Leukemia Research Vol. 20, No. 8, pp. 665-676, 1996.

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ALL-TRANS AND 9-CIS RETINOIC ACID ENHANCE

1,25-DIHYDROXYVITAMIN DJ-INDUCED MONOCYTIC
DIFFERENTIATION OF U937 CELLS

Hideaki Nakajima, Masahiro Kizaki, Hironori Ueno, Akihiro Muto, Nobuyuki Takayama,
Hiromichi Matsushita, Akira Sonoda and Yasuo Ikeda
Division of Hematology and Laboratory Medicine, Keio University School of Medicine, Tokyo 160, Japan

(Received 30 October 1995. Revision accepted 8 February 1996)

Abstract-Retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (D3) are well known for inducing
differentiation in many leukemic cell lines. The nuclear signalling pathways of RA and D3 are
mediated through their cognate receptors, the retinoic acid receptor (RAR) and vitamin D3
receptor (VDR), respectively. Retinoid X receptor (RXR) is an auxiliary factor that forms a
heterodimer with RAR and VDR, enabling their efficient transcriptional activation. 9-cis RA, a
high-affinity ligand for RXR, greatly enhanced D3-induced CD14 expression in U937 cells, while
RA alone did not induce CD14 expression. 9-cis RA also resulted in morphological changes of
U937 cells to macrophage-like cells when combined with D3, while RA alone resulted in
granulocyte-like cells. RA and D3 together enhanced c-fms expression, phagocytic activity, and
acted synergistically to promote nitroblue tetrazolium reduction activity and inhibit prolifera-
tion. Northern analysis showed that U937 cells constitutively expressed RAR-z, VDR and RXR-cr
mRNAs. RA or Ds alone or in combination did not affect RAR-a and VDR expression, while 9-cis
RA and 9-cis RA plus all-Pans RA significantly reduced RXR-a expression. Interestingly, D3
could restore the down-regulation of RXR-cx mRNA by 9-cis RA. These findings suggest that
there is crossover of the nuclear signalling pathways of RA and DB. This may have clinical
implications in that RA and D3 may be used in combination for differentiation-inducing therapy
in acute myelogenous leukemia and myelodysplastic syndrome. Copyright c 1996 Elsevier
Science Ltd.

Key words: All-trans retinoic acid, 9-cis retinoic acid, vitamin D3, U937, differentiation.

Introduction vitamin D3 (D3) is a biologically active form of vitamin

Retinoic acid (RA) and vitamin D3 have profound
effects on cellular growth and differentiation [l-3]. All-
tram RA (ATRA) and its stereoisomer,9-cis RA, induce
differentiation and inhibit proliferation in vitro in some
leukemic cell lines and in fresh leukemic cells from
patients with acute promyelocytic leukemia (APL) [4-
6]. Several clinical studies have recently reported
complete remissions induced by ATRA in a high
proportion of patients with APL [7-lo]. 1,25Dihydroxy-
Abbreviations: RA, retinoic acid; Dj, 1,25-dihydroxyvitamin
D3; RAR, retinoic acid receptor; RXR, retinoid X receptor;
VDR, vitamin D3 receptor; RARE, RA responsive element;
VDRE, vitamin D3 responsive element; ATRA, all-trans
retinoic acid; APL, acute promyelocytic leukemia; AML, acute
myelogenous leukemia; MD& myelodysplastic syndrome.
Correspondence to: Dr Masahiro Kizaki, Division of
Hematology, Keio University School of Medicine,
Shinanomachi, Shinjuku-ku, Tokyo 160, Japan (Tel: 81 3
3353 1211 (Ex 2385); Fax: 81 3 3353 3515).
D3 that functions primarily in calcium homeostasisin
vivo. D3 also plays an important role as an immuno-
hematopoietic regulatory hormone [ 1l] that can induce
some myeloid leukemic cell lines to differentiate along
the monocyte/macrophage pathway [12] and can pro-
long the survival of leukemic mice [13].
RA and D3 exert their effects through their respective
intracellular receptors, retinoic acid receptor (RAR) and
vitamin D3 receptor (VDR). They are membersof the
steroid receptor superfamily and act as ligand-inducible
transcription factors by binding to their cognate
responsive DNA elements, termed the retinoic acid
responsive element (RARE) and vitamin D3 responsive
element (VDRE) [14-161. RAR and VDR heterodimer-
ize with the retinoid X receptor (RXR) to bind DNA
with high affinity and effect efficient transcriptional
activation of target genes [17-211. VDR also forms
35 heterodimerswith RAR and mediatesresponsivenessto
ATRA on VDRE [22].
Although differentiation-inducing therapy for acute

