signal of the EGFP– M1 and EGFP–M2 fusion proteins was only slightly lower than that of EGFP itself [2.1 ± 0.1, 1.7 ± 0.1, and 1.8 ± 0.1 (· 103 arbitrary units) for EGFP, EGFP–M1, and EGFP–M2, respectively], indicating that the EGFP moiety of the fusion proteins retained its activity.Fluores-cent microscopy revealed that EGFP and the EGFP–M1 and EGFP–M2 fusion proteins localized in the cytosol GST activity in lysates of the transfected COS-7 cells was assayed by measuring GSH–CDNB conjugate formation
As shown in Fig.1A, total GST activity in COS-7 cells was increased 3.4- and 1.9-fold after transfection with pEGFP–
Trang 4M1 and pEGFP–M2, respectively.The observed differences
in CDNB conjugation between pEGFP–M1- and pEGFP–
M2-transfected cells can be explained by the lower catalytic
activity of the GSTM2-2 protein towards CDNB compared with the activity of the GSTM1-1 protein [22].The size of the fusion proteins was 50 kDa, as determined by Western blotting with an EGFP-specific primary antibody (Fig.1B).This value is in close agreement with the expected size, calculated by summation of the molecular weights of EGFP (25 kDa) and GSTM (27 kDa)
Colocalization of ODN and pEGFP For proper evaluation of antisense effects, it is important that the AS-ODNs and the EGFP-expressing plasmids are transfected into the same cells.This was accomplished by the cotransfection of plasmid and AS-ODN.By FACS analysis, it was shown that after cotransfection of COS-7 cells with a fluorescently labelled ODN and pEGFP, ODN and plasmid colocalized in the same target cells as > 90% of the EGFP-positive cells were also positive for the TRITC-labelled ODN (Fig.2).The observations suggest that the uptake of ODN is far more efficient than the uptake of the EGFP plasmid, because almost all cells were positive for TRITC-labelled ODNs, whereas only 33% of the cells were expressing EGFP
Screening of ODNs for their antisense activity
To identify AS-ODNs that are potent and sequence-specific inhibitors of GSTM1 and/or GSTM2 expression, 15 PS-ODNs were screened for their ability to inhibit EGFP– GSTM fusion protein expression.The ODNs were targeted against different regions in the mRNA of GSTM1 and GSTM2 (Table 2).Some of the ODNs (i.e.AS-1, AS-6, AS-7, AS-8, AS-12 and AS-15) were designed to inhibit selectively GSTM1 expression, whereas others (i.e AS-5, AS-10, AS-11, AS-13 and AS-14) were designed to inhibit selectively GSTM2 expression.A third group of ODNs (i.e AS-2, AS-3, AS-4 and AS-9) was directed against homol-ogous regions in the GSTM1 and GSTM2 mRNAs, and should therefore inhibit the expression of both isoforms.An unrelated AS-ODN (AS-ctrl) with no sequence homology with EGFP, GSTM1 or GSTM2 was taken as a negative control.An EGFP-specific PO-ODN (AS-GFP), with two PS-linkages at either end for protection against nuclease activity, was taken as a positive control.It has been reported that this ODN inhibits GFP expression in HeLa cells transiently transfected with pEGFP [23]
Initially, COS-7 cells were transfected with 1.6 lg of the AS-ODNs (final concentration in the medium: 0.5 lM) and 0.5 lg of pEGFP, pEGFP-M1 or pEGFP-M2.After a 4-h transfection period and culture for a further 18 h, cells were analysed for EGFP expression by flow cytometry Propidium iodide was added to the cell suspensions to exclude nonviable cells from the analysis.The percentage
of propidium iodide-positive cells increased from 3% in cell cultures that were transfected with plasmid only, to
9% in cell cultures cotransfected with ODN and plasmid.This is probably due to cytotoxicity of DOTAP,
as a larger amount of DOTAP was used for cotransfection than for transfection of plasmid alone.The number of propidium-iodide positive cells was the same for all cotransfected PS-ODNs
Clear differences were found in the ability of the various AS-ODNs to inhibit EGFP expression.The control
Fig 1 Expression of EGFP–GSTM fusion proteins in COS-7 cells.
