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Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2225-2233
1,25-Dihydroxyvitamin D3 Induces Differentiation of a
Retinoic Acid-Resistant Acute Promyelocytic Leukemia Cell Line (UF-1)
Associated With Expression of p21WAF1/CIP1 and
p27KIP1
By
Akihiro Muto,
Masahiro Kizaki,
Kenji Yamato,
Yohko Kawai,
Maiko Kamata-Matsushita,
Hironori Ueno,
Masahiro Ohguchi,
Tatsuji Nishihara,
H. Phillip Koeffler, and
Yasuo Ikeda
From the Division of Hematology and Clinical Laboratories, Keio
University School of Medicine, Tokyo; the Department of Molecular
Cellular Oncology/Microbiology, Faculty of Dentistry, Tokyo Medical and
Dental University, Tokyo; the Division of Oral Science, The National
Institute of Infectious Diseases, Tokyo, Japan; and the Division of
Hematology/Oncology, Cedars-Sinai Medical Center, University of
California-Los Angeles School of Medicine, Los Angeles, CA.
 |
ABSTRACT |
Retinoic acid (RA) resistance is a serious problem for patients with
acute promyelocytic leukemia (APL) who are receiving all-trans
RA. However, the mechanisms and strategies to overcome RA resistance by
APL cells are still unclear. The biologic effects of RA are mediated by
two distinct families of transcriptional factors: RA receptors (RARs)
and retinoid X receptors (RXRs). RXRs heterodimerize with
1,25-dihydroxyvitamin D3
[1,25(OH)2D3] receptor (VDR), enabling their
efficient transcriptional activation. The cyclin-dependent kinase (cdk)
inhibitor p21WAF1/CIP1 has a vitamin
D3-responsive element (VDRE) in its promoter, and 1,25(OH)2D3 enhances the expression of
p21WAF1/CIP1 and induces differentiation of
selected myeloid leukemic cell lines. We have recently established a
novel APL cell line (UF-1) with features of RA resistance.
1,25(OH)2D3 can induce growth inhibition and G1
arrest of UF-1 cells, resulting in differentiation of these cells
toward granulocytes. This 1,25(OH)2D3-induced
G1 arrest is enhanced by all-trans RA. Also,
1,25(OH)2D3 (10 10 to
107 mol/L) in combination with RA markedly inhibits
cellular proliferation in a dose- and time-dependent manner. Associated
with these findings, the levels of
p21WAF1/CIP1 and
p27KIP1 mRNA and protein increased in these cells.
Northern blot analysis showed that p21WAF1/CIP1 and
p27KIP1 mRNA and protein increased in these cells.
Northern blot analysis showed that p21WAF1/CIP1 and
p27KIP1 transcripts were induced after 6 hours'
exposure to 1,25(OH)2D3 and then decreased to
basal levels over 48 hours. Western blot experiments showed that
p21WAF1/CIP1 protein levels increased and became
detectable after 12 hours of 1,25(OH)2D3
treatment and induction of p27KIP1 protein was much
more gradual and sustained in UF-1 cells. Interestingly, the
combination of 1,25(OH)2D3 and RA markedly
enhanced the levels of p27KIP1 transcript and
protein as compared with levels induced by
1,25(OH)2D3 alone. In addition, exogenous
p27KIP1 expression can enhance the level of CD11b
antigen in myeloid leukemic cells. In contrast, RA alone can induce G1
arrest of UF-1 cells; however, it did not result in an increase of
p21WAF1/CIP1 and p27KIP1
transcript and protein expression in RA-resistant cells. Taken together, we conclude that 1,25(OH)2D3 induces
increased expression of cdk inhibitors, which mediates a G1 arrest, and
this may be associated with differentiation of RA-resistant UF-1 cells
toward mature granulocytes.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
RETINOIC ACID (RA) and vitamin
D3 have profound effects on the growth and differentiation
of hematopoietic cells, as well as other cell types.1-3
All-trans RA and its stereoisomer, 9-cis RA, induce
differentiation and inhibit proliferation of human leukemic cell lines
such as HL-60 cells and fresh leukemic cells from patients with acute
promyelocytic leukemia (APL).3-6 1,25-Dihydroxyvitamin
D3 [1,25(OH)2D3] is a biologic
active form of vitamin D3 that functions primarily in
calcium homeostasis in vitro. 1,25(OH)2D3 can
also induce some myeloid leukemic cell lines to differentiate along the
monocyte/macrophage pathway and can prolong the survival of leukemic
mice.7,8 These biologic effects of RA and
