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Aphidicolin, an Inhibitor of DNA Replication, Blocks the TPA-Induced
Differentiation of a Human Megakaryoblastic Cell Line, M E G - 0 1
By Takashi Murate, Tomomitsu Hotta, Keitaro Tsushita, Motoshi Suzuki, Tomoaki Yoshida, Shinsuke Saga,
Hidehiko Saito, and Shonen Yoshida
The commitment process of a human megakaryoblastic cell
line (MEG-01) induced with phorbol ester, TPA, was investigated with special reference t o glycoprotein (GP) Ilb/ llla
expression, multinuclear formation, and DNA replication.
TPA (lo-’ mol/L) completely inhibited cellular division in
MEG-01. but did not suppress de novo DNA synthesis. Two
days’ culture with lo-’ mol/L TPA was sufficient for MEG-01
cells to initiate an irreversible commitment process. These
cells could not resume cell growth and expressed GP Ilb/llla
antigen; some of them showed multinuclear form and DNA
polyploidy even after removal of TPA from the culture
medium. DNA histogram analysis showed that, upon treatment with TPA, the percentage of cells whose DNA ploidy
was more than 8N was 5 t o 10 times higher than that of
control cells. Precise analysis using cell size fractionation by
centrifugal elutriation method showed that there was strong
correlation between the percentage of multinuclear cells and
DNA polyploidy in TPA-treated cells. The percentage and
staining intensity of GP Ilb/llla and other megakaryocytic
phenotypes such as von Willebrand factor and PAS staining
were highest in large multinuclear cell populations, suggesting that these cells are the most differentiated population in
this system. In TPA-treated cells, the activity of DNA polymerase a, a marker for cell growth, remained at the same
level as in control cells. Aphidicolin, a specific inhibitor of
DNA polymerase a, completety inhibited the differentiation
induction of MEG-01 cells with TPA measured by either GP
Ilb/llla expression or multinuclear cell formation. Therefore,
DNA replication appears t o be involved in the process of
phenotypic expression as well as endomitosis in megakaryocyte differentiation of MEG-01 cells. Aphidicolin was also
effective in inhibiting megakaryocytic differentiation of other
leukemia cell lines such as human erythroleukemia (HEL) and
K562 cell lines induced with TPA, suggesting the close
interplay of DNA replication and phenotypic expression in
megakaryopoiesis.
o 1991by The American Society of Hematology.
A
induced to differentiate by TPA to exhibit multiple
megakaryocytic phenotypes. To examine the commitment
process of the megakaryocytic differentiation of MEG-01
by TPA, we adopted the methods used for mouse erythroleukemia cell line (MELC) and human K562 cell line.21-27
We report here the relationship among the DNA replication, ploidy, and the expression of megakaryocytic phenotypes such as platelet glycoprotein (GP) IIb/IIIa antigen”.“
and production of polysaccharides in the cytoplasm (PAS
stainingz6).It was shown that aphidicolin, an inhibitor of
DNA polymerase a,inhibits the polyploid formation as well
as megakaryocytic phenotypic expression of MEG-01,
human erythroleukemia cells (HEL),” and K562 cells. So, it
was found that DNA replication was essential for the
coordinated megakaryocytic differentiation (phenotypic expression and endomitosis) of various leukemia cell lines.
MEGAKARYOCYTE is derived from the hematopoietic multipotential stem cell and produces platelets.’,’
The megakaryocyte differentiation process is unique in that
the nuclear DNA ploidy increases (endomitosis) with its
cytoplasmic maturation and the megakaryocyte releases
platelets from its plasma membra ne^.^.^ However, cytologic
as well as biochemical analysis of this process has been
hampered because of the paucity of megakaryocytes (less
than 0.1% of the nucleated cells) in the bone marrow (BM),
although several methods have been developed to collect
megakaryocyte population^."^^ To analyze the process of
megakaryocyte differentiation, therefore, established cell
lines that have a capacity to differentiate into megakaryocytic lineage are useful as a model system.”-” We have
previously established and characterized a human megakaryoblastic cell line, MEG-01, from the bone marrow cells
of a patient with the megakaryoblastic crisis of chronic
myelogeneous leukemia.’’,L8Phorbol 12-myristate 13 acetate (TPA) is known to enhance normal megakaryocytic
colony formation in vitro,lY~Zo
and MEG-01 cells can also be
From The First Department of Internal Medicine, Research Institute
for Disease Mechanism and Control, The Department of Immunology,
and The Second Department of Pathology, Nagoya University School
of Medicine, Showa-ku, Nagoya, Japan.
Submitted December 7,1990; accepted August 26,1991.
Supported in part by a Grant-in-Aidfor Cancer Research from the
Ministiy of Education, Science and Culture of Japan.
