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1991 78: 451-457
Establishment and characterization of a new human leukemia cell line
derived from M4E0
K Yanagisawa, T Horiuchi and S Fujita
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Establishment and Characterization of a New Human Leukemia Cell Line
Derived From M,E,
By Kohsuke Yanagisawa, Takahiko Horiuchi, and Shigeru Fujita
A new human leukemia cell line, designated as ME-1, was
established from the peripheral blood leukemia cells of a
patient with acute myelomonocytic leukemia with eosinophilia (M,E,). This cell line has the characteristic chromosome
abnormality of M,E,, inv(l6) (p13q22).When cultured in RPMI
1640 medium containing 10% fetal calf serum, ME-1 cells
were monoblastoid, but with the addition of cytokines such
as interleukin-3 (IL-3). granulocyte-macrophage colonystimulatingfactor (GM-CSF), IL-4, or medium conditioned by
phytohemagglutinin-stimulated human peripheral leukocytes (PHA-LCM), the cells exhibited differentiationto macrophage-like cells. PHA-LCM also promoted eosinophiliclineage differentiation of this cell line, although IL-5 did not
do so. To elucidate the mechanism of proliferation and
differentiationof ME-1 cells, we studied the effect of a potent
inhibitor of protein kinase C, 1-(5-isoquinolinyl-suIfonyl)-2methylpiperazine (H-7). on colony formation of ME-1 cells.
H-7 inhibited colony formation of ME-1 cells by IL-3 or
GM-CSF dose dependently, but had little inhibitory effect on
colony formation by IL-4. These results indicate that the
proliferation and differentiation of ME-1 cells by IL-3 or
GM-CSF were related to the activation of protein kinase C,
while those by IL-4 involved other regulatory systems. ME-1
cells should be useful for studying the pathogenesis of M,E,
and the mechanisms of proliferation and differentiation of
leukemic and normal progenitorsby cytokines.
0 1991by The American Society of Hematology.
S
at 37°C in a humidified atmosphere of 5% CO, in air. The medium
was changed two to three times a week. After cell growth was
confirmed in the wells, the cells were transferred to 24-well
microplates (No. 25820; Coming Glass Works, Corning, NY), and
finally, the growing cells were maintained in culture flasks (No.
3013; Falcon).
Cytochemical staining. Cells were centrifuged in a Shandon
cytospin (Shandon Southern Products Ltd, Cheshire, UK) and
stained with May-Griinwald-Giemsa solution, peroxidase, a-naphthy1 butyrate esterase, naphthol AS-D chloroacetate esterase,
Luxol-fast-blue, and toluidine blue.
Cellsurface markers. Cells were tested by direct immunofluorescence for membrane antigens using various monoclonal antibodies
(MoAbs): OKDR, T l 1 (CD2), Leu9 (CD7), J5 (CDlO), B1
(CD20), Mol (CDllb), Mo2 (CD14), MY4 (CD14), MY7 (CD13),
and MY9 (CD33). T11, J5, B1, Mol, Mo2, MY4, MY7, and MY9
were purchased from Coulter Immunology (Hialeah, FL), and
OKDR and Leu9 were purchased from Becton Dickinson (Oxnard,
CA).
Epstein-Barr virus nuclear antigen (EBNA). The presence of
EBNA was tested by the method of Reedman and Klein.’
Lysozyme and phagocytic activity. Lysozyme activity in the
culture medium was assayed according to the lysoplate technique:
and the phagocytic activity of ME-1 cells was tested using latex
particles.
Chromosome analysis. Chromosome analysis was performed on
ME-1 cells in the logarithmic growth phase. Cells were treated with
0.075 m o m KC1 hypotonic solution for 30 minutes at 37”C, and
fixed by methanol-acetate (311, vol/vol). Chromosomes were banded
by the trypsin-Giemsa method.’
Ultrast~cturaleuamation. Cell pellets were fixed in cacodylatebuffered 2.0% glutaraldehyde for 2 hours and postfixed in cacodylate-buffered 1.0% osmium tetroxide for l hour. Specimens were
INCE THE FIRST report by Arthur and Bloomfield,’
acute myelomonocytic leukemia with eosinophilia
(M,E,) has been known to be characterized by abnormal
eosinophils and the chromosome abnormality inv( 16)
(p13q22),’., and patients with M,E, have been reported to
have a favorable prognosis? No cell line derived from M,E,
has been established to date and the characteristics of the
leukemic cells of M,E, are not yet well known. We previously reported that the eosinophils of a patient with M,E,
arose from leukemic precursors.6
In this article we report the establishment and some
properties of a new leukemia cell line, designated as ME-1,
from peripheral blood leukemia cells of a patient with
M,E,.
