1. No category

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1993 82: 456-464
Establishment and characterization of an erythropoietin-dependent
subline, UT-7/Epo, derived from human leukemia cell line, UT-7
N Komatsu, M Yamamoto, H Fujita, A Miwa, K Hatake, T Endo, H Okano, T Katsube, Y Fukumaki
and S Sassa
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Establishment and Characterization of an Erythropoietin-Dependent Subline,
UT-7/Epo, Derived From Human Leukemia Cell Line, UT-7
By Norio Komatsu, Masayuki Yamamoto, Hiroyoshi Fujita, Akiyoshi Miwa, Kiyohiko Hatake, Toshiyasu Endo,
Hiromitsu Okano, Takanori Katsube, Yasuyuki Fukumaki, Shigeru Sassa, and Yasusada Miura
UT-7 is a human leukemic cell line capable of growing in
interleukin-3 (IL-3). granulocyte/macrophagecolony-stimulating factor (GM-CSF), or erythropoietin (Epo) (Komatsu
etal, CancerRes51:341,1991).Tostudytheeffectof Epo
on proliferationand differentiation of UT-7, w e maintained
the UT-7 cell culture for more than 6 months in the presence of Epo. As a result, a subline, UT-7/Epo, was established. The growth of UT-7/Epo could be supported by Epo
but not by GM-CSF or IL-3. UT-7/Epo showed a greater
level of heme content and ratio of benzidine-positivestaining cells than did UT-7. Butyric acid promoted the synthesis of hemoglobin in UT-7/Epo, but not UT-7. Further, the
mRNA concentrationsof the c-myb oncogene and GM-CSF
receptor @-subunitwere decreased substantially in UT-7/
Epo cells. These findings showed that UT-7/Epo cells had
progressed further in erythroid development than UT-7
cells, and suggestedthat long-term culture in Epo had promoted this differentiation. Whereas availability of the Epo
receptor (Epo-R) for binding of Epo was reduced in UT-7/
Epo cells compared with UT-7 cells, the Epo-R showed a
similar affinity for Epo. This observation suggested that
change(s)in postreceptorsignaling step might be involved
in the establishment and maintenance of the UT-7/Epo
phenotype.
0 1993 by The American Society of Hematology.
T
of the molecular mechanisms by which Epo promotes the
growth and differentiation of hematopoietic cells. The
abundant expression of the Epo binding sites expressed on
UT-7 cells encouraged us to attempt a long-term culture of
the cells in the presence of Epo. A subline, UT-7/Epo, was
finally established that could proliferate continuously in medium containing only Epo as the growth factor; neither
GM-CSF nor IL-3 could support the growth of these cells.
The expression of erythroid lineage markers in UT-7/Epo
cells was then analyzed to determine whether the culture
conditions promoted erythroid development of the cells.
We also examined the expression of the c-myh oncogene
and GM-CSF receptor (GM-CSF-R) in UT-7/Epo by RNA
blot hybridization analysis, because downregulation of the
expression of these genes was reported to be an essential
event for Epo-induced erythroid differentiati~n.~.’
Transcription factor GATA- 1 has been reported to be essential
for the transcriptional activation of erythroid, mast cell, and
megakaryocyte-specific
Therefore, we examined
the expression of GATA- 1 mRNA in UT-7 and UT-7/Epo
cells. The results of these analyses clearly demonstrated that
UT-7/Epo cells were committed to the erythroid lineage.
The availability of Epo-R on the surface of UT-7/Epo cells
was significantly decreased, suggesting that changes in postreceptor signaling events were responsible for the establishment and maintenance of the UT-7/Epo phenotype.
HE PRINCIPAL EFFECT of erythropoietin (Epo) is to
promote the erythroid differentiation of cells in hematopoietic lineages and support the proliferation of erythroid
cells.’ Epo also stimulates in vitro megakaryopoiesis.’ Epo
initially binds to a specific cell-surface receptor (Epo receptor [Epo-RI) in the process of its signal transduction. However, intracellular events provoked by signals from the Epo/
Epo-R system have not yet been elucidated. We previously
established a human leukemic cell line, UT-7, based on its
ability to be maintained in granulocyte/macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (1L-3),3
Using a proliferation assay, we found that UT-7 cells could
also respond to Epo. The UT-7 cell has a large number
(7,200 to 13,0OO/ceil) of Epo binding sites425and is one of
the best available human cell lines to study the expression
and function of Epo-R. We also found that this cell line
could differentiate to megakaryocytes in response to phorbo1 myristate acetate (PMA).3
A cell line in which growth is totally dependent on the
presence of Epo would provide great help in further analysis
~~
~~
~
~
From the Division of Hematology, Department ofMedicine, Jichi
Medical School, Tochigi, Japan; the Department of Biochemistry,
and Department of Applied Physiology and Molecular Biology, Tohoku University School ofMedicine, Sendai, Japan; the Department
of School-Nurse Training, Kyushu Women’s Jr College, Fukuoka,
Japan; Kyushu Jr College ofKinki University, Fukuoka, Japan; the
Research Laboratory for Genetic Information, Kyushu University,
Fukuoka, Japan; and the Laboratory of Metabolism. and Pharmacology, The Rockcfiller University, New York, NY.
