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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3929-3938
NEOPLASIA
From The University of Texas M. D. Anderson Cancer Center, Houston,
TX.
The antiapoptotic proteins, Bcl-2 and Bcl-XL, are
expressed in most cases of acute myeloid leukemia (AML) and may
contribute to drug resistance in AML. We tested the hypothesis that
down-regulation of Bcl-2 alone by antisense oligodeoxynucleotides
(Bcl-2-AS) induces apoptosis, even in the presence of other
antiapoptotic genes. We tested Bcl-2-AS in myeloid leukemic HL-60
cells, in Bcl-2 and Bcl-XL overexpressing HL-60-DOX cells,
and in primary AML samples. Down-regulation of Bcl-2 by Bcl-2-AS
reduced the viability of HL-60 cells and, less effectively, HL-60-DOX
cells and increased ara-C cytotoxicity in both cell lines. Incubation
of primary AML blasts with Bcl-2-AS decreased Bcl-2 expression in
CD34+ blast cells after induction of apoptosis and
enhancement of ara-C cytotoxicity in 11 of 19 primary AML samples. In 8 samples in which Bcl-2-AS did not induce apoptosis, baseline Bcl-2
levels were found to be strikingly high. The expression of other
antiapoptotic proteins (Bcl-XL, Bag-1, A1, and Mcl-1) did
not prevent Bcl-2-AS-induced apoptosis. Bcl-2-AS also inhibited colony
formation of AML progenitor cells. Low concentrations of Bcl-2-AS
induced significant increases in S-phase cells (P = .04).
Results establish Bcl-2 as a critical target for AS strategies in AML
in which the baseline levels predict response to Bcl-2-AS. Bcl-2 exerts
both antiapoptotic and antiproliferative functions in AML. Because
early normal hematopoietic stem cells do not express Bcl-2, Bcl-2-AS
therapy should be highly selective for AML cells.
(Blood. 2000;95:3929-3938)
Physiologic cell death (apoptosis) is controlled by an
intrinsic genetic program that is remarkably conserved in evolution. All currently available cytotoxic drugs induce tumor death by triggering apoptosis. However, many tumors have defects in the regulation of genes that control apoptosis. This may contribute to
their growth and also render them resistant to chemotherapeutic agents.
Members of the Bcl-2 family regulate a distal step in the cell death
pathway. Although its mechanism of action is still unclear, Bcl-2
appears to function as a suppressor of cell death that can be triggered
by a variety of signals. In gene transfection experiments,
overexpression of Bcl-2 and its homolog Bcl-XL can render
neoplastic cells resistant to the induction of apoptosis by a variety
of chemotherapeutic drugs.1-5
Likewise, down-regulation of the Bcl-2 protein has been shown to
reverse chemoresistance in several experimental systems.6-9 In acute myeloid leukemia (AML), high Bcl-2 levels are associated with
resistance to chemotherapy, decreased rates of complete remission, and
shortened survival.10-14
Recently, it was demonstrated that Bcl-2 not only inhibits apoptosis
but also restrains cell cycle entry15-17 and that these 2 functions can be genetically dissociated.18 This
antiproliferative effect could provide additional cytoprotection
because proliferating cells are more vulnerable to apoptotic stimuli.
Therefore, agents that can overcome the inhibitory effects of Bcl-2 on
cell cycle entry could be a useful adjunct to currently available
chemotherapeutic drugs.
Inhibiting the function of Bcl-2 might have a more pronounced effect on
neoplastic cells than on normal cells, that is, the loss of cell cycle
control mechanisms drives cells into the cell cycle, despite
drug-induced damage.19 Inactivation of the G1 cell cycle checkpoint occurs when p53 is inactivated, either by mutation or deletion.20 Cells, then, do not arrest and
repair DNA damage but proceed through the cell cycle and undergo
programmed cell death. It is known that the most primitive
hematopoietic precursors express Bcl-XL but not
Bcl-2.21,22 Bcl-2 is universally expressed in AML
progenitors cells, and a subset of patients with AML have higher levels
of expression than normal CD34+ cells.10,14
Results from our group and others indicate that retinoids down-regulate
the expression of Bcl-2, and can enhance the effect of chemotherapy in
vitro.22,23 Recent data suggest the ability of all-trans
retinoic acid to inactivate Bcl-2 by phosphorylation.24
Antisense oligodeoxynucleotides targeted against Bcl-2 have been used
to induce apoptosis of malignant cells or to sensitize them to
conventional chemotherapeutic drugs,6-8 and the first
clinical study of Bcl-2 antisense therapy in patients with recurrent
non-Hodgkin's lymphoma was recently reported.25 Other
studies suggest that liposomal delivery of oligodeoxynucleotides may
circumvent the poor cellular uptake and delivery of antisense oligodeoxynucleotides alone.26 We have recently
demonstrated that the p-ethoxy modification of phosphodiesters enhances
nuclease resistance and increases incorporation efficiency into
liposomes.27 Experimental animal models indicate a lack of
significant adverse effects such as autoimmunity and organ toxicity.
