Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 975-981
December 31, 1990
EFFECT OF K252a, THE PROLIFERATION Kazuhiro
Ohmi,
A PROTEIN KINASE INHIBITOR, ON OF V A S C U L A R SMOOTH MUSCLE CELLS
Shigeru
Yamashita
and Yoshiaki
Nonomura
Department of Pharmacology, Faculty of Medicine, The U n i v e r s i t y of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received November 14, 1990 SUMMARY: In the growth arrested cultures of bovine carotid smooth muscle, K252a (i0 - i00 ng/ml), a protein kinase inhibitor with wide spectrum suppressed the cell p r o l i f e r a t i o n induced by TPA and increase of serum. K252a was more potent in the antiproliferative activity than H7, a C - k i n a s e - s p e c i f i c inhibitor, but less than staurosporine, another w i d e - s p e c t r u m protein kinase inhibitor. Since C-kinase plays an important role in the signal t r a n s d u c t i o n leading to the cell p r o l i f e r a t i o n and K252a inhibits C-kinase in vitro, the a n t i p r o l i f e r a t i v e effect of K252a to carotid smooth muscle cells is likely to be exerted through C-kinase dependent pathway. ©1990AcademlcPre~s.Znc.
It is well the
cultured
of serum with
known state
contains
is a potent
the native
state.
revert
from
cell
to know
smooth
culture.
state
we prepared arteries
are found
of aorta has b e e n
However,
since
type of VSMC the cultured
as the source smooth
muscle
state used
some
muscle
PDGF roles
cells
in
to be in a quiesent
one.
SMC
Thus,
it
cultured
by stimulation. as the source
in aortic
it more
of
smooth
suitable
of cultured cell
(i).
among which
the quiescent
myofilaments
we considered
is accompanied
has occurred,
to a p r o l i f e r a t i v e that
in
to increase
to have
Smooth
atherogenesis
the m e c h a n i s m
(VSMC)
to synthetic
factors,
(2).
into a p r o l i f e r a t i v e
muscle
state
is considered
media
cells
in response
growth
disease
once
are not abundant,
the muscular
and
of arterial
However,
are entering
muscle
for VSMC
a quiescent
is important
muscle
from musclar
mitogenic
of vascular
state
growth
Usually
to p r o l i f e r a t e
of phenotype
mitogen
smooth
This p r o l i f e r a t i v e
various
in p a t h o g e n e s i s
VSMC
start
concentration.
the change
Serum
that vascular
to use
cells.
from bovine
Thus,
carotid
in this report.
Abbreviations: VSMC, vascular smooth muscle cells; TPA, 12-O-tetradecanoylphorbol-13-acetate; PDGF, p l a t e l e t - d e r i v e d growth factor; FBS, fetal bovine serum; TCA, tri chloro acetic acid; EDTA, ethylene diamine tetra acetic acid. 0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
976
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
As for growth of VSMC, ed to be one of factors of cultured VSMC H7,
to suppress
protein kinase C
involved
in PDGF-stimulated
(3) and C-kinase
inhibitor,
the growth of cultured VSMC
phorbol
esters has both p r o l i f e r a t i v e
actions
through C-kinase pathways
Recently K252a has been antibiotics.
It has been
ses as follows; muscle myosin dependent
(C-kinase) was report-
C-kinase,
staurosporine (4, 5).
considered
G-kinase
in relation
(i0).
iden-
And then, K252a
is
in culture,
the effect of K252a on the using carotid
artery as the
type of the cell and aimed at clarifing
on the signal
It was
inhibiter with wide spectrum.
we investigated
of VSMC
(9).
of the contraction of arterial
to be a useful kinase
proliferation contractile
inhibitor
(7), and smooth
(8) as well as calmodulin-
induced by several agents
In this report,
(6).
