|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 852-862
Oligodeoxyribonucleotide Uptake in Primary Human Hematopoietic
Cells Is Enhanced by Cationic Lipids and Depends on the
Hematopoietic Cell Subset
By
Ralf Kronenwett,
Ulrich Steidl,
Michael Kirsch,
Georg Sczakiel, and
Rainer Haas
From Klinische Kooperationseinheit Molekulare
Hämatologie/Onkologie and Forschungsschwerpunkt Angewandte
Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany;
and Medizinische Klinik und Poliklinik V, Universität
Heidelberg, Heidelberg, Germany.
 |
ABSTRACT |
The use of antisense oligodeoxyribonucleotides (ODN) is a potential
method to switch off gene expression. The poor cellular uptake of ODN
in primary cells still is a limiting factor that may contribute to the
lack of functional efficacy. Various forms of cationic lipids have been
developed for efficient delivery of nucleic acids into different cell
types. We examined the two cationic lipids DOTAP and DOSPER to improve
uptake of ODN into primary human hematopoietic cells. Using a
radiolabeled 23-mer, ODN uptake into blood-derived mononuclear cells
could be increased 42- to 93-fold by DOTAP and 440- to 1,025-fold by
DOSPER compared with application of ODN alone. DOTAP was also effective
for delivery of ODN into leukocytes within whole blood, which may
resemble more closely the in vivo conditions. As assessed by
fluorescein isothiocyanate-conjugated ODN both cationic lipids
enhanced cytoplasmic accumulation of ODN in endosome/lysosome-like
structures with a partial shift of fluorescence to the whole cytoplasm
and the nucleus following an incubation of 24 hours. ODN uptake by
cationic lipids into different hematopoietic cell subsets was examined by dual-color immunofluorescence analysis with subset-specific monoclonal antibodies. We found a cell type-dependent delivery of ODN
with greatest uptake in monocytes and smallest uptake in T cells.
CD34+ cells, B cells, and granulocytes took up ODN at an
intermediate level. Uptake of ODN into isolated CD34+
cells could be increased 100- to 240-fold using cationic lipids compared with application of ODN alone. Stimulation of
CD34+ cells by interleukin-3 (IL-3), IL-6, and stem cell
factor did not significantly improve cationic lipid-mediated ODN
delivery. Sequence-specific antisense effects in clonogenic assays
could be shown by transfection of bcr-abl oncogene-directed
antisense ODN into primary cells of patients with chronic myelogenous
leukemia using this established protocol. In conclusion, cationic
lipids may be useful tools for delivery of antisense ODN into primary hematopoietic cells. These studies provide a basis for clinical protocols in the treatment of hematopoietic cells in patients with
hematologic malignancies and viral diseases by antisense ODN.
| |
INTRODUCTION |
ANTISENSE oligodeoxyribonucleotides (ODN)
are capable of downregulating gene expression and are used for the
assessment of gene function and for therapeutic
purposes.1-3 Several clinical trials are ongoing with
antisense ODN directed to hematopoietic cells for the treatment of
hematologic malignancies and viral diseases. For instance, a human
immunodeficiency virus-1 (HIV-1)-directed antisense ODN is used for
systemic in vivo administration in patients with aquired
immunodeficiency syndrome to protect normal T lymphocytes and
macrophages.4 In other clinical trials bcr-abl-,
c-myb-, or p53-directed antisense ODN were used for systemic
therapy or for ex vivo treatment of hematopoietic cells in patients
with acute and chronic myelogenous leukemia or advanced myelodysplastic syndrome.5-8 Functional efficacy of ODN requires not only
the selection of an appropriate target sequence9-12 but
also a sufficient intracellular concentration. The latter one depends
on the degree of cellular uptake, the intracellular distribution, and
the rate of degradation of ODN by serum and cytoplasmic
nucleases.1 Anionic ODN cannot diffuse through cell
membranes, but are actively taken up by endocytosis.13-15
As a result, only a small amount of extracellular ODN is available in
the cytosol due to the limited capacity of the endocytotic process and
the lysosomal degradation. In the light of the ongoing clinical
studies, an increased stability and uptake of ODN into hematopoietic
cells could improve the efficacy of antisense nucleic acid-based
therapies. A variety of cationic lipids with low toxicity are
protective against degradation and can facilitate the transport of ODN
into different cell types mainly by endocytosis even in the presence of
human serum.16-19
In this study, we examined the delivery of phosphorothioate-modified
ODN by cationic lipids into primary human hematopoietic cells,
including CD34+ cells using the cationic lipids DOTAP and
DOSPER under ex vivo culture conditions as well as under conditions
resembling the in vivo situation. For quantitative analyses the
experiments were performed with 32P-labeled ODN followed by
liquid scintillation counting and gel electrophoresis of cellular
extracts. In addition, fluorescein-labeled ODN were used to examine
subcellular localization and cell subset-dependent uptake. For
demonstration of functional effects of cationic lipid-mediated ODN
delivery primary cells of patients with chronic myelogenous leukemia
(CML) were treated by bcr-abl-directed ODN and suppression of
clonogenic growth was examined.
| |
MATERIALS AND METHODS |
Cells.
Human peripheral blood mononuclear cells (MNC) were obtained from
patients with hematologic malignancies or solid tumors in complete
remission. Cells were obtained by leukapheresis using a Fenwal CS3000
(Baxter Deutschland, Munich, Germany) after cytotoxic chemotherapy
supported with recombinant human G-CSF (R-metHuG-CSF; Amgen, Thousands
Oaks, CA). Primary cells from patients with CML were obtained from
peripheral blood by vein puncture. Red blood cells and cell debris were
removed by density centrifugation using the lymphocyte separation
medium Lymphoprep (Nycomed Pharma, Oslo, Norway) as previously
described.20 Separation of CD34+ cells from
leukapheresis products was performed using the miniMACS immunomagnetic
separation system (Miltenyi Biotec, Bergisch Gladbach, Germany)
according to the manufacturer's instructions as previously described.20 For transfection experiments cells were
cultured in RPMI-1640-medium (CC Pro GmbH, Neustadt/Weinstrasse,
Germany) supplemented with 10% heat-inactivated fetal calf serum
(Biochrom, Berlin, Germany), 100 IU/mL penicillin (Life Technologies,
Eggenstein, Germany), 100 µg/mL streptomycin (Life Technologies), 2 mmol/L L-glutamine (Life Technologies). For culture of
CD34+ cells, interleukin-3 (IL-3) (20 ng/mL), stem cell
factor (SCF) (50 ng/mL), and IL-6 (20 ng/mL) were added to the culture
medium in some experiments. Cytokines were obtained from PromoCell
(Heidelberg, Germany).
Oligodeoxyribonucleotide synthesis and labeling.
The ODN used for transfection studies were derived from bcr-abl
oncogene-directed antisense nucleic acids and had the following sequences: antisense-b3a2(A):
5 -GCTGAAGGGCTTTTGAACTCTGC-3,11 scrambled-b3a2(A): 5-TTATTGAGGGTGATCCGCTAGCC-3,
anti-sense-b3a2(B): 5-GCTGAAGGGCTTTTGAACTCTGCTTAAA-3,11
scrambled-b3a2(B): 5-AGAGGTCACGCTTTTAGAGATTGCTTCA-3, antisense-b2a2:
5-CGCTGAAGGGCTTCTTCCTTATTGAT-3,21
scrambled- b2a2: 5-TGGTCATACAGGCCTATTTCGTCTTG-3.
