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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 2057-2066
Effects of Novel RAR- and RXR-Selective Retinoids on Myeloid Leukemic
Proliferation and Differentiation In Vitro
By
Masaaki Shiohara,
Marcia I. Dawson,
Peter D. Hobbs,
Nobukuni Sawai,
Tsukasa Higuchi,
Kenichi Koike,
Atsushi Komiyama, and
H. Phillip Koeffler
From the Department of Pediatrics, Shinshu University School of
Medicine, Matsumoto Japan; Retinoid Program, SRI International, Menlo
Park, CA; the Division of Hematology/Oncology, Cedars-Sinai Medical
Center, University of California at Los Angeles School of Medicine, Los
Angeles, CA.
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ABSTRACT |
Retinoids such as all-trans-retinoic acid (ATRA) and
9-cis-retinoic acid (9-cis-RA) have an important role
in many aspects of proliferation and differentiation of hematopoietic
cells. They exert their effects by binding to retinoic acid receptors
(RARs) and/or retinoid X receptors (RXRs). We studied the
effects of novel retinoids on proliferation and differentiation of
HL-60 and NB4 myeloid leukemic cells, as well as acute promyelocytic leukemia (APL) cells from patients. RXR-selective SR11345 (Retinoid C)
had little ability to inhibit the clonal growth and to induce the
differentiation of either HL-60 or NB4 cells. However, SR11276 (Retinoid E), which activated both the RAR and RXR classes, and SR11278
(Retinoid D), which activated the RAR subtypes  ,  , and  , could
inhibit clonal growth of both cell types, as well as leukemic cells
from APL patients. The combination of ATRA and either SR11276 or
SR11278 additively inhibited APL cell proliferation. SR11302 (Retinoid
A), with reported anti-AP-1 activity and no activation of RARs and RXR
and SR11363 (Retinoid B), which selectively activated RAR and ,
were inactive. The clonal proliferation of both HL-60 and NB4 cells
that were pulse-exposed to 10-9 mol/L ATRA, SR11276,
SR11278, or SR11345 for 3 days, washed, and plated in methylcellulose
culture were inhibited by 0%, 51%, 21%, and 1% for HL-60 cells and
43%, 41%, 35%, and 1% for NB4, respectively, compared with
nontreated control cells. When the HL-60 cells were pulse-exposed to
10-9 mol/L of either SR11278 or SR11276, plus
10-9 mol/L ATRA for 3 days, colony numbers were reduced by
46% and 64%, respectively. Induction of leukemic cell differentiation as determined by the nitroblue tetrazolium (NBT) assay showed that the
combination of 10-7 mol/L of either SR11278 or SR11276 with
10-7 mol/L ATRA had additive effects on HL-60 cells, NB4
cells, and fresh APL cells. Induction of CD11b expression on both HL-60
and NB4 cells occurs during their differentiation. Expression of this antigen was synergistically augmented by the combination of either 10-7 to 10-8 mol/L SR11278 or 10-7
to 10-9 mol/L SR11276 with 10-9 mol/L ATRA
compared with either analog alone in HL-60 cells. Expression of the
novel myeloid specific transcription factor C/EBP was increased by
SR11278 and SR11276 in both the HL-60 and NB4 cell lines. We conclude
that retinoids or combination of retinoids with specificities for both
RAR and RXR may markedly enhance the ability of ATRA to inhibit clonal
growth and induce differentiation of HL-60 and NB4 leukemic cells. This
occurs in the absence of continuous contact with retinoids.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ALL-TRANS-RETINOIC ACID (ATRA)
inhibits proliferation and induces granulocytic differentiation of
leukemic cells including cell lines (eg, HL-60),1,2 as well
as fresh acute promyelocytic leukemia (APL) cells.1-3 A
high proportion of APL patients achieved complete remission after ATRA
therapy.4,5 Also, ATRA enhances the clonal growth of normal
human myeloid and erythroid precursors.6-8
Retinoic acids (RAs) exert their effects through their binding and
activation of specific nuclear receptors, retinoic acid receptors
(RAR , RAR, RAR) and retinoid X receptors (RXR, RXR, RXR), who are members of the steroid/thyroid nuclear hormone receptor superfamily and form heterodimeric RAR/RXR and homodimeric RXR/RXR complexes.9-12 Both heterodimers of RAR/RXR and
homodimers of RXR are ligand inducible trans-regulators that
modulate the transcription of target genes by interacting with
cis-acting specific sequences (RA-response elements [RAREs])
of cellular genes.9-11 The consensus DNA sequences
recognized by RAR/RXR are represented by a tandem repeat of the
sequence AGGTCA separated by five nucleotides. In contrast, the
consensus sequence recognized by RXR/RXR is composed of the same tandem
repeats separated by only one nucleotide.13-15 The effect
of ATRA is mediated by its binding to a RAR/RXR
heterodimer.16 On the other hand, the effect of
9-cis-retinoic acid (9-cis-RA), which is a stereoisomer
of ATRA, is mediated by its binding to either a RAR/RXR heterodimer or
a RXR/RXR homodimer.17,18 We have shown that
9-cis-RA is slightly more potent than ATRA in inducing
differentiation and inhibiting proliferation of acute myelogeneous
leukemia cell lines and fresh myeloid leukemic cells.19 Moreover, we showed that 9-cis-RA in combination with ATRA was an effective inducer of differentiation of a RA-resistant HL-60 variant
cell line.20 Novel classes of synthetic retinoids have been
synthesized that selectively interact with RAR/RXR heterodimers and
RXR/RXR homodimers.21 In this study, we examined the
effects of these retinoids on inhibiting proliferation and inducing
differentiation of acute myeloid leukemic cells.
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MATERIALS AND METHODS |
Cells.