665
 666 H. Nakajima
leukemia is currently restricted to ATRA therapy for
APL, the combination of differentiation-inducing agents
may have a role in other types of leukemia. Taimi et al.
have reported that ATRA combined with D3 has
synergistic effects on the functional properties of U937
cell lines [23]. We have reported previously that 9-cis
RA, a high-affinity ligand for both RAR and RXR [24],
induces differentiation in leukemic cell lines and fresh
leukemic cells, and 9-cis RA is often more potent than
ATRA in differentiating capacity and inhibition of
colony formation [5]. 9-cis RA may, therefore, have a
greater synergistic effect with D3 than ATRA. In
addition, 9-cis RA is the high-affinity ligand for RXR,
a heterodimerizing partner for RAR and VDR that is
indispensable for efficient DNA binding and transcrip-
tion. Therefore, it may have more potent and profound
effects on differentiation when combined with ATRA
and Da.
In this paper, we report that RA, particularly 9-cis
RA, enhances monocytic differentiation in Da-induced
U937 cells. RA enhances the Da induction of macro-
phage-like morphology in U937 cells, while RA alone
induced them to granulocyte-like cells. These results
demonstrate interaction between the nuclear signalling
pathways of RA and Da.
Materials and Methods
Cells
U937 cells were cultured (37°C 5% COz) in a-
minimum essential media (a-MEM, Gibco, Santa Clara,
CA, U.S.A.) with 10% fetal bovine serum (Cytosystems,
NSW, Australia) and 1% penicillin and streptomycin
(Gibco). Induction of differentiation was evaluated on
cytospin slide preparations with Giemsa staining.
Chemicals
ATRA (Sigma Chemical Co., St Louis, MO, U.S.A.)
and 9-cis RA (kindly provided by Dr M. Klaus, F.
Hoffmann-La Roche, Basel, Switzerland) were dis-
solved in 100% ethanol at concentrations of 10K2 M
and stored at - 20°C in the dark. Ds, kindly provided by
Chugai Pharmaceutical Co. (Tokyo, Japan), was stored
in 100% ethanol (lop4 M) at -20°C in the dark. None
of the cultures contained more than 0.3% ethanol.
Cellular proliferation assays
Cellular proliferation was measured by the MTT [3-
(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide] assay and 3H-thymidine incorporation. U937 cells
were cultured for 5 days in 96-well plates with various
concentrations of RA and D3. The number of viable cells
was determined by the MTT assay as follows. Briefly,
10 ul of MTI (5 @ml) was added to each well. The
reaction was stopped after 4 h of incubation by adding
et al.
100 pl of 0.04 N HCl in isopropanol, and the OD570 was
determined. For the 3H-thymidine incorporation studies,
U937 cells were incubated with RA and Da for 5 days,
and 3H-thymidine (1 pi/well) (6.7 Ci/mmol, New Eng-
land Nuclear, Boston, MA, U.S.A.) was added for the
last 4 h of incubation. Cells were harvested and
precipitated in 5% trichloroacetic acid (TCA, 30 mM
Na2HP04) at 4°C for 1 h, filtered on to glass microfiber
membranes (Whatman, Hillsboro, OR, U.S.A.) and
washed in 3% TCA (30 mM Na2HP04). Samples were
assayed by liquid scintillation counting.
Flow cytometry
U937 cells were treated with various concentrations
of RA and Da for 4 days, harvested, and stained with
anti-CDllb and CD14 monoclonal antibody (Becton-
Dickinson, Mountain View, CA, U.S.A.). Cells were
washed twice with phosphate-buffered saline (PBS;
Gibco BRL, Gaithersburg, MD, U.S.A.) containing
0.3% bovine serum albumin (Sigma) and 0.05% NaN3.
Surface c-fms expression was determined by incubation
with an anti-c@ antibody (Oncogene Science, Union-
dale, NY, U.S.A.) followed by a fluorescein isothiocya-
nate (FITC)-conjugated anti-rat IgG antibody (Cappel
Research Products, Durham, NC, U.S.A.). Cells were
analyzed by a Cytron Absolute Flow Cytometer (Ortho
Diagnostic Systems, Raritan, NJ, U.S.A.).
Phagocytosis assay
U937 cells were cultured for 5 days with various
concentrations of RA and Da in 24-well plates. Cells
were incubated with 1 pl of FITC-labeled fluorescent
microspheres (1.0 pm in diameter, Polysciences, War-
rington, PA, U.S.A.) for 4 h, harvested, and washed once
with PBS. The percentage of phagocytic cells was
analyzed by flow cytometry as above.
Nitroblue tetrazolium (NBZ) reduction activity
Following 5 days of culturing with various concen-
trations of RA and Da, U937 cells (2 x lo5 /ml) were
mixed with an equal volume of a solution containing
nitroblue tetrazolium (1.25 mg/ml) (NBT, Sigma),
bovine albumin (17 mg/ml), and 120tetradecanoyl-
phorbol 13-acetate (1 mg/ml) (TPA, Sigma) for 30 min
at 37°C. Following incubation, the medium was
discarded and formazan deposits were dissolved by
adding 100 pl of dimethyl sulfoxide (DMSO, Sigma).
Optical density was measured at 570 nm.
RNA extraction and Northern analysis
U937 cells were harvested and total RNA was
extracted by the method of Chomczynski and Sacchi
[25]. RNA samples (20-30 pg) were electrophoresed
and size-fractionated on a 1.0% agarose gel (Gibco
BRL) containing 17% formaldehyde and then trans-
 Effects of retinoic acid and vitamin D3 on U937 cells 667