MATERIALS AND METHODS
Case report. A 38-year-old Japanese man was admitted to
Ehime University Hospital (Ehime, Japan) because of fever and
swelling of the cervical lymph nodes in August 1986. On admission,
his peripheralwhite blood cell (WBC) count was 6.6 X 1041kL,with
22.0% myeloblasts, 47.0% monoblasts, 1.5% eosinophils, and 6.0%
basophils. His bone marrow was hypercellular with 12.0% myeloblasts, 47.0% monoblasts, 25.0% eosinophils, and 5.0% basophils
(Fig IA). Chromosome analysis of the bone marrow cells showed
46 XY, inv(l6) (p13q22). The patient was diagnosed as having
M,E, on the basis of these data. Remission induction chemotherapy with the BHAC-DMP regimen resulted in the complete
remission. His first relapse occurred in June 1987, but the chemotherapy soon induced another complete remission. In May 1988, he
was admitted again because of a second relapse. His peripheral
WBC count was 3.3 X 104/kLwith 60.0% blasts, 3.0% eosinophils,
and 6.0% basophils. Chromosome analysis of bone marrow cells
showed 47 XY, +8, inv(l6) (p13q22), del(17) ( ~ 1 2 ~ 1 3Despite
).
receiving chemotherapy, he died of sepsis and bleeding.
Cell culture. At the second relapse, mononuclear cells were
collected from the patient’s heparinized peripheral blood by
density gradient centrifugation using the Ficoll-Conray method.
Mononuclear cells were suspended in RPMI 1640 (GIBCO, Grand
Island, NY) supplemented with 10% fetal calf serum (FCS;
GIBCO) in plastic culture dishes (No. 1008; Falcon, Oxnard, CA)
and incubated for 2 hours at 37°C in a humidified atmosphere of
5% CO, in air. Then the nonadherent cells were collected, washed
twice with RPMI 1640 containing 5% FCS, and cultured at 1 x
1061mLin 96-well tissue culture plates (No. 3072; Falcon) using
RPMI 1640 supplemented with 20% FCS. The cells were cultured
Blood, VOI 78, NO 2 (July 15). 1991: ~ ~ 4 5 1 - 4 5 7
From the First Department of Intemal Medicine, School of Medicine, Ehime University, Ehime, Japan.
Submitted October 29, 1990; accepted February 28, 1991.
Address reprint requests to Kohsuke Yanagisawa, MD, The First
Department of Intemal Medicine, School of Medicine, Ehime University, Shigenobu, Ehime 791-02, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
0 I991 by The American Society of Hematology.
0006-4971I9117802-01I1$3.0010
45 1
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452
YANAGISAWA, HORIUCHI, AND FUJITA
.--.
.
..
D
Fig 1. (A) May-Griinwald-Giemsa (MGG)-stainedpreparation of the patient‘s bone marrow cells containing blasts, eosinophils, and basophils
(originalmagnification ~ 1 , 0 0 0 )(. 8 )MGG-stained ME-1 cells cultured under standard conditions (originalmagnification ~1,000).(C) MGG-stained
ME-1 cells cultured with 10 U/mL of IL-4 for 7 days (original magnification x 1.000). (D) An MGG-stained ME-1 colony stimulated by 10%
PHA-LCM. The colony is composed of eosinophils and macrophage-like cells (originalmagnification x 1.0001.
sectioned and examined under a Hitachi HU-12A electron microscope (Hitachi, Tokyo, Japan).