(7) from bacteria as one of
cyclic nucleotide p h o s p h o d i e s t e r a s e
smooth muscle
TPA, one of
found to inhibit various protein kinaA-kinase,
light chain kinase
tified as a potent
and
and a n t i p r o l i f e r a t i v e
in cultured VSMC
isolated
proliferation
transduction pathway of growth
K252a action
factor especially
to C-kinase.
MATERIALS
AND METHODS
Materials: K252a was a gift from Kyowa Hakko, Co., Ltd. H7 was purchased from Seikagaku Kogyo Co. Ltd. Staurosporine was a gift from Prof. Ohmura, Kitazato University. TPA was purchased from Sigma chemical Co. [3H]thymidine was purchased from Amersham. Bovine carotid artery was prepared freshly at the Meat Market of Tokyo Central W h o l e Sale Market. Cell culture: Preparation of primary culture of VSMC were carried out from bovine carotid arteries according to Ross (i). The explants from the medial layers were placed in 60 mm culture dishes with MEM containing 10 % FBS. Medium was changed every 4 days. After 3 weeks, the cells having migrated from the explants were trypsinized and seeded. The cells in secondary cultures maintained in MEM containing i0 % FBS. The cells were always cultured at 37°C in a humidified atmosphere of 5 % CO 2 and 95 % air. Growth assays: VSMC in secondary cultures were trypsinized and seeded into 12 well plates in MEM containing 0.5 % FBS at a density of 2 x 104 cells/well for count of cell number or 5 x 104 c e l l s / 3 5 m m dish for d e t e r m i n a t i o n of [3H] thymidine incorporation into acid insoluble fractions. For cell counts, cells were washed twice with 2 ml PBS, treated with 0.2 ml of 0.05 % t r y p s i n - 0 . 0 1 % EDTA at room temperature, and rinsed from the well with 0.8 ml medium containing 10 % FBS. The number of the trypsinized cells was counted with a heamocytometer. For d e t e r m i n a t i o n of [3H] thymidine incorporation, growth arrested cells were cultured in medium containing 1 uCi/ml [JH] thymidine (20 Ci/mmol) and 10 % FBS or i0 ng/ml TPA plus 0.5 % FBS in the absence or presence of various concentrations of K252a. After 24 hours, the cells were washed 3 times with 2 ml PBS, lysed by the addition of 1.0 ml of 0.2 N NaOH and neutralized 977
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
w i t h 0.2 m l of 1 N HCI. The precipitates were collected on w h a t m a n G F / C f i l t e r s , and the f i l t e r s w e r e w a s h e d w i t h c o l d TCA, ethanol and acetone. A f t e r d r i n g , the r a d i o a c t i v i t y on the filters was determined with a Beckman liquid scintillation s y s t e m , LS 1 8 0 0 .
RESULTS We
confirmed
sisted lial
of
culturing not
and were
DISCUSSION
immunohistologically
smooth
cells
AND
muscle
cells
that
without
fibroblasts,
because
stained
anti-alpha
with
all
the
VSMC
contamination
all
the
cells
smooth
used
of
just
muscle
con-
endotheafter
actin
(data
shown). When
the
the
FBS
concentration
growth-arrested
cell
number
tion
as
became
that
of
cells,
as
non-stimulated
this
condition
bited
such
a serum-induced
dose-dependent
cell
3 times
under
was
manner
as
was
about
increased
proliferation
high
at
96
after
counterpart. 48 hrs.
in Fig.
The
Addition of i.
0.5
% to
i0
and
the
started
hrs
proliferation shown
from
the
stimula-
doubling
time
of
inhi-
K252a
cultured The
% in
cell
VSMC numbers
in
a of
o
o~120
7.0~o
~100
6.0-
|
so
=
60
5.0.9o
4.0i
.0-
e
2.0-
~ 2o
t.o! 0.0
Q
~-a
~ ....
