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Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3163-3171
By
From the Department of Hematology and the Department of Anatomy and
Developmental Biology, Tokyo Women's Medical College; the Japanese Red
Cross Center; the Department of Pathology, the Research Institute of
Tuberculosis; the Institute of Molecular and Cellular Bioscience,
University of Tokyo; and the Department of Veterinary Anatomy, Tokyo
University of Agriculture and Technology, Tokyo, Japan.
To overcome the problem of multidrug resistance, we investigated the
effectiveness of phosphrothioate antisense oligonucleotides (MDR1-AS)
in suppressing multidrug resistance gene (mdr1) expression in
drug-resistant acute myelogenous leukemia (AML) blast cells and the
K562 adriamycin-resistant cell line K562/ADM. The percentage of cells
with the mdr1 gene product P-glycoprotein (P-gp) was decreased
from 100% to 26% by 20 µmol/L MDR1-AS in the K562/ADM cells, and
from 48.1% to 10.2% by 2.5 µmol/L MDR1-AS in the AML blast cells.
Western blot analysis also showed a decrease in the amount of P-gp in
the MDR1-AS-treated K562/ADM cells. This effect was specific to
MDR1-AS, and not observed with sense or random control
oligonucleotides. The expression of mdr1 mRNA in K562/ADM and
AML blast cells treated with MDR1-AS was decreased compared with the
random control. Intracellular rhodamine retention and [3H]daunorubicin also increased after antisense
treatment. Chemosensitivity to daunorubicin increased in
MDR1-AS-treated blast cells up to 5.9-fold in the K562/ADM cells and
3.0- to 6.4-fold in the AML blast cells. The expression of mdr1
mRNA derived from colony cells decreased in the MDR1-AS-treated
groups. No inhibitory effect of the oligonucleotides on normal bone
marrow progenitors was observed. These findings suggest that MDR1-AS is
useful to overcome multidrug resistance in the treatment of
leukemia.
RESISTANCE TO CHEMOTHERAPY is a major
obstacle in cancer treatment and has been closely associated with
treatment failure. This phenomenon is termed multidrug resistance
(MDR). One of the mechanisms of MDR involves the overexpression of the
multidrug resistance gene (mdr1) product, P-glycoprotein
(P-gp).1 P-gp is a 170-kD membrane
glycoprotein capable of the adenosine triphosphate (ATP)-dependent
cellular efflux of a variety of compounds across the plasma membrane.
It has been shown that the overproduction of P-gp causes not only an
increase in drug excretion from cells, but also a decrease in
intracellular drug accumulation, thereby reducing the intracellular
drug contents such as daunorubicin (DNR) and adriamycin. We previously
reported that the intracellular DNR content is lower in
P-gp+ acute myelogenous leukemia (AML) blast cells than in
P-gp Three strategies have been developed for the eradication of
P-gp-expressing MDR cells: (1) chemical compounds such as
verapamil,5 cyclosporin-A,6 and
MS-2092; (2) monoclonal antibodies such as
MRK167 or immunotoxins against P-gp8; and (3)
antisense (AS) oligonucleotides for the specific inhibition of P-gp
gene expression.9-11 AS oligonucleotides are short
sequences of DNA that form a specific hydrogen bond with complementary
single-strand mRNA sequences, allowing the regulation of specific gene
expression.12,13 The possible use of AS oligonucleotides in
suppressing the expression of certain genes is now under investigation
by several groups.14,15
We addressed the question of whether AS oligonucleotides to
mdr1 mRNA (MDR1-AS) could suppress the overexpression of P-gp in AML blast cells. In the present study, we showed that incubation with AS oligonucleotides to mdr1 mRNA reduced the in vitro
expression of P-gp in drug-resistant K562 cells and AML blast cells.
Furthermore, P-gp function of the AS-treated blast cells was inhibited,
as shown by intracellular rhodamine (Rh123) and intracellular DNR retention assays, and the AS-treated blast cells recovered their sensitivity to DNR, as shown by a leukemic blast colony assay.