In a data base research using the softwares of the Heidelberg Unix
Sequence Analysis Resources (HUSAR, version 4.0; DKFZ, Heidelberg,
Germany) no homology of the used scrambled ODN with any human sequence
of the EMBL data bank was found. The ODN were obtained from Interactiva
(Ulm, Germany). They were synthesized using standard phosphoramidate
chemistry. The two internucleotide linkages at the 3 and
5 end of the ODN were phosphorothioates, and the internal
deoxyribonucleotides were connected by phosphodiesters. For fluorescent
labeling the last coupling step was performed using fluorescein
isothiocyanate (FITC)-labeled amidite. The ODN were purified by
reverse-phase high-performance liquid chromatography and lyophilized
after synthesis by the manufacturer. Before use ODN were resolved in
HEPES-buffer (20 mmol/L, pH 7.4). Radioactive labeling of the 5-ends
was performed by phosphorylation as described.22 Briefly,
40 pmol of ODN was incubated for 45 minutes at 37°C with 70 mmol/L
Tris-HCl, pH 7.6, 10 mmol/L MgCl2, 5 mmol/L dithiothreitol,
150 µCi of [ -32P]ATP (3,000 Ci/mmol; Amersham,
Braunschweig, Germany) and 30 U of T4 polynucleotide kinase (New
England Biolabs, Schwalbach/Taunus, Germany) in a final volume of 40 µL. After heating to 68°C for 10 minutes the ODN were purified by
gel filtration (Sephadex G-50; Pharmacia, Freiburg, Germany). After
precipitation with ethanol the ODN were dissolved in TE-buffer (10 mmol/L Tris-HCl, pH 7.6; 1 mmol/L EDTA). Chemicals were obtained from
Merck (Darmstadt, Germany).
Treatment of cells by ODN and cationic lipids.
Before transfection, ODN were incubated with the respective cationic
lipid for 15 minutes at room temperature for formation of ODN/cationic
lipid complexes. Using DOTAP
(N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate;
Boehringer Mannheim, Mannheim, Germany), 3 µg ODN (corresponding to a
final concentration of a 23-mer ODN in 500 µL cell suspension of 0.84 µmol/L) was mixed with 15 µL cationic lipid (1 µg/µL) and
HEPES-buffer (20 mmol/L, pH 7.4) to a final volume of 75 µL. Using
DOSPER (1,3-Di-Oleoyloxy-2-(6-Carboxy-spermyl)-propyl-amid; Boehringer
Mannheim), 5.4 µg ODN (final concentration: 1.5 µmol/L) was mixed
with 14 µL cationic lipid (1 µg/µL) and HBS-buffer (HEPES 20 mmol/L, pH 7.4; NaCl 150 mmol/L) to a final volume of 100 µL. When
using an amount of ODN differing from that given above, the amount of
cationic lipid was altered correspondingly. For transfection of
radioactively labeled ODN, the specific activity of oligonucleotides was 3 × 109 cpm/µmol. After 15 minutes the
ODN/cationic lipid mixture was added dropwise to the cell suspension
which was incubated at 37°C for at least 30 minutes before
transfection. MNC as well as CD34+ cells were incubated in
a final volume of 500 µL at a concentration of 1 × 106 cells/mL. Cells were incubated with the ODN between 30 minutes and 27 hours at 37°C. After incubation, the cells were
washed at 4°C once with PBS-buffer (KCl 0.2 g/L;
KH2PO4 0.2 g/L; NaCl 8.00 g/L;
Na2HPO4 1.15 g/L) and with PBS-buffer
containing 1% bovine serum albumin, respectively. To remove ODN bound
to the cell membrane the acid-salt elution method was used as
described.23 Briefly, the cell pellet was suspended in an
ice-cold solution containing 0.5 mol/L NaCl and 0.2 mol/L acetic acid
(pH 2.5), incubated at 4°C for 10 minutes and centrifuged at
1,000g for 5 minutes. After washing and acid-salt elution, the
cells were either treated with proteinase K lysis buffer (10 mmol/L
Tris-HCl, pH 8.3; 50 mmol/L KCl; 1.5 mmol/L MgCl2; 1.5%
Tween-20; 1 µg/mL Proteinase K) for measurement of cell-associated
radioactivity or washed with PBS-buffer for flow cytometry.
For assessment of cellular uptake of the FITC-molecule cells were
incubated for 2 and 24 hours with 0.3 µmol/L FITC (Aldrich, Steinheim, Germany) in the presence or absence of cationic lipids.
For transfection of ODN into cells within whole blood, 500 µL of
blood anticoagulated by EDTA was incubated in the presence or absence
of cationic lipids. The incubation was performed using a rotator to
avoid sedimentation of blood cells. After incubation, 100 µL of
samples was mixed with 1.9 mL Becton Dickinson 1 × FACS lysing
solution (Becton Dickinson, Heidelberg, Germany) for 5 minutes at room
temperature for lysis of erythrocytes. The cells were washed,
resuspended in proteinase K lysis buffer, and analyzed by scintillation
counting.
Quantification of cell-associated ODN.
To determine cell-associated radioactivity cell pellets were incubated
with 40 µL of the proteinase K lysis buffer for 1 hour at 56°C.
Twenty microliters was analyzed by counting in a liquid scintillation
counter (Beckman Instruments, Fullerton, CA) with 1 mL of liquid
scintillation cocktail (Ready Safe; Beckman Instruments). Cellular ODN
uptake was expressed as pmol/106 cells. Lysates were
examined by polyacrylamide gel electrophoresis under denaturing
conditions (12% polyacrylamide gel containing 7 mol/L urea in 89 mmol/L Tris-borate buffer, pH 8.3) to determine the proportion of
degraded ODN as well as of removed 5 phosphate residues. Gels
were dried and exposed to x-ray film. Gels were scanned with a
PhosphorImager (Molecular Dynamics, Krefeld, Germany) and band
intensities were measured using the IMAGE QUANT software (Molecular
Dynamics).
Time-dependent decrease of cell-associated full-length ODN was
computer-fitted by nonlinear regression curves with single- and
double-exponential decays. Best fit was achieved using a double exponential function. The curves were compatible to an at least two-phase or compartment model which can be described by a function of
the form: C(t) = Ae-k1t + Be-k2t. C(t) is the amount of full-length
ODN at time t, while A and B indicate the amount of
full-length ODN at t = 0 in each phase or compartment. The coefficients
k1 and k2 are the rate
constants for the decay of full-length ODN from the respective compartment.24 The half-life of ODN in each compartment was obtained from the equation: t1/2 = ln2 /
k.25 The overall half-life of intracellular
full-length ODN was calculated from the double-exponential function
assuming that C(t1/2) = C(0)/2.
Immunofluorescence staining and flow cytometry.
After washing 1 × 106 transfected cells were
stained with the phycoerytherin (PE)-conjugated monoclonal antibodies
(MoAbs) CD7 (clone 3A1; Sigma, Deisenhofen, Germany), CD13 (clone L138; Becton Dickinson, Heidelberg, Germany), CD15 (clone 80H5; Immunotech, Marseille, France), CD19 (clone 467; Becton Dickinson), or CD34 (clone
8612; Becton Dickinson) in 500 µL PBS at 4°C for 30 minutes. Isotype-identical MoAbs served as control (IgG1-PE, Becton Dickinson; IgG2a-PE, Becton Dickinson; IgM-PE, Immunotech). After antibody staining, cells were washed and suspended in PBS-buffer. For
determination of the proportion of viable cells, propidium iodide
(Sigma) was added at a final concentration of 10 µg/mL before
analysis. The cells were analyzed using a Becton Dickinson FACScan with
a 2-W argon ion laser. Fluorescence was measured using 530/15 nm (FITC) and 575/36 nm (PE) band pass filter. Data were analyzed using the
Becton Dickinson Lysis II software after gating on viable cells.
For fluorescence microscopy, FITC-ODN transfected cells were
transferred onto slides by centrifugation (1,000 rpm, 5 minutes) with a
Shandon Cytospin3 (Life Sciences International, Frankfurt am Main,
Germany). In some experiments cells were fixed using 2%
paraformaldehyde for 20 minutes. For staining of nuclei, slides were
washed once for 5 minutes with 2 × SSC (20 × stock
solution: 3 mol/L NaCl; 0.3 mol/L sodium citrate, pH 7.0), once for 10 minutes with 2 × SSC containing 0.2 µg/mL
4,6-Diamidino-2-Phenylindole (DAPI; Sigma), and once for
5 minutes with 2 × SSC containing 0.05% Tween 20. After
staining, cells were embedded under cover slips in Vecta-Shield
mounting medium (Vector Laboratories, Burlingame, CA). Analysis was
performed using a fluorescent microscope (Zeiss Axioskop, Jena,
Germany).
