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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4543-4553
A New Cytokine-Dependent Monoblastic Cell Line With t(9;11)(p22;q23)
Differentiates to Macrophages With Macrophage Colony-Stimulating Factor
(M-CSF) and to Osteoclast-Like Cells With M-CSF and Interleukin-4
By
Takashi Ikeda,
Kazunori Sasaki,
Kazuma Ikeda,
Genji Yamaoka,
Koichi Kawanishi,
Yasunori Kawachi,
Tatsumi Uchida,
Jiro Takahara, and
Shozo Irino
From the First Department of Internal Medicine, the Department of
Transfusion Medicine, and the Department of Clinical Laboratory
Medicine, Kagawa Medical University, Kagawa, Japan; and the Department
of Internal Medicine, Takamatsu Red Cross Hospital, Kagawa, Japan.
 |
ABSTRACT |
Monocytes/macrophages exert a series of important functions in vivo.
To facilitate detailed investigation of their functional capacity and
the mechanism leading to their differentiation, several cell lines have
been established from primary material. We present here a new human
monoblastic cell line, designated UG3. UG3 cells are characterized by
the following features. (1) UG3 cells harbor the t(9;11)(p22;q23)
translocation that results in fusion of the MLL and the AF9 genes and
produce the corresponding AF9-MLL and MLL-AF9 fusion transcripts. (2)
UG3 cells rely on the presence of exogenous growth factors for
viability and proliferation, such as interleukin-3 (IL-3),
granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte
colony-stimulating factor (G-CSF), or macrophage colony-stimulating
factor (M-CSF). (3) When cultured in the presence of G-CSF, UG3 cells
differentiate along the granulocytic lineage, as evidenced by
segmentation of nuclei and positive staining for neutrophilic alkaline
phosphatase and peroxidase. (4) When cultured in the presence of GM-CSF
or M-CSF, UG3 cells differentiate into mature macrophages while
preserving surface expression of CD14 and CD68 and also start to
release cytokines into cell-culture supernatants. Under these culture
conditions, UG3 cells also take up acetylated LDL. (5) When cultured in
the presence of M-CSF and IL-4, UG3 cells differentiate into
osteoclast-like multinucleated giant cells capable of bone resorption
and display tartrate-resistant acid phosphatase (TRAP) activity. UG3
cells thus provide features to qualify them as a useful model to
further investigate the mechanism underlying these processes and also
to further elucidate the functional role of mature
monocytes/macrophages or osteoclasts.
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INTRODUCTION |
MONOCYTES/MACROPHAGES exert a variety of
important functions, including phagocytosis, release of proinflammatory
cytokines, antigen presentation, and incorporation of lipids. Upon
appropriate stimulation, they are also capable of differentiating into
osteoclasts or dendritic cells. Several cell lines have been
established from primary material and serve as models to further study
function as well as differentiation of the monocyte/macrophage lineage. THP-1 cells,1 for example, when exposed to phorbol ester
(phorbol myristate acetate [PMA]) express
CD142 and CD36,3 display phagocytotic
ability,4 and produce tumor necrosis factor  (TNF).5 U937 cells6 incubated with PMA
express CD363 and produce TNF,5
interleukin-6 (IL-6),7 and macrophage colony-stimulating
factor (M-CSF).8 Furthermore, the combination of vitamin
D3 and a lymphokine, such as IL-2, -interferon, or  -interferon, promotes differentiation of U937 cells into
osteoclast-like cells.9 Similarly, HL60 cells incubated
with vitamin D3 or PMA also differentiate into
osteoclast-like cells.10,11 Recently, it has been pointed
out that monocytes/macrophages induced to differentiate by M-CSF differ
from those induced by granulocyte-macrophage colony-stimulating factor
(GM-CSF) in terms of terminal differentiation and
function.12-17 It has also been reported that GM-CSF spurs differentiation of peripheral blood monocytes (PBMs) into dendritic cells when combined with IL-4 and/or TNF.18,19
In contrast, M-CSF and IL-4 promote differentiation of PBMs into
osteoclasts.18 To date, no human cell line has been
established that can be maintained and induced to differentiate into
monocytes/macrophages by physiological stimuli alone. Therefore, a
detailed analysis of the molecular mechanism of differentiation into
either dendritic cells or osteoclasts has been hampered by the lack of
a suitable cell model.
We present here a novel human cell line, designated UG3, that, unlike
previous established human cell lines, including THP-1, U937, or HL60
cells, has preserved the ability to differentiate along either lineage
upon exposure to physiologic stimuli. Thus, UG3 cells may prove useful
to study the mechanism underlying the differentiation process as well
as to further elucidate the functional role of monocytes/macrophages,
even though these are established from leukemic cells. Moreover, UG3
cells carry the chromosomal translocation t(9;11)(p22;q23) and
synthesize the corresponding AF9-MLL as well as the MLL-AF9 fusion
transcripts. They may thus also represent a valuable tool to further
investigate leukemogenesis resulting from the 11q23 anomaly.
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MATERIALS AND METHODS |
Case history.