Our studies used the HL-603 and NB4 myeloid leukemic cell
lines,22 as well as fresh leukemic samples from bone marrow
from APL patients, which were collected in heparinized tubes before any
therapy. The percentage of blasts and promyelocytes from these individuals was more than 80% of the mononuclear population at the
time of harvesting the cells. The diagnosis was established according
to French-American-British (FAB) criteria.23 The leukemic cells were isolated by Ficoll-Hypaque (Pharmacia, Inc, Piscataway, NJ)
gradient centrifugation and washed twice in phosphate-buffered saline
(PBS). The blast cells were immediately placed into suspension culture
containing RPMI 1640 medium with 10% fetal bovine serum (FBS; HyClone
Laboratories, Inc, Logan, UT), 100 U/mL penicillin, and 100 µg/mL
streptomycin in humidified air with 5% CO2.
Retinoids and transfection assays.
ATRA was purchased from Sigma Chemical Co (St Louis, MO). Synthetic
retinoids used in this study included SR11302 (Retinoid A), SR11363
(Retinoid B), SR11345 (Retinoid C), SR11278 (Retinoid D), and SR11276
(Retinoid E), which were described by Dawson et al.24
Retinoids were dissolved in 100% ethanol to a stock concentration of
10-2 mol/L, stored at  20°C, and protected from
light. In each experiment, controls were performed using the same
concentration of ethanol as was present in the experimental plates.
This diluant had no effect on proliferation and differentiation of cells.
Transient transfections of CV-1 cells were performed using the calcium
phosphate precipitation procedure, as described
previously.25 Approximately 5 × 105 cells
were transfected with 50 ng of an expression vector for either human
RAR, RAR, RAR, or RXR,25 100 ng of a reporter gene (TREpal) 2-tk-CAT, and 150 ng of the
-galactosidase expression plasmid pCH110. After transfection, cells
were grown in the presence or absence of retinoids for 20 hours26 before determination of the levels of the reporter
chloramphenicol acetyl transferase (CAT). Results were corrected for
control -galactosidase expression.
Clonogenic assay in soft gel culture.
HL-60 and NB4 cells were plated at 2× 103 cells per
plate in six-well culture dishes in methylcellulose according to
previously described methods.24 For analysis of myeloid
leukemic cell clonal growth, 1 × 105 blast cells were
plated and retinoids were added as indicated. After incubation for 10 days, colonies (> 40 cells) were counted using an inverted
microscope. At day 10, more than 95% of the colonies consisted of more
than 40 cells. All experiments were performed using triplicate plates
per experimental point; each experiment was performed at least three
times. The results were expressed as the mean percentage of clonal
growth in plates containing retinoids as compared with the number of
colonies in control dishes without retinoids.
Assays for cellular differentiation.
Induction of differentiation of either HL-60, NB4 cells, or fresh
leukemic cells from patients was measured by reduction of nitroblue
tetrazolium dye (NBT) and expression of CD11b antigen. For NBT
reduction, each cell suspension (2 × 105 cells per
mL) was mixed with an equal volume of solution containing 1.25 mg/mL
NBT (Sigma), 17 mg/mL bovine serum albumin (fraction V; Sigma), and 1 mg/mL 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma) for 30 minutes at 37°C. After incubation, the medium was discarded, and
the formazan deposits were dissolved by adding 0.1 mL of dimethyl
sulfoxide (DMSO; Sigma) and measured by optical density (OD) at 580 nm.
All experiments were performed using triplicate plates per experimental point.
For analysis of cell-surface antigens, a direct immunofluorescence
staining technique was used. Cells were exposed to phycoerythrin (PE)-conjugated murine antihuman CD11b (DAKO Corp, Carpinteria, CA).
Control studies were performed with a nonbinding control murine
IgG1 isotype antibody (DAKO Corp). Analysis of fluorescence was performed on a FACScan flow cytometer (Beckton Dickinson, Mountain
View, CA).
RNA isolation and Northern blot analysis.
Total cellular RNA was extracted by the acid guanidine
thiocyanate-phenol-chloroform method.27 Total RNA (10 µg/lane) was electrophoresed on 1% formaldehyde-agarose gels, and
transferred to positively charged nylon membranes (Hybond
N+, Amersham Corp, Arlington Heights, IL).
DNA-probes for C/EBP 28 and -actin29 were
labeled with [-32P]-deoxycytidine triphosphate
(dCTP) (3,000 µCi/mmol; Amersham Corp) using a random
priming kit (Takara Shuzo Co, Ltd, Tokyo, Japan).28
Hybridization of blots was previously described.29 Briefly,
the labeled probe was hybridized for 16 hours at 68°C in 2X SSC (pH
7.0; 1X SSC = 150 mmol/L NaCl, 15 mmol/L sodium citrate), 5X
Denhardt's solution, 0.1% sodium dodecyl sulfate (SDS). Filters were
washed to a stringency of 0.25X SSC at 68°C and exposed to Kodak
XAR film (Eastman-Kodak, Rochester, NY). Autoradiograms were exposed
for 48 hours. Blots were sequentially hybridized with labeled DNA for
C/EBP and -actin. The levels of C/EBP mRNA were calculated by
normalizing signal densities to -actin mRNA by densitometric analysis.
Analysis of effects of combination of drugs.
Isobologram analysis was used to evaluate the effect of combinations of
drugs on leukemic cells.30 Dose-dependent activities were
determined separately for each compound, and then the effects were
determined for the combination of one compound held at a fixed
concentration and the other at different dilutions. The interaction of
two compounds was quantified by determining the combination index (CI)
according to the classical isobologram equation:
CI=(D)1/(Dx)1
+(D)2/(Dx)2, where Dx is the dose of one compound alone required to produce an effect, and (D)1 and (D)2 are the
doses of both compounds that produce the same effect. From this assay,
the combined effects of two analogs can be assessed as either summation
(additive or zero interaction), indicated as CI=1; synergism, indicated
as CI<1; or antagonism, indicated as CI>1. Other statistical data
were handled using the Student's t test.
