(Received for publication, May 5, 1995; and in revised form, September 6, 1995)
From the
The aim of this study was to selectively inhibit human
mitochondrial aldehyde dehydrogenase (ALDH) gene expression
by triple helix assembly. Eight 21-mer oligodeoxyribonucleotides were
designed to bind to two purine-rich sequences in the 5`-flanking region
of the human ALDH
gene. Gel mobility shift assays showed
that triplex formation is sequence-specific for the target duplex and
the third strand oligonucleotide. In the presence of
Mg
, but absence of K
,
triplex-forming oligonucleotides bind to their target sites with
apparent dissociation constants (K
) in
the 10
to 10
M range.
Potassium cation virtually suppressed the triplex formation of G-C-rich
duplex DNA with natural oligonucleotides, but did not prevent triplex
formation with phosphorothioate-modified oligonucleotides.
Phosphorothioate-modified oligonucleotides were delivered into human
hepatoma Hep G2 cells by cationic liposomes. The reduction in
ALDH
mRNA levels in the cells was determined by the
competitive reverse transcription-polymerase chain reaction. One of the
phosphorothioate-modified oligonucleotides designed to form an
antiparallel triplex with a target in the 5`-flanking region of human
ALDH
gene (-105 to -125 from the translation
initiation codon ATG) reduced by 80-90% the ALDH
mRNA
levels without affecting albumin mRNA levels. Data suggest that
triple-helix formation may provide a means to selectively inhibit
hepatic ALDH
gene expression for therapeutic use.
Liver aldehyde dehydrogenases (ALDHs) play an
important role in the in vivo detoxification of aldehydes.
Based on enzymological(1, 2) ,
metabolism(3, 4) , and human genetics (5) studies, it is believed that the mitochondrial isozyme
(ALDH
) (6) is mainly responsible for the oxidation
of acetaldehyde generated during alcohol oxidation in vivo.
Approximately 40% of Orientals have a defective ALDH (ALDH
(-)), which has greatly diminished
catalytic activity, while virtually all Caucasians examined thus far
have the active liver ALDH
(ALDH
(+))(7, 8) . Individuals
presenting low ALDH
activity refrain from excessive
drinking, likely due to an aversive reaction caused by elevated blood
acetaldehyde levels(9) . The discomfort caused by this effect
may be responsible for the low prevalence of alcoholism in subjects who
present the ALDH
(-) allele in Japan and
China(10, 11, 12) . We have recently
investigated the relative role of the ALDH
deficiency on
alcohol consumption in North American-born Canadian and Americans of
Oriental origin and found that marked protection against alcohol abuse
by this allele also occurs in North America and is independent of the
degree of acculturation(13) . ALDH
(-)
homozygotes are virtual abstainers(13) .
Disulfiram
(Antabuse), which inhibits ALDH has been used for treatment of
alcoholics for many years(14) . However, disulfiram has a
number of side effects(15) . The marked difference in the
prevalence of alcohol abuse and alcoholism of ALDH(+)
and ALDH
(-) individuals suggests that a drug able to
specifically inhibit the expression of the normal ALDH
gene
for prolonged periods could be of value as a therapeutic agent for
alcohol abuse and alcoholism.
Oligonucleotide-directed triplex formation of double-stranded DNA has been shown to selectively inhibit gene transcription and subsequent protein synthesis in both in vitro(16, 17, 18, 19, 20, 21, 22) and in ex vivo systems(23, 24, 25, 26, 27, 28) . However, other investigators have been unable to demonstrate triplex formation at any of several different G-rich, polypurine DNA target under physiological potassium ion concentrations. Studies revealed that high potassium ion concentrations, such as those found within cells, can virtually suppress the antiparallel triplex formation by G-rich oligonucleotides (29, 30) .
Milligan et al. (29) used 7-deaza-2`-deoxyxanthosine to replace thymidine in a triplex-forming oligonucleotide and found that the substitution of 7-deaza-2`-deoxyxanthosine for T causes a greater than 100-fold increase in affinity in the presence of 140 mM KCl for this sequence. Gee et al. (31) and Olivas et al.(32) independently reported that site-selective substitution of 2`-deoxy-6-thioguanosine for guanosine (G) in the triplex-forming oligonucleotide results in an increase in triplex formation in the presence of physiological levels of potassium ion.
We sought another way to enhance the ability of oligonucleotides to
form triplexes with their targets under physiological potassium
conditions. This report provides evidence that submicromolar
concentrations of phosphorothioate-modified oligonucleotides designed
to bind to the specific area of 5`-flanking region of human ALDH gene are able to form stable triplex with their corresponding
targets under physiological potassium ion concentrations in
vitro, and to selectively inhibit the expression of human
ALDH
gene in human hepatoma Hep G2 cells, without affecting
the steady-state albumin mRNA levels.