= o
a ~
.1= Ii
i
i
i
24 48 72 Time after stimulation (hrs)
i
,,r
96
Fig. i. Effect of K252a on VSMC proliferation induced by serum. Growth arrested cuitures were stimulated by i0 % FBS without (O) or with 1 ng/ml (O), i0 ng/ml (~), i00 ng/ml (A) of K252a and no stimulation (i). At the times indicated, cells were counted. In allthe figures other details are described in MATERIALS AND METHODS and results are the means ± SE of quadruplicate dishes. Fig. 2. Effect of K252a on DNA synthesis of VSMC induced by serum or TPA. Growth arrested cultures were stimulated by i0 % FBS or 0.5 % FBS with i0 ng/ml TPA in the presence or absence of various concentrations of K252a. 1 uCi/ml of [3H] thymidine was added at 0 time, and the cultures were incubated at 37°C for 24 hours, then DNA synthesis was assayed. Control means the cell stimulated by increase of serum or addition of TPA without K252a treatment. The value of [3H] thymidine incorporation in the control cell is expressed as i00 % and the values to other cells are described as relative value to that of the control. Notice the effect of stimulant as compared with no stimulation cells. 978
Vol. 173, No. 3, 1990
the
culture
% and ml
50
treated
% of
of K 2 5 2 a Next,
was
that
using
incorporation at
24 hrs
of
after
VSMC.
In this
could
not have
incubation by
serum
presence was
of
Furthermore, of K 2 5 2 a
by we
by
with
synthesis
stimulants.
which
was
on DNA
carotid
VSMC of
induced
the
smooth
of
the
was
cultured cells
regulation
or e l o n g a t i o n
DNA
%
fraction
muscle
the d o w n
i0
the
time p o i n t
synthesis
by K 2 5 2 a
dose-dependently.
serum-or
TPA-induced
difference
synthesis
when
(x104 )
DNA
we
of
of
induced In the synthesis
of
K252a
the was
a
inhibitory added
at v a r i o u s
(x104 )
b
2-
80
O"
111
,o
8.
,o
6-
Q.
20
~
cultured
insoluble
of
1 ug/
toxicity.
in i n c r e a s e
5100 |
than
%, r e s p e c t i v e l y .
the
i120
of
not shown).
90
studied
phase
by 25
Addition
This
concentrations
ng/ml)
40 % or
2).
growing
inhibited
(i00
cell
the acid
(Fig.
any p h e n o m e n a
(data
of
reflected
into
cultured
of
cell
TPA
as
reduced
More
its
on DNA
logarismic
of T P A
of K 2 5 2 a
of cells
thymidine
increase
or T P A was
of K 2 5 2 a
respectively.
stimulations
observed by
suppressed
effect
[3HI
study
time
and
ng/ml
to the g r o w t h - a r r e s t e d
both
the m i d s t
synthesis
of K 2 5 2 a serum
i00
K252a,
damage
synthesis
almost
DNA
the
TPA
of D N A
i0 and
without
the e f f e c t
i0 ng/ml
increase
with
induced
examined
FBS or
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
4"
o I
2"
E
-~ o
Q
o Addition time of K252a
0
•
none
Q
8
7
,
•
•
O"
"
6 5 4 none 8 Concentratlonsofdrugs(-IogM)
.
.
7
.
.
6
Fig. 3. Time dependent effect of addition of K252a on the i0 % FBS or TPA induced DNA synthesis. Growth arrested cultures of VSMC were incubated with i0 % FBS or 0.5 % FBS with i0 ng/ml TPA. DNA synthesis was assayed 24 hours incubation, K252a was added at the indicated time at the final concentration of I00 ng/ml. An essential explanation of the figure is the same as that of Fig. 2. Fig. 4. comparison of antiproliferative activity of K252a with that of Staurosporine and H7. Growth arrested cultures were stimulated by i0 % FBS (a) or TPA (b) in the absence or presence of various concentrations of Staurosporine (m), K252a (O) and H7 (A) as described in fig. 2.