Cell Line
AML Blast Cells
Oligonucleotides
Oligonucleotide Treatments of Cells With Lipopolyamine Lipopolyamine, a cationic liposome (Transfectam; Biospra, Marlbourough, MA) was used to increase the oligonucleotide uptake of cells according to the method reported by Behr et al.19 The oligonucleotide/lipid complex was obtained by adding various concentrations of oligonucleotide solution to lipopolyamine solution (when the DNA content of the oligonucleotides was x µg, 0.5x µL of lipopolyamine solution was added according to the manufacturer's protocol). In every experiment, the oligonucleotide/lipid complex thus prepared was added every 12 hours to the cell suspension (1 ~ 2 × 106 cells/mL) in RPMI medium with 7% FCS, and incubated at 37°C in a humidified atmosphere with 5% CO2. After 96 hours, the cells were washed with phosphate-buffered saline (PBS), then analyzed for the determination of P-gp positivity, functional assay, and drug sensitivity.Efficiency of Oligonucleotide Uptake by Cells Oligonucleotides were treated with 3 end-labeled
[ -32P] dATP using terminal deoxynucleotidyl
transferase (Amersham, Little Chalfort, UK) in 0.14 mol/L sodium
cacodylate (pH 7.2), 1 mmol/L CoCl2, and 0.1 mmol/L
dithiothreitol for 1 hour at 37°C. After elution through a Sephadex
G-50 column to remove unincorporated ATP, labeled oligonucleotides (1 µmol/L) and lipopolyamine were mixed and added to K562/ADM cells. At
various times after incubation (from 10 minutes to 24 hours), cells
were washed, and the cell-bound radioactivity was measured by a
scintillation counter.
Determination of P-gp by Fluorescence Microscopy The P-gp of the leukemic blast cells was stained according to the method described by Schinkel et al20 with some modifications. The cytospin preparations of leukemic blast cells were fixed with 3% formaldehyde, then incubated with 100 µg/mL MRK16 antibody. After washing, the preparations were incubated with goat anti-mouse IgG-conjugated fluorescein isothiocyanate (dilution, 1:25; Tago, Burlingame, CA), then examined by fluorescence microscopy. The percentage of P-gp+ cells was determined for 1,000 cells counted.Determination of P-gp by Flow Cytometry The leukemic blast cells were stained according to the method reported previously using MRK16 monoclonal antibody,7 and analyzed using an Epics-Profile II flow cytometer (Coulter Co, Miami, FL). P-gp positivity was determined by the Epics Elite software Immuno 4 computer program21 (Coulter), and the sample was considered to be P-gp+ when more than 20% of the cells were stained.3Determination of P-gp by Western Blot Analysis Plasma membrane fractions from random- or MDR1-AS-treated K562/ADM cells were isolated as described by Kawai et al.22 Membrane-enriched fractions (10 µg proteins) were electrophoresed in a 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel and then electrotransferred to polyvinylfluoride paper. Western blot analysis was performed with monoclonal anti-P-gp antibody C219 (Centcor, Malvern, PA) using peroxidase-conjugated goat anti-mouse IgG (dilution, 1:1,500; Sigma Chemical Co, St Louis, MO) and enhanced chemiluminescence (Amersham).Detection of mdr1 mRNA Expression by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis Total RNA from oligonucleotide-treated cells (K562/ADM 5 µmol/L, AML blast cells 2.5 µmol/L) was extracted using acid-guanidium-phenol-chloroform as described23 and converted to single-strand cDNA using a random primer and reverse transcriptase under reaction conditions described previously.24 PCR was performed on the cDNA after the addition of Taq DNA polymerase, dNTP, and each primer using a Perkin-Elmer Cetus thermal cycler as described previously.24 An aliquot of each reaction mixture was then analyzed by electrophoresis on a 2% agarose gel, and the amplified DNA band was visualized by ethidium bromide staining. The amplification products of mdr1 and 2-microglobulin
were 309 bp and 261 bp, respectively.