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RESULTS |
Effects of retinoids on transactivation of a reporter gene having RAR
and RXR response sequences.
Table 1 shows the retinoid receptor
transcriptional activities on the synthetic palindromic response
element (TREpal) of ATRA and five synthetic retinoids in the presence
of RAR, RAR, RAR, and RXR. The TREpal response element,
which was used in the transfection assay, is responsive to both RAR/RXR
and RXR/RXR dimer complexes that have been activated by retinoids. The
retinoids show a range of activities for these retinoid receptors. For
example, retinoid SR11345 (Retinoid C) selectively activates RXR.
Retinoid SR11278 (Retinoid D) activates RARs (>>).
Retinoid SR11276 (Retinoid E) is a panagonist for the RARs and RXR.
Retinoid SR11363 (Retinoid B) activates RAR. SR11302 (Retinoid A)
does not activate these receptors, but is reported to inhibit AP-1
activity.31
Effects of retinoids on proliferation of myeloid leukemic cells in
methylcellulose culture.
The retinoids were examined for their effect on either HL-60 or NB4
clonogenic proliferation (Fig 1). Retinoids
A, B, and C at 10-6 mol/L were poor inhibitors (less than
20%) of leukemic colony formation. Retinoids D and E effectively
inhibited colony formation by 50% (ED50) at approximately
7 × 10-8 mol/L and 7 × 10-9 mol/L
in HL-60 and 7 × 10-8 mol/L and 6 × 10-9 mol/L in NB4, respectively. ATRA had an
ED50 of about 3 × 10-8 mol/L for
inhibition of clonal growth of HL-60 cells and 1 × 10-9 mol/L for NB4 cells.
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| Fig 1.
Effects of synthetic retinoids on HL-60 and NB4 clonal
proliferation. HL-60 and NB4 cells (2 × 103 per plate)
were cultured with various concentrations of retinoids in
methylcellulose. Colonies (>40 cells) were counted after 10 days of
incubation. Results are expressed as the percentage of clonal growth in
retinoid-treated plates compared with the number of colonies in control
plates not containing retinoids. Data represent mean ± standard
deviation (SD) of triplicate cultures. This figure shows representative
findings of three independent experiments, each of which had similar
results.
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The inhibitory effects of ATRA and the other retinoids on proliferation
of fresh APL cells from two individuals paralleled those observed with
HL-60 cells (Fig 2, upper panel). For
example, Retinoids A, B, and C alone at 10-7 mol/L had
little inhibitory effect on leukemic blast cell growth and were unable
to enhance the potency of 10-7 mol/L ATRA in sample No. 1. In contrast, Retinoids D and E at 10-7 mol/L inhibited the
proliferation of leukemic cells by 35% and 54%, respectively, whereas
their combination with ATRA (10-7 mol/L) had a subadditive
inhibitory effect. ATRA plus Retinoid E significantly reduced the
colony formation compared with ATRA alone (P < .05). These
retinoids showed similar effects on fresh acute leukemic cells from
sample No.2 (lower panel).
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| Fig 2.
Effects of retinoids (10-7 mol/L) on clonal
proliferation of fresh APL cells. Samples from two patients were
analyzed and shown in upper and lower panels. Results are expressed as
the percentage of control plates not exposed to retinoids. Data
represent mean ± SD of triplicate cultures.
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Effects of pulse-exposure of retinoids on clonal growth of HL-60 and
NB4 cells.
HL-60 or NB4 cells were cultured in liquid medium for 3 days with
10-11 to 10-7 mol/L experimental analog alone
or combined with 10-9 mol/L ATRA, and then washed
extensively with medium to remove retinoid before being plated in
methylcellulose soft-gel culture. Colonies were counted at day 10 (Fig 3). For HL-60 cells exposed to low
concentrations of ATRA (10-11 to 10-9 mol/L),
clonal proliferation was not inhibited; whereas, higher concentrations
of ATRA (10-8 to 10-7 mol/L) inhibited clonal
proliferation by 38% to 63% (Fig 3). Pulse-exposure to
10-7 mol/L Retinoids B or C did not inhibit HL-60 clonal
growth. However, pulse-exposure to 10-7 mol/L Retinoids D
or E inhibited clonal growth by 50% and 76%, respectively. When the
cells were cultured in liquid culture with Retinoid D at either
10-10 mol/L, 10-9 mol/L, 10-8
mol/L, or 10-7 mol/L plus 10-9 mol/L ATRA for 3 days, thoroughly washed and plated in methylcellulose, colony numbers
were reduced by 27% (P < .02, compared with the same
concentration of Retinoid D), 48% (P < .01), 56% (P < .01, CI<1), and 65% (CI<1), respectively. Similarly, when the
cells were cultured with Retinoid E at either 10-10,
10-9, 10-8, or 10-7 mol/L plus
10-9 mol/L ATRA for 3 days, colony numbers were reduced by
55% (P < .01), 66% (P < .01, CI<1), 75%
(P < .05, CI<1), and 84% (CI<1), respectively.
For NB4 cells, ATRA produced an ED50 of 6 × 10-9 mol/L (Fig 3). Pulse-exposure to 10-7
mol/L Retinoid B or C did not inhibit NB4 clonal growth. Pulse-exposure to 10-7 mol/L Retinoid D or E inhibited clonal growth by
59% and 69%, respectively. When the NB4 cells were cultured in liquid
culture with Retinoid D at either 10-11 or
10-10 mol/L plus 10-9 mol/L ATRA for 3 days,
colony numbers were reduced by 20% (P < .01) and 30%
(P < .02), respectively. When the NB4 cells were cultured
with Retinoid D at 10-7 mol/L plus 10-9 mol/L
ATRA, colony numbers were reduced by 63% (CI<1). Similarly, when the
NB4 cells were cultured with Retinoid E at either 10-11 or
10-10 mol/L plus 10-9 mol/L ATRA for 3 days,
colony numbers were reduced by 23% (P < .01) and 33%
(P < .05)), respectively. When the NB4 cells were cultured
with Retinoid E at either 10-8 or 10-7 mol/L
plus 10-9 mol/L ATRA, colony numbers were reduced by 61%
and 70% (CI<1), respectively. These results suggest that HL-60 and
NB4 cells irreversibly lost the ability to form colonies after
pulse-exposure to Retinoids D or E and the addition of ATRA increased
their inhibitory effects.