1,25(OH)2D3 are mediated by their respective nuclear receptors, retinoic acid receptor (RAR), retinoid X receptor (RXR), and vitamin D3 receptor (VDR).9 They are
members of the steroid receptor superfamily and act as ligand-inducible
transcription factors by binding to their cognate responsive DNA
elements, termed the RA-responsive element (RARE) and vitamin
D3-responsive element (VDRE).10-12 RAR and VDR
heterodimerize with RXR to bind DNA with high affinity and effect
efficient transcriptional activation of target genes.13-15
Inhibition of the G1/S transition induces growth arrest and monocytic
differentiation of both HL-60 and U937 cells, which is mediated by a
block of cell cycle progression at the G1 phase.16,17 The
p21WAF1/CIP1 protein is a cyclin-dependent kinase
(cdk) inhibitor that is one of the regulators of cell cycle progression
in the G1/S transition. Exposure of HL-60 cells to
1,25(OH)2D3 causes transient overexpression of
p21WAF1/CIP1.17 Further studies showed
that the p21WAF1/CIP1 promoter contains a VDRE, and
exposure of U937 myelomonoblasts to 1,25(OH)2D3
results in transcriptional activation of
p21WAF1/CIP1, probably through a ligand/VDR/VDRE
interaction.18 Another report has suggested that
p27KIP1 protein is also a strong candidate as a
cell cycle regulator that blocks the entry into S phase of
1,25(OH)2D3-treated HL-60 cells.19
Several clinical studies have recently reported that all-trans
RA achieved a complete remission in most patients with APL via an
induction of differentiation of the leukemic cells in
vivo.20-23 Nevertheless, most patients who received
continuous treatment with RA relapsed and developed RA-resistant
disease.24 However, the mechanisms and strategies to
overcome RA resistance by APL cells are still unclear. Recently, we
established a novel APL cell line (UF-1) with RA-resistant features
that can be used as a model for studies on RA resistance in APL
cells.25 In this study, we report that
1,25(OH)2D3 can overcome RA resistance in this
model system of APL.
| |
MATERIALS AND METHODS |
Cells and chemicals.
The RA-resistant UF-1 promyelocytic leukemia cell line was established
in our laboratory from a patient with APL in relapse who had received
all-trans RA.25 HL-60 and NB4 promyelocytic cells
are RA-responsive leukemic cells (the latter being a gift from Dr M. Lanotte, Hôpital St Louis, Paris, France).26 The cells were maintained in RPMI 1640 medium (GIBCO-BRL; Grand Island, NY)
with 10% fetal calf serum ([FCS] Hyclone Laboratories, Logan, UT),
100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified atmosphere with 5% CO2. All-trans RA was purchased
from Sigma Chemical Co (St Louis, MO), and
1,25(OH)2D3 was a generous gift from Chugai
Pharmaceutical Co (Tokyo, Japan). They were dissolved in 100% ethanol
to a stock concentration of 1 mmol/L, stored at  20°C, and
protected from light. The morphology and cytochemistry of the cells
were evaluated from cytospin slide preparations with Giemsa and
naphthol AS-D chloroacetate esterase staining.
Assays for cellular proliferation.
Cellular proliferation was measured by cell viability and a
nonradioactive cell proliferation assay system (MTT assay;
Boehringer-Mannheim, Indianapolis, IN). Cells (2 × 103)
were incubated with chemicals for 4 days in 96-well plates (Flow Laboratories, Irvine, CA). Ten microliters of MTT
(3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide, 5 µg/mL) was added to each well. The reaction was stopped after 4 hours
of incubation by adding 100 µL 0.04N HCl in isopropanol, and
absorbance at 570 nm was determined.
Flow cytometric analysis.
For analysis of cellular differentiation, expression of cell-surface
antigens was determined by an immunofluorescence staining technique.
Cells were incubated for 30 minutes with human AB serum (Sigma) to
block Fc receptors and then stained using fluorescein isothiocyanate
(FITC)-conjugated mouse anti-human CD14 and phycoerythrin (PE)-conjugated mouse anti-human CD11b antibodies (Becton Dickinson, Mountain View, CA). Control studies were performed with nonbinding control mouse IgG1 and IgG2a isotype antibodies
(Becton Dickinson). Cells were analyzed by a Cytron Absolute Flow
Cytometer (Ortho Diagnostic Systems, Raritan, NJ).
Cell cycle analysis.