A 26-year-old Japanese woman was admitted with gingival swelling and
skin rashes. She had had no known exposure to cytotoxic drugs or
radiation and no prior history of hematological disorders. On
admission, her peripheral blood counts were 6,500/µL white blood
cells with 26.5% blast cells, 3.9 × 106/µL red
blood cells, 13.4 g/dL hemoglobin, and 8.7 × 104/µL
platelets. Bone marrow aspirate showed a hypercellular marrow infiltrated with 96.8% monoblasts. Surface markers analyzed by fluorocytometry were as follows: CD11a, 88.9%; CD11b, 38.3%; CD11c, 92.3%; CD13, 10.9%; CD14, 1.1%; CD33, 90.8%; and HLA-DR, 58.3%. Cytogenetic studies showed a consensus karyotype of 45XX,  7, t(9;11)(p22;q23). The patient was subsequently diagnosed as AML(M5). On
postchemotherapy nadir, the patient received 75 µg/d granulocyte colony-stimulating factor (G-CSF), which resulted in a marked increase
in the leukocyte count, which increased from 1,000/µL to 23,200/µL
with 84.5% blast cells. After terminating G-CSF treatment, the white
blood cell count decreased immediately to the level before G-CSF
injection. Upon G-CSF-induced leukocytosis, a peripheral blood sample
was obtained with informed consent.
Cytokines and antibodies.
Recombinant human stem cell factor (SCF), IL-3, and IL-6 were provided
by Kirin Brewery Co, Ltd (Tokyo, Japan). Recombinant human
M-CSF20 was provided by Morinaga Milk Industry Inc (Tokyo,
Japan). Recombinant human GM-CSF, G-CSF, IL-1, IL-2, IL-4, IL-5, IL-7,
and flt3 ligand were purchased from Genzyme Corp (Cambridge, MA).
Fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies (MoAbs)
against CD14, CD36, CD64, CD71, and HLA-DR and phycoerythrin
(PE)-labeled MoAbs against CD13, CD16, and CD33 were purchased from
Coulter Corp (Hialeah, FL). PE-labeled MoAb against CD11c and CD54,
biotin-conjugated MoAb against G-CSF receptor (G-CSFR),
biotin-conjugated murine IgG1 as a control, and PE-labeled avidin were
obtained from Pharmingen (San Diego, CA). PE-labeled MoAb against
CD11b, MoAb against CD68, FITC-conjugated rabbit antimouse IgG1 Ab, and
mouse IgG1 as a control were purchased from Dako Japan (Kyoto, Japan).
FITC-labeled MoAb against c-kit was obtained from Nichirei Co Ltd
(Tokyo, Japan).
Cell culture.
Fresh leukemic blasts were obtained from the peripheral blood of the
patient at marked blastosis after G-CSF injection. Cells were separated
by Ficoll-Hypaque (d = 1.077; Pharmacia LKB, Uppsala, Sweden) density
gradient centrifugation at 400g for 30 minutes. The interphase
containing mononuclear cells was harvested and then washed twice with
phosphate-buffered saline (PBS) and once with fresh Iscove's modified
Dulbecco's medium (IMDM; GIBCO BRL, Gaithersburg, MD). Cells were
cultured at a density of 2.5 × 105/mL in IMDM
supplemented with 5% fetal calf serum (FCS; CSL Ltd, Victoria,
Australia) and IL-3 (5 ng/mL) at 37°C in an atmosphere of 5%
CO2 in air. The medium was exchanged every 4 to 7 days
depending on the rate of cell growth. After 8 weeks of culture, the
cells were cloned by hand-picking a colony from semisolid culture. Some of the cells maintained with IL-3 were washed twice with PBS and then
moved to IMDM containing 5% FCS and 100 ng/mL M-CSF, 10 ng/mL G-CSF,
or 1 ng/mL GM-CSF, respectively. In all experiments, cells were
cultured for 2 weeks in the presence of one of the cytokines and then
further analyzed. Half of the culture medium was exchanged every 4 days
to fresh medium containing a cytokine at the described concentration.
In selected experiments, a combination of 100 ng/mL SCF, 20 pg/mL IL-1,
and 10 ng/mL IL-6 were also added to the culture.
Growth response to cytokines.
To measure growth response of UG3 cells under various experimental
conditions as outlined in the legends to the respective figures, MTT
assay21 was performed. Briefly, 180 µL of cells (1 to 2 × 105 cells/mL) were cultured for a period of 7 days
in the presence or absence of cytokines as indicated in the respective
figure legends in 96-well microplate. Twenty microliters of sterilized MTT [5 mg/mL; 3-(4,5-dimethyl-2,5-diphenyl-2H tetrazolium
bromide); Wako Pure Chemical Industries Ltd, Osaka,
Japan] was added to each culture well and incubated for an additional
4 hours at 37°C in a CO2 incubator. After
centrifugation of the cell culture wells, the cell-free culture
supernatants were discarded and cells were resuspended with 200 µL of
dimethyl sulfoxide (DMSO). The plates were read by a MTP-120 Microplate
Reader (Corona Electric Co, Ibaragi, Japan) at a wave length of 570 nm.
In selected experiments, cell number was determined with a
hematocytometer after trypan blue dye exclusion.
Morphological analysis.