Determination of Intracellular Rh123 Retention Rh123 (Sigma) studies were performed according to our previous study2 with some modifications. Briefly, oligonucleotide-treated cells (2 × 105) (K562/ADM 5 µmol/L, AML blast cells 2.5 µmol/L) were loaded with 150 ng/mL of Rh123 in 5 mL of PBS for 10 minutes at 37°C. Rh123 retention was assayed by allowing the dye efflux in 5 mL of PBS with 10% FCS for 3 hours at 37°C. After two washes, intracellular Rh123 fluorescence intensity was quantitated by flow cytometry using a 530-nm (Rh123) long band-pass filter and determined by the Epics Elite software Immuno 4 computer program.21Determination of Intracellular [3H]DNR Retention A [3H]DNR retention assay was performed according to the method described by Tsuruo et al16 with some modifications. Briefly, oligonucleotide-treated cells (2 × 105) (K562/ADM 5 µmol/L, AML blast cells 2.5 µmol/L) were incubated at 37°C with [3H]DNR (1 µmol/L; specific activity 4.4 Ci/mmol; Daiichi Pure Chemical Co Ltd, Tokyo, Japan) for 1 hour in 1 mL of RPMI medium. Then, each cell suspension was washed and resuspended in 1 mL of medium at 37°C for 3 hours. The amount of intracellular DNR was measured with liquid scintillation counter after the cells were lysed with 0.5 mL of 0.2 N NaOH. The [3H]DNR retention/accumulation ratio was calculated as follows: intracellular retention was divided by intracellular accumulation.Measurement of Cellular Sensitivity to DNR DNR sensitivity of leukemic blast cells from patients and K562/ADM was examined. The effect of oligonucleotides was also examined in K562/ADM (5 µmol/L) and in four AML samples (2.5 µmol/L). Leukemic blast cells (5 × 105 cells/mL) were exposed to different concentrations of DNR (10 7 mol/L
~5 × 105 mol/L) for 1 hour at 37°C according to
the method reported previously.17 After being washed three
times with PBS, the cells were suspended with 0.8% methylcellulose and
20% FCS, and plated in a 96-microwell plate (Linbro; Flow
Laboratories) at a concentration of 5 × 104 cells/0.1 mL.
A mixture of recombinant human granulocyte-macrophage colony-stimulating factor (rGM-CSF; 103 U/mL), recombinant
human granulocyte CSF (rG-CSF; 104 U/mL), and recombinant
human interleukin-3 (102 U/mL) was added to the culture as
a CSF. All of the CSFs were obtained from Kirin-Brewery Co Ltd (Tokyo,
Japan). Culture plates were incubated at 37°C in a humidified
atmosphere with 5% CO2. After 7 days, colonies consisting
of more than 20 cells were counted under an inverted microscope. The
cellular sensitivity to DNR was determined by calculating the dose of
DNR that reduced the colony numbers to 10% of the control
(D10 value).17 To examine the effect of the
three oligonucleotides on the sensitivity to DNR, the D10
ratio was calculated as follows: the D10 value without oligonucleotides was divided by the D10 value with
oligonucleotides.
Persistence of P-gp Suppression After AS Treatment To examine the persistence of P-gp suppression after MDR1-AS treatment, oligonucleotide-treated cells (K562/ADM 5 µmol/L, AML blast cells 2.5 µmol/L) were washed and incubated in oligonucleotide-free RPMI medium with 10% FCS for 1 to 10 days, and then the P-gp was examined.Effect of Oligonucleotides on Bone Marrow Progenitors Bone marrow cells were obtained from normal volunteers who had given their informed consent, and mononuclear cells were separated using a Ficoll-Conray density gradient. The colony assay for bone marrow progenitors was performed by the method described by Fauser and Messner25 and Dover et al26 with slight modifications. Bone marrow mononuclear cells (2 × 106 cells/mL) were incubated with or without oligonucleotide/lipid complex added every 12 hours for 96 hours at the concentration of 2.5 µmol/L, washed, then plated in 35-mm tissue culture dishes (Nunc Inc, Naperville, IL) containing 1 mL of a mixture of 0.9% methylcellulose, 30% FCS, 1% bovine serum albumin, 1 U/mL recombinant human erythropoietin (Kirin), and 5 × 106 U/mL rG-CSF. Granulocyte-macrophage colony-forming units (CFU-GM) and erythroid colony-forming units (CFU-E) were scored on day 7, and erythroid burst-forming units (BFU-E) and pluripotent hemopoietic progenitors (CFU-GEMM) were scored on day 14, respectively. The criteria for positive colony formation for each different lineage were defined as described by Fauser and Messner25 and Dover et al26 The numbers of colonies were counted in three dishes in all experiments and compared with the untreated controls.Statistical Analysis Student's t-test was used to evaluate the differences between two groups.
