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NEOPLASIA
From the H. Lee Moffitt Cancer Center and Research
Institute, University of South Florida, Tampa, FL; and Emory University
School of Medicine, Atlanta, GA.
The differentiation and apoptosis-sensitizing effects of the
Bcr-Abl-specific tyrosine kinase inhibitor CGP57148B, also known as
STI-571, were determined in human Bcr-Abl-positive HL-60/Bcr-Abl and
K562 cells. First, the results demonstrate that the ectopic expression
of the p185 Bcr-Abl fusion protein induced hemoglobin in the acute
myeloid leukemia (AML) HL-60 cells. Exposure to low-dose cytosine
arabinoside (Ara-C; 10 nmol/L) increased hemoglobin levels in
HL-60/Bcr-Abl and in the chronic myeloid leukemia (CML) blast crisis
K562 cells, which express the p210 Bcr-Abl protein. As compared with
HL-60/neo, HL-60/Bcr-Abl and K562 cells were resistant to apoptosis
induced by Ara-C, doxorubicin, or tumor necrosis factor- The dysregulated activity of the tyrosine kinase
(TK) encoded by the bcr-abl fusion gene is responsible for
the malignant phenotype of the Bcr-Abl-expressing chronic myeloid
leukemia (CML) and acute lymphoid leukemia (ALL) blasts.1
The fusion gene encodes for either the p210 or p185 TK implicated in
the pathogenesis of CML or ALL, respectively.2 Leukemic
blasts expressing Bcr-Abl display arrested differentiation, as well as
resistance to apoptosis, even when exposed to high doses of
antileukemic drugs.3-5 Recent studies from our laboratory
demonstrated that Bcr-Abl also exerts its antiapoptotic effect against
apoptosis triggered by sphingosine or C2-ceramide or Fas (CD95)
receptor-mediated death signaling.6 Ectopic or endogenous
expression of Bcr-Abl in HL-60/Bcr-Abl or K562 cells, respectively,
blocked the mitochondrial permeability transition ( In the present studies, we investigated the roles of potential
downstream effectors of Bcr-Abl TK and its pharmacologic inhibition by
CGP57148B in modulating drug-induced differentiation and apoptosis of
Bcr-Abl-positive leukemic blasts. Surprisingly, Bcr-Abl expression was
noted to induce hemoglobin (Hb) production in HL-60/Bcr-Abl cells. This
was further augmented by treatment with low-dose Ara-C (LODAC).
Inhibition of NF Reagents
Cells and transfection of the bcr-abl
gene
Generation of plasmids and I  B 2N, and CMVt-IB2N 4
plasmids were created as previously described.15,16 These
were kindly provided by Dr John Hiscott of McGill University, Montreal, Quebec, Canada. First, K562 cells were transfected with the
CMVt-rtTA plasmid DNA. Then puromycin-resistant clones were
transfected with CMVt-Neo, CMVt-2N, and
CMVt-2N4 plasmids by a previously described
method.5 Transformants were analyzed for inducible IB expression, and 3 clones from each transformant pool were selected for Western analyses and further studies.15,16
Stable transfectants of HL-60/Bcr-Abl cells containing IB2N4
were also created for Western analyses and further studies, as
previously described.5
NF B2N4, as
well as K562/rtTA, K562/ IB2N4, or K562/ IB2N4 were
transfected, using Lipofectamine, with the reporter plasmid for pNFB
(Clontech, Palo Alto, CA), which has 3 repeats of the NFB
site upstream of a minimal thymidine kinase promoter and the
luciferase gene. After exposure to 2.0 µg/mL doxycycline
alone or cotreatment with doxycycline and TNF-, cells were harvested
in phosphate-buffered saline (PBS) and lysed in a luciferase lysis
buffer. The lysates were assayed for luciferase using a
luminometer.17
Growth inhibitory effects of Ara-C Logarithmically growing HL-60/neo versus HL-60/Bcr-Abl cells were exposed to low concentrations (10 nmol/L) of Ara-C for 7 days. Following these treatments, aliquots of cells were withdrawn and the cell numbers were determined using a Coulter particle count and size analyzer (Coulter Inc, Hialeah, FL). Suspension culture growth inhibition by Ara-C was determined, as previously described.18Flow cytometric analysis of apoptosis The flow cytometric evaluation of apoptosis was performed according to a modification of a previously described method.19 Briefly, untreated or drug-treated cells were centrifuged, washed in Hanks' balanced saline solution, and fixed in 70% ethanol. The tubes containing the cell pellets were stored at 20°C for at least 24 hours. Following this, the cells were