Fig. 1. Morphological changes in U937 cells treated with retinoic acid (RA) and 1,2Sdihydroxyvitamin D3 (D3). U937 cells Lvere
cult ured for 5 days with various combinations of RA and D3 and examined by light microscopy (Giemsa staining, orig ;inal
mag:nification xl000 2. (A) Control, (B) all-tmns RA (ATRA) 10K6 M, (C) 9-cis RA lop6 M, (D) ATRA IOK M + 9-k RA
lo- .‘M,(E)D310P M,(F)D~1O-7M+ATRA10-6M,(G)D~10~7M+9-~I~RA10-6M,(H)D~10-7M+ATRA10~6 M+
9-cis RA lop6 M.
668 H. Nakajima
(A)
Fig. 2A.
ferred to nylon membranes (Hybond N+, Amersham,
Arlington Heights, IL, U.S.A.). cDNA probes for
Northern analysis were labeled with a-32P-dCTP using
a random primer DNA labeling kit (Takara Shuzo Co.,
Tokyo, Japan). Membranes underwent hybridization for
24-48 h at 42°C in 50% formamide, 2 x SSC
(1 x SSC = 150 mM NaCl, 15 mM sodium citrate, pH
7.0) 5 x Denhardt’s, 0.1% SDS, 10% dextran sulfate
(Pharmacia, Piscataway, NJ, U.S.A.), and 100 @ml
salmon sperm DNA (Sigma). Filters were washed with
0.1 x SSC at 65°C and exposed to Kodak XAR film
(Eastman Kodak, Rochester, NY, U.S.A.). Autoradio-
grams were exposed for periods of 24 h-7 days.
Reverse transcription-polymerase chain reaction (RT-
PCR)
Total RNA (1 pg) was reverse-transcribed with
random hexanucleotide primers (Takara Shuzo Co.)
and 200 units of Superscript II reverse transcriptase
(Gibco BRL) for 60 min at 37°C. For amplification of c-
jins, 30 cycles of PCR (94°C for 45 set, 60°C for 45 set,
et al.
72°C for 2 min) followed by 7 min of extension at 72°C
were conducted using a human macrophage colony-
stimulating factor receptor (M-CSF) amplimer set
(CLONTECH, Palo Alto, CA, U.S.A.) according to the
manufacturer’s protocol. To amplify the granulocyte
colony-stimulating factor (G-CSF) receptor, 30 cycles of
PCR (95°C for 1 min, 55°C for 2 min, 72°C for 3 min)
followed by 7 min of extension at 72°C were carried out
using the following primers: sense 5’-AGTA-
CAGTCCTCACCCTGAT-3’ (residues2000-2019) and
antisense5’-AACTGCTCTTGGGTCCCCCTG-3’ (resi-
dues 2377-2357) [26].
DNA probes
cDNA probes for human RAR-c( and RXR-c( were the
Kpn I-EcoRI fragment (1.9 kb) from plasmid pXM [15]
and the EcoRI-EcoRI fragment (1.9 kb) from plasmid
pSG1 [27], respectively. A human 1,25-dihydroxyvita-
min D3 receptor cDNA probe (EcoRI-PstI, 1.1 kb) was
purified from plasmid pGEM-7Zf(+) [16].
 Effects of retinoic acid and vitamin D3 on U937 cells 669