Address reprint requests to Takashi Murate, MD, The First Department of Internal Medicine, Nagoya University School of Medicine, 65
Tsurumai-cho,Showa-ku, Nagoya 466, 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 1734 solely to
indicate this fact.
o 1991 by The American Society of Hematology.
0006-4971191 17812-0027$3.0010
3168
MATERIALS AND METHODS
Cells and culture conditions. The origin and initial characterization of MEG-01 cells were described by Ogura et al.” To harvest
cells, 1% trypsin and 0.2% ethylenediamine tetraacetate (EDTA)
in phosphate-buffered saline (PBS) were used. The cells were
cultured in Falcon 3013 plastic flasks (Becton Dickinson, Oxnard,
CA) containing RPMI 1640 medium (GIBCO, Grand Island, NY)
supplemented with 10% heat-inactivated fetal calf serum (FCS;
Hazelton Research Products, Inc, Lenexa, KS) at 37°C in 5% CO,
and air in a humidified incubator. As MSG-01 cells began
adhering to culture wells after TPA treatment, Lab Tek chamber
slides 4804 (Nunc Inc, Napeville, IL) were used in some experiments for the direct staining procedure of the cells. HEL cell line”
was obtained from Japanese Cancer Research Resources Bank
(Tokyo, Japan) and was maintained in RPMI 1640 containing 10%
FCS. The characterization and culture condition of U 6 2 cells were
described previously.22
Chemicals and reagents. TPA was purchased from Sigma Co (St
Louis, MO) and the stock solution was described previously.I8
Propidium iodide (PI), RNase, aphidicolin, cycloheximide, and
actinomycin D were purchased from Sigma. A monoclonal antiBlood, Vol78, No 12 (December 15), 1991: pp3168-3177
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APHlDlCOLlN BLOCKS DIFFERENTIATION OF MEG-01
body (MoAb) against GP IIb/IIIa was the generous gift of Dr T.
Naoe (Branch Hospital, Nagoya University School of Medicine).z8
Mouse MoAbs against GP IIIa and rabbit polyclonal antibodies
against factor V and von Willebrand factor (vWF), respectively,
were purchased from Dako Co (Glostrup, Denmark). ’H-TdR
(specificactivity, 2.23 to 3.33 MBq/mmol) was purchased from ICN
Biomedicals Inc (Costa Mesa, CA). 2,3-Dideoqcytidine (DDC)
and 2,3-dideoqthymidine (DDT) were obtained from Pharmacia
(Uppsala, Sweden).
Histochemical staining. Specimens were air-dried and fixed
with 4% paraformaldehyde (PFA) at 4°C overnight. Enzyme
immunostaining with MoAbs against GP IIb/IIIa or GP IIIa was
performed using Dako’s APAAP staining kit (Carpinteria, CA)
according to the manufacturer’srecommendation.Briefly, cytospin
slides were incubated with MoAb against GP IIbiIIIa or GP IIIa
for 30 minutes and then washed twice with PBS for 5 minutes.
Linking antibody was then overlaid on the specimen for 30 minutes
and then washed with PBS twice for 5 minutes. APAAP complex
was added for another 30 minutes, washed twice with PBS.
Substrates were then added and the specimen was incubated for an
additional 15 minutes. Background staining with normal mouse
serum or incidental IgG was less than 2% under our experimental
conditions. Staining of cytospin slides with rabbit antihuman factor
V antibody or anti-vWF was described previously.18Normal rabbit
serum was used as a negative control of these experiments.At least
200 cells were counted and repeated three times. PAS staining was
performed according to Wislocki et al?9 For the quantitative
expression of PAS staining intensity, a DVS 3000 system image
processor equipped with CCD camera (Hamamatsu Photonics,
Hamamatsu, Japan), which was connected to microscope, was
used. Each cell was visualized on the video screen and the cell’s
total staining intensity and its surface area were measured with the
accompanying computer program of this processor. The mean
staining intensity was calculated from total staining intensity and
surface area.
Centrifugal elutriation. Centrifugal elutriation was performed
using a Sanderson chamber with Beckman J-6MIE centrifuge
(Beckman, Palo Alto, CA). All operations were performed at 0 to
4°C in 0.2% bovine serum albumin (BSA) in PBS. Cells (1 X lo’) in
5 mL of RPMI 1640 containing 10% FCS were loaded into an
elutriation rotor running at 2,000 rpm. Eight fractions of 100 mL
each were collected as follows. The first fraction was collected at a
flow rate of 22 mL/min. From the collection of the second fraction,
the flow rate was increased to 34 mL/min. The centrifuge speed
was changed from 2,000 rpm (fraction 2) to 1,480 (fraction 3), 1,200
(fraction 4), 1,000 (fraction 5), 750 (fraction 6), and 600 rpm
(fraction 7 and 8), successively. The cell number in each fraction
was determined with a hemocytometer using aliquots and the rest
was collected and used for further study.