COS-7 cells were transfected with pEGFP-M1, pEGFP-M2, or
pEGFP (0.5 lg DNA per well).After a further 18 h of culture, the cells
were lysed.(A) Total GST activity in 50 lL of protein lysate was
measured by following CDNB–GSH conjugate formation over time.
The increase in absorption at 340 nm was recorded with lysis buffer as
a blank.The GST activity is expressed as a percentage of the activity in
pEGFP-transfected cells (0.46 DA 340 Æmin)1Æmg protein)1).Means of
12 determinations in three separate experiments ± SEM are shown.
**P < 0.0001 (unpaired student t-test).(B) A Western blot was
per-formed on 5 lg protein lysate of pEGFP-M1 (lane 1), pEGFP-M2
(lane 2) and pEGFP (lane 3) transfected cells.The samples were
denatured, and separated by SDS/15% PAGE, together with a
Bio-Rad prestained kaleidoscope protein marker.Subsequently,
proteins were blotted onto a nitrocellulose membrane, and the blot was
incubated consecutively with a rabbit anti-EGFP antibody and a
peroxidase-labelled goat anti-rabbit secondary
antibody.EGFP-containing proteins were visualized with enhanced chemiluminescence.
The positions and molecular weights of the marker proteins are
indi-cated in the left margin.In the right margin, the estimated sizes of the
protein bands are shown.
Trang 5AS-ODN did not have any effect on the expression of
EGFP, EGFP–M1 or EGFP–M2, indicating that
cotrans-fection of PS-ODNs per se does not influence EGFP
expression.AS-6 and AS-8, directed against GSTM1,
inhibited EGFP–M1 expression by 95 ± 1% and
81 ± 6%, respectively (Fig.3A).The expression of EGFP
and the other isoform, EGFP–M2, were also affected, but
the inhibitory effect on expression of pEGFP–M1 was
significantly greater (P < 0.05) than the effect on
expres-sion of EGFP or EGFP–M2.AS-1, however, inhibited the
expression of all three proteins, and the expression of EGFP
even by > 95%.This is probably not caused by
sequence-specific hybridization with the EGFP mRNA, because the
maximal continuous homologous region with the EGFP
sequence was eight nucleotides long.AS-7 also displayed
some nonspecific inhibition of protein synthesis: its effect on
EGFP–M1 expression, although greater, was not
signifi-cantly different from its effect on the expression of EGFP or
EGFP–M2.Two other AS-ODNs against GSTM1, AS-12
and AS-15, were completely ineffective in the
down-regulation of protein synthesis
Similar results were found for AS-ODNs targeted at
GSTM2.AS-5 and AS-10 inhibited EGFP–M2 expression
by 82 ± 4% and 85 ± 0.4%, respectively, and affected
the expression of the control proteins EGFP and EGFP– M1 by < 10% (Fig.3B).For AS-10, the isoform-specif-icity was remarkably good, as this AS-ODN contains only three mismatches with respect to the sequence of GSTM1 Again, one AS-ODN, AS-11, displayed nonspecific effects
on the expression of all three proteins, whereas two other ODNs, AS-13 and AS-14, were not able to affect protein expression
AS-2 and AS-3, targeted against the coding sequence of both GSTM1 and GSTM2, inhibited the expression of the EGFP–GSTM isoforms by 95% (AS-2) and 80% (AS-3), while expression of the control EGFP was inhibited
by 45 ± 12% and 18 ± 13%, respectively (Fig.3C).AS-9 demonstrated severe nonspecific effects on EGFP expres-sion, as the expression of EGFP, alone or in a fusion construct, was inhibited by > 95%.The effects on EGFP expression were not due to sequence-specific hybridization because a significant homology with the sequence of EGFP was not found.AS-4 showed less severe, but significant, nonspecific effects on EGFP expression.Surprisingly, the AS-GFP, which was reported to down-regulate EGFP expression [23], did not have any effect on the expression of either of the EGFP proteins at the tested concentration of 0.5 l
Fig 2 FACS analysis of COS-7 cells cotransfected with TRITC-ODN and pEGFP Untransfected COS-7 cells (A), cells trans-fected with 0.2 l M TRITC-ODN (B), cells transfected with 0.5 lg pEGFP (C), and cells transfected with 0.2 l M TRITC-ODN and 0.5 lg pEGFP (D) were analysed by flow cytometry for EGFP expression (FL-1, x-axis) and TRITC-ODN uptake (FL-3, y-axis) Single cells were gated in the forward–side-ward scatter plot (gate R1, not shown).The following gates were applied: R2, nontrans-fected; R3, TRITC-positive; R4, GFP-posit-ive; R5, TRITC-positive and GFP-positive (A–D) provide representive examples of multiple FACS analyses.The table gives the amounts of cells (expressed as percentage of the total amount of cells) counted in each gate under the different incubation conditions.