Effect of cyrokines on ME-I cells in liquid culture. To examine
the effect of cytokines on cell differentiation, ME-1 cells were
seeded at 4 x lo’ cells/mL in 10 mL RPMl 1640 containing 10%
FCS and the following cytokines at optimal concentrations: interleukin-3 (IL-3) (100 U/mL),“’ granulocyte-macrophage colonystimulating factor (GM-CSF) (100 U/mL),” G-CSF (100 U/mL),”
IL-4 (10 U/mL),I3 IL-5 (10 U/mL),I4 or medium conditioned by
phytohemagglutinin-stimulated human peripheral leukocytes (PHALCM) (l0%).l5 IL-3, GM-CSF, and IL-5 were purchased from
Amersham Japan Co (Tokyo, Japan); G-CSF was kindly provided
by Chugai Pharmaceutical Co (Tokyo, Japan); and IL-4 was kindly
provided by Ono Pharmaceutical Co (Tokyo, Japan). After incubation for 7 days, viable cells were counted by the trypan blue dye
exclusion test. For morphologic and cytochemical analysis, cytospin
slides were prepared in a Shandon cytospin and stained with
May-Griinwald-Giemsa solution and by cytochemical methods.
Effect of cytokines on ME-I cells in semisolid culture. ME-1 cells
were cultured in semisolid medium with various cytokines. ME-1
cells (1 x 10‘ cells) were cultured with 1 mL of a-medium (Flow
Laboratories, McLean, VA) containing 0.8% methylcellulose, 20%
FCS, and IL-3, IL-4, GM-CSF, or PHA-LCM. Cells were then
plated onto a plastic dish (No. 1008; Falcon). After incubation for 7
days, colonies having more than 20 cells were counted under an
inverted microscope. Smears of cells from each colony were
prepared by cytospin and stained with May-Griinwald-Giemsa
solution. For estimation of the secondary plating efficiency, primary colonies were picked up and washed twice with a-medium.
Then 1 x lo’ ME-1 cells were replated in 1 mL of a-medium
containing 0.8% methylcellulose, 20% FCS, and IL-3 or GM-CSF.
After incubation for 7 days, colonies having more than 20 cells were
counted.
Effect of protein kinase C inhibitor on ME-I cells. H-7 (Seikagaku Kogyo, Tokyo, Japan)” was used as the protein kinase C
inhibitor. Various concentrations of H-7 were added in the culture
medium, and the effects on colony formation of ME-1 cells and
their protein kinase C activity were evaluated.
Protein kinase C acrivily assay of ME-I cells. Protein kinase C
from a cytosolic preparation of ME-1 cells was partially purified
using a diethylamino ethanol (DEAE)-cellulose column. The
protein kinase C activity was measured by the incorporation of ”P
from y-’2P-adenosine triphosphate (y--”P-ATP) into histone H-1
(Sigma Chemical Co, St Louis, MO), as described by Kikkawa et
al.” The reaction mixture was a final volume of 250 WLconsistingof
80 p g h L phosphatidylserine, 20 mmol/L Tris, 0.1 mmol/L CaCIZ,5
mmol/L MgCI,, 2.5 nmol y-”P-ATP, 50 p,g of histone H-1.1 ng/mL
12-0-tetradecanoylphorbol-13-acetate
(TPA), and 50 WLenzyme
preparation. After incubation for 3 minutes at 30°C. the reaction
was stopped by the addition of 25% trichloroacetic acid. The
acid-precipitable material was collected on Millipore filters (Massachusetts) and counted in a liquid scintillation spectrometer.
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HUMAN LEUKEMIA CELL LINE DERIVED FROM M,E,
453
Table 1. Characteristics of ME-1Cells
negative for a-naphthyl butyrate esterase. The cells contained granules positive for Luxol-fast-blue and toluidine
blue (Table 1).
Cellsurfacemarkzrs. The reactions to the MoAbs showed
ME-1 cells had surface antigens specificfor the myelomonocytic lineage: MY4, MY7, MY9, and OKDR (Table 1).
Almost all of the cells showed no reaction to J5, B1, T11, or
Leu9. EB virus-associated nuclear antigen was not detected
in ME-1 cells.
Lysozyme activity and phagocytosis. Lysozyme activity of
6.1 bg/mL was detected in the supernatant obtained from a
7-day culture of ME-1 cells when 1 x lo5 cells/mL were
seeded. Five percent to 10% of ME-1 cells had phagocytic
activity, defined as cells ingesting more than five latex
particles.