centrifuged at 800g for 15 minutes and supernatant was
discarded to remove ethanol completely. The pellets were resuspended in
40 µL (for 2-3 × 106 cells) of phosphate-citrate
buffer at room temperature for 30 minutes. Following this incubation,
cells were washed with 4 to 5 mL PBS and stained with propidium iodide
(PI) solution (20 µg/mL PI and 20 µg/mL RNAse A in PBS) for 30 minutes. The samples were read on a Coulter Elite flow cytometer using
Elite software program 4.0 for 2-color detection. The percentage of
cells in the apoptotic sub-G1 phase was calculated using
Multicycle software (Phoenix Flow Systems, San Diego, CA).
Apoptosis assessment by annexin-V staining After drug treatment, 5 × 105 to 1 × 106 cells were washed in PBS and resuspended in 100 µL staining solution (containing annexin-V fluorescein and PI in a HEPES buffer, Annexin-V-FLUOS Staining Kit, Boehringer-Mannheim). Following incubation at room temperature for 15 minutes, cells were analyzed by flow cytometry. Annexin V binds to those cells that express phosphotidylserine on the outer layer of the cell membrane, and PI stains the cellular DNA of those that have a compromised cell membrane. This allows for the discrimination of live cells (unstained with either fluorochrome) from apoptotic cells (stained only with annexin V) and necrotic cells (stained with both annexin V and PI).20Western analyses Western analyses of Bcl-2, Bcl-xL, Bax, Fas receptor (CD95), FasL, Bcr-Abl, IB, caspase-8, tyrosine phosphorylated
proteins, and  -actin were performed using specific antisera or
monoclonal antibodies (see above), as described
previously.19,21 Horizontal scanning densitometry was
performed on Western blots by using acquisition into Adobe Photo Shop
(Apple, Inc, Cupertino, CA) and analysis by the NIH Image Program
(National Institutes of Health, Bethesda, MD). The expression of
-actin was used as a control.
Immunophenotyping for differentiation markers and Hb production The HL-60/neo, HL-60/Bcr-Abl, and K562 cells were treated with Ara-C for 7 days. Cells were then washed with PBS, and resuspended in 100 µL FACS wash buffer (PBS, 0.2% NaN3, 0.1% bovine serum albumin [BSA], 2.0% human AB+ serum, filtered by suction at 0.45 µm). Ten microliters phycoerythrin (PE) antihuman CD11b, CD33, or CD34 antibody (Pharmingen) was added,22,23 and the cells were incubated in the dark at 4°C for 30 minutes. The samples were then analyzed by flow cytometry. Alternatively, untreated or drug-treated cells were washed in PBS and intracellular Hb levels were determined by a spectrophotometric assay, as previously described.5Akt kinase assay In untreated and CGP57148B-treated cells, Akt kinase activity was determined by using an immunoprecipitation-kinase assay with reagents provided in a commercially available kit (New England Biolabs). Briefly, cell lysates were used to immunoprecipitate Akt utilizing a polycolonal Akt antibody. Immunoprecipitates were then incubated with GSK-3 fusion protein in the presence of adenosine triphosphate (ATP) and kinase buffer, allowing immunoprecipitated Akt to phosphorylate GSK-3, which was analyzed by Western blotting using a phospho-GSK-3/ (serine 21/9) antibody.24
Morphology of apoptotic cells After treatment with or without Ara-C, 50 × 103 cells were washed with PBS (pH 7.3) and resuspended in the same buffer. Cytospin preparations of the cell suspensions were fixed and stained with Wright stain. Cell morphology was determined by light microscopy. In all, 5 different fields were randomly selected for counting 500 cells. The percentage of apoptotic cells was calculated for each experiment, as described previously.25Statistical analysis Significant differences between values obtained in a population of leukemic cells treated with different experimental conditions were determined by paired t test analyses. A one-way ANOVA was also applied to the results of the various treatment groups, and post hoc analysis was performed using the Bonferroni adjustment method.