D3 10-W
(B) I 1

cm4

+Qcis RA
Dsl O-‘M 1 OgM 1 o-7M 1O”M
1 I I ,o,r- I -
I I I

CD14 cm4 cm4

+ATRA+S-cis RA
10-w lO-‘M 1 O*M

(C) CD1 4 Expression

loo-

go- ATRA+S-cis RA

80-

70-

80-

50-

30

20

10

Fig. 2. Flow cytometric analysis of CDllb and CD14 expression. (A) U937 cells were cultured with various concentrations of all-
bans retinoic acid (ATRA), 9-cis RA and ATRA + 9-k RA for 4 days and subjected to flow cytometric analysis. (B) U937 cells
were cultured with 1,25dihydroxyvitamin Ds (Da) alone or various combinations of RA and D3 for 4 days. (C) Percentages of CD14
positive cells in (B).
670 H. Nakajima et al.

c-fms Expression

w..,
12345678 9 10 11 12 13 14
D3(M) - - - - 10-7 lo-7 10-7 10-7 10-7 10-7 lo-7 10-7 10‘7 10-T
ATRA(M) - lO-6 - 10-6 _ 10-9 10-6 to-7 - - - 10-g 10-a 10-7
g-cjsRA(M) _ _ 10-6 lo-6 _ - - - 1O-g 1O-8 1C7 1O-g 1O-8 1O-7

1 2 3 4 5 6 7 8 9 10 11 12 13 14

D3 (Ml - - - - 10-710-710-710-710-710-710-710-710-710-7
ATRA (M) - 1o-7 - 1o‘7 - lo-gl()-*lo-7 - - - l(jgl(j*lo-7
9 - cis RA (M) - - lo-71(j7 - - - - 10-g10-810-710-g10-810-7

c - fms

G - CSF receptor

P - actin

Fig. 3. Effects of retinoic acid (RA) and 1,25dihydroxyvitamin D3 (Ds) on the expression of the c-fins/M-CSF receptor. (A) Flow
cytometry: U937 cells were cultured with various concentrations of RA and D3 for 4 days and subjected to flow cytometric analysis
using anti-c-fms antibody. Data represent the mean of triplicate experiments, and the SD was within 10% of the mean. (B) Reverse
transcription-polymerase chain reaction (RT-PCR): the semi-quantitative analysis of c-fis and G-CSF receptor mRNA expression
was performed by RT-PCR as described in Materials and Methods. b-actin mRNA was analyzed as the control.