979
5
4
Vol. 173, No, 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
times after stimulation of serum or TPA. synthesis
more effectively
stimulation
K252a repressed
by its addition within
by either stimulant as shown in Fig.
the DNA
12 hrs after 3.
Addition of
K252a at 18 hrs after both stimulation by serum and TPA scarcely inhibited DNA synthesis.
On the other hand,
that almost the cells might enter
into S phase before
the cell cycle.
The results
required
synthesis were completed
for ~ A
after stimulation Finally,
we compared
which were considered
between
in steps
12 to 18 hrs
the inhibitory activity of K252a on DNA inhibitors,
staurosporine
intermediate
induced by serum or TPA.
and H7
effect on the growth
inhibition on C-kinase
4, staurosporine was most potent,
synthesis
18 hrs
that K252a-sensitive
to have the suppresive
of VSMC perhaps through in Fig.
suggested
showed
in most of the cells.
synthesis with those of other
and K252a was
this evidence
(4, 5).
H7 was
As shown
least effective
in the inhibition of the DNA synthesis K252a acted more effectively
on the DNA
induced with TPA than with serum.
These evidences kinase of K252a
and the works reporting
in vitro
(7) and
the inhibition on C-
important role of C-kinase
for
the p r o g r e s s i o n of both from GO to G1 and from G1 to S in a cell cycle
(ii) suggest
VSMC by blocking C-kinase
that K252a suppressed
the entry
to S phase
the p r o l i f e r a t i o n
of
in the cell cycle through
inhibition.
Acknowledgments W e thank Drs. Matsuda and Nakanishi, Kyowa Hakko, Co, Ltd. for preparing K252a and for helpful advice. We also thank Dr. Shiraishi for kindly providing bovine carotid. This work was supported by G r a n t s - i n - A i d for Scientific Research from the Ministry of Education, Science and Culture of Japan, the Ministry of Health and W e l f a r e of Japan and the Research Program on Cell Calcium Signal in Cardiovascular System.
REFERENCES !. Chamly-Campbell, J., Campbell, G.R..and Ross, R. (1979) Physiol. Rev. 59, I- 61. 2. Ross, R. (1986) N. Engel. J. Med. 314, 488-500. 3. Kariya, K., Kawahara, Y., Tsuda, T., Fukuzaki, H. and Takai, Y. (1987) Atherosclerosis 63, 251-255. 4. Matsumoto, H. and Sasaki, Y. (1989) Biochem. Biophpys. Res. Commun. 158, 105-109. 5. Takagi, Y., Hirata, Y., Takata, S., Yoshimi, H., Fukuda, Y., Fujita, T. and Hidaka, H. (1988) Atherosclerosis 74, 227-230.
980
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
6. Kariya, K., Fukumoto, Y., Tsuda, T., Kawahara, Y., Fukuzaki, H. Yamamoto, T. and Takai, Y. (1987) FEBS LETTERS 217, 69-73. 7. Kase, H°, Iwahashi, K°, Nakanishi, S., Matsuda, Y., Yamada, K., Takahashi, M., Murakata, C., Sato, A. and Kaneko, M. (1987) Biochem. Biophysic. Res. Commun. 142, 436-440. 8. Nakanishhi, S., Yamada, K., Kase, H., Nakamura, S. and Nonomura, Y. (1988) J. Biol. Chem. 263, 6215-6219. 9. Matsuda, Y., Nakanishi, S., Nagasawa, K., Iwahashi, K. and Kase, H. (1988) Biochem. J. 256, 75-80 i0. Yamada, K., Tanaka, H., Kubo, K. and Kase, H. (1987) Jpn. J. Pharmacol. 43, 284. ii. Kawahara, Y., Kariya, K., Fukumoto, Y., Fukuzaki, H. and H. and Takai, Y. (1989) Proc. "Current Concept of Critical Gene Expression in Carcinogenesis" pp 102-117 IARC Scientific Pub.
