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NEOPLASIA
From the Divisione di Ematologia, Ospedali Riuniti di
Bergamo, Bergamo, Italy; and Laboratorio di Biologia Molecolare, Centro
Catullo e Daniela Borgomainerio, Istituto di Ricerche Farmacologiche
"Mario Negri," Milano, Italy.
The 2-phenylaminopyrimidine derivative STI571 is a selective
inhibitor of c-Abl, c-kit, and platelet-derived growth factor-receptor tyrosine kinases and is presently in phase II-III clinical studies. Here, this study reports on a novel pharmacologic activity of the
compound, ie, enhancement of the cyto-differentiating,
growth-inhibitory, and apoptogenic actions of
all-trans-retinoic acid (ATRA). Whereas STI571 is not a
cytodifferentiating agent by itself, the compound interacts with ATRA
and enhances the myeloid maturation program set in motion by the
retinoid in the PML-RAR All-trans-retinoic acid (ATRA)
represents the sole example of clinically useful cytodifferentiating
agent.1 Treatment of patients with acute promyelocytic
leukemia (APL) with ATRA alone or in combination with chemotherapy
results in high rates of complete clinical remission.1,2
However, the therapeutic use of this compound is limited by a number of
problems, which include serious systemic toxicity3 and
induced ATRA resistance.4,5 In addition, the use of ATRA
is limited to the APL subset of acute myelogenous leukemia, as all the
other French-American-British subtypes are generally refractory to the
cytodifferentiating action of the retinoid. One way to circumvent some
or all of the aforementioned problems is to identify agents capable of
enhancing the pharmacologic activity of ATRA. In previous reports, we
and others demonstrated that agents such as granulocyte
colony-stimulating factor, cell-permeable analogs of cyclic adenosine
3',5'-monophosphate (cAMP), and interferons enhance the
cytodifferentiating action that ATRA exerts on APL and other myeloid
cell lines.6-11
The 2-phenylaminopyrimidine derivative STI571 is a tyrosine kinase
inhibitor, acting on a restricted number of target
proteins,12,13 ie, c-Abl and its pathologic derivative
BCR-Abl,12,14,15 c-kit16,17, and the
platelet-derived growth factor (PDGF) receptor.17,18 In
vitro, the compound selectively inhibits the growth and
induces apoptosis of chronic myelogenous leukemia (CML) blasts
synthesizing p210 BCR-Abl19,20 and lymphoblastic leukemia
cells expressing p185 BCR-Abl.21 In vivo, the tyrosine
kinase inhibitor causes regression of human CML xenografts in nude
mice.22 The compound is currently undergoing phase II-III
clinical trials for the treatment of patients with CML with very
promising results.23
In this study, we report on an unexpected pharmacologic effect of
STI571 in acute myelogenous leukemia cells, which do not express the
p210 or p185 BCR-Abl oncogenic protein. We observe that STI571 enhances
the cyto-differentiating, antiproliferative, and apoptogenic activity
of ATRA. In addition, in certain APL cell lines made resistant to the
retinoid, the tyrosine kinase inhibitor is capable of relieving
resistance. These effects are associated with a decrease in
ATRA-induced degradation of RAR Chemicals
Cell culture and treatment
Cellular proliferation, apoptosis, and cytodifferentiation The total number of cells and cell viability were determined by manual counting in Burker chambers following staining with erythrosin (Sigma). To prevent apoptosis, control cultures were kept in the logarithmic phase of growth (3-6 × 105 cells/mL) by dilution in fresh medium every 2 days. Cultures treated in parallel were similarly diluted in drug-containing medium to keep the cell density between 3-6 × 105 cells/mL. For the determination of the apoptotic index (percentage of cells with fragmented nuclei), cells were stained with 4'-6-diamidine-2-phenylindole (DAPI), as described.33 The surface markers CD11b, CD11c, and CD33 were measured by flow cytometry as previously described.34 The nitrobluetetrazolium (NBT) reduction assay was performed on extracts of PMA-stimulated cells as detailed in a previous study.35 In some experiments, we determined the percentage of NBT positivity by counting (3 microscopic fields/experimental point, in double blind) the number of cells reducing NBT to formazans (blue cells) and that of cells devoid of NBT-reducing activity (white cells). We expressed the data as the percentage of the following ratio: blue cells/blue + white cells.Western blot analysis immunoprecipitation and determination of the proteasome activity Total extracts from NB4, HL60, or U937 cells (approximately 3 × 106 cells) were subjected to Western blot analysis as reported.33 The experiments were performed with antibodies against the F-domain of RAR (RPF'36),
RXR, 4RX1F6,37 CEBP (C-22; Santacruz Biotechnology,
Santa Cruz, CA), STAT1 p91 (C-24; Santacruz), c-kit (C-14; Santacruz),
c-Abl (24-11; Santacruz), phosphotyrosine (PY20; Signal Transduction
Laboratories, Lexington, KY), or  -actin (C-11; Santacruz). Bands
were visualized with the enhanced chemiluminescence detection kit
(Amersham, Little Chalfont, United Kingdom). Immunoprecipitations with
antibodies recognizing c-kit (C-19; Santacruz) or c-Abl (K-12; Santacruz) were performed as described by the manufacturer, using 2 × 107 cells. The in vitro activity of the proteasome
was determined according to the fluorimetric method of Grune et
al38 on cell lysates (2.5 × 106 cells).