Flow cytomehic analysis of DNA ploidy and GP IIbIIIIA staining.
DNA staining using propidium iodide was described previously2’
using Epics profile cell sorter (Coulter Corporation, Hialeah, FL)
with its accompanying DNA analysis program. The procedure to
analyze membrane GP IIbIIIIa with fluorescent cell sorter was
described before.I8
De novo DNA synthesis. De novo DNA synthesis was studied
according to the method described previously?’ Briefly, 1 mL of
MEG-01 cells was labeled with 5 KCi of [3H]dThdat 37°C for 6
hours. After collection, the cells were washed once, lysed, and the
acid-precipitableradioactivitywas measured.
Assays for DNA polymerase a, p, and y. The measurement of
DNA polymerase a, p, and y in crude extracts of cells was
described previo~sly.~~
Activity was expressed as the acidprecipitable radioactivity per 5 x lo4 cells under the conditions
used.
3169
Statistics. Student’st-test was performed to evaluate the statistical significance between the two groups.
RESULTS
Cell proliferation and differentiation markers. Figure 1A
shows the cell proliferation curves of M E G - 0 1 cells in the
absence and presence of various concentrations of TPA.
TPA showed or inhibited MEG-01 cell growth in a dosemol/L
dependent manner. For further analysis, 1 x
TPA was selected because the percentages of GP IIb/IIIa
expression and multinuclear cells were maximum under this
mol/L TPA-treated
condition (Fig 1B). The viability of
cells was about 75% on day 5, because TPA of more than
lo-’ mol/L slightly decreased the cell viability. In our
experimental conditions, control unstimulated cells grew
continuously until day 4 and reached a plateau thereafter.
More than half of them are in suspension form, and the rest
of them attached loosely to the culture vessels. More than
99% of cells were mononuclear, but there were also a few
large, multinuclear cells. Upon TPA treatment, MEG-01
cells rapidly attached to the surface of the culture vessels
within a few hours. The absolute number of large and
multinuclear cells began to increase with the increasing
culture period (Fig 1D). Immunohistochemical staining
using anti-GP IIb/IIIa MoAb showed that cells (especially
multinuclear cells) in the TPA-treated group were positive
for GP IIb/IIIa antigen (Figs 1B and 2B). Concomitantly,
these multinuclear cells were strongly positive for PAS
staining. Although a small fraction of mononuclear cells in
the control group were also weakly positive for these
platelet-megakaryocyte markers (PAS and GP IIb/IIIa),
the frequency of GP IIb/IIIa staining and PAS staining
increased even with mononuclear cells in the TPA-treated
group (Figs lB, 2B, 3A, and data not shown).
M E G - 0 1 cells ceased cell division in the presence of 1 x
lo-’ mol/L of TPA. However, they continued to synthesize
D N A for the experimental period at a constant rate that
was lower than that of control cells (Fig 1C). Washout
studies showed that exposure to TPA for 1 or 2 days was
sufficient for most M E G - 0 1 cells to lose their capacity to
proliferate. Even after the removal of TPA from culture
medium, the percentage of G P IIb/IIIa-positive cells did
not decrease significantly as compared with cells with
continuous TPA exposure (data not shown). Hence, it is
suggested that M E G - 0 1 cells were committed to megakaryocytic differentiation by a transient exposure to TPA.
DNA content. Figure 4 shows the amount of D N A in
cells measured by a fluorescence-activated cell sorter
(FACS) after D N A staining with PI. In the TPA-treated
group, an increase in a population whose D N A ploidy was
more than 8N D N A (area 4) was observed on day 5.
Calculation showed that polyploid cells increased four to six
times compared with the control group. These values are
consistent with the percentage of multinuclear cells in
May-Giemsa staining, suggesting that multinuclear cells
correspond to cells with D N A polyploidy.
Cell size fractionation. To analyze the commitment process of M E G - 0 1 cells induced with TPA, we fractionated
M E G - 0 1 cells according to the cell size using the centrifu-
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MURATE ET AL
3170
A
x1
Cell growth curve
B
c
phenotypic expression
/ ml
3H-TdR uptake
C P M / l X105
Cells
,
I
1
2
!
