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Molecular Biology and Genetics |
by Granulocyte Macrophage Colony-stimulating Factor in Human Myeloid Leukemia Cell Lines1
Department of Hygiene-Chemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Tokyo 162-0826, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mRNA and protein and stimulates the binding of
nuclear proteins to direct repeat 5, a consensus sequence with high
affinity for RAR-RXR heterodimers. Furthermore, expression of CD38 mRNA
mediated through RAR is induced synergistically by treatment with
ATRA + GM-CSF. These results suggest that GM-CSF stimulates
transcriptional activity mediated via RAR in ML-1 cells. The
induction of RAR by GM-CSF may therefore be a mechanism for
stimulation by GM-CSF. The induction of RAR by GM-CSF was also
detected in other myeloid leukemia cell lines (THP-1 and KG-1) that
showed a synergistic effect similar to that seen in ML-1 cells in
response to ATRA + GM-CSF. We also found that GM-CSF induced
the expression of RAR in blood cells obtained from patients with
acute myeloid leukemia. This activity of GM-CSF may serve as a useful
adjunct to differentiation therapy for retinoic acid-nonresponsive
leukemias. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene was found to be RA resistant (5)
.
Similarly, in HL-60 cells, RA resistance was shown to be associated
with a dominant negative, structurally altered RAR in a resistant
subclone (6)
. Therefore, RARs and RXRs play an important
role in the regulation of RA signal transduction.
RA is a known inducer of cell differentiation into granulocytes. In
particular, it is known that ATRA promotes granulocytic maturation of
the human cell line HL-60 (7)
and of hematopoietic cells
from patients with APL (8
, 9)
. ATRA is effective in
differentiation therapy for APL (10
, 11)
. However,
retinoids generally do not promote granulocytic maturation in AML
subtypes, with the exception of APL. Study of the mechanism underlying
the cellular responses to ATRA in AML cells may lead to the application
of differentiation therapy using ATRA. ML-1 cells, derived from a
patient with acute myeloblastic leukemia, are at an earlier stage of
differentiation than HL-60 cells and can be easily differentiated into
the macrophage but not the granulocytic pathway by various inducers
such as tumor necrosis factor (12)
, IFN-
(13)
, and 12-O-tetradecanoylphorbol-13-acetate
(14
, 15)
. We recently succeeded in inducing
differentiation toward granulocytes in ML-1 cells by treatment with
ATRA + GM-CSF (16)
. On the basis of this result
and the results of other previous reports (7, 8, 9)
, we
speculate that GM-CSF may promote the differentiative effects of RA. We
predicted that activation of RARs and RXRs is the mechanism underlying
this promoting effect; therefore, we investigated the effect of GM-CSF
on RARs and RXRs in ML-1 cells. We found that GM-CSF induces the
expression of RAR and stimulates the binding of nuclear proteins to
RAREs in these cells. Using other AML cell lines, we found a
correlation between induction of RAR and the synergistic effect of
ATRA and GM-CSF on granulocytic differentiation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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NBT-reducing Ability.
NBT-reducing activity was assayed microscopically by the modified
method of Bachner and Nathan (17)
. Briefly,
3 x 105 cells were suspended in
96-well microplates with reagents and then incubated at 37°C. After 2
days of incubation, 0.1% NBT dye and 20 ng of
12-O-tetradecanoylphorbol-13-acetate were added to each well
and incubated at 37°C for 30 min in a 5% CO2
incubator. After incubation, the reaction was terminated by adding 50
µl of 2 N HCl to each well of a 96-well
microplate, which was then cooled on ice for 30 min. The medium was
then discarded, the formazan deposits were dissolved by adding 0.1 ml
of DMSO, and the dissolved formazan measured at 595 nm by a
spectrophotometer for the 96-well microplates (Microplate Reader model
550; Bio-Rad, Tokyo, Japan).
Morphological Changes.
The appearance of morphological changes was assessed until day 9 of
incubation. Cells were prepared on glass slides by centrifugation in a
Cytospin (Shandon Southern Products, Ltd., Cheshire, United Kingdom).
The cells were stained with May-Grüenwald’s solution for 3 min
and then stained with diluted Giemsa solution for 30 min. Cells with
the morphological characteristics of granulocytes were counted under
the microscope.
RT-PCR.
Total cellular RNA was isolated using Isogen (Wako, Osaka, Japan).