Clonogenic assay of primary CML cells transfected by ODN.
Mononuclear cells from peripheral blood from patients with CML were
incubated two times with an interval of 16 hours by ODN (final
concentrations: 1 µmol/L in first and 0.5 µmol/L in second incubation) using the cationic lipid DOTAP as described above. After
transfection the cells were seeded in methylcellulose growth medium
(MethoCult H4433; StemCell Technology, Vancouver, Canada) at a density
between 5 × 104 and 2 × 105
cells/mL.21,26 The colony numbers (colony-forming unit
granulocyte/macrophage [CFU-GM], burst-forming unit erythrocyte
[BFU-E]) were counted after 2 weeks. The type of bcr-abl
fusion point in each sample was determined by polymerase chain reaction
following reverse transcription as described.27
| |
RESULTS |
The use of radioactively labeled ODN provides a suitable method to
study ODN uptake into cells in vitro, as it permits to measure the
amount and the proportion of internalized full-length ODN.28 Still, it cannot be distinguished between uptake
into living and dead cells or specific uptake into the different cell types of blood-derived MNC. FACS analysis permits the assessment of
uptake of FITC-labeled ODN on a single cell level, while the intracellular localization and time-dependent distribution can be
examined by fluorescence microscopy.16,29-31 In this study, these methods were used to address quantitative as well as qualitative aspects of ODN uptake into primary human hematopoietic cells.
Improvement of cellular uptake of ODN by DOTAP and DOSPER.
The radioactively labeled phosphorothioate-modified 23-mer
scrambled-b3a2(A) ODN was used for quantitative analysis of ODN uptake
into blood-derived MNC by means of DOTAP and DOSPER. The following
parameters were evaluated: (1) the weight ratio of ODN to cationic
lipid,32 (2) the dependency on ODN concentration, and (3)
the time course of cationic lipid-mediated ODN uptake.
Cells were incubated for 2 hours with 0.3 µmol/L radioactively
labeled ODN complexed to different amounts of DOTAP resulting in
lipid/ODN ratios (µg/µg) between 1:2 and 10:1. Before analysis, cells were washed according to the acid-salt method to remove extracellularly membrane-bound ODN.23 The cell-associated
radioactivity varied between 3 and 30 pmol/106 cells with a
maximum when the DOTAP/ODN ratio was between 5:1 and 7:1 (µg:µg)
(data not shown). This corresponds to a charge ratio between 2:1 and
3:1 (+/ ). Having defined the best DOTAP/ODN ratio, cells were
incubated with increasing amounts of ODN/DOTAP complexes up to the
toxicity limit of 30 µg DOTAP/mL (Fig
1A). The cell-associated radioactivity was dose-dependent without
reaching a plateau. The peak value of 120 pmol/106 cells
was observed at 0.84 µmol/L ODN which corresponds to 7.2 × 107 ODN molecules per cell. The increase of DOTAP-mediated
ODN uptake varied from 42- to 93-fold (mean, 70-fold; standard
deviation [SD], 26) in comparison with the application of ODN alone.
DNA extracts of transfected cells were analyzed by polyacrylamide gel
electrophoresis to measure the proportion of full-length ODN (Fig 1C).
As shown by x-ray films and PhosphorImager analysis, the proportion of
full-length ODN to whole cell-associated radioactivity was more than
80%.
View larger version (18K):
[in this window]
[in a new window]
| Fig 1.
Effect of cationic lipids on cell-associated ODN.
Blood-derived mononuclear cells were incubated for 2 hours with
increasing amounts of radiolabeled ODN in the absence ( ) or presence
( ) of DOTAP (A) or DOSPER (B). The amount of cell-associated ODN was
measured by liquid scintillation counting of cellular extracts. (C)
Analysis of extracts from cells incubated with ODN/DOSPER complexes by
denaturing 12% polyacrylamide gel electrophoresis. Full-length 23-mer
ODN are indicated.
|
|
For the assessment of the time course of ODN uptake, cells were
incubated between 30 minutes and 27 hours with 0.3 µmol/L ODN
complexed to DOTAP (Fig 2). Approximatly
two thirds of the maximum cell-associated radioactivity was observed
within the first 2 hours of incubation. During the following 22 hours
intracellular ODN accumulated at a significantly lower rate, suggesting
that an incubation time between 2 and 8 hours is sufficient for
delivery of ODN by DOTAP into blood-derived MNC.
View larger version (14K):
[in this window]
[in a new window]
| Fig 2.
Time course of cationic lipid-mediated ODN uptake in
blood-derived mononuclear cells. Cells were incubated with 0.3 µmol/L radiolabeled ODN complexed to DOTAP () or DOSPER () for the indicated times. Means and standard deviations of three independent experiments are presented.
|
|
Using DOSPER the uptake of ODN in MNC could also be improved
significantly, but some parameters were different from the results obtained with DOTAP. The maximum of cell-associated radioactivity was
found at a DOSPER/ODN ratio of 2.6:1 (µg:µg) which corresponds to a
charge ratio of 3:1 (+/). There were 800 pmol ODN associated with 106 cells (4.8 × 108 molecules/cell)
when the ODN concentration was 1.5 µmol/L (Fig 1B), and the
DOSPER-mediated increase of uptake varied between 440- and 1,025-fold
(mean, 690-fold; SD, 220) compared with the application of ODN alone.
The time course of ODN uptake by DOSPER was also different in
comparison with DOTAP, as the cell-associated radioactivity reached a
maximum after 2 hours of incubation (Fig 2) followed by a decrease as a
function of time. In addition, uptake of the radioactively labeled
26-mer scrambled-b2a2 as well as the 28-mer scrambled-b3a2(B) ODN with
different base compositions was measured to look for a sequence
dependency of ODN delivery. No differences in cationic lipid-mediated
uptake were observed between the 23-mer, the 26-mer, and the 28-mer ODN
(data not shown).
Subcellular localization of ODN after transfection with cationic
lipids.
The functional activity of antisense ODN depends on their access to the
cellular compartment of the biological target. Therefore, the
subcellular localization and intracellular trafficking of ODN was
evaluated by fluorescence microscopy using FITC-labeled 23-mer
scrambled-b3a2(A) ODN (Fig 3). Incubation
of blood-derived MNC with ODN/DOTAP complexes resulted in a faint
cytoplasmic stain of lymphoid cells at a circumscript perinuclear area
on one side of the cell (Fig 3A). In contrast, approximately 90% of
cells with monocyte appearance showed a bright fluorescent staining with a spotted distribution within the cytosol (Fig 3B). Although there
was no nuclear fluorescence found after 2 hours of incubation, a
homogenous nuclear stain with a pronounced accumulation in the nucleoli
was observed after 24 hours (Fig 3C). In approximately 80% of cells
the cytoplasmic stain persisted in spotted endosome/lysosome-like structures after 24 hours (Fig 3D), whereas the other cells developed a
homogenous cytoplasmic fluorescence (Fig 3E). Similar intracellular distributions of ODN were obtained with DOSPER except for the difference that the proportion of cells with a homogenous cytoplasmic fluorescence after 24 hours reached 50% (data not shown). There was no
difference between fixed and unfixed cells, indicating that fixation
does not influence subcellular localization of ODN.
View larger version (78K):
[in this window]
[in a new window]
| Fig 3.
Subcellular localization of FITC-labeled ODN. After
transfection of 0.3 µmol/L FITC-labeled ODN using DOTAP, cells were
transferred on slides by centrifugation, stained by DAPI, and analyzed
by fluorescence microscopy. The upper row shows the DAPI stained nuclei, the lower row the intracellular distribution of FITC-labeled ODN. (A and B) Representative cells after incubation with ODN/DOTAP complexes for 2 hours. (C through E) Representative cells after an
incubation time of 24 hours.
|
|
Transfection of ODN by cationic lipids using whole peripheral blood.