Submitted September 15, 1992; accepted March 10, 1993.
Supported by Grants-in-Aid for Cancer Research and Scientific
Research from the Ministry of Education, Science and Culture of
Japan.
Address reprint requests to Norio Komatsu, MD, Division of Hematology, Department of Medicine, Jichi Medical School, Minamikawachi-machi, Kawachi-gun, Tochigi-ken. 329-04, Japan.
The publication costs of this article were defiayed 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.
0 1993 by The American Society of Hematology.
0006-4971/93/8202-00I 3$3.00/0
456
MATERIALS AND METHODS
Reagents. Recombinant human Epo, with a specific activity of
1.7 X IO5 U/mg, was a gift of the Life Science Research Institute of
Snow Brand Milk Products Company (Tochigi, Japan). Recombinant human GM-CSF, with a specific activity of IO9 U/mg, was
provided by Sumitomo Pharmaceutical Company (Osaka, Japan).
Recombinant human IL-3, with a specific activity of IO8 U/mg, was
a gift of Genetics Institute (Cambridge, MA). Recombinant human
IL-6 was purchased from Genzyme CO(Boston, MA). Butyric acid
(BA), hexamethylene-his-acetamide (HMBA), N-methylnicotinamide (NMN), and phorbol 12-myristate 13-acetate (PMA) were
purchased from Sigma Chemical CO (St Louis, MO). Plasmid
DNAs for human c-myb cDNA and human ribosomal genomic
DNA (5’ portion) were provided by the Japanese Cancer Research
Resources Bank. A human Epo-R cDNA was newly cloned from
UT-7.” Human GATA-1 and GATA-2 cDNAs were kindly proBlood, VOI 82,NO 2 (July 15).1993:pp 456-464
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
457
ERYTHROID DIFFERENTIATION OF UT-7
vided by Dr S.H. Orkin.12.” cDNAs encoding cy- and 8-subunit of
human GM-CSF-R were kindly provided by Dr K. Hayashida.
Cell culture. The UT-7 cell line was established from marrow
cells obtained from a patient with acute megakaryoblastic leukemia.3 The original UT-7 cells were maintained in liquid culture
with Iscove’s Modified Dulbecco’sMedium (IMDM;GIBCO Laboratories, Grand Island, NY) supplemented with 10% fetal calf
serum (FCS; Hyclone Laboratories, Logan, UT) and 1 ng GMCSF/mL, by replacement of one half of the medium with fresh
medium every 3 or 4 days. UT-7/Epo was maintained with Epo 1
U/mL and 10%FCS by replacement ofthree fourths ofthe medium
every 3 or 4 days.
Colorimetric MTT assay. Cell growth was estimated by a modified colorimetric MTT assay of M o ~ m a n n . ’Briefly,
~
20 pL of a
sterilized 5 mg/mL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma] solution were added to each
culture well containing a final volume of 0.1 mL of cell suspension.
After 5 hours of incubation, 100 pL of 10%sodium dodecyl sulfate
(SDS) solution was added to the wells and mixed thoroughly. Each
culture well of the plate was examined using a microplate reader
(model 3550; Bio-Rad Laboratories, Richmond, CA) at a wavelength of 595 nm.
Analysis of cell surface markers by immunofluorescence. Cellsurface antigens were detected by immunofluorescence staining
with the monoclonal antibodies (MoAbs) P2 (CD4la, specific for
platelet glycoprotein [GP] IIb/IIIa) and SZ2 (CD42b, specific for
platelet GP Ib). These MoAbs were purchased from Immunotech
(Marseilles, France). Surface marker studies were camed out as
follows: UT-7 cells were incubated for 30 minutes at 4°C with the
appropriatelydiluted MoAbs. After washing, the cells were reincubated with fluorescein-labeledgoat antimouse IgG for 30 minutes
at 4°C. After a second washing, fluorescence analysis was performed using a Becton Dickinson Flow Cytometer (Facstar;Becton
Dickinson, Mountain View, CA), using 10,000 cells for each antibody. The proportion of positive cells was determined by comparison with cells that were reacted with the fluorescein isothiocyanate
(F1TC)-conjugated secondary antibody alone.