Cells were washed twice and moved to IMDM containing cytokine and
cultured for 2 weeks in Slide Flasks (Nunc A/S, Roskilde, Denmark) to
allow for in situ evaluation of adherent cells. Nonadherent cells were
evaluated in situ on cytospin slides using Cytospin 2 (Shandon Southern
Products Ltd, Cheshire, UK). Slides were stained with
May-Grünwald-Giemsa, peroxidase, neutrophilic alkaline
phosphatase (NAP), -naphthyl butyrate esterase
(NB), and naphthol AS-D chloracetate esterase (NAC). Some slides
were also stained for tartrate-resistant acid phosphatase (TRAP) using
a commercially available kit (Sigma Diagnostics, St Louis, MO)
according to the manufacturer's instructions.
Cytogenetic analysis.
Chromosomes were analyzed using standard techniques and Giemsa Trypsin
G (GTG) bandings.
Reverse transcription-polymerase chain reaction (RT-PCR) and
sequencing.
RT-PCR and Southern blot analysis were performed to detect cytokine
receptor mRNA and fusion transcripts resulting from t(9;11). Total
cellular RNA was isolated with RNAzolB according to the manufacturer's
instructions (Biotecx Laboratories, Inc, Houston, TX). RNA
concentrations were measured spectrophotometrically using a GeneQuant
(Pharmacia LKB, Uppsala, Sweden). Complementary DNA (cDNA) was
synthesized by reverse transcription in an 80-µL reaction mixture
containing 1 µg total cellular RNA, 150 µg/mL random
hexanucleotide, and 50 U reverse transcriptase (Seikagaku Co, Tokyo,
Japan). As a control, we prepared templates containing the same
components except for reverse transcriptase. A volume of 0.5 µL cDNA
reaction mixture or control template was amplified in the presence of 1 U Thermus aquaticus DNA polymerase (Takara, Shiga, Japan), 25 mmol/L
dNTP, and 10 mmol/L of each specific primer
(Table 1) in a total volume of 20 µL in a
Thermal Cycler (Perkin-Elmer Cetus, Norwalk, CT).22 One PCR
cycle consisted of denaturation at 95°C for 1 minute, primer
annealing at 62°C for 1 minute, and extension at 72°C for 2 minutes. This cycle was repeated 40 times. An aliquot of 6 µL of each
PCR reaction was electrophoresed in 3% agarose (NuSieve; FMC
BioProducts, Rockland, ME), stained with ethidium bromide, and
transferred to a nylon membrane (Hybond N; Amersham Japan, Tokyo,
Japan). After hybridization with specific biotin-labeled oligonucleotides, the membrane was incubated with avidin-alkaline phosphatase and visualized by chemiluminescence of alkaline
phosphatase-lumigen PPD (Boehringer Mannheim Japan, Tokyo, Japan)
reaction. The nucleotide sequences of the specific primers and probes
are shown in Table 1. Nucleotide sequences of some of the amplified
fragments were determined by the cycle sequence method using ABI PRISM
Dye Primer Cycle Sequencing Core Kit (Perkin-Elmer Cetus, Norwalk, CT)
according to the manufacturer's instructions.
Clonogenic assay.
UG3 cells were moved to IMDM containing 5% FCS supplemented with 100 ng/mL M-CSF, 10 ng/mL G-CSF, or 1 ng/mL GM-CSF, respectively, after
being washed twice with PBS. After cell culture for 0, 7, or 14 days in
the presence or absence of each cytokine, cells were suspended in
semisolid IMDM containing 0.9% methyl cellulose, 30% FCS, and 1%
bovine serum albumin. One milliliter of culture medium containing 1,000 cells was incubated in duplicate in 35-mm dishes at 37°C in 5%
CO2 in a humidified incubator. After 1 week, cell
aggregates of more than 30 cells were counted as colonies. Some
colonies were picked from each dish, suspended in IMDM, and evaluated
in situ on cytospin slide preparations as described in
"Morphological analysis."
Immunophenotyping.
Most cell surface antigens were detected by standard direct
immunofluorescence assay. In brief, cells were maintained for 2 weeks
in the presence of IL-3, GM-CSF, G-CSF, or M-CSF; then incubated at
4°C for 40 minutes with FITC- or PE-conjugated mouse MoAbs; and
washed twice with PBS. After washing, 20,000 cells per sample were
analyzed using a fluorocytometric analyzer (EPICS XL; Coulter Corp,
Hialeah, FL). For G-CSFR analysis, cells were first incubated with
biotin-labeled mouse anti-G-CSFR MoAbs for 40 minutes at 4°C.
After repeated washings, cells were then incubated with
avidin-conjugated PE at 4°C for an additional 40 minutes. After
additional washings, cells were analyzed by fluorocytometry as
described above for direct immunofluorescence assay. To analyze for
CD68 expression, immunohistochemistry was performed using the Histofine
detection system (Nichirei Co Ltd, Tokyo, Japan). Three hundred cells
were counted and the percentage of cells staining positive for CD68 was
determined.
Detection of cytokines in the culture supernatant.