The data presented are helpful for the design of ex vivo transfection
protocols of ODN into MNC. On the other hand, antisense ODN can also be
used for systemic intravenous administration. Therefore, we measured
uptake of radioactively labeled ODN in cells of whole blood which may
resemble more closely the in vivo conditions. The peripheral blood was
anticoagulated by EDTA, because heparin is a negatively charged
macromolecule that may interfere with cationic lipids.33,34
In comparison with the use of ODN alone, the uptake into leukocytes was
approximately 30-fold and 20-fold greater using DOTAP and DOSPER,
respectively (Fig 4). Polyacrylamide gel
analyses of DNA extracted from transfected cells were performed to
exclude that the radioactive label was displaced from the ODN by serum
phosphatases and nucleases. PhosphorImager analyses of gels showed that
the proportion of full-length ODN to whole cell-associated
radioactivity was more than 80% in each sample (data not shown). As
shown by FACS analysis with FITC-labeled ODN complexed to cationic
lipids, the fluorescence staining was only found in leukocytes, whereas
the erythrocytes were negative (data not shown).
View larger version (16K):
[in this window]
[in a new window]
| Fig 4.
Effect of DOTAP and DOSPER on ODN uptake into leukocytes
using whole blood. Cells were incubated for 2 hours with 0.3 µmol/L radiolabeled ODN complexed to cationic lipids. After lysis of erythrocytes, radioactivity associated with white blood cells was
measured by liquid scintillation counting. The results from two
experiments are indicated.
|
|
Cell subset-dependent ODN uptake.
Data obtained from uptake studies using radioactively labeled ODN
provide mean values of cell-associated ODN of all cell subsets in the
MNC fraction. Therefore, DOTAP-mediated uptake of FITC-labeled ODN was
examined by dual-color immunofluorescence analysis with subset specific
PE-conjugated MoAbs. The results of one representative experiment of
three experiments are shown in Figs 5 and
6. As assessed by propidium iodide
staining, the proportion of dead cells was less than 5%, reflecting
the low toxicity of the transfection. The mean FITC-fluorescence of all
cells was approximately 35-fold greater compared with ODN treatment
alone (Fig 5). As assessed by sideward scatter gating, greatest uptake
of ODN into monocytes could be seen in both samples, with and without
DOTAP. This was confirmed by the antibody staining of lineage-specific
antigens (Fig 6). Monocytes, as assessed by gating on CD13+
and CD15 cells, had the greatest cell-associated
fluorescence intensity. The smallest uptake was observed in
CD7+ T cells, while the uptake was intermediate in
CD15+ myeloid cells, in CD19+ B cells, as well
as in CD34+ hematopoietic progenitor and stem cells.
View larger version (19K):
[in this window]
[in a new window]
| Fig 5.
Analysis of ODN uptake into blood-derived mononuclear
cells by flow cytometry. Cells were incubated for 2 hours with 0.3 µmol/L FITC-labeled ODN in the absence (B) or presence (C) of DOTAP. (A) Background fluorescence of cells without ODN incubation. The relative mean fluorescence intensities (Ym) are indicated.
|
|
View larger version (27K):
[in this window]
[in a new window]
| Fig 6.
Subset dependent ODN uptake into primary blood-derived
mononuclear cells. Cells were incubated for 2 hours with 0.3 µmol/L FITC-labeled ODN complexed to DOTAP and stained with the indicated PE-conjugated lineage-specific antibodies before FACS-analysis. Cells
were analyzed after gating on the lineage-specific antibody staining.
The background fluorescence with an isotype-specific control antibody
is shown in the first dot blot. The relative mean FITC fluorescence
intensities (Ym) are indicated.
|
|
After a 24-hour incubation of cells with ODN/DOTAP complexes, the mean
FITC-fluorescence further increased approximately 4-fold in
CD13+ and CD15+ cells and approximately
2.5-fold in CD34+ cells. In contrast, uptake of ODN into
CD7+ and CD19+ lymphocytes was not enhanced
following the longer incubation time (data not shown). Small and large
cells were gated within each subpopulation to exclude that the
different efficiency of uptake among the subsets was related to cell
volume. The proportion of cell subsets was 78% for CD13+
cells, 52% for CD15+ cells, 24% for CD19+
cells, 20% for CD7+ cells, and 1.5% for CD34+
cells. We found no correlation between the proportion of each cell
subset and ODN uptake. The same subset-specific ODN uptake was seen
using mononuclear cells from patients with CML in chronic phase as well
as using DOSPER instead of DOTAP (data not shown). Incubation of cells
with FITC in the presence or absence of cationic lipids showed no
increased cellular fluorescence after 2 hours as well as after 24 hours
in comparison with untreated cells.
ODN uptake in CD34+ cells.
For application of antisense ODN in ex vivo treatment protocols uptake
into enriched CD34+ hematopoietic progenitor cells is of
special interest. Therefore, delivery of ODN by cationic lipids into
isolated blood-derived CD34+ cells was examined using
radioactively labeled ODN. After an incubation of CD34+
cells with the ODN/cationic lipid complexes for 2 hours, the cell-associated radioactivity was increased 100-fold (SD, 6) using DOTAP and 240-fold (SD, 32) using DOSPER compared with application of
ODN alone, respectively (data not shown). Isolated CD34+
cells were also cultured in the presence of IL-3, IL-6, and SCF for 48 hours before transfection to examine whether ODN uptake is greater
after cellular activation. The cytokines were chosen, as they stimulate
proliferation of CD34+ cells without loss of long-term
culture potential.35 Still, stimulation with growth factors
did not significantly improve the cationic lipid-mediated ODN uptake in
comparison with unstimulated CD34+ cells
(Fig 7). As assessed by trypan blue
staining and a colony-forming assay in semisolid culture medium, DOTAP
and DOSPER had no toxic effects on CD34+ cells after an
incubation time of 10 hours with the cationic lipid/ODN complexes (data
not shown).
View larger version (16K):
[in this window]
[in a new window]
| Fig 7.
Influence of cellular activation of CD34+
cells on cationic lipid-mediated ODN uptake. Cells were incubated for 2 hours with 0.3 µmol/L radiolabeled ODN complexed to DOTAP ( ) or
DOSPER ( ) with or without a 48-hour preculture in IL-3, IL-6, and
SCF supplemented medium. The amount of cell-associated ODN was measured
by liquid scintillation counting. The results of two experiments are
indicated.
|
|
Kinetic analyses of cell- and medium-associated radioactivity were
performed to determine the intracellular half-life of ODN in
CD34+ cells and the efflux rate of cell-associated
radioactivity. Radioactively labeled ODN/DOSPER complexes were added to
CD34+ cells for 2 hours, the medium was removed and the
cells were incubated with fresh medium devoid of ODN. The time courses
of cell-associated as well as medium-associated radioactivity were measured. The cell-associated radioactivity decreased, while the extracellular radioactivity increased as a function of time
(Fig 8A). As shown by PhosphorImager
analysis of polyacrylamide gels of cellular DNA extracts, the
cell-associated ODN degraded during incubation. The proportion of
full-length ODN to whole cell-associated radioactivity decreased from
80% to 55% after 24 hours (Fig 8B). Degradation products were more
than 95% mononucleotides or phosphate residues without successively
shortened ODN (data not shown). These results indicate a very fast
degradation after removal of the 5 or 3
cap-phosphorothioates. Multiplication of the functions of
cell-associated radioactivity and of proportion of full-length ODN
resulted in a curve reflecting the kinetics of full-length ODN (Fig
8B), which is a relevant parameter with regard to the efficacy of
antisense ODN. The kinetics best fit to a bi-exponential function in
which the exponential terms represent at least two compartments or
phases.24,25 The ODN half-lives of the individual phases or
compartments were calculated from the computer fit. The first phase was
rapid with a half-life of approximately 2 hours, whereas the second
phase was slow, reflected by a half-life of approximately 30 hours.