I
3
4
days
I
5
0 1
x
v
x
)
v
)
y
2
3
4
days
I
TPA (M)
D
Con t ro t
I
Fig 1. Cell proliferation, differentiation, and DNA synthesis of
MEG-01 cells. (A) Cell proliferation. MEG-01 cells (1 x 10' cells/mL)
were cultured in the absence or presence of various concentrations of
TPA. After trypsin and EDTA treetment, cells were collected, washed
with PBS, and counted with a hemocytometer. Cell viability was
examined with a 0.4% trypan blue dye exclusion test. (B) GP llblllla
immunohistochemical staining. Cells were harvested on day 5, washed,
and cytospin preparations made. Immunohistochemical staining using
an MoAb against GP Ilb/llla was performed as described in Materials
and Methods. The ordinate denotes the percentage of GP llb/lllapositive cells. At least 200 cells were evaluated. (C) '[HI dThd incorporation. De novo DNA synthesis was measured by the method described in
Materials and Methods. MEG-01 cells (1 x 10'lmL) were plated into
culture medium with or without 10 ' mol/L of TPA. Another set of
culture flasks was prepared simultaneously t o measure cell concenhations during the culture period. Every day, 5 pCi of '[HI dThd was
directly added t o 1 mL of culture medium. Six hours later, cells were
collected and washed with PBS twice. The acid-precipitable radioactivity was measured. Experiments were performed in triplicate and the
mean value is shown. SD was less than 10% of the mean value. The data
were expressed as the radioactivity (cpm)/l x 10' cells. This experiment was repeated and produced a similar result. (D) Photomicrograph.
The photographs show May-Giemsa staining of control and 10 mol/L
TPA-treated MEG-01 cells (day 5) cultured in Lab-Tek chamber slides
(original magnification ~200).
'
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APHlDlCOLlN BLOCKS DIFFERENTIATION OF MEG-01
3171
diameter
diameter
- 50
-40
- 30
-20
-10
- 90
-80
6P I l b / I l I a
‘70
.
L?
f
‘60
M.N.C.
-50
5
I?
-40
1
1
3
4
5
Fraction
6
1
1
1
3
4
5
6
7
Fraction
B
Fig 2. (A) Centrifugal elutriation of MEG-01 cells.
Aliquots of 1 x lo’ of the control and TPA-treated
MEG-01 cells (day 5) were applied t o Beckman
elutriator according t o the procedure described in
Materials and Methods. Mean cell size under this
elutriation condition was calculated according t o the
manufacturer and indicated in the upper part of the
figure. (0)Cell yield per fraction. ( 0 )The percentage
of multinuclear cells. (A)The percentageof GP llb/illapositive cells. (6) The immunohistochemical staining
of typical fractions (2 and 4) from each sample are
shown. Top left, control fraction 2; top right, TPA
fraction 2; bottom left, control fraction 4; bottom
right, TPA fraction 4. Other conditions are described
in Materials and Methods.
-
gal elutriation method. Figure 2A showed the result of a
typical experiment. The elutriation patterns of both groups
were apparently similar except that the yield of fraction 4
(containing large cells) was always higher in the TPAtreated group than in the control group. The large and
multinuclear cells consisting of at most 5% to 10% of total
population was concentrated in fractions 3 and 4 (Fig 2B
and C). The control cells also contained a very small
number of differentiated cells (mainly large and multinuclear cells) (Fig 2B), probably because of spontaneous
differentiation. GP IIb/IIIa staining by the APAAP method
of cytospin slides of typical fraction is shown in Fig 2B. To
y.
analyze more precisely the relationship between expression
of GP IIb/IIIa, multinuclear formation, and DNA ploidy,
each fraction was processed for flow cytometric analysis of
DNA content and GP IIb/IIIa staining. A clear correlation
between DNA polyploidy and the increase of GP IIb/IIIa
expression was observed in the large cell fraction of TPA
treated cells as shown in Fig 2C. In control fraction 2, DNA
ploidy of most cells are 2N, while, in control fraction 4,
some 4N cells and small percentage of 8N cells were
observed. In the TPA-treated fraction 2, the percentage of
4N ploidy was increased but that of 8N was very few. In the
TPA-treated fraction 4, a great increase in 8N was ob-
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3172
C
MURATE ET AL
DNA ploidy
Fr2
A : 14.4%
I:5.6%
:I[,
6P I I b l I a
,
V
H I
21
4I
i
I1
1
21
41
in
.
,
,
FLUORESCENCE
Cont
Fr4
-
,
TPA Fr2
1
0
FLUORESCENCE
A : 52.1%
B: 19.7%
E: 3.0%
a
u
0
21
41
IN
21
4i
IN
Fig 2. (Cont'd) (C) Flow cytometric analysis of
DNA ploidy and GP Ilb/llla staining. Left, the result of
DNA staining; right, GP Ilb/llla staining. Control
fractions 2 and 4 and TPAfractions2 and 4 are similar
to (A) and (B). The ordinate shows the arbitrary units.
A, the percentage of DNA 2N population; 6, the
percentage of DNA 4N population; C, the percentage
of cells with DNA EN or more; D, the background
level of GP Ilb/llla staining; E and E', the peak
position of positive staining of TPA fractions 2 and 4,
respectively.
lL!!L
FLUORESCENCE
served. So, the percentage of multinuclear cells (as shown
in Fig 2A and B) and DNA ploidy are positively correlated.