Liposomal oligodeoxynucleotides are mainly distributed to the liver,
spleen, and bone marrow, which are the major organs of leukemic
manifestation.28 Thus, liposomal delivery of anti-Bcl-2
oligodeoxynucleotides (Bcl-2-AS) in combination with chemotherapeutic
agents and biologic response modifiers may potentially be used as novel
treatment modalities for hematologic malignancies.
In this study, we used liposomally delivered Bcl-2-AS to induce
down-regulation of the Bcl-2 protein in AML cells. Similar approaches
in different tumor models have resulted in decreased cell
survival,29,30 the induction of apoptotic cell
death,31 and increased drug sensitivity in vitro and in
vivo.6,32 Because other antiapoptotic proteins are
expressed in AML,22,33 we wished to investigate whether
Bcl-2 was critical for AML survival and if apoptosis could be induced,
in particular, despite high levels of antiapoptotic Bcl-XL,
which is expressed in most AML.22,34,35 Also, we wished to
investigate what levels of cellular Bcl-2 are critical for the survival
and chemoresistance of primary AML cells.
Cell lines
Subjects
Preparation of liposomal oligodeoxynucleotides P-ethoxy oligodeoxynucleotides (ODN) (Oligos Etc, Willsonville, OR) were chosen because this modification makes ODN nuclease resistant and can be efficiently incorporated into liposomes. Liposomal oligodeoxynucleotides were prepared as previously described.38 Briefly, ODN dissolved in DMSO were added to phospholipids (Avanti Polar Lipids, Alabaster, AL) in the presence of excess tert-butanol. The mixture was frozen in a dry ice/acetone bath, lyophilized overnight, and finally hydrated with 0.9% normal saline at a final oligodeoxynucleotide concentration of 0.1 mmol/L. For Bcl-2-AS, we used a sequence, which is complimentary to the Bcl-2 translation initiation site (5'-CAGCGTGCGCCATCCTTCCC-3').39 Scrambled sequence (nonsense, NS) oligodeoxynucleotides (5'-TCGCCACTCGATCCTGCCCG-3') and empty liposomes were used as controls.Suspension culture of leukemic cells HL-60 and HL-60-DOX cells were cultured at 2.5 × 104 cells/mL, and AML mononuclear cells were seeded at 5 × 105 cells/mL. Cells were cultured in complete media (RPMI supplemented with 10% fetal calf serum [FCS]) in the presence or absence of liposomal Bcl-2-AS or NS at an appropriate concentration (see below). Empty liposomes were also included as controls. Granulocyte colony-stimulating factor (G-CSF) (200 U/mL) was added to cultures of fresh AML cells. In previous studies, we demonstrated that G-CSF and granulocyte-macrophage colony-stimulating factor (GM-CSF) support proliferation and block spontaneous apoptosis of AML blasts without affecting average Bcl-2 expression levels.40 For protein and apoptosis studies, a final concentration of 8 µmol/L ODN was used. These studies were repeated 3 times.Quantitation of viability (MTS assay) To determine cell viability, leukemic cells were seeded at a density of 2.5 × 103 cells per well in 96-well plates (Costar, Cambridge, MA). Six hours later, liposomal Bcl-2-AS, NS, or empty liposomes were added to the cells at a final concentration of 2 to 20 µmol/L. After 5 days of culture, cell viability was measured using the Cell Titer 96 AQ Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI). This assay is based on the ability of viable cells only to reduce MTS to formazan, which can be measured with a spectrophotometer at an absorbance of 490 nm. MTS solution (2 mL) was mixed with 100 µL of phenazine methosulfate (PMS) immediately before being added to the cells in the culture plate. The MTS/PMS solution (20 µL) was than added to each well to maintain a ratio of 20 µL MTS/PMS to 100 µL medium. After 1 hour, the reduction product was measured at an absorbance of 490 nm and compared with a standard curve.Quantitation of Bcl-2 protein by flow cytometry The cellular content of Bcl-2 was measured in conjunction with the CD34 antigen in AML blast cells. Briefly, after staining with phycoerythrin (PE)-conjugated anti-CD34 monoclonal antibody (HPCA-2; Becton Dickinson, San Jose, CA), cells were washed twice and fixed in 1% formaldehyde (Sigma) for 15 minutes on ice, followed by permeabilization with 0.1% Triton X in phosphate-buffered albumin (1% albumin, 0.1% NaN3) for 10 minutes at 4°C. Cells were then washed in cold phosphate-buffered saline (PBS) before being added to 10 µL of fluorescein isothiocyanate (FITC)-conjugated anti-Bcl-2 or isotype IgG1 monoclonal antibody (DAKO, Carpinteria, CA). Dead cells were eliminated by gating, based on their scatter (high side scatter and/or low forward scatter) characteristics, and Bcl-2 expression was measured selectively on live cells. The intensity of Bcl-2-associated fluorescence was measured on a logarithmic scale. Bcl-2 was quantitated using Quantum Simply Cellular microbeads with QuickCal Software (Flow Cytometry Standard, Triangle Park, NC), as previously described,41,42 and expressed as the antibody-binding capacity (ABC), which is an estimate of the number of antibody molecules bound per cell. A FACScan flow cytometer (Becton Dickinson) equipped with an argon laser (488 nm) was used to measure fluorescence. For each sample, 10 000 cells were analyzed; cells were live-gated for CD34 positivity. Data were analyzed using Lysys software (Becton Dickinson).Detection of apoptotic cells and cell kinetics studies Cell cycle kinetics were determined by staining cells with acridine orange for cellular DNA and RNA content, followed by flow cytometric analysis. This method is able to discriminate cells in G0, G1, S, and G2M phases and determines the mean RNA content per cell during each phase of the cell cycle.43 A minimum of 30 000 cells were analyzed and the percentage of cells in the S-phase was determined using ModFit software (Verity Software House, Inc, Topsham, ME).Detection of apoptotic cells based on DNA fragmentation Aliquots (80 µL) of cells were mixed with 100 µL of solution containing 0.1% (vol/vol) Triton X-100, 0.05 mol/L HCl, 0.15 mol/L NaCl, and 8 µg/mL acridine orange (Polysciences, Warrington, PA). The cell fluorescence was measured within 5 minutes of staining, using the logarithmic scale of the FACScan flow cytometer with a 488-nm excitation of a 15-mW argon laser and filter settings for green (530 nm) (DNA) and red (585 nm) (RNA) fluorescence. Ten thousand events were stored in the list mode for analysis. The percentage of cells in the "sub G1 region" defined the proportion of apoptotic cells in the tested populations. Cell debris was defined as events in the lowest 10% range of fluorescence and eliminated from analysis.Acute myeloid leukemia blast colony assay A previously described method was used to assay AML blast colony formation.44,45 Bone marrow mononuclear cells containing more than 80% blasts from the bone marrow of patients with AML were incubated for 24 hours in Iscove's modified Dulbecco medium (IMDM) supplemented with 10% FCS and 50 ng/mL recombinant human GM-CSF (Immunex Inc, Seattle, WA). Bcl-2-AS was added at the initiation of cultures at concentrations of 4, 6, 8, and 12 µmol/L. Untreated cells and cells that were incubated for 24 hours in the presence of 12 µmol/L NS and empty liposomes were used as controls. After extensive washing, 1 × 105 AML cells were plated in 0.8% methylcellulose in IMDM with 10% FCS and 50 ng/mL GM-CSF. Duplicate cultures were incubated in 35-mm petri dishes for 7 days at 37°C in a humidified atmosphere of 5% CO2 in air. AML blast colonies were microscopically evaluated on day 7. A blast colony was defined as a cluster of 40 or more cells. Individual colonies were plucked, smeared on glass slides, and stained to confirm leukemic cellular composition. As previously described, the AML blast colonies grown in this assay contained only blasts and no normal progenitors.46Western blot analysis Cells were lysed in protein lysis buffer. An equal amount of protein lysate was placed on 8% SDS-PAGE for 2 hours at 100 V, followed by transfer of the protein to a Nytran membrane (S&S, Heween, NH). Immunoblotting was performed at room temperature for 2 hours with 5% milk before incubation with the first antibody in 1:1000 dilution for another 2 hours, followed by 3 washings in phosphate-buffered saline (PBS). The procedure was repeated for the secondary antibody. Blots were then soaked in ECL buffer for 1 minute and exposed to ECL films. Polyclonal rabbit antibodies to Bcl-2, Bcl-XL, Bax, Bak, A1, Mcl-1,47-49 and a murine monoclonal antibody to Bag-150 were used at 1:1000 dilution (kindly provided by Dr J.C. Reed).Statistical analysis The statistical analysis was performed using the 2-tailed Student t test and the Spearman rank correlation coefficient. Statistical significance was considered when P < .05. Unless otherwise indicated, average values were expressed as mean ± SEM. The chi-square test was used to compare effects of Bcl-2-AS on AML cells in vitro with several clinical features of AML.