Cells (1 × 106) were suspended in hypotonic solution
(0.1% Triton X-100, 1 mmol/L Tris hydrochloride, pH 8.0, 3.4 mmol/L
sodium citrate, and 0.1 mmol/L EDTA) and stained with 5 µg/mL
propidium iodide. Analysis was performed immediately after staining
using the CELLFIT program (Becton Dickinson).
RNA isolation and Northern blotting.
Total RNA was extracted using the Isogen kit (Nippon Gene, Tokyo,
Japan) according to the manufacturer's instruction. Total RNA (15 µg
per lane) was electrophoresed on formaldehyde-agarose gels (GIBCO-BRL)
and transferred to nitrocellulose membranes (Hybond N+;
Amersham Japan, Tokyo, Japan). The filters were hybridized with 32P-labeled probe for 24 hours at 42°C in 50% formamide,
2X SSC (1X SSC is 150 mmol/L NaCl plus 15 mmol/L sodium citrate, pH
7.0), 5X Denhardt solution, 0.1% sodium dodecyl sulfate (SDS), 10%
dextran sulfate, and 100 µg/mL salmon sperm DNA. Filters were washed
to a stringency of 0.1X SSC at 65°C and exposed to Kodak XAR film (Eastman Kodak, Rochester, NY). Autoradiograms were exposed for 24 hours to 3 days. Densitometry was performed for signal intensity on
autoradiograms of Northern blots using a digital densitometer.
DNA probes.
The plasmid containing human p21WAF1/CIP1 cDNA
(NotI-NotI; 2.1 kb) was kindly provided by Dr B. Vogelstein (Johns Hopkins University, Baltimore, MD),27 and
the human p27KIP1 cDNA was purified from the
pBluescript II SK plasmid (EcoRI-EcoRI, 1.5 kb; a gift
from Dr J. Massagué, Memorial-Sloan Kettering Cancer Center, New
York, NY).28 These probes were labeled with [ -32P]dCTP using the rediprime DNA labeling
system (Amersham, Arlington Heights, IL). The specific
activity was approximately 2 × 108 cpm/µg DNA.
Western blotting.
Cells were collected by centrifugation at 1,200 rpm for 10 minutes, and
then the pellets were resuspended in lysis buffer (1% Nonidet P-40, 1 mmol/L, 40 mmol/L Tris hydrochloride, pH 8.0, and 150 mmol/L NaCl) at
4°C for 15 minutes. Protein concentrations were determined using the
Bio-Rad protein assay system (Bio-Rad, Richmond, CA). Cell lysates (10 µg protein per lane) were fractionated in 12.5% SDS-polyacrylamide
gels before transfer to the membrane (Immobilon-P membranes; Millipore,
Bedford, MA) by standard protocol. The membrane was blocked overnight
with 5% defatted milk in phosphate-buffered saline (PBS) at 4°C. The
anti-p21WAF1/C1P1 -p27KIP1,
-p15INK4B, and -p16INK4A
antibodies (Calbiochem, Cambridge, MA) were used at 1:1,000 dilution in
5% defatted milk in PBS for 1 hour at 25°C. Subsequently, membranes were incubated with anti-rabbit Ig conjugated with horseradish peroxidase (1:3,000; Amersham) for 1 hour at 25°C. All steps were followed by three washes for 5 minutes in PBS, 1% defatted milk, and
0.2% Tween 20. Antibody binding was detected using the enhanced chemiluminescence kit for Western blotting detection with hyper-ECL film (Amersham). Blots were stained with Coomassie brilliant blue and
confirmed to contain a similar amount of protein extract on each lane.
Plasmid and transfection.
The full-length human p27KIP1 cDNA plasmid
(pBluescript II SK-hp27) was used as the template in a polymerase chain
reaction to amplify the human p27KIP1 cDNA fragment
(codons 2 to 199) that was cloned into the BglII and
BamHI sites of the pEGFP-C1 expression vector (Clontech, Palo Alto, CA), which contains a cytomegalovirus (CMV) IE
promoter and green fluorescent protein (GFP) reporter gene. The
resultant plasmid was designated pEGFP-hKIP1. The pEGFP-neo was used as a control plasmid. Exponentially growing cells were suspended in RPMI
1640 containing 10% FCS at a density of 2 × 107/mL.