Chromosome anabsis. Chromosome analysis was performed on more than 100 metaphases of ME-1 cells. All of
them showed the same chromosome abnormality: 47 X Y ,
+8, inv(l6) (p13q22), del(17) (p12p13) (Fig 2). This was
identical to that of the patient's bone marrow cells at the
second relapse.
Response to cytokines in liquid culture condition. When
ME-1 cells were cultured with IL-3, IL-4, GM-CSF, or
PHA-LCM, the cells were induced to differentiate (Table
2). The cells adhered to culture flasks, formed clusters, and
showed an increase in size. May-Griinwald-Giemsa-stained
cytospin preparations demonstrated that ME-1 cells had
differentiated into macrophage-like cells. They showed a
significant decrease in the nuclear-cytoplasmic ratio, and an
increase in cytoplasmic vacuoles (Fig IC). The a-naphthyl
butyrate esterase reaction became positive in macrophagelike cells, whereas peroxidase.reactions became negative in
these cells. Differentiation of ME-1 cells into macrophagelike cells was most marked in cultures containing PHALCM (Table 2). Surface marker analysis showed that the
expression of some surface antigens for the monocytemacrophage lineage was increased by PHA-LCM. The
percentage of cells reactive with the MoAbs OKDR and
MY7 increased markedly after treatment with PHA-LCM
(Table 3). PHA-LCM also induced ME-1 cells to differen-
Growth pattern:
Suspension
Doubling time:
4-5 days
Morphology:
Monoblastoid
Chromosome analysis: 47 XY, +8, inv(16)(p13q22),de1(17)(p12p13)
Cytochemical staining
Peroxidase:
80.4%
a-Naphthyl-butyrateesterase:
1.6%
Naphthol AS-D chioroacetateesterase:
10.6%
Luxol-fast-blue:
1.2%
Toluidine blue:
0.6%
EB virus-associated nuclear antigen (-)
Surface marker
J5 (CD10)
3.0%
MY4 (CD14)
12.8%
E1 (CD20)
1.4%
MY7 (CD13)
17.9%
Leu 9 ( 0 7 )
0.8%
MY9 (CD33)
36.3%
Mol (CDllb)
6.2%
OKT 11 (CD2)
0.9%
OKDR
13.9%
Mo2 (CD14)
7.5%
Lysozymeproduction:
6.1 pg/mL
(supernatantobtained from a 7-day culture when 1 x lO'cells/mL
were seeded)
RESULTS
Establishment of cell line. About 6 months after the
culture of the mononuclear cells from the patient's peripheral blood was initiated, gradual proliferation of the cells
was observed. Cells were subsequently maintained in continuous culture for 18 months. They grew in suspension with a
little adhesion and aggregation and were designated as
ME-1 cells. The ME-1 cell line has since been maintained
in RPMI 1640 supplemented with 10% FCS and has a
doubling time of 4 to 5 days.
Morphologic and cytochemical characteristics of the cell
line. In May-Griinwald-Giemsa-stained preparations,
ME-1 cells were almost all monoblastoid, with a small
percentage of the cells being eosinophils, basophils, or
macrophage-like cells. The blasts had round or lobulated
nuclei with fine chromatin and one to three nucleoli and
slightly basophilic cytoplasm with a few vacuoles (Fig 1B).
The blasts were significantly positive for peroxidase, weakly
positive for naphthol-AS-D chloroacetate esterase, and
1
2
7
6
13
Fig2. G-bsndingkaryotypeof
ME-1 cells: 47 XY, +8, inv(l6)
(p13q22). del(17) ( ~ 1 2 ~ 1 3 ) .
k
19
14
4
3
8
15
t
9
10
t 16
5
11
t 17
12
18
#
20
21
22
X
Y
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454
YANAGISAWA, HORIUCHI, AND FUJITA
Table 2. Differentiation of ME-1 Cells by Cytokines
(cytochemical analysis)
Table 4. Colony Formation of ME-1 Cells by Cytokines
Cytokine
Percentage of Positive Cells
Cell Density
After 7 Days
( x lo5 cellslml) Peroxidase
Cytokine
(-1
PHA-LCM
IL-3
GM-CSF
G-CSF
IL-4
IL-5
9.1 f 0.7
5.8 ? 1.7
10.9 f 1.1
12.2 f 2.5
8.9 f 1.0
6.8 f 0.2
8.9 f 0.1
80.2
9.2
73.8
50.4
83.6
28.0
79.6
(-1
u-Naphthyl-
Butyrate
Esterase
LuxolToluidine
Fast-Blue
Blue
1.8
74.4
25.8
36.2
7.6
53.4
3.0
1.2
7.4
0.6
0.8
0.8
0.8
1.0
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
For induction of cell differentiation, ME-1 cells were seeded at an
initial concentration of 4 x l o 5 cellsimL with or without cytokines. The
percentage of positive cells was determined by counting 500 cells.