Quantitative analysis ofplatelet factor 4 (PF4) and p-thromboglobulin (8-TG). Enzyme-linked immunosorbent assay (ELISA)kits
(Asserachrom PF4 and Asserachrom 8-TG; Diagnostica Stago, Asnieres, France) were used for quantitative determinations of PF4
and 0-TG concentrations in UT-7 and UT-7IEpo cells as described.’ Briefly, cell lysate and international standards of PF4 and
0-TG were incubated in a multiwell ELISA plate coated with antihuman PF4 or antihuman 0-TG rabbit antibodies;second antibody
conjugated with horseradish peroxidase was added to each well.
After completion of the reaction, the substrate solution (0.4 mg/mL
orthophenyleneand 0.015% H,O,) was added. The peroxidase reaction was stopped with sulfuric acid and the absorbency at 490 nm
was measured using an immunoplate reader (Immuno-Reader NJ2,000 InterMed, Tokyo, Japan). The concentrations of PF4 and
8-TG in the cell lysates were determined by comparison of results
with those obtained with the standards.
RNA preparation and RNA blot hybridization analysis. UT-7
cells (1 X IO6 cells/mL) in IMDM supplemented with 10%FCS in
the presence or absence of 10 ng PMA/mL were incubated at 37°C
for the indicated period. Total cellular RNA was prepared from
cells using a guanidinium isothiocyanate procedure described by
Chromczynski et al.” RNA was electrophoresed on an agarose
formaldehyde gel, transferred to nylon membranes in lox standard
sodium citrate (SSC), and hybridized to human Epo-R cDNA fragment, c-myb cDNA fragment, bovine @-actincDNA fragment, human GATA- 1 and GATA-2 cDNA fragments, GM-CSF-R a-and
0-subunit cDNA fragments, and human ribosomal genomic DNA
fragment. Those cDNA fragmentswere labeled with 32P-aCTPby a
random priming method. Filters were autoradiographed using Kodak XAR-5 film (Eastman Kodak, Rochester, NY) with intensifying screens at -70°C.
Fluorometric assay of heme. Suspensions containing 1 X IO’
cells were transferred to 6 X 50 mm disposable glass tubes. The
tubes were centrifuged at 680g for 5 minutes. Cell pellets were
washed once with 500 pL of Eagle’s buffer devoid of Ca2+and Mg2+
(pH 7.4). Five hundred microliters of 2 mol/L oxalic acid were then
added to the cell pellets. The suspension was mixed, and the mixture was immediately heated for 30 minutes at 100°C. Fluorometric analysis of heme was performed as described previously.16
Characterization of hemoglobin. Hemoglobin fractions were
analyzed by isoelectric focusing (IEF) accordingto the method previously described.” In brief, samples were prepared for IEF using
0.05% KCN and LKB ampholines with a pH 6 to 9 gradient, and
separated on an acrylamide slab gel. After electrophoresis, benzidine staining was performed to detect the peroxidase activity of
hemoglobin. To characterizethe hemoglobins, adult red blood cells
(HbA) and cord blood cells (HbF) were used as controls.
Epo iodination. Labeling of Epo with ”’I was done by the solid
phase method with the IODO-GEN reagent ( 1,2,4,6-tetrachloro3a,6a-diphenylglycouril; Pierce Chemical CO,Rockford, IL) as described previously.’8The specific activity of the labeled Epo was 50
to 100 pCi/ng and there was no loss of biologic activity.
Binding studies with lZsI-labeledEpo. UT-7 cells grown in the
presence of 1 ng GM-CSF/mL were washed thoroughly with phosphate-buffered saline (PBS) and suspended in 50 pL of IMDM supplemented with 10%FCS. For binding studies, 50 pL of a suspension containing 2 to 5 X IO6 cells was mixed with 50 p L PBS
containing 60 mmol/L HEPES (pH 7.2), 0.3% bovine serum albumin (BSA), 0.6% NaN,, and 12’I-Epo with or without a 200-fold
excess of unlabeled Epo. After 4 hours of incubation at 15”C, the
cells were transferred to a microtube and binding was terminated by
sedimentingthe cells through a mixture of dibutyl phthalate oil (60
pL; Sigma) and bis(3,5,5-trimethylhexyl) phthalate oil (40 pL;
Fluka Chemical, Ronkonkoma, NY) for 1.5 minutes in a microfuge
(8,OOOg). The tube was rapidly frozen at -8O”C, and the tip containing the cell pellet was cut off. The radioactivity of the tip was
counted on a gamma counter. Specific binding at a given concentration of radioactive Epo was defined as the difference in bound
radioactivity between samples incubated in the absence or in the
presence of excess unlabeled Epo. Scatchardanalysis of Epo equilibrium binding data was performed as described. Epo data analysis
and curve fitting were performed using the computerized “ENZFITTER’ program (Sigma).