Basal levels of Bcl-2 and Bcl-XL expression in leukemic cell lines The level of protein expression was examined in the HL-60 myeloid cell line and its drug-resistant counterpart, HL-60-DOX, by quantitative flow cytometry and Western blot analysis. As shown in Figure 1A, HL-60-DOX cells express significantly higher levels of Bcl-2 than the parental HL-60 cells. Using a series of calibrated FITC microbeads (see "Materials and methods"), the antibody-binding capacity per cell was calculated. HL-60 cells had 49 × 103 ABC per cell and HL-60-DOX had 86 × 103 ABC per cell. By Western blot analysis, HL-DOX cells overexpressed both Bcl-2 and Bcl-XL (Figure 1B).
Cytotoxic effects of Bcl-2-AS on HL-60 and HL-60-DOX cell lines The effect of Bcl-2-AS on the growth of leukemic cell lines was tested using the MTS viability assay. Leukemic cells were incubated with increasing concentrations of Bcl-2-AS or NS for 5 days (Figure 2). The viability of HL-60 cells was effectively reduced by Bcl-2-AS in a dose-dependent fashion (IC50 = 4 µmol/L). NS exhibited only a modest effect with all concentrations used. HL-60-DOX-resistant cells with a higher Bcl-2 and Bcl-XL content required a higher concentration of Bcl-2-AS to achieve equivalent toxicity (IC50 = 10 µmol/L), but were not protected by Bcl-XL. Empty liposomes did not inhibit cell growth under similar conditions, and NS only affected cell viability of HL-60-DOX cells when used in concentrations greater than or equal to 16 µmol/L. Interestingly, antisense oligonucleotides targeting the Bcl-XL translation initiation site were much less effective in killing HL-60 cells, with IC50 = 10 µmol/L in 2 independent experiments (unpublished observations).
Bcl-2-AS induces apoptosis of leukemic cells. To investigate the mechanism of dose-dependent inhibition of cell growth, apoptotic cells were identified by flow cytometry based on differential staining with acridine orange ("sub G1 region"). HL-60 and HL-60-DOX cells were treated with 8 µmol/L Bcl-2-AS or NS for 5 days. After permeabilization, cells were stained with acridine orange and analyzed by flow cytometry in 3 independent experiments. The number of apoptotic HL-60 and HL-60-DOX cells increased to 24.3% ± 5.1% and 25.3% ± 3.2%, respectively, after treatment with Bcl-2-AS, compared with treatment with NS (HL-60, 12.6% ± 4.8%; HL-DOX, 3.9 ± 0.9, P = .01) and control cultures (HL-60, 5.8% ± 1.7%; HL-DOX, 2.3% ± 0.6%, P = .005). Bcl-2-AS reduces Bcl-2 protein levels in leukemic cells.
