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Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4715-4726
Induction of Differentiation in Acute Promyelocytic Leukemia Cells by
9-cis Retinoic Acid  -Tocopherol Ester (9-cis
Tretinoin Tocoferil)
By
Makoto Makishima,
Kazuhiko Umesono,
Koichi Shudo,
Tomoki Naoe,
Kenji Kishi, and
Yoshio Honma
From the Department of Chemotherapy, Saitama Cancer Center Research
Institute, Saitama, Japan; the Institute for Virus Research, Kyoto
University, Kyoto, Japan; the Faculty of Pharmaceutical Sciences,
University of Tokyo, Tokyo, Japan; the Department of Infectious
Diseases, Nagoya University School of Medicine, Nagoya, Japan; and the
First Department of Internal Medicine, Niigata University School of
Medicine, Niigata, Japan.
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ABSTRACT |
Acute promyelocytic leukemia (APL) has a specific genetic
rearrangement between the retinoic acid receptor (RAR)- gene
and the pml nuclear protein gene. All-trans retinoic
acid (ATRA) induces granulocytic differentiation of APL-derived cells
and is used to treat APL patients. However, ATRA interacts with normal
cells with RAR throughout the entire body, and when used at high doses or over a long duration, it induces several adverse effects. The development of drugs that selectively act on APL cells may contribute to increasing the therapeutic efficacy of APL treatment as well as
elucidating the mechanisms of response to ATRA. In this study, 9-cis retinoic acid -tocopherol ester (9CTT) inhibited the
proliferation of APL-derived NB4 and HT93 cells and induced
differentiation markers, such as granulocytic maturation, nitroblue
tetrazolium reduction, and CD11b expression, in these cells. The
effects of 9CTT on non-APL cells, including HL-60 and U937 cells, were
much weaker than those on APL cells, and tretinoin tocoferil (TT), which is an -tocopherol ester of ATRA, did not induce the
differentiation of APL cells as effectively as 9CTT. The
differentiation-inducing effects of 9CTT were inhibited by RAR
antagonists. 9CTT and TT similarly induced the transactivating activity
of RARs, but were not effective on RXRs. 9CTT downregulated the
expression of PML/RAR- protein more effectively than TT, which
suggests that it may be involved in the selectivity of 9CTT against APL
cells. Interestingly, 9CTT enhanced the differentiation of APL cells
induced by ATRA, 9-cis retinoic acid, and synthetic
retinobenzoic acids. Combined with 1,25-dihydroxyvitamin
D3 (VD3), 9CTT also more than additively induced the differentiation of APL cells. Thus, 9CTT, alone or in
combination with other retinoids or VD3, may be useful for the treatment of APL.
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INTRODUCTION |
ACUTE PROMYELOCYTIC leukemia (APL; M3 in
the French-American-British [FAB] classification) has a specific
chromosomal abnormality t(15;17), which involves a genetic
rearrangement between the retinoic acid receptor (RAR)- gene
and the pml gene coding a nuclear protein,1,2 and
can be successfully treated with all-trans retinoic acid
(ATRA).3 ATRA induces granulocytic differentiation of
APL-derived cells.4,5 The biological actions of retinoic
acid are mediated through specific nuclear receptors. ATRA binds to
three types of receptors; RAR-, RAR- , and RAR- .6,7
Another natural retinoid, 9-cis retinoic acid (9CRA), binds to
RARs and other receptors: retinoid X receptor (RXR)-, RXR-, and
RXR-.6,7 The PML/RAR- fusion protein retains the
ligand-binding and DNA-binding domains of RAR- and can induce the
expression of responsive genes in response to ATRA.1,2 However, the PML/RAR- protein exhibits a dominant negative action on
RAR-, and expression of the pml/RAR- gene that has been
introduced into human myeloid leukemia U937 and HL-60 cells interferes
with the induction of their differentiation by ATRA.8,9 The
PML protein is physiologically localized within nuclear organella called nuclear bodies10,11 and has been reported to
suppress oncogenic transformation.12 In APL cells,
PML/RAR- protein delocalizes the wild-type PML protein and disrupts
nuclear bodies; treatment with ATRA restores the structure of nuclear
bodies.10,11 Thus, the PML/RAR- fusion protein has been
suggested to contribute to leukemogenesis and responsiveness to ATRA,
although their precise mechanisms have not been elucidated.
Retinoids are involved in several biological phenomena, such as
embryogenesis and the control of cell proliferation and
differentiation. Although ATRA induces remission in APL patients
without significant toxicities, the administration of retinoids at
higher doses or over a long duration causes several adverse effects in
other organs, including the skin, liver, and central nervous
system.13-15 Because retinoids are teratogenic, their use
in fertile women is limited.16,17 Several retinoid
analogues have been synthesized, but their biologic activities are
associated with toxicities.18 Because expression of the
oncogenic PML/RAR- protein is limited to APL cells, the fusion
protein might be a good target for chemotherapeutic drugs in APL
treatment. The development of retinoid derivatives that selectively act
on APL cells may overcome retinoid-related drawbacks and contribute to
improving the effects of therapy in APL as well as elucidating the
mechanisms for response to retinoids.
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MATERIALS AND METHODS |
Materials.