Bcr-Abl expression induced Hb but inhibited LODAC-induced myeloid differentiation Ectopic and stable expression of p185 Bcr-Abl in HL-60 and endogenous expression of p210 Bcr-Abl in K562 cells are associated with high Bcl-xL and barely detectable levels of Bcl-2. These effects of Bcr-Abl expression have been previously reported, although the precise mechanism(s) underlying these effects has not been elucidated.5,6 As previously reported, there was no significant difference in Bax expression in the control HL-60/neo versus HL-60/Bcr-Abl and K562 cells.6 Surprisingly, the ectopic expression of Bcr-Abl induced Hb production (Figure 1A), imparting a red color to the pellet of the centrifuged HL-60/Bcr-Abl cells. Hb was barely detectable in HL-60/neo cells and their pellet was colorless. As compared to HL-60/neo, there was no significant alteration in the expression of CD33 or CD34 in HL-60/Bcr-Abl cells (data not shown). Figure 1A also demonstrates that treatment with 10 nmol/L Ara-C (LODAC) for 7 days significantly increased Hb levels in both HL-60/Bcr-Abl and K562 cells (P < .05). This was associated with other morphologic features of erythroid differentiation such as the loss of cytoplasmic granularity and nuclear condensation in approximately 40% of HL-60/Bcr-Abl cells (data not shown). Exposure to LODAC for 7 days markedly increased the expression of the late myeloid differentiation marker CD11b in HL-60/neo (from 33.7% ± 3.8% to 62.7% ± 6.4%), but not in HL-60/Bcr-Abl cells (Figure 1B). This was associated with morphologic features of myeloid differentiation in approximately 60% of HL-60/neo but not in HL-60/Bcr-Abl and K562 cells (data not shown). The effect of the ectopic expression of Bcr-Abl on the differentiation response to phorbol esters was not investigated and remains to be determined. It is noteworthy that K562 cells did not express CD11b, and the ectopic expression of Bcr-Abl was associated with inhibition of CD11b expression in HL-60/Bcr-Abl cells (Figure 1B). Treatment with LODAC for 7 days produced 95% and 97.6% growth inhibition as well as 38% and 5% apoptosis in HL-60/neo and HL-60/Bcr-Abl cells, respectively (means of 2 experiments performed in duplicate).
Inhibition of constitutively high NF B activity in the IL-3-dependent murine
myeloid cells.7 K562 and HL-60/Bcr-Abl cells also had higher NFB activity than HL-60/neo cells, which was reduced by the
doxycycline-inducible or stable expression of the transdominant repressor of IB, that is IB2N or IB2N4,
respectively (data not shown). Clear evidence for the
doxycycline-inducible or stable expression of the transdominant
repressors of IB is provided in the immunoblot analysis presented
in Figure 3. In Figures
2 and 3, the inducible expression of
IB2N or IB2N4 in K562 cells is representative of 3 separate clones of stable transfectants. The effect of the repression
of NFB activity on LODAC-induced Hb generation was also determined
in K562 and HL-60/Bcr-Abl cells. Doxycycline-induced expression of
IB2N or IB2N4 significantly inhibited LODAC-induced Hb
levels in K562 cells (P < .05) (Figure 2A). Stable
expression of IB2N4 had a similar effect in HL-60/Bcr-Abl cells (Figure 2B). We next examined the effects of IB2N or
IB2N4 expression on TNF--, Ara-C-, or doxorubicin-induced
apoptosis of K562 and HL-60/Bcr-Abl cells. Table
1 shows that treatment of the control
HL-60/Bcr-Abl and K562 cells (K562/rtTA) with TNF- (100 ng/mL for 24 hours) only slightly increased the percentage of apoptotic cells.