Although the yield of multinuclear cells varied between
experiments, more than 70%of the multinuclear cells in the
large cell fraction had 8N DNA ploidy or more. The result
of the FACS analysis of GP IIb/IIIa expression was
consistent with light microscopic observation (Fig 2B), as
well as quantitative analysis of staining intensity per cell
using the DVS 3000 image processor (data not shown). It
was confirmed that GP IIb/IIIa expression of the large cell
fraction of the TPA-treated cells (fraction 4) was higher
than that of the small cell fraction (fraction 2). In PAS
staining and immunofluorescent staining of vWF, multinuclear cells showed the strongest staining intensity (data not
shown). It can be said, therefore, that the multinuclear cells
in the TPA-treated MEG-01 are the most differentiated
population in this system.
We then cultured each fractionated cell of both the
control and TPA-treated groups, with or without TPA, and
examined their properties. Table 1 shows that, in the
control group, fractions 1 and 2 grew well and were
uncommitted, while fractions 3 to 5 exhibited a moderate
degree of differentiation (spontaneous differentiation). In
contrast, even the small cells (fractions 1 and 2) in the
TPA-treated group lost their self-renewal capacity and
expressed the markers for differentiation (GP IIb/IIIa and
PAS) even after removal of TPA. These results confirmed
that TPA induced an irreversible commitment of MEG-01
cells.
Activities of DNA polymerases. DNA polymerase a, p,
and y were measured in cells during TPA-induced differentiation (Fig 5). DNA polymerase (Y activity per cell showed
no significant change during treatment with TPA. DNA
polymerase p and y per cell increased approximately
twofold compared with the control group. Because these
cell cultures consisted of heterogeneous populations (Fig
lD), we also measured the DNA polymerase a activity in an
almost homogeneous population of cells obtained by centrifugal elutriation. Extracts from comparable fractions of both
groups again showed similar levels of DNA polymerase
activities (data not shown).
Effects of aphidicolin and other DNA polymerase inhibitors
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APHlDlCOLlN BLOCKS DIFFERENTIATION OF MEG-01
A
B
PAS
Van Willebrand Factor
3173
C
Table 1. The Effects of TPA Treatment on Size-Fractionated
MEG-01 Cells
Factor V
Fraction
20
10
10
Cont T P A I t A P H
Cant T P A I t A P H
Fig 3. Effects of TPA and/or aphidicolin on PAS staining, vWF, and
factor V expression of MEG-01 cells. MEG-01 cells were cultured with
TPA in the presence and absence of aphidicolin for 4 days. Their
cytospin preparations were stained with PAS (A) or with antibodies
against vWF (6) or factor V (C). PAS-stained preparations were
analyzed with a DVS 3000 system image processor as described in
Materials and Methods. The control experiment was performed
without TPA. Each cell was scanned, and the mean PAS intensity/cell
was calculated from the data of total cell staining intensity and cell
surface area. The ordinate denotes the arbitrary unit of staining
intensity. The background level (66.9 f 0.53) was subtracted and
values were expressed as mean ? SD from the analysis of 200 cells.
The concentrations of TPA and aphidicolin were
mol/L and 5 x
mol/L, respectively. (6) and (C) show the effect of TPA and/or
aphidicolin on cytoplasmic vWF and factor V expression measured
according t o the method described previously.18 In the case of factor
V, samples were incubated with the first antibody overnight, and
excessive washing was performed between each step, because
fluorescence intensity was weak as compared with vWF or GP llb/llla.
At least 200 cells were examined with the fluorescence microscope.
Values are the means f SD of day 4 samples of three separate
mol/L of TPA; T+APH, lo-' mol/L of TPA and
experiments. TPA,
5 x lo-' mol/L of aphidicolin.
1
Cont Do
1
Fr 1
-
a
0
72.2
2 13.8
3 12.7
4 1.2
%
4
1
1
Fr 1
75.9 '"
4
Control cells
TPA (-)
Cell proliferation
GP Ilb/llla (%)
Multinuclear cells (%)
TPA (+)
Cell proliferation
GP Ilb/llla (%)
Multinuclear cells (%)
TPA-treated cells
TPA (-)
Cell proliferation
GP Ilb/llla (%)
Multinuclear cells (%)
TPA (+)
Cell proliferation
GP Ilb/llla (%)
Multinuclear cells (%)
1
2
++
++
+
+
-
5
1
5
1
14
7
30
25
37
28
4N
8N
5
-
-
-
-
-
80
12
90
25
95
45
95
35
f
68
7
-
-
-
-
82
8
89
30
95
50
93
45
-
-
-
-
-
80
15
92
15
90
35
92
48
95
ND
As shown in Fig 2, control and TPA-treated cells (day 5) were
size-fractionated by centrifugal elutriation. Each fraction was cultured
again with or without
mol/L TPA. After 5 days, cell counts, the
percentages of multinuclear cells, and the percentages of GP llb/lllapositive cells were measured. The grade of cell proliferation was: -,
cell density on day 5 was less than 1.2 times of the initial cell density; +,
cell density on day 5 was between 1.2 and 1.5 times of the initial cell
density; + and + cell density on day 5 was between 1.5 and 4 times
of the initial cell density, or more, respectively. The data denote the
mean of two separate measurements. SD was less than 10% of the
mean. Other conditions are described in Materials and Methods.