981
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 975-981
December 31, 1990
EFFECT OF K252a, THE PROLIFERATION Kazuhiro
Ohmi,
A PROTEIN KINASE INHIBITOR, ON OF V A S C U L A R SMOOTH MUSCLE CELLS
Shigeru
Yamashita
and Yoshiaki
Nonomura
Department of Pharmacology, Faculty of Medicine, The U n i v e r s i t y of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received November 14, 1990 SUMMARY: In the growth arrested cultures of bovine carotid smooth muscle, K252a (i0 - i00 ng/ml), a protein kinase inhibitor with wide spectrum suppressed the cell p r o l i f e r a t i o n induced by TPA and increase of serum. K252a was more potent in the antiproliferative activity than H7, a C - k i n a s e - s p e c i f i c inhibitor, but less than staurosporine, another w i d e - s p e c t r u m protein kinase inhibitor. Since C-kinase plays an important role in the signal t r a n s d u c t i o n leading to the cell p r o l i f e r a t i o n and K252a inhibits C-kinase in vitro, the a n t i p r o l i f e r a t i v e effect of K252a to carotid smooth muscle cells is likely to be exerted through C-kinase dependent pathway. ©1990AcademlcPre~s.Znc.
It is well the
cultured
of serum with
known state
contains
is a potent
the native
state.
revert
from
cell
to know
smooth
culture.
state
we prepared arteries
are found
of aorta has b e e n
However,
since
type of VSMC the cultured
as the source smooth
muscle
state used
some
muscle
PDGF roles
cells
in
to be in a quiesent
one.
SMC
Thus,
it
cultured
by stimulation. as the source
in aortic
it more
of
smooth
suitable
of cultured cell
(i).
among which
the quiescent
myofilaments
we considered
is accompanied
has occurred,
to a p r o l i f e r a t i v e that
in
to increase
to have
Smooth
atherogenesis
the m e c h a n i s m
(VSMC)
to synthetic
factors,
(2).
into a p r o l i f e r a t i v e
muscle
state
is considered
media
cells
in response
growth
disease
once
are not abundant,
the muscular
and
of arterial
However,
are entering
muscle
for VSMC
a quiescent
is important
muscle
from musclar
mitogenic
of vascular
state
growth
Usually
to p r o l i f e r a t e
of phenotype
mitogen
smooth
This p r o l i f e r a t i v e
various
in p a t h o g e n e s i s
VSMC
start
concentration.
the change
Serum
that vascular
to use
cells.
from bovine
Thus,
carotid
in this report.
Abbreviations: VSMC, vascular smooth muscle cells; TPA, 12-O-tetradecanoylphorbol-13-acetate; PDGF, p l a t e l e t - d e r i v e d growth factor; FBS, fetal bovine serum; TCA, tri chloro acetic acid; EDTA, ethylene diamine tetra acetic acid. 0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
976
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
As for growth of VSMC, ed to be one of factors of cultured VSMC H7,
to suppress
protein kinase C
involved
in PDGF-stimulated
(3) and C-kinase
inhibitor,
the growth of cultured VSMC
phorbol
esters has both p r o l i f e r a t i v e
actions
through C-kinase pathways
Recently K252a has been antibiotics.
It has been
ses as follows; muscle myosin dependent
(C-kinase) was report-
C-kinase,
staurosporine (4, 5).
considered
G-kinase
in relation
(i0).
iden-
And then, K252a
is
in culture,
the effect of K252a on the using carotid
artery as the
type of the cell and aimed at clarifing
on the signal
It was
inhibiter with wide spectrum.
we investigated
of VSMC
(9).
of the contraction of arterial
to be a useful kinase
proliferation contractile
inhibitor
(7), and smooth
(8) as well as calmodulin-
induced by several agents
In this report,
(6).