Reverse transcription of total RNA into cDNA was performed using Bulk
First-Strand cDNA Reaction Mix (Amersham Pharmacia Biotech, San
Francisco, CA). Briefly, 1 µg of total RNA was reverse transcribed
using pd(N)6 primer in a final volume of 15 µl. The total reaction
product was amplified by PCR for each cycle at 94°C for 30 s,
55°C for 1 min, and 72°C for 2 min. Amplification was carried out
for 32 cycles for RAR and 28 cycles for glyceraldehyde-3-phosphate
dehydrogenase. PCR primers were as follows: (a) RAR,
5'-CAACAGCTCAGAACAACGTG-3' (sense primer) and
5'-GATCTCCATCTTCAGCGTGA-3' (antisense primer); and (b)
glyceraldehyde-3-phosphate dehydrogenase, 5'-ATCATCAGCAATGCCTCCTG-3'
(sense primer) and 5'-CTGCTTCACCACCTTCTTGA-3' (antisense primer).
Northern Blot Analysis.
Total cellular RNA was isolated using Isogen.
Poly(A)+ RNA was purified using Oligotex dT30
(Takara, Kyoto, Japan). The poly(A)+ RNA (4 µg)
was electrophoresed on 1.2% agarose-formaldehyde gels and transferred
to Hybond-N membranes (Amersham Pharmacia Biotech). Hybridizations were
carried out according to the manufacturer’s instructions. DNA
fragments used as probes were amplified by RT-PCR using the following
primers: (a) RAR, 5'-CAACAGCTCAGAACAACGTG-3' (sense
primer) and 5'-GATCTCCATCTTCAGCGTGA-3' (antisense primer);
(b) RARß, 5'-TACTGTCGACTCCAGAAGTG-3' (sense primer) and
5'-GTGCTTCCAGCAATGGTTCT-3' (antisense primer); (c) RAR,
5'-CATCACCAAGGTCAGCAAAG-3' (sense primer) and
5'-TCTCCAGCATCTCTCGGATT-3' (antisense primer); (d) RXR,
5'-TCAATGGCGTCCTCAAGGTC-3' (sense primer) and
5'-CGCCTCCAGCATCTCCATAA-3' (antisense primer); (e) RXRß,
5'-GAAGATGTGAGCCACCAGT-3' (sense primer) and 5'-GCTCAGGGTACTTCTGTTTG-3'
(antisense primer); (f) RXR, 5'-ATGAACTACCCATCCACCAG-3'
(sense primer) and 5'-CGGATACTTCTGCTTGGTGT-3' (antisense primer); and
(g) CD38, 5'-AGTTGGGAACTCAGACCGTA-3' (sense primer) and
5'-TCCTGGC ATAAGTCTCTGGA-3' (antisense primer).
Preparation of Nuclear Protein Extracts.
Nuclear extracts were isolated according to the following method. The
cells were lysed in lysis buffer [10 mM Tris-HCl (pH 7.5),
3 mM MgCl2, 10 mM NaCl,
and 0.5% NP40] and the nuclei were collected, resuspended in nuclear
resuspension buffer [250 mM Tris-HCl (pH7.8), 60
mM KCl, 1 mM DTT, and 1 mM
phenylmethylsulfonyl fluoride], and lysed by three cycles of
freeze/thaw. The nuclear extracts were spun, and supernatants were
frozen immediately and stored at -80°C. Protein concentration was
determined by using the Bio-Rad Protein Assay.
Western Blot Analysis.
Western blot analysis was performed using the method described
previously (18)
. The denatured nuclear extracts were
analyzed by 10% SDS-PAGE and transferred to a polyvinylidene
difluoride membrane. The blots were blocked at 4°C using 5% nonfat
milk overnight in PBST (10 mM PBS and 0.05% Tween 20) and
incubated for 2 h at room temperature with the monoclonal
anti-RAR antibody from Affinity Bioreagents and the polyclonal
anti-RXR antibody (D-20) from Santa Cruz Biotechnology.
The blots were then washed three times and incubated for 1 h with
horseradish peroxidase-conjugated secondary antibodies. Proteins were
visualized using the enhanced chemiluminescence detection system
(Amersham Pharmacia Biotech).
Gel Mobility Shift Assay.
