Vol. 181, No. 3, 1991 December 31, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1042-1047
EFFECT OF ERYTHROID DIFFERENTIATION FACTOR ON MEGAKARYOCYTIC DIFFERENTIATION OF L8057, A MURINE MEGAKARYOBLASTIC LEUKEMIA CELL LINE Manabu Nishimura, Kohei Kaku*, Yoichi ,Makoto Shiozaki**. Hideki Yuzuru Etoh** Toshio Kaneko Departments University,
Received
October
Azuno, Koichiro Okafuji, Sasaki’, Tohru Inoue2 and
of ‘Pediatrics and 2Pathology, Yokohama City School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, Japan 29,
1991
SUMMARY : To assess the potent effect of erythroid differentiation factor (EDF) on megakaryocytopoiesis, effect of EDF on megakaryocytic differentiation of L8057, a murine megakaryoblastic cell line, was examined. EDF potentiated AchE induction of L80.57 in a dose dependent manner. The potency of EDF on megakaryocytic differentiation is comparable to that on erythroid differentiation reported previously. The present results suggest that EDF may play a regulatory role in megakaryocytopoiesis as well as in erythropoiesis. 0 1991Academic Press, Inc.
Erythroid differentiation factor (EDF) was first found in the culture fluid of human monocytic cell (THPI-cell) as a differentiation inducing activity toward Friend murine erythroleukemia (MEL) cell [l]. EDF is a non species-specific protein with a molecular weight of 25000, which induces the differentiation of MEL cell in a dose dependent manner. Since molecular cloning of EDF has revealed that activin-A, a gonadal protein with pituitary FSH releasing activity, is identical to EDF, it is also named FSH releasing protein (FRP) [2-41. In addition to the differentiation effect in MEL cell, EDF is also known to induce hemoglobin synthesis *
**
To whom correspondence should be addressed at The Third Department The University of Yamaguchi, Internal Medicine, School of Medicine, Kogushi1144, Yamaguchi 755, Japan.
Central Research Laboratory, Ajinomoto, Kawasaki-ku, Kawasaki 2 10, Japan.
0006-291x/91 Copyright AI1 rights
$1.50
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
1042
Co., Suzuki-cho
of Ube
Vol.
181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in a human erythroleukemia cell line K562 [5], and to potentiate the proliferation and differentiation of CFU-E and BFU-E in human bone of EDF on hematomarrow culture [6]. Thus, the potent effects poiesis reported previously have been restricted in erythropoiesis. In order to assess EDF as a potentiator of megakaryocytopoiesis, effects of EDF on megakaryocytic differentiation was investigated using a murine megakaryoblastic cell line, L8057.
MATERIALS
AND METHODS
EDF was provided from Ajinomoto Co. (Central Research Laboratory, Kawasaki, Japan). 12-0-tetradecanoylphorbol-13acetate (TPA) and acetylcholine iodide were purchased from Sigma Chemical Co. (St. Louis, MO). 5,5’-dithio-bis-2-nitrobenzoic acid (DTNB) was from Wako Pure Chemical Industries, LTD. (Osaka, Japan). All other chemicals were purchased from standard sources. Cell Culture : L8057 cell line was maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum, penicillin streptomycin (5Opg/ml) and 0.2% (w/v> sodium (50U/ml), bicarbonate in a 5%CO2/95% humidified air, and was routinely passaged with medium changes twice a week. To determine a biological effect of EDF toward L8057, cells at I .0x106/ml were cultured in 10% FCS RPMI-1640 medium using a 24 well plastic plate with various concentrations of TPA or EDF over three days period without medium changes. TPA is known to induce megakaryocytic differentiation of L8057 [8]. Viability of cells was over 90% as judged by trypan blue dye exclusion test, and cell numbers were counted in duplicate. AchE activitv of L8057 : The degree of differentiation of L8057 cells was evaluated by measuring an AchE activity. which can be used as a positive marker for megakaryocyte maturation in rodents [9], as described previously [7]. After three days period of incubation, cells were washed twice with phosphate buffered saline, pH7.2. They were resuspended in 750~11 of PBS, followed by the addition of 250~1 of DTNB (1.25mg/ml) in 0.1% sodium citrate containing 2% Triton X-100 and 80mM MgCl2. The reaction was then started by the addition of lO0l.1 I of 7.5mM acetylcholine iodide. The absorbance (OD 690-405nm) of cell suspension was measured after 60 min incubation at 37’C. AchE activity of L8057 cells was also determined by a histochemical examination of cell suspension.