RESULTS
Establishment of UT-7/Epo cell line. To establish
growth factor-dependent sublines of UT-7, we cultured
UT-7 cells in the presence of IL-3 (10 U/mL), GM-CSF (1
ng/mL), or Epo (1 U/mL) for more than 6 months. As a
result, UT-7 cells proliferated continuously in all these culture conditions. We tentatively designated these cells UT-7/
IL-3, UT-7/GM-CSF, and UT-7/Epo, respectively. Figure 1
shows the result of the MTT assay of these UT-7 cells in
response to these growth factors. Whereas UT-7/IL-3 and
UT-7/GM-CSF showed almost the same pattern of response to the growth factors as the original UT-7, UT-7/Epo
completely lost its response to both IL-3 and GM-CSF.
Therefore, we concluded that UT-7/Epo was a subline of
UT-7 whose growth was totally dependent on the presence
of Epo. Supporting this conclusion, the growth profile of
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458
KOMATSU ET AL
1.01
IC
B
0
0.01
0.1
1.0
10
la,
o
0.01
0.1
1.0
i o iw o
0.01
UT-7/Epo in liquid culture was significantly different from
that of the original UT-7 (Fig 2 ) . UT-7 had a saturation
density of 3 to 5 X 105/mL and a doubling time of 36 to 48
hours in log growth phase, whereas UT-7/Epo showed a
saturation density of 1 x I 06/mL and a doubling time of 18
to 24 hours.
UT-7/Epo has committed more to erythroid lineage but
less to megakaryocytic lineage compared with original UT7. The ratio of the benzidine-positive to benzidine-negative cells was routinely monitored during the course of UT7/Epo culture to follow erythroid differentiation. The
frequency of the benzidine-positive cells increased to 3% to
4% in UT-7/Epo compared with less than 1% in the original
UT-7. We previously observed that original UT-7 grew as
dispersed cells in su~pension,~
but UT-7/Epo formed aggregates of 10 to 20 cells (Fig 3A). The cytoplasm of the original
UT-7 cells was relatively basophilic and the nuclear chro-
IO
-8
E
L
2
0
X 6
v
L
Q,
n 4
5c
6 2
0
0
1
2
3
4
5
Day of Culture
6
7
Fig 2. Growth curves of UT-7 and UT-7/Epo cells in tissue culture flasks in the absence or presence of Epo. Growth curves of
UT-7 (circles) and UT-7/Epo (squares) cells in tissue culture flasks
were examined in the absence (solid symbols) or presence of 1 U/
mL Epo (open symbols). Cell numbers and viability were assessed
by the trypan blue dye exclusion test.
0.1
1.0
io
io0
Fig 1. Proliferative responses of UT-7 cell lines
to recombinant human Epo, GM-CSF, IL-3, or IL-6.
ProliferativeresponsesofUT-7 cell linesto recombinant human Epo (U; U/mL), GM-CSF (0;
ng/mL),
IL-3 (A; U/mL). or IL-6 (m; ng/mL) were analyzed by
MTT assay. The cells had been maintained for 1
year in the presenceof (A) 1 ng GM-CSF/mL, (B) 10
U IL-3/mL. or (C) 1 U Epo/mL at the time of this
assay. Cells were plated at 1O*/well in I M D M in the
presence of increasing concentrations of growth
factors. MTT incorporation was measured after 3
days of culture as described in Materials and Methods.
matin was not c o n d e n ~ e dIn
. ~contrast, cytoplasm of UT-7/
Epo was stained relatively eosinophilic and chromatin condensation was observed (Fig 3B). These observations
suggest that UT-7/Epo cells attained a more mature stage of
erythroid differentiation than the original UT-7.
We also examined UT-7/Epo for changes in the expression of megakaryocyte-specific antigens, and also for its response to PMA. As a result, we found that UT-7lEpo expressed lower levels of GPIIb/IIIa and GPIb than original
UT-7 (Fig 4). This showed marked contrast to UT-7/IL-3
and UT-7/GM-CSF, in which abundant megakaryocytespecific antigens were expressed in similar concentration to
that of original UT-7 (data not shown). PMA promoted the
production of P-TG and PF4 in original UT-7, but not in
UT-7/Epo (Table I). PMA-treated UT-7 cells adhered to
plastic dishes and extended pseudopods. Conversely, UT-71
Epo did not show morphologic changes by PMA treatment
(Fig 5). These observations indicated that the megakaryocyte differentiation program was repressed in the UT-7/Epo
cell line.
BA induced erythroid diflerentiation of UT-7/Epo but not
UT-7. Cell pellets of UT-7IEpo showed slightly red in
color upon centrifugation, suggestingthe existence of hemoglobin. To confirm this, we quantitated the heme content in
UT-7/Epo, as shown in Table 2. The basal level of heme in
UT-7/Epo was three times higher than that in original UT-7
cells.