RNA extraction, Northern blot analysis, and microarray screening Northern blot analysis was performed on total RNA,6 using complementary DNA (cDNA) probes for cEBP, IRF-1, STAT1, CD11b, p22/phox, p47/phox, p67/phox, gp91/phox, rac1, rac2, and
-actin.35 Except for -actin, all the cDNA probes
were obtained by reverse transcriptase-polymerase chain reaction
(RT-PCR) with the use of specific amplimers: CD11b (sense primer,
nucleotide 2471-2490; antisense primer, nucleotide
3271-329039); cEBP (sense primer, nucleotide 196-215;
antisense primer, nucleotide 940-970); p22/phox (sense primer,
nucleotide 131-150; antisense primer, nucleotide 651-67040); p47/phox (sense primer, nucleotide 191-210;
antisense primer, nucleotide 931-95041); p67/phox (sense
primer, nucleotide 821-840; antisense primer, nucleotide
1631-165042); gp91/phox (sense primer, nucleotide 911-930;
antisense primer, nucleotide 1641-166043); rac1 (sense
primer, nucleotide 141-160; antisense primer, nucleotide
911-93044); rac2 (sense primer, nucleotide 11-30; antisense
primer, nucleotide 491-51045). For the microarray experiment, we used the ATLAS Human Cytokine/Receptor Array (cat. no.
7744-1; Clontech, Palo Alto, CA). Imaging and quantitation of Northern
blots were performed with the STORM 460 phosphoimager (Amersham-Pharmacia Instrumentation, Little Chalfont, United Kingdom).
STI571 enhances the growth-inhibitory, apoptogenic, and cyto-differentiating effects of ATRA on APL cells As shown in Figure 1A, treatment of NB4 cells with STI571 (5 × 10 6 M) slows down the
logarithmic growth of NB4 cells. The effect is of the same order of
magnitude as that observed following treatment with a suboptimal
concentration of ATRA (107 M). When cells are
simultaneously treated with STI571 and ATRA, a more than additive
effect is observed. As illustrated in Figure 1B, from the fourth day
on, the STI571 + ATRA combination causes a significant decrease in
cell viability relative to what is observed in cultures treated with
vehicle alone, ATRA, or STI571. The loss of viability is accompanied by
a significant increase in the number of apoptotic cells (Figure 1C).
More importantly, STI571 accelerates the process of NB4 blasts
differentiation, as assessed by determining the percentage of cells
expressing NBT-reducing activity, a phenotypic marker of granulocytic
maturation (Figure 1D). This is associated with a stronger induction of
the marker enzyme in STI571 + ATRA than in
ATRA-differentiated cells, as indicated by the data obtained following
measurement of NBT-reducing activity on whole cell extracts (Figure
1E). With reference to this last point, when the majority of cells is
NBT+ following treatment with ATRA + STI571 and ATRA,
cell-associated NBT-reducing activity is significantly higher in the
former than in the latter experimental group (compare the results at 4 and 5 days in Figure 1D,E).