Oligonucleotides were delivered
into cultured Hep G2 cells by cationic liposomes (Lipofectamine, Life
Technologies, Inc.) according to the manufacturer's
recommendations. Oligonucleotides (0-4 µg) and liposomes (24
µg) were diluted separately in 100 µl of OptiMEM (Life
Technologies, Inc.). These two solutions were then gently mixed and
incubated at room temperature for 45 min to form
oligonucleotide-liposome complex. The cultured cells at an 80%
confluence were rinsed twice with OptiMEM prior to the addition of a
mixture of 800 µl of OptiMEM and 200 µl of
oligonucleotide-liposome complex. The cells were exposed to the complex
for 6 h at 37 °C, 5% CO, 100% humidity, and then
returned to the growth medium. Antibiotics were not present during the
liposome-mediated delivery. After 24 h of incubation, the cells were
rinsed twice with phosphate-buffered saline (Life Technologies, Inc.)
prior to total RNA preparation.
To determine the delivery efficiency
of oligonucleotides into Hep G2 cells by Lipofectamine, cells grown in
35-mm culture dishes were washed twice with OptiMEM prewarmed to 37
°C. P-End-labeled oligonucleotides were added to the
cells at a final concentration of 1 µM in the absence or
presence of 24 µg/ml Lipofectamine. At the indicated times, the
medium was removed and the cell monolayer was washed four times with
ice-cold phosphate-buffered saline. Cells were detached from the dish
and harvested from 0.2 M glycine buffer (pH 2.8). Cell- or
medium-associated radioactivity was quantified by liquid scintillation.
To quantitate the amount of PCR product,
[-
P]dCTP at a final concentration of 50
µCi/ml was added to PCR master mix without adjusting the
concentration of individual dNTPs in the dNTP stock(37) . After
amplification, an aliquot of each sample was subjected to
electrophoresis on a 2.0% agarose gel, and two bands were seen by
ethidium bromide stain: 222 bp from cDNA and 722 bp from gDNA
amplification in the case of ALDH
mRNA determination (see Fig. 1), or 224 bp for cDNA and 436 bp for gDNA amplification in
the case of albumin mRNA determination (data not shown). The labeled
bands were cut out and counted. The ratio of gDNA/cDNA PCR products was
plotted as a function of the amount of known gDNA. The counts of gDNA
fragment was divided by the ratio of gDNA fragment bp/cDNA fragment bp
to correct for the greater number of
[
-
P]dCTP incorporation in the large
fragment. The point of equivalence (i.e. where there is 1:1
ratio) is where cDNA equals gDNA and represents the concentration of
cDNA in the unknown (37) .
Figure 1: Quantitation of mRNA by competitive RT-PCR. A fixed amount of cDNA, which is equivalent to the amount of mRNA generated by reverse transcription, was co-amplified with various amounts of human genomic DNA (termed gDNA, 0.01-100 ng) by PCR. Lanes 1-13, sample tubes; lane 14, 100-bp ladder (Pharmacia).
Figure 2:
Selection and synthesis of target sites
for triplex formation. Two target sites (A and B)
were chosen from the 5`-flanking region of human ALDH gene(34) . Two fragments bearing each target site were
synthesized by PCR using two pairs of primers listed under
``Experimental Procedures.'' The CAAT box, TATA box,
initiation codon ATG, and codon for Val 17 are marked in the boxes.
For each target site (A or B), eight different types of triplex-forming oligonucleotides were designed (Table 1). All the oligonucleotides are 21-mer. Oligos A-1 and B-1 have the same sequences but opposite orientation (antiparallel) to the purine-rich strand of Target sites A and B, respectively; oligos A-2 and B-2 have the same sequences and same orientation (parallel) as the purine-rich strands; oligos A-3 and B-3 and oligos A-4 and B-4 use thymidines (T) to replace adenosines (A), and guanidines (G) to replace cytosines (C) in the sequences and have different orientation from each other; oligos A-5 and B-5 and oligos A-6 and B-6 use thymidines (T) to replace both adenosines (A) and cytosines (C) in their sequences; oligos A-7 and B-7 and oligos A-8 and B-8 only use thymidines (T) to replace adenosines (A) in their sequences.
Figure 3:
Determination of the apparent dissociation
constant (K) using gel mobility shift
analysis. Increasing concentrations of the unlabeled 400-bp
double-stranded fragment bearing target site A were incubated with a
constant amount of radiolabeled PS-oligo A-5 in a buffer containing 140
mM KCl and 10 mM MgCl
as described under
``Experimental Procedures.'' The gel was dried and submitted
to autoradiography. The apparent dissociation constant K
(3-10
10
M) occurs at the concentration of duplex where
oligonucleotide and triplex are equal as determined by
densitometry.