tiate into eosinophils. The percentage of eosinophils positive for Luxol-fast-blue increased from 1.8% to 7.4% in
cultures with PHA-LCM. Neither G-CSF nor IL-5 induced
ME-1 cells to differentiate, and no combination of the
cytokines induced ME-1 cells to differentiate into eosinophils except PHA-LCM.
Response to cytokines in semisolid culture condition. In
semisolid culture conditions, ME-1 cells formed few colonies without cytokines, but they formed substantial numbers of colonies by the addition of IL-3, IL-4, GM-CSF, or
PHA-LCM (Table 4). Colonies formed by IL-4 or PHALCM contained less than 40 cells and colonies formed by
IL-3 or GM-CSF contained more than 50 to 100 cells.
Morphologic analysis of 100 colonies formed in the presence of PHA-LCM by May-Griinwald-Giemsa staining
showed 84 macrophage-like cell colonies, 14 macrophagelike cell-eosinophil colonies (Fig lD), 1 macrophage-like
cell-basophil colony, and 1macrophage-like cell-eosinophilbasophil colony. Colonies formed by IL-4 consisted almost
entirely of macrophage-like cells, and colonies formed by
IL-3 or GM-CSF consisted of blasts and macrophage-like
cells. Secondary plating efficiency was estimated for cells
from colonies formed by IL-3 or GM-CSF. Cells from
colonies formed by IL-4 had a very low plating efficiency
(Table 5).
Colony-stimulating activity was not found in the supernatant of ME-l cells (data not shown). By Northern blot
analysis, no messages for GM-CSF or G-CSF were found in
ME-1 cells (data not shown).
Electron microscopy findings. Electron microscopy
showed that ME-1 cells had clear large nuclei with welldefined nucleoli and a few mitochondria, rough endoplasmic reticula, and Golgi apparatuses in the cytoplasm (Fig
3A). On the other hand, almost all the differentiated ME-1
cells induced by PHA-LCM showed a significant decrease
Table 3. Differentiation of ME-1 Cells by PHA-LCM
(surface marker analysis)
Colony Size
(cells)
4 f 1
268 ? 78
140 f 17
115 f 27
22f 13
57 f 14
5 40
5 40
250
100
250
100
5 40
5 40
in the nuclear-cytoplasmic ratio and had many cytoplasmic
components (Fig 3B). Phagocytosis was sometimes seen in
some of these differentiated cells. Other differentiated cells
had many granules in the cytoplasm. The granules were
large and spherical, and appeared as membrane-limited
vacuoles partially filled with electron-dense material. The
granules had similar morphologic characteristics to those of
normal immature eosinophils, but they lacked the central
crystalloids (Fig 3C and D), which are characteristic of
normal eosinophils.
Effect of the protein kinase C inhibitor H-7 on colony
formation of ME-1 cells. The effect of the protein kinase C
inhibitor H-7 on colony formation of ME-1 cells by IL-3,
IL-4, or GM-CSF was examined (Fig 4). H-7 inhibited the
colony formation by IL-3 or GM-CSF dose dependently,
while it showed little effect on colony formation by IL-4.
Effect of H-7 on the protein kinase C activity of ME-1 cells.
To determine whether the inhibitory effect on colony
formation of ME-1 cells by H-7 was related to protein
kinase C activity, we examined the effect of H-7 on the
protein kinase C activity of ME-1 cells. As shown in Fig 5,
H-7 inhibited the protein kinase C activity of ME-1 cells
dose dependently. This dose-dependent decrease in protein kinase C activity corresponded to the effect of H-7 on
colony formation of ME-1 cells by IL-3 or GM-CSF.