Nuclear protein (2 µg) was incubated with binding buffer [10
mM Tris-HCl (pH 7.5), 50 mM NaCl, 1
mM DTT, 0.5% glycerol, and 3 µg of salmon sperm DNA] in
a total volume of 20 µl at 20°C for 15 min. One ng of
32P-labeled double-stranded probe DR5
(5'-AGCTTCAGGTCACCAGGAGGTCAGAGAGCT-3') was added, and the
reaction mixture was incubated for another 20 min. For the competition
experiment, a 50-fold excess of unlabeled RAR gel shift
oligonucleotides (sc-2559 or sc-2560; Santa Cruz Biotechnology) was
added to incubations with the labeled fragments. For antibody
experiments, samples were preincubated with the anti-RAR or
anti-RXR antibody (Santa Cruz Biotechnology) for 15 min before the
addition of probe. DNA-protein complexes were electrophoresed on native
6% polyacrylamide gels in 0.25x Tris-borate EDTA buffer. Gels were
vacuumed, dried, and exposed to Fuji X-ray film at -70°C.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the expression of RAR markedly increased (3-fold) 3 h after
treatment with ATRA + GM-CSF. RAR and RXR also
increased (1.4- and 2-fold, respectively) at 3 h, returning to
control levels after 24 h. RARß was not detected under control
conditions but was induced slightly after treatment for 2 days. RXRß
was expressed basally and decreased slightly during differentiation.
RXR was not detected in differentiation-induced cells or in
untreated cells (data not shown).
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mRNA and Protein Expression in ML-1 Cells., RAR, and RXR
in ML-1 cells treated with GM-CSF or ATRA. The expression of RAR and
RXR genes was increased by GM-CSF but not by ATRA. In contrast,
RAR expression was increased by ATRA but was minimally altered by
treatment with GM-CSF (Fig. 2A
). To determine whether the increased gene expression by
GM-CSF results in corresponding increases in the respective proteins,
we performed Western blot analysis with specific antibodies, and we
detected induction of RAR but not RXR protein 3 h after
treatment with GM-CSF (Fig. 2B
).
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in ML-1 cells, it is
likely that the amount of receptor bound to RARE is also modulated
after GM-CSF treatment. To investigate the effect of GM-CSF on the
binding of nuclear receptors to RAREs, nuclear proteins were extracted
from untreated and GM-CSF-treated ML-1 cells for gel mobility shift
assay. The DR5 sequence that encodes two copies of AGGTCA in a DR
separated by 5 nucleotides was used as a probe. DR5 is
preferentially bound by RAR/RXR heterodimers. As shown in Fig. 3
, the total amount of nuclear protein bound to DR5 was increased after
3 h of GM-CSF treatment. GM-CSF-stimulated binding was blocked by
the addition of excess cold RAR gel shift oligonucleotide but not by
the addition of a mutant oligonucleotide. Furthermore, the DNA and
protein complexes were shifted to a position indicating slower
migration when the extracts were treated with specific antibodies
against RAR and RXR. This shift was not detected when normal
rabbit serum was used. Thus, these observations suggest that GM-CSF
increases the amount of RAR-RXR binding to DR5 in ML-1 cells.
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.
CD38 induction may be a useful marker for studying retinoid action in
myeloid cells. To investigate the effect of GM-CSF on transcriptional
activity via RAR, we examined the expression of the CD38 gene in
ML-1 cells treated with GM-CSF and/or ATRA. CD38 gene expression was
induced synergistically by treatment with GM-CSF + ATRA in
these cells. Induction of gene expression began within 3 h of
combined treatment and occurred faster than that induced by treatment
with ATRA alone. Moreover, the level of CD38 mRNA in ML-1 cells treated
with both regents was 2–4-fold higher than that seen in response to
ATRA alone (Fig. 4
). These observations indicate the possibility that GM-CSF stimulates
transcriptional activity via RAR in ML-1 cells.