(7) from bacteria as one of
cyclic nucleotide p h o s p h o d i e s t e r a s e
smooth muscle
TPA, one of
found to inhibit various protein kinaA-kinase,
light chain kinase
tified as a potent
and
and a n t i p r o l i f e r a t i v e
in cultured VSMC
isolated
proliferation
transduction pathway of growth
K252a action
factor especially
to C-kinase.
MATERIALS
AND METHODS
Materials: K252a was a gift from Kyowa Hakko, Co., Ltd. H7 was purchased from Seikagaku Kogyo Co. Ltd. Staurosporine was a gift from Prof. Ohmura, Kitazato University. TPA was purchased from Sigma chemical Co. [3H]thymidine was purchased from Amersham. Bovine carotid artery was prepared freshly at the Meat Market of Tokyo Central W h o l e Sale Market. Cell culture: Preparation of primary culture of VSMC were carried out from bovine carotid arteries according to Ross (i). The explants from the medial layers were placed in 60 mm culture dishes with MEM containing 10 % FBS. Medium was changed every 4 days. After 3 weeks, the cells having migrated from the explants were trypsinized and seeded. The cells in secondary cultures maintained in MEM containing i0 % FBS. The cells were always cultured at 37°C in a humidified atmosphere of 5 % CO 2 and 95 % air. Growth assays: VSMC in secondary cultures were trypsinized and seeded into 12 well plates in MEM containing 0.5 % FBS at a density of 2 x 104 cells/well for count of cell number or 5 x 104 c e l l s / 3 5 m m dish for d e t e r m i n a t i o n of [3H] thymidine incorporation into acid insoluble fractions. For cell counts, cells were washed twice with 2 ml PBS, treated with 0.2 ml of 0.05 % t r y p s i n - 0 . 0 1 % EDTA at room temperature, and rinsed from the well with 0.8 ml medium containing 10 % FBS. The number of the trypsinized cells was counted with a heamocytometer. For d e t e r m i n a t i o n of [3H] thymidine incorporation, growth arrested cells were cultured in medium containing 1 uCi/ml [JH] thymidine (20 Ci/mmol) and 10 % FBS or i0 ng/ml TPA plus 0.5 % FBS in the absence or presence of various concentrations of K252a. After 24 hours, the cells were washed 3 times with 2 ml PBS, lysed by the addition of 1.0 ml of 0.2 N NaOH and neutralized 977
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
w i t h 0.2 m l of 1 N HCI. The precipitates were collected on w h a t m a n G F / C f i l t e r s , and the f i l t e r s w e r e w a s h e d w i t h c o l d TCA, ethanol and acetone. A f t e r d r i n g , the r a d i o a c t i v i t y on the filters was determined with a Beckman liquid scintillation s y s t e m , LS 1 8 0 0 .
RESULTS We
confirmed
sisted lial
of
culturing not
and were
DISCUSSION
immunohistologically
smooth
cells
AND
muscle
cells
that
without
fibroblasts,
because
stained
anti-alpha
with
all
the
VSMC
contamination
all
the
cells
smooth
used
of
just
muscle
con-
endotheafter
actin
(data
shown). When
the
the
FBS
concentration
growth-arrested
cell
number
tion
as
became
that
of
cells,
as
non-stimulated
this
condition
bited
such
a serum-induced
dose-dependent
cell
3 times
under
was
manner
as
was
about
increased
proliferation
high
at
96
after
counterpart. 48 hrs.
in Fig.
The
Addition of i.
0.5
% to
i0
and
the
started
hrs
proliferation shown
from
the
stimula-
doubling
time
of
inhi-
K252a
cultured The
% in
cell
VSMC numbers
in
a of
o
o~120
7.0~o
~100
6.0-
|
so
=
60
5.0.9o
4.0i
.0-
e
2.0-
~ 2o
t.o! 0.0
Q
~-a
~ ....