Abbreviation: ND, not done.
+,
on the process of differentiation and endomitosisprocess. To
determine the role of each species of DNA polymerase in
the commitment process, especially the endomitosis process of MEG-01 differentiation, the effects of aphidicolin
and other DNA polymerase inhibitors were tested. Figure
6A and B showed that aphidicolin, a potent inhibitor of
DNA polymerase a,strongly suppressed the appearance of
multinuclear cells in a dose-dependent manner and also
inhibited the phenotypic expression (ie, the appearance of
.
2N
4
70
6
Meg 01
4000
3000
2N
3
4N
8N
Fig 4. FACS analysis of DNA staining. DNA staining of the control
and TPA-treated cells and FACS analysis were performed according t o
the method described in Materials and Methods. Fractions 1,2, and 3
correspond t o the cell population in G,/Gl(2N), S, and GJM(4N).
respectively. Fraction 4 denotes the cell population containing DNA
polyploidy (mainly 8N). Cont Do, control untreated sample. Cont D,
control culture on day 5. TPA D, lo-' mol/L TPA-treated MEG-01
cells after 5 days of incubation. The locations of 2N. 4N. and 8N are
shown on the abscissa. The ordinate shows arbitrary units.
1000-
TPA-'
3000
2000
2000
IO00
?-TxT- days
-
0
0
days
1
2
3
1
5
days
Fig 5. Activity of DNA polymerase a, p, and y. The enzymatic
activity of DNA polyploidy a, p, and y of crude extracts was measured
according t o the method described in Materials and Methods. The
data were expressed as the acid-precipitable radioactivity (cpm) per 5
x 10' cells. The ordinate denotes the radioactivity incorporated into
DNA. The abscissa indicates the culture periods.
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MURATE ET AL
3174
B
A
I
I
1
p, : 0.010,
p2
: 0.009,
P,<O.OOl
C
1
ltOU1
T
P2<0.001
D
z
100
cells induced with TPA. However, we could not make a
definitive conclusion because the long incubation of cells
with 1 x lo-' mol/L of TPA in combination with more than
25 nmol/L of actinimycin D also inhibited DNA synthesis
and was strongly cytotoxic (data not shown).
Other markers for megakaryocytic differentiation. Figure
3 shows other megakaryocytic markers (PAS staining,
factor V, and vWF) of TPA-induced MEG-01 cells and the
effects of aphidicolin on these markers. It is clear that TPA
could induce phenotypes other than GP IIb/IIIa and that
these could be inhibited with aphidicolin, too.
Other leukemia cell lines. Table 2 shows the results of
similar experiments using other leukemia cell lines, HEL
and K562 cells, that were known to have megakaryocytic
differentiation capacity. TPA could induce endomitosis as
well as megakaryocytic phenotypes (GP IIb/IIIa or GP
IIIa) in these cell lines, and aphidicolin inhibited the
phenotypic expression as well as endomitosis in the same
manner as observed in MEG-01 cells.
DISCUSSION
Fig 6. Effect of aphidicolin on differentiation and endomitosis of
MEG-01 cells induced with TPA. MEG-01 cells (1 x 10S/mL) were
cultured with or without lo-' mol/L of TPA in Lab-lek chamber slides.
In the cultures containing
mol/L of TPA, various doses of
aphidicolin or 1 Wg/mL of cycloheximide were added as indicated.
After 5 days, medium was removed and the slides were air-dried and
stained with May-Giemsa or antibodies as described in Materials and
Methods. The percentages of mukinuclear cells and GP Ilb/llla
positive cells were counted in triplicate using at least three different
fields. At least 200 cells were counted for each group. Data are shown
as a percentage of the total cells. (A) The percentage of multinuclear
cells; ( 6 )the percentage of GP Ilb/llla-positive cells. Con, the control
group; 1 . 1 x lo-' mol/L of TPA-treated cells; T+A, 10.' mol/L of TPA
mol/L of aphidicolin-treatedgroup; TtCY, lO-'mol/Lof
plus5 x
TPA plus 1 Fg/mL of cycloheximide-treated group; T+DDC, lo-'
mol/L of TPA plus 40 pmol/L of DDC; T+DDT,
mol/L of TPA plus
40 pmol/L of DDT. (C and D) The dose-response curve of the effect of
aphidicolin. The ordinates show the percentage of multinuclear cells
(C) or GP Ilb/llla-positive cells (D). T, TPA-treated cells on day 5; IO-',
and 1 x 10.' M of
5 x lo-', and 1 x lo-' A denote lo-', 5 x
aphidicolin addition, respectively. The data are the sum of the two
experiments. P value of student's t-test between the group in question and the TPA-treated group.