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in Other Leukemia Cells Treated with GM-CSF. by GM-CSF
and the synergistic effect of granulocytic differentiation by ATRA + GM-CSF, we examined the alteration of NBT-reducing activity by
ATRA and/or GM-CSF and the induction of RAR by GM-CSF in other
leukemia cell lines. As shown in Fig. 5A
, the KG-1 and THP-1 cell lines showed a synergistic effect
in response to ATRA + GM-CSF. In contrast, HL-60, a human
promyelocytic leukemia cell line that shows granulocytic
differentiation in response to treatment with RA or DMSO, showed high
NBT-reducing activity in response to treatment with ATRA alone and no
synergistic effect in response to treatment with ATRA + GM-CSF. K562 cells did not show induction of NBT-reducing activity
after treatment with ATRA and/or GM-CSF. To investigate the
differentiation levels of KG-1 and THP-1 cells, we examined the
morphological change induced by GM-CSF and/or ATRA in KG-1 and THP-1
cells. As shown in Table 1
, treatment with ATRA + GM-CSF synergistically induced
morphological changes in KG-1 and THP-1 cells similar to those seen in
ML-1 cells after ATRA + GM-CSF treatment. This result
suggests that treatment with ATRA + GM-CSF synergistically induces
differentiation of KG-1 and THP-1 cells to granulocytes. We also
examined the expression of RAR in these cell lines after treatment
with GM-CSF alone (Fig. 5B
). Interestingly, the KG-1 and
THP-1 cell lines that showed synergistic differentiation were induced
to express the RAR gene by GM-CSF. In contrast, HL-60 cells that did
not show a synergistic effect were induced slightly to express the gene
as compared to KG-1 and THP-1 cells. K562 cells were not induced to
express the gene. These results raise the possibility that the
expression of the RAR gene by GM-CSF is associated with a
synergistic effect of ATRA + GM-CSF. Finally, to investigate
the effect of GM-CSF on primary AML cells, we examined the induction of
RAR by GM-CSF in short-time cultured peripheral blood cells from two
AML patients (FAB-M2 and FAB-M5a) using RT-PCR. As in ML-1
cells, GM-CSF induced the expression of RAR mRNA in these two
primary AML cell cultures (Fig. 6
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in
these cells, and we showed a correlation between the induction of
RAR and retinoid sensitivity in various cell lines. Data indicating
a correlation between the expression level of RARs and cellular
retinoid sensitivity have been reported previously. High levels of
RAR in estrogen receptor-positive mammary carcinoma cells are
correlated with stronger retinoid-mediated growth-inhibitory effects
when compared with estrogen receptor-negative breast carcinoma cells
(19)
. In addition, RA-resistant HL-60 cells that carry a
defective RAR can be rendered RA responsive by the introduction of
RAR, RARß, RAR, or RXR (20)
. These results
suggest that GM-CSF induction of RAR in ML-1 cells increases
cellular retinoid sensitivity and substantially advances
differentiation toward the granulocytic pathway in combination with
ATRA. We also demonstrated that GM-CSF increases the binding of nuclear
proteins to RAREs and stimulates expression of CD38 mRNA via RAR in
a ligand-dependent manner in ML-1 cells. These results indicate the
possibility that GM-CSF stimulates RAR-RXR transcriptional
activity in a ligand-dependent manner through the induction of RAR
expression. This may provide a novel pathway for GM-CSF action in
granulocytic differentiation. We also demonstrated the induction of
RAR by GM-CSF in peripheral blood cells from AML patients. This
activity of GM-CSF may therefore be useful as an adjunct for
differentiation therapy using ATRA in various AML patients.
Investigation of the mechanism of induction of the RAR gene by
GM-CSF is important because the signal or molecules related for
induction may provide targets for the stimulation of cellular response
to RA. We have little information regarding the mechanism of induction
of the RAR gene by GM-CSF. Chen et al. (21)
reported that AP-1, a transcription factor, plays an important role in
signaling the expression of the RAR, RAR, and RXR genes
stimulated by transforming growth factor ß1. The induction of RAR
by GM-CSF may be associated with AP-1 because GM-CSF induces c-fos and
c-jun, which form AP-1. The expression of RAR is regulated by
tyrosine kinase signaling pathways (22)
, and estrogen
increases the level of RAR mRNA and protein (23)
. To
examine whether the induction is transcriptional or
posttranscriptional, we performed a preliminary examination using a
run-on assay. However, we could not detect the increase in the RAR
transcription to account for the large increase in RAR mRNA. We have
not examined the alteration of RAR mRNA half-time by treatment with
GM-CSF; however, it may be that RAR induction by GM-CSF is due to
the effect on the posttranscriptional level of the mRNA.