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| Fig 3.
Clonal inhibition of HL-60 and NB4 cells after
pulse-exposure (3 days) to retinoids. HL-60 and NB4 cells were exposed
in liquid culture to 10-11 to 10-7 mol/L of
either ATRA, Retinoids B, C, D, E ( for HL-60,  for NB4, A
through E), or a combination of 10-11 to 10-7
mol/L of either Retinoids D or E plus 10-9 mol/L ATRA ( for HL-60, for NB4, D and E), washed, plated in methylcellulose,
and the resulting colonies counted. Each point represents a mean ± SD
of triplicate dishes. This figure shows representative findings of
three independent experiments, each of which had similar results.
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Effects of retinoids on differentiation of leukemic cells.
Induction of differentiation of the myeloid cell lines and fresh
leukemic cells from two patients with APL (FAB classification M3) into
more mature granulocyte-like cells by these novel retinoids either
alone or in combination with ATRA was assayed for NBT reduction and
CD11b antigen expression. Retinoids A, B, and C at 10-7
mol/L did not induce HL-60 cells to reduce NBT
(Fig 4, top panel). The addition of each of
these analogs to ATRA at 10-7 mol/L had little additional
effect on the ability of ATRA to induce HL-60 cells to reduce NBT as
compared with the effect of ATRA alone, although Retinoid C did have a
small subadditive effect. Retinoids D and E at 10-7 mol/L
induced HL-60 cell differentiation with about 90% to 100% and 135%
to 140% of the activity of ATRA, respectively. Combinations of either
Retinoids C, D, or E at 10-7 mol/L with 10-7
mol/L ATRA significantly reduced NBT as compared with ATRA alone (P < .01, P < .01, and P < .01, respectively). The same tendency was observed with NB4 cells (Fig 4,
middle panel), and combination of Retinoid E at 10-7 mol/L
with 10-7 mol/L ATRA significantly reduced NBT as compared
with ATRA alone (P < .02). The reduction of NBT by leukemic
cells from two patients with APL using this same series of synthetic
retinoids either alone or in combination with ATRA showed comparable
results with those observed with HL-60 cells (Fig 4, bottom panel).
Combinations of either Retinoids C, D, or E at 10-7 mol/L
with 10-7 mol/L ATRA significantly reduced NBT as compared
with ATRA alone (P < .05, P < .01, and P < .01, respectively). Two independent experiments using samples from
each patient had similar results, and Fig 4, bottom panel, shows
representative results.
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| Fig 4.
Comparison of the differentiation-inducing activity (NBT
reduction) of retinoids. HL-60 cells (top panel), NB4 cells (middle
panel), and fresh APL cells (lower panel) were cultured with
10-7 mol/L of either ATRA, Retinoids A, B, C, D, E, or
10-7 mol/L ATRA combined with 10-7 mol/L of one
of the other retinoids for 5 days. Differentiation was determined by
NBT reduction. Results are expressed as the percentage of control
dishes that contained no retinoids (100% activity) and represent the
mean ± SD of three independent experiments performed in triplicate
dishes.
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The expression of CD11b increases as myeloid cells differentiate
towards granulocytes.20 Exposure of HL-60 cells to
increasing concentrations of ATRA (10-12 to
10-8 mol/L for 2 days) increased dose dependently the CD11b
expression, with 10-8 mol/L ATRA producing an approximately
10-fold greater expression of CD11b compared with that of untreated
HL-60 cells (Fig 5). Neither anti-AP-1
Retinoid A, RAR-selective Retinoid B, nor RXR-selective retinoid
C increased the expression of CD11b (Fig 5) and did not enhance the
ability of ATRA (10-12 to 10-8 mol/L) to
increase CD11b expression (data not shown). In contrast, Retinoids D
and E at 10-8 mol/L alone increased CD11b expression
(sevenfold to 10-fold), compared with untreated HL-60 cells.
Experiments with ATRA were very similar (Figs 5C and D). However, equal
molar concentrations of either Retinoids D or E and ATRA markedly
increased CD11b expression. For example, at 10-11 mol/L,
Retinoids D and E increased CD11b expression by 2.1-fold and 2.3-fold,
respectively, compared with untreated HL-60 cells; but when either
Retinoids D or E at 10-11 mol/L were combined with ATRA at
10-11 mol/L, expression of CD11b increased by 8.3-fold and
12.2-fold, respectively. Forty-seven percent of untreated NB4 cells
expressed CD11b, and neither Retinoids A, B, nor C increased the
expression of CD11b (data not shown). ATRA increased in a
dose-dependent fashion the CD11b expression on NB4 cells, with
10-8 mol/L ATRA producing approximately 95% positive CD11b
cells (Fig 5E and F). Retinoids D and E at 10-8 mol/L alone
increased CD11b expression (approximately twofold, respectively),
compared with untreated NB4 cells. Equal molar concentrations
(10-12 and 10-11 mol/L) of either Retinoids D
or E and ATRA increased CD11b expression.
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| Fig 5.
Effects of retinoids on CD11b expression on HL-60 (A, B,
C, and D) and NB4 cells (E and F). Cells were cultured for 48 hours
with 10-12 to 10-8 mol/L ATRA (A, C, and D in
HL-60, E and F in NB4 cells), Retinoid A (B), Retinoid B (B), Retinoid
C (B), Retinoid D (C and E), or Retinoid E (D and F), or a combination
of equal molar concentrations of Retinoids D or E with ATRA (C, E and
D, F, respectively) and cells then were analyzed by FACScan for
expression of CD11b. This figure shows representative findings of three
independent experiments, each of which had similar results.