Tretinoin tocoferil [TT;
tocoretinate/(±)-3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraenoate] and its Z-isomer, 9-cis retinoic acid -tocopherol
ester [9CTT; 9-cis tretinoin
tocoferil/(±)-3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl (2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraenoate], were synthesized and donated by Pharmaceutical Research Laboratories, Nisshin Flour Milling Co (Saitama, Japan). The chemical structures of
TT and 9CTT are shown in Fig 1. The
concentration of both stock solutions was 3.5 × 10 3 mol/L in ethanol. ATRA was purchased from Sigma
(St Louis, MO), 9CRA was from Biomol Research Laboratories (Plymouth
Meeting, PA), and 1,25-dihydroxyvitamin D3
(VD3) was from Wako Pure Chemical Industry (Osaka, Japan).
The stock solutions for ATRA and 9CRA were 4 × 103 mol/L and that for VD3 was 1.2 × 103 mol/L in ethanol. Stock solutions for
synthetic retinoids (Am80, Am580, Ch55, Re80, Am555s, and LE540) were 1 × 102 mol/L and that for LGD1069 was 3 × 103 mol/L in ethanol. The highest concentration of
9CTT in this study was 1.5 × 105 mol/L and the
maximal final concentrations of ethanol was 0.5%, which did not affect
cell proliferation or differentiation.
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| Fig 1.
Chemical structures of tretinoin tocoferil (TT) and its
Z-isomer (9-cis isomer), 9-cis retinoic acid
-tocopherol ester (9CTT). TT and 9CTT are -tocopherol esters of
ATRA and 9CRA, respectively.
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Cell lines and cell culture.
Human myeloid leukemia HL-60, U937, ML-1, THP-1, P39/TSU, P31/FUJ,
NB4,5 and HT93 cells19 were cultured in
suspension in RPMI 1640 medium containing 10% fetal bovine serum and
80 µg/mL gentamicin at 37°C in a humidified atmosphere of 5%
CO2 in air.20 We confirmed the expression of
the pml/RAR- gene in NB4 and HT93 cells but not in HL-60,
U937, ML-1, THP-1, P39/TSU, or P31/FUJ cells by a previously described
reverse transcription-polymerase chain reaction (RT-PCR)
technique1 (data not shown). Monkey kidney CV-1 and COS-7
cells were cultured in Dulbecco's modified Eagle medium or RPMI 1640 medium containing 10% fetal bovine serum and 80 µg/mL gentamicin.
Cell growth and differentiation.
Suspensions of cells were cultured with or without the test compounds
in multidishes. The cells were counted in a Model ZM Coulter Counter
(Coulter Electronics, Luton, UK). Nitroblue tetrazolium (NBT) reduction
was assayed colorimetrically by a method reported by Takuma et
al21 and modified by our laboratory.20 Briefly, cells were incubated with 1 mg/mL NBT (Sigma) and 100 ng/mL
phorbol-12-myristate 13-acetate (Sigma) in RPMI1640 medium at 37°C
for 30 minutes, and the reaction was stopped by adding HCl. Formazan
deposits were solubilized in dimethyl sulfoxide (Wako), and absorption of the formazan solution at 560 nm per 107 cells was
measured in a spectrophotometer (U-2000; Hitachi, Tokyo, Japan).
Lysozyme activity in the conditioned medium was determined using a
lysoplate containing 1% agar, 66.7 mmol/L sodium phosphate buffer (pH
6.6), 50 mmol/L NaCl, and 0.5 mg/mL heat-killed Micrococcus lysodeikticus (Sigma).20 One unit is equivalent to 1 µg/mL egg-white lysozyme. Cell morphology was examined in cell smears
stained with May-Grünwald and Giemsa solutions (Merck, Darmstadt,
Germany) by examination of more than 200 cells. -Naphthyl acetate
esterase was determined cytochemically with an esterase kit (Sigma).
Flow cytometry.
Expression of the granulocyte- and macrophage-specific antigens CD11b
and CD14 on the cell surface was determined by indirect immunofluorescent staining and flow cytometry.22 Mouse
monoclonal antibodies to CD11b (2LPM19c), CD14 (TÜK4), and
control mouse IgG1 and IgG2a were obtained from Dako (Glostrup,
Denmark). Cells were treated with the mouse monoclonal antibody in IFA
buffer (10 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 4% fetal bovine
serum, and 0.1% NaN3) plus 2% Block Ace (Snow Brand Milk
Products, Sapporo, Japan) and stained with fluorescein isothiocyanate
(FITC)-conjugated F(ab )2 fragment of goat antimouse
IgG (Dako) in IFA buffer plus 2% Block Ace. The stained cells were
assayed using a flow cytometer (Epics XL; Coulter Electronics). The
mean fluorescence intensity was calculated using the Immuno-4 histogram
analysis program (Coulter) with mouse Ig of the same isotype as a
negative control. The Immuno-4 program subtracts a control histogram
from a test histogram to calculate the percentage of positive cells and
mean fluorescence intensity in the test histogram.23
Cell cycle analysis.
The cell cycle was analyzed using propidium iodide
staining.24 Briefly, cells were fixed by the addition of
cold ethanol, suspended with 250 µg/mL RNase A in 1.12% sodium
citrate at 37°C for 30 minutes, and stained with 50 µg/mL
propidium iodide (Sigma) on ice for more than 30 minutes. The stained
nuclei were analyzed with a Epics XL flow cytometer (Coulter).