Cells were transfected with plasmids by electroporation using a Gene
Pulser II (Bio-Rad) set at 240 V, 1,000 µF. A pulse was delivered to
300 µL cell suspension and 30 µg plasmid DNA. After 48 hours of
electroporation, cells were stained with PE-conjugated mouse anti-human
CD11b antibody (Becton Dickinson) and analyzed for CD11b positive cells
in the GFP-positive fraction by two-color flow cytometry.
| |
RESULTS |
Morphologic changes induced in RA-resistant APL cells (UF-1) cultured
with 1,25(OH)2D3.
UF-1 cells in control cultures showed large round nuclei and numerous
granules in the cytoplasm, as found with hypergranulocytic promyelocytes (Fig 1A). Incubation with
107 mol/L 1,25(OH)2D3 for 4 days
resulted in differentiation of UF-1 cells, as evidenced by nuclear
maturation and lobulation, and a diminished number of granules in the
cytoplasm (Fig 1B). Naphthol AS-D chloroacetate esterase was positive
for both control and 1,25(OH)2D3-treated UF-1
cells (Fig 1C and D), consistent with these cells being of the
granulocytic lineage.
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| Fig 1.
Morphologic and cytochemical changes in RA-resistant APL
cells by 1,25(OH)2D3. UF-1 cells were cultured
with either 107 mol/L
1,25(OH)2D3 (B and D) or culture media alone (A
and C) for 4 days, and cytospin slides were prepared and stained with
either Giemsa (A and B) or naphthol AS-D chloroacetate esterase (C and
D). Original magnification ×1,000.
|
|
Effects of 1,25(OH)2D3 on cellular
proliferation of UF-1 cells.
UF-1 cells were cultured in the presence of various concentrations of
1,25(OH)2D3 (1010 to
107 mol/L) with or without all-trans RA
(107 mol/L) for 4 days (Fig
2A) or treated with the indicated agents for different times (Fig 2B). As previously reported,25
all-trans RA alone did not effect growth of UF-1 cells, as
reflected by a lack of change in their absorbance in the MTT assay. In
contrast, 1,25(OH)2D3 inhibited the cellular
proliferation and decreased the number of viable cells in a dose
(1010 to 107 mol/L)- and time (0 to 96 hours)-dependent manner. Interestingly, the combination of
107 mol/L all-trans RA and
1,25(OH)2D3 markedly inhibited cell growth in a
dose- and time-dependent manner (Fig 2A and B). We next
examined the effect of 1,25(OH)2D3 and
all-trans RA on cell cycle progression in UF-1 cells. The cells
were incubated with either 107 mol/L all-trans
RA, 1,25(OH)2D3, or a combination of both for 48 hours and analyzed for cell cycle distribution by flow cytometry (Table 1). Cultivation with
1,25(OH)2D3 for 48 hours increased the
population of cells in G1 phase from 80.2% to 93.3% with a reduction
of cells in S phase from 13.8% to 5.0%. Consistent with the cellular
proliferation assay, the combination of all-trans RA and
1,25(OH)2D3 significantly increased the number
of cells in the G1 phase of the cell cycle (98.4%) compared with
control cells (80.2%), with a concomitant decrease in the S phase
(Table 1). These increased populations of G1 phase in
1,25(OH)2D3-treated cells represent G1-arrested
cells.
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| Fig 2.
Dose- and time-dependent effects of
1,25(OH)2D3 and/or all-trans RA
on cellular proliferation of UF-1 cells. RA-resistant UF-1 cells were
cultured in 96-well plates with various concentrations
(1010 to 107 mol/L) of
1,25(OH)2D3 for 4 days (A) or for different
times (0-96 hours) with the indicated compounds (B), and then MTT
incorporation was measured. Absorbance at 570 nm (OD570)
was recorded using an enzyme-linked immunosorbent assay plate reader.
Results are presented as the mean of triplicate experiments; the SD was
within 10% of the mean.
|
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Functional evidence for differentiation of UF-1 cells to granulocytes
by 1,25(OH)2D3.
Induction of the differentiation of UF-1 cells to granulocytes by
1,25(OH)2D3 was assessed by the expression of
CD11b and CD14 antigens (Figs 3 and
4). All-trans RA
(107 mol/L) did not alter the expression of CD11b and
CD14 antigens by fluorescent-activated cell sorter (FACS) analysis
compared with control cells. 1,25(OH)2D3
(109 to 107 mol/L) induced CD11b positive
cells in a dose-dependent manner from 1% to 66%. The combination of
various concentrations of 1,25(OH)2D3 plus
all-trans RA (107 mol/L) greatly enhanced the
expression of CD11b antigen as compared with either agent alone.