Trang 6Determination of the concentration/activity profile
of the AS-ODNs
To compare the potency and specificity of some of the
effective AS-ODNs, COS-7 cells were transfected with
different concentrations of AS-1, AS-2, AS-5, AS-6, and
AS-9.Figure 4 shows the effects of cotransfection with 0.01,
0.1 and 0.5 lM AS-ODN on the expression of pEGFP,
pEGFP–M1, and pEGFP–M2.Both the true antisense
effects and the non-target-specific effects appeared to be
highly concentration-dependent.AS-5 was a potent and
selective inhibitor of EGFP–M2 (IC50value 0.2 lM), and
did not have any effect on the expression of EGFP–M1 or
EGFP at the highest concentration tested.AS-2, directed
against both GSTM isoforms, and AS-6, directed at
GSTM1, potently inhibited the expression of their
respect-ive targets with IC50 values slightly above 0.1 lM.At
0.1 lM, the inhibition was specific.However, at the highest
concentration tested also the expression of EGFP was
affected, indicating that sequence-specific antisense effects
occur at lower concentrations than non-target-specific
effects.AS-9 was the most potent inhibitor of both
EGFP–M1 and EGFP–M2 expression with estimated
IC50 values < 0.1 lM.However, the EGFP expression
was also inhibited, although to a slightly lesser extent.AS-1,
targeted to GSTM1, was another nonspecific inhibitor of
EGFP expression as the IC50values for inhibition of EGFP
expression and of EGFP–M1 were both in the same range
Analysis of the effect of AS-ODNs on GST activity
To examine whether the inhibitory effects of the AS-ODNs
on EGFP–GSTM fusion protein expression were associated
with a decrease in GST activity, we determined the effect of
cotransfection with AS-ODNs on the GST activity in lysates of COS-7 cells transfected with pEGFP, pEGFP-M1, or pEGFP-M2.In the GST activity assay, we tested AS-5 and AS-10, directed against GSTM2, and AS-6, directed against GSTM1, which were found to be specific inhibitors of either GSTM1 or GSTM2 expression in the EGFP assay.The results are shown in Table 3.None of these ODNs affected the CDNB conjugation in cells transfected with pEGFP, indicating that the AS-ODNs were specific for the rat GSTM isoforms, and did not inhibit the activity of endogenous GSTs present in lysates of COS-7 cells.The rates of CDNB conjugation in the lysates of pEGFP-M1 and pEGFP-M2 transfected cells were correc-ted for the endogenous GST activity, determined in COS-7 cells transfected with pEGFP.AS-6 appeared to be a very potent inhibitor of GSTM1-1 enzyme activity (95 ± 2% decrease).AS-5 and AS-10 were somewhat less potent inhibitors of GSTM2-2 enzyme activity (77 ± 6%, and
70 ± 6% decrease, respectively).However, conjugation by the nontargeted isoform was also affected by 40–50% Nonetheless, the effects of the different AS-ODNs on the activity of the targeted GSTM isoform were significantly greater than on the nontargeted GSTM isoform (P < 0.002 for all tested AS-ODNs)
D I S C U S S I O N Most of currently available screening assays for the selection
of effective AS-ODNs are based on cell-free assays, e.g RNAse H digestion screens and oligonucleotide scanning arrays [9].As activity in cell-free assay may not always correlate with activity in cellular systems, we developed in the present study a novel cellular screening assay for the selection of effective AS-ODNs with a sequence-specific
Table 2 Antisense ODN sequences.