Repression of NFB activity by inducible (in K562) or stable
expression of IB2N4 (in HL-60/Bcr-Abl) by itself did not alter
the percentage of apoptotic cells. However, it significantly increased
TNF--induced apoptosis of HL-60/Bcr-Abl and K562 cells (P < .05) (Table 1), although the increment was modest in
K562 cells (Table 1). In contrast, the expression of IB2N4 did not sensitize K562 or HL-60/Bcr-Abl cells to Ara-C (5-100 µmol/L), etoposide (50 µmol/L), or doxorubicin-induced (0.25-1.0 µmol/L) apoptosis (data not shown). These results suggest that in HL-60/Bcr-Abl and K562 cells NFB activity contributes to the resistance to apoptosis due to TNF- but not due to Ara-C, etoposide, or
doxorubicin. Immunoblot analyses shown in Figure 3 clearly demonstrate
that exposure to 2.0 µg/mL doxycycline induced the levels of either IB2N, which has the same molecular weight as IB (40 kd), or the levels of the smaller p37 IB2N4 in K562/IB2N or
K562/IB2N4 cells, respectively. Inducible (K562) or stable
expression of IB2N4 (HL-60/Bcr-Abl) reduced the endogenous
IB levels in K562 and HL-60/Bcr-Abl cells (Figure 3A), since
NFB is known to transactivate its own repressor
IB.16 Expression of IB2N or IB2N4 did
not alter the levels of Bcr-Abl, Abl, Fas, FasL, Bcl-xL,
and Bax in K562 or HL-60/Bcr-Abl cells (Figure 3). Contrary to the
reported findings from other cell types,9 intracellular cIAP1 levels were also not affected by inhibition of NFB activity (data not shown).
Bcr-Abl TK inhibitor CGP57148B induces Hb and apoptosis of K562 and HL-60/Bcr-Abl cells Recent reports had indicated that the ATP binding-site antagonist, Bcr-Abl-specific TK inhibitor CGP57148B or STI-571 can suppress growth and induce apoptosis of Bcr-Abl-positive leukemic cells.11,12,26 In the present studies, we determined whether CGP57148B would induce differentiation and apoptosis, as well as sensitize HL-60/Bcr-Abl and K562 cells to high-dose Ara-C (HIDAC)- and doxorubicin-induced apoptosis. First, we examined the effect of 0.25 µmol/L CGP57148B, a dose previously shown to be the IC50 value for the autophosphorylation of vAbl or Bcr-Abl,26 on Bcr-Abl, Abl, Bcl-xL, Bcl-2, Bax, and cellular protein tyrosine phosphorylation levels in HL-60/Bcr-Abl and K562 cells. Twenty-four to 72 hours of exposure to 0.25 µmol/L (or 0.5 µmol/L, not shown) CGP57148B did not alter the intracellular levels of Bcr-Abl, Abl, Bcl-2, and Bax (Figure 4A,D,E). CGP57148B treatment also did not affect the intracellular levels of Apaf-1 (not shown). In contrast, exposure to CGP57148B for 48 to 72 hours produced approximately a 5-fold decline in Bcl-xL levels (Figure 4C) and inhibited the tyrosine phosphorylation of cellular proteins (Figure 4B). Leukemic transformation mediated by Bcr-Abl is known to involve an increase in Akt kinase activity that inhibits apoptosis.27 Therefore, we examined Akt kinase activity in untreated and CGP57148B-treated (0.5 µmol/L for 48 hours) HL-60/neo, HL-60/Bcr-Abl, and K562 cells. As shown in Figure 5, the latter 2 cell types demonstrated higher activity