Transactivation assays for retinoid receptors.
To assay ligand-binding specificity, CV-1 cells were transfected with
100 ng of receptor plasmid (pCMX-hRARs, pCMX-hRXR-, or
pCMX-PML-RAR-), 250 ng of reporter plasmid, 200 ng of pCMX--gal, and 450 ng of carrier pGEM using Lipofectin reagent (GIBCO BRL, Gaithersburg, MD).1,25 The reporters used were
TK-TREpx2-LUC25 and mRARx2-LUC, which contains the
promoter region of the mouse RAR- gene,2 for
RARs and TK-CRBPII-LUC for RXR-.26 The cells were
transfected for 18 hours, and after removing the DNA-containing medium,
they were incubated with a test compound in medium containing 10%
resin-charcoal-stripped fetal bovine serum for 24 hours. Luciferase and
-galactosidase activities were analyzed using a luminescence reader
(BLR-201; Aloka, Tokyo, Japan) and a spectrophotometer (Hitachi),
respectively. All transfection data were normalized using an internal
-galactosidase marker. To investigate the effect of PML/RAR-
protein on transactivation by RAR, CV-1 cells were cotransfected with
100 ng of pCMX-PML-RAR-, 250 ng of reporter plasmid (TK-TREpx2-LUC
for exogenous receptor or TK-REx3-LUC1,2 for endogenous
receptor), and pGEM carrier to give 1 µg of DNA/well. A GAL4-receptor
hybrid system was also used to assay ligand-binding specificity.
Chimeric GAL4-receptor expression plasmids (CMX-GAL4-hRARs and
CMX-GAL4-mRXRs) were constructed by fusion of the ligand-binding domains of retinoid receptor and the DNA-binding domain of the yeast
GAL4 to pCMX vector.25,27 CV-1 cells were transfected with
TK-GAL4-UASx4-LUC reporter, pCMX--gal, each receptor expression plasmid, and pGEM carrier in medium containing 10%
resin-charcoal-stripped fetal bovine serum by the calcium phosphate
coprecipitation technique.25 To assay the transactivating
activity of endogenous retinoid receptor in NB4 and U937 cells, cells
were transfected with 5 µg of TK-REx3-LUC reporter, 5 µg of
pCMX--gal, and 10 µg of pGEM carrier by electroporation using a
Gene Pulser (Bio-Rad, Hercules, CA).1
Western blot for PML/RAR- protein.
PML/RAR- protein was expressed in COS-7 cells using Lipofectin
(GIBCO BRL). After the cells were treated with a test compound for 2 days, total cellular protein was extracted28 and separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and
electrophoretically transferred from the gel onto a polyvinylidene
difluoride microporous membrane (Immobilon; Millipore, Bedford, MA).
After blocking with Block Ace (Snow Brand Milk Products), the membrane
was immunoblotted with a rabbit polyclonal antibody to RAR- (Santa
Cruz Biotechnology, Santa Cruz, CA) and visualized with a
biotin-avidin-alkaline phosphatase system (Vectastain ABC system;
Vector, Burlingame, CA). To examine the expression of PML/RAR-
protein in NB4 cells, crude nuclear extracts were prepared and a rabbit
polyclonal antibody to PML was used.29
Statistical evaluation.
Statistical analyses were performed using an unpaired two-tailed
Student's t-test.
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RESULTS |
Effects of 9CTT on the growth and differentiation of human leukemia
cells.
TT, an -tocopherol ester of ATRA, induces granulocytic
differentiation in human promyelocytic leukemia HL-60
cells.20 Because ATRA is clinically used to treat APL, we
studied the effects of TT on the differentiation of APL-derived NB4 and
HT93 cells. TT at concentrations of up to 1.5 × 105mol/L only slightly affected proliferation and
NBT reduction, which is a marker of myelomonocytic differentiation, in
NB4 cells, but did not affect either proliferation or NBT reduction in
HT93 cells (Table 1 and
Fig 2A). Next, we examined the effect of
9CTT, which is an -tocopherol ester of 9CRA and a Z-isomer of TT
(Fig 1), on the proliferation and differentiation of APL cells. 9CTT at
1.5 × 105 mol/L inhibited the proliferation of
NB4 and HT93 cells to 44% and 36% of that in the control,
respectively, and increased the percentage of cells in G1 phase (Table
1). In contrast to TT, 9CTT at up to 1.5 × 105
mol/L effectively and concentration-dependently induced the
NBT-reducing activity of NB4 and HT93 cells (Fig 2A). Morphologically,
9CTT induced the differentiation of these cells into myelocyte- and metamyelocyte-like cells, in that the cytoplasm became less basophilic, nucleoli disappeared, and nuclei were slightly lobulated, whereas TT
did not affect the morphology (Table 1). The expression of CD11b
antigen, another marker of myelomonocytic differentiation, was assayed
by a flow cytometer with a very sensitive Immuno-4 histogram analysis
program.23 TT and 9CTT increased CD11b expression by NB4
and HT93 cells, and 9CTT was more potent than TT in both cell lines
(Table 1). Thus, 9CTT is more potent in inducing the differentiation of
APL-derived NB4 and HT93 cells than TT.
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| Fig 2.