UG3 cells, maintained in the presence of IL-3, GM-CSF, or M-CSF for 2 weeks, were washed twice with PBS and moved to IMDM containing 5% FCS
without any cytokines, with the final cell concentration being 2 × 105/mL. After 6 hours, culture
supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA)
using commercially available kits according to the manufacturer's
instructions. The kits used for analysis were as follows: IL-1
(minimal detectable concentration [MDC], 3.9 pg/mL), IL-6 (MDC, 3.1 pg/mL), IL-8 (MDC, 31.3 pg/mL), IL-12 (MDC, 7.8 pg/mL), TNF (MDC,
15.6 pg/mL), monocyte chemoattractant protein-1 (MCP-1; MDC, 31.3 pg/mL), G-CSF (MDC, 39.1 pg/mL), M-CSF (MDC, 31.3 pg/mL), and GM-CSF
(MDC, 7.8 pg/mL), which were all supplied by R&D Systems, Inc
(Minneapolis, MN); IL-10 (MDC, 14 pg/mL) and interferon (IFN;
MDC, 0.1 U/mL), which were supplied by Medgenix (Fleurus, Belgium);
IL-15 (MDC, 10 pg/mL), which was purchased from Genzyme; and lysozyme
(MDC, 1.5 g/mL), which was obtained through Sigma.
Uptake of acetylated LDL.
UG3 cells were cultured for 2 weeks in the presence of IL-3, GM-CSF, or
M-CSF. Then,
1,1 -dioctadecyl-1-1-3,3,33-tetramethyl-indo-carbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-Ac-LDL; Biomedical Technologies Inc, Stoughton, MA) was added to the culture medium at a final concentration of 10 µg/mL. The cells were incubated for 4 hours at 37°C, washed with PBS, and then fixed and analyzed using a fluorescence microscope. To determine the percentage of cells
having taken up DiI-Ac-LDL, 300 cells were counted.
Osteoclast differentiation assay.
UG3 cells were cultured in IMDM containing 5% FCS and 100 ng/mL M-CSF
for 2 weeks, washed twice with PBS, and thereafter exposed to IMDM
supplemented with 5% FCS, M-CSF (100 ng/mL), and IL-4 (100 U/mL) for
an additional 2 weeks. Then, UG3 cells were stained for TRAP as
detailed in "Morphological analysis." To analyze for bone
resorption activity, cells were first cultured for 2 weeks in IMDM
containing 5% FCS and M-CSF (100 ng/mL) and then seeded on glass
slides covered with hydroxyapatite (Osteologic; Millennium Biologix
Inc, Kingston, Ontario, Canada) in IMDM with 5% FCS
supplemented with a combination of IL-4 (100 U/mL) and M-CSF (100 ng/mL) or IL-3 (5 ng/mL) for an additional 3 weeks before being
subjected to Von Kossa staining.23
Statistical analyses.
Statistical analyses were performed with the Student's t-test,
except for the clonogenic assay, which was evaluated with the paired
t-test.
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RESULTS |
Establishment of the cell line.
Primary mononucleated interphase cells were maintained in liquid
culture supplemented with IL-3 for 8 weeks, during which time cell
growth stabilized with a doubling time of 4 to 7 days. A cell aliquot
was then transferred to semisolid cultures supplemented with IL-3 (5 ng/mL). Individual cells were obtained by hand-picking from semisolid
cultures and were moved again to suspension culture. One cell line,
designated UG3, grew as a single-cell suspension with a doubling time
of 60 to 70 hours in the presence of IL-3. UG3 cells were round with
basophilic cytoplasm that contained azurophilic granules and had
soy-bean like nuclei with a few nucleoli (Fig 1).
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| Fig 1.
Morphology of UG3 cultured in the presence of
IL-3 (5 ng/mL). May-Grünwald-Giemsa staining. Original
magnification, 300-fold.
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Response to cytokines.
Proliferative activity was observed in the presence of IL-3, GM-CSF,
G-CSF, or M-CSF, but not in the presence of IL-1, IL-2, IL-5, IL-6,
IL-7, SCF, or flt3 ligand or in the absence of any cytokine (data not
shown). The proliferative response induced by IL-3, GM-CSF, G-CSF, or
M-CSF was dose-dependent and reached plateau at 3 ng/mL in IL-3-, 0.5 ng/mL in GM-CSF-, 10 ng/mL in G-CSF-, and 125 ng/mL in
M-CSF-supplemented cultures (data not shown). Based on our data,
including unpublished results, we decided on a
concentration for IL-3, GM-CSF, M-CSF, and G-CSF in all experiments of
5 ng/mL, 1 ng/mL, 100 ng/mL, and 10 ng/mL, respectively. Growth curves
showed a doubling time of about 60 to 70 hours in cell cultures
supplemented with IL-3 or GM-CSF and about 4 to 5 days in cell cultures
supplemented with G-CSF or M-CSF (Fig 2A).
The combination of SCF, IL-1, and IL-6 potentiated M-CSF-induced
growth of UG3 cells (Fig 2B), but not that of UG3 cells cultured with IL-3, GM-CSF, or G-CSF (data not shown). The viability of UG3 cells in
the presence of IL-3, GM-CSF, G-CSF, or M-CSF was more than 95%. The
viability of UG3 in the absence of any cytokine decreased daily and was
less than 20% at day 7 in culture.
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| Fig 2.
(A) Growth curve of UG3 cells cultured in the presence or
absence of IL-3 (5 ng/mL), GM-CSF (1 ng/mL), G-CSF (10 ng/mL), or M-CSF
(100 ng/mL). Cells were enumerated with a hematocytometer after trypan
blue dye exclusion. Error bars indicate standard deviation of cell
numbers. (B) Growth of UG3 cells cultured in the presence of M-CSF (100 ng/mL) with or without a combination of SCF (100 ng/mL), IL-1 (20 ng/mL), and IL-6 (10 ng/mL). Cells were enumerated with a
hematocytometer after trypan blue dye exclusion. Error bars indicate
standard deviation of cell numbers. *P < .01 compared with
M-CSF. Results are representative of three independent experiments.