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Changes in expression of C/EBP after exposure of HL-60 cells to
retinoids.
C/EBP is a newly identified CCAAT/enhancer-binding transcriptional
factor whose expression is restricted to myeloid
cells.28,32 Expression of C/EBP occurs in HL-60 cells
induced to differentiate to granulocytes by ATRA.28 On the
other hand, levels of this gene decreased when HL-60 and KG-1
myeloblasts were induced to differentiate to macrophages.28
We studied the effect of retinoids (10-7 mol/L, 48 hours)
alone or in the presence of ATRA on the expression of C/EBP in HL-60
cells and NB4 cells as examined by Northern blot
(Fig 6). Our previous experiments showed
that maximal induction of C/EBP mRNA occured at 48 hours exposure to
retinoid (10-7 mol/L)28; and therefore, similar
culture conditions were used for these experiments. After hybridization
with 32P-labeled C/EBP, the same blot was then
rehybridized with a -actin probe and the intensity of each signal
was examined by densitometric analysis, and signals of C/EBP were
normalized against the -actin band. Untreated HL-60 cells (lane 1)
and NB4 cells (lane 8) expressed low levels of C/EBP mRNA, and ATRA
(lane 2, lane 9) induced C/EBP expression by 5.0-fold and 6.8-fold,
respectively, compared with each untreated cells. RXR-selective
Retinoid C (lane 3, lane 10) induced lower levels of C/EBP mRNA
compared with ATRA-treated HL-60 cells and NB4 cells, respectively.
RAR-selective Retinoid D (lane 4, lane 11) induced C/EBP by 2.0-fold
and 5.3-fold of that unstimulated HL-60 and NB4, respectively.
Panagonist Retinoid E (lane 5, lane 12) induced C/EBP by 4.5-fold
and 6.0-fold compared with unstimulated control. The combination of
10-7 mol/L of either Retinoids D or E with 10-7
mol/LATRA enhanced the expression of C/EBP 7.0-fold (lane 6) and
7.5-fold (lane 7) in HL-60 and 7.8-fold (lane 13) and 7.5-fold (lane
14) in NB4, respectively, compared with control. We also examined
C/EBP mRNA expression after HL-60 cells were exposed for 72 hours to
the same retinoids, and similar results as those obtained by a 48-hour
exposure was obtained (data not shown). Cyclin-dependent kinase
inhibitors (CDKIs) are important negative regulators of the cell
cycle.33-36 The p21WAF1 protein, which is the
first reported CDKI,37-40 inhibits the kinase activity of
cyclin A/CDK2, cyclin B/CDC2, cyclin E/CDK2, and cyclin D/CDK4
complexes in vitro and slows progression of the cell
cycle.41 The p27KIP1 is also a CDKI, which
binds to a variety of cyclin/CDK complexes, inhibits the kinase
activities of these complexes, and halts cell cycle
progression.42 Recently, these CDKIs were reported to play
an important role in cell differentiation. We examined the effects of
retinoid analogs on the expression of p21WAF1 and
p27KIP1. HL-60 cells were cultured for 72 hours in the
presence of ATRA, Retinoids A, B, C, D, E (10-7 mol/L),
either alone or in combination with ATRA (10-7 mol/L) and
modulation of p21WAF1 and p27KIP1 expression
was examined by Western blot analysis (data not shown). Neither
wild-type HL-60 cells nor those cultured with a retinoid either alone
or combined with ATRA (10-7 mol/L) expressed detectable
levels of p21WAF1. Wild-type HL-60 cells expressed
p27KIP1, and the levels of this CDKI did not change when
the cells were cultured with retinoids. These results suggest that
these CDKIs may not play an important role in induction of
differentiation down the granulocytic pathway.
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| Fig 6.
Modulation of C/EBP mRNA expression by retinoids in
HL-60 and NB4 cells. Upper panel: cells were treated for 48 hours with
10-7 mol/L retinoid, either alone or in combination with
10-7 mol/L ATRA. Total RNA was extracted and analyzed by
Northern blot technique (10 µg/lane) and hybridized with
[32P]-labeled C/EBP cDNA as described in Materials and
Methods. The same blot was rehybridized with
[32P]-labeled -actin probe to show RNA loading in each
lane; results for HL-60 and NB4 were independently normalized such that
expression in wild-type cells equaled one. HL-60 cells (lanes 1 through
7) and NB4 cells (lanes 8 through 14) were cultured for 48 hours with
10-7 mol/L retinoid as follows: untreated control (lanes 1, 8); ATRA (lanes 2, 9); Retinoid C (lanes 3, 10); Retinoid D (lanes 4, 11); Retinoid E (lanes 5, 12); ATRA plus Retinoid D (lanes 6, 13); ATRA
plus Retinoid E (lanes 7, 14). Lower panel: Densitometric quantitation
of upper panel. Signal intensity of C/EBP in untreated HL-60 cells
(lane 1) and NB4 cells (lane 8) were used as the control.
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DISCUSSION |
We have shown that ATRA, which binds and activates RAR/RXR
heterodimers, and 9-cis-RA, which binds and activates RAR/RXR
heterodimers and RXR/RXR homodimers, efficiently inhibited the
proliferation and induced differentiation of HL-60 cells,19
and our data suggested that the RAR/RXR pathway is more important than
the RXR/RXR pathway for differentiation and proliferation of myeloid
leukemic cells.21 In this study, we showed that
RAR-selective analogs are more potent than RXR-selective analogs in
inhibiting the proliferation and inducing the differentiation of HL-60
and NB4 cells. Several of these analogs appeared to have prominent
activity when combined with ATRA.