Results tion and lobulation (Fig. lB-D). Incubation with Da

RA enhancesthe D3-induced morphological changesof
U937 cells to macrophage-like cells
To clarify the effects of combining RA and Da, we
first examined the effect of these agents on U937 cell
morphology. Cells in the control culture demonstrateda
monoblast-like morphology (Fig. 1A). Incubation with
RA alone resulted in the differentiation of U937 cells to
granulocyte-like cells as evidenced by nuclear matura-
resulted in the differentiation to macrophage-like cells
characterized by the appearance of cytoplasmic va-
cuoles, an enlargedcytoplasm, and a ruffled cytoplasmic
membrane (Fig. 1E). RA enhanced the formation of
vacuoles and enlargementof the pale cytoplasm induced
by Da (Fig. lF-H). This suggests that RA acts in
cooperation with D3 in inducing differentiation of U937
cells to macrophage-like cells. This enhancing effect of
RAs was more significant with 9-cis RA and 9-cis RA
 Effects of retinoic acid and vitamin D3 on U937 cells 671

Phagocytic Activity

looti

-.,
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Ds(M) - - - - 10-7 10-7 lo-’ lo-’ 10-7 lo-’ 10-7 10-7 10-7 10-7
ATFlA(M) _ 10-6 _ 10-6 _ ‘o-9 ‘o-8 lo-7 _ _ _ 10-g 10-e 10-7
9-d FlA(M) - - lo-6 10-6 - - - - 10-g 10-e 10-7 10-g 10-a 10-7

Fig. 4. Phagocytosis assay. Phagocytic activity was analyzed by flow cytometry using fluorescein isothiocyanate-conjugated latex
beads. U937 cells were cultured with various concentrations of retinoic acid and 1,25-dihydroxyvitamin Da for 5 days.

NBT Reduction Activitv

0.9
1 ATFlA10%+9-cis FlAlO-‘M+Ds

0.8
94s RAlO-‘M+Ds
0.7

0.6

’ .,

0.1 j+/ , I 1 I
0 1 o-‘0 10 -9 10-0 10.’
1,25(OH)zDa (M)

Fig. 5. Effects of retinoic acid (RA) and 1,25 hydroxyvitamin Da (D3) on nitroblue tetrazolium (NBT) reduction activity. U937 cells
were cultured with various combinations of RA and Ds for 4 days, and NBT reduction activity was analyzed as described in
Materials and Methods.

plus ATRA than when compared with ATRA alone by Effects of RA and D3 on c-fms expressionby U937 cells
counting the number of cells presenting macrophage- Flow cytometric analysis, which we have repeated
like cells (data not shown). three times, showedthat ATRA, 9-cis RA, and 9-cis RA
plus ATRA slightly enhanced c-fms expression when
RA greatly enhances CD14 expression induced by D3 usedat very high concentrations (10K6 M) (Fig. 3A). D3
treatment of U937 cells (lop7 M) alone also slightly enhancedc-fms expression.
Flow cytometric analysis showed that ATRA, 9-cis The combination of RA and D3 cooperatively enhanced
RA and 9-cis RA plus ATRA induced CDllb expres- c-fms expression, and this effect was more significant
sion in a dose-dependentmanner. They did not induce when 9-cis RA was combined with Ds. RT-PCR analysis
CD14 expression (Fig. 2A). These effects were more was also performed to semiquantitatethe c-fms mRNA.
potent with 9-cis RA or 9-cis RA plus ATRA than with This revealed that RA in combination with D3 (lop7 M)
ATRA alone. Da (1O-7 M) induced expression of both enhancedc-jins mRNA expression in a dose-dependent
the CDllb (94%) and CD14 (28%) antigens. Interest- manner (Fig. 3B). This effect on mRNA expressionwas
ingly, 9-cis RA and 9-cis RA plus ATRA greatly also more significant when 9-cis RA was combined with
enhanced the induction of CD14 expression by D3 Ds. RA or Ds alone did not affect c-fms mRNA
(84%) (Fig. 2B, C). expression. An analysis of G-CSF receptor mRNA by
672 H. Nakajima et al.