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UG3 cells synthesize transcripts for IL-1R, IL-3R, IL-6R, IL-6R gp130,
IL-7R, c-kit, flt3, GM-CSFR, G-CSFR, and c-fms. No transcripts of EPO-R
or c-mpl were detected in UG3 cells (data not shown).
Karyotype and analysis for t(9;11) fusion transcripts.
At 9 weeks of culture after cloning of UG3 cells from semisolid
culture, cytogenetic analysis of UG3 cells showed the consensus karyotype of 46XX, 7, +8, t(9;11)(p22;q23)
(Fig 3). To detect transcripts of MLL-AF9
and AF9-MLL fusion genes resulting from t(9;11)(p22;q23), we performed
RT-PCR using various primers (Table 1 and
Fig 4A) and sequenced the amplified
fragments. Amplified fragments were detected with the following
primer-pairs: AF9s-MLLex12as, AF9s-MLLex14as, and MLLex9s-AF9as (Fig
4B). Sequencing showed that the AF9 gene was deleted at nucleotide 1320 and fused to exon 12 of the MLL gene in the AF9-MLL fusion transcript
and that the 3 end of exon 9 of the MLL gene was fused to
nucleotide 1321 of the AF9 gene in the MLL-AF9 fusion transcript.
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| Fig 3.
Karyotype of UG3 cells. The consensus karyotype of UG3
cells was 46XX, 7, +8, t(9;11)(p22;q23). Chromosome analysis was
performed twice.
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| Fig 4.
(A) MLL and AF9 break points in UG3 cells. Break points
in UG3 cells as determined by sequencing the RT-PCR products are shown with vertical black arrow heads. Primers are shown as horizontal arrow
heads; ex, exon; bps, base pairs. (B) RT-PCR analysis for AF9-MLL and
MLL-AF9 fusion transcripts in UG3 cells. RT-PCR analysis was performed
as detailed in the Materials and Methods. Amplification reactions
performed with all primer combinations failed to produce any PCR
product, with the exception of the following primer pairs: AF9s-MLLex12as (lane 1), AF9s-MLLex14as (lane 3), and MLLex9s-AF9as (lane 5). As a control, RT-PCR analysis was also performed with reversely transcribed RNA obtained from K562 cells using these primer
pairs, and reaction products were loaded into lanes 2, 4, and 6, respectively. Lane M depicts pBR322 DNA digested with Msp I as
a molecular weight marker. Shown is one representative result of four
independent experiments.
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Morphological analysis.
Specific staining showed that UG3 cells cultured with IL-3 were 98%
positive for NB esterase, 13% double-positive for NB and NAC
esterase (Fig 5A), and
negative for peroxidase and NAP (data not shown). UG3 cells cultured
with GM-CSF showed increased NB activity and decreased NAC activity
(Fig 5B) as compared with UG3 cells maintained in the presence of IL-3.
A few of the cells cultured in the presence of GM-CSF were of
macrophage-like appearance, having wide and irregular cytoplasm, and
grew adherently. In contrast, about 45% of UG3 that were cultured in
the presence of M-CSF grew adherently. Nonadherent cells obtained from
M-CSF-supplemented culture showed the same morphological
characteristics as UG3 cells that had been maintained in the presence
of IL-3, but stained 100% positive for NB and negative for NAC
esterase (Fig 5C). Adherent cells obtained from cultures maintained in
the presence of M-CSF showed macrophage-like morphology with widely
spread cytoplasm containing vacuoles (Fig
6A) and also displayed strong positivity for NB esterase (Fig 6B).
In contrast, UG3 cells cultured in the presence of G-CSF featured a
slightly irregular cytoplasm, contained granules, and showed increase
of NAC esterase positivity (Fig 5D). These cells were 4% positive for
peroxidase (Fig 7A) and 6% for NAP (Fig
7B). Some UG3 cells maintained in the presence of G-CSF had a segmented
nucleus (Fig 7A).
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| Fig 5.
Double staining for -naphthyl butyrate
esterase and naphthol AS-D chloracetate esterase of UG3 cells cultured
with IL-3 (5 ng/mL; A), GM-CSF (1 ng/mL; B), M-CSF (100 ng/mL; C), and
G-CSF (10 ng/mL; D) for 2 weeks. Original magnification, 200-fold.
Histochemical analyses were performed using samples taken from four
independent cell cultures.
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| Fig 6.
Adherent UG3 cells cultured in the presence of M-CSF (100 ng/mL) for 2 weeks. Double staining for a-naphthyl butyrate esterase and naphthol AS-D chloracetate esterase (A; original magnification, 100-fold) and phase-contrast microscopy (B; original magnification, 60-fold). Morphological analysis was performed of cells obtained from
two independent cultures.
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| Fig 7.
Peroxidase (A) and neutrophilic alkaline phosphatase (B)
staining of UG3 cells cultured in the presence of G-CSF (10 ng/mL) for
2 weeks. Original magnification, 300-fold. Histochemical analysis was
performed with samples obtained from three independent cultures.
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Immunophenotyping.
Results obtained from all surface marker expression as analyzed by
fluorocytometry are summarized in Table 2.
