Effects of okadaic acid on mouse hemopoietic cells

Vol.

165,

November

No.

1, 1989

30,

BIOCHEMICAL

BIOPHYSICAL

AND

RESEARCH

COMMUNICATIONS

Pages

1989

539-546

EFFFaCI'SOF OKADAIC ACID ON MOUSEHEMOPOIETIC CELLS Syuichi

Oka1* , Kazuya Nakagomi', Hideoki and Takeshi Yasumoto2

Agency of Industrial Science Tsukuba, Ibaraki 305, Japan

' Fermentation Research Institute, and Technology, Higashi l-l-3, 2

TanakaI,

Faculty of Agriculture, Tsutsumi-dori, Sendai,

Tohoku University, Miyagi 980, Japan

Received October 25, 1989 Effects of okadaic acid, a potent non-12-O-tetradecanoyl-phorbol13-acetate(TPA)-type tumor promoter, on mouse hemopoietic cells were investigated. Okadaic acid stimulated mouse bone marrow cells to form granulocyte-macrophage colony-forming unit(CFU-GM) colonies without added colony stimulating factors(CSFs). At the concentration of 1.82 x 10e8M, colony formation of 77 +_14 colonies/l x IO5 bone marrow cells was observed. .Cbservations on the effects of other cells on the CSF induction suggested that okadaic acid primarily stimulated the functions of macrophages, and the CSF production from macrophages might be attributed to the CFU-GM colony formation. On the other hand, the erythroid colony-forming unit(CFU-E) colony formation stimulated by erythropoietin was inhibited by the addition of okadaic acid. 0 1989Academic Press.1°C.

Granulocytes,

monocyte-macrophages,

from the proliferation cell.

Such a process of hemopoiesis

factors(CSFs) regulators,

that

and differentiation

and erythropoietin

and erythrocytes

are derived

of a common hemopoietic

requires

as proliferation

colony

stem

stimulating

and differentiation

respectively.

In the investigations

on bioactive

affect

we found that

hematopoiesis,

mouse bone marrow cells * To whom correspondence

substances of microbial the teleocidins

to form granulocyte-macrophage

origin

stimulated colony-forming

should be addressed.

Abbreviations: TPA, 12-0-tetradecanoyl-phorbol-13-acetate; CSF, colony stimulating factor; CFU-GM, granulocyte-macrophage colony-forming unit; CFU-E, erythroid colony-forming unit; IL-l, interleukin 1. 0006-291X/89$1.50 539

Copyright 0 1989 by Academic Press, Inc. Ali rights oj. reproduction in any form reserved.

Vol.

165,

No.

1, 1989

unit(CFU-GM)

BIOCHEMICAL

colonies

teleocidins

in the

absence

bound to phorbol

activated

protein

kinase

ester

acid,

be a product Recently,

of the benthic okadaic

phosphatases tumor

bind

to the

stimulate

ester

promoting

Consequently,

receptors activity

cells

from

sponges

of okadaic

and

(3),

was

Prorocentrum inhibitor

not acid

whether

okadaic activate was

and found

that

to form

CFU-GM colonies

to

lima

(4).

non-TPAacid

did

protein

acid

okadaic without

acid

not

kinase

comparable

okadaic

formation

found

of

to be a potent

teleocidins,

and did

membranes

(2).

revealed the

the

as 12-O-tetradecanoyl-

to be a potent

we investigated

CFU-GM colony

to that

could stimulated

added CSFs,

as

teleocidins.

In this mouse

shown

in cell

dinoflagellate

Unlike

(II).

phorbol

mouse bone marrow do the

was

COMMUNICATIONS

Because

classified

isolated

marine

RESEARCH

(1).

promoters

and was further

promoter

The tumor

of TPA.

acid

(S-lo),

type

C.

first

of CSF

were

tumor

though

BIOPHYSICAL

receptors

C, these

phorbol-13-acetate(TPA)-type Okadaic

AND

paper,

hemopoietic

we will cells

describe

the

and possible

effects

mechanisms

of okadaic for

acid

on

the CFU-GM colony

formation.

MATERIALS

AND

MEXHODS

Male ICR mice, 6- to IO-weeks-old, were Animals and Materials: Okadaic acid was isolated from purchased from Charles River Japan, Inc. cultured cells of the dinoflagellate Prorocentrum lima as described Horse serum was obtained from Gibco Laboratories. previously (4). CSF-CHUGAI, a Fetal calf serum was obtained from HyClone Laboratories. partially purified preparation from a human squamous cell carcinoma cell Erythropoietin line, was a product of Chugai Pharmaceutical Co., Ltd. The (from human urine, 72 U/mg) was purchased from Toyobo CO., Ltd. other chemicals used were of analytical grade. Assay for CFU-GM and CFU-E colony formation: CFU-GM and CFU-E colony formation was assayed as described in the previous paper (I). Methylcellulose cultures based on the modified methods of Iscove et were carried out using bone marrow cells (1 x 105) obtained from 2al (12) ICI? mice. 540

Vol.