Retinoid B and C alone were very weak inhibitors of proliferation and
inducers of differentiation of myeloid leukemic cells. Retinoid B
activates RAR, and Retinoid C activates RXR/RXR homodimer. These
results suggest that neither a RAR-selective nor a RXR-selective retinoid has a prominent effect on growth and differentiation of
leukemic cells. In our previous study, other ligands selective for
RXR/RXR homodimers (SR11236, SR11246, and SR11269) also had little
effect on inducing the differentiation and inhibiting the clonal growth
of myeloid leukemic cells.21 In contrast, Retinoid D, which
activates RAR, RAR and RAR, and Retinoid E, which activates
both the RARs and RXR, inhibited the clonal growth and induced the
differentiation of HL-60, NB4 cells, and fresh APL cells. Also, the
panagonist Retinoid E was slightly more potent than Retinoid D. Retinoid D most readily activated RAR>>RAR>>RAR (Table
1); and some investigators43,44 have found weak expression of RAR in HL-60 cells, which possibly could explain why Retinoid E
is more potent than Retinoid D. Furthermore, inhibition of colony formation and induction of differentiation were markedly augmented by
the combination of these analogs with ATRA. These results are reminiscent of our prior study19,21 showing that the
panagonist 9-cis-RA was more potent than ATRA, which activates
the RARs. These findings may be explained by the work of Nagy et
al,45 who suggested that activation of the RAR pathway
induced differentiation, thereby making the cells responsive to
induction of apoptosis through activation of the RXR pathway. However,
we show that Retinoid E (panagonist) plus ATRA (RAR selective) markedly
and synergistically induced differentiation of HL-60 cells as measured
by expression of CD11b. Thus, the enhanced effect of these analogs does
not rely on one retinoid stimulating differentiation and the other causing apoptosis.
We found that Retinoid A, which is reported to inhibit selectively the
AP-1 activity, but not activate transcription from a
RARE,31 had very little effect on either the clonal
proliferation or the differentiation of either HL-60, NB4 cells or
fresh APL cells. Those results indicate that AP-1 may not be involved
in the signaling pathway of proliferation and differentiation of HL-60
and NB4 cells by retinoids.
C/EBP is a member of the C/EBP gene family that includes C/EBP,
C/EBP, C/EBP , C/EBP, and C/EBP ,28,32 and these
proteins have been implicated in the differentiation of a variety of
mammalian cells, including myeloid cells, adipocytes, and
hepatocytes.46-48 Myeloid progenitors have high levels of
C/EBP, which decreases during granulocytic
differentiation,46 while the levels of C/EBP and
C/EBP are low in early myeloid stem cells and increases during granulocytic differentiation.46 Expression of C/EBP is
highly restricted to late myeloblasts and more mature granulocytic
cells.28,49 Results of experiments using cotransfection of
the human C/EBP expression constructs with CAT-reporter vectors
containing myeloid-specific c-mim and human myeloperoxidase promoters
suggested its role as a transcription factor in the regulation of a
subset of myeloid-specific genes.28 Our data showed that
ATRA, Retinoids D and E induced the expression of C/EBP, and the
combination of either Retinoids D or E with ATRA augmented the
expression of C/EBP. These results suggest that the RAR/RXR pathway
has a more important role in the expression of this myeloid-specific
transcription factor than the RXR/RXR pathway. In other experiments, we
found that retinoids can directly enhance the transactivation of
C/EBP,49 because upstream of the C/EBP gene is a
retinoic acid response element, which can bind RAR/RXR, and when this
sequence was placed before a reporter gene, C/EBP increased
transactivation in the presence of retinoid agonists (data not shown).
We have also investigated the antiproliferative potencies of the
synthetic retinoids in different cancer cell subtypes. For example, the
highly RXR-selective Retinoid C had no effect on HL-60 promyelocytic
leukemic cells, but markedly inhibited the clonal growth of LNCaP
prostate cancer cells (data not shown). Furthermore, the
RAR-selective Retinoid B prominently inhibited growth of MCF-7
breast cancer cells, but had little activity against either HL-60 or
LNCaP cells (data not shown). Furthermore, the DU-145 prostate cancer
cells were recalcitrant to all the retinoids (data not shown). Prior
studies have shown that each of these cell lines express each of the
retinoid receptors.50,51 The reason for the differential
sensitivity of these cells to this array of analogs requires additional
analysis. These results indicated that a different class of retinoids
may have selective therapeutic efficacy for different types of cancer
cells. In summary, we showed that several retinoid combinations may
offer greater therapeutic activity than when the retinoids are used alone.
| |
ACKNOWLEDGMENT |
We thank Dr Susumu Ito (Shinshu University) for help with the FACS
analysis. We also thank Dr Tatsuya Kinoshita, Dr Kazuo Sakashita, Dr
Kouichi Takeuchi (Shinshu University), Dr Adrian F. Gombart
(Cedars-Sinai Medical Center), and Dr Tsuyoshi Nakamaki (Showa
University) for generous technical assistance.
| |
FOOTNOTES |
Submitted March 11, 1998; accepted November 13, 1998.
Supported in part by grants from the National Institutes of Health
(NIH) and the U.S. Army, the Parker Hughes Trust, and the C. and H. Koeffler Fund. H.P.K. is a member of the UCLA Jonsson Comprehensive
Cancer Center and holds an endowed Mark Goodson Chair of Oncology
Research at Cedars-Sinai Medical Center/UCLA School of Medicine.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to H. Phillip Koeffler, MD, Division of
Hematology/Oncology, Cedars-Sinai Medical Center/UCLA School of
Medicine, 8700 Beverly Blvd, B-213, Los Angeles, CA 90048.
| |
REFERENCES |
1.
Koeffler HP:
Induction of differentiation of human acute myelogenous leukemia cells: Therapeutic implication.
Blood
62:709, 1983[Abstract/Free Full Text]
2.
Sporn MB, Roberts AB, Goodman DS:
The Retinoids. Biology, Chemistry, and Medicine (ed 2). New York, NY, Raven, 1994.