The low frequency of megakaryocytes in BM and the
difficulty in obtaining a pure population always pose a
problem for further studies. Thus, MEG-01 may provide a
useful model in analyzing this peculiar process, because this
system can mimic normal megakaryopoiesis, at least in
part.I8
Commitment to terminal cell differentiation is defined as
the irreversible loss of self-replicating capacity and lineagespecific phenotype e x p r e s ~ i o n . MEG-01
~ ~ ~ ~ . ~ cell
~ division
was irreversibly inhibited by TPA in a dose-dependent
manner (Fig 1 and Table 1). As for the phenotypic
expression, we chose mainly two phenotypes, GP IIb/IIIa
and PAS staining, in addition to vWF and factor V. GP
IIb/IIIa is a membrane protein uniquely expressed in a
mature megakaryocyte series.24325
PAS staining is used as the
maturation index of megakaryocyte lineage and is supposed
to appear very early.z6 It was shown that TPA greatly
increased GP IIb/IIIa expression (Figs 1B and 2 ) and PAS
staining of MEG-01 cells (Fig 3). DNA staining of TPAinduced MEG-01 showed that the frequency of cells with
hyperploidic nuclei was in the range of 5% to 10% of the
total cells, as observed previously.'8 This value is consistent
with the percentage of large and multinuclear cells obTable 2. The Effect of Aphidicolin on Megakaryocytic Differentiation
of HEL and K562 Cells
Treatment
Cell Line
HEL
GP IIb/IIIa-positive cells). On the contrary, DDC and
DDT, precursors of inhibitors for DNA polymerase p and
y, did not produce any significant change in the percentage
of multinuclear cells and phenotypic expression at the
concentrations of 5, 20, and 40 kmol/L. Cycloheximide, an
inhibitor of protein synthesis, also inhibited both the
appearance of GP IIb/IIIa-positive cells and multinuclear
cells. We also analyzed the effect of actinomycin D, a
transcription inhibitor, on the differentiation of MEG-01
K562
Index
Control
TPA
TPA+APH
MNC (%)
GP Ilb/llla (%)
MNC(%)
GP llla (%)
7.0k 2.0
6.3? 1.5
1.0 -t 0.5
0.7 2 0.6
29 -t 4.5
88 ? 1.7
25k 3.0
88 2 3.5
8 ? 2.0
18 ? 1.5
3.0? 1.5
13 -c 2.5
HEL or K562 cells were cultured at the initial cell density of 1 x
105/mLwith or without treatment as indicated. After culture for 4 days,
the percentages of multinuclear cells (MNC) and the percentages of GP
llb/llla- or GP Illa-positive cells (GP Ilb/llla or GPllla) were measured.
TPA, lo-' mol/L of TPA; TPA APH, 1 x 10." mol/L of TPA and 5 x
mol/L of aphidicolin. Data are mean 2 SD from three separate
experiments.
+
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3175
APHlDlCOLlN BLOCKS DIFFERENTIATION OF MEG-01
served with May-Giemsa staining of cytospin preparations,
suggesting that multinuclear cells contain DNA polyploidy.
This finding was further supported by the analysis of DNA
ploidy of multinuclear cells separated by centrifugal elutriation (Fig 2C). It was estimated that 70% or more of the
multinuclear cells have 8N or more DNA ploidy. Recently,
it was reported that MEG-01 cells release platelet-like
particles upon TPA treatment?' These results indicate that
the MEG-01 system reflects normal megakaryopoiesis.
However, the frequency of endomitosis observed in the
TPA-MEG-01 system (up to 10%) is much lower than that
of endomitosis in normal megakaryopoiesis (65%),33while
the induction of GP IIb/IIIa phenotype is well attained
(80%). After TPA treatment, the major part of mononuclear cells became GP IIb/IIIa-positive, but only small
population underwent endomitosis, indicating that the
induction of GP IIb/IIIa was not completely coupled with
multinuclear phenotype in the present system. The apparent dissociation of these two criterion raised an important
question: what is the role of endomitosis or DNA polyploidy
in megakaryocytic differentiation? In preliminav experiments, we have used another reagent, cytochalasin B, to
induce polyploidy in 90% of the MEG-01 or HEL cells.
Under this condition, the expression of GP IIb/IIIa was
observed, but to a limited extent, as a mirror image of that with
TPA (unpublished observations). A combination of these two
reagents might improve the induction system that, at
present, mimics the normal megakaryopoiesis only in part.