As documented by Figure 2A, when STI571
is incubated for 4 days in the presence of ATRA (10
Interaction between STI571 and ATRA is not limited to NB4 blasts but is also observed in retinoid-responsive HL-60 and U937 myeloblastic cells as well as in retinoid-resistant NB4 clones HL-60 and U937 are PML-RAR myeloblastic cell
lines that respond to the cyto-differentiating action of
ATRA.46,47 As illustrated by Figure
3A, following 4 days of treatment, STI571
has an inducing effect on NBT-reducing activity similar to that of ATRA
in both HL-60 and U937 blasts. When both cell lines are incubated with STI571 and ATRA simultaneously, NBT-reducing activity is significantly increased relative to treatment with the tyrosine kinase inhibitor or
the retinoid alone. As expected, NB4.306 and NB4.007 cells are
refractory to the cyto-differentiating action of ATRA. When NB4.306
blasts are treated with STI571 alone, a slight but reproducible increase in NBT-reducing activity is observed, whereas this
differentiation marker is left substantially unaffected by the tyrosine
kinase inhibitor in NB4.007 cells. In both cell lines, the STI571 + ATRA combination has a stronger inducing effect than STI571 or ATRA alone. However, the most interesting results are observed in
ATRA-resistant NB4.R1 blasts (Figure 3B). In this cell line,
coadministration of STI571 and ATRA results in a significant induction
of NBT-reducing activity relative to what is observed following
treatment with vehicle, STI571, or ATRA alone. The cyto-differentiating
effect of combinations between STI571 and ATRA is further confirmed by increased expression of the 2 membrane markers CD11b and especially CD11c. These phenomena are accompanied by cell growth inhibition, which
is largely the result of the STI571 activity, although the effect is
slightly augmented by addition of ATRA to the growth medium.
STI571 interacts with RAR and PML-RAR agonist with strong
cyto-differentiating activity in APL cells.24 Figure
4A demonstrates that peak induction of
NBT-reducing activity in NB4 cells is already observed following 4 days
of treatment with AM580 at concentrations as low as 108
M. STI571 enhances AM580-dependent stimulation of the marker enzyme,
indicating that activation of RAR and/or PML-RAR is necessary and
sufficient for the STI571-dependent enhancement of the granulocytic
maturation program triggered by retinoids.
Combinations of cell-permeable analogs of cAMP and RXR agonists have been recently shown to cause granulocytic maturation of NB4 cells through an intracellular pathway that is entirely different from that activated by ATRA and RAR agonists.48 As shown in Figure 4B, we confirm this with the use of the cell-permeable analog of cAMP, 8-CPT-cAMP, and CD2915, a powerful and selective RXR ligand.25 The level of NBT-reducing activity observed following treatment with cAMP + CD2915 is higher than that observed in NB4 cells treated with cAMP + ATRA or STI571 + ATRA. Thus, we determined whether STI571 could substitute for or complement cAMP and activate the RXR-dependent pathway leading to granulocytic maturation of NB4 cells. Combinations between STI571 and CD2915 are as ineffective as the single agents in inducing cyto-differentiation in NB4 cells. In addition, the tyrosine kinase inhibitor has no significant effect on the induction of NBT-reducing activity triggered by cAMP + ATRA. These data indicate that RXRs do not mediate the interaction between STI571 and retinoids. Other structurally unrelated c-Abl inhibitors, such as AG957, NSC68410, and NSC676448,26-28 have no significant effects on the induction of NBT-reducing activity caused by ATRA (Figure 4C), although, in the presence or absence of the retinoid, they inhibit cell growth to variable extents (Figure 4D). Thus, STI571 is unique among c-Abl tyrosine kinase inhibitors in enhancing the cyto-differentiating effect of ATRA. This is likely to be the result of the fact that AG957, NSC68410, and NSC676448 are cytotoxic to NB4 cells and can be used only at concentrations below the 50% inhibitory concentration, presumably affording only limited intracellular inhibition of the target tyrosine kinase(s). STI571 enhances the expression of other ATRA-induced granulocytic differentiation markers acting at the messenger RNA level With the use of optimal concentrations of STI571 (5 × 106 M) and variable amounts of ATRA, we
determined the effects of these combinations on markers of myeloid
differentiation other than NBT staining (Figure
5). In control conditions, approximately 35% of NB4 cells are CD11b+ and less than 10% are
CD11c+, whereas the whole cell population is positive for
the early myeloid marker CD33. As expected,34 ATRA
increases the number of CD11b+ and CD11c+ cells
in a dose-dependent manner, whereas it does not alter the proportion of
CD33-synthesizing cells to a significant extent. ATRA-dependent
increases in the number of CD11b+ and CD11c+
cells are accompanied by a corresponding elevation in the amount of
cell-associated fluorescence. When given in combination with the
retinoid, STI571 augments the ATRA-induced surface expression of both
CD11b and CD11c, leaving unaffected CD33. Notice that the enhancing
effect on CD11c is limited to an increase in cell-associated fluorescence. Taken together, these results indicate that the tyrosine
kinase inhibitor has positive effects on various phenotypic markers of
the granulocytic differentiation program set in motion by ATRA in
NB4 cells.