We chose several chemicals known to induce erythroid
differentiation of cultured erythroleukemia cell lines and
tested whether they could induce hemoglobin synthesis in
UT-7/Epo cells. We added BA, dimethylsulfoxide (DMSO),
HMBA, and NMN to the culture medium. Of these chemicals, only BA induced an increase in heme content of UT-71
Epo cells. None ofthe chemicals could induce an increase in
the heme level of original UT-7 cells (Table 2).
We next analyzed the types of hemoglobin expressed in
UT-7/Epo cells. IEF analysis of the cell lysates showed the
and Hb
synthesis of HbF (a2yz),HbA (azP2),HbX (t2a2),
Bart's (y4) in UT-7/Epo cells, but not in UT-7. The BAtreatment of UT-7/Epo cells markedly induced hemoglobin
synthesis in the cells without changing the expression pattern of hemoglobin subtypes (Fig 6). UT-7/Epo cells seemed
to have acquired an ability to produce large quantities of
heme and globin, an essential character of erythroid lineage
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ERYTHROID DIFFERENTIATION OF UT-7
459
I
4
Fig 3. Morphologic characteristics of UT-7IEpo cells in liquid culture. (A) Phase contrast
photomicrograph of UT-7/Epo
(original magnification X 10).
(B) Cytospin smear of UT-7/Epo
cells stained with May-Giemsa
(original magnification X 400).
cells. This observation supported the theory that UT-7/Epo
had progressed further in erythroid development than had
UT-7.
Expression of Epo-R, c-myb, and GATA-I in UT-7/
Epo. We also examined the expression of Epo-R, c-myb
oncogene, and GATA- 1 transcription factor in UT-7/Epo
cells by using RNA blot hybridization analysis. As shown in
Fig 7, the level of Epo-R transcript was decreased in UT-71
Epo cells compared with that in the original UT-7 cells. BA
treatment of the UT-7/Epo cells strongly decreased the level
of Epo-R mRNA initially; the mRNA level partially recovered by 5 days of treatment (Fig 7) and recovered to almost
the basal level by 7 days of the treatment (data not shown).
This was a somewhat surprising result because the growth of
the UT-7/Epo cell line was totally dependent on the presence of Epo. Therefore, we analyzed Epo-R expression in
UT-7/Epo in detail, as will be seen in the next section.
4001
GPI Ib/IIIa
4001
The GATA-I transcription factor had been reported to be
important for transcriptional activation of erythroid, megakaryocytic, and mast cell-specific genes.*-" The original
UT-7 cells expressed GATA-I mRNA as abundantly as
UT-7/Epo (Fig 7). This suggested that UT-7 cells had already committed to erythroid and/or megakaryocytic lineages. Steady-state level of the GATA-I mRNA was markedly decreased by BA treatment of the UT-7/Epo cells, but
the level partially recovered by 3 to 5 days of the treatment.
This pattern of response was similar to that of the Epo-R
mRNA level to BA treatment. In contrast, mRNA level of
GATA-2, another member of the GATA transcription factor family, did not show recovery by 3 to 5 days of the
treatment. Because a number of GATA consensus sequences (binding sites for GATA transcription factors)''
have already been found in the regulatory region of the EpoR gene, this result suggested that Epo-R expression might be
GPIb
0
FlTC Fluorescence Intensity
Fig 4. Comparison of the expression of megakaryocyte-specific antigens GPllb/llla and GPlb in
UT-7 and UT-7/Epo. A total of 10,000 cells were
analyzed for the antibody bindingagainst each antigen. Antibodies used in this analysis were as described in Materials and Methods.
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460
KOMATSU ET AL
Table 1. Comparison of PF4 and 8-TG Production Between UT-7 and UT-7/Epo
p-TG (ng/lO’ cells)
PF4 (ng/lO’ cells)
UT-7
UT-7/Epo
Od
5d
10 d
Od
1.4k0.1
1 .O f 0.4
431.4 2 9.9
1.5 k 0.5
612.8k 13.3
0.7 ? 0.4
2.0 1.o
1.6 2 0.2
*
5d
10 d
465.6 ? 17.2
1.3 2 0.2
637.5 k 25.2
0.6 2 0.2
UT-7 or UT-7/Epo cells were cultured in the presence of PMA (10 ng/mL) for 5 or 10 days, harvested, and prepared for the quantitationof PF4 and
8-TG as described in Materials and Methods. Cell viability was greater than 80% over 10 days of exposure to PMA.
regulated by the GATA-I factor. However, it is not clear
from this analysis whether GATA-I alone can direct cell
type-specific expression of the Epo-R gene (see Discussion).