However, expression of CD14 antigen was unchanged after exposure to
either 1,25(OH)2D3 alone or the combination of
RA and 1,25(OH)2D3 (Figs 3 and 4). These
results are consistent with the morphologic and cytochemical studies,
suggesting that 1,25(OH)2D3 induces
differentiation of UF-1 cells down the granulocytic pathway and RA can
enhance this effect.
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| Fig 3.
Expression of CD11b and CD14 antigens by FACS analysis.
UF-1 cells were treated with either 107 mol/L
all-trans RA, various concentrations (109 to
107 mol/L) of 1,25(OH)2D3, or
combinations of both for 4 days. Cells were incubated for 30 minutes
with human AB serum to block Fc receptors and then stained with direct
immunofluorescence using FITC-conjugated mouse anti-human CD14 and
PE-conjugated mouse anti-human CD11b antibodies. Control studies were
performed with nonbinding control mouse IgG1 and
IgG2a isotype antibodies.
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| Fig 4.
Graphic representation of the FACS analysis of CD11b and
CD14 antigens shown in Fig 3. Results show the differentiation-inducing
activities of all-trans RA,
1,25(OH)2D3, and the combination of both
chemicals.
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Expression of p21WAF1/CIP1 and
p27KIP1 mRNA in HL-60, NB4, and UF-1 cells.
1,25(OH)2D3 induced the differentiation of UF-1
cells in association with a block of cell cycle progression at the G1
phase. Therefore, to address the mechanisms of action of
1,25(OH)2D3 on RA-resistant APL cells, we
examined the expression of cdk inhibitors in UF-1 cells.
1,25(OH)2D3 can induce HL-60 cells to
differentiate into monocyte/macrophage-like cells. Forty-eight hours'
incubation of 1,25(OH)2D3 (107
mol/L) was sufficient to commit HL-60 cells to differentiate along the
monocyte/macrophage pathway (data not shown). HL-60 cells were cultured
for 48 hours in the presence of 107 mol/L
1,25(OH)2D3, and then the expression of
p21WAF1/CIP1 and p27KIP1
transcripts was examined by Northern blotting (Fig
5). Low levels of
p21WAF1/CIP1 mRNA were detected in the control
cells, whereas the accumulation of p21WAF1/CIP1
mRNA increased after the cells were exposed to
1,25(OH)2D3, suggesting that the induction of
p21WAF1/CIP1 was associated with the induction of
differentiation of HL-60 cells to monocytes/macrophages. NB4 cells are
genetically different from HL-60 cells in that they possess a
reciprocal chromosomal translation, t(15;17), which is characteristic
of APL cells.26 NB4 cells respond to
1,25(OH)2D3 differently from HL-60 cells. Recent reports have shown that 1,25(OH)2D3
alone failed to induce the differentiation of NB4 cells to
monocytes/macrophages.29,30 In NB4 cells,
p21WAF1/CIP1 mRNA was not detectable
constitutively, and 1,25(OH)2D3 did not enhance
the expression of these transcripts. In contrast, constitutive expression of p21WAF1/CIP1 and
p27KIP1 transcripts was observed in RA-resistant
UF-1 cells, and the levels of p21WAF1/CIP1 and
p27KIP1 transcripts were not changed after 48 hours' exposure to all-trans RA (107 mol/L) but
were slightly increased after 48 hours' exposure to 1,25(OH)2D3 (107 mol/L) (Fig 5).
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| Fig 5.
Expression of p21WAF1/CIP1 mRNA in
HL-60 and NB4 cells, and p21WAF1/CIP1 and
p27KIP1 transcripts in UF-1 cells. Cells were
cultured with either 107 mol/L
1,25(OH)2D3 (A) or 107 mol/L
all-trans RA (B) for 48 hours. Total RNA (15 µg per lane) was
extracted and analyzed by Northern blot with
[32P]-labeled p21WAF1/CIP1 or
p27KIP1 cDNA probe. Bottom panel shows the ethidium
bromide-stained formaldehyde gel before Northern blotting; the levels
of 28S and 18S ribosomal RNA are comparable in each lane.
|
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Time course of p21WAF1/CIP1 and
p27KIP1 induction by
1,25(OH)2D3.