= o
a ~
.1= Ii
i
i
i
24 48 72 Time after stimulation (hrs)
i
,,r
96
Fig. i. Effect of K252a on VSMC proliferation induced by serum. Growth arrested cuitures were stimulated by i0 % FBS without (O) or with 1 ng/ml (O), i0 ng/ml (~), i00 ng/ml (A) of K252a and no stimulation (i). At the times indicated, cells were counted. In allthe figures other details are described in MATERIALS AND METHODS and results are the means ± SE of quadruplicate dishes. Fig. 2. Effect of K252a on DNA synthesis of VSMC induced by serum or TPA. Growth arrested cultures were stimulated by i0 % FBS or 0.5 % FBS with i0 ng/ml TPA in the presence or absence of various concentrations of K252a. 1 uCi/ml of [3H] thymidine was added at 0 time, and the cultures were incubated at 37°C for 24 hours, then DNA synthesis was assayed. Control means the cell stimulated by increase of serum or addition of TPA without K252a treatment. The value of [3H] thymidine incorporation in the control cell is expressed as i00 % and the values to other cells are described as relative value to that of the control. Notice the effect of stimulant as compared with no stimulation cells. 978
Vol. 173, No. 3, 1990
the
culture
% and ml
50
treated
% of
of K 2 5 2 a Next,
was
that
using
incorporation at
24 hrs
of
after
VSMC.
In this
could
not have
incubation by
serum
presence was
of
Furthermore, of K 2 5 2 a
by we
by
with
synthesis
stimulants.
which
was
on DNA
carotid
VSMC of
induced
the
smooth
of
the
was
cultured cells
regulation
or e l o n g a t i o n
DNA
%
fraction
muscle
the d o w n
i0
the
time p o i n t
synthesis
by K 2 5 2 a
dose-dependently.
serum-or
TPA-induced
difference
synthesis
when
(x104 )
DNA
we
of
of
induced In the synthesis
of
K252a
the was
a
inhibitory added
at v a r i o u s
(x104 )
b
2-
80
O"
111
,o
8.
,o
6-
Q.
20
~
cultured
insoluble
of
1 ug/
toxicity.
in i n c r e a s e
5100 |
than
%, r e s p e c t i v e l y .
the
i120
of
not shown).
90
studied
phase
by 25
Addition
This
concentrations
ng/ml)
40 % or
2).
growing
inhibited
(i00
cell
the acid
(Fig.
any p h e n o m e n a
(data
of
reflected
into
cultured
of
cell
TPA
as
reduced
More
its
on DNA
logarismic
of T P A
of K 2 5 2 a
of cells
thymidine
increase
or T P A was
of K 2 5 2 a
respectively.
stimulations
observed by
suppressed
effect
[3HI
study
time
and
ng/ml
to the g r o w t h - a r r e s t e d
both
the m i d s t
synthesis
of K 2 5 2 a serum
i00
K252a,
damage
synthesis
almost
DNA
the
TPA
of D N A
i0 and
without
the e f f e c t
i0 ng/ml
increase
with
induced
examined
FBS or
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
4"
o I
2"
E
-~ o
Q
o Addition time of K252a
0
•
none
Q
8
7
,
•
•
O"
"
6 5 4 none 8 Concentratlonsofdrugs(-IogM)
.
.
7
.
.
6
Fig. 3. Time dependent effect of addition of K252a on the i0 % FBS or TPA induced DNA synthesis. Growth arrested cultures of VSMC were incubated with i0 % FBS or 0.5 % FBS with i0 ng/ml TPA. DNA synthesis was assayed 24 hours incubation, K252a was added at the indicated time at the final concentration of I00 ng/ml. An essential explanation of the figure is the same as that of Fig. 2. Fig. 4. comparison of antiproliferative activity of K252a with that of Staurosporine and H7. Growth arrested cultures were stimulated by i0 % FBS (a) or TPA (b) in the absence or presence of various concentrations of Staurosporine (m), K252a (O) and H7 (A) as described in fig. 2.