3.
Breitman TR, Selonick SE, Collins SJ:
Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid.
Proc Natl Acad Sci USA
77:2936, 1980[Abstract/Free Full Text]
4.
Huang ME, Ye YC, Chen SR, Chai JR, Lu JX, Zhoa L, Gu HT, Wang ZY:
Use of all-trans-retinoic acid in the treatment of acute promyelocytic leukemia.
Blood
72:567, 1988[Abstract/Free Full Text]
5.
Chomienne C, Ballarini P, Balitrand N, Amar M, Bernard JF, Boivin P, Daniel MT, Berger R, Castaigne S, Degos L:
Retinoic acid therapy for acute promyelocytic leukemia.
Lancet
1:746, 1989[Medline]
[Order article via Infotrieve]
6.
Douer D, Koeffler HP:
Retinoic acid enhances colony-stimulating factor-induced clonal growth of normal human myeloid progenitor cells in vitro.
Exp Cell Res
138:193, 1982[Medline]
[Order article via Infotrieve]
7.
Douer D, Koeffler HP:
Retinoic acid; inhibition of the clonal growth of human myeloid leukemia cells.
J Clin Invest
69:277, 1982
8.
Douer D, Koeffler HP:
Retinoic acid enhances growth of human early progenitor cells in vitro.
J Clin Invest
69:1039, 1982
9.
Giguere V, Ong ES, Segui P, Evans RM:
Identification of a receptor for the morphogen retinoic acid.
Nature
330:624, 1987[Medline]
[Order article via Infotrieve]
10.
Petkovich M, Brand NJ, Krust A, Chambon P:
A human retinoic acid receptor which belongs to the family of nuclear receptors.
Nature
330:444, 1987[Medline]
[Order article via Infotrieve]
11.
Mangelsdorf DJ, Ong ES, Dyck JA, Evans RM:
A nuclear receptor that identifies a novel retinoic acid receptor pathway.
Nature
345:224, 1990[Medline]
[Order article via Infotrieve]
12.
Leid M, Kastner P, Lyons R, Nakshatri H, Saunders M, Zacharewski T, Chen JY, Staub A, Garnier JM, Mader S, Chambon P:
Purification, cloning, and RXR identify of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently.
Cell
68:377, 1992[Medline]
[Order article via Infotrieve]
13.
Umesono K, Murakami KK, Thompson CC, Evans RM:
Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors.
Cell
65:1255, 1991[Medline]
[Order article via Infotrieve]
14.
Mangelsdorf DJ, Umesono K, Kllewer SA, Borgmeyer U, Ong ES, Evans RM:
A direct repeat in the cellular retinol-binding protein type II gene confers differential regulation by RXR and RAR.
Cell
66:555, 1991[Medline]
[Order article via Infotrieve]
15.
Kliewer SA, Umesono K, Heyman RA, Mangelsdorf DJ, Dyck JA, Evans RM:
Retinoid X receptor-COUP-TF interactions modulate retinoic acid signaling.
Proc Natl Acad Sci USA
89:1448, 1992[Abstract/Free Full Text]
16.
Yang N, Schule R, Mangelsdorf DJ, Evans RM:
Characterization of DNA binding and retinoic acid binding properties of the retinoic acid receptor.
Proc Natl Acad Sci USA
88:3559, 1992[Abstract/Free Full Text]
17.
Levin AA, Sturzenbecker LJ, Kazmer S, Bosakowski T, Huselton C, Allenby G, Speck J, Kratzeisen CL, Rosenberger M, Lovey A, Grippo JF:
9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR.
Nature
355:359, 1992[Medline]
[Order article via Infotrieve]
18.
Heyman RA, Mangelsdorf DJ, Dick JA, Stein RB, Eichele G, Evans RM, Thaller C:
9-cis retinoic acid is a high affinity ligand for the retinoid X receptor.
Cell
68:397, 1992[Medline]
[Order article via Infotrieve]
19.
Sakashita A, Kizaki M, Pakkala S, Schiller G, Tsuruoka N, Tomosaki R, Cameron JF, Dawson MI, Koeffler HP:
9-cis-retinoic acid: Effects on normal and leukemic hematopoiesis in vitro.
Blood
81:1009, 1993[Abstract/Free Full Text]
20.
Kizaki M, Nakajima H, Mori S, Koike T, Morikawa M, Ohta M, Saito M, Koeffler HP, Ikeda Y:
Novel retinoic acid, 9-cis retinoic acid, in combination with all-trans retinoic acid is an effective inducer of differentiation of retinoic acid-resistant HL-60 cells.
Blood
83:3289, 1994[Abstract/Free Full Text]
21.
Kizaki M, Dawson MI, Heyman R, Elstner E, Morosetti R, Pakkala S, Chen D-L, Ueno H, Chao W-R, Morikawa M, Ikeda Y, Heber D, Pfahl M, Koeffler HP:
Effects of novel retinoid X receptor-selective ligands on myeloid leukemic differentiation and proliferation in vitro.
Blood
87:1997, 1996[Abstract/Free Full Text]
22.
Lanotte M, Martin-Thouvenin V, Najman S, Balerini P, Valensi F, Berger R:
NB4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (3M).
Blood
77:1080, 1991[Abstract/Free Full Text]
23.
Bennet JM, Catavsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR, Sultan C:
Proposed revised criteria for the classification of acute myeloid leukemia.
Ann Intern Med
103:620, 1985
24.
Dawson MI, Elstner E, Kizaki M, Chen D-L, Pakkala S, Kerner B, Koeffler HP:
Myeloid differentiation mediated through retinoic acid receptor/retinoic X receptor (RXR) not RXR/RXR pathway.
Blood
84:446, 1994[Abstract/Free Full Text]
25.
Lehmann JM, Zhang X-K, Pfahl M:
RAR2 expression is regulated through a retinoic acid response element embedded in Sp1 sites.