RESULTS
AND DISCUSSION
L8057 cells were cultured with various concentrations EDF for 3 days. Fig.1 shows the dose dependent effects 1043
of TPA of TPA
and and
Vol.
181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
O-
control
10-g
10-B
lo-7
10-a
TPA (M)
10-10
10-g
10-E
10-T
EDF (M)
Fig.1. Effects of TPA and EDF on L8057 cell growth. The LX057 cells were seeded 1.0~10~ cell per ml in 10% FCS RPMI-1640 medium with various concentrations of TPA or EDF and cultured for 3 days without medium changes. Results are the mean of duplicate experiments.
EDF
on L8057
cell growth.
Continuous
exposure of L8057 cells to
EDF
appeared to stimulate the cell growth but not significant whereas TPA showed an inhibition on L8057 cell statistically, growth.
As shown in Fig.2A. exposure of L8057 cells to TPA increased both the percentage of AchE positive cells and the absorbance (OD) of cell suspension in a dose dependent manner. An increase in the
ratio of AchE positive cells was directly proportional to an increase in the absorbance (OD). This result indicates that the photometric analysis can be used as substitute for the histochemical A time course of the absorbance of cell suspension examination. treated with TPA (IOOnM) showed that the ratio of AchE positivity of L8057 cells was readily increased within 60 min after the addition of acetylcholine iodide (Fig2B). Thus, in the following experiments, the incubation for the assay of AchE activity was completed within 60 min. To evaluate the effects of TPA and EDF on megakaryocyte differentiation of L8057 cells, effects of these stimulators on AchE positivity of the cells were examined. As shown in Fig.3, both TPA and EDF stimulated the megakaryocyte differentiation of L8057 1044
Vol. 181, No. 3, 1991
A
0.66
1 -c-a-
BIOCHEMICAL
OD (690-405nm) Percem of AChE
positlve
cell
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
100 =?I 80
.-$! .z
0.6 -
60
E g w
E :: *g6 0.4 -
40
-6
g
E 8 20
0
control
.
TPAlOOnM
0.2 -
i
,
0 IO
0.0-c
I 60
0
I 120
TPA (r&t)
I 160 time (min)
I 240
0 300
, 360
Fig2A. Relationship between histochemical examination of AchE activity and photometric analysis of L8057 cell suspension in the presence of various concentrations of TPA. Fia.2B. Time course of the absorbance (photometric analysis) of L8057 cell suspension in the presence of IOOnM TPA.
cells activity
in
a dose dependent
manner.
on megakaryocytic
TPA. A half that of TPA megakaryocyte concentration
EDF
showed
differentiation
a more
compared
with
potent that
of
maximal effect 01’ EDF was observed at 0.2nM while was at >50nM. The maximal effect of EDF on the differentiation of L8057 was observed at 1 nM. The of EDF inducing maximum differentiation of L8057
-Q-
0.15-t
.‘.....I 0
.l
. . . . ..I 1
.