To investigate the effect of other cytokines on RAR, we examined the
expression of RAR in ML-1 cells treated with other cytokines related
to granulocytic differentiation. Expression did not change after
treatment with various cytokines including G-CSF, macrophage
colony-stimulating factor, interleukin 3, and interleukin 6 (data not
shown). However, these observations may result from differences in
expression of each cytokine receptor in these cells because regulation
of RARs and RXRs by some cytokines has been reported. Chen et
al. (21)
reported that transforming growth factor
ß1 transcriptionally stimulates the expression of RAR, RAR, and
RXR genes. Another group of researchers (24)
observed
that IFN- increases the expression of the RAR gene. These results
suggest that the expression of RAR and RXR genes is stimulated by some
cytokines. Regulation of nuclear hormone receptors by cytokines may be
a way to control cell growth and differentiation.
In this report, we noted the possibility that induction of the RAR
gene by GM-CSF is part of the mechanism underlying synergistic
granulocytic differentiation by ATRA + GM-CSF. The
synergistic effect of cytokines and hormones has been investigated by
others, who have reported similar results concerning the mechanism of
synergistic effect. Widschwendter et al.
(24)
proposed that an IFN--mediated increase in RAR
may play a role in the synergistic inhibition of proliferation in
breast cancer cell lines after treatment with retinoids + IFN-. Another group suggested that up-regulation of the G-CSF
receptor by RA may account for the synergistic effect of G-CSF and RA
in the differentiation of APL cells (25)
. Cell growth and
differentiation are probably regulated by a complicated interaction
between cytokines and hormones. In future studies, the synergistic
effect of combinations of various cytokines and hormones should be
studied. It is also important to examine the expression of various
receptors in cancer cells. Because the sensitivity of the cell to
cytokines and hormones is affected by the expression level of
appropriate receptors, further information on the expression pattern of
receptors in target cells and on the dosages of cytokines and hormones
that are optimum for expression will probably be required for advances
in clinical treatment of APL and other forms of leukemia or,
eventually, all types of cancers. We expect the application of combined
treatment to lead to improved clinical efficacy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by a Grant-in Aid for
Scientific Research from the Ministry of Education, Science, Sports and
Culture of Japan. 
2 To whom requests for reprints should be
addressed, at Department of Hygiene-chemistry, Faculty of
Pharmaceutical Sciences, Science University of Tokyo, 12 Ichigaya
Funagawara-Machi Shinjuku-ku, Tokyo 162-0826, Japan.
3 The abbreviations used are: RA, retinoic acid;
GM-CSF, granulocyte macrophage colony-stimulating factor; ATRA,
all-trans retinoic acid; APL, acute promyelocytic
leukemia; RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE,
retinoic acid response element; AML, acute myeloid leukemia; NBT,
nitroblue tetrazolium; RT-PCR, reverse transcription-PCR; DR, direct
repeat; poly(A)+ RNA, polyadenylated RNA; G-CSF,
granulocyte colony-stimulating factor.
Received 12/ 6/99. Accepted 6/ 7/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, a promiscuous partner of retinoic acid and thyroid hormone receptors. EMBO J., 11: 1409-1418, 1992.[Medline]
retinoic acid receptor gene in a retinoic acid-nonresponsive embryonal carcinoma cell. Mol. Cell. Biol., 10: 6445-6453, 1990.). Mol. Cell. Biol., 10: 2154-2163, 1990. in induction of differentiation of human myeloid leukemia cell lines ML-1 and HL-60. Biochem. Biophys. Res. Commun., 124: 100-105, 1984.[Medline]
1 isoform is induced by estradiol and confers retinoic acid sensitivity in human breast cancer cells. Mol. Cell. Endocrinol., 109: 77-86, 1995.[Medline]
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1 gene expression: role of estrogen receptor-sp1 complex. Mol. Endocrinol., 12: 882-890, 1998.-interferon on expression of retinoic acid receptor and cellular retinoic acid-binding protein in breast cancer cells. Cancer Res., 55: 2135-2139, 1995.This article has been cited by other articles:
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  K. Kim, H. H. Kim, J. H. Kim, Y. H. Choi, Y. H. Kim, and J. Cheong Chemokine stromal cell-derived factor-1 induction by C/EBP{beta} activation is associated with all-trans-retinoic acid-induced leukemic cell differentiation J. Leukoc. Biol., November 1, 2007; 82(5): 1332 - 1339. [Abstract] [Full Text] [PDF]
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