Mol Cell Biol
12:2976, 1992[Abstract/Free Full Text]
26.
Lehmenn JM, Jong L, Fanjul A, Cameron JF, Lu XP, Haefner P, Dawson MI, Pfahl M:
Retinoids selective for retinoid X receptor response pathways.
Science
258:1944, 1992[Abstract/Free Full Text]
27.
Chomczynski P, Sacchi N:
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:156, 1987[Medline]
[Order article via Infotrieve]
28.
Chumakov AM, Grillier I, Chumakova E, Chih D, Slater J, Koeffler HP:
Cloning of the novel human myeloid-cell-specific C/EBP-epsilon transcription factor.
Mol Cell Biol
17:1375, 1997[Abstract]
29.
Shiohara M, Akashi M, Gombart AF, Yang R, Koeffler HP:
Tumor necrosis factor : Posttranscriptional stabilization of WAF1 mRNA in p53-deficient human leukemic cells.
J Cell Physiol
166:568, 1996[Medline]
[Order article via Infotrieve]
30.
Chen Z-X, Breitman TR:
Tributyrin: A prodrug of butyric acid for potential clinical application in differentiation therapy.
Cancer Res
54:3494, 1994[Abstract/Free Full Text]
31.
Fanjul A, Dawson MI, Hobbs PD, Jong L, Cameron JF, Harlev E, Graupner G, Lu X-P, Pfahl M:
A new class of retinoids with selective inhibition of AP-1 inhibits proliferation.
Nature
372:107, 1994[Medline]
[Order article via Infotrieve]
32.
Antonson P, Stelleon P, Yamanaka R, Xanthopoulos KG:
A novel human CCAAT/enhancer binding protein gene, C/EBP-, is expressed in cells of lymphoid and myeloid lineages and is localized on chromosome 14q11.2 close to the T-cell receptor / locus.
Genomics
35:30, 1996[Medline]
[Order article via Infotrieve]
33.
Hunter T:
Braking the cycle.
Cell
75:839, 1993[Medline]
[Order article via Infotrieve]
34.
Sherr CJ:
G1 phase progression: Cycling on cue.
Cell
79:551, 1994[Medline]
[Order article via Infotrieve]
35.
Nurse P:
Ordering S phase andMphase in the cell cycle.
Cell
79:547, 1994[Medline]
[Order article via Infotrieve]
36.
Hirama T, Koeffler HP:
Role of the cyclin-dependent kinase inhibitors in the development of cancer.
Blood
86:841, 1995[Free Full Text]
37.
Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D:
p21 is a universal inhibitor of cyclin kinases.
Nature
366:701, 1993[Medline]
[Order article via Infotrieve]
38.
Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge S:
The p21 cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases.
Cell
75:805, 1993[Medline]
[Order article via Infotrieve]
39.
El-Deiry WS, Tokino T, Velculescu V, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B:
WAF1, a potent mediator of p53 tumor suppression.
Cell
75:817, 1993[Medline]
[Order article via Infotrieve]
40.
Noda A, Ning Y, Venable SF, Pereira-Smith OM, Smith JR:
Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen.
Exp Cell Res
211:90, 1994[Medline]
[Order article via Infotrieve]
41.
Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D:
p21 is a universal inhibitor of cyclin kinases.
Nature
366:701, 1993
42.
Polyak K, Lee M-H, Erdjument-Bromaga H, Koff A, Roberts JM, Tempst P, Massague J:
Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals.
Cell
78:59, 1994[Medline]
[Order article via Infotrieve]
43.
Hashimoto Y, Petkovic M, Gaub MP, Kagechika H, Shudo K, Chambon P:
The retinoic acid receptors alpha and beta are expressed in the human promyelocytic leukemia line HL-60.
Mol Endocrinol
3:1046, 1989[Abstract/Free Full Text]
44.
Kizaki M, Ikeda Y, Tanosaka R, Nakajima H, Morikawa M, Sakashita A, Koeffler HP:
Effects of novel retinoic acid compound, 9-cis retinoic acid, on proliferation, differentiation, and expression of retinoic acid receptor (RAR)- and retinoid X receptor (RXR)- RNA by HL-60 cells.
Blood
82:3592, 1993[Abstract/Free Full Text]
45.
Nagy L, Thomazy VA, Shipley GL, Fesus L, Lamph W, Heyman RA, Chandraratna RAS, Davies PJA:
Activation of retinoid X receptors induces apoptosis in HL-60 cell lines.
Mol Cell Biol
15:3540, 1995[Abstract]
46.
Scott LM, Civin CI, Rorth P, Friedman AD:
A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells.
Blood
80:1725, 1992[Abstract/Free Full Text]
47.
Cao Z, Umek RM, McKnight SL:
Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells.
Genes Dev
5:1538, 1991[Abstract/Free Full Text]
48.
Friedman AD, Landschulz WH, McKnight SL:
CCAAT/enhancer binding protein activates the promotor of the serum albumin gene in cultured hepatoma cells.
Genes Dev
3:1314, 1989[Abstract/Free Full Text]
49.
Chih DY, Chumakov AM, Park DJ, Silla AG, Koeffler HP:
Modulation of mRNA expression of a novel human myeloid-selective CCAAT/enhancer binding protein gene (C/EBP).
Blood
90:2987, 1997[Abstract/Free Full Text]
50.
Dahiya R, Park HD, Cusick J, Vessella RL, Fournier G, Narayan P:
Inhibition of tumorigenic potential and prostate-specific antigen expression in LNCaP human prostate cancer cell line by 13-cis-retinoic acid.
Int J Cancer
59:126, 1994[Medline]
[Order article via Infotrieve]
51.
Roman SD, Clarke CL, Hall RE, Alexander IE, Sutherland RL:
Expression and regulation of retinoic acid receptors in human breast cancer cells.
Cancer Res
52:2236, 1992[Abstract/Free Full Text]
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ABSTRACT
INTRODUCTION