Target siteb
Mismatch (number of bases)
c
AS-ctrl TGAGAGCTGAAAGCAGGTCCAT Unrelated – –
a All sequences were PS-ODNs except for AS-GFP.The sequence of AS-GFP is taken from literature [23]: it is a PO-ODN with PS-modified internucleotide linkages at the 3¢- and 5¢-ends, indicated by asterisks b
The region in the mRNA against which the ODNs are indicated as follows: CDS, coding sequence; STOP, STOP codon; 3¢-UTR, 3¢-untranslated region.The target sites in the GSTM1 or GSTM2 mRNAs are indicated, nucleotide 1 being the ATG start site c The number of mismatches in the corresponding region of the nontargeted isoform are given.Mismatches are underlined in the sequence.
Trang 7mode of action.In the present assay, antisense activity is
directly correlated with EGFP-derived fluorescence by
constructing fusion proteins of the target protein and EGFP
Unlike in conventional target-specific screens, in the current
assay specific antibodies need not be available and
isoform-specific assays for the determination of enzyme activity need
not be developed.Furthermore, the measurement of
EGFP-derived fluorescence by flow cytometry has excellent
quantitative properties and offers good reproducibility.This
is probably due to the elimination of variation in transfection efficiencies as a complicating factor in the assessment of antisense effectiveness.Our experiments in which an EGFP-containing plasmid was cotransfected with fluorescently labelled ODNs, suggest that all EGFP-positive cells had taken up ODNs.Therefore, in the present assay the antisense effects are determined in the whole population of cells that express the target gene.In other cellular assays, including a luciferase reporter gene-based assay [24], antisense effects may be underestimated because not all cells that express the gene of interest are transfected with AS-ODNs.An EGFP-based approach has been used previously for the selection of ribozymes against the c-erbB-2 oncogene [25].However, in this earlier study the plasmid coding for the c-erb-B-2 EGFP fusion protein, was cotransfected with a ribozyme expressing plasmid and not with an exogenously added antisense molecule.Cotransfection with the ribozyme-expressing plasmid resulted in a reduction of EGFP expression to a maximum of 70%, whereas we observed a > 90% reduction with our most potent ODNs.Possibly, a significant part of the c-erbB-2-EGFP transfected cells had not taken up a ribozyme construct
A C-terminal fusion construct and not an N-terminal fusion construct, was used because AS-ODNs against the 3¢-untranslated region of the mRNA of the gene of interest, which has been shown to be a favourable region for antisense action [7], can only be tested in C-terminal fusion constructs
The newly developed screening assay was used for the selection of effective AS-ODNs against rat GSTM1 and GSTM2 out of a set of 15 PS-ODNs.Some ODNs were designed to specifically inhibit either GSTM1 or GSTM2 expression, which show a sequence identity of 80% at the DNA level.For these ODNs, the nontargeted isoform served as a mismatch target control with 1–13 mismatches Other ODNs were targeted against homologous regions in both isoforms.As a control for the true antisense nature of the observed effects on protein expression, the effects of the ODNs on the expression of EGFP without a fusion construct were evaluated.Three ODNs (AS-3, AS-5 and AS-10) were found to inhibit gene expression with very high sequence specificity.These ODNs reduced at 0.5 lM the expression of their target isoforms by > 80%, whereas the nontargeted isoform and/or EGFP control were not affected significantly.Other ODNs (AS-2, AS-6, AS-7 and AS-8) displayed a combination of target sequence-specific and nontarget-specific inhibitory effects on EGFP levels These ODNs inhibited the targeted isoform to a signifi-cantly greater extent than the nontargeted isoform and/or EGFP control, but also attenuated the expression of the controls by 40–60%.Three ODNs (AS-1, AS-9 and AS-11) had severe non-sequence-specific effects on EGFP expres-sion.In these cases, the expression of EGFP without a GSTM fusion was affected to a similar extent as the expression of the targeted proteins.Five ODNs (AS-4, AS-12, AS-13, AS-14 and AS-15) did not demonstrate major effects on EGFP–GSTM fusion protein expression Our results indicate once again that, when evaluating antisense effects, identification of false-positives is common
As stated before by others [26], it is crucial to analyse the effects on the expression of target-related control proteins, which is easily accomplished in our screening assay
Fig 3 Effects of AS-ODNs on EGFP and EGFP–GSTM fusion
protein expression COS-7 cells were transfected with 0.5 lg pEGFP
(open bars), pEGFP-M1 (hatched bars) or pEGFP-M2 (closed bars),
together with 0.5 l M of the indicated AS-ODNs.The ODNs were
directed against GSTM1 (A), GSTM2 (B), or both GSTM isoforms
(C).AS-ctrl is a control ODN without sequence homology with
GSTM or EGFP.AS-GFP is an AS-ODN against EGFP, taken from
[23].At 22 h after transfection, cells were analysed for EGFP
expres-sion (FL-1) and propidium iodide uptake (FL-3) by flow cytometry.