of Akt kinase than HL-60/neo cells. CGP57148B inhibited Akt kinase activity in HL-60/Bcr-Abl and K562 but not in HL-60/neo cells (Figure 5). Although not shown, CGP57148B did not affect Akt levels in any of the cell types. CGP57148B treatment also significantly lowered XIAP levels (Figure 5). As shown, exposure to CGP57148B also lowered cIAP1 levels in HL-60/Bcr-Abl cells. Collectively, XIAP and cIAP1 are known to inhibit the activity of caspase 3, 7, 8, and 9.28,29 Although a shorter exposure (24 hours) produced only modest effects, exposure to higher doses of CGP57148B did not augment these effects of CGP57148B (data not shown). Although the mechanism underlying this remains to be firmly established, treatment with CGP57148B (0.25 µmol/L) also modestly inhibited NFB activity by a mean of 20% (2 experiments) in
HL-60/Bcr-Abl and K562 cells (data not shown). There was no effect of
CGP57148B on Akt kinase (Figure 5) or NFB activity (data not shown)
in HL-60/neo cells.
Figure 6 demonstrates that treatment with
0.25 µmol/L CGP57148B for 7 days also induced Hb levels in
HL-60/Bcr-Abl and K562 cells. This increase in Hb was more than that
observed following exposure to LODAC (P < .05). A
combined treatment with CGP57148B and LODAC for 7 days did not increase
Hb levels over those induced by treatment with CGP57148B alone
(P > .05) (Figure 6). CGP57148B (0.25 µmol/L for 7 days) also increased the percentage of cells expressing CD11b from
1.0% to 27.4% in HL-60/Bcr-Abl and from 0.4% to 37.5% in K562 cells
(mean of 3 experiments; Figure 6). Exposure to LODAC alone for 7 days
only modestly increased CD11b expression in K562 cells. In addition,
cotreatment with LODAC did not augment the expression of CD11b induced
by treatment with CGP57148B alone (Figure 6).
Data in Table 2 describe the apoptotic
effects of CGP57148B, Ara-C, and doxorubicin in HL-60/Bcr-Abl and K562
cells. Following an exposure to 0.25 µmol/L CGP57148B for 48 hours
approximately 15% of HL-60/Bcr-Abl and 18% of K562 cells were
apoptotic as determined by annexin V staining and flow cytometry.
CGP57148B had no effect on HL-60/neo cells (data not shown). In Table
2, the differences in apoptotic rates detected by the various methods
may be attributable to a disparate feature detected by each method to
characterize apoptosis. Cotreatment with CGP57148B (0.25 µmol/L) also
significantly increased the percentage of apoptotic cells following
exposure to Ara-C (5.0 µmol/L) or doxorubicin (0.25 µmol/L) for 48 hours (P < .05; Table 2). This was not observed in
HL-60/neo cells (data not shown), which are known to be highly
sensitive to apoptosis induced by Ara-C and doxorubicin. Cotreatment
with CGP57148B also significantly increased apoptosis of HL-60/Bcr-Abl
cells induced by etoposide (5.0 µmol/L) (etoposide: 8.0% ± 0.9%
versus CGP57148B plus etoposide: 24.1% ± 1.5%) or HIDAC (100.0 µmol/L) for 4 hours, where Ara-C was added in the final 4 hours of
exposure to CGP57148B, versus Ara-C alone (Ara-C: 6.5 ± 1.0% versus
CGP7148B plus Ara-C: 28.4% ± 2.2%) (P < .01).