This resulted in an overall half-life of full-length ODN in
CD34+ cells of about 10 hours.
View larger version (20K):
[in this window]
[in a new window]
| Fig 8.
(A) Time course of extracellular () and
cell-associated () radioacivity in CD34+ cells. After
a 2-hour incubation with radiolabeled ODN/DOSPER complexes the medium
was removed and cells were resuspended in fresh medium. Radioactivity
was measured at indicated time points by liquid scintillation counting.
(B) Time course of cell-associated full-length ODN (). The
proportion of full-length ODN to the whole cell-associated
radioactivity as determined by PhosphorImager analysis of a
polyacrylamide gel is shown as a function of time by ( ).
Multiplication of this function with the function of cell-associated radioactivity () resulted in the curve indicating the time course of
cell-associated full-length ODN.
|
|
Using DOTAP a similar bi-exponential curve with the same overall
half-life of full-length ODN was found (data not shown).
Functional effects of DOTAP-mediated ODN delivery.
ODN used in the experiments presented were derived from antisense
sequences directed against the CML-related bcr-abl
oncogene. The CML can serve as a model for antisense ODN-mediated
inhibition of leukemic cell growth, because the bcr-abl
rearrangement of the Philadelphia-chromosome (Ph+) that
results from a reciprocal translocation between chromosomes 9 and 22 leads to the expression of a pathological p210 bcr-abl fusion
protein in more than 90% of patients. Depending on whether the second
exon of the abl gene fuses with bcr exon 2 or 3, two different types of bcr-abl mRNA (b2a2 and b3a2) are found in
most cases of CML.36
The functional effects of ODN transfected by DOTAP was examined using
primary hematopoietic cells from 9 patients with CML. Seven patients
were in chronic phase without previous treatment, 1 patient was in
accelerated phase, and 1 in blast crisis. Mononuclear cells were
obtained from peripheral blood and the type of bcr-abl fusion
point was determined by polymerase chain reaction following reverse
transcription of cellular RNA. The b3a2 fusion point-directed antisense-b3a2(B) ODN and the b2a2-directed antisense-b2a2 ODN as well
as the respective control ODN with the same nucleotide compositions in
scrambled sequence (scrambled-b3a2(B), scrambled-b2a2) were used to
assess the antiproliferative activity on the patients' cells in
clonogenic assays. ODN were transfected two times with an interval of
16 hours at concentrations of 1 µmol/L and 0.5 µmol/L,
respectively. The b3a2 antisense sequence was chosen based on kinetic
in vitro selection to achieve specific hybridization with the b3a2
bcr-abl fusion sequences while sparing the wild-type sequences bcr or abl.11 In 3 of 9 cases,
fusion point-specific inhibition of colony formation was observed
ranging between 39% and 72% at a low variation within the experiments
performed in duplicates (Table 1). In none
of the 9 cases, any inhibitory effects were observed with scrambled
control sequences or alternative junctional antisense ODN; ie,
inhibition was only measured with bcr-abl junction-specific
antisense ODN in cells with the appropriate target mRNA. Colony
formation of bcr-abl cells was not
influenced after transfection of ODN by DOTAP. The transfection
procedure alone had also no effect on cell proliferation.
View this table:
[in this window]
[in a new window]
|
Table 1.
Fusion Point-Specific Inhibition of Clonogenic Growth of
Primary Cells From Three Patients With CML by
bcr-abl-Directed Antisense ODN
|
|
| |
DISCUSSION |
In this report we show that ODN uptake into human blood-derived MNC can
be greatly improved by cationic lipids. Because primary hematopoietic
cells are target cells for the treatment of patients with hematologic
diseases and viral infections by antisense ODN,4-8 an
efficient delivery of nucleic acids is required to obtain functional effects. We found an increase of ODN uptake by the cationic lipids DOTAP and DOSPER between 30- and 800-fold in comparison to the values
achieved with ODN alone. This enhancement is significantly greater than
that observed for other cell types. For instance, Capaccioli et
al17 showed a 25-fold increase of ODN uptake into a
lymphoblastic cell line when DOTAP was added. Other groups described a
2-to 10-fold greater ODN uptake by cationic lipids into primary endothelial cells as well as leukemic and cervical cancer cell lines16,28,37 when compared with an incubation of ODN
alone. On the other hand, there was no effect of cationic lipids on the uptake and inhibitory activity of antisense ODN in primary
keratinocytes.29 Using FITC-labeled ODN, we found that ODN
delivery was dependent on the cell type with smallest uptake into T
cells. Uptake into monocytes was 30-fold, into CD34+ cells,
B cells, and granulocytes about 2-fold greater compared with T cells,
while there was no measurable uptake into erythrocytes. Still, an exact
quantification of FITC-labeled ODN uptake is difficult because the
emission intensity of FITC is pH dependent.24,38 Therefore,
in a cellular compartment with low pH, such as lysosomes, the amount of
intracellular FITC-labeled ODN might be underestimated by FACS
analysis.
The great variability of uptake obtained with different cell types and
lipids may be related to two essential steps of ODN uptake into cells:
(1) The interaction of the positively charged cationic lipid/DNA
complex with anionic residues on the cell surface39 and (2)
the endocytosis of the complex by the cells.18,40 There is
some evidence that membrane-associated sulfated proteoglycans are
involved in the transfection by cationic lipids.41
Proteoglycans consist of a core protein covalently linked to one or
more sulfated glycosaminoglycans.42 Hematopoietic cells
have a different expression pattern of the hematopoietic proteoglycan
core protein (HpPG) dependent on the lineage and state of
differentiation.43 Monocytes, eosinophils, and basophils
express the HpPG at higher levels than lymphocytes, neutrophils, and
immature myeloid cells. Therefore, a variable expression of
proteoglycans on different cell types may influence the susceptibility
of cells to transfection. Assuming that ODN/cationic lipid complexes
are taken up by endocytosis,18,40 the differences observed
between the cell subsets could also be explained by a greater
endocytosis activity of monocytes compared with T or B
lymphocytes.44 A similar cell subset-dependent ODN delivery without the use of transfection reagents was described for
human and murine hematopoietic cells.31,45 In contrast to
the data of Zhao et al,31 who used ODN alone, we could not observe an increased cellular uptake of ODN/cationic lipid complexes into CD34+ cells following incubation with a cocktail of
the cytokines IL-3, IL-6, and SCF. This could indicate that uptake of
cationic lipid/ODN complexes in contrast to ODN alone is independent
from cellular activation of CD34+ cells. The subset
dependent uptake is important with respect to clinical application of
antisense ODN. DOTAP and DOSPER may be less efficient in delivery of
ODN into HIV-1-infected T cells compared with macrophages and
monocytes. Additionally, our data suggest a significant benefit of
cationic lipids for uptake into silent and activated CD34+
cells which is of relevance for ex vivo purging protocols of hematopoietic stem cells.
The difference of the transfection efficacy between DOTAP and DOSPER
may be related to the tetravalent cationic structure of the DOSPER
molecule, while DOTAP is a monocationic liposomal reagent. The
polycationic lipid may lead to a better formation of the lipid/ODN
complex and improved interaction between the complex and the negatively
charged cell surface. The initial peak after 2 hours with subsequent
decrease of cell-associated radioactivity using DOSPER in comparison
with continuous increase of ODN uptake using DOTAP may be related to a
lower stability of the DOSPER molecule compared with DOTAP. Therefore,
an efficient uptake can be observed during the first 2 hours of the
transfection procedure, but afterwards metabolization and excretion of
cell-associated radioactivity may overcome the uptake of extracellular
ODN as a result of destabilization of ODN/DOSPER complexes. This
hypothesis is supported by the reduced efficacy of ODN uptake by DOSPER
compared with DOTAP using whole blood conditions instead of the
isolated mononuclear cell fraction. Differences in transfection
efficacies depending on the chemical structure of the cationic lipids
have been also described for other liposomes.46
Nucleic acid/cationic lipid complexes are mainly internalized via
endocytosis.18,40 The mechanism of release of the nucleic acid from the endosome to the cytoplasm as well as the intracellular trafficking are poorly understood. Recently, a model for intracellular release of DNA from cationic liposomes into the cytoplasm was proposed.33,34 After internalization via endocytosis the
DNA/cationic lipid complex destabilizes the endosomal membrane as a
result of a flip-flop exchange between anionic and cationic lipids.