A
MlT Assav 3H - Thymidine Incorporation
0.7

0.6

0.5

ATRAlo-'M
0.4

i 0.3
0

0.2

0.1

a I I
7zP 1o.9’ 1o-8 lo-'
1,25(0H)zD3 (M) 1,25(0H)zD3 (M)

Fig. 6. Effects of retinoic
various concentrations of
D3 slightly inhibited the
thymidine incorporation.
acid (RA) and 1,25-hydroxyvitamin D3 (Ds) on cellular proliferation. U937 cells, cultured with RA and
Da for 5 days, were examined using MIT assay (A) and 3H-thymidine incorporation (B). (A) MlT assay.
proliferation of U937 cells. RA in combination with D3 synergistically inhibited their growth. (B) 3H-
D3 alone minimally reduced the incorporation of 3H-thymidine. RA combined with D3 synergistically
reduced incorporation.

12345678
D3 (M) - - - - 1o-6 1o-7 10.’ 1o-7
ATRA(M) - 1O-6 - 1O-6 - lo+ - 1O-6
9 - cisRA(M) - - 10m610m6 - - 10s6 10m6

VDR - 4.6 kb

- 4.5 kb
- 3.4 kb

Fig. 7. Analysis of vitamin D3 receptor (VDR), retinoid X receptor (RXR)-a and retinoic acid receptor @AR)-a mRNA expression.
U937 cells, cultured with various combinations of RA and D3 for 5 days, underwent RNA extraction and Northern analysis as
described in Materials and Methods.
 Effects of retinoic
RT-PCR revealed constant mRNA levels under various
RA and Da conditions. /3-actin mRNA was similarly
analyzed as a control (Fig. 3B).
Effects of RA and D3 on phagocytic activity and NBT
reduction activity in U937 cells
Incubation with D3 alone moderately enhanced
phagocytic activity in U937 cells (Fig. 4). RA combined
with D3 (lop7 M) markedly enhanced phagocytic
activity and NBT reduction activity in a dose-dependent
manner, while D3 alone had a minimal effect on NBT
reduction activity (Fig. 5). NBT reduction activity was
made more prominent by combining 9-cis RA with D3
and ATRA plus 9-cis RA with Ds (Fig. 5).
Effects of RA and 03 on the proliferation of U937 cells
Incubation with D3 alone slightly inhibited the
proliferation of U937 cells in a dose-dependentmanner,
as measured by the MIT assay and 3H-thymidine
incorporation (Fig. 6A, B). ATRA (lop7 M), 9-cis RA
(10K7 M) and 9-cis RA plus ATRA combined with D3
resulted in an enhancement of its inhibitory effect on
proliferation. The enhancing effect of ATRA plus 9-cis
RA was greater than 9-cis RA alone. ATRA alone had
the least significant effect.
Effects of RA and D3 on mRhCAexpression of VDR,
RXR-a, and RAR-a genes
Northern analyses were conducted to analyze the
expression of receptor genes during the treatment of
U937 cells with RA and Da. RA and D3 alone or in
combination did not affect mRNA expression of the
VDR and RAR-a genes(Fig. 7). Interestingly, 9-cis FU
(lop6 M) and ATRA (lop6 M) plus 9-cis FW (10K6 M)
down-regulated RXR-a expression, and the combination
of D3 with 9-cis RA and ATRA plus 9-cis RA resulted in
the recovery of RXR-a expression to control levels.
ATRA and D3 did not affect the expressionof the RXR-
tl gene (Fig. 7).
Discussion
The U937 cell line has several monoblast-like
characteristics and can be induced to a macrophage-like
cell by several differentiation-inducing agents such as
TPA and Da [28,29]. We have shown that RA,
particularly 9-cis RA, enhancesD3-induced differentia-
tion along the monocyte/macrophage lineage, while RA
alone induced differentiation along the granulocytic
pathway. Flow cytometric analysis revealed that RA
induced CDllb expression but not CD14 expression.
Morphological changesobserved in RA-treated cells in
this study were totally distinct from those of cells treated
with Ds, a compound well known to induce macrophage
differentiation [29]. RA-treated U937 cells can undergo