. . . . ..I 10
TPA(nM) EDF(nM)
. . ..rrl 100
TPA or EDF (nhn)
Fie.3. Effects of TPA and EDF on megakaryocytic differentiation of L8057. The L8057 cells were cultured with various concentrations of TPA or EDF for 3 days. The AChE activity was assayed as described in the text. The data were corrected by cell numbers (OD/I O6 cells) and expressed as the mean + SD. (* :p
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1042-1047
EFFECT OF ERYTHROID DIFFERENTIATION FACTOR ON MEGAKARYOCYTIC DIFFERENTIATION OF L8057, A MURINE MEGAKARYOBLASTIC LEUKEMIA CELL LINE Manabu Nishimura, Kohei Kaku*, Yoichi ,Makoto Shiozaki**. Hideki Yuzuru Etoh** Toshio Kaneko Departments University,
Received
October
Azuno, Koichiro Okafuji, Sasaki’, Tohru Inoue2 and
of ‘Pediatrics and 2Pathology, Yokohama City School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, Japan 29,
1991
SUMMARY : To assess the potent effect of erythroid differentiation factor (EDF) on megakaryocytopoiesis, effect of EDF on megakaryocytic differentiation of L8057, a murine megakaryoblastic cell line, was examined. EDF potentiated AchE induction of L80.57 in a dose dependent manner. The potency of EDF on megakaryocytic differentiation is comparable to that on erythroid differentiation reported previously. The present results suggest that EDF may play a regulatory role in megakaryocytopoiesis as well as in erythropoiesis. 0 1991Academic Press, Inc.
Erythroid differentiation factor (EDF) was first found in the culture fluid of human monocytic cell (THPI-cell) as a differentiation inducing activity toward Friend murine erythroleukemia (MEL) cell [l]. EDF is a non species-specific protein with a molecular weight of 25000, which induces the differentiation of MEL cell in a dose dependent manner. Since molecular cloning of EDF has revealed that activin-A, a gonadal protein with pituitary FSH releasing activity, is identical to EDF, it is also named FSH releasing protein (FRP) [2-41. In addition to the differentiation effect in MEL cell, EDF is also known to induce hemoglobin synthesis *
**
To whom correspondence should be addressed at The Third Department The University of Yamaguchi, Internal Medicine, School of Medicine, Kogushi1144, Yamaguchi 755, Japan.
Central Research Laboratory, Ajinomoto, Kawasaki-ku, Kawasaki 2 10, Japan.
0006-291x/91 Copyright AI1 rights
$1.50
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
1042
Co., Suzuki-cho
of Ube
Vol.
181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in a human erythroleukemia cell line K562 [5], and to potentiate the proliferation and differentiation of CFU-E and BFU-E in human bone of EDF on hematomarrow culture [6]. Thus, the potent effects poiesis reported previously have been restricted in erythropoiesis. In order to assess EDF as a potentiator of megakaryocytopoiesis, effects of EDF on megakaryocytic differentiation was investigated using a murine megakaryoblastic cell line, L8057.
MATERIALS
AND METHODS
EDF was provided from Ajinomoto Co. (Central Research Laboratory, Kawasaki, Japan). 12-0-tetradecanoylphorbol-13acetate (TPA) and acetylcholine iodide were purchased from Sigma Chemical Co. (St. Louis, MO). 5,5’-dithio-bis-2-nitrobenzoic acid (DTNB) was from Wako Pure Chemical Industries, LTD. (Osaka, Japan). All other chemicals were purchased from standard sources. Cell Culture : L8057 cell line was maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum, penicillin streptomycin (5Opg/ml) and 0.2% (w/v> sodium (50U/ml), bicarbonate in a 5%CO2/95% humidified air, and was routinely passaged with medium changes twice a week. To determine a biological effect of EDF toward L8057, cells at I .0x106/ml were cultured in 10% FCS RPMI-1640 medium using a 24 well plastic plate with various concentrations of TPA or EDF over three days period without medium changes. TPA is known to induce megakaryocytic differentiation of L8057 [8]. Viability of cells was over 90% as judged by trypan blue dye exclusion test, and cell numbers were counted in duplicate. AchE activitv of L8057 : The degree of differentiation of L8057 cells was evaluated by measuring an AchE activity. which can be used as a positive marker for megakaryocyte maturation in rodents [9], as described previously [7]. After three days period of incubation, cells were washed twice with phosphate buffered saline, pH7.2. They were resuspended in 750~11 of PBS, followed by the addition of 250~1 of DTNB (1.25mg/ml) in 0.1% sodium citrate containing 2% Triton X-100 and 80mM MgCl2. The reaction was then started by the addition of lO0l.1 I of 7.5mM acetylcholine iodide. The absorbance (OD 690-405nm) of cell suspension was measured after 60 min incubation at 37’C. AchE activity of L8057 cells was also determined by a histochemical examination of cell suspension.