Thereby, the DNA is displaced from the complex allowing the ODN to
diffuse into the cytoplasm. Alternatively, full-length ODN could also be released immediately from endosomes to the extracellular compartment by exocytosis24,25,47 or the DNA/cationic lipid complex is transferred to lysosomes, where the nucleic acid is rapidly degraded by
nucleases. Our results on the intracellular localization of ODN as well
as the kinetic data are consistent with these views. Accumulation of
ODN in cytoplasmic granules which presumably represent endosomes and
lysosomes was observed after 2 hours of incubation with FITC-labeled
ODN. The nuclear localization of fluorescence after 24 hours indicated
a significant release of ODN from endocytic vesicles into the cytosol
and subsequent transport to the nucleus, suggesting that ODN may reach
the target RNA. Additionally, the kinetic data on the efflux of
cell-associated radioactivity from ODN-transfected CD34+
cells can be described in a mathematical model for ODN trafficking with
at least two phases or compartments.1,24 The first phase or
compartment with a short half-life of 2 hours may reflect the rapid
transfer of ODN from endosomes to the cell surface or to lysosomes with
subsequent degradation and exocytosis. The second and further phases
with slow turnover may reflect the release of ODN from endosomes to
other cellular compartments followed by metabolization. The overall
decrease of cell-associated ODN in CD34+ cells resulted in
a half-life of full-length ODN of 10 hours. These kinetic data indicate
that for targeting a gene which codes for a RNA and a protein with long
half-lives a single transfection of antisense ODN may not be sufficient
and further transfections after intervals of 10 hours using the half
ODN dose may be useful.
The results obtained in ex vivo experiments may differ from ODN uptake
in vivo. Although DOTAP and DOSPER are effective in the presence of
human serum,17 transfection can be hampered by charged
components of the blood such as heparin.34 Therefore, the
efficacy of ODN delivery was evaluated under conditions mimicking an in
vivo administration. Using DOSPER the enhancement of ODN uptake was
significantly decreased from 800-fold to about 20-fold when whole blood
was used instead of defined ex vivo conditions. The efficacy of DOTAP
was less influenced by components of peripheral blood and still an
30-fold increase of ODN was observed. This indicates that DOTAP may be
suitable for systemic in vivo application.
Still, one has to take into account that the FITC label or the sequence
of the ODN may influence uptake and intracellular trafficking of the
nucleic acid.48 However, we did not find differences in the
extent of uptake using a 26-mer and a 28-mer ODN with various
sequences, suggesting that the data obtained with the 23-mer ODN may be
representative for other ODN. Nevertheless, it is important to ensure
for each individual ODN an efficient cationic lipid-mediated delivery
into the respective cell type before using the ODN in cell culture
experiments or clinical trials.
A great uptake of ODN does not necessarily translate into biological
activity, because antisense ODN may lack an efficient binding with the
target RNA or they could remain in endosomes and lysosomes without
reaching their target. Because the uptake studies were performed with
ODN derived from bcr-abl oncogene-directed antisense sequences,
primary cells from patients with CML were used to look for functional
effects of ODN delivered by cationic lipids. Reports on the inhibitory
effects of bcr-abl-directed antisense
oligodeoxyribonucleotides on the proliferation of Ph+
primary leukemic cells are heterogeneous and even contradictory with
respect to sequence specificity and efficacy in some
instances.21,49-54 This heterogeneity may be related to
differences in cell-culture conditions, to the target sequence, as well
as to the high ODN concentrations ranging between 5 µmol/L and 20 µmol/L, which could result in unspecific effects. We found in one
third of the cases a fusion point-specific inhibition of clonogenic
growth by ex vivo treatment of primary CML cells with bcr-abl
antisense ODN/DOTAP complexes using an ODN concentration of only 1 µmol/L, which indicates a benefit of DOTAP for functional effects of
ODN. The lack of growth inhibition observed with alternative junctional
and scrambled control ODN implies that the antiproliferative activity
is due to a specific antisense effect. A correlation between inhibition of colony formation in these cases and any patients' characteristics such as disease status or type of fusion point was not found. Thus, the
antisense ODN may only be beneficial for a subset of patients with
Ph+ CML. The lack of efficacy in the other patients remains
unclear. There might be additional genomic alterations or alternative
signal transduction pathways that make the cells independent from
bcr-abl expression.55,56
In conclusion, DOTAP and DOSPER have a significant advantage over the
use of ODN alone for delivery of ODN into primary hematopoietic cells.
Thus, using cationic lipids in clinical studies the ODN can be applied
at a lower dose to reduce side effects and cytotoxicity. The data
obtained in this study, including those obtained with the primary cells
of patients with CML, can serve as basis for ongoing and future
clinical ex vivo purging protocols of hematopoietic stem cells in the
treatment of hematologic diseases such as CML as well as for systemic
in vivo administration of antisense ODN in leukemias and viral
infections.
| |
FOOTNOTES |
Submitted May 29, 1997;
accepted September 29, 1997.
Address reprint requests to Rainer Haas, MD, Medizinische Klinik und
Poliklinik V, Universität Heidelberg, Hospitalstr. 3, 69115 Heidelberg, Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
REFERENCES |
1.
Stein CA,
Chang YC:
Antisense oligonucleotides as therapeutic agents: Is the bullet really magical?
Science
261:1004,
1993[Abstract/Free Full Text]
2.
Crooke ST,
Bennett CF:
Progress in antisense oligonucleotide therapeutics.
Annu Rev Pharmacol Toxicol
36:107,
1996[Medline]
[Order article via Infotrieve]
3.
Wagner RW,
Flanagan WM:
Antisense technology and prospects for therapy of viral infections and cancer.
Mol Med Today
3:31,
1997 [Medline]
[Order article via Infotrieve]
4.
Lisziewicz J,
Sun D,
Weichold FF,
Thierry AR,
Lusso P,
Tang J,
Gallo RC,
Agrawal S:
Antisense oligodeoxynucleotide phosphorothioate complementary to Gag mRNA blocks replication of human immunodeficiency virus type 1 in human peripheral blood cells.
Proc Natl Acad Sci USA
91:7942,
1994[Abstract/Free Full Text]
5.
De Fabritiis P,
Amadori S,
Petti MC,
Mancini M,
Montefusco E,
Picardi A,
Geiser T,
Campbell K,
Calabretta B,
Mandelli F:
In vitro purging with BCR-ABL antisense oligodeoxynucleotides does not prevent haematologic reconstitution after autologous bone marrow transplantation.
Leukemia
9:662,
1995[Medline]
[Order article via Infotrieve]
6.
Hanania EG,
Kavanagh J,
Hortobagyi G,
Giles RE,
Champlin R,
Deisseroth AB:
Recent advances in the application of gene therapy to human diseases.
Am J Med
99:537,
1995[Medline]
[Order article via Infotrieve]
7.
Bishop MR,
Iversen PL,
Bayever E,
Sharp JG,
Greiner TC,
Copple BL,
Ruddon R,
Zon G,
Spinolo J,
Arneson M,
Armitage JO,
Kessinger A:
Phase I trial of an antisense oligonucleotide OL(1)p53 in hematologic malignancies.
J Clin Oncol
14:1320,
1996[Abstract/Free Full Text]
8. (abstr, suppl 1)
Gewirtz AM,
Luger S,
Sokol D,
Gowdin B,
Stadtmauer E,
Reccio A,
Ratajczak MZ:
Oligodeoxynucleotide therapeutics for human myelogenous leukemia: Interim results.