Positivity for CD13 was highest when UG3 cells had been cultured with
G-CSF and lowest when UG3 cells had been exposed to IL-3. CD14
expression was relatively low but increased when UG3 cells were
cultured with M-CSF or G-CSF. A majority of UG3 cells expressed CD54
when cultured with IL-3, GM-CSF, or M-CSF, but CD54 expression was low
on UG3 cells cultured in the presence of G-CSF. UG3 cells failed to
display CD68 surface expression when maintained in the presence of
IL-3, whereas CD68 was detectable on UG3 cells exposed to GM-CSF and
became strongly positive on UG3 cells that had been exposed to M-CSF
(Table 2 and Fig 8). In addition,
nonadherent large-sized cells obtained from cultures supplemented with
M-CSF were 98.6% positive for CD14 and negative for CD71 (Table 2, percentages in parentheses).
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| Fig 8.
CD68 expression was determined by immunohistochemistry of
UG3 cells cultured with IL-3 (5 ng/mL; A), GM-CSF (1 ng/mL; B), or
M-CSF (100 ng/mL; C) for 2 weeks. Original magnification, 200-fold. Histochemical analysis was performed with samples taken from three independent cultures.
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Uptake of acetylated LDL.
To detect scavenger receptor-mediated incorporation of Ac-LDL in UG3
cells, we analyzed incorporation of Ac-LDL using DiI-Ac-LDL. UG3 cells
incorporated Ac-LDL when cultured in the presence of IL-3. However,
even more Ac-LDL was incorporated in UG3 cells obtained from cultures
supplemented with either GM-CSF or M-CSF. UG3 cells that had been
exposed to M-CSF incorporated the highest amount of Ac-LDL and some of
these cells resembled foam cells. The percentages of UG3 cells that
incorporated Ac-LDL in all cells are summarized in Table
3.
Cytokine production by UG3 cells.
The concentrations of various cytokines secreted into the cell culture
supernatant were analyzed by ELISA as described in the Materials and
Methods. A notable amount of IL-8 and MCP-1 was secreted into the
supernatant when UG3 cells were cultured in the presence of either
IL-3, GM-CSF, or M-CSF (Fig 9). UG3 cells exposed to
these cytokines also released IL-6, although the production was three
times higher when cultured in the presence of IL-3 or M-CSF as compared
with GM-CSF (P < .01). Production of M-CSF and G-CSF was
below the minimum detectable concentration when UG3 cells were exposed
to GM-CSF or IL-3, but UG3 cells cultured with M-CSF produced a
significant amount of M-CSF and G-CSF (P < .01). Production
of GM-CSF was below the detectable limit when UG3 cells were cultured
in the presence of either IL-3, GM-CSF, or M-CSF (data not shown).
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| Fig 9.
Concentration of various cytokines released into culture
supernatant by UG3 cells cultured for 2 weeks in the presence of IL-3
(5 ng/mL), GM-CSF (1 ng/mL), or M-CSF (100 ng/mL). Error bars indicate
the standard deviation of cytokine concentration. *P < .01. Results are representative of three independent experiments.
|
|
Clonogenic assay.
Clonogenic assay showed that, in the absence of cytokines, UG3 cells
did not form colonies (data not shown). When UG3 cells were maintained
in the presence of IL-3 for 2 weeks and then transferred to the
semisolid media, IL-3, GM-CSF, M-CSF, or G-CSF stimulated formation of
33.51.5, 39.01.0, 14.01.0, or 13.01.0 colonies/1,000 cells,
respectively. The ratio of spread to compact colonies was 3:7, 3;7,
4:6, and 6:4 when UG3 cells maintained in IL-3- were subsequently
transferred to semisolid media containing IL-3, GM-CSF, G-CSF, or
M-CSF, respectively. IL-3- or GM-CSF-stimulated colonies tended to be
larger in size than M-CSF- or G-CSF-stimulated colonies. Individual
cells comprising colonies stimulated by each cytokine were similar in
morphology to those cultured with the respective cytokine in liquid
media, although the cells comprising spreaded colonies tended to have
higher positivity for NB than those obtained from compact colonies
(data not shown). Preculture of UG3 cells in liquid culture
supplemented with IL-3 or GM-CSF for a period of 2 weeks did not alter
the number of cells capable of forming colonies in semisolid media
supplemented with IL-3, GM-CSF, M-CSF, or G-CSF as compared with UG3
cells that had not been precultured (Table 4). In
contrast, preculture of UG3 cells for 2 weeks in the presence of M-CSF
reduced the capacity of UG3 cells to form colonies in semisolid media
supplemented with IL-3 or G-CSF. Under these conditions, colony forming
capacity of UG3 cells was preserved in semisolid media supplemented
with M-CSF or GM-CSF (Table 4). Only UG3 cells responding to GM-CSF
could preserve their colony-forming potential after 2 weeks of liquid
culture in the presence of G-CSF (Table 4).
Differentiation into osteoclast-like cells.
UG3 cells preincubated with 100 ng/mL M-CSF for 2 weeks formed
multinucleated giant cells displaying TRAP activity when cultured for
an additional 2 weeks in the presence of both 100 ng/mL M-CSF and 100 U/mL IL-4
(Fig
10). UG3 cells did not form multinucleated giant cells when cultured
with M-CSF alone for the whole observation period of 4 weeks (not
shown). UG3 cells cultured in osteologic slide flasks in IMDM with 5%
FCS supplemented with M-CSF and IL-4 resorbed larger amounts of
hydroxyapatite (Fig 11A) than those cultured with
IL-3 and IL-4 (Fig 11B).
