Vo1.182, No. 1,1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 379-387
Januaw 15,1992
A NOVEL COMPOUND, DEPUDECIN, INDUCES PRODUCTION OF TRANSFORMATION TO TIlE FLAT PHENOTYPE OF NIIBT3 CELLS TRANSFOP3/IED BY RAS-ONCOGENE K e n j i SugiLa*, H i r o s h i Yoshida,
MakoLo Matsumoto, and S h i g e r u
Matsutani Division ofOneology,
Department o f M i c r o b i o l o g y , S h i o n o g i
Research Laboratories, Sagisu,
S h i o n o g i & Co., L t d . ,
Fukushima-ku,
4-12,
5-chome,
Osaka 553, J a p a n
Received December 4, 1991
SUMMARY: A n o v e l compound, d e p u d e c i n , i n d u c e d p r o d u c t i o n o f t h e f l a t phenoLype o f K i - r a s - L e a n s f o e m e d NIH3TB c e l l s a t t h e low c o n c e n t r a t i o n o f 1 vg/ml. T h i s e f f e e t was r e v e r s i b l e . A e t i n s t r e s s f i b e r was d e t e c t e d in t h e s e c e l l s a f t e r d e p u d e c i n t r e a t m e n t . Almost c o m p l e t e r e v e r s i o n t o t h e f l a t p h e n o t y p e was observed at 6 h after depudeein addition. The s y n t h e s i s o f Pas-~RNA d i d n o t d e c r e a s e enough w i t h d e p u d e e i n t r e a t m e n t a t t h e c o n c e n t r a t i o n o f 10 #g/ml t o r e v e r s e t h e t r a n s f o r m e d morphology. © 1992 A c a d e m i c
Press,
Inc.
r a s - O n e o g e n e h a s been r e p o r t e d human tumors and tumor c e i l
lines
ko be e x p r e s s e d in many (1).
The p r o d u c t o f r a s -
oncogene i s a k i n d o f GTP-binding p r o / e i n , activity
can be c o r r e l a t e d
GTPase
with /he transforming a c t i v i t y
There have been o n l y a few r e p o r t s r a s - o n e o g e n e (6, 7).
and i t s
on i n h i b i t o e s
(2-5).
against
Oxanosine was found t o i n h i b i t
the function
* To whom c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d . A b b r e v i a t i o n s u s e d : TSA, t r i e h o s t a t i n A; MTT, 3 - ( 4, 5 - d i m e t h y l t h i a z o l - 2 - y l )-2, 5 - d i p h e n y l t e t r a z o l ium bromide; PEG, p h o s p h a t e - b u f f e r e d s a l i n e ( 0 . 1 5 M NaC1); D-MEM, D u l b e c e o ' s m o d i f i e d minimum e s s e n t i a l medium; ICs0, c o n c e n t r a t i o n o f d r u g r e q u i r e d f o r 50% i n h i b i L i o n o f c e l l growth; DMSO, d i m e t h y l s u l f o x i d e ; HMBA, h e x a m e L h y l e n e - b i s - a c e L a m i d e ; Pas/NIH3T3, r a s - t r a n s f o r ~ a e d N I HBT3. 0006-291Xd92 $1.50
379
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
V o l . 182, N o . 1, 1 9 9 2
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
o f r a s - o n c o g e n e by d e c r e a s i n g t h e pool o f g u a n i n e n u c l e o t i d e s i n s i d e t h e c e l l s by i n h i b i t i n g
IMP d e h y d r o g e n a s e (6).
Azatyrosine
was n o t e d Co r e v e r s e t h e p h e n o t y p e o f t h e r a s - t r a n s f o r m e d c e l l s , g i v i n g a f l a L r e v e F t a n L a f L e r long-Lerm culLune (7).
As t h e r e a r e
many GTP-binding pFoLeins which acL a s normal s i g n a l LFansduceFs, an i n h i b i t o r
o f r a s - o n c o g e n e mighL modulaLe t h e normal c e l l
funcLion. To a v o i d s e l e c L i n g s u c h an i n h i b i L o r ,
we d e v i s e d a
s c r e e n i n g syshem u s i n g NItBT3 c e l l s d o u b l y h r a n s f o r m e d by r a s and s r c - o n c o g e n e s , b a s e d on Lhe h y p o t h e s i s Lhak Lhere would be a pathway beLween t h e s i g n a l LransducLion a f L e r s i g n a l s of
common
ras
and
src
functions
phenotH3e
of such
iL should
be an
afLer
and
from
ras
depudecin,
phenotype We describe
inhibitor
cells
broth
of
was
can
AfLer
fungi,
isolated
It could
which
induce
of cells
doubly
here
the action
reverses
be detected
in the pathway
functions.
which
brassicicola.