Blood
88:270a,
1996
9.
Rittner K,
Burmester C,
Sczakiel G:
In vitro selection of fast-hybridizing and effective antisense RNAs directed against the human immunodeficiency virus type 1.
Nucleic Acids Res
21:1381,
1993[Abstract/Free Full Text]
10.
Ho SP,
Britton DH,
Stone BA,
Behrens DL,
Leffet LM,
Hobbs FW,
Miller JA,
Trainor GL:
Potent antisense oligonucleotides to the human multidrug resistance-1 mRNA are rationally selected by mapping RNA-accessible sites with oligonucleotide libraries.
Nucleic Acids Res
24:1901,
1996[Abstract/Free Full Text]
11.
Kronenwett R,
Haas R,
Sczakiel G:
Kinetic selectivity of complementary nucleic acids: bcr-abl-directed antisense RNA and ribozymes.
J Mol Biol
259:632,
1996[Medline]
[Order article via Infotrieve]
12.
Lima WF,
Brown-Driver V,
Fox M,
Hanecak R,
Bruice TW:
Combinatorial screening and rational optimization for hybridization to folded hepatitis C virus RNA of oligonucleotides with biological antisense activity.
J Biol Chem
272:626,
1997[Abstract/Free Full Text]
13.
Loke SL,
Stein CA,
Zhang XH,
Mori K,
Nakanishi M,
Subasinghe C,
Cohen JS,
Neckers LM:
Characterization of oligonucleotide transport into living cells.
Proc Natl Acad Sci USA
86:3474,
1989[Abstract/Free Full Text]
14.
Yakubov LA,
Deeva EA,
Zarytova VF,
Ivanova EM,
Ryte AS,
Yurchenko LV,
Vlassov VV:
Mechanism of oligonucleotide uptake by cells: Involvement of specific receptors?
Proc Natl Acad Sci USA
86:6454,
1989[Abstract/Free Full Text]
15.
Beltinger C,
Saragovi HU,
Smith RM,
LeSauteur L,
Shah N,
DeDionisio L,
Christensen L,
Raible A,
Jarett L,
Gewirtz AM:
Binding, uptake, and intracellular trafficking of phosphorothioate-modified oligodeoxynucleotides.
J Clin Invest
95:1814,
1995
16.
Bennett CF,
Chiang M-Y,
Chan H,
Shoemaker JEE,
Mirabelli CK:
Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides.
Mol Pharmacol
41:1023,
1992[Abstract]
17.
Capaccioli S,
Di Pasquale G,
Mini E,
Mazzei T,
Quattrone A:
Cationic lipids improve antisense oligonucleotide uptake and prevent degradation in cultured cells and in human serum.
Biochem Biophys Res Commun
197:818,
1993[Medline]
[Order article via Infotrieve]
18.
Zhou X,
Huang L:
DNA transfection mediated by cationic liposomes containing lipopolylysine: Characterization and mechanism of action.
Biochim Biophys Acta
1189:195,
1994[Medline]
[Order article via Infotrieve]
19.
Lewis JG,
Lin K-Y,
Kothavale A,
Flanagan WM,
Matteucci MD,
DePrince RB,
Mook RA Jr,
Hendren RW,
Wagner RW:
A serumresistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA.
Proc Natl Acad Sci USA
93:3176,
1996[Abstract/Free Full Text]
20.
Deichmann M,
Kronenwett R,
Haas R:
Expression of the human immunodeficiency virus type-1 coreceptors CXCR-4 (fusin, LESTR) and CKR-5 in CD34+ hematopoietic progenitor cells.
Blood
89:3522,
1997[Abstract/Free Full Text]
21. Szczylik C, Skorski T, Nicolaides NC, Manzella L, Malaguarnera
L, Venturelli D, Gewirtz AM Calabretta B: Selective inhibition of
leukemia cell proliferation by BCR-ABL antisense oligodeoxynucleotides.
Science 253:562, 1991
22. Sambrook J, Fritsch EF, Maniatis F: Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1989
23.
Gao W-Y,
Jaroszewski JW,
Cohen JS,
Cheng Y-C:
Mechanisms of inhibition of herpes simplex virus type-2 growth by 28 mer phosphorothioate oligodeoxycytidine.
J Biol Chem
265:20172,
1990[Abstract/Free Full Text]
24.
Tonkinson JL,
Stein CA:
Patterns of intracellular compartmentalization, trafficking and acidification of 5-fluorescein labeled phosphodiester and phosphorothioate oligodeoxynucleotides in HL60 cells.
Nucleic Acids Res
22:4268,
1994[Abstract/Free Full Text]
25.
Besterman JM,
Airhart JA,
Woodworth RC,
Low RB:
Exocytosis of pinocytosed fluid in cultured cells: kinetic evidence for rapid turnover and compartmentation.
J Cell Biol
91:716,
1981[Abstract/Free Full Text]
26. Sutherland HJ, Eaves AC, Eaves CJ: Quantitative assays of human
hemopoietic progenitor cells, in Gee AP (ed): Bone Marrow Processing
and Purging: A Practical Guide. Boca Raton, FL, CRC, 1991, p 155
27.
Maurer J,
Janssen JWG,
Thiel E,
van Denderen J,
Ludwig W-D,
Aydemir Ü,
Heinze B,
Fonatsch C,
Harbott J,
Reiter A,
Riehm H,
Hoelzer D,
Bartram CR:
Detection of chimeric BCR-ABL genes in acute lymphoblastic leukaemia by the polymerase chain reaction.
Lancet
337:1055,
1991[Medline]
[Order article via Infotrieve]
28.
Lappalainen K,
Urtti A,
Söderling E,
Jääskeläinen I,
Syrjänen K,
Syrjänen S:
Cationic liposomes improve stability and intracellular delivery of antisense oligonucleotides into CaSki cells.
Biochim Biophys Acta
1196:201,
1994[Medline]
[Order article via Infotrieve]
29.
Nestle FO,
Mitra RS,
Bennet CF,
Chan H,
Nickoloff BJ:
Cationic lipid is not required for uptake and selective inhibitory activity of ICAM-1 phosphorothioate antisense oligonucleotides in keratinocytes.
J Invest Dermatol
103:569,
1994[Medline]
[Order article via Infotrieve]
30.
Zhao Q,
Waldschmidt T,
Fisher E,
Herrera CJ,
Krieg AM:
Stage-specific oligonucleotide uptake in murine bone marrow B-cell precursors.
Blood
84:3660,
1994[Abstract/Free Full Text]
31.
Zhao Q,
Song X,
Waldschmidt T,
Fisher E,
Krieg AM:
Oligonucleotide uptake in human hematopoietic cells is increased in leukemia and is related to cellular activation.
Blood
88:1788,
1996[Abstract/Free Full Text]
32.
Jääskeläinen I,
Mönkkönen J,
Urtti A:
Oligonucleotide-cationic liposome interactions. A physicochemical study.
Biochim Biophys Acta
1195:115,
1994[Medline]
[Order article via Infotrieve]
33.
Xu Y,
Szoka FC Jr:
Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection.
Biochemistry
35:5616,
1996[Medline]
[Order article via Infotrieve]
34.
Zelphati O,
Szoka FC Jr:
Mechanism of oligonucleotide release from cationic liposomes.
Proc Natl Acad Sci USA
93:11493,
1996[Abstract/Free Full Text]
35.
Di Giusto DL,
Lee R,
Moon J,
Moss K,
O'Toole T,
Voytovich A,
Webster D,
Mule JJ:
Hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood progenitor cells evaluated in vitro and in vivo.
Blood
87:1261,
1996[Abstract/Free Full Text]
36.
Kurzrock R,
Guttermann JU,
Talpaz M:
The molecular genetics of Philadelphia chromosome-positive leukemias.
N Engl J Med
319:990,
1988[Medline]
[Order article via Infotrieve]
37.