To gain insight into the molecular mechanisms underlying the
interaction between STI571 and ATRA, we determined the steady-state levels of the transcripts coding for CD11b and the multiple components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase responsible for NBT-reducing activity. Following treatment of NB4 cells
with medium alone or STI571 for 4 days, very low levels of CD11b
messenger RNA (mRNA) are evident. ATRA up-regulates the transcript, and
this effect is enhanced a further 3-fold if STI571 is added to the
retinoid (Figure 6A). This is similar to
what is observed in the case of the mRNAs encoding p47/phox and
gp91/phox, 2 of the NADPH oxidase components. By contrast, the tyrosine
kinase inhibitor has no significant effects on the induction of rac1 and p22/phox mRNAs by ATRA. As to the remaining constituents of the
NADPH oxidase enzymatic complex, the basal levels of expression of
p67/phox and rac2 mRNAs are left unaltered by all the treatments considered.
To investigate whether enhanced cyto-differentiation by STI571 + ATRA is the result of a modulation in the expression of genes coding
for transcription factors that are involved in the process of myeloid
maturation, we measured the levels of the mRNAs for cEBP STI571 alone or in combination with ATRA alters the expression profile of many other genes With the use of a cDNA microarray containing clones corresponding to cytokine and cytokine receptors, we studied the differential expression of almost 400 genes in NB4 cells. As indicated in Table 1, on treatment with STI571, ATRA, or STI571 + ATRA for 2 days, approximately 6% of the cDNAs represented in the array show reproducible alterations in their basal levels of expression. These cDNAs can be divided into 4 groups according to their expression profile. The first group contains genes (the myeloid chemokine monocyte chemotactic protein 1 precursor [MCP-1], migration-inhibitory factor-related protein 8 [MRP-8], MRP-14, and corticotropin-releasing factor receptor type 1), which are expressed at low levels in control conditions, are induced by ATRA and further augmented by the combination of ATRA + STI571. The second one consists of genes, such as those for the transcription factor interferon regulatory factor 1 (IRF-1) and the ligand/receptor couple brain-derived neurotrophic factor/neuromodulin, whose expression is induced by ATRA but not further enhanced by ATRA + STI571. The genes for thymosine3, the neurotrophic growth factor receptor, and
heparin-binding neurite outgrowth-promoting factor 1, whose expression
is inhibited by STI571 regardless of the presence or absence of ATRA,
are gathered in a third group. A fourth subset combines genes,
including predominantly those coding for cytokines (such as granulocyte
macrophage colony-stimulating factor, interferon  , IL-4, and IL-13),
which are up-regulated by STI571 and whose induction is inhibited by
the addition of ATRA. At present, tumor necrosis factor (TNF) is
the only example of a gene that is down-regulated by ATRA and further
inhibited in its expression by STI571 + ATRA. The profile of
expression of a number of genes modulated by STI571 was confirmed by
RT-PCR and Northern blot experiments (data not shown except for IRF-1 in Figure 6B) or by immunoassay, as in the case of the MCP-1 protein (data not shown).