Oligonucleotide Uptake Efficiency The uptake of oligonucleotides into K562/ADM cells reached the maximum value after 30 minutes of incubation (9%), and the overall uptake efficiency was 5%~9%.Effect of MDR1-AS on K562/ADM Cells Expression of P-gp and mdr1 mRNA in MDR1-AS-treated K562/ADM cells. The number of K562/ADM cells did not decline after exposure to MDR1-AS, and the viability of the blast cells remained at 80%~90%. Figure 1A shows the percentages of P-gp+ cells among K562/ADM cells after incubation with MDR1-AS, as detected by fluorescence microscopy. The percentages of P-gp+ cells were reduced in proportion to the dose of MDR1-AS used (at 2.5 µmol/L, 69%; 5 µmol/L, 58%; 10 µmol/L, 55%; 20 µmol/L, 26%). The maximum effect of MDR1-AS was observed at the highest concentration (20 µmol/L), and the percentage of P-gp+ cells decreased from 100% to 26% at this concentration. The suppression of P-gp was observed when leukemic blast cells were treated with MDR1-AS, but was not observed with the MDR1-S or random oligonucleotides in low (2.5 µmol/L ~ 10 µmol/L) concentrations. Only slight inhibition was seen in the random- and MDR1-S-treated groups at the high (20 µmol/L) concentration. Because a reduction of P-gp positivity was observed after treatment with 5 µmol/L MDR1-AS, we used this concentration for further experiments. The reduction of P-gp expression on K562/ADM cells was also observed by Western blot analysis, as shown in Fig 2.Only scarce P-gp expression was detected on K562/ADM cells treated with MDR1-AS (5 µmol/L), whereas P-gp from random-treated K562/ADM cells was definitely observed. P-gp expression from untreated K562/ADM cells was similar to that of random-treated cells, and a slight decrease was seen in MDR1-S-treated cells (data not shown).
P-gp function in MDR1-AS-treated K562/ADM cells.
As shown in Fig 1B and Table 2,intracellular Rh123 retention in the MDR1-AS-treated K562/ADM cells (5 µmol/L, 37.4%) increased compared with untreated cells (13.3%),
indicating Rh123 efflux is inhibited. In contrast, intracellular Rh123
retention did not change in random- (13.4%) or MDR1-S-treated cells
(11.7%). As shown in Table 2, the [3H]DNR
retention/accumulation ratio of K562/ADM cells after MDR1-AS treatment
(5 µmol/L) was 25.0% in the MDR1-AS-treated group, and this value
was higher than that of untreated, random-treated and MDR1-S-treated
cells.
Sensitivity to DNR of leukemic blast progenitors in MDR-1-AS-treated
K562/ADM cells.
Figure 4 shows the DNR dose-response curve
of oligonucleotide treated (5 µmol/L) K562/ADM cells for colony
formation. The numbers of leukemic blast colony cells incubated with
DNR was decreased remarkably in the MDR1-AS-treated group compared
with those of the untreated, random-treated, and MDR1-S-treated
groups. In the MDR1-AS-treated cells, the D10 value was
1.7 × 10
mdr1 expression in leukemic blast colony cells after
MDR1-AS treatment.
K562/ADM cells from pooled colonies formed after incubation with DNR
were examined for mdr1 mRNA expression. As shown in Fig 3B, the
cells of colonies formed from MDR1-AS-treated cells did not express
mdr1 mRNA, whereas the cells of colonies formed from random-treated cells expressed mdr1 mRNA. The levels of
Persistence of P-gp suppression by MDR1-AS treatment.
Figure 5 shows the percentage of
P-gp+ cells which were treated with oligonucleotides, then
incubated in oligonucleotide-free medium for various durations. After 4 days incubation in oligonucleotide-free medium, the percentage level of
P-gp+ cells (93%) recovered to those of the random-
(100%) and MDR1-S- (99%) treated groups.
Effect of MDR1-AS on AML Blast Cells
Expression of P-gp and mdr1 mRNA in MDR1-AS-treated AML blast
cells.
The numbers of blast cells did not decline after exposure to MDR1-AS;
the viability of the blast cells remained at 80%~90%. Figure
6 shows a typical dose-response curve for
oligonucleotide treatment of AML blast cells (Case No. 8) as detected
by flow cytometry. The P-gp positivity was reduced in proportion to the dose of MDR1-AS added, and the maximum effect of MDR1-AS was observed at the concentration of 2.5 µmol/L. The P-gp positivity at this concentration decreased from 49.4% to 19.5%. The P-gp positivity was
specifically suppressed when AML blast cells were treated with MDR1-AS,
and was not suppressed when the cells were treated with the MDR1-S or
random oligonucleotides. These findings were also observed in Case No.
9 AML blast cells (data not shown), and confirmed by fluorescence
microscopy in both AML blast cells (data not shown). Therefore, we used
this concentration for further experiments. The P-gp positivity of
blast cells from 10 AML patients before and after treatment with
MDR1-AS are listed in Table 1. The P-gp positivity was greatly
decreased by the treatment with MDR1-AS; the mean P-gp positivity of
AML blast cells declined from 48.1% (±15.1%) to 10.2% (±6.8%).
The maximum reduction of P-gp positivity was observed in Case No. 3, from 76.1% to 5.2% (about one-fifteenth).