979
5
4
Vol. 173, No, 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
times after stimulation of serum or TPA. synthesis
more effectively
stimulation
K252a repressed
by its addition within
by either stimulant as shown in Fig.
the DNA
12 hrs after 3.
Addition of
K252a at 18 hrs after both stimulation by serum and TPA scarcely inhibited DNA synthesis.
On the other hand,
that almost the cells might enter
into S phase before
the cell cycle.
The results
required
synthesis were completed
for ~ A
after stimulation Finally,
we compared
which were considered
between
in steps
12 to 18 hrs
the inhibitory activity of K252a on DNA inhibitors,
staurosporine
intermediate
induced by serum or TPA.
and H7
effect on the growth
inhibition on C-kinase
4, staurosporine was most potent,
synthesis
18 hrs
that K252a-sensitive
to have the suppresive
of VSMC perhaps through in Fig.
suggested
showed
in most of the cells.
synthesis with those of other
and K252a was
this evidence
(4, 5).
H7 was
As shown
least effective
in the inhibition of the DNA synthesis K252a acted more effectively
on the DNA
induced with TPA than with serum.
These evidences kinase of K252a
and the works reporting
in vitro
(7) and
the inhibition on C-
important role of C-kinase
for
the p r o g r e s s i o n of both from GO to G1 and from G1 to S in a cell cycle
(ii) suggest
VSMC by blocking C-kinase
that K252a suppressed
the entry
to S phase
the p r o l i f e r a t i o n
of
in the cell cycle through
inhibition.
Acknowledgments W e thank Drs. Matsuda and Nakanishi, Kyowa Hakko, Co, Ltd. for preparing K252a and for helpful advice. We also thank Dr. Shiraishi for kindly providing bovine carotid. This work was supported by G r a n t s - i n - A i d for Scientific Research from the Ministry of Education, Science and Culture of Japan, the Ministry of Health and W e l f a r e of Japan and the Research Program on Cell Calcium Signal in Cardiovascular System.
REFERENCES !. Chamly-Campbell, J., Campbell, G.R..and Ross, R. (1979) Physiol. Rev. 59, I- 61. 2. Ross, R. (1986) N. Engel. J. Med. 314, 488-500. 3. Kariya, K., Kawahara, Y., Tsuda, T., Fukuzaki, H. and Takai, Y. (1987) Atherosclerosis 63, 251-255. 4. Matsumoto, H. and Sasaki, Y. (1989) Biochem. Biophpys. Res. Commun. 158, 105-109. 5. Takagi, Y., Hirata, Y., Takata, S., Yoshimi, H., Fukuda, Y., Fujita, T. and Hidaka, H. (1988) Atherosclerosis 74, 227-230.
980
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
6. Kariya, K., Fukumoto, Y., Tsuda, T., Kawahara, Y., Fukuzaki, H. Yamamoto, T. and Takai, Y. (1987) FEBS LETTERS 217, 69-73. 7. Kase, H°, Iwahashi, K°, Nakanishi, S., Matsuda, Y., Yamada, K., Takahashi, M., Murakata, C., Sato, A. and Kaneko, M. (1987) Biochem. Biophysic. Res. Commun. 142, 436-440. 8. Nakanishhi, S., Yamada, K., Kase, H., Nakamura, S. and Nonomura, Y. (1988) J. Biol. Chem. 263, 6215-6219. 9. Matsuda, Y., Nakanishi, S., Nagasawa, K., Iwahashi, K. and Kase, H. (1988) Biochem. J. 256, 75-80 i0. Yamada, K., Tanaka, H., Kubo, K. and Kase, H. (1987) Jpn. J. Pharmacol. 43, 284. ii. Kawahara, Y., Kariya, K., Fukumoto, Y., Fukuzaki, H. and H. and Takai, Y. (1989) Proc. "Current Concept of Critical Gene Expression in Carcinogenesis" pp 102-117 IARC Scientific Pub.
981