If an agent
transfor~ned
src
a culture
(8).
of signal
screening
we
found
from
the
a novel
Lransformation
Iransduction samples
compound, AILernaria
into
by ras-
of depudecin
by screening,
numerous
fungus
transformed
the
and
against
the
flat
src-oncogenes. Fas/NIH3T3
cells.
MATERI ALS AND METHODS Chemicals. Depudecin (Fig. 1) and TSA (9) were p r e p a r e d i n o u r l a b o r a t o r i e s . DMSO was p u r c h a s e d from Nakarai Tesque Inc. (Kyoto, J a p a n ) , h e x a m e t h l e n e - b i s - a c e t ~ m i d e (HMBA) and MTT from Wako Pure Chemical Co. (Osaka, J a p a n ) , and v - K i - r a s probe from Takara Shuzo Co., LLd. (Kyoto, J a p a n ) . H
.,,\\ 0
,~,.\0
_
R
~ H
Fig.
=-
C
"
H
~
"~.,~ OH
"~
"
OH
I.
SLrucLure
380
of depudecin.
3
Vol. 182, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Cells. v - K i - r a s - t r a n s f o r m e d NItBT3 (ras/NIH3TB) c e l l s were a g i f t from Dr. K. g a n a g i h a r a a t t h e R e s e a r c h I n s t i t u t e f o r N u c l e a r M e d i c i n e and B i o l o g y , H i r o s h i m a U n i v e r s i t y . These c e l l s were grown i n D u l b e c e o ' s m o d i f i e d minimum e s s e n t i a l medium (D-MEM) s u p p l e m e n t e d w i t h 10% f e t a l b o v i n e serum. MTT a s s a y I n h i b i t i o n o f c e l l growth by d e p u d e c i n was d e t e r m i n e d a c c o r d i n g t o a method d e s c r i b e d p r e v i o u s l y u s i n g MTT a s a dye (10). Staining of aetin stress fiber with rhodamine-phalloizin. F o r f l u o r e s c e n c e m i c r o s c o p y , t h e a e t i n s t r e s s f i b e r in c e l l s was o b s e r v e d a f t e r s t a i n i n g w i t h r h o d a m i n e - p h a l l o i z i n a s r e p o r t e d p r e v i o u s l y ( 11 ). N o r t h e r n bl o t t i n g . P r e p a r a t i o n o f t o t a l RNA from r a s - t r a n s f o r m e d and normal NIHBTB c e l l s w i t h o r w i t h o u t d e p u d e e i n t r e a t m e n t , e l e c t F o p h o r e t i e s e p a r a t i o n o f RNA and N o r t h e r n b l o t a n a l y s i s were done a s r e p o r t e d p r e v i o u s l y (12, 1B).
RESULTS E f f e c t o f d e p u d e c i n on t h e growth and morphology o f r a s t r a n s f o r m e d NItBTB c e l l s . Depudeein a t more than 1 ~g/ml r e v e r s e d t h e t r a n s f o r m e d p h e n o t y p e o f ras/NIHBTB e e l l s for this aetivity
(Fig.
2).
The e o n c e n t r a t i o n n e e d e d
was mueh l o w e r t h a n t h a t
for inhibition
t h e i r growth ( t h e v a l u e f o r IC50 was 10 gg/ml, assay)
( d a t a n o t shown).
when t h e c e l l after
its
The e f f e e t
removal (Fig.
shape,
4).
The t r a n s f o r m e d c e l l s
(14).
Therefore,
the morphologieal
a d d i t i o n o f d e p u d e e i n t o ras/NIHBTB
T h i s ehange was o b s e r v e d a f t e r
bundles of actin stress cells
o f d e p u d e e i n was r e v e r s i b l e
B). To f i n d t h e l e n g t h o f d e p u d e e i n
change was f o l l o w e d a f t e r
(Fig.
d e t e r m i n e d by MTT
c u l t u r e was i n e u b a t e d f o r more t h a n 24 h a t B7°C
e x p o s u r e n e e d e d go i n d u e e t h e f l a t
cells.