RESULTS
AND DISCUSSION
L8057 cells were cultured with various concentrations EDF for 3 days. Fig.1 shows the dose dependent effects 1043
of TPA of TPA
and and
Vol.
181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
O-
control
10-g
10-B
lo-7
10-a
TPA (M)
10-10
10-g
10-E
10-T
EDF (M)
Fig.1. Effects of TPA and EDF on L8057 cell growth. The LX057 cells were seeded 1.0~10~ cell per ml in 10% FCS RPMI-1640 medium with various concentrations of TPA or EDF and cultured for 3 days without medium changes. Results are the mean of duplicate experiments.
EDF
on L8057
cell growth.
Continuous
exposure of L8057 cells to
EDF
appeared to stimulate the cell growth but not significant whereas TPA showed an inhibition on L8057 cell statistically, growth.
As shown in Fig.2A. exposure of L8057 cells to TPA increased both the percentage of AchE positive cells and the absorbance (OD) of cell suspension in a dose dependent manner. An increase in the
ratio of AchE positive cells was directly proportional to an increase in the absorbance (OD). This result indicates that the photometric analysis can be used as substitute for the histochemical A time course of the absorbance of cell suspension examination. treated with TPA (IOOnM) showed that the ratio of AchE positivity of L8057 cells was readily increased within 60 min after the addition of acetylcholine iodide (Fig2B). Thus, in the following experiments, the incubation for the assay of AchE activity was completed within 60 min. To evaluate the effects of TPA and EDF on megakaryocyte differentiation of L8057 cells, effects of these stimulators on AchE positivity of the cells were examined. As shown in Fig.3, both TPA and EDF stimulated the megakaryocyte differentiation of L8057 1044
Vol. 181, No. 3, 1991
A
0.66
1 -c-a-
BIOCHEMICAL
OD (690-405nm) Percem of AChE
positlve
cell
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
100 =?I 80
.-$! .z
0.6 -
60
E g w
E :: *g6 0.4 -
40
-6
g
E 8 20
0
control
.
TPAlOOnM
0.2 -
i
,
0 IO
0.0-c
I 60
0
I 120
TPA (r&t)
I 160 time (min)
I 240
0 300
, 360
Fig2A. Relationship between histochemical examination of AchE activity and photometric analysis of L8057 cell suspension in the presence of various concentrations of TPA. Fia.2B. Time course of the absorbance (photometric analysis) of L8057 cell suspension in the presence of IOOnM TPA.
cells activity
in
a dose dependent
manner.
on megakaryocytic
TPA. A half that of TPA megakaryocyte concentration
EDF
showed
differentiation
a more
compared
with
potent that
of
maximal effect 01’ EDF was observed at 0.2nM while was at >50nM. The maximal effect of EDF on the differentiation of L8057 was observed at 1 nM. The of EDF inducing maximum differentiation of L8057
-Q-
0.15-t
.‘.....I 0
.l
. . . . ..I 1
.
. . . . ..I 10
TPA(nM) EDF(nM)
. . ..rrl 100
TPA or EDF (nhn)
Fie.3. Effects of TPA and EDF on megakaryocytic differentiation of L8057. The L8057 cells were cultured with various concentrations of TPA or EDF for 3 days. The AChE activity was assayed as described in the text. The data were corrected by cell numbers (OD/I O6 cells) and expressed as the mean + SD. (* :p