The c-myh mRNA level in UT-7/Epo was far lower than
that in original UT-7 cells (Fig 7). BA treatment of UT-7
cells further decreased the expression ofc-mvh mRNA. This
pattern, an initial decrease followed by a gradual increase in
the mRNA level in response to BA. was similar to that observed for Epo-R and GATA- 1. This observation agreed
with the previous report that downregulation of c-mvh oncogene expression is an essential event for Epo-induced erythroid differentiation6 and, therefore, provided another line
of evidence supporting the conclusion that UT-7/Epo represents a later stage of erythroid development.
Change in postreceptor signaling events may he involved
in the eslablishment and maintenance ofthe UT-7/Epophenotype. We examined UT-7 and UT-7 Epo cells to determine the number of cell-surface Epo receptors, and their
affinity for Epo (Table 3). UT-7IEpo had 3,000 Epo-binding sites per cell, whereas UT-7 had 13,000 binding sites per
cell. Dissociation constants of Epo binding were 68 pmol/L
and 100 pmol/L for UT-7/Epo and UT-7, respectively. This
decrease in the concentration of Epo-R in UT-7/Epo cells
correlated well with the observed decrease in the level of
Epo-R mRNA (Fig 7). These results clearly showed that
proliferation of UT-7/Epo was not maintained by any stimuli provided by either an increased number of Epo-R or an
increased affinity of the Epo-R to Epo. Therefore, it was
suggested that there might be some modifications of postreceptor signaling pathways in UT-7/Epo cells that led the cell
to acquire the ability to proliferate in medium containing
Epo as the sole growth factor.
UT-7/Epo cells lose their response to GM-CSF and IL-3
because ? f a decrease afthe GM-CSF-R P-subunit expression. UT-7/Epo cells lose their response to both GM-CSF
and IL-3. Receptors for these growth factors share a common P-subunit” and the signals seem to be transduced
through a common intracellular pathway.2’.22The P-subunit generates high-affinity binding sites through combining a-subunit (low-affinity binding chain). In our preliminary binding analysis, UT-7/Epo was found to have
GM-CSF binding sites on the surface of the cells, but number of high-affinity binding sites on UT-7/Epo cells was
clearly decreased compared with that on UT-7 cells (data
not shown). This suggested that UT-7/Epo might have a
defect in a common component ofGM-CSFand IL-3 signal
Fig 5. Morphologic characteristics of UT-7 and UT-7/Epo
by the treatment with PMA.
(A) UT-7 cells and (B) UT-7/
Epo cells were treated with 10
ng/mL of PMA for 10 days. (A)
The majority of PMA-treated
UT-7 cells adhered to plastic
dishes and extended pseudopods. (E)Conversely, the UT7/Epo cells did not show significant changes in morphology
with PMA treatment. (Original
magnification X 20.)
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46 1
ERYTHROID DIFFERENTIATION OF UT-7
Table 2. Heme Content in Chemical-Induced UT-7 and UT-7/Epo
(medium)
DMSO
(1.5%)
HMBA
(5 mmol/L)
BA
(1.3 mmol/L)
NMN
(8 mmol/L)
15.0 2 0.4
40.0 ? 3.4
11.5? 1.5
36.5 t 1.0
11.221.7
33.2 t 0.8
19.9 2 1.3
121.5 2 1.1
14.2 t 0.2
48.5 t 0.8
Control
UT-7
UT-7/EDO
UT-7 or UT-7/Epo cells were incubated with any one of the chemicals for 5 days and prepared for heme content analysis. Values are the heme
content (pmol/106 cells).
transduction pathway, perhaps the receptor &subunit.
Therefore, we examined the expression ofa- and &subunits
of GM-CSF-R by RNA blot hybridization analysis using
The
cDNA clones encoding receptor a- and
result is shown in Fig 8. Whereas the level of 0-subunit in
UT-7/Epo cells was almost the same as that in the UT-7
cells, the level of &subunit in UT-7/Epo was markedly reduced compared with that in UT-7. This suggested that the
downregulation of the common &subunit made UT-7/Epo
refractory to both GM-CSF and IL-3 stimulation.
DISCUSSION
To study the effect of Epo on the proliferation and differentiation of UT-7 in long-term culture, we maintained UT7 cells in the presence of Epo and, after 6 months, a subline
designated UT-7/Epo was established. The UT-7/Epo phenotype differed from that of the parent cells (UT-7) in the
following respects: ( I ) UT-7/Epo lost the response to IL-3
and GM-CSF, resulting in complete dependence on Epo for
maintenance and growth: (2) it lost the ability to express the
megakaryocytic program in response to PMA; and (3) it
acquired the ability to respond to BA, resulting in increased
hemoglobin synthesis. Among the several human and murine Epo-dependent cell
UT-7/Epo cells are
unique in that growth can be maintained solely by Epo and
in that the cells have a large number of Epo binding sites on
the surface. Therefore, taking advantage of this fact, the cell
line may serve as a useful system for the bioassay of Epo and
modified Epo molecules because MTT reduction paralleled
the concentration of Epo in the range of 0.01 to 1 U/mL of
Epo (Fig 1).