In our study the abilities
of the oligonucleotides to form triplex with their respective Target
sites A or B are in the following order: oligo 5 > oligo 6 >
oligo 3 > oligo 4 > oligo 1 and 2 > oligo 7 and 8. These
results indicate that the best triplex-forming oligonucleotides should
be those which can lead to GG-C, T
A-T, or T
C-G
pairing and are antiparallel to the purine-rich strand of the DNA
target duplex (for example, oligos A-5 and B-5). Change in the
orientation of oligonucleotides (in the case of oligos A-6 and B-6) was
found to reduce the ability of triplex formation. It was also observed
that if C, rather than G, is used to form a triplex with C-G
(C
C-G) only A (in the cases of oligos A-1, B-1 and A-2, B-2),
rather than T (in the cases of oligos A-7, B-7 and A-8, B-8), is
permissible in the same oligonucleotide to form a triplex with A-T
(A
A-T).
To further determine the specificity of triplex formation, an oligonucleotide (PS-oligo A-5) designed for Target site A was incubated with the 300-bp fragment bearing Target site B, and an oligonucleotide (PS-oligo B-5) designed for Target site B was incubated with the 400-bp fragment bearing Target site A as described above. It was found that these two oligonucleotides are not able to form triplex with the 300- or 400-bp fragments containing mismatch target sites (data not shown). Overall, the results above indicate that the triplex formation is highly sequence-specific in term of both the target duplex and the third strand oligonucleotide. In the presence of 140 mM KCl, except for PO-oligo A-5, none of the natural oligonucleotides are not able to form triplexes with the corresponding duplex targets (Table 2).
As shown in Table 2, the
phosphorothioate modification greatly reduces the interference of
triplex formation by potassium ion. The fully and partially
phosphorothioate-modified oligonucleotides are able to form triplexes
with their corresponding target fragments in the presence of 140 mM KCl, with K values in the 10
to 10
M range.
To determine whether cationic
liposomes are able to increase the delivery of oligonucleotides into
the target cells, 1 µMP-labeled PS-oligo A-5
was exposed to cultured Hep G2 cells in the absence and presence of 24
µg/ml Lipofectamine for various times as described under
``Experimental Procedures.'' Oligonucleotide uptake is
markedly enhanced by liposomes. At virtually all times, the
cell-associated radioactivity in the presence of liposomes was 10-fold
higher than that obtained without liposome (data not shown). After 6 h
of treatment in the presence of liposomes, the intracellular
concentration of delivered oligonucleotides reached a concentration of
approximately 80 pmol/10
cells.
It was reported that cationic liposomes may be toxic to some cell lines (41) , and that oligonucleotides containing four contiguous G residues may be antiproliferative(43) . In our study at the concentration used, neither Lipofectamine nor phosphorothioate-modified oligonucleotides containing contiguous G residues (PS-oligo A-5) affected the total amount of proteins in Hep G2 cultures (data not shown).
The steady-state levels of ALDH mRNA in Hep G2
cells were quantitatively determined after oligonucleotide/liposome
complex treatment. As shown in Fig. 4a, both 5`-PS-3`
oligo A-5 and PS-oligo A-5 inhibited the expression of human ALDH
gene in a dose-dependent manner, while PO-oligo A-5 and PS-oligo
B-5 did not (see ``Discussion''). The concentrations of
PS-oligo A-5 and 5`-PS-3` oligo A-5 for 50% inhibition are the order of
150 and 300 nM, respectively. As shown in Fig. 4b, triplex-forming oligonucleotides did not
affect albumin mRNA levels in the same experiments.
Figure 4:
Effect of triplex-forming oligonucleotides
on steady-state levels of human ALDH mRNA (a) and
albumin mRNA (b) in Hep G2 cells. One unmodified
oligonucleotide (PO-oligo A-5) and three oligonucleotides fully or
partially phosphorothioate-modified (PS-oligo B-5; 5`-PS-3` oligo A-5
and PS-oligo A-5,) were delivered into cultured Hep G2 cells by
Lipofectamine as described under ``Experimental Procedures.''
After treatment, total RNA of each dish was extracted and the
steady-state mRNA levels were quantitated by competitive RT-PCR (Fig. 1). The data are plotted as the measured mRNA level
relative to that derived from the determination of mRNA in untreated
cells (0 nM). Error bars refer to standard deviations of the
average values from four experiments. Oligonucleotide concentrations
are indicated below the bars. Bars from left to right: empty bar, PS-oligo B-5; squares,
PO-oligo A-5; stippled, 5`-PS-3` oligo A-5; cross-hatched, PS-oligo A-5.