1,25(OH)2D3-induced expression of cdk inhibitor
p21WAF1/CIP1 was examined more closely by comparing
transcript and protein levels over a time frame of 0 to 72 hours
following the addition of 1,25(OH)2D3 either
with or without all-trans RA to UF-1 cells (Figs
6, and 8A and B). The related cdk inhibitor
p27KIP1 was also included in this analysis (Figs
7 and 8A and B). The levels of
p21WAF1/CIP1 and p27KIP1
transcripts increased about twofold after 6 hours of exposure to
1,25(OH)2D3, and then the expression decreased
toward baseline levels over the remaining 42 hours (Figs 6 and 7).
Western blot experiments showed that p21WAF1/CIP1
protein levels became detectable after 6 hours and peaked at 12 hours
of 1,25(OH)2D3 (107 mol/L)
treatment. The level of p27KIP1 protein increased
by 3 hours and continued to increase at 72 hours of culture with
1,25(OH)2D3 (107 mol/L) (Fig
8A). The dissociation between the early and
transient mRNA induction of p27KIP1 and the late
and sustained appearance of the corresponding protein suggests that
posttranscriptional regulation is imposed on this cdk inhibitor.
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| Fig 6.
Time course of p21WAF1/CIP1 mRNA
induction of expression by either 1,25(OH)2D3
alone or combined with RA in UF-1 cells. Cells were cultured for
various durations (0 to 48 hours) with 107 mol/L
1,25(OH)2D3 (left) or 107 mol/L
of both RA and 1,25(OH)2D3 (right). Northern
blot analysis of p21WAF1/CIP1 mRNA was
performed by blotting total RNA (15 µg per lane). Equal loading of
RNA in each lane was confirmed by ethidium bromide staining of the
formaldehyde gel; thus, the densitometric reading for the relative
levels of p21WAF1/CIP1 transcript was compared with
that of untreated cells.
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| Fig 7.
Time-dependent effects of either
1,25(OH)2D3 alone or in combination with RA on
levels of p27KIP1 mRNA in UF-1 cells. Cells were
cultured for the indicated durations with either 107
mol/L 1,25(OH)2D3 alone or in combination with
107 mol/L RA. Total RNA (15 µg per lane) was blotted
and hybridized with a p27KIP1 cDNA probe. Bottom
panel shows the ethidium bromide-stained gel with 28S and 18S
ribosomal RNA demonstrating equivalent RNA-loading per lane. Expression
of p27KIP1 transcripts relative to the expression
in control cells was determined by densitometry.
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| Fig 8.
Western blot analysis of p21WAF1/CIP1
and p27KIP1 protein. (A) Total cellular protein (10 µg per lane) from UF-1 cells treated for 3-72 hours with
107 mol/L 1,25(OH)2D3 was
separated on a 12.5% SDS-polyacrylamide gel and transferred to the
membrane. p21WAF1/CIP1 and
p27KIP1 protein levels were detected by Western
blotting using antibodies directed against
p21WAF1/CIP1 and p27KIP1, and
then the blots were stained with Coomassie brilliant blue to confirm
that equal amounts of protein were present in each lane. (B) Enhanced
expression of p27KIP1 protein by exposure of cells
to the combination of 107 mol/L all-trans RA and
1,25(OH)2D3 for 48 hours.
|
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All-trans RA combined with 1,25(OH)2D3
did not further enhance the expression of
p21WAF1/CIP1 mRNA and protein (Figs 6 and
8B). However, p27KIP1 transcripts and proteins were
strongly induced after 6 hours' exposure to the combination of
1,25(OH)2D3 and all-trans RA compared with 1,25(OH)2D3 alone (Figs 7 and 8B). These
findings paralleled the synergistic ability of these two ligands to
induce the differentiation of UF-1 cells toward granulocytes. We also
examined the ankyrin family of cdk inhibitors that includes
p15INK4B and p16INK4A. However,
these INK4 proteins were not detectable by Western blot analysis
following 1,25(OH)2D3 treatment (data not shown).
Forced expression of p27KIP1 induces cellular
differentiation of myeloid cells.
The results of our expression studies suggest an association of
p27KIP1 expression and myeloid cell differentiation
induced by 1,25(OH)2D3. To further explore this
association, we constructed a GFP-p27KIP1 fusion
protein-expressing plasmid (pEGFP-hKIP1) and a control plasmid
(pEGFP-neo) and transfected them into myeloid leukemic cells. The
slow-growing promyelocytic NB4 and UF-1 cells were impossible to
transfect effectively; therefore, our experiments focused on HL-60
cells. This expression vector contains the GFP reporter gene, and thus,
the plasmid-introduced cell population was positive for green
fluorescence. The cell population that expressed green fluorescence was
evaluated for differentiation by analysis of expression of the CD11b
antigen by two-color flow cytometry. The transient expression of
p27KIP1 in HL-60 cells resulted in a rightward
shift of the fluorescence intensity compared with control
vector-transfected cells (Fig 9). This
clearly suggests that forced expression of p27KIP1
can result in cellular differentiation of leukemic cells.