P-gp function in MDR1-AS-treated AML blast cells.
As shown in Table 2, intracellular Rh123 retention in MDR1-AS-treated
AML blast cells (2.5 µmol/L) increased 1.4- and 1.2-fold, respectively, compared with the untreated cells. These values were
apparently higher than those of untreated, random-treated, and
MDR1-S-treated cells. As shown in Table 2, the [3H]DNR
retention/accumulation ratio of MDR1-AS-treated AML blast cells (2.5 µmol/L) increased 1.8- and 1.2-fold for Cases No. 8 and 9, respectively, and these values were higher than those of untreated,
random-treated, and MDR1-S-treated cells.
Sensitivity to DNR of AML blast progenitors in MDR1-AS-treated AML
blast cells.
The sensitivity to DNR of oligonucleotide-treated leukemic progenitors
were examined in four cases. As shown in Table 1, MDR1-AS-treated AML
blast cells (2.5 µmol/L) were more sensitive to DNR than were
untreated blast cells and those treated with random or MDR1-S. The
D10 ratios of the MDR1-AS-treated blast cells were between
3.0 and 6.4; in contrast, those of the random- and MDR1-S-treated
cells were from 0.7 to 1.3, except for MDR1-S-treated cells in case
No. 9 (2.2).
mdr1 expression in AML blast colony cells after MDR1-AS
treatment.
AML cells from pooled leukemic colonies formed after incubation with
DNR were examined for mdr1 mRNA expression. As shown in Fig 7B
(Case No. 9), cells in colonies formed from MDR1-AS-treated cells did
not express mdr1 mRNA, whereas those from random-treated cells
expressed mdr1 mRNA. The levels of
Persistence of P-gp suppression after AS treatment.
To determine the persistence of P-gp suppression in MDR1-AS-treated
AML blast cells (2.5 µmol/L), oligonucleotide-treated cells were
washed, incubated at various durations in oligonucleotide-free medium,
and analyzed for P-gp expression (Fig 8).After 3 days incubation in oligonucleotide-free medium, P-gp positivity
(40.9%) recovered to the levels of P-gp with random- (41.6%) and
MDR1-S- (49.6%) treated groups (Case No. 8).
Effect of MDR1-AS on Bone Marrow Progenitors
In the present study, we showed that a lipopolyamine-coated antisense
oligonucleotide complementary to mdr1 (MDR1-AS) was effective
in inhibiting P-gp expression in AML blast cells. The effectiveness of
MDR1-AS use with lipopolyamine was observed not only in
adriamycin-resistant K562/ADM cells, but also in blast cells taken from
patients with acute leukemia. The maximum suppression of P-gp was
obtained with 20 µmol/L MDR1-AS in the K562/ADM cells, and with 2.5 µmol/L MDR1-AS in AML blast cells. The reduction of P-gp was also
observed in the K562/ADM cells by Western blot analysis using C219
monoclonal antibody. This is considered to be caused by the reduction
of P-gp expression, because K562/ADM did not express mdr3 (our
observation). The suppression effect of MDR1-AS was observed not only
on P-gp but also on mdr1 mRNA. In the AML blast cells, P-gp
positivity was reduced by MDR1-AS treatment to less than 20%, which
was considered to be P-gp Submitted February 28, 1997;
accepted December 30, 1997.
We thank Dr K. Takeuchi of the Nara Institute of Science and Technology
(NAIST), Graduate School of Biological Science for useful technical
advice, and I. Yamato for technical assistance. We also thank Dr T. Mekawa of The Institute of Medical Science, University of Tokyo for his
critical advice, and David Brown for checking the manuscript.
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Abstract
Introduction
Abstract
12 to +12, 5
), MDR1-S (
), and MDR1-AS (
) treatment at various
concentrations. The results are expressed as the percentage of treated
cells compared with the untreated control, and represent the mean ± the SD of the three experiments. (B) Intracellular Rh123 retention in
K562/ADM cells after oligonucleotide treatment (5 µmol/L).
Intracellular Rh123 retention of untreated, random-, MDR1-S-, and
MDR1-AS-treated cells was measured using flow cytometry, and the
figure represents untreated and MDR1-AS-treated cells.
), random- (
), MDR1-S- (
), and MDR1-AS-treated cells (
). The results are expressed as the
percentage of DNR treated colonies compared with the DNR untreated
control, and represent the mean ± the SD of colony numbers of three
dishes.
Is the bullet really magical?
Science
261:1004,
1993