There are two possible explanations for the derivation of
UT-7/Epo from UT-7. One is that a subline that responds
exclusively to Epo has spontaneously replaced the parent
UT-7 cells as a result of long-term exposure to Epo. The
other explanation is that Epo has promoted the erythroid
differentiation of UT-7. We note that UT-7 cultured with
Epo for 3 months responds to BA and begins to lose its
response to GM-CSFand IL-3 (data not shown). A concomitant decrease in the number of Epo-R starts at this stage
(5,50O/cell. Table 2). These observations suggest that the
latter possibility is likely. Epo seems to promote the ery-
'HbX ( ~ 2 ~ 2 )
' HbBarl's (y4)
Fig 6. BA-induced hemoglobinsynthesis in UT7 and UT-7/Epo. UT-7 or UT-7/Epo cells were
treated with 1.3 mmol/L BA for 5 days and prepared for isofocus electrophoresis as described in
Materials and Methods.
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KOMATSU ET AL
462
a ba b2
@
Table 3. Binding of '2sl-Epoto UT-7 Sublines
@
UT-?
0rigina1
GM-CSF
Original Epot
EPO
EPO-R
GATA-'
GATA-:
c-myb
18s
Fig 7. RNA blot hybridization analysis of Epo-R, GATA-1,
GATA-2. and c-my6 mRNAs in UT-7, UT-7/Epo, and BA-treated
UT-7/Epo cells. UT-7/Epo cells were treated with 1.3 mmol/L BA
for 1, 3. or 5 days. Cells were harvested and RNA was extracted
from each cell sample. Total cellular RNA (20 fig) isolated from UT7 (lane 1). UT-7/Epo (lane 2).or BA-treated UT-7/Epo (lane 3, 1
day; lane 4,3 days; lane 5, 5 days) was subjected to agarose/formaldehyde gel electrophoresis and then was hybridized to "P-labeled Epo-R. GATA-1, GATA-2, or c-my6 cDNA probes. 1 8 s ribosomal RNA was hybridized to "P-labeled human ribosomal DNA
probe as described in Materials and Methods.
throid differentiation of UT-7, resulting in subline UT-7/
Epo.
It is of interest that UT-7/Epo loses its response to both
IL-3 and GM-CSF because receptors for these growth factors share a common fi subunit.*' In the present analysis, we
found that UT-7/Epo cells contained a much lower level of
the @-subunitmRNA than did UT-7 cells (Fig 8). This suggests that UT-7/Epo has lost its ability to respond to GMCSF and IL-3 because the cells cannot generate high-affinity
binding sites for GM-CSFand IL-3. However, further analysis using specific antibody is necessary to clarify the molecular mechanism underlying this phenomenon. Liboi et al'
recently reported that Epo-induced differentiation of murine erythroid cell lines was accompanied by downregulation of AIC29, a murine counterpart of the @-subunit of
human GM-CSF-R. This observation suggests that the
downregulation of the common P-subunit in UT-7/Epo
cells is the result of Epo-mediated erythroid development of
the UT-7/Epo cells. An alternative explanation for the
downregulation of the &subunit mRNA level is that a point
mutation that generates a termination codon makes the
No. of Binding
SiteslCell
Dissociation Constant
No.
1
2
Average
1
2
Average
1
2
Average
1
2
Aver age
11,879 f 217
13,761 ? 318
12,820
10,764 f 263
10,757 f 156
10,761
4,795 f 56
6,184 f 86
5,490
2,392 ? 26
4.240 f 92
3.3 16
112f8
83 f 8
97
130f 1
97 f 6
113
122 f 54
94 f 9
108
66 ? 3
65 ? 6
66
Experiment
b
(r"lIL1
UT-7 cells have been culturedfor 6 months in the presence of GM-CSF
or Epo and tentatively named UT-7/GM-CSF or UT-7/Epo, respectively.
In this study, UT-7 sublines were cultured in the absence of hematopoietic growth factors for 12 to 16 hours. Then, each subline was prepared
for Epo binding assays and Scatchard analyses were performed. The
data shown for each experiment represent the mean value r SE from
duplicate cultures.
* Original UT-7 cells were newly thawed and prepared for Epo binding
assays.
t The original UT-7 cells were continuouslyculturedwith Epo (1 U/mL)
for 3 months and prepared for Epo binding assays.
mRNA unstable and reduces the steady-state level of the
P-subunit mRNA.