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| Fig 9.
Flow cytometric analysis for markers of cellular
differentiation on p27KIP1-transfected HL-60 cells.
Cells were transiently transfected with either pEGFP-hKIP1 or pEGFP-neo
control plasmid by electroporation. Transfected cells were cultured for
48 hours and then stained with PE-conjugated mouse anti-human CD11b
antibody. Expression of CD11b antigen in the GFP-positive fraction was
analyzed by two-color flow cytometry.
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| |
DISCUSSION |
To date, the mechanisms of RA resistance in APL cells are still
unclear, and most approaches have not been successful in overcoming RA
resistance in vivo.31,32 Cell differentiation is regulated in a cell cycle-dependent manner; for example, differentiation of
hematopoietic cells is associated with a loss of cell cycling capacity,
and the cells become arrested in the G0/G1 phase of the cell
cycle.33 Also, the principal block to cell cycle
progression in 1,25(OH)2D3-treated human cells
is known to occur in the G1 phase.16,34 In the present
study, we showed that RA-resistant UF-1 cells differentiated toward
granulocytes when cultured with 1,25(OH)2D3,
and this was associated with the G1 arrest of these cells and an
increased expression of the cdk inhibitors
p21WAF1/CIP1 and p27KIP1.
Consistent with the induction of p21WAF1/CIP1 and
p27KIP1 transcripts in UF-1 cells after a short
exposure to either 1,25(OH)2D3 or
1,25(OH)2D3 combined with all-trans RA,
the inhibition of cellular proliferation occurred within 6 to 12 hours.
These data suggest that an increase of p21WAF1/CIP1
and p27KIP1 transcripts associates with cyclin-cdk
complexes during the first 12 hours of exposure to
1,25(OH)2D3 and is capable of inhibiting the
kinase activities with these complexes, leading to G0/G1 arrest and
differentiation of the myeloid leukemic cells.
1,25(OH)2D3 transduces its signal through the
DNA-binding transcription factor, VDR.9 Therefore,
ligand-inducible effects on cell growth and differentiation are
initiated through the direct activation or repression of target genes
by VDR. Many genes are reportedly regulated by
1,25(OH)2D3; however, none of these genes have
been shown to be direct targets for VDR regulation. Recently, Liu et
al18 reported that the induced differentiation of the myelomonocytic leukemia cell line U937 by
1,25(OH)2D3 was facilitated by transcriptional
stimulation of the p21WAF1/CIP1 gene by VDR. We
demonstrated that 1,25(OH)2D3, but not
all-trans RA, upregulated the expression of
p21WAF1/CIP1 and p27KIP1
transcripts and proteins in RA-resistant UF-1 cells during granulocytic differentiation. Also, prior studies showed that
1,25(OH)2D3 did not increase the accumulation
of p21WAF1/CIP1 mRNA in RA-responsive NB4 cells;
indeed, other biologic effects of 1,25(OH)2D3
in NB4 cells may occur independently of VDR/VDRE action.35
Furthermore, a transient elevation in the expression of
p21WAF1/CIP1 and/or
p27KIP1 in U937 cells in the absence of
1,25(OH)2D3 enhanced the expression of CD11b
and CD14 antigens.18 Consistent with our study, several studies have linked the enhanced expression of
p21WAF1/CIP1 in myeloid leukemic cells to their
induced differentiation.16-18,34 These data suggest that
1,25(OH)2D3 induces increased expression of
p21WAF1/CIP1 and p27KIP1, which
mediates a G1 arrest; this may be closely associated with the terminal
differentiation of myeloid leukemic cells.
Other studies have suggested that the cdk inhibitor
p27KIP1 is the principal mediator of the
antiproliferative action of 1,25(OH)2D3 on
HL-60 cells, by showing that the induction of
p27KIP1 but not p21WAF1/CIP1
correlated with the onset of G1 arrest.19 We found in UF-1 cells that p21WAF1/CIP1 protein levels increased
and became detectable after 12 hours of
1,25(OH)2D3 treatment, while the induction of
p27KIP1 protein was much more gradual and sustained
in UF-1 cells. Also, all-trans RA alone did not modulate the
expression of p21WAF1/CIP1 and
p27KIP1 transcripts. The combination of RA and
1,25(OH)2D3 cooperatively enhanced the levels
of p27KIP1 transcript and protein but not
p21WAF1/CIP1 mRNA and protein in UF-1 cells.