Effects of TT and 9CTT on the NBT-reducing activity of
myeloid leukemia cells. Effects of TT ( ,  ) and 9CTT ( ,  ) on
APL-derived NB4 (, ) and HT93 (, ) cells (A) are compared
with those of ATRA (, ) and 9CRA (, ) on NB4 (, ) and
HT93 (, ) cells (B). Cells (5 × 104 cells/mL) were
treated with TT, 9CTT, ATRA, or 9CRA for 4 days. Values represent the
means ± SD of three separate experiments.
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Because TT and 9CTT are derivatives of ATRA and 9CRA, respectively, the
effects of ATRA and 9CRA on the differentiation of APL-derived cells
were compared. As previously reported,30 ATRA and 9CRA
similarly induced NBT reduction in both cell lines, and a drastic
difference in inducing this activity, as observed between TT and 9CTT,
was not observed between ATRA and 9CRA (Fig 2B).
The effects of TT and 9CTT on other leukemia cells were examined. TT
and 9CTT inhibited the proliferation of monoblastic leukemia U937 cells
and induced morphologic differentiation into myelocytic cells without
-naphthyl acetate esterase activity or CD14 expression (Table 1 and
data not shown). The expression of CD11b and NBT-reducing activity in
U937 cells were only slightly induced by 9CTT (Table 1 and Fig 2C).
HL-60 cells are promyelocytic leukemia cells that do not possess
t(15;17) chromosomal rearrangement and are derived from
FAB-M2.31 TT and 9CTT at 1.5 × 105
mol/L minimally affected the differentiation of these cells (Table 1
and Fig 2C). 9CTT only slightly induced the NBT-reducing activity of
myeloblastic ML-1, monoblastic THP-1, P39/TSU, and P31/FUJ cells, and
their induction was marginal, compared with that in APL-derived NB4 and
HT93 cells (Fig 2C). Therefore, 9CTT was a potent and selective inducer
of differentiation in APL-derived leukemia cells.
Effects of RAR antagonists on the differentiation of NB4 cells
induced by 9CTT.
We examined the effect of LE540, which is an antagonist for
RAR,32 on the differentiation of NB4 cells induced by 9CTT. LE540 inhibited the transactivation induced by ATRA through RAR- and
RAR- on TK-TREpx2-LUC reporter (data not shown). As shown in
Fig 3A and B,
LE540 inhibited the NBT-reducing activity of NB4 cells induced by ATRA
or 9CRA and slightly counteracted growth inhibition. LE540 effectively
reversed the growth inhibition induced by 9CTT and completely inhibited
the NBT-reducing activity induced by 9CTT (Fig 3C and D). The reversing
action of LE540 on growth inhibition and NBT reduction in NB4 cells
treated with 9CTT was more potent than that in cells treated with ATRA
or 9CRA. 9CRA at 3 × 109 mol/L and 9CTT at
6 × 106 mol/L induced a similar mean
intensity of CD11b expression in NB4 cells (3.0 U). LE540 also
inhibited the CD11b expression (Fig 3E). The NBT-reducing activity and
CD11b expression induced by 9CTT were also inhibited by another RAR
antagonist, Ro41-525333 (data not shown). These findings
suggest that the action of 9CTT in APL cells is mainly mediated by RAR.
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| Fig 3.
Effects of the RAR antagonist LE540 on growth inhibition
and differentiation in NB4 cells induced by ATRA, 9CRA, TT, or 9CTT. The effects of LE540 on growth inhibition (A) and NBT-reducing activity
(B) in NB4 cells induced by ATRA (6 × 109 mol/L
[] or 6 × 108 mol/L []) or 9CRA (6 × 109 mol/L [] or 6 × 108 mol/L
[]) were examined. Next, the effects of LE540 on growth inhibition
(C) and NBT-reducing activity (D) induced by 9CTT at 6 × 106 mol/L ( ) or 9 × 106 mol/L
( ) were examined. LE540 alone () did not affect growth or NBT
reduction. *P < .05, compared with values in the absence of
LE540 in (A) through (D). The effects of LE540 on CD11b expression that
was induced equally by 3 × 109 mol/L 9CRA and 6 × 106 mol/L 9CTT are shown in (E). *P < .05, compared with 9CRA. Cells (5 × 104 cells/mL) were treated
with ATRA, 9CRA, TT, or 9CTT in the absence or presence of LE540 for 4 days. Values represent the means ± SD of three separate
experiments.
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Activation of retinoid receptors by 9CTT.