acid and vitamin D3 on U937 cells 673
differentiation to granulocyte-like cells as demonstrated
by morphology and flow cytometry. A previous study
has reported that RA induced monocytic differentiation
in U937 cells. This observation was based on the
findings that RA slightly increased the number of
phagocytic cells (up to 14% of cells) and induced
morphological changessuch as nuclear lobulation [30].
It is possible that the U937 cells used in our study had
different characteristics from those used by Olsson and
Breitman [30]. Previous studiesindicated that transcripts
encoding the M-CSF receptor and its protein are induced
along the Ds-induced monocytic pathway, but not the
DMSO-induced granulocytic pathway in HL-60 cells
[31,32]. Thus, c-&s is a lineage specific marker for the
monocytic differentiation. We analyzed, therefore, three
different subclones of U937 cells obtained from the
JapaneseCancer Research ResourcesBank (JCRB) by
the expressionof c-fms transcripts and morphology, and
obtained similar results (data not shown). RA thus
clearly induces granulocytic differentiation in U937
cells in most instances.
Brown et al. [33] have shown that HL-60 cells fail to
differentiate in responseto ATRA in serum-free media.
However, ATRA acts synergistically with Da in
promoting the differentiation of HL-60 cells to mono-
cytes. It is of interest that under the conditions in which
RA retains the capacity to promote granulocytic
differentiation in our system, it also enhances Da-
induced monocytic differentiation. Masciulli et al. [34]
have also reported combined effects of RA and D3
which resulted in the differentiation to monocytes in
HL-60 cells or granulocytes in AML-193 cells. They
have speculatedthat one of the chemical inducers more
strongly affects cell morphology when RA and D3 are
added together. However, RA did not enhance the
effects of D3 in their study. It may be partly becausethe
dosage of Da that they used induces near maximal
differentiation in HL-60 and AML-193 cells and there-
fore obscuresthe effect of RA. Defacque et al. [35] have
reported that the treatment of U937 cells with ATRA
and D3 increasesRXR-a expressionat the protein level,
and they proposed that RXR-a may have an important
role in this setting. However, we observed down-
regulation of RXR-c( mRNA by the treatment of 9-cis
RA or 9-cis RA plus ATRA that was restored to normal
levels by the addition of Ds. Our observation also
supportsthe importance of RXR-c( in the differentiation
of leukemic cells.
The mechanismby which RA enhancesthe effects of
Da remains to be clarified. However, there should be a
direct interaction and crossover between nuclear signal-
ling by RA and D3. D3 activates setsof genesthat lead to
monocytic differentiation through binding to VDR, a
ligand-inducible transcription factor. RA may modulate
this nuclear signalling pathway of Da. ATRA and 9-cis
674 H. Nakajima et al.
RA, for example, may enhance the transactivation
capacity of VDR by enhancing the receptor hetero-
dimerization of RAR-VDR by binding to RAR or of
RXR-VDR by binding to RXR. If this is the case,
RAR-VDR or RXR-VDR heterodimers must play an
important role in activating genesthat lead to monocytic
differentiation. Alternatively, ATRA and 9-cis RA may
divert unbound RAR or RXR away from the pre-existing
RAR-VDR or RXR-VDR heterodimer on VDRE,
enhancing the formation of VDR-VDR homodimers
[36] and leading to the efficient activation of monocyte-
specific sets of genes. The pathway through which