DISCUSSION
In the present study, the ME-1 cell line derived from the
peripheral blood leukemia cells of a patient with acute
myelomonocytic leukemia with eosinophila (M,E,) has
been established. Cytogenetic analysis of the ME-1 cells
showed a karyotype of 47 XY, +8, inv(l6) (p13q22), del(17)
(p12p13), which was identical to that of the patient's bone
marrow cells and demonstrated that this cell line originated
from the patient's leukemic blasts. Most of ME-1 cells had a
blastic appearance, but a small percentage of the cells were
macrophage-like cells, eosinophils, or basophils. IL-3, GMTable 5. Secondary Colony Formation of ME-1 Cells
Primary Colony Stimulator
Secondary
OKDR
MY7
MY9
Mol
18.4
79.5
26.0
67.5
16.3
35.6
10.0
5.2
-
'Values are the means f SD to triplicate cultures.
Colony Stimulator
Percentage of Positive Cells
ME-1 cells
PHA-LCM-stimulated ME-1 cells (7 d)
PHA-LCM
IL-3
GM-CSF
G-CSF
IL-4
No. of Colonies"
(per i o 5 cells)
IL-3
GM-CSF
IL-3
GM-CSF
IL-4
1 1 6 ? 11
56 ? 39
53 f 11
4424
3?2
0
Values represent the secondary colony numbers per l o 5 cells from
colonies formed by each primary colony stimulator. Colony numbers
are the means ? SD to triplicate cultures.
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HUMAN LEUKEMIA CELL LINE DERIVED FROM M,E,
455
Fig 3. (A) Ultrastructuralappearance of an ME-1 cell cultured under standard conditions (original magnification ~6,000).(E) Ultrastructural
appearance of a macrophage-like ME-1 cell cultured with 10% PHA-LCMfor 7 days (original magnification~4,000).(C) Ultrastructuralappearance
of an ME-1 cell with granules cultured with 10%PHA-LCM for 7 days (originalmagnification ~ 6 , 0 0 0 )(D)
. Higher magnification of granules in an
ME-1 cell cultured in 10%PHA-LCM for 7 days (originalmagnification ~ 1 5 , 0 0 0 ) .
CSF, IL-4, or PHA-LCM, which contains many cytokines,
induced the differentiation of ME-1 cells. They mainly
induced differentiation into macrophage-like cells. PHALCM also induced differentiation to eosinophils, but IL-5,
which has been reported to induce eosinophilic differentiadon,'4 did not do so in this cell line. IL-3 and GM-CSF are
known to stimulate monocytic precursors and induce differentiation to monocyte-macr~phage,".'~
and the ability of
IL-4 was recently reported to transform monocytes into
macrophages." ME-1 cells exhibited responsiveness to
those cytokines and differentiated into macrophage-like
cells as do normal macrophage precursors. In this respect,
the ME-1 cell line appears to be valuable as a model cell
line to study the mechanism of cell differentiation by
cytokines, because myeloid leukemia cell lines such as
HL-60 and U937 have little or no differentiation response
to these cytokines?' IL-5,IL-3, and GM-CSF have been
reported to induce eosinophilic differentiati~n,'~.''.'~but all
of these cytokines had no effect of eosinophilic differentiation on ME-l. PHA-LCM induced a small number of ME-l
blasts to differentiate into eosinophils, but PHA-LCM
induced most of the ME-1 cells to macrophage-like cells.
PHA-LCM contains IL-3, GM-CSF, and other cytokines,u
and it has been reported to have an "eosinophil-stimulating
a~tivity."~.*~
In the present study, it can be suggested that
the differentiation of ME-1 cells into eosinophils was
related to this eosinophil-stimulatingactivity, which was not
IL-5, IL-3, or GM-CSF. Ultrastructural examination of
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456
YANAGISAWA, HORIUCHI, AND FUJITA
100
% 60
x
v
.-12
40
C
0
5 20
0
U
M I L - 3 , H-7
HGM-CSF, H-7
W I L - 4 , H-7
I!
5
10
Concentration of H - 7 ( y M)
1
Fig 4. Effect of H-7 on colony formation of ME-1 cells in response
to IL-3, IL-4, or GM-CSF. Results are given as the percentage of the
number of colonies in each control culture.