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AND

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Peritoneal exudate cells, Preparation of peritoneal macrophages: obtained from male ICR mice 4 days after they had been intraperitoneally were centrifuged at 140 x g injected with 2 ml of 10% proteose peptone, The pellets were suspended in Hanks' balanced salt solution for 5 min. The peritoneal cells were and washed twice with the same medium. resuspended in RPM1 1640 medium supplemented with 5% fetal calf serum and incubated in 16 mm wells at 1 x 106cells/well for 2 hrs at 37OC in 5% co2. The adherent macrophages were washed twice with the same medium and used for the culture experiments. Macrophages were cultivated in 1 ml of the medium with or without test samples at 37OC in a humidified atmosphere of 5% C02. The aliquots were centrifuged and assayed for CFU-GM colony formation. Preparation of T cell-enriched fractions: The splenocytes obtained from male ICR mice (7- to 8-weeks-old) were suspended in Eagle's minimum essential medium and mashed on a stainless steel mesh (# 150). Then, a solution of 0.83% NH4C1 / 0.17M Tris HCl buffer (pH 7.65) (9/l) was added and maintained at 4OC for 5 min to remove erythrocytes. After the centrifugation at 140 x g for 5 min, the cells were washed twice with Eagle's medium containing 5% fetal calf serum and passed through a Sephadex G-10 column . The cell fractions were centrifuged and then loaded on a column of nylon fiber equilibrated with Eagle's medium supplemented with 10% fetal calf serum. T cell-enriched fractions were collected and washed twice with RPM1 1640 medium supplemented with 5% fetal calf serum. The culture experiments were performed with 1 x IO6 cells/l ml of the same medium with or without test samples at 37OC for 3 days in a humidified atmosphere of 5% C02. The thus obtained cells reacted with anti-mouse Thy-l and complement. Preparation of B cell enriched fractions: The splenocytes that had been passed through a Sephadex G-10 column were treated with anti-mouse Thy-l at 30°C for 30 min and followed by treatment with complement(l/5 dilution). Then, the cells were washed and used for the culture experiments. The thus obtained cells reacted with anti-mouse B cell.

REZSULTS

and

DISCUSSIONS

The effects examined.

As shown

formation IO-'M x

10

in the

in

in Fig.

while

colonies/l

the

IO5

10W7M okadaic effect

acid

on --in vitro okadaic

1,

of CSF.

5 units/ml x

acid

concentration

the absence

-8M stimulated

cells,

cytotoxic

of okadaic

acid

range

between

Okadaic

acid

formation

cells.

to the culture of okadaic

acid

formation

stimulated 1.82

at the

induced However,

produced at this 541

the

were

CFU-GM colony

x 10m8M and

1.82

concentration

of 77 +_ 14 colonies/l

of CSF-CHUGAI

bone marrow

colony

x

formation

the addition no colonies

concentration.

of

x

1.82

bone marrow

IO5

of 27 t 2 of

1.82

x

of

the

because Although

the

Vol.

165,

tumor

No.

1, 1989

promoting

teleocidin

activity

(5),

formation

BIOCHEMICAL

concentration

of

1.82

colonies,

while

formation

of 56 +

10

dual staining)

BIOPHYSICAL

acid

of okadaic

was acid

x 10m8M resulted A-l

at

RESEARCH

comparable to

than h a t of teleocidin

teleocidin

Morphological

okadaic

of okadaic

the ability

was greater

AND

to that

A-l.

x

of

CFU-GM

stimulate

colony

Okadaic acid at the

in the formation

5.00

COMMUNICATIONS

10m8M merely

of 77 +

14

stimulated

the

colonies.

analysis

showed that

(non-specific

and chloroacetate

the colonies

esterase

induced by the addition

acid were composed of granulocytes

of

and macrophages at 7 days of

culture. Because a number of mechanisms can be hypothesized effects to

of okadaic first

examine

acid on myeloid the stimulatory

As macrophages and lymphoid cells

were prepared

colony effects

cells

formation

01

40-

5 1 ? 0

zo-

are the main producers

and examined for

the secretion

12

10

8

we decided

of CSF, these

of CSF.

P 0

o-

in vitro,

the

on endogenous CSF production.

;i 60' .%! 6 a 0

to explain

(92

nM)

0

6

thwantration ( -logMI 02

0

1 Cultivation

2 Time

3 ( day

Effect of okadaic acid on CFU-GM colony formation. Fig. 1. in the absence Mouse bone marrow cells were cultured at 1~10~ cells/dish acid ( 0 ) or ( q ) or presence of CSF ( H ) at 5 U/ml, okadaic Each circle represents teleocidin A-l ( 0 ) at various concentrations. the mean of triplicate data with S. E. Fig. 2. macrophages. with okadaic acid 250 nM ) or RPM1 Fifty-microliter circle represents

Effect of okadaic acid Peritoneal macrophages ( 0 , 9 nM, 92 nM, ) at 1640 medium ( q aliquots were assayed the mean of triplicate 542

on CSF production from peritoneal were cultured (1~10~ cells/well) 465 nM ), teleocidin A-l ( 0 37OC in 5% CO2 for 3 days. Each for CFU-GM colony formation. data with S. E.