It is noteworthy that UT-7/Epo loses its response to PMA
and acquires erythroid properties on long-term exposure to
a subunit
B subunit
18s
B
Fig 8. RNA blot hybridization analysis of U- and B-subunit
mRNAs of GM-CSF-R in UT-7 and UT-7/Epo cells. RNA blot hybridization analysis was performed as describedin Fig 7. As a standard,
1 8 s ribosomal RNA was probed with 3Z-labeledhuman ribosomal
DNA.
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ERYTHROID DIFFERENTIATION OF UT-7
Epo. Because the importance of protein kinase C (PKC)
activity in the Epo signal transduction pathway has been
rep~rted,~'
Epo-induced erythroid differentiation of UT-7
should be associated with the activation of PKC. On the
other hand, PMA, an activator of PKC, can induce UT-7
but not UT-7/Epo to differentiate into mature megakaryocytes. Therefore, the activation of PKC may also be essential for the megakaryocytic differentiationof UT-7. Because
PKC is reported to have isoforms that have distinct tissue
distribution, localization, and requirements for activati01-1,~'the PKC isoform activated by Epo may be different
from that activated by PMA. Although we do not have experimental evidence, we speculate that different isoforms of
PKC may be involved in erythroid versus megakaryocytic
differentiation.
We find that expression of GATA- 1 and Epo-R mRNAs
is closely coordinated during BA-induced erythroid differentiation of UT-7/Epo. This result suggests that GATA- 1
may have an important role in the regulation of Epo-R gene
expression. GATA sequenceshave been found in the regulatory region of both murine and human Epo-R gene, and
Epo has been reported to induce GATA-1 mRNA level in
murine erythroid cell
However, changes in Epo-R
mRNA expression are not fully explained by the changes in
GATA- 1 expression, because the levels of GATA- 1 expression in UT-7 are almost the same as UT-7/Epo, although
Epo-R mRNA is expressed at a much higher level in UT-7
than in UT-7/Epo. Heberlein et a134recently reported that a
correlation between Epo-R and GATA- I gene expression
could not be found in embryonal stem cells. We find that
Epo downregulates Epo-R gene transcription without
changes in GATA- 1 mRNA concentration (unpublished
observation). These results imply an additional regulatory
mechanism(s) for Epo-R transcription. The binding of
GATA-I to the GATA consensus sequence of the Epo-R
gene may be affected by other transcription factors including myb, ets, myc, Spl, and GATA-2. These oncogenes and
transcription factors are suggested to be important for erythroleukemia cell d i f f e r e n t i a t i ~ n , and
~~-~
homologies
~
to the
consensus binding motifs for these factors are found in the
Epo-R gene r e g i ~ n .It~is~of, ~interest
~
to study the relationship between Epo-R and these transcription factors using
UT-7 and UT-7/Epo.
The present study suggests that there might be a close
relationship between megakaryopoiesis and erythropoiesis.
This is consistent with previous observations that a specific
class of progenitor has the capacity to give rise to both megakaryocytic and erythroid progeny.38This idea raises a question as to the regulation of expression of these two differentiation programs. At least two transcription factors,
GATA- 1 and GATA-2, are candidates for the lineage determinant transcription factors, because these factors exist in
both megakaryocytic and erythrocytic cells. GATA binding
motifs are found in the promoter regions of megakaryocytespecificgenes such as GP IIb and platelet factor-4, as well as
in erythroblast-specific genes, such as Epo-R and globin.
GATA factors are probably necessary for the activation of
both erythrocyte- and megakaryocyte-specificgenes. However, the Epo-R gene is downregulated, and the PF-4 and
463
0-TG genes are upregulated during megakaryocyticmaturat i ~ n . Thus,
~ . ~ the on/off switching mechanism of gene expression during hematopoietic cell differentiation remains
to be clarified. The UT-7 and UT-7/Epo cell lines, which
mimic the differentiation patterns of erythroid and megakaryocyte progenitors, could serve as model systems for resolving these problems.
ACKNOWLEDGMENT
The helpful discussions and critical comments of Dr John W.
Adamson (The New York Blood Center) are gratefully acknowledged. We thank Dr Stuart H. Orkin for his generous gift of the
human GATA- 1 and GATA-2 cDNAs, Dr Kazuhiro Hayashida for
cDNAs encoding a-and &subunits of human GM-CSF-R, and the
Japanese Cancer Resources Center for c-myb and ribosomal gene.
We also thank Dr Seiji Okada and Yukie Urabe for their technical
assistance, Dr Susan J. Stamler for advice, and Motoko Yoshida for
the manuscript preparation.
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