STI571 delays the ATRA-dependent degradation of RAR and RAR,
and this phenomenon has been related to the ability of the retinoid to
induce granulocytic differentiation of APL cells.52-55 As
shown in Figure 7A, treatment of NB4
cells with ATRA for 12 and 24 hours causes the disappearance of the
bands corresponding to PML-RAR and a remarkable decrease in the
levels of those corresponding to RAR. These effects are the result
of specific degradation of the 2 proteins, as the amounts of the
relative mRNAs are left unaltered by the tyrosine kinase inhibitor or
the retinoid (data not shown). Whereas STI571 alone does not modify the
quantities of RAR or PML-RAR observed in control conditions,
combinations between the tyrosine kinase inhibitor and ATRA lead to a
decrease in the degradation of the 2 receptors that is evident at least until 24 hours of treatment. As shown in Figure 7B, inhibition of
RAR and PML-RAR degradation is observed at the same
concentrations of STI571 that induce growth inhibition and granulocytic
maturation. The protective effect on ATRA-dependent RAR proteolysis
afforded by STI571 is not limited to NB4 but is also observed in HL-60 and U937 cells (Figure 7C). Because the retinoid-dependent catabolism of nuclear retinoic acid receptors is largely, although not
exclusively, mediated by the proteasome complex,55,56 we
evaluated the effect of STI571 and ATRA on the proteolytic activity of
this subcellular organelle, using a fluorogenic peptide substrate. As
shown in Figure 7D, treatment of NB4 cells with the combination
STI571 + ATRA results in a significant inhibition of the
proteolytic activity of the proteasome. The effect is already observed
at 24 hours, a time at which neither STI571 nor ATRA affects proteasome activity. This interaction is progressively lost at 36 and 48 hours,
since ATRA alone starts to exert an inhibitory action that is not
further modified by the tyrosine kinase inhibitor. The proteolytic
activity measured in our experimental conditions is largely due to the
activity of the proteasome as it is more than 80% inhibited by the
specific proteasome inhibitor
z-Ile-Glu(Obut)-Ala-Leu-H(aldehyde) (data
not shown).
STI571 inhibits c-Abl but not c-kit tyrosine phosphorylation As shown in Figure 8A, in basal conditions, NB4 cells express significant amounts of the STI571 targets, c-Abl and c-kit. Concentrations of the tyrosine kinase inhibitor (5 × 106 M) capable of enhancing the
maturation effect of ATRA do not affect c-Abl or c-kit protein levels
significantly. Similarly, either alone or in combination with STI571,
ATRA does not alter the levels of the 2 proteins. As shown in Figure
8B, both c-Abl and c-kit are constitutively tyrosine-phosphorylated, as
indicated by Western blot experiments conducted with
antiphosphotyrosine antibodies on immunoprecipitates of the 2 proteins.
Following 18 hours of continuous treatment, STI571 decreases
tyrosine-phosphorylation of c-Abl (54% in the experiment shown),
whereas it has no significant effect on c-kit. ATRA is ineffective in
modulating the level of c-Abl (50% inhibition) or c-kit
tyrosine-phosphorylation observed in the absence or presence
of STI571.
In this article, we demonstrate that STI571 enhances and
accelerates the cyto-differentiating activity of ATRA in
retinoid-sensitive cell lines derived from APL and other types of
myeloblastic leukemia. In addition, the compound relieves ATRA
resistance in NB4 sublines selected for their insensitivity to the
retinoid. The interaction between STI571 and ATRA is mediated by
PML-RAR Stimulation of granulocytic maturation by STI571 + ATRA is
observed at the level of many myeloid markers, whose expression is
up-regulated by the retinoid alone. These include CD11b, CD11c, MRP-8,
MRP1-4, MCP-1, and some of the components of the enzymatic complex,
NADPH oxidase, which is responsible for NBT-reducing activity.
Elevation of these markers is primarily the result of transcriptional
or post-transcriptional events, as the phenomenon is associated with an
increase of the steady-state levels of the corresponding transcripts.
Treatment of NB4 cells with STI571, ATRA, or the combination of the 2 compounds has also significant effects on the expression profile of a
substantial number of genes coding for cytokine and cytokine receptors.