Furthermore, we determined the effect of cotreatment with CGP57148B
plus Ara-C or doxorubicin on the processing of caspase-8 and cytosolic
Bid, as well as the accumulation of cytosolic cyt c and the generation
of PARP cleavage activity of caspase-3.10 Figure
7 demonstrates that treatment with Ara-C alone did not result in the processing of procaspase-8 and Bid or cause
cytosolic accumulation of cyt c and caspase-dependent cleavage of PARP.
Doxorubicin treatment produced modest accumulation of cyt c in the
cytosol associated with partial cleavage of p116 PARP into its p85
fragment (Figure 7). Treatment with CGP57148B alone clearly induced the
processing of procaspase-8 and Bid and was associated with an increase
in cytosolic cyt c (Figure 7). Accumulation of cyt c in the cytosol
triggers Apaf-1-mediated cleavage and activity of caspase-9, followed
by activation of caspase-3.13,30 Caspase-3 results in the
cleavage of PARP and expression of phosphatidyl serine on the cell
membrane, detected by increased staining by annexin V,13
as shown in Figure 7 and Table 2, respectively. Cotreatment with
CGP57148B and Ara-C (5.0 µmol/L) or doxorubicin (0.25 µmol/L) for
48 hours produced procaspase-8 and Bid processing, resulting in an
increase in the cytosolic cyt c. The results of the immunoblot
analyses presented in Figure 7 are representative of 3 separate
experiments. Collectively, these data indicate that CGP57148B not only
induces phenotypic markers of differentiation and apoptosis but also
sensitizes Bcr-Abl-positive cells to antileukemic drugs such as Ara-C,
etoposide, and doxorubicin.
Previous reports had indicated that Bcr-Abl-mediated
transformation and resistance of mouse myeloid cells to apoptosis is associated with constitutively increased activities of Akt kinase and
NF Bcr-Abl enhances NF Studies have shown that in K562 cells LODAC treatment induces
Hb.5 In contrast, in HL-60 cells, LODAC treatment
increases the expression of the myeloid differentiation marker
CD11b.5,22 Surprisingly, enforced expression of Bcr-Abl
induced Hb in HL-60/Bcr-Abl cells, imparting a red color to their cell
pellet (Figure 1). A recent report has indicated that Bcr-Abl TK can
fully support erythroid development and maturation even in those
progenitor cells that lack erythropoietin receptors.41
Bcr-Abl expression also sensitized HL-60/Bcr-Abl cells to erythroid but
not myeloid differentiation, as evidenced by LODAC-induced Hb but not
CD11b or the morphologic features of myeloid differentiation (data not shown). In contrast, the ectopic expression of Bcr-Abl also inhibited LODAC-induced apoptosis of HL-60 cells. Inhibition of NF Conventional chemotherapy with Ara-C, doxorubicin, and etoposide does
not have major clinical efficacy against Bcr-Abl-positive acute
leukemia or the blast crisis of CML.2,42 This may be because, as compared with HL-60/neo, HL-60/Bcr-Abl and K562 cells are
relatively resistant to antileukemic drug-induced mitochondrial In summary, data presented here indicate that specific inhibition of
Bcr-Abl TK by CGP57148B results in the down-regulation of
Bcl-xL, XIAP, and cIAP1 levels as well as inhibition of Akt kinase and NF
Submitted July 8, 1999; accepted May 17, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Kapil Bhalla, H. Lee Moffitt Cancer Center, 12902 Magnolia Drive, MRC3E, Room 3056D, Tampa, FL 33612; e-mail: bhallakn{at}moffitt.usf.edu.
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Top
Abstract
Introduction
m) and release
of cytochrome c (cyt c), thereby inhibiting the activation of caspase-3
and apoptosis.
mediated resistance to apoptosis had not been established.
Recent studies have shown that the inhibition of Bcr-Abl activity by a
relatively specific Bcr-Abl TK inhibitor CGP57148B causes in vitro and
in vivo eradication of Bcr-Abl-positive human leukemia
cells.