To examine the postulated sequence-specific down-regulation of target
protein, we used Western blot analysis and flow cytometry to determine
Bcl-2 protein levels after treatment with Bcl-2-AS in leukemic cell
lines. Western blot analysis of HL-60 cells after 5 days of treatment
with 8 µmol/L Bcl-2-AS (Figure 3A)
demonstrated that the Bax protein was equally expressed in treated and
untreated cells, whereas expression of the Bcl-2 protein decreased in
cells treated with Bcl-2-AS but not in those treated with NS. By flow cytometry, Bcl-2 levels in HL-60 cells decreased slightly on day 3 of
incubation with 8 µmol/L Bcl-2-AS and decreased further on day 5 (Figure 4A). Bcl-2 levels were quantitated
using microbeads with QuickCal Software and were expressed as
antibody-binding capacity (ABC). In HL-60 cells, Bcl-2 expression
decreased from 49 × 103 on day 0 to
30 × 103 ABC per cell on day 3, and to
20 × 103 ABC per cell on day 5. In HL-60-DOX cells
treated with Bcl-2-AS, Bcl-2 decreased from
85.7 × 103 on day 0 to
43.4 × 103 ABC per cell on day 5 (Figure 4B). In
cells treated with NS, only an insignificant decrease was observed on
day 5 (HL-60, 46 × 103 ABC per cell; HL-60-DOX,
73 × 103 ABC per cell). Importantly, Bcl-2-AS
decreased viability and down-regulated Bcl-2 levels in
Bcl-XL--overexpressing HL-DOX cells. Interestingly, an increase in
Bcl-XL expression was observed (Figure 3B).
Bcl-2-AS enhances ara-C induced cytotoxicity.
We then evaluated the sensitivity of HL-60 and HL-60-DOX cells to the
combination of Bcl-2-AS with ara-C. HL-60 cells were more sensitive to
1 µmol/L ara-C (38.9% ± 4.2 viable cells, MTS assay) than
HL-60-DOX cells (51.7% ± 3.5 viable cells). As shown in Figure
5, the combination of ara-C with Bcl-2-AS
for 72 hours significantly enhanced killing of HL-60 sensitive and
resistant cells by Bcl-2-AS. Treatment with NS had no effect on
viability, whereas the combination of ara-C and Bcl-2-AS in HL-60 cells
significantly reduced the amount of Bcl-2-AS necessary to reach maximal
cytotoxicity from 12 to 4 µmol/L. In HL-60-DOX cells with higher
baseline levels of Bcl-2, Bcl-2-AS alone was not able to eliminate all
viable cells, regardless of the dose. In contrast, the combination
of ara-C and Bcl-2-AS exhibited maximal cytotoxicity at 16 µmol/L. NS did not enhance cytotoxicity of ara-C,
even at high levels.
Bcl-2-AS reduces viability of AML blasts in vitro.
Effect of Bcl-2-AS on the cell growth of leukemic blasts in vitro was
investigated in samples from patients with AML by cell count with
trypan blue-dye exclusion. The patients' characteristics are shown in
Table 1. In the majority of the patients, a
decrease in cell numbers was documented after 5 days of culture in the presence of 8 µmol/L Bcl-2-AS, compared with the number of cells in
culture after 5 days without oligodeoxynucleotides
(2.3 ± 0.3 × 105 cells/mL vs
3.6 ± 0.4 × 105 cells/mL;
P < .001). In contrast, very little toxicity was
observed in cultures with NS (3.1 ± 0.3 × 105
cells/mL).
Bcl-2-AS induces apoptosis in primary AML samples.
We examined the effect of Bcl-2-AS on the induction of apoptosis in
leukemic cells using flow cytometry to determine the apoptotic "sub
G1" cells by staining with acridine orange. Examples of
flow cytometric results are represented in Figure
6.
Bcl-2-AS increases proliferation of AML blasts.
To investigate the potential effect of Bcl-2-AS on proliferation of
leukemic cells, we performed cell cycle analysis of AML blasts treated
with different concentrations of Bcl-2-AS. In 5 of 6 cases, we observed
an increase in the percentage of S-phase cells after treatment with low
concentrations of Bcl-2-AS (4 samples: 4 µmol/L; 1 sample: 2 µmol/L) (Table 2). In 5 of 6 samples,
increases in cells in S-phase were observed. For the entire group, the
increase was significant (P = .04). No significant change in
the percentage of cells in G2M was observed. At higher
concentrations of Bcl-2-AS that induced apoptotic changes in leukemic
cells, no cell cycle effect was observed (data not shown).
Representative examples of DNA histograms are shown in Figure
9.
Bcl-2-AS inhibits colony-formation ability of AML blasts.
To determine the effect of Bcl-2-AS on myeloid progenitor cells, we
studied the colony-forming ability of leukemic blasts from 5 patients
with newly diagnosed AML in which the blast count was in excess of 80%
(before density gradient separation). Results demonstrated that in 4 of
the 5 patient samples, treatment with Bcl-2-AS significantly
(P = .01) inhibited the growth of AML colony-forming cells at
8 and 12 µmol/L (Figure 10). NS at the
highest concentration (12 µmol/L) and empty liposomes had no
statistically significant effect on the colony-forming capacity of
AML progenitors.