Recently, Casaccia-Bonnefil et al36 reported that
oligodendrocyte progenitor cells derived from
p27KIP1-knockout mice showed impaired growth arrest
and differentiation after removal of a mitogen in the culture, thus
demonstrating for the first time that p27KIP1 is an
important component of the machinery required for the G0/G1 transition.
In addition, we demonstrated that exogenous expression of
p27KIP1 in HL-60 cells can enhance the level of
expression of the CD11b antigen, demonstrating directly the role of
p27KIP1 in cellular differentiation. Taken
together, p27KIP1 may be more important than
p21WAF1/CIP1 for the G1 arrest and
granulocytic differentiation of UF-1 cells induced by
1,25(OH)2D3.
Interestingly, all-trans RA greatly enhanced
1,25(OH)2D3-induced differentiation of UF-1
cells to granulocytes, whereas RA alone did not induce differentiation
of the cells. Although all-trans RA alone was able to induce G1
arrest of UF-1 cells (Table 1), the reason it did not inhibit the
cellular growth is unclear. All-trans RA alone did not
upregulate the expression of p21WAF1/CIP1 and
p27KIP1 in UF-1 cells, and therefore, induction of
G1 arrest may be a necessary, but insufficient, condition for cellular
differentiation in UF-1 cells. Cell cycle arrest and activation of cdk
inhibitors may be important for induction of cellular differentiation.
The mechanisms underlying acquired RA resistance in APL patients have remained elusive. Recent studies have shown that mutations in the
ligand-binding domain of RAR or PML/RAR genes are related to the
development of RA resistance in some patients with
APL.37,38 Moreover, it has been reported that RA can
dissociate the nuclear receptor corepressor/histone deacetylase complex
from PML/RAR, which relieves transcriptional repression and leads to
activation of genes involved in the terminal differentiation of APL
cells.39-42 Since p21WAF1/CIP1 might be
a transcriptional target of RARs, a RARE in the promoter region of the
p21WAF1/CIP1 gene was required to confer RA-induced
cellular differentiation.43 Taken together, our results
suggest that the failure to induce cdk inhibitors by the
ligand-activated RAR through RARE in the promoter region of their
target genes may explain, in part, the RA resistance in UF-1 cells.
Further studies will be needed to clarify the mechanisms of RA
resistance and regulation of cdk inhibitors.
The combination of 1,25(OH)2D3 and
all-trans RA enhanced p27KIP1 mRNA and
protein in RA-resistant UF-1 cells. Our group and others have already
reported the synergistic differentiation of myeloid leukemic cells by
RA and 1,25(OH)2D3, which was probably mediated through their cognate nuclear receptors.44-46 Also, we
previously found that the combination of a novel series of vitamin
D3 analogs (20-epi vitamin D3 compounds) and
9-cis RA synergistically decreased proliferation and induced
differentiation and apoptosis in APL cells.47 The mechanism
by which RA enhances the effects of 1,25(OH)2D3 remains to be clarified. RA may enhance the transactivation capacity of
VDR by receptor heterodimerization of RAR/VDR or RXR/VDR. This heterodimerization may lead to efficient transactivation of cdk inhibitors. Moreover, the role of the APL-specific PML/RAR fusion protein in these processes should be explored.
In summary, 1,25(OH)2D3 can inhibit growth in
the G1 phase of the cell cycle and induce differentiation of UF-1
RA-resistant APL cells. These effects are associated with an increased
expression of several cdk inhibitors. Furthermore, the combination of
RA and 1,25(OH)2D3 cooperatively enhances the
expression of cdk inhibitors, G0/G1 cell cycle arrest, and
differentiation of UF-1 cells.
| |
FOOTNOTES |
Submitted December 3, 1997; accepted November 19, 1998.
Supported by grants from the Ministry of Education, Science, and
Culture of Japan, the National Grant-in-Aid for the Establishment of a
High-Tech Research Center in a Private University and the Keio
University Special Grants as well as grants from the National Institutes of Health, US Army, and Parker Hughes Fund.
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 Masahiro Kizaki, MD, Division of
Hematology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; e-mail:
makizaki{at}mc.med.keio.ac.jp.
| |
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ABSTRACT
INTRODUCTION