CV-1 cells were transfected with receptor plasmid and luciferase
reporter plasmid to examine the luciferase-inducing activity of TT and
9CTT on the transactivation of retinoid receptors. TK-TREpx2-LUC and
TK-mRARx2-LUC respond to RAR1,2,25 and TK-CRBPII-LUC
responds to RXR.26 We used ATRA and LGD106934
as positive controls for RAR and RXR, respectively. As shown in
Table 2, when using TK-TREpx2-LUC as a
reporter, TT and 9CTT showed agonistic action to RAR-, RAR-, and
RAR-. TT and 9CTT also stimulated the transactivating activity of
RAR- and RAR- on the TK-mRARx2-LUC reporter and that of
endogenous RAR on the TK-REx3-LUC reporter. The effect of 9CTT on
the activation of RARs in these systems was similar to that of TT. We
also examined the receptor selectivity of TT and 9CTT by using
GAL4-chimeric receptor. Because the TK-GAL4-UASx4-LUC reporter does not
respond to mammalian nuclear factors, the interaction between a ligand and a GAL4-chimeric receptor can be analyzed without
interference.27 TT interacted with GAL4-RAR-,
GAL4-RAR-, and GAL4-RAR-, whereas 9CTT activated GAL4-RAR- and
GAL4-RAR-, but to a lesser extent than TT (Table 2). 9CTT did not
effectively affect RXR- under TK-CRBPII-LUC reporter or GAL4-RXRs
under GAL4-UASx4-LUC reporter. Thus, although there are some
differences among the receptor-reporter systems, TT and 9CTT are
agonistic for RARs and not effective on RXRs. Next, we investigated the
interaction of TT and 9CTT with PML/RAR- fusion protein. TT and 9CTT
activated the transactivating action of PML/RAR- on the
TK-TREpx2-LUC reporter, but less than that of RAR- (Table 2). When
PML/RAR- was cotransfected with RAR- or RAR-, the fusion
protein inhibited the transactivating function of RAR- and RAR-
induced by TT and 9CTT. The PML/RAR- protein also inhibited the
transactivating activity of endogenous receptors stimulated by TT and
9CTT on the TK-REx3-LUC reporter. Thus, as previously reported for
ATRA,9 the fusion protein acts in a dominant negative
manner on RARs in stimulation by TT and 9CTT. These findings indicate
that the interaction with retinoid receptors is not involved in the
predominance of 9CTT over TT in inducing the differentiation of
APL-derived cells.
We examined the luciferase-inducing activity of TT and 9CTT on
transactivation via endogenous retinoid receptors in leukemia cells.
The TK-REx3-LUC reporter was used because it is highly sensitive in
response to the addition of a ligand via endogenous RARs without the
cotransfection of expression plasmids for RARs.1 TT and
9CTT concentration-dependently induced luciferase activity in
APL-derived NB4 and non-APL U937 cells (Fig
4). In these cells, the effect of 9CTT on transactivation in the
RAR-responsive reporter system was similar to that of TT.
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| Fig 4.
Induction of the transactivation of endogenous RARs on an
RAR-responsive element by TT or 9CTT in APL-derived NB4 (A) and non-APL
U937 (B) cells. Cells were transfected with TK-REx3-LUC reporter by
an electroporation technique and treated with TT or 9CTT for 24 hours.
Luciferase activities were adjusted for the efficiency of transfection
by cotransfected -galactosidase activities. ATRA and 9CTA at 1 × 10-6 mol/L induced the luciferase activity to 7.7 and 16.6 U, respectively, in NB4 cells and 24.8 and 54.8 U, respectively, in
U937 cells. Values represent the means of three separate experiments.
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Interaction of 9CTT with PML/RAR- protein in transfected COS-7
cells and NB4 cells.
TT and 9CTT similarly stimulated transactivation via RARs in assay
systems of transient transfection, but 9CTT induced the differentiation
of APL-derived cells more effectively than TT. Recently, the
PML/RAR- protein was reported to be downregulated by treatment with
ATRA, and this phenomenon was suggested to be one of the mechanisms for
the action of ATRA on APL cells.28,29 COS-7 cells were
transfected with PML/RAR- expression vector and the interaction of
the fusion protein with TT and 9CTT was examined. As shown in
Fig 5A, 9CTT concentration-dependently
downregulated the expression of the PML/RAR- protein, whereas TT had
only a slight effect. 9CTT at 3 × 106 mol/L
obviously decreased the expression of PML/RAR- protein and at higher
concentrations of 1.2 to 1.5 × 105 mol/L
markedly decreased it to faint levels. In contrast, the decrease
induced by TT at 1.5 × 105 mol/L was slight.
In NB4 cells, 9CTT also downregulated the PML/RAR- protein more
strongly than had TT (Fig 5B). Thus, the downregulation of
the expression of the PML/RAR- protein by 9CTT may lead to its
selective induction of differentiation in APL cells.
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| Fig 5.
Expression of PML/RAR- protein in
pml/RAR--introduced COS-7 cells (A) and NB4 cells (B) that
had been treated with TT or 9CTT. (A) COS-7 cells were transfected with
a pml/RAR- expression vector, pCMX-PML-RAR-, by
lipofection and treated with various concentrations of TT or 9CTT for 2 days. Total cellular protein from 105 cells was separated
on a 10% sodium dodecyl sulfate-polyacrylamide gel and blotted onto an
Immobilon membrane. The PML/RAR- protein was detected with a rabbit
polyclonal antibody to RAR- and visualized with a
biotin-avidin-alkaline phosphatase system. The amount of the fusion
protein was quantified with a densitometer and compared with that in
the untreated cells. Two additional experiments showed similar results.
(B) NB4 cells (2 × 105/mL) were treated with TT or 9CTT
for 4 days and the crude nuclear extracts (20 µg) were separated on a
gel. The fusion protein was detected with an anti-PML antibody.
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Effects of 9CTT on the differentiation of NB4 cells induced by ATRA,
9CRA, and other retinoid analogues.