Quattrone A,
Papucci L,
Schiavone N,
Mini E,
Capaccioli S:
Intracellular enhancement of intact oligonucleotide steady-state levels by cationic lipids.
Anti Cancer Drug Design
9:549,
1994[Medline]
[Order article via Infotrieve]
38.
Maxfield FR:
Weak bases and ionophores rapidly and reversibly raise the pH of endocytic vesicles in cultured mouse fibroblasts.
J Cell Biol
95:676,
1982[Abstract/Free Full Text]
39.
Rädler JO,
Koltover I,
Salditt T,
Safinya CR:
Structure of DNA-cationic liposome complexes: DNA intercalation in multilamellar membranes in distinct interhelical packaging regimes.
Science
275:810,
1997[Abstract/Free Full Text]
40.
Zabner J,
Fasbender AJ,
Moninger T,
Poellinger K,
Welsh MJ:
Cellular and molecular barriers to gene transfer by a cationic lipid.
J Biol Chem
270:18997,
1995[Abstract/Free Full Text]
41.
Mislick KA,
Baldeschwieler JD:
Evidence for the role of proteoglycans in cation-mediated gene transfer.
Proc Natl Acad Sci USA
93:12349,
1996[Abstract/Free Full Text]
42.
Ruoslahti E:
Structure and biology of proteoglycans.
Annu Rev Cell Biol
4:229,
1988
43. Stellrecht CM, Mars WM, Miwa Hiroshi, Beran M, Saunders GF:
Expression pattern of a hematopoietic proteoglycan core protein gene
during human hematopoiesis. Differentiation 48:127, 1991
44. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD:
Transport from the plasma membrane via endosomes: Endocytosis, in:
Molecular Biology of the Cell (ed 3). New York, NY, Garland, 1994, p
618
45.
Iversen PL,
Crouse D,
Zon G,
Perry G:
Binding of antisense phosphorothioate oligonucleotides to murine lymphocytes is lineage specific and inducible.
Antisense Res Dev
2:223,
1992[Medline]
[Order article via Infotrieve]
46.
Balasubramaniam RP,
Bennet MJ,
Aberle AM,
Malone JG,
Nantz MH,
Malone RW:
Structural and functional analysis of cationic transfection lipids: The hydrophobic domain.
Gene Therapy
3:163,
1996[Medline]
[Order article via Infotrieve]
47.
Thierry AR,
Dritschilo A:
Intracellular availability of unmodified, phosphorothioated and liposomally encapsulated oligodeoxynucleotides for antisense activity.
Nucleic Acids Res
20:5691,
1992[Abstract/Free Full Text]
48. Hughes JA, Avrutskaya, Juliano RL: Influence of base composition
on membrane binding and cellular uptake of 10-mer phosphorothioate
oligonucleotides in chinese hamster ovary (CHRC5) cells.
Antisense Res Dev 4:211, 1994
49.
Nichols GL:
Antisense oligonucleotides as therapeutic agents for chronic myelogenous leukemia.
Antisense Res Dev
5:67,
1995[Medline]
[Order article via Infotrieve]
50.
Käbisch A,
Perenyi L,
Seay U,
Lohmeyer J,
Pralle H:
Unmodified phosphodiester antisense oligodeoxynucleotides to the BCR-ABL junction do not suppress Philadelphia-positive clonogenic cells.
Acta Haematol
92:190,
1994[Medline]
[Order article via Infotrieve]
51.
Mahon FX,
Ripoche J,
Pigeonnier V,
Jazwiec B,
Pigneux A,
Moreau JF,
Reiffers J:
Inhibition of chronic myelogenous leukemia cells harboring a BCR-ABL B3A2 junction by antisense oligonucleotides targeted at the b2a2 junction.
Exp Hematol
23:1606,
1995[Medline]
[Order article via Infotrieve]
52.
Chasty R,
Whetton A,
Lucas G:
A comparison of the effect of bcr/abl breakpoint specific phosphothiorate oligodeoxynucleotides on colony formation by bcr/abl positive and negative, CD34 enriched mononuclear cell populations.
Leuk Res
20:391,
1996[Medline]
[Order article via Infotrieve]
53.
Smetsers TFCM,
Linders EHP,
van de Locht LTF,
de Witte TM,
Mensink EJBM:
An antisense Bcr-Abl phosphodiester-tailed methylphosphonate oligonucleotide reduces the growth of chronic myeloid leukaemia patient cells by a non-antisense mechanism.
Br J Haematol
96:377,
1997[Medline]
[Order article via Infotrieve]
54.
Vaerman JL,
Lammineur C,
Moureau P,
Lewalle P,
Deldime F,
Blumenfeld M,
Martiat P:
BCR-ABL antisense oligodeoxyribonucleotides suppress the growth of leukemic and normal hematopoietic cells by a sequence-specific but nonantisense mechanism.
Blood
86:3891,
1995[Abstract/Free Full Text]
55.
Ratajczak MZ,
Hijiya N,
Catani L,
DeRiel K,
Luger SM,
McGlave P,
Gewirtz AM:
Acute- and chronic-phase chronic myelogenous leukemia colony-forming units are highly sensitive to the growth inhibitory effects of c-myb antisense oligodeoxynucleotides.
Blood
79:1956,
1992[Abstract/Free Full Text]
56.
Tauchi T,
Broxmeyer HE:
BCR/ABL signal transduction.
Int J Hematol
61:105,
1995[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
I. Bruns, U. Steidl, J. C. Fischer, A. Czibere, G. Kobbe, S. Raschke, R. Singh, R. Fenk, M. Rosskopf, S. Pechtel, et al.
Pegylated granulocyte colony-stimulating factor mobilizes CD34+ cells with different stem and progenitor subsets and distinct functional properties in comparison with unconjugated granulocyte colony-stimulating factor
Haematologica,
March 1, 2008;
93(3):
347 - 355.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
U. Steidl, S. Bork, S. Schaub, O. Selbach, J. Seres, M. Aivado, T. Schroeder, U.-P. Rohr, R. Fenk, S. Kliszewski, et al.
Primary human CD34+ hematopoietic stem and progenitor cells express functionally active receptors of neuromediators
Blood,
July 1, 2004;
104(1):
81 - 88.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
U. Steidl, R. Kronenwett, U.-P. Rohr, R. Fenk, S. Kliszewski, C. Maercker, P. Neubert, M. Aivado, J. Koch, O. Modlich, et al.
Gene expression profiling identifies significant differences between the molecular phenotypes of bone marrow-derived and circulating human CD34+ hematopoietic stem cells
Blood,
March 15, 2002;
99(6):
2037 - 2044.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C.-C. J. Chang, A. Wright, and J. Punnonen
Monocyte-Derived CD1a+ and CD1a- Dendritic Cell Subsets Differ in Their Cytokine Production Profiles, Susceptibilities to Transfection, and Capacities to Direct Th Cell Differentiation
J. Immunol.,
October 1, 2000;
165(7):
3584 - 3591.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. J. Klasa, M. B. Bally, R. Ng, J. H. Goldie, R. D. Gascoyne, and F. M. P. Wong
Eradication of Human Non-Hodgkin's Lymphoma in SCID Mice by BCL-2 Antisense Oligonucleotides Combined with Low-Dose Cyclophosphamide
Clin. Cancer Res.,
June 1, 2000;
6(6):
2492 - 2500.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
J. G. Karras, K. McGraw, R. A. McKay, S. R. Cooper, D. Lerner, T. Lu, C. Walker, N. M. Dean, and B. P. Monia
Inhibition of Antigen-Induced Eosinophilia and Late Phase Airway Hyperresponsiveness by an IL-5 Antisense Oligonucleotide in Mouse Models of Asthma
J. Immunol.,
May 15, 2000;
164(10):
5409 - 5415.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. M. Gewirtz, D. L. Sokol, and M. Z. Ratajczak
Nucleic Acid Therapeutics: State of the Art and Future Prospects
Blood,
August 1, 1998;
92(3):
712 - 736.
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract