From the Department of Biochemistry and Molecular
Biology, Ferrara University, Via L.Borsari n.46, 44100 Ferrara, Italy,
§ Institute of Biostructure and Bioimaging, CNR,
80134 Napoli, Italy, and ¶ Biotechnology Centre, Ferrara
University, Via Fossato di Mortara 8/A, 44100 Ferrara, Italy
Received for publication, July 8, 2002, and in revised form, November 19, 2002
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ABSTRACT |
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Peptide nucleic acids (PNAs) are DNA-mimicking
molecules in which the sugar-phosphate backbone is replaced by a
pseudopeptide backbone composed of N-(2-aminoethyl)glycine
units. We determined whether double-stranded molecules based on PNAs
and PNA-DNA-PNA (PDP) chimeras could be capable of stable interactions
with nuclear proteins belonging to the Sp1 transcription factor family
and, therefore, could act as decoy reagents able to inhibit molecular interactions between Sp1 and DNA. Since the structure of PNA/PNA hybrids is very different from that of the DNA/DNA double helix, they
could theoretically alter the molecular structure of the double-stranded PNA-DNA-PNA chimeras, perturbing interactions with
specific transcription factors. We found that PNA-based hybrids do not
inhibit Sp1/DNA interactions. In contrast, hybrid molecules based on
PNA-DNA-PNA chimeras are very effective decoy molecules, encouraging
further experiments focused on the possible use of these molecules for
the development of potential agents for a decoy approach in gene
therapy. In this respect, the finding that PDP-based decoy molecules
are more resistant than DNA/DNA hybrids to enzymatic degradation
appears to be of great interest. Furthermore, their resistance can even
be improved after complexation with cationic liposomes to which PDP/PDP
chimeras are able to bind by virtue of their internal DNA structure.
In vitro transfection of cis elements that decoy
against nuclear factors leads to alteration of gene expression and was
recently proposed in molecular medicine as a novel tool for the
possible therapy of several disorders (1-12). One of the most
effective decoy approaches so far described involves nuclear proteins
belonging to the NF- As to the molecular targets for the decoy approach, proteins belonging
to the Sp1 family are of great interest since these transcription
factors are involved in the regulation of the expression of several
genes relevant to human pathologies, including those encoding vascular
endothelial growth factor, plasminogen-activator inhibitor type-1
(PAI-1), COL1A2, urokinase-type plasminogen activator (uPA), and uPA
receptor (3, 21-25). Sp1 binding sites are also present in the HIV-1
LTR (26). Thus, the development of experimental approaches based on Sp1
decoys to modulate the transcription of Sp1-dependent genes
appears to be of great interest (3).
Recently, Ishibashi et al. (3) demonstrated that
transfection of oligodeoxynucleotides (ODNs) carrying the consensus
sequence for Sp1 binding (Sp1 decoy ODNs) was able to inhibit
the tumor necrosis factor- The activity of decoy molecules carrying Sp1 binding sites was also
studied by Motojima et al. (22) and by Hata et
al. (25). Motojima et al. (22) demonstrated that
Sp1 decoys are able to inhibit angiotensin II-induced up-regulation of
PAI-1 gene expression in mesangial cells (22). Hata et
al. (25) showed that Sp1 decoys reduced expression of kinase
domain receptor, a high affinity, endothelial cell-specific,
autophosphorylating tyrosine kinase receptor for vascular endothelial
growth factor. This transcriptionally regulated receptor is a critical
mediator of endothelial cell growth and vascular development (25).
In a recent study, we have investigated the possible use of peptide
nucleic acids (PNAs) (27-30) as alternative reagents in experiments
aimed at the control of gene expression involving the decoy approach
(20, 31). In PNAs, the pseudopeptide backbone is composed of
N-(2-aminoethyl)glycine units (27). PNAs hybridize with high
affinity to complementary sequences of single-stranded RNA and DNA,
forming Watson-Crick double helices (28, 29), are resistant to both
nucleases and proteases (30, 32), and were found to be excellent
candidates for antisense and antigene therapies (33-35). We
demonstrated that NF- More recently, PNA-DNA chimeras have been described as reagents of
great interest in gene therapy. PNA-DNA chimeras are PNA-DNA (30,
36-39) covalently bonded hybrids and were designed on the one hand to
improve the poor cellular uptake and solubility of PNAs and on the
other hand to exhibit biological properties typical of DNA, such as the
ability to stimulate RNaseH activity and to act as substrate for
cellular enzymes (for instance DNA polymerases). Finally, PNA-DNA
chimeras in which the PNA part is attached to 3'- or 5'-ends of the DNA
are expected to be particularly stable against degradation by 3'-
and/or 5'-exonucleases, which represent the major type of
oligonucleotide degrading enzymes found in serum (30).
In the present report, we determined whether PNA/PNA and PNA/DNA hybrid
could act as decoy molecules for transcription factor Sp1. Furthermore,
we investigated binding to Sp1 transcription factor(s) and biological
activity of PNA-DNA-PNA chimeras mimicking Sp1 binding sites.
Synthetic Oligonucleotides and Peptide Nucleic Acids--
The
synthetic oligonucleotides used in this study were purchased
from Amersham Biosciences. HPLC-purified PNAs were purchased from
ISOGEN Biosciences (Maarssen, the Netherlands).
PNA-DNA-PNA Oligomer Assembly--
Chimera synthesis proceeded
by sequential elongation of the PNA fragment, to which DNA first and
then PNA were attached. Aminomethyl-polystiren-NH2 resin
(loading 37 µmol/g), resin functionalized with an
hexamethylene bisacetamide linker bound through an ester bond to
a Gly, was used. The DNA part of the chimera was prepared on an
Amersham Biosciences Gene Assembler. Chain elongation was performed
with 15 eq of methyl DNA phosphoramidites using
5-(o-nitrophenyl)tetrazole (8 eq) as the activator. Standard
DNA capping, washing, oxidation, and detritylation cycles were used.
Coupling yields were gauged spectrophotometrically (254 nm) by the
absorption of the released trityl cation after each deprotection step.
In the last DNA elongation step, cyanoethyl 5-amino-5-deoxythymidine
phosphoramidite was used (38, 39). The PNA part of the chimera was
prepared on a full automated PerSeptive Biosystems Expedite 8900 Nucleic Acid synthesizer (PerSeptive Biosystems, Foster City, CA) using
standard (designed for 2-µmol scale) PNA coupling cycles and
solutions. Fmoc (Bz, benzyl)/(iBu, isobutyl)-protected PNA was used. To
improve the coupling efficiency of the first PNA moiety, a double
coupling cycle was employed (40). Upon completion of the last
elongation cycle, the terminal Fmoc group was cleaved by piperidine
treatment, and the primary amine was acetylated. The methyl groups were
removed from the phosphate functions by treatment of the resin with
0.25 ml of thiophenol in 0.5 ml of tetrahydrofuran and 0.5 ml of
triethylamine for 45 min. The resin was washed consecutively with
tetrahydrofuran, methanol, acetonitrile, and water (5 × 1 ml for
each solvent). The oligomers were cleaved from the support with
concomitant deprotection of the remaining protective groups by
treatment with 0.1 M sodium hydroxide in water/dioxane
(1/1, v/v, 1.5 ml) at 55 °C for 16 h. The reaction mixtures
were neutralized by the addition of acetic acid, concentrated, and
redissolved in 0.15 M ammonium bicarbonate. Desalting was
performed using a Sephadex G-25 (superfine, DNA grade) gel filtration
column with 0.15 M ammonium bicarbonate buffers. Samples
were filtered and then purified by reverse-phase-HPLC on a
LiCrosphere 100 RP-18 endcapped column (4 × 250 mm) on a Jasco
HPLC system. Gradient elution was performed at 40 °C, building up
gradient starting with buffer A (50 mM triethylammonium
acetate in water) and applying buffer B (50 mM
triethylammonium acetate in acetonitrile/water 1/1 v/v) with a flow
rate of 1 ml/min. Matrix-assisted laser desorption ionization
time-of-flight (MALDI-TOF) analyses were performed on all chimeras as
follows: (a) 5842.6 (M+H)+ and (b)
5602.0 (M+H)+; (a) cag- *TGA GGC GTG GCCA
-ggg-Gly and (b) ccc- *TGG CCACGC CTCA -ctg-Gly.
Electrophoretic Mobility Shift Assay--
The electrophoretic
mobility shift assay (41, 42) was performed using the double-stranded
synthetic oligonucleotides mimicking the Sp1 (nucleotide sequences are
presented in Table I and in Fig.1). The DNA stretches of the target
molecules were 5'-end-labeled using [ Cell Lines and Culture Conditions--
Human erythroleukemia
K562(S) cells (44) were cultured in a humidified atmosphere at 5%
CO2 in RPMI 1640 (Flow Laboratories) supplemented with 10%
fetal bovine serum (CELBIO), 50 units/ml penicillin, and 50 µg/ml
streptomycin (45, 46). Cell growth was studied by determining the cell
number/ml after different days of in vitro cell culture
(46). Stock solutions of ara-C (100 µM) were stored at
Stability of Decoy Molecules--
The stability of decoy
molecules was evaluated after incubation of DNA and PNA-DNA-PNA-based
decoys with 3' Liposome Preparation--
Egg phosphatidyl choline was purchased
from Lipid Products (Surrey, England). Tetralysine cationic lipids,
tetralysine-cholesterol (Lys4-Chol), and
tetralysine-palmitate (Lys4-Palm) were a generous gift of
Prof. M. Marastoni (Department of Pharmaceutical Sciences, University
of Ferrara, Ferrara, Italy). Positively charged liposomes were produced
by a protocol based on reverse phase evaporation followed by extrusion
of the liposome suspension through polycarbonate filters with
homogeneous pore size. Liposomes were subjected to one extrusion cycle
through two stacked 400-nm pore size filters followed by three
extrusion cycles through two stacked 200-nm pore size membranes in
order to obtain unilamellar liposomes with a homogeneous size
distribution. Different cationic detergents were alternatively
used for the production of the liposomes, namely Lys4-Chol and Lys4-Palm (47). The resulting
liposomal formulations were named as follows:
lipo-Lys4-Chol and lipo-Lys4-Palm. The morphological and dimensional analysis of the produced liposomes was
performed by freeze-fracture electron microscopy technique and photo
correlation spectroscopy (Zetasizer, Malvern, UK). The freeze-fracture
electron micrographs (47, 48) confirmed that the extruded liposomal
suspension was mainly constituted by unilamellar vesicles. Photon
correlation spectroscopy studies demonstrated that the extruded
vesicles present a narrow size distribution with an average diameter
reflecting the pore size of the employed membrane.
For analysis of protective effects, 32P-labeled DNA/DNA and
DNA/PNA hybrids were incubated with increasing amounts (2-25
µg/reaction) of lipo-Lys4-Chol and
lipo-Lys4-Palm for 30 min at room temperature. A further
overnight incubation period was then performed in the absence or in the
presence of a 25-µl reaction of serum, and 32P-labeled
material was phenol-extracted, ethanol-precipitated, and analyzed by
polyacrylamide gel electrophoresis and autoradiography.
Design of Synthetic Oligonucleotides, PNAs and PNA-DNA
Chimeras--
The design of the Sp1 oligonucleotides, PNA, and
PNA-DNA-PNA chimeras was conducted, taking into account possible
solubility and self-annealing problems related to the chemical
properties of the molecules. Indeed, it has been reported that PNAs
could exhibit low solubility at high G+C/A+T ratios (29, 30). In addition, oligonucleotides mimicking Sp1 binding sites but exhibiting intramolecular self-complementary stretches should not be considered in
order to avoid self-hybridization of the molecules to be used for
production of a double-stranded decoy. For these reasons, we decided to
synthesize oligonucleotides, PNAs, and PNA-DNA-PNA chimeras mimicking a
genomic region present within the HIV-1 LTR and containing Sp1
binding sites exhibiting a G+C/A+T ratio lower than Sp1 binding sites
present in the promoter sequences of most eukaryotic genes (Table
I and data not shown).
The nucleotide sequences of the employed HIV-1 Sp1 oligonucleotides are
shown in Table I, together with the other Sp1 oligonucleotides, PNAs,
and PNA-DNA chimeras employed in this study. We synthesized two
oligonucleotides, HIV-Sp1 (14-mer) and HIV-Sp1-L (19-mer), whose
sequences are identical to HIV-Sp1(PNA) and HIV-Sp1(PDP), respectively.
The PNA-DNA-PNA chimera carries a DNA sequence, identical to HIV-Sp1
DNA and HIV-Sp1(PNA), flanked by two PNA stretches at both ends. Since
we wanted to define the decoy activity of these molecules in the
double-stranded configuration, the complementary oligonucleotides,
PNAs, and PNA-DNA chimeras were also synthesized. Two classes of
oligonucleotides were also produced: (a) three DNA molecules
carrying the Sp1 binding sites present in the promoters of the human
urokinase-type plasminogen activator receptor (uPAR-Sp1),
Fig. 1 shows all the decoy molecules
analyzed in our study. The DNA/DNA, PNA/PNA, and PDP/PDP hybrids were
produced after annealing of the complementary DNA, PNA, and PNA-DNA-PNA
molecules. DNA/PNA and PNA/DNA hybrids were produced after annealing of
HIV-Sp1 14-mer DNA and HIV-Sp1(PNA). DNA/PDP and PDP/DNA hybrids were produced after annealing of the HIV-Sp1-L 19-mer DNA and
HIV-Sp1(PDP).
The Double-stranded PNA/DNA, DNA/PNA, and
PNA/PNA Hybrids Carrying Sp1 Binding Sites Are Unable to
Inhibit the Interactions between Sp1 Nuclear Factors and Target
DNA/DNA Molecules--
When 12 µg of crude nuclear
extracts from human leukemic K562 cells were incubated for 20 min in
the presence of the cold double-stranded 14- and 19-bp Sp1 DNA/DNA
hybrids, a concentration-dependent inhibition of
interactions between 32P-labeled Sp1 14-mer and nuclear
factors was observed (Fig.
2A). As expected, the longer
double-stranded HIV-Sp1 oligonucleotide displayed higher decoy
efficiency with respect to the 14-mer oligonucleotide. 50% inhibition
of Sp1-DNA interactions was obtained with 25 ng of the 14-mer
oligonucleotide and 6 ng of the 19-mer oligonucleotide (Fig.
2A, lower part of the panel). The
experiments shown in Fig. 2B, left side of the
panel, and Fig. 2C, lower side of the
panel, clearly indicate that DNA/PNA, PNA/DNA, or PNA/PNA
HIV-Sp1 molecules are unable, even when added at 100 ng/reaction, to
inhibit the interactions between 32P-labeled Sp1 14-mer and
nuclear factors.
The Double-stranded DNA/PDP, PDP/DNA, and
PDP/PDP Molecules Carrying PNA-DNA-PNA Chimeras Mimicking
Sp1 Binding Sites Efficiently Inhibit the Interactions between Sp1
Nuclear Factors and Target DNA/DNA Molecules--
The
effects of putative decoy molecules based on PNA-DNA chimeras were at
first determined on the molecular interactions between 32P-labeled Sp1 14-mer and nuclear factors (Fig. 2,
B and C); subsequently, their effects were also
assayed using the 32P-labeled Sp1 19-mer (Fig.
3). The results obtained clearly indicate that DNA/PDP and PDP/DNA hybrids are efficient decoys (in particular, see Fig. 2C). PDP/PDP molecules are also able to suppress
the interactions between 32P-labeled Sp1 14-mer and nuclear
factors, but when added to the binding reaction at a concentration of
25 ng/reaction. These effects were reproducibly obtained also using the
32P-labeled Sp1 19-mer. In this case, the binding of
nuclear factors to the target DNA originates three retarded bands (Fig.
3, arrows). We first performed control experiments
demonstrating the decoy effects of the double-stranded HIV-Sp1 19-mer
DNA/DNA (Fig. 3A); then, we demonstrated that NF-IL2A and
GATA-1 DNA/DNA hybrids had no inhibitory effects on the binding of
nuclear factors to the 32P-labeled Sp1 19-mer (Fig.
3B). Fig. 3C demonstrates that DNA/PDP, PDP/DNA,
and PDP/PDP hybrids are able to efficiently inhibit the interactions
between the 32P-labeled Sp1 19-mer and nuclear factors.
Fig. 3C (right side of the panel)
shows that DNA/PNA, PNA/DNA, and PNA/PNA hybrids carrying Sp1 binding
sites do not exhibit any decoy activity even when added at 200 ng/reaction. Finally, HIV-Sp1 PDP/DNA and PDP/PDP hybrids exhibit low
ability to inhibit the generation of the fast mobility band. This was
reproducibly obtained in independent experiments and tentatively
explained by a different affinity of PNA-DNA-based decoy molecules for
different proteins (or protein complexes) belonging to the Sp1
family.
The Double-stranded DNA/PDP, PDP/DNA, and
PDP/PDP Molecules Carrying PNA-DNA-PNA Chimeras Mimicking
HIV-1 Sp1 Binding Sites Do Not Inhibit the Interactions between STAT-1,
NF-IL2A, and GATA-1 Transcription Factors to the Relative Target
DNA-DNA Sequences--
The results reported in Fig.
4, which were performed using
32P-end-labeled STAT-1, NF-IL2A, and GATA-1 DNA-DNA target
molecules and nuclear factors isolated from the K562 cell line, firmly
establish that the effects of PNA-DNA chimera-based decoy molecules are sequence-specific. In fact, although STAT-1, NF-IL2A, and GATA-1 cold
oligomers suppress the binding of nuclear factors to the relative
32P-end-labeled DNA/DNA target molecules, no inhibitory
activity was determined by addition of double-stranded PDP/DNA,
DNA/PDP, and PDP/PDP chimera mimicking the HIV-1 Sp1 binding sites.
Binding of Nuclear Factors to 32P-labeled
DNA/PDP, PDP/DNA, and PDP/PDP
Chimeras--
To directly confirm that PNA-DNA chimera-based molecules
are recognized by Sp1 factors, we carried out another set of
experiments. DNA strands were labeled with [ The Double-stranded DNA/PDP, PDP/DNA, and
PDP/PDP Molecules Carrying PNA-DNA-PNA Chimeras Mimicking
the HIV-1 Sp1 Binding Sites Inhibit the Interactions between Nuclear
Factors and Sp1 Binding Sites Present within the Promoter of Genes
Coding
The same extent of inhibition was reached using 6 and 12 ng of
Ex Vivo Effects of PDP-based Decoy Hybrids: Inhibition of
ara-C-induced Erythroid Differentiation of K562 Cells--
The finding
that DNA/PDP, PDP/DNA, and PDP/PDP HIV-Sp1 molecules are able to
inhibit the binding of nuclear factors to 32P-end-labeled
Therefore, we determined the activity of HIV-Sp1 DNA/PDP, PDP/DNA, and
PDP/PDP molecules on ara-C-treated K562 cells and compared this
activity with that of HIV-Sp1 DNA/DNA molecules. Cells were treated for
6 days in the presence or absence of the indicated concentration of Sp1
decoy molecules, and the proportion of benzidine-positive cells was
determined. The results obtained are shown in Fig. 7D and
demonstrate that all PDP-based Sp1 decoys are able to reduce the
ara-C-induced increase of benzidine-positive cells. Among PDP-based
decoy molecules, the most active ones on the inhibition of erythroid
differentiation were found to be DNA/PDP and PDP/DNA. On the contrary,
only slight inhibition was found using PDP/PDP decoy molecules. Control
experiments demonstrated that unrelated DNA/DNA molecules were almost
uneffective in inhibiting ara-C-mediated increase of the proportion of
benzidine-positive cells (data not shown and Fig. 7D). These
experiments suggest that PNA-DNA chimera-based decoy molecules for Sp1
nuclear factors are active on ex vivo experimental cell
systems, their efficiency being very similar to that of Sp1 DNA/DNA
decoy molecules, at least in the case of DNA/PDP and PDP/DNA hybrids.
Stability of the Decoy Molecules Based on PNA-DNA-PNA
Chimeras--
The results of the experiments shown in Figs. 2, 3, and
7 suggest that double-stranded biomolecules based on PNA-DNA chimeras might be considered efficient decoy molecules. To propose these biomolecules for gene therapy, however, two points should be, in our
opinion, addressed: stability of the decoy molecules in serum and
possible delivery with commonly used vectors, such as liposomes.
To determine the stability of the decoy molecules based on PNA-DNA-PNA
chimeras mimicking the Sp1 binding sites, unlabelled PDP/PDP molecules
or 32P-end-labeled DNA/PDP and PDP/DNA HIV-Sp1 hybrid
molecules were incubated (a) with increasing amounts of 3'
Fig. 8 clearly demonstrates that Sp1 PDP/PDP decoy molecules are
resistant to ExoIII 3' Complexation of PDP-based Decoy Molecules to Cationic
Liposomes--
Different cationic detergents were used, namely
Lys4-Chol and Lys4-Palm, for the production of
the liposomal formulation (47, 48). The resulting liposomal
formulations were named as follows: lipo-Lys4-Chol and
lipo-Lys4-Palm. The complexation of PNA-DNA chimeras to
liposomes was performed just before the analysis, simply mixing the
nucleic acid to "preformed" cationic vesicles, resulting in a
quantitative association yield.
Fig. 9 (A and B)
demonstrates that Lys4-Chol and Lys4-Palm are
able to complex to 32P-end-labeled HIV-Sp1 DNA/PDP and
PDP/DNA hybrids. This is demonstrated by the formation of complexes
(identified as bound material) unable to migrate into the gels when
high concentrations of liposomes were used (1-2 µg/reaction). It
should be noted that complexation of Sp1 DNA/PDP and PDP/DNA to both
Lys4-Palm and Lys4-Chol is similar to
complexation of Sp1 DNA/DNA hybrids. Both Lys4-Chol and
Lys4-Palm are also able to complex with PDP/PDP HIV-Sp1
(Fig. 9C). In this case, due to the difficulty in obtaining
32P-labeled PDP/PDP chimeras (56), unlabelled PDP/PDP
molecules were used, and the gels were stained with ethidium bromide.
The data presented in Fig. 9 conclusively demonstrate that Sp1 decoy molecules based on PNA-DNA chimeras can be complexed to cationic liposomes.
These results are relevant for the delivery of PDP-based decoys, as
well as for a possible increase of their resistance to nucleases. This
could be particularly important for PDP/DNA and DNA/PDP molecules. To
determine whether liposome complexation leads to an increase of
resistance of PDP/DNA molecules to degradation in serum, the experiment
shown in Fig. 9D was performed. Free or liposome-complexed
32P-end-labeled DNA/PDP HIV-Sp1 hybrid molecules were
incubated in the presence of serum concentrations causing complete
degradation of the PNA/DNA molecules (as shown in Fig. 8C,
lane d). The results obtained clearly suggest that
complexation of Sp1 DNA/PDP molecules to both
Lys4-Chol and Lys4-Palm liposomes leads to a
protective effect against enzymatic degradation.
The transcription factors belonging to the Sp1 superfamily are of
great importance for the control of expression of a variety of genes.
Members of this family of regulatory proteins bind with varying
affinities to sequences designated as "Sp1 sites" (for instance GC
and CACCC boxes), making up a transcriptional network playing an
important role in the fine-tuning of gene expression. Sp1-dependent transcription can be growth-regulated, and
the activity, expression, and/or post-translational modification of
multiple family members is altered with cell growth. Furthermore, Sp1
factors are involved in many growth-related signal transduction
pathways, apoptosis, and angiogenesis and, therefore, in several
aspects of tumorigenesis (51).
With respect to gene therapy, the decoy approach against Sp1
transcription factors has been proposed as a useful tool to alter Sp1-dependent gene expression (3, 21, 25). This was
achieved by using synthetic ODNs carrying Sp1-specific cis elements as decoy molecules.
In fact, Ishibashi et al. (3) demonstrated that Sp1 decoy
ODNs are able to inhibit the tumor necrosis factor- In a recent study, we proposed PNAs as alternative reagents in
experiments aimed at the control of gene expression involving the decoy
approach (20). In PNAs, the pseudopeptide backbone is composed of
N-(2-aminoethyl)glycine units (27-30). PNAs hybridize with
high affinity to complementary sequences of single-stranded RNA and
DNA, forming Watson-Crick double helices (27), and are resistant to
both nucleases and proteases (32). We demonstrated that NF- More recently, we determined whether PNA-DNA chimeras mimicking NF- In the present report, our main focus was to determine whether decoy
molecules based on PNA and PNA-DNA chimeras are capable of stable
interactions with Sp1 and, therefore, could act as decoy reagents able
to inhibit molecular interactions between Sp1 and DNA. The results
obtained firmly indicate that Sp1-molecules based on PNA-DNA-PNA
chimeras are powerful decoy reagents (Figs. 2, 3, and 6). This effect
is specific since these decoys do not affect binding of NF-IL2A,
GATA-1, and STAT-1 to their binding elements.
A second conclusion to be drawn from the results of this report is that
PDP/DNA and DNA/PDP Sp1 decoy molecules exhibit the ability to inhibit
ara-C-induced erythroid differentiation of human leukemia K562 cells to
an extent similar to that of Sp1 DNA/DNA decoys. This finding supports
the hypothesis of a biological activity of decoy molecules based on
PNA-DNA chimeras.
Our results are expected to have practical implications. The finding
that DNA-PNA chimeras stably interact with Sp1 transcription factors
encourages further experiments focused on the possible use of these
molecules for the development of potential agents for a decoy approach
in gene therapy. In this respect, the finding that PDP-based decoy
molecules are more resistant than DNA/DNA hybrids to enzymatic
degradation (Fig. 8 and data not shown) appears to be of great
interest. Furthermore, their resistance can even be improved, and their
delivery could be facilitated after complexation with cationic
liposomes or microspheres (56) to which PDP/PDP chimeras are able to
bind by virtue of their internal DNA structure.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B1
superfamily (7-9, 13-20). Decoy molecules against NF-
B inhibit the
expression of NF-
B regulated genes (MHC complex genes,
IL2 receptor
, Igk, IL6,
opioid receptor, preprogalanin,
adhesion molecule-1) (20). More recently, dumbell DNA decoy elements against NF-
B were demonstrated to be active in inhibiting ex vivo transcription driven by the long terminal repeat (LTR) of human immunodeficiency type-1 virus (HIV-1) (19). In addition to
proteins belonging to the NF-
B superfamily, decoy molecules for
other target transcription factors, such as HNF-1, RFX1, nuclear factor YB, E2F, cAMP-response element, and Sp1, were found to be
effective (1-6, 10-12, 21).
-mediated expression of both vascular
endothelial growth factor and transforming growth factor
1. These
results are appealing since it is well known that the expression of
these genes is an important aspect in growth and metastasis of solid tumors. In addition, it was found that the in vitro
invasiveness, synthesis of mRNA for uPA, and cell proliferation
were also inhibited by the transfection of Sp1 decoy ODNs, suggesting
that Sp1 decoy strategy could be effective for regulating tumor growth
by reducing in cancer cell (a) angiogenic growth factor
expression, (b) proliferation, and (c)
invasiveness. In another report, Verrecchia et al. (21) found that decoy Sp1-binding ODNs inhibited COL1A2
promoter activity both in cultured fibroblasts and in vivo,
in the skin of transgenic mice, which have integrated a mouse
COL1A2 promoter/luciferase reporter gene construct,
indicating that targeting Sp1 efficiently blocks extracellular matrix
gene expression, and suggest that such an approach may represent an
interesting therapeutic alternative toward the treatment of fibrotic disorders.
B p52 is able to bind to both DNA/DNA and
DNA/PNA hybrids mimicking the NF-
B target sites present in the HIV-1
LTR. On the contrary, low binding of NF-
B p52 to PNA/PNA hybrids was
found (20). We have also reported a conformational study to explain
these binding data using a molecular dynamics approach. These data have
underlined that the loss of charged phosphate groups and the different
shape of the helix in PNA/DNA and PNA/PNA hybrids drastically reduce
binding efficiency to NF-
B transcription factor (31).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP and T4
polynucleotide kinase (MBI Fermentas, Milano, Italy) in the case
of DNA/DNA, DNA/PNA, and DNA/PDP hybrids. 32P-labeled
PDP/PDP molecules were obtained by nick translation, using low
concentrations (0.1-0.01 units/reaction) of DNase I and
[
-32P]dCTP. Binding reactions were set up as described
elsewhere (43) in a total volume of 25 µl of binding buffer plus 5%
glycerol, 1 mM dithiothreitol, and 0.25 ng of
32P-labeled oligonucleotides. 12 µg of crude nuclear
extracts isolated from human cell lines were used, and the binding
reaction was carried out in the presence of 1 µg of the nonspecific
competitor poly(dIdC)·poly(dIdC) (43). After 20 min of binding at
room temperature, the samples were electrophoresed at constant voltage (200 V) under low ionic strength conditions (0.25× TBE buffer = 22 mM Tris borate, 0.4 mM EDTA) on 6%
polyacrylamide gels. Gels were dried and subjected to standard
autoradiographic procedures (43). In competition experiments, the
competitor molecules carrying HIV-1 Sp1 binding sites (DNA/DNA,
PNA/PNA, DNA/PNA, PDP/PDP, and DNA/PDP) were preincubated for 20 min
with nuclear extracts, before the addition of labeled target DNA.
Nuclear extracts were prepared according to Dignam et al.
(42). The nucleotide sequences of competitor double-stranded target
DNAs used as controls were 5'-TAA TAT GTA AAA ACA TT-3' (sense strand,
NF-IL2A), 5'-CAC TTG ATA ACA GAA AGT GAT AAC TCT-3' (sense strand,
GATA-1), and 5'-CAT GTT ATG CAT ATT CCT GTA AGT G-3' (sense strand,
STAT-1).
20 °C in the dark and diluted immediately before use. Treatment
with the indicated concentrations of DNA- and PNA-based molecules was
carried out by adding the appropriate concentrations of the compounds
at the beginning of the experiment (cells were usually seeded at 30,000 cells/ml). The medium was not changed during the induction period. K562
cells containing heme or hemoglobin were detected by specific reaction
with a benzidine/hydrogen peroxide solution as reported elsewhere (45,
46). The final concentration of benzidine was 0.2% in 5 M
glacial acetic acid, 10% H2O2 (45, 46).
5'-exonuclease III, 5'
3'-
-exonuclease, and
DNase I. ExoIII,
-exonuclease, and DNase I were purchased from MBI
Fermentas (ExoIII and
-exonucleases) and Promega Corp., Madison, WI
(DNase I). In addition, serum (fetal calf serum, Eurobio, 30 g/liter of
protein concentration) was also employed. After incubation with
increasing amounts of the enzymes (for 10 min in the case of ExoIII,
for 30 min in the case of
-exonuclease and DNase I), the decoy
molecules were layered on the top of a 2% agarose gel and detected by
ethidium bromide staining. Disappearance of the decoy molecule was
considered as an evidence of degradation by the employed enzymes.
Results were presented as percentage of recovery with respect to
control untreated reaction mixtures.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Synthetic oligonucleotides, PNA, and PNA-DNA chimeras
-globin (
-glob-Sp1), and
-globin (
-glob-Sp1) genes and
(b) additional oligonucleotides carrying unrelated binding
sites for NF-IL2A, STAT-1, and GATA-1 transcription factor proteins.
The sequences of these oligonucleotides are shown in Table I or stated under "Materials and Methods."
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Fig. 1.
Structure and sequences of
the decoy molecules employed. DNA stretches are in
green, PNA stretches are in dark blue.
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Fig. 2.
Effects of DNA-, PNA-, and PDP-based hybrids
on molecular interactions between nuclear proteins and
32P-labeled HIV-1 Sp1 14-mer DNA/DNA target molecules.
12 µg of K562 nuclear extracts were incubated for 20 min in binding
buffer in the absence ( ) or in the presence of the indicated amounts
of decoy molecules. After this incubation period, a further 20-min
incubation step was performed in the presence of
32P-labeled HIV-1 Sp1 14-mer DNA/DNA target molecules.
Protein/DNA complexes were separated by polyacrylamide gel
electrophoresis, and autoradiography was performed. Arrows
indicate protein/DNA complexes. * indicates the free
32P-labeled Sp1 mer.
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Fig. 3.
Effects of DNA-, PNA-, and PDP-based hybrids
on molecular interactions between nuclear proteins and
32P-labeled HIV-1 Sp1 19-mer DNA/DNA target molecules.
12 µg of K562 nuclear extracts were incubated for 20 min in binding
buffer in the absence ( ) or in the presence of the indicated amounts
of decoy molecules. After this incubation period, a further 20-min
incubation step was performed in the presence of
32P-labeled HIV-1 Sp1 19-mer DNA/DNA target molecules.
Protein/DNA complexes were separated by polyacrylamide gel
electrophoresis, and autoradiography was performed. Arrows
indicate protein/DNA complexes. * indicates the free
32P-labeled Sp1 mer. Control DNA/DNA competitors carrying
binding sites for GATA-1 and NF-IL2A were used, as indicated.
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Fig. 4.
Effects of DNA/DNA, DNA/PDP, PDP/DNA, and
PDP/PDP hybrids carrying Sp1 binding sites on the interaction between
crude nuclear extracts from human leukemic K562 cells and
32P-labeled STAT-1 (A), NF-IL2A
(B), and GATA-1 (C) DNA-DNA target
molecules. 12 µg of nuclear factors were incubated for 20 min in
binding buffer in the absence ( ) or in the presence of 100 ng of
DNA/DNA, DNA/PDP, PDP/DNA, and PDP/PDP molecules. After this incubation
period, a further 20-min incubation step was performed in the presence
of 32P-labeled DNA/DNA target molecules. Protein/DNA
complexes were separated by polyacrylamide gel electrophoresis, and
autoradiography was performed. Arrows indicate protein/DNA
complexes. * indicates the free 32P-labeled NF-IL2A, GATA-1
and STAT-1 mers. Control DNA/DNA competitors carrying binding sites for
STAT-1, NF-IL2A, and GATA-1 were used (100 ng) to verify the
specificity of protein/DNA interactions observed.
-32P]ATP
before annealing to the PNA-DNA-PNA chimera or PNA-relative counterparts (Fig. 5). This allows the
generation of 32P-labeled Sp1 DNA/PNA, PNA/DNA, DNA/PDP,
and PDP/DNA molecules. In addition, PDP/PDP molecules were labeled by
nick translation reaction, using low amounts of DNase I, as described
under "Materials and Methods" and reported elsewhere (56). When
these PNA and PDP-based 32P-labeled hybrids were added to
12 µg of nuclear extracts from K562 cells, only DNA/PDP and PDP/DNA
molecules generated a clearly detectable retarded band (Fig.
5A). The retarded band generated by PDP/PDP hybrids is
barely detectable (Fig. 5A, right panel), as
expected in consideration of the low specific activity of these 32P-labeled molecules (56). On the contrary, Sp1 DNA/PNA
and PNA/DNA hybrids were unable to generate retarded bands, as shown in
Fig. 5B. In this experimental approach, Sp1 PNA/PNA hybrids
were not considered due to the difficulty of obtaining radioactive
molecules. The data shown in Fig. 5 confirm on the one hand that Sp1
PNA/DNA hybrids are not efficiently recognized by Sp1 nuclear factors, whereas PDP-based hybrids are recognized by these nuclear proteins. Despite the slightly different retarded patterns obtained, the data
shown in Fig. 5A support the hypothesis that the decoy
activity of Sp1 PDP-based hybrids (Figs. 2 and 3) is due to the ability of these molecules to interact with transcription factors belonging to
the Sp1 family.
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Fig. 5.
Direct binding of nuclear factors to PDP- and
PNA-based hybrids. 32P-labeled Sp1 DNA/DNA
(A and B), DNA/PDP (A), PDP/PDP
(A), DNA/PNA (B), and PNA/DNA (B)
hybrids were incubated for 20 min in binding buffer in the absence ( )
or in the presence (+) of 12 µg of nuclear extracts from K562 cells.
Protein/DNA complexes were separated by polyacrylamide gel
electrophoresis, and autoradiography was performed. Arrows
indicate complexes between proteins and target molecules.
-Globin,
-Globin, and uPAR--
The experiment reported
in Fig. 6 (A-C) was performed
using 32P-end-labeled
-globin Sp1 DNA-DNA
target molecules and nuclear factors isolated from K562 cells. The
results obtained firmly establish that DNA/PDP, PDP/DNA, and PDP/PDP
HIV-Sp1 molecules are able to inhibit the binding of nuclear factors to
32P-end-labeled
-globin Sp1 DNA-DNA target molecules
when added at high concentrations (50 ng/reaction; Fig. 6C,
left side of the panel).
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Fig. 6.
Effects of DNA-, PNA-, and PDP-based hybrids
on molecular interactions between nuclear proteins and
32P-labeled Sp1 binding sites present in the
-globin (A-C),
-globin (D), and uPAR
(E) promoters. 12 µg of K562 nuclear
extracts were incubated for 20 min in binding buffer in the absence
(
) or in the presence of the indicated amounts of decoy molecules.
After this incubation period, a further 20-min incubation step was
performed in the presence of 32P-labeled
-glob-Sp1,
-glob-Sp1, and uPAR
target molecules. Protein/DNA complexes were separated by
polyacrylamide gel electrophoresis, and autoradiography was performed.
Arrows indicate protein/DNA complexes. * indicates the free
32P-labeled Sp1 mer. Control DNA/DNA competitor carrying
binding sites for GATA-1 was used, as indicated.
-globin Sp1 DNA/DNA and HIV-Sp1 DNA/DNA hybrids, respectively (Fig.
6, A and B). Inhibitory effects of DNA/PDP,
PDP/DNA, and PDP/PDP HIV-Sp1 molecules were also found on the binding
of nuclear factors to 32P-end-labeled
-globin
(Fig. 6D) and uPAR (Fig. 6E) Sp1
DNA-DNA target molecules. These data demonstrate that the HIV-Sp1 decoy is able to inhibit the binding of Sp1 factors to other promoter elements carrying Sp1-like binding sites.
-globin and
-globin Sp1 DNA-DNA target
molecules prompted us to determine their effects on the human leukemic
K562 cells. This cell line does not produce large amounts of hemoglobin (Fig. 7A) but, after treatment
for 5-7 days with a variety of chemical inducers, among which were
5-azacytidine (45), mithramycin (43), tallimustine (44), and cytosine
arabinoside (ara-C) (44), they undergo erythroid differentiation,
becoming positive to the benzidine stain (benzidine-positive cells as
shown in Fig. 7B). Erythroid differentiation of K562 cells
is associated with accumulation of Hb Portland
(
2
2) and Hb Gower 1 (
2
2) (43). Therefore, activation of both
-globin and
-globin genes is operated in
erythroid-induced K562 cells, as also determined and elsewhere verified
by Northern blotting and quantitative reverse transcription-PCR analysis (44). Interestingly, if ara-C-treated K562 are cultured in the
presence of HIV-Sp1 DNA/DNA molecules, erythroid differentiation is
inhibited (Fig. 7C). In contrast, unrelated double-stranded oligonucleotides are unable to exert this inhibitory effect (Fig. 7C). The finding that Sp1 double-stranded oligonucleotides
are able to inhibit erythroid differentiation of K562 cells is in agreement with a number of reports pointing out that Sp1 transcription factor could be involved in transcriptional regulation of the expression of erythroid-specific genes, including the human globin genes (49-54).
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Fig. 7.
Effects of DNA-, PNA-, and PDP-based hybrids
on ara-C-mediated induction of erythroid differentiation of K562
cells. A and B, benzidine
staining of uninduced (A) and ara-C-induced K562
(B) cells. C, increase in the proportion of
benzidine-positive cells cultured with 1 µM ara-C in the
absence (closed symbols) or in the presence (open
symbols) of 5 µg/ml HIV-1 Sp1 DNA/DNA molecules. D,
effects of DNA/DNA, DNA/PDP, PDP/DNA, and PDP/PDP molecules on increase
of benzidine-positive cells after 6 days of cell culture in the
presence of 5 µg/ml decoy molecules. ( ), ara-C-treated K562
cells.
5'-exonuclease (Fig. 8, A
and B) and (b) with serum (Fig. 8C).
After incubations, the decoy molecules were isolated, layered on the
top of a polyacrylamide (the 32P-labeled decoy molecules)
or an agarose gel (the unlabelled decoy molecules), and
electrophoresed, and autoradiography was performed in the case of
DNA/PDP or DNA/DNA molecules (Fig. 8A). In the case of
PDP/PDP hybrids (Fig. 8B) or when serum was employed (Fig. 8C), gels were stained with ethidium bromide. This was
necessary due to the 5'-phosphatase activity present in serum, leading
to a rapid removal of 5'-32P (data not shown).
Disappearance of the 32P-end-labeled or ethidium
bromide-stained bands indicates that degradation of the decoy molecules
has occurred. The observed stabilities of PDP/PDP hybrids or DNA/PDP
molecules were compared with those of DNA/DNA decoy molecules.
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Fig. 8.
Stability of decoy molecules.
A and B, experiments showing the effects of Exo
III on DNA/DNA (A and B), DNA/PDP (A),
and PDP/PDP (B) decoy molecules. 250 ng of HIV-1 Sp1
PDP/PDP, DNA/PDP, and DNA/DNA decoys were incubated for 10 min in the
absence (a) or in the presence of 0.001 (b), 0.01 (c), 0.1 (d), 1 (e), 10 (f), and 100 (g) units of Exo III in a 20-µl
reaction mixture. After incubation, the decoy molecules were layered on
the top of a 20% polyacrylamide gel and detected by autoradiography
(A) or on 2% agarose gels and detected by ethidium bromide
staining (B). C, differential effects of serum
(a = no serum; b = 3 µl/reaction;
c = 12.5 µl/reaction; d = 25 µl/reaction) on DNA (upper part of the panel)
and PNA-DNA-PNA (middle and lower parts of the
panel)-based decoys. Incubations were conducted at 37 °C
for 3 h in a 50-µl reaction mixture. After incubation, the decoy
molecules were layered on the top of a 2% agarose gel and detected by
ethidium bromide staining. DNA/DNA and DNA/PDP hybrids were
32P-labeled in panel A. In panels B
and C, unlabelled decoy molecules were used. Disappearance
of the decoy molecules was considered as an evidence of degradation by
the employed enzymes.
5'-exonuclease (Fig. 8B), unlike the corresponding DNA/DNA hybrid. PDP/PDP chimeras are also resistant to serum (Fig. 8C, lower part of the
panel). When experiments were conducted using DNA/PDP
hybrids, the results obtained demonstrated that DNA/PDP hybrids exhibit
a high level of resistance to both 3'
5'-exonuclease (Fig.
8A) and serum (Fig. 8C, middle part of
the panel) as compared with HIV-Sp1 DNA/DNA decoy
oligonucleotides. These results were consistently reproduced in three
independent experiments; in addition, results similar to those obtained
with DNA/PDP molecules were also obtained using PDP/DNA decoy hybrids (data not shown). Furthermore, DNA/PDP and PDP/DNA molecules were consistently found more resistant than DNA/DNA hybrids to 5'
3'-exonuclease and DNase I and when exposed to cellular
extracts.2 Taken together,
these data demonstrate that decoy molecules based on PNA-DNA-PNA
chimeras exhibit higher levels of resistance to nucleases with respect
to decoy molecules based on DNA/DNA hybrids.
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Fig. 9.
Comparison of complexation efficiencies of
Sp1 DNA/DNA, DNA/PDP and PDP/DNA hybrids to the different liposomal
formulations and protective effects. As shown in A-C,
the indicated amounts of liposomes were incubated in the presence of
radiolabeled hybrid molecules, and formed complexes were
electrophoresed through an agarose gel and exposed to autoradiographic
procedure. Complexation of Sp1 DNA/DNA, DNA/PDP, and PDP/DNA hybrids
with liposomes were performed using the multivalent cationic lipid
Lys4-Chol (lipo-Lys4-Chol) (A) or
Lys4-Palm (lipo-Lys4-Palm) (B).
C, complexation of Sp1 PDP/PDP molecules to
lipo-Lys4-Chol (Lys4-Chol)
and lipo-Lys4-Palm
(Lys4-Palm). In this case, detection was
performed on unlabelled PDP/PDP hybrids by ethidium bromide staining
of molecules after agarose gel electrophoresis.
D, protective effects of lipo-Lys4-Chol and
lipo-Lys4-Palm. 32P-labeled DNA/DNA and DNA/PDP
hybrids were incubated for 30 min with or without the indicated amounts
of liposomes (final reaction volume = 50 µl). A further
overnight incubation period (final reaction volume = 75 µl) was
then performed in the absence or in the presence of serum (25 µl/reaction), as indicated. 32P-labeled material was
phenol-extracted, ethanol-precipitated, and analyzed by polyacrylamide
gel electrophoresis and autoradiography.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mediated expression of vascular endothelial growth factor, transforming growth
factor
1, and tissue factor by cancer cells. In another report,
Verrecchia et al. (21) found that decoy Sp1-binding oligonucleotides inhibited COL1A2 promoter activity
both in cultured fibroblasts and in vivo. Motojima et
al. (22) demonstrated that Sp1 decoys are able to inhibit
angiotensin II-induced expression of PAI-1 gene in mesangial
cells. Finally, Hata et al. (25) showed that treatment with
Sp1 decoys reduced expression of kinase domain receptor, a tyrosine
kinase receptor for vascular endothelial growth factor. This
transcriptionally regulated receptor is a critical mediator of
endothelial cell growth and vascular development. Unfortunately,
synthetic ODNs are not stable and, therefore, should be extensively
modified to be used in vivo or ex vivo
(1-7).
B p52 is
able to bind to both NF-
B DNA/DNA and DNA/PNA hybrid, mimicking the
NF-
B target sites present in the HIV-1 LTR. However, the binding of
the NF-
B DNA-PNA to NF-
B transcription factors was found to
exhibit low stability and, therefore, this reagent is expected to be
unsuitable for a decoy approach (20).
B
binding sites are capable of stable interactions with proteins
belonging to the NF-
B family (55). DNA-PNA chimeras were originally
proposed to improve the poor cellular uptake and solubility of PNAs
(30). More recently, they were found to exhibit biological properties
typical of DNA, such as the ability to stimulate RNaseH activation and
to act as substrate for cellular enzymes (for instance, DNA
polymerases) (30). Molecular modeling was employed for the design of
PNA-DNA chimeras; prediction of molecular interactions between chimeras
and NF-
B nuclear proteins were investigated by molecular dynamics
simulations, and interactions between PNA-DNA chimeras and NF-
B
proteins were studied by gel shifts. We found significant differences
between the structure of duplex NF-
B PNA-DNA chimera and duplex
NF-
B DNA-DNA (31). However, it was found that these differences do
not prevent the duplex PNA-DNA chimera from binding to NF-
B
transcription factors, being able to suppress the molecular
interactions between HIV-1 LTR and p50, p52, and nuclear factors from
B-lymphoid cells. Therefore, these results demonstrate that the
designed NF-
B DNA-PNA chimeras could be used for a decoy approach in
gene therapy.
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ACKNOWLEDGEMENTS |
---|
Giuseppe Perretta is acknowledged for technical assistance. We thank to Prof. J. H. van Boom and J. C. Verhejien for giving the possibility of synthesizing the chimeras in their laboratory. We thank Dr. Susan Treves (Departments of Anesthesia and Research, Hebelstrasse 20, Kantonsspital, 4031, Basel, Switzerland) for checking the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by CNR-P.F. Biotecnologie, Ministero dell'Università e della Ricerca Scientifica e Tecnologica-PRIN-2002, CNR Agenzia 2000, Finalized Research funds (year 2001) from the Italian Ministry of Health, and Ministero dell'Istruzione, Università e Ricerca-Contributo Straordinario Decreto 4-10-2001.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
39-532-291448; Fax: 39-532-202723; E-mail: gam@unife.it.
Published, JBC Papers in Press, November 20, 2002, DOI 10.1074/jbc.M206780200
2 M. Borgatti, I. Lampronti, A. Romanelli, C. Pedone, M. Saviano, N. Bianchi, C. Mischiati, and R. Gambari, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NF-B, nuclear factor
B;
Sp1, promoter-specific transcription factor Sp1;
HIV-1, human immunodeficiency virus type 1;
LTR, long terminal repeat;
ODN, oligodeoxyribonucleotide;
PNA, peptide nucleic acid;
PDP, PNA-DNA-PNA chimera;
uPA, urokinase-type plasminogen activator;
uPAR, urokinase-type plasminogen activator receptor;
ara-C, cytosine
arabinoside;
HPLC, high pressure liquid chromatography;
Lys4-Chol, tetralysine-cholesterol;
Lys4-Palm, tetralysine-palmitate;
lipo-Lys4-Chol, liposomal
formulation of Lys4-Chol;
lipo-Lys4-Palm, liposomal formulation of Lys4-Palm;
Fmoc, N-(9-fluorenyl)methoxycarbonyl;
glob, globin.
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REFERENCES |
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