It is remarkable that DNA synthesis continued in the
TPA-treated MEG-01 cells despite their apparently constant cell number (Fig 1A and C). This DNA synthesis
might be related to the endomitosis induced with TPA. The
maintenance of DNA polymerase a level in the TPAtreated group as compared with the control (Fig 5) is
apparently consistent with DNA synthesis, and it is suggested that DNA polymerase a is mainly involved in the
endomitosis process. This finding is in sharp contrast to
HL-60 differentiation induced with dimethyl sulfoxide
(DMSO) or TPA, in which DNA polymerase activity per
cell rapidly declined (Geis et
and our data). The DNA
polymerase p and y per cells was higher in the TPA-treated
MEG-01 than in the control cells. The high level of DNA
polymerase y might be correlated with proliferation of
mitochondria, which is an essential component of platelets.
At present, the reason for the high level of DNA polymerase p is unknown, but this result may be consistent with
the previous observation indicating that the level of DNA
polymerase p correlated with functional levels of cells,
especially endocrine organs" and testis.36
Recently, we reported a high level of DNA polymerase a
per cell in aphidicolin-induced K562 cellsn that could
differentiate without DNA replication. However, in the
MEG-01 system, aphidicolin completely inhibited the production of multinuclear cells as well as the expression of GP
IIb/IIIa (Fig 6). It is noted that aphidicolin blocks the
appearance of GP IIb/IIIa even in mononuclear MEG-01
cells after TPA treatment. Other megakaryocytic phenotypes, ie, PAS, vWF, and factor V of MEG-01 cells induced
with TPA, were also inhibited with aphidicolin (Fig 3).
These results clearly indicate that DNA synthesis is a
prerequisite not only for endomitosis but also for the
expression of megakaryocytic markers. The strong correlation between DNA polyploidy measured with FACS ( 2 8N)
and the percentage of morphologic multinuclear cells (Fig
2C) supplies the basis of the data interpretation of the
change in the percentage of multinuclear cells shown in Fig
6 and Table 1. Although the percentage of multinuclear
cells is not completely the same as that of cells with DNA
polyploidy, it can be said that cells with DNA polyploidy
( 2 8N) disappeared or were inhibited considerably with
aphidicolin treatment. Inhibitors for DNA polymerase p or
y, 2,3-dideoxynucleosides, did not affect the differentiation
commitment of MEG-01 cells induced with TPA, in
agreement with our previous ob~ervation.~~
At present,
however, it is not certain whether actions of DNA polymerase p and y are essential for megakaryocyte differentiation. Meanwhile, cycloheximide (CY), an inhibitor for
protein biosynthesis, blocked the differentiation of MEG-01
cells induced with TPA. However, protein synthesis is
known to be indispensable for nuclear division and DNA
~ynthesis.~'
So, it is possible that CY inhibited differentiation through the inhibition of DNA replication. Recently,
several leukemia cell lines have been reported to show
megakaryocytic differentiation with some inducer^.",'^,'^,^^^^
Both HEL and K562 cells expressed the megakaryocytic
phenotypes upon treatment with TPA (Table 2), in accordance with previous report^.'^*^ It was noted that the
percentage of multinuclear cells in the TPA-treated HEL
and K562 cells was higher than that in the MEG-01 cells.
The analysis of DNA ploidy confirmed the clear relationship between DNA polyploidy and the percentage of
multinuclear cells in these systems (data not shown), which
is consistent with a previous report.17These results support
our data using MEG-01 cells (Fig 2). Aphidicolin also
inhibited their megakaryocytic differentiation in either
endomitosis or GP IIb and/or IIIa expression of HEL and
K562 cells.
It is known that TPA binds to its receptor, protein kinase
C (PKC), and that the activated PKC mediates intracellular
signal transduction. This pathway also has a role in the
MEG-01-TPA system?' Aphidicolin is known to be a
specific inhibitor of DNA polymerase a and would not
affect the TPA-PKC system. Considering the different
localizations of target points of TPA43and aphidicolin," the
complete blockage of phenotypic expression by aphidicolin
may indicate that aphidicolin might block the TPA-PKC
signal transduction system at the nucleus through inhibition
of DNA replication. This characteristic entirely differs from
those of erythropoietic differentiation in which inhibition of
DNA replication does not block but rather induces cell
differentiation."
Taken together, our present results show the close
interplay of DNA replication and phenotypic expression in
the megakaryocytic differentiation of leukemia cell lines.
ACKNOWLEDGMENT
The authors are grateful to Dr M. Homma, Nagoya University,
for his assistance in operating the DVS 3000 system image
processor, and to Drs M. Ogura and Y. Morishima for their useful
advice.
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3176
MURATE ET AL
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1991 78: 3168-3177
Aphidicolin, an inhibitor of DNA replication, blocks the TPA-induced
differentiation of a human megakaryoblastic cell line, MEG-O1
T Murate, T Hotta, K Tsushita, M Suzuki, T Yoshida, S Saga, H Saito and S Yoshida
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