View larger version (132K):
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| Fig 10.
Multinucleated giant cells formed after culture of UG3
cells for 2 weeks in the presence of M-CSF (100 ng/mL) and IL-4 (100 U/mL) stained for TRAP. Original magnification, 75-fold. Histochemical analysis was performed on cells obtained from three independent cultures.
|
|
View larger version (141K):
[in this window]
[in a new window]
View larger version (126K):
[in this window]
[in a new window]
| Fig 11.
Resorption of hydroxyapatite by osteoclast-like giant
cells exposed to M-CSF and IL-4. Hydroxyapatite was stained black by Von Kossa staining. UG3 cells that had been cultured in the presence of
M-CSF (100 ng/mL) and IL-4 (100 U/mL) had pericellular clear areas (A)
resulting from hydroxyapatite resorption, whereas those that had been
cultured in the presence of IL-3 (5 ng/mL) and IL-4 (100 U/mL) did not
(B). Original magnification, 30-fold. Histochemical analysis was
performed with cells obtained from three independent cultures.
|
|
| |
DISCUSSION |
UG3 is a novel human leukemic cell line derived from a patient with AML
(M5). UG3 cells have several unique characteristics. (1) They rely on
the presence of IL-3, GM-CSF, G-CSF, or M-CSF for growth. (2) They
resemble immature monocytes/macrophages, but are induced to display
features characteristic of granulocytes on exposure to G-CSF. (3) They
are induced to differentiate into the monocyte/macrophage lineage on
exposure to M-CSF. (4) They are induced to further differentiate into
the osteoclast-like cells on exposure to M-CSF and IL-4. (5) They carry
the t(9;11)(p22;q23) and express the MLL-AF9 and AF9-MLL fusion
transcript.
Translocation of chromosome involving 11q23 is known to be closely
associated with leukemogenesis.24-26 Yu et al27
reported that MLL heterozygous (+/) mice display retarded
growth, hematopoietic abnormalities, and bidirectional homeotic
transformations of the axial skeleton as well as sternal malformations.
Corral et al28 found that chimeric mice carrying the
MLL-AF9 fusion gene developed tumors, which were restricted to acute
myeloid leukemias despite the widespread activity of the MLL promoter.
Translocations involving 11q23 occur frequently in patients with
secondary leukemia subsequent to chemotherapy, including topoisomerase
II antagonist against primary malignancy.29 Both the MLL
and the AF9 gene bear putative topoisomerase II binding
sites.30 The mechanism of leukemogenesis underlying MLL
gene abnormality has yet to be elucidated. UG3 cells may prove useful
to dissect further how MLL gene abnormality may be operational in
leukemogenesis.
UG3 cells transcribe an AF9-MLL fusion transcript in which the AF9 RNA
is fused at nucleotide 1320 to exon 12 of the MLL transcript. The
MLL-AF9 fusion transcript in UG3 cells fused exon 9 of MLL to
nucleotide 1321 of AF9. Because no PCR analysis of the genomic DNA of
UG3 cells has been performed, it is unclear at present whether exon 10 and 11 of the MLL gene have been spliced out during transcriptation of
the AF9-MLL and MLL-AF9 fusion genes, respectively, or whether exon 10 and 11 were deleted as a result of the translocation. DNA sequencing of
the genomic breakpoints of the AF9-MLL and MLL-AF9 fusion genes should
also help to determine whether putative topoisomerase II binding sites,
which have previously been identified in the MLL breakpoint cluster
region, are also present in the breakpoint area of UG3
cells.30,31
UG3 cells grew faster when exposed to IL-3 or GM-CSF than when
stimulated with G-CSF or M-CSF. UG3 cells preserve similar viability
when cultured in the presence of IL-3, GM-CSF, G-CSF, or M-CSF,
respectively. However, both number and size of IL-3- or
GM-CSF-stimulated colonies was increased as compared with G-CSF- or
M-CSF-stimulated colonies. These findings suggest that the prolonged
doubling time of UG3 cells maintained in the presence of G-CSF or M-CSF
was due to a slower progression of cells through the cell cycle rather
than accelerated cell death in a fraction of UG3 cells. The number of
colony forming cells responding to IL-3 or G-CSF decreased after 2 weeks of liquid culture period in the presence of M-CSF. Likewise, the
number of colony-forming UG3 cells responding to IL-3, G-CSF, or M-CSF
also decreased after 2 weeks of liquid culture in the presence of
G-CSF. These findings suggest that irreversible commitment was induced
in some UG3 cells during the 2 weeks of exposure to M-CSF or G-CSF.
IL-3- or GM-CSF-stimulated colonies obtained after previous liquid
culture in the presence of M-CSF or G-CSF were similar in morphology to
those obtained after liquid culture supplemented with IL-3 or GM-CSF.
This may suggest that a fraction of UG3 cells remains undifferentiated or, alternatively, that a fraction of UG3 cells differentiated reversibly while exposed to G-CSF or M-CSF for 2 weeks.