We examined the effects of 9CTT on the differentiation of NB4 cells
treated with ATRA or 9CRA. TT slightly enhanced the differentiation of
NB4 cells induced by ATRA, and the differentiation-enhancing effect of
9CTT was slightly greater than that of TT
(Fig 6A). TT also enhanced the NBT-reducing
activity of NB4 cells induced by 9CRA. Interestingly, 9CTT enhanced the
activity induced by 9CRA significantly more than TT (Fig 6B).
9CRA-induced CD11b expression by NB4 cells was also augmented by 9CTT
(Fig 6C).
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| Fig 6.
Effects of TT and 9CTT on the differentiation induced by
ATRA or 9CRA in NB4 cells. The enhancing effects of TT and 9CTT on the
NBT reduction induced by ATRA (A) and 9CRA (B) and those on CD11b
expression in the cells induced by 9CRA (C) were examined. Cells (5 × 104 cells/mL) were cultured with ATRA or 9CRA in the
absence () or presence of 3 × 106 mol/L TT () or
9CTT ( ) for 4 days. Values represent the means of three separate
experiments. *P < .05, compared with the effects of TT.
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Next, the effects of 9CTT in combination with synthetic retinoids on
the differentiation of NB4 cells were examined. Am80 and Am580 are
specific for RAR- and RAR- and Ch55 binds to RAR-, RAR-,
and RAR-, but not to cytoplasmic retinoic acid binding protein
(CRABP).18,35 Re80 is a very strong ligand for RAR-, RAR-, and RAR- and Am555s binds to RAR- more effectively than to RAR-.18,35 All of these synthetic retinoids inhibited
the proliferation of NB4 cells and induced NBT-reducing activity and CD11b expression in these cells (Table 3).
Combined with these retinoids at low concentrations, 9CTT at 3 × 106 mol/L, which alone had only marginal effects,
augmented growth inhibition and differentiation as reflected by NBT
reduction and CD11b expression (Table 3). Thus, 9CTT also enhanced the
differentiation of NB4 cells induced by synthetic retinoids.
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Table 3.
Effects of Combinations of 9CTT and Retinoid Analogues
on Growth Inhibition, NBT Reduction, and CD11b Expression in NB4
Cells
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Effects of the combination of 9CTT and VD3 on the
differentiation of leukemia cells.
Because TT enhanced the differentiation of several myelomonocytic
leukemia cells induced by VD3,20 the effects of
9CTT plus VD3 on the differentiation of leukemia cells were
examined. VD3 at concentrations from 3 × 109 to 3 × 107 mol/L was
combined with 3 × 106 mol/L TT or 9CTT. TT and
9CTT at this concentration barely induced NBT-reducing activity in NB4,
HT93, U937, and HL-60 cells (Fig 7).
VD3 at up to 3 × 107 mol/L was not
effective in inducing NBT-reducing activity in NB4 cells (Fig 7A). TT
slightly induced this activity in NB4 cells in combination with 3 × 107 mol/L VD3. On the other
hand, 9CTT effectively induced this activity in cells treated with
VD3 even at 3 × 109 mol/L (Fig
7A). TT and 9CTT at 3 × 106 mol/L did not
inhibit the proliferation of NB4 cells, and VD3 at 3 × 107 mol/L inhibited proliferation to 73% of
that in the control. TT plus VD3 inhibited proliferation to
40% and 9CTT plus VD3 inhibited proliferation to 19%
(Table 4). TT and 9CTT did not induce
lysozyme activity, which is a marker of monocytic differentiation, in
NB4 cells, but 9CTT enhanced the activity induced by VD3
more effectively than TT. VD3 at 3 × 107 mol/L increased CD11b expression, but 9CTT
decreased the CD11b expression induced by VD3. 9CTT plus
VD3 effectively increased CD14 expression in NB4 cells
(Table 4). In cells treated with 9CTT plus VD3, the nucleus
was slightly indented, nucleoli disappeared, and cytoplasm became
abundant and less basophilic and contained some vacuoles. The activity
of -naphthyl acetate esterase was increased in NB4 cells
that had been treated with 9CTT plus VD3 (Table 4). These
findings indicated that 9CTT plus VD3 induced monocytic
differentiation of NB4 cells more effectively than TT plus
VD3. 9CTT plus VD3 also effectively induced
NBT-reducing activity in HT93 cells, whereas TT plus VD3
did not (Fig 7B). TT enhanced the NBT-reducing activity of HL-60 and
U937 cells induced by VD3, as previously
reported,20 and 9CTT enhanced the activity by
VD3 slightly more effectively than TT (Fig 7C and D). The
enhancing effect of 9CTT on the differentiation induced by
VD3, compared with that of TT, was more prominent in NB4
and HT93 cells than in U937 and HL-60 cells. Therefore, in combination with VD3, 9CTT also selectively induced the differentiation
of APL-derived cells.
View larger version (17K):
[in this window]
[in a new window]
| Fig 7.
Effects of the combination of TT or 9CTT with
VD3 on the NBT-reducing activity of APL-derived NB4 (A) and
HT93 cells (B) and non-APL-derived HL-60 (C) and U937 cells (D). Cells
(5 × 104 cells/mL) were treated with VD3 in
the absence () or presence of 3 × 106 mol/L TT
() or 9CTT () for 4 days. Values represent the means ± SD of
three separate experiments. *P < .05, where the enhancing effects of 9CTT on VD3 were compared with those of TT.
|
|
View this table:
[in this window]
[in a new window]
|
Table 4.