The number of living (i.e propidium iodide-negative), EGFP-positive
cells was counted and is expressed as the percentage of EGFP-positive
cells in cultures transfected with plasmid, but without AS-ODN.
Means of three independent experiments ± SEM are shown.
*P < 0.05; **P < 0.005 (one group student t-test compared to
con-trol without AS-ODN).
Trang 8Table 3 Effects of AS-ODNs on GST activity COS-7 cells were transfected with 0.5 lg pEGFP, pEGFP-M1 or pEGFP-M2, together with 0.1 l M
of the indicated AS-ODNs.AS-5 and AS-10 are directed against GSTM2; AS-6 is directed against GSTM1.At 22 h after transfection, GST activity
in the protein lysates was determined by assaying CDNB conjugation over time.Conjugation rates are expressed as percentages of EGFP controls (column 2, 3 and 5), or percentages of the additional EGFP-M1-dependent (column 4) or EGFP-M2-dependent (column 6) GST activity, calculated by subtraction of the endogenous GST activity, which was determined in pEGFP-transfected cultures.Means of 10–12 determinations in three separate experiments ± SEM are shown.Statistical significance of the difference between AS-ODN-treated and untreated cultures are indicated: a P < 0.005, b P < 0.0001 Statistical significance of the difference between the effect of the AS-ODNs on EGFP–M1 and EGFP–M2 expression are indicated:cP ¼ 0.0013 (AS-5), d
P < 0.0001 (AS-6),eP ¼ 0.0002 (AS-10).
CDNB conjugation rate
AS-ODN
% of EGFP
control
% of EGFP control
% of EGFP-M1 control
% of EGFP control
% of EGFP-M2 control
Fig 4 Concentration-dependent inhibition of
EGFP and EGFP–GSTM fusion protein
expression by AS-ODNs COS-7 cells were
transfected with 0.5 lg of pEGFP (n),
pEG-FP-M1 (j) or pEGFP-M2 (d), together with
the indicated concentrations of AS-1 (A),
AS-2 (B), AS-5 (C), AS-6 (D) or AS-9
(E).AS-1 and AS-6 are directed against GSTM(E).AS-1, AS-5
is directed against GSTM2, whereas AS-2 and
AS-9 are complementary to both GSTM1 and
GSTM2.At 22 h after transfection, cells were
analysed for GFP expression (FL-1) and
propidium iodide uptake (FL-3) by flow
cytometry.The number of living
(i.e.propi-dium iodide-negative), EGFP-positive cells
was counted and is expressed as the percentage
of EGFP-positive cells in cultures transfected
with plasmid, but without AS-ODN.Means
of three independent experiments ± SEM are
shown.An unpaired student t-test was used to
determine whether the effect on EGFP–M1 or
EGFP–M2 expression was significantly
dif-ferent from the effect on EGFP expression:
*P < 0 05; **P < 0.005.