1

Vol.

165,

No.

Figure induced

2 shows

the

by proteose

macrophage

cultures

The addition

doses

reduced hand,

showed

the

macrophages

acid

to the

CSF production.

The

concentration

of

CSF production

the

COMMUNICATIONS

on peritoneal

of okadaic

stimulated with

RESEARCH

acid

The addition

On the other cultures

BIOPHYSICAL

of okadaic

obtained

of higher

macrophage

AND

significantly was

cytotoxicity.

effect

peptone.

induction

maximal

2.5

BIOCHEMICAL

1, 1989

addition

9.2 x 10V8M.

because

of

of teleocidin

no stimulatory

effect

fractions

prepared

A-l

to the

at the concentration

of

x 10-7M. Next,

T cell-enriched

splenocytes

and cultured

atmosphere

of 5% CO2 for

at the

with

of

x

bone marrow

IO5

okadaic

3 days.

concentration

colonies/l

were

9.20

acid

at

Although

murine

37OC in a humidified

the addition

x 10T8M stimulated cells,

from

the

no significant

of okadaic

formation

acid

of

production

14 t 5

was

observed. Because

B lymphocytes

stimulatory

effects

stimulation

was

only

by the

to the that

that

okadaic

acid

obtained

at

produced

shown).

ug/ml

CSF

examined.

However,

acid

colony

acid

primarily

formation.

inhibited monocytes

IL-l for

from

Recently,

secretion

(15).

24- and 48-hr

the maximum stimulation

l(IL-1)

lower

obserbed

cultures.

These

the

macrophages

secretion used

may be

concentrations

exposures.

from

in the

tumor of

Maximum stimulation

was almost 543

was

Hokama -2 et al

at the concentrations of the

no

stimulates

interleukin

the addition

(13,14),

formation

to the macrophage

and the CSF production

while

stimulated 0.05

not

okadaic

acid

blood

studies,

were

to produce

of myeloid

CFU-GM colony

okadaic

human peripheral

acid

of okadaic

of macrophages,

attributed

that

(data

reported

stimulation

suggest

functions

promotion

observed

addition

observations

showed

of okadaic

a marked

Thus,

were

was

The concentrations the

same

as

that

Vol. 165, No. 1, 1989

producing

BIOCHEMICAL

the maximal CSF secretion

On the other

hand, it

myeloid

mechanism for

cells

colony

formation

the CFU-GM colony

to the bone marrow culture

acid

induces CSF and IL-l synergistically

to the participation

that

IL-l

with

Therefore,

(18).

formation

macrophages.

induced CSF production

and synergized

(16,17)

acid

cytokines

from mouse peritoneal

has been reported

from marrow accessory stimulate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

various

a possible

by the addition

of okadaic

could be deduced as follows:

production

from marrow cells

enhance CFU-GM colony of phosphatase activities,

CSFs to

Okadaic

and then,

formation. further

these

With respect investigations

are in progress. Then, the effects examined.

of okadaic

The addition

showed no stimulatory

acid

inhibited

stimulated

erythroid

by 0.1

acid

in vitro

formation

colony

(data

colony-forming

unit(CFU-E) in vitro --

was significantly

,

formation

not shown).

range between 3.60 x lo-'M

14

progenitors

to the methylcellulose

on erythroid

U/ml of erythropoietin

In the concentration CFU-E colony

of okadaic effect

absence of erythropoietin

acid on erythroid

10 Concentration

However,

okadaic

formation

as shown in Fig. and 1.82

inhibited,

8 ( -log

culture in the

colony

6

M )

Fig. 3. Effect of okadaic acid on CFU-E colony formation. Mouse bone marrow cells (1~10~ cells/dish) were cultured with 0.1 U/ml erythropoietin (Epo) in the absence ( q ) or presence of okadaic acid ( 0 ) or teleocidin A-l ( 0 ) at various concentrations. Each circle represents the meanof triplicate data with S. E. 544

3.

x 10m8M, the

whereas CFU-GM

I I I a+-;-.,

12

were

Vol. 165, No. 1, 1989

colony

formation

Although

was markedly

teleocidin

inhibited potent

BIOCHEMICAL

A-l

erythropoietin

reported

receptors

erythropoietin

is interesting

progenitors.

concentration

added to the methylcellulose CFU-E colony

(19).

that

cultures

formation

(I),

range. also it

was less

in the number of

altering

of the receptor

without

TPA caused a decrease

to elucidate

Further

the affinity

Because the action

presumably mediated by a receptor it

in this

acid.

Broudy -et al.

for

stimulated

erythropoietin-induced than okadaic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

its

experiments

distinct

of okadaic

acid

is

from the TPA receptor

mode of action

(IO),

on erythroid

are under way.

ACKNOWLEDGMENT We thank Dr. N. Imai of Chugai Pharmaceutical valuable

1. 2. 3.

4. 5. 6. 7. 8. 9.

10.

Co., Ltd.

for

discussions.

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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