Effects of TT and 9CTT in Combination With
VD3 on Growth Inhibition and Differentiation of Human
Promyelocytic Leukemia NB4 Cells
|
|
| |
DISCUSSION |
We previously reported the effects of TT on the differentiation of
myelomonocytic leukemia cells,20 and in this study we investigated the effects of its Z-isomer, 9CTT. TT and 9CTT are -tocopherol esters of ATRA and 9CRA, respectively. Although TT has
been reported to be stable in vivo and in in vitro treatment with
esterase,36 it is still unclear whether 9CTT acts on
leukemia cells by being catabolized to 9CRA. Zhu et al30
showed that ATRA and 9CRA similarly inhibit the proliferation of APL
cells and induce differentiation, with 9CRA being only slightly more potent than ATRA,30 and we confirmed this similarity
between ATRA and 9CRA in NB4 and HT93 cells, as shown in Fig 2. On the other hand, 9CTT effectively induced the differentiation of APL-derived cells, whereas TT had little or no effect. In the presence of VD3, a difference was also observed between TT and 9CTT in
inducing the differentiation of APL cells. 9CTT enhanced the
differentiation induced by 9CRA more effectively than TT on 9CRA or
9CTT on ATRA. These findings suggest that 9CTT acts on leukemia cells
differently than 9CRA, although we cannot completely rule out the
possibility that a very small amount of 9CTT is converted to 9CRA in
the cells. TT increases the migration of guinea pig peritoneal
macrophages, whereas ATRA does not, and TT stimulates, whereas ATRA
inhibits, the proliferation of human skin fibroblasts.36
Thus, -tocopherol esterification of retinoic acids may produce
different biological effects.
Because RAR antagonists inhibited the differentiation of NB4 cells
induced by 9CTT, 9CTT may act on the cells through RARs. 9CTT and TT
similarly activated RARs when TK-TREpx2-LUC or TK-mRARx2-LUC was
used as a reporter. 9CTT also activated GAL4-RAR chimeric receptors,
but less effectively than TT. Because members of the nuclear hormone
receptor family, including RARs, interact with several
cofactors,37,38 the interaction between a ligand and the
receptor complex may change in the GAL4 chimera system. These transient
expression experiments showed that 9CTT is an agonist for RARs, but the
predominance of 9CTT over TT with regard to their effects on RARs was
not observed. TT did not activate RXRs and 9CTT had only a marginal
effect on RXRs. -Tocopherol esterification of 9CRA leads to the loss
of its agonistic action on RXRs. The synthetic arotinoids Ro 13-7410 and St80 are potent activators of RARs, but the corresponding Z-isomers
(9-cis isomers), Ro 18-8093 and St88, respectively, are less
active on RARs and not active at all on RXRs.39
Z-Isomerization of RAR agonists may not simply correlate with the
acquisition of a response to RXRs. Recently, geranyl geranoic acid and
its derivatives were shown to activate RARs and RXRs.40 The
relationship between the structure of retinoids and their selectivity
for receptors is too complex to be elucidated.
APL cells have a specific fusion protein, PML/RAR-, which retains
the ligand-binding and DNA-binding domains of RAR-.1,2 TT and 9CTT similarly induced the transactivating activity of PML/RAR- on TK-TREpx2-LUC and TK-mRARx2-LUC reporters (Table 2).
On the other hand, PML/RAR- exhibited a dominant negative effect on
RAR- under stimulation with ATRA,1,2 and transfection of
the pml/RAR- gene to HL-60 cells makes the cells resistant to ATRA.9 PML/RAR- inhibited the transactivation induced
by TT and 9CTT via RAR- and RAR- on TK-TREpx2-LUC and
TK-mRARx2-LUC and via endogenous receptors on TK-REx3-LUC.
Recently, Gianni et al41 reported that Am580 has selective
differentiating effects on APL cells and induces the transactivating
activity of PML/RAR- more effectively than ATRA. However, Am580 also
induces the differentiation of non-APL HL-60 cells more potently than
ATRA.18 In testing 43 retinoids, including ATRA and Am580,
for their differentiation-inducing activities in HL-60 and NB4 cells, a
good linear correlation was found for these cells, and selectivity of
Am580 for NB4 cells was not observed.42 Grignani et
al43 showed that, in an experiment using deletion mutants
of PML/RAR-, transactivating activity does not necessarily correlate
with the capacity to block differentiation. The fusion protein shows
both positive and negative results with regard to transactivation
depending on the assay system, including cells, receptors, and
reporters. Thus, the induction of transactivation through PML/RAR-
by a ligand, which may appear to be a possible mechanism, is simply not
considered to lead to selectivity for APL cells.
9CTT induced transactivation on RAR-responsive reporter as effectively
as TT in a transient expression system in CV-1 cells cotransfected with
the pml/RAR- gene and in APL-derived NB4 cells. Recently,
PML/RAR- protein was reported to be downregulated in treatment with
ATRA, and it suggests that this is a mechanism for the induction of
differentiation in APL cells by ATRA.28,29 We investigated
the interaction of 9CTT with PML/RAR- protein and found that 9CTT
diminished the expression of PML/RAR- protein more effectively than
TT. These findings suggest that the destruction of PML/RAR- protein
is important in the selectivity for the induction of differentiation.