PHA-LCM-stimulated cells showed the existence of “abnormal eosinophils” with granules containing electron-dense
material and lacking central crystalloids. These characteristics of eosinophils have also been reported in other patients
with M4E0.2.4In semisolid culture, ME-1 cells formed
colonies in response to IL-3, IL-4, GM-CSF, or PHA-LCM.
Colonies formed by PHA-LCM contained various combinations of macrophage-like cells, eosinophils, and basophils.
These results demonstrated that clonogenic ME-1 cells
with inv( 16) were able to differentiate to macrophage-like
cells, eosinophils, and basophils. It has been shown that
patients with M4E, had an inversion of chromosome 16,’-’
and some cases of M,E, with basophilia have been reported
recently.””6 Thus, the eosinophils and basophils in M,E,
Concentration of H - 7 ( p M)
Fig 5. Effect of H-7 on the protein kinase C activity of ME-1 cells.
Results are given as the percentage of control assays in the presence
of TPA, CaCI,, and phosphatidylserine. Protein kinase C activity is
expressed as picomoles of
incorporated into histone H-l/min/lQ
cells. The activity was 31.0 pmol
and 0.4 pmol ”P in the presence
and absence of TPA, CaCI, and phosphatidylserine,respectively.
patients appear to be part of the neoplastic process, but no
direct evidence for this hypothesis had been obtained. In a
previous report, we have recently demonstrated the neoplastic involvement of monocytic, eosinophilic, and basophilic
lineages in the patient with M,E, from whose leukemic cells
the ME-1 cell line was established, by a cytogenetic,
cytochemical, and morphologic analysis of colonies derived
from leukemic precursors.6 The data we obtained for the
ME-1 cell line also confirm that eosinopils and basophils in
M,E, are neoplastic cells.
CSFs stimulate the proliferation and differentiation of
hematopoietic precursors. CSFs also act to promote the
differentiation and self-renewal of the leukemic progenitors of acute myeloblastic leukemia (AML) blast
In
the ME-1 cell line, IL-3 or GM-CSF promoted differentiation from blasts to mature macrophage-like cells, but they
also stimulated self-renewal of clonogenic ME-1 cells, as
shown by the secondary plating efficiency study. On the
other hand, IL-4, which was first named B-cell-stimulating
factor-1, induced ME-1 cells to macrophage-like cells and
the cells from colonies formed by IL-4 had very low
secondary plating efficiency. Thus, it was concluded that
IL-4 induced the differentiation of ME-1 cells without
stimulating their self-renewal capacity. These results suggest the possibility that IL-4 may become a useful therapeutic agent for AML.
Protein kinase C appears to have an important role in the
signal transduction responses in various cell^.'^,^' For hematopoietic cells, using a hematopoietic growth factordependent cell line and human myeloid progenitor cells, it
has also been suggested that activation of protein kinase C
was involved in the proliferation of progenitor cells stimulated by CSFS.~’.~~
We examined the effect of the protein
kinase C inhibitor H-7 on the stimulation of colony formation of ME-l cells by IL-3, GM-CSF, or IL-4. Colony
formation of ME-1 cells by IL-3 or GM-CSF was inhibited
by H-7 dose dependently. In contrast, colony formation by
IL-4 was affected little, suggesting that the inhibitory effect
of H-7 was not due to cytotoxicity. In addition, H-7
inhibited the protein kinase C activity of ME-1 cells dose
dependently in a manner corresponding to its effect of H-7
on the response of ME-1 cells to IL-3 or GM-CSF. These
results indicate that the proliferation and differentiation of
ME-1 cells by IL-3 or GM-CSF were related to the
activation of protein kinase C, whereas those by IL-4
involved other regulatory systems. Recent
indicating that IL-4 did not induce protein kinase C translocation
from cytosol to membrane in resting murine B lymphocytes
support our results.
ME-1 is a unique leukemia cell line derived from M,E,
that responds to CSFs and IL-4. ME-1 can provide a useful
model for studying the pathogenesis of M,E, and the
mechanisms of proliferation and differentiation of leukemic and normal progenitors by cytokines.
ACKNOWLEDGMENT
The authors express their great appreciation to Prof Yuzuru
Kobayashi for his continuous support of this work and the valuable
comments.
From www.bloodjournal.org by guest on November 24, 2014. For personal use only.
457
HUMAN LEUKEMIA CELL LINE DERIVED FROM M,E,
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