In this study, two target sites were chosen from the
5`-flanking region of human ALDH gene. In vitro experiments showed that both these two target sites are able to
form triplex with their corresponding phosphorothioate-modified
oligonucleotides. However, after being delivered into Hep G2 cells,
only PS-oligo A-5, which forms triplex with Target site A, inhibited
human ALDH
gene expression. This suggests that Target site
A, but not Target B, is located in a positive regulatory area in human
ALDH
gene. After these studies had been completed, Dipple et al.
reported that the -160 to -75
of the 5`-flanking region of the gene contains several positive
regulatory sites. Target A duplex in our study corresponds to
-125 to -105 in the 5`-flanking region. Thus, it is
possible that PS-oligo A-5 blocked one of these positive regulatory
sites. On the other hand, a negative regulatory site was found
by Dipple et al. in the -600 to -490 of the
5`-flanking region of the gene, in line with our finding that PS-oligo
B-5, which binds to Target B in -536 to -516 of the
5`-flanking area, did not inhibit gene expression. The fact that the
latter oligonucleotide did not activate gene expression may be due to
its relatively short (21-mer) length, compared to the 110-mer long
segment where the inhibitory sites are located. Thus, the negative
regulatory site may not be masked by the oligonucleotide.
The
natural phosphodiester oligonucleotide PO-oligo A-5 was found not to
inhibit human ALDH gene expression (Fig. 4a). This may not only be due to its weaker
affinity to Target A than that of the modified counterpart PS-oligo
A-5, in the presence of potassium ions (Table 2), but also due to
its lability toward intracellular nucleases in the cells(44) .
As reported by other laboratories(29, 30, 31, 32) , we also found that potassium ions strongly suppress the antiparallel triplex formation of G-rich DNA duplex. The mechanism of this inhibition is unclear. Potassium ion is known to promote the formation of inter- or intramolecular guanine tetraplex, species with G-rich oligonucleotides (31, 45, 46, 47) . The formation of these stable structures would reduce the amount of oligonucleotide available to form triplex structure. However, attempts to overcome the suppression by substitution of 7-deaza-2`-deoxyguanosine for G failed(29) . Milligan et al. reported that the oligonucleotides containing 7-deaza-2`-deoxyguanosine, which are capable of triplex formation but not self-association by Hoogsteen base pairing, were equally suppressed by 140 mM KCl as their corresponding G-rich oligonucleotide counterparts(29) . On the other hand, the substitution of 7-deaza-2`-deoxyxanthosine for T causes a greater than 100-fold increase in affinity in the presence of 140 mM KCl(29) . The authors suggested that this substitution could decrease the repulsion forces between the phosphodiester backbone in the third strand and that in the duplex.
Phosphorothioate
oligonucleotide is one of the most commonly employed modified
oligonucleotide in the antisense strategy (see (48) ). The
substitution for one of the nonbridging oxygen atoms of the
internucleotide phosphate by sulfur produces a compound that is highly
resistant to degradation by nucleases (49) and also creates a
new stereogenic center(50, 51) .
Phosphorothioate-modified oligonucleotide prepared by existing
methodologies consists of the mixture of 2 diastereomers
where n is a number of phosphorothioate internucleotide
linkage. This fact results in averaging of many characteristics, which
are noticeably different in stereoregular oligomers, especially
``all R
'' and ``all S
.'' The differences are observed in
hydrophobicity, hybridization parameters, and charge
density(52) . Hacia et al.(53) observed that
in the absence of potassium ions, the purine-rich phosphorothioate
oligonucleotides were able to form triplex with their corresponding
duplex, but the pyrimidine-rich counterparts were not.
Analyses of
experimental evidence regarding bond order and charge delocalization in
thiophosphate anions have led to the conclusion that the negative
charge is localized mainly on the sulfur, which can be represented by
the partial valence-bond structure O=P-S (see (54) ). Since O=P-S
has less
electronegativity than O=P-O
, we reasoned that
the substitution would reduce the repulsion forces between
phosphodiester backbone in the third strand and that in the duplex,
thus increasing the affinity of triplex-forming oligonucleotides to
their duplex targets.
On the other hand, inspection of molecular
models clearly indicates that the P-S bond-axis for
the R
configuration is oriented into the major
groove, whereas in the S
configuration this
bond-axis points away from helix with sulfur thus being positioned on
the face of the sugar-phosphate backbone(54) . It is
conceivable that some of phosphorothioate-modified oligonucleotide
distereomers may favor the antiparallel triplex formation under
physiological conditions. From the observation that the fully
phosphorothioate-modified oligonucleotide inhibits the expression of
human ALDH
gene in a stronger manner than the partially
modified one does, it could be concluded that the more substitutions an
oligonucleotide has the more would the antiparallel triple-helix
formation be favored.
It should be noted that we have not measured transcription rates per se. Therefore, the hypothesis that triplex-forming oligonucleotides act by gene-specific inhibition of transcription initiation has not been answered conclusively. A nuclear run-on assay, which is often used to measure transcription rates, could not be used in this study due to its lesser sensitivity.