Among these, down-regulation of the TNF In APL cells, the process of granulocytic maturation triggered by ATRA is likely to depend on the activation of a complex network of genes controlled directly or indirectly by retinoic acid receptors and other transcription factors. Thus, it is conceivable that STI571 enhances the pharmacologic action of ATRA by modulating the levels or the state of activation of this type of nuclear proteins. As to retinoic acid receptors, the APL blast expresses
significant amounts of PML-RAR In retinoid-sensitive APL cells, ATRA and synthetic retinoids degrade the nuclear retinoic acid receptors through activation of a caspase- and/or a proteasome-dependent pathway.52-55 Within the first 4 days of culture, caspase activity is very low in NB4 cells and is left unaffected by treatment with ATRA,25 STI571, or the combination of the 2 compounds (M.G. and E.G., unpublished results, January 2000). This suggests that inhibition of caspases is not involved in the stabilization of nuclear retinoic acid receptors observed following treatment with STI571 + ATRA. By contrast, when given in association with ATRA, STI571 has a significant inhibitory effect on the activity of the proteasome complex. The phenomenon is likely to explain the stabilization effect on nuclear retinoic acid receptors and is of a complex nature, since it requires the contemporaneous presence of the tyrosine kinase inhibitor and the retinoid. As to the transcription factors other than retinoic acid receptors,
potentially involved in ATRA-induced myeloid maturation of the APL
blast, we took into consideration STAT1, cEBP A key question in the observed interaction between the tyrosine kinase
inhibitor and ATRA is whether enhanced cyto-differentiation, growth
inhibition, apoptosis, as well as retarded PML-RAR In conclusion the observed interaction between STI571 and ATRA could be
exploited at the clinical level in the first- or second-line treatment
of APL and perhaps other types of PML-RAR
We are thankful to Prof Silvio Garattini and Dr Mario Salmona for critical reading of the manuscript. We would like to thank Dr Julia Szeberenyi and Mr Massimiliano Marini for skillful experimental work performed. We would also like to thank Prof P. Chambon and Dr Cecile Rochette-Egly (IGBMC, Strasbourg, France) for the kind gift of anti-RAR and anti-RXR antibodies.
Submitted August 29, 2000; accepted January 22, 2001.
Supported by grants to E.G. from the Associazione Italiana per la Ricerca contro il Cancro (AIRC), from the "Istituto Superiore di Sanità," and from the Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST). I.P. is the recipient of a fellowship from "La Via di Natale."
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: Enrico Garattini, Laboratorio di Biologia Molecolare, Centro Catullo e Daniela Borgomainerio, Istituto di Ricerche Farmacologiche "Mario Negri," via Eritrea 62, 20157 Milano, Italy; e-mail: egarattini{at}irfmn.mnegri.it.
1. Warrell RP Jr, Frankel SR, Miller WH Jr, et al. Differentiation therapy of acute promyeloc |
and PML-RAR
Top
Abstract
Introduction
) or medium containing ATRA (10
), STI571 (5 × 10
), and STI571 + ATRA (
). Aliquots of the cultures were withdrawn at each time point
and subjected to counting, following erythrosin staining for the
determination of the total number of viable cells (A), the percentage
of viable cells (B), the degree of apoptosis as assessed by DAPI
staining (C), and the level of granulocytic maturation, as assessed by
measuring the percentage of NBT-positive cells (D) or cell-associated
NBT-reducing activity (E). Results are the mean ± SD of 3 separate culture dishes and are representative of at least 3 independent experiments. *, significantly higher or lower than the
untreated control (P < .01 according to the Student
t test). °, significantly higher or lower than the sum of
the effects observed in the corresponding STI571- and ATRA-treated
groups, following 2-way analysis of variance and measurement of the F
of interaction (P < .01 according to Tukey
test).
), STI571 (
,
5 × 10
, 10
). (A) The level of NBT-reducing activity was measured in
the indicated cell line. (B) The level of NBT-reducing activity (left
panel); the percentage of CD11b, CD11c, or CD33 (middle panel); and the
total number of viable cells (right panel) were determined in NB4.R1
cells (the percentage of cell viability is shown above columns).
Results are the mean ± SD of 3 separate culture dishes and are
representative of at least 2 independent experiments. *, significantly
lower than the corresponding group treated with medium alone
(P < .01 according to the Student t test).
°°, significantly higher than the sum of the effects observed in
the corresponding STI571- and ATRA-treated groups, following 2-way
analysis of variance and measurement of the F of interaction
(P < .01 according to Tukey test).
O.D. at 540 nm: control = 0.21; STI571 = 0.21;
ATRA = 0.41; STI571 + ATRA = 0.92.