Of interest is the ability of UG3 cells to differentiate in the
presence of physiological cytokines not requiring the presence of
chemical compounds such as PMA. UG3 cells exposed to G-CSF displayed
specific markers of granulocytic in addition to monocytic differentiation, which included strong positivity for NAC esterase. Furthermore, UG3 cells cultured in the presence of G-CSF were 4%
positive for peroxidase (Fig 7A) and 6% positive for NAP (Fig 7B).
CD14 is usually considered a monocyte/macrophage marker. However,
expression of CD14 is also enhanced in response to G-CSF in
neutrophils.32 Strong positivity for CD14 observed in UG3 cells after exposure to G-CSF might reflect differentiation and activation of UG3 cells in response to G-CSF. UG3 cells may thus represent a valuable tool to further investigate both the mechanism and
the regulation of granulocytic differentiation, even though UG3 cells
are established from leukemic cells.
A few murine cell lines that can be induced to differentiate along the
monocyte/macrophage lineage in response to physiological substances
have been established. NFS-60 cells, for example, differentiate into
neutrophils and macrophages in the presence of IL-3 and
GM-CSF.33 Nakoinz et al34 described two
sublines of NFS-60, one of which grows slowly in the presence of M-CSF
and shows properties of differentiated macrophages. The other subline
grows rapidly as round nonadherent cells in response to M-CSF but does
not respond to GM-CSF.34 GM-CSF- and IL-3-responsive
FDC-P1 cells become M-CSF-responsive after transfection of c-fms
expression vector. Some c-fms-expressing FDC-P1 cells are induced to
myeloid differentiation by M-CSF, but the other undifferentiated
c-fms-expressing FDC-P1 cells retain responsiveness to
IL-3.35 UG3 cells cultured in the presence of GM-CSF or
M-CSF displayed the following features characteristic for
monocytes/macrophages: (1) strong positivity for NB esterase, (2)
expression of CD68, (3) production of various cytokines, and (4) uptake
of Ac-LDL. Furthermore, UG3 cells cultured in the presence of M-CSF
also exhibited characteristics of differentiated macrophages such as
spread morphology, even greater production of cytokines, and increased
expression of CD14 and CD68 as compared with UG3 cells cultured in the
presence of IL-3 or GM-CSF. A subpopulation of UG3 cells cultured in
the presence of M-CSF became relatively large in size and showed almost
100% positivity for CD14 and negativity for CD71, a specific marker of
macrophages induced to differentiate by GM-CSF.17 To our
knowledge, UG3 cells might be the first human cell line inducible to
differentiate along the monocyte/macrophage lineage in response to
physiological stimuli alone. UG3 cells thus may represent a suitable
model to further elucidate the different characteristics of M-CSF- and
GM-CSF-induced macrophages under limited conditions.
Furthermore, UG3 cells differentiated into functional osteoclast-like
cells, capable of bone resorption, in the presence of both M-CSF and
IL-4 similarly. HL60 cells differentiate into osteoclast-like cells in
response to vitamin D3.10 Recent studies
suggest that peripheral blood monocytes mature into osteoclast-like
cells in the presence of M-CSF and IL-4.18 Various other
factors, such as IL-1, IL-6, IL-11, or leukemia inhibitory factor
(LIF), either stimulated or inhibited osteoclast formation of bone
marrow mononuclear cells.36 GM-CSF inhibited M-CSF and
IL-4-induced differentiation into osteoclasts,18 but was
also reported to support M-CSF- and IL-4-induced osteoclast-like cell
formation from monocytes in another study.37 Such
conflicting findings may be explained by the use of different cell
sources or factors or by a divergent definition of osteoclasts. Because
more UG3 cells were induced to osteoclast-like cells in response to
M-CSF and IL-4 after 2 weeks of preincubation with M-CSF than without
that preincubation (data not shown), UG3 cells might differentiate into
osteoclast-like cells through mature monocytes/macrophages. UG3 cells
could be useful in elucidating the mechanisms regulating not only
differentiation of monocytes into osteoclasts, but also to further
dissect the function of osteoclasts under limited conditions.
In summary, UG3 is a human monoblastic cell line that requires
exogenous growth factors, including IL-3, GM-CSF, G-CSF, or M-CSF, for
growth. Optimum proliferative response is achieved by exposure of UG3
cells to IL-3 or GM-CSF. IL-3- or GM-CSF-stimulated UG3 cells
developed a more differentiated phenotype than has previously been
reported for other myelomonocytic cell lines, including
FDC-P135 and NFS-60.34 UG3 cells were induced
to display characteristics of mature granulocytes and
monocytes/macrophages after exposure to G-CSF or M-CSF, respectively,
and further differentiated into functional osteoclast-like cells in the
presence of M-CSF and IL-4.
| |
FOOTNOTES |
Submitted May 5, 1997;
accepted February 10, 1998.
Address reprint requests to Takashi Ikeda, MD, First Department of
Internal Medicine, Kagawa Medical University, 1750-1 Ikenobe, Miki-cho,
Kita-gun, Kagawa 761-07, Japan; e-mail: takikeda{at}mailbox.kms.ac.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.
| |
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Genes on chromosomes 4, 9, and 19 involved in 11q23 abnormalities in acute leukemia share sequence homology and/or common motifs.
Proc Natl Acad Sci USA
90:4631,
1993[Abstract/Free Full Text]
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Abstract
Introduction
Abstract