Reduction of Bcl-2 expression in acute myeloid leukemia progenitor
cells by Bcl-2-AS
Inhibition of Bcl-2 expression increases the cytotoxicity of ara-C
in sensitive AML cells.
We next examined the sensitivity of AML blasts treated with Bcl-2-AS to
the cytotoxic drug ara-C. AML blasts from 13 patients were
simultaneously treated with 1 µmol/L ara-C and 8 µmol/L Bcl-2-AS for 72 hours and analyzed for apoptosis by DNA content (Table 3). Ara-C in the presence of Bcl-2-AS
significantly increased apoptosis in all samples that were responsive
to treatment with Bcl-2-AS alone (P < .05). No significant
difference was seen in blasts cultured with ara-C alone or cultured
with ara-C and NS (P > .4). Interestingly, in these
samples, Bcl-2-AS induced the same degree of apoptosis as ara-C alone
(43.8% ± 8.1% vs 42.3% ± 5.8%, respectively). In
Bcl-2-AS nonresponsive blasts, Bcl-2-AS sensitized leukemic cells to
ara-C treatment in only 1 of 6 samples. Finally, the level of Bcl-2
expression did not change in responsive or nonresponsive blasts after
the addition of ara-C (Table 3).
Bcl-2-AS induces apoptosis in primary AML in the presence of other
antiapoptotic genes.
As shown in Table 4, several other
proapoptotic and antiapoptotic genes were found to be expressed in the
AML samples tested. Besides Bcl-2, the antiapoptotic genes
Bcl-XL, Bag1, A1 and
Mcl-1 were expressed as determined by Western blot analysis, at
various levels, in 5 of 6 samples studied (Figure
12). By RT-PCR, all AML samples (10 of
10) tested expressed these antiapoptotic proteins (data not shown).
This was also true when the samples sensitive to the induction of
apoptosis by Bcl-2-AS (group I) were analyzed separately: Antiapoptotic
genes were expressed in 6 of 6 samples by RT-PCR and in 3 of 4 samples
by Western blot analysis (Table 4).
In vitro response to Bcl-2-AS correlates with response to
chemotherapy in vivo.
We then compared the ability to induce apoptosis in AML cells cultured
in vitro with Bcl-2-AS with the response to induction chemotherapy in
patients. All patients were treated with high-dose ara-C in
combination with idarubicin or topotecan on University of Texas M. D. Anderson Cancer Center protocols. Complete remissions were
achieved in 7 of 9 patients whose blasts responded in vitro to
Bcl-2-AS, but only in 2 of 7 patients whose blasts did not respond to
Bcl-2-AS (P = .04). No correlation with percentage of blast
cells or cytogenetics was found.
The emergence of resistance to chemotherapeutic agents remains a
major problem in the treatment of AML, despite the fact that patients
usually have a good initial response to chemotherapy. The Bcl-2 protein
can block apoptosis by most chemotherapeutic agents,6,51
and has been found to be expressed at high levels in AML blasts capable
of autonomous growth in vitro.52 Our own data indicate that
virtually all AML samples express Bcl-2.53 This ubiquitous
expression of Bcl-2 in AML cells may be an important survival factor
for those cells. Several studies, including our own, have identified
Bcl-2 levels as prognostic in AML.10-14,53,54 The
prognostic impact depends on the cytogenetic abnormalities of the AMLs
studied.53 An earlier study by Keith et al29
demonstrated induction of apoptosis by Bcl-2-AS in vitro in fresh AML
samples as well as increased chemosensitivity to ara-C. However, they reported decreased levels of Bcl-2 expression after Bcl-2-AS in fewer
than 50% of the samples studied. This was thought to be related to the poor bioavailability of the
phosphorothioate oligonucleotides used.29 In
this study, we studied the induction of apoptosis in AML by
quantitating cellular Bcl-2 levels before and after Bcl-2-AS and by
also determining other antiapoptotic proteins. We also investigated
mechanisms of resistance of leukemic cells to Bcl-2-AS.
Submitted March 30, 1999; accepted February 11, 2000.
Supported by grants from NIH CA 55164, CA 49639, CA 16672, a
grant from Gabriella Rich Leukemia Fund to G.L.B. and the Stringer Professorship for Cancer Treatment and Research to M.A.
Reprints: Michael Andreeff, Section of Molecul |
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Abstract
Introduction