Esterification of 9CRA with -tocopherol may disturb its fixing into
a binding pocket in the receptor, as suggested from the chemical
structure of 9CTT, and could contribute to making PML/RAR- protein
susceptible to degradation. The differentiation of NB4 cells induced by
9CTT was inhibited by RAR antagonists and enhanced by potent RAR
agonists. Recently, Chen et al44 reported that arsenic
trioxide downregulates PML/RAR- protein and induces apoptosis of APL
cells, but the differentiation of these cells is only partial. These
findings suggest that both downregulation of PML/RAR- protein and
RAR activation are necessary to induce the differentiation of APL
cells. PML/RAR- forms a complex with itself, PML, or RXR, and, in
addition to the dominant negative effect on RAR, blocks VDR function by
sequestering RXR in the cytoplasm.45 The downregulation of
PML/RAR- protein by 9CTT may contribute to its potent enhancement of
differentiation induced by VD3 in NB4 and HT93 cells. The
interaction of a ligand with a nuclear receptor is very complicated.
For example, an RXR ligand influences the function of LXR in the
RXR/LXR heterodimer without liganding to LXR, and an RXR antagonist
behaves as a phantom ligand for RAR in RXR/RAR.38,46
Further studies are required to elucidate the functions of the
PML/RAR- complex and its interaction with various drugs. Although
9CTT activated the transactivation of RAR-responsive promoter in U937
cells as well as in NB4, its effects on differentiation in non-APL
cells were much less than those in APL-derived cells. This may be
because leukemogenesis in APL cells is mainly due to the abnormal
protein PML/RAR-, whereas that in non-APL cells is due to other
mechanisms.
-Tocopherol esterification of ATRA reduces retinoid-related
toxicities, including teratogenicity.36 -Tocopherol has
been reported to ameliorate the toxicity of 13-cis retinoic
acid in a trial against myelodysplastic syndrome.47 These
findings suggest that the retinoid-related toxicities of 9CTT are
weaker than those of ATRA and 9CRA and should be further examined in
animal models. U937 cells into which the pml/RAR- gene was
introduced and APL-derived NB4 cells are reportedly resistant to
VD3, and this sensitivity is restored in combined treatment
with ATRA.48 APL-derived NB4 and HT93 cells are more
resistant to VD3 than HL-60 and U937 cells, and 9CTT
drastically restored the sensitivity to VD3 in APL cells, whereas TT had little or no additional effect. Furthermore, 9CTT enhanced the differentiation of APL cells induced by other retinoids, such as ATRA, 9CRA, and synthetic retinoids, as shown in Fig 6 and
Table 3. 9CTT enhanced the differentiation induced by ATRA and 9CRA
more effectively than TT. ATRA is successfully used in the treatment of
APL,3,13 and 9CRA has pharmacologic advantages over ATRA
because it is ineffective in inducing its own catabolism and it also
induces a complete remission in APL patients.49 Am80 and
Am580 are strong agonists for RAR- and RAR- and induce granulocytic differentiation of HL-60 cells more effectively than ATRA.18,35 Am80 reportedly induced a complete remission in an APL patient relapsing after ATRA treatment and was less toxic on
skin than ATRA because it does not interact with RAR-.50 Am580 is reportedly selective for APL cells,41 and the
enhancing effect of 9CTT on the differentiation of NB4 cells by Am580
is interesting. The different mechanisms of Am580, which preferentially activates PML/RAR-,41 and 9CTT, which downregulates
PML/RAR- protein, may contribute to their synergy in inducing
differentiation in APL cells. Ch55 binds to RAR-, RAR-, and
RAR- but not to CRABP, which regulates the serum concentration of
ATRA, and induces the differentiation of HL-60 and NB4 cells more
effectively than Am80.18,42 Re80 is also a very potent
inducer of the differentiation of HL-60 and NB4 cells.18,42
Am555s activates RAR- more effectively than RAR- and, in addition
to its antileukemic activity, was recently reported to inhibit the
liver metastasis of intrasplenically transplanted human gastric cancer
in nude mice.51 Because these synthetic retinobenzoic acids
have strong agonistic activity for RARs, the enhancing effects of 9CTT
may not be due to the same agonistic activity for RARs but to other
mechanisms such as the downregulation of PML/RAR- protein.
Therefore, 9CTT, alone or in combination with VD3 or other
retinoids, may be useful for the treatment of APL.
| |
FOOTNOTES |
Submitted July 29, 1997;
accepted February 10, 1998.
Supported in part by Grants for Cancer Research from the Ministry of
Education, Science, Sports and Culture and from the Ministry of Health
and Welfare, Japan.
Address reprint requests to Yoshio Honma, PhD, Department of
Chemotherapy, Saitama Cancer Center Research Institute, 818 Komuro, Ina-machi, Kita-adachi, Saitama 362, Japan; e-mail:
honma{at}saitama-cc.go.jp.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Drs Kohei Inomata, Toshihiro Takahashi, and Takao
Kishie (Pharmaceutical Research Center, Nisshin Flour Milling Co,
Saitama, Japan) for synthesizing and kindly providing TT and 9CTT and
Dr Shin-ichi Hayashi (Department of Biochemistry, Saitama Cancer Center
Research Institute) for his helpful discussions regarding the
electroporation technique.
| |
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Abstract
Introduction
Abstract