RXR-VDR, RAR-VDR and VDR-VDR act in mono-
cytic differentiation induced by DQ,is unknown. Further
studieswill be neededto clarify the molecular mechan-
ism of differentiation induced by RA and Ds.
ATRA is now being usedin the treatment of APL asa
differentiation-inducing agent [7-lo]. APL is character-
ized by the t(15;17) translocation that fuses the PML
gene on chromosome 15 to the RAR-a gene on
chromosome 17 [37]. This PML-RAR-a fusion tran-
script may be involved in the leukemogenesisof this
type of leukemia, and the rate of responseto ATRA in
the patients demonstratedby molecular analysis to have
the chimeric protein is very high [37]. Although a high
proportion of patients with APL achieve complete
remission (CR) with ATRA, a minority of patients are
primarily resistant to ATRA, and relapsed patients are
also frequently resistant to re-induction therapy with
ATRA [38]. In studies relevant to overcoming ATRA
resistance, we have previously shown that 9-cis RA
combined with ATRA was an effective inducer of
differentiation in RA-resistant HL-60 cells [38]. Why
the combination of both retinoids could induce the
differentiation of RA-resistant HL-60 cells is unclear.
Potentially, the two ligands together bind receptors that
attach to novel RA responsive elements @ARES) that
might activate a new set of differentiation-related genes.
Alternatively, the combination of both retinoids may
make the existing transactivation pathway much more
efficient. Our present study demonstrates that the
combination of D3 and RA, particularly ATRA plus 9-
cis RA, acts cooperatively or synergistically in inducing
differentiation and inhibiting the proliferation of U937
cells. These findings introduce the possibility that RA
combined with D3 may be able to induce differentiation
in some monocytic leukemias. D3 and RA have been
previously reported to be effective in the treatment of
selected patients with myelodysplastic syndrome
(MDS), improving anemia in these patients [39]. Thus,
the combination of RA and Ds may also be effective in
these patients. A problem in applying these agents in a
clinical situation, however, is the very low serum
concentration (10-10-10-9 M) of Ds achieved by oral
administration [40]. The cooperative effects of RA and
Ds were observedat concentrationsof 10-8-10-6 M for
each agent. Although the serumconcentration of RA can
readily reach 10K7 M by oral administration [41], the
serum concentration of D3 required to exert the
cooperative effect with RA is not easily achieved as it
would result in significant toxicity such as hypercalce-
mia. This problem may be resolved by using a vitamin
D3 derivative, such as la,25-dihydroxy-20-epi-vitamin
D3 [42], 1,25-(OH)2-A16-yne-vitamin Ds, or 1,25-(OH)*-
23-yne-vitamin D3 [43-45], all of which have a high
differentiation-inducing capacity with fewer side-effects
than Ds. These agents may also cooperate efficiently
with R4 even at low concentrations.
In summary, we have demonstratedthat RA, particu-
larly 9-cis RA, enhancesDs-induced monocytic differ-
entiation. These observations reveal interaction in the
nuclear signalling pathways of RA and D3 through RAR,
RXR and VDR. They also suggest the possible clinical
application of 9-cis R4 combined with D3 in differ-
entiation-inducing therapy for acute myelogenous leu-
kemia (AML) and MDS.
Acknowledgement-This study was supported in part by a
grant from the Ministry of Education, Science and Culture of
Japan.
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