Trang 9The sensitivity of the inhibition towards mismatches in
the target sequences appeared to be high.One mismatch
(AS-7) was not sufficient to achieve complete
isoform-specificity (the expression of the targeted and nontargeted
isoform was reduced by 77 ± 6% and 45 ± 15%,
respect-ively).The presence of three mismatches (AS-10), however,
resulted in isoform-specifc inhibition of EGFP–GSTM
fusion protein expression (85 ± 0.4% and 10 ± 0.3%
reduction of targeted and nontargeted isoform,
respect-ively).Interestingly, an AS-ODN against EGFP, described
to be effective in HeLa cells [23], was totally ineffective in
inhibiting the expression of either of the EGFP-containing
proteins in our study.This may be attributed to the fact that
the ODN was a phosphorothioate-capped PO-ODN.The
sensitivity of these chimeras towards nucleolytic
degrada-tion is higher than that of PS-ODNs, and depends on the
cell-type used [27,28]
True antisense effects and nonantisense effects elicited by
the ODNs were both found to be concentration-dependent
The IC50 values of the most potent, specifically acting
AS-ODNs were 0.2 lM.It should be noted that for most
AS-ODNs, with the exception of AS-5, the concentration
window where sequence-specific antisense effects were
observed, was narrow.This was also found in other studies
where PS-ODNs were used, and may be explained by the
relatively low affinity of PS-ODNs for their target mRNA
sequences together with the high incidence of nonantisense
effects [1,24].It is therefore of highest importance to evaluate,
in each antisense study, the concentration–activity profile
The nature of the nonspecific effects elicited by PS-ODNs
remains to be clarified.With the possible exception of AS-11,
which contained only five mismatches with respect to the
EGFP sequence, neither of the AS-ODNs against GSTM
showed significant sequence homology with EGFP.Thus,
the observed effects on EGFP expression are probably not
caused by partial hybridization of the AS-ODNs with the
EGFP mRNA.We cannot exclude that some of the
nonspecific AS-ODNs decrease the transfection efficiency
of the EGFP plasmids.However, from earlier studies it
became apparent that sequence-dependent variations in
cationic lipid-mediated transfection efficiencies were small,
unless homo-oligonucleotides, such as A18, were applied
[29,30].More likely, sequence-dependent aptameric effects
play a role.The negative charge on the sulfur atom may
cause avid binding of the ODNs to key cellular proteins, e.g
proteins involved in mRNA translation [31,32]
The inhibition of EGFP–GSTM fusion protein
expres-sion was reflected by a decrease in GST enzyme activity, as
determined in a CDNB conjugation assay.AS-6 displayed
potent and specific inhibition of GSTM1-1 enzyme activity
AS-5 and AS-10, directed against GSTM2, inhibited
GSTM2-2 enzyme activity but showed also some effect on
GSTM1-1 enzyme activity.This was not expected because
the effects of these AS-ODNs on EGFP fusion protein
expression were highly isoform-specific.The inhibitory
effects cannot be explained by a general inhibition of GST
activity, because the ODNs did not affect endogenous GST
activity in COS-7 cells.Possibly, the presence of four (AS-5)
and three (AS-10) mismatches with respect to the GSTM1
sequence results in partial hybridization with the GSTM1
mRNA and in some hindrance of the synthesis of full-length
EGFP–M1 fusion proteins without induction of RNAse
H-mediated cleavage and subsequent degradation of
EGFP–M1 mRNA.In that case, the formation of the EGFP moiety is not affected, whereas the formation of the GSTM1-1 is.This would explain the higher isoform specificity of AS-5 and AS-10 in the EGFP assay, compared
to the GST assay
In summary, we selected several effective antisense ODNs against rat GSTM1 and GSTM2 from a set of 15 PS-ODNs
in a novel, sensitive screening assay.The assay discriminates between effective AS-ODNs and ODNs that are ineffective
or inhibit protein expression by nonantisense mechanisms The effectiveness of the selected AS-ODNs will be evaluated further in rat hepatocytes and in vivo, potentially allowing the study of the effect of decreased GSTM expression on the toxicity and carcinogenicity of xenobiotics
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