of
fiber,
e x p o s u r e f o r more t h a n 6 h
have been r e p o r t e d t o l o s e whieh a r e o b s e r v e d i n t h e normal
we examined t h e a p p e a r a n c e o f t h e a e k i n 381
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. 2. E f f e c t o f d e p u d e e i n on t h e morphology o f ras/NIHBT3 c e l l s , ras/NIH3T3 c e l l s were i n o e u l a L e d i n t o p e L r i d i s h e s ( 6 6 em, F a l c o n ) a t 2 . 5 x 10" e e I I s / m l in % mI o f D-MEM (10% FBS) and c u l t u r e d o v e r n i g h t in a 57; C02 i n c u b a t o r . The c e i l s were i n c u b a t e d f o r a f u r t h e r 24 h i n the a b s e n c e (a) o r t h e p r e s e n c e (b) o f d e p u d e e i n a t 5 ~g/ml, and t h e n s t a i n e d w i t h h e m a g o x y l i n and e o s i n , and p h o t o g r a p h e d .
Fig. 3. Reversible effects of depudeein on the morphological change o f ras/NIH3T3 c e l l s , ras/NIH3T3 c e l l s were i n c u b a t e d in t h e a b s e n c e (a) o r t h e p r e s e n c e (b) o f d e p u d e c i n a t 5 ~g/ml. A f t e r d e p u d e e i n t r e a t m e n t , t h e c u l t u r e was washed and i n c u b a t e d f o r a f u r t h e r 48 h in t h e a b s e n c e o f d e p u d e c i n (e), and photographed.
382
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. 4. KineLies o f act.ion o f depudeein in r e v e r s i n g the phenot.ype o f Pas/NIH3T3 e e l l s . The procedure was the same as t h a t in Fig. 2, exeepL Lhat t.he morphology o f the e e l l s was observed at. 0 h (a), 2 h (b), 4 h (e), 6 h (d), 8 h (e), and 10 h (f) a f t e r the a d d i t i o n o f depudeein, and photographed. stress
fiber
i n ras/NIHBT3 c e l l s
with depudecin treatment.
Clear
b a n d s were o b s e r v e d by f l u o r o s c e n e e m i c r o s c o p y i n ras/NIHBTB ceils
after
depudeein treatment
f o r 24 h ( F i g .
whether the morphological reversion o f ras-mRNA s y n t h e s i s , Northern blotting
To d e t e r m i n e
was c a u s e d by t h e i n h i b i t i o n
t h e amount o f ras-mRNA was m e a s u r e d by
u s i n g a r a s - o n e o g e n e p r o b e i n Pas/NIH3TB c e l l s
with or withouL depudeein treatment. significantly
5).
affect
the synthesis
Depudecin d i d n o t
o f ras-nRNA a t
10 ~g/ml,
a
c o n e e n L r a t i o n a t which t h e p h e n o t y p e t r a n s f o r m a h i o n was c l e a r l y rever~oed ( F i g .
6). DISCUSSION
We h a v e d e s c r i b e d reverse
the phenotype transformation
v e r y low c o n c e n t r a t i o n ~g/ml).
how t h e n o v e l compound d e p u d e c i n c a n o f ras/NIHBTB c e l I s
(1 v g / m l ) compared t o a z a C y r o s i n e
Depudeein indueed the transformation 383
at a (500
of ras/NItBTB cells
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. 5. R e s t o r a t i o n o f a c t i n s t r e s s f i b e r in ras/NIHBT3 c e l l s wiLh d e p u d e c i n ~ e a t m e n L . AcLin s t r e s s f i b e r was s t a i n e d w i t h r h o d a m i n e - p h a l l o i z i n . (a) UnLreated c e l l s , (b) d e p u d e c i n - t r e a t e d cells.
Fig. 6. E f f e c t s o f d e p u d e c i n on t h e s y n t h e s i s o f ras-mRNA. Normal (a, b) and s i s - t r a n s f o r m e d (c, d) NIHBTB c e l l s were i n o c u l a t e d i n t o a 75 cm= b o t t l e ( F a l c o n ) a t 5 x 10" c e l l s / m l i n 15 ml o f D-MEM (10% FBS) and i n c u b a t e d f o r 24 h i n t h e a b s e n c e (a, f) o r t h e p r e s e n c e o f d e p u d e c i n a t 1.0 gg/ml (b, g), 2 . 5 gg/ml (e, h), 5 . 0 ~g/ml (d, i ) , and 10.0 ug/ml (e, j ) . (A) v - K - r a s prx3be, (B) a c t i n probe. 384
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
t o t h e f l a t p h e n o t y p e when t h e c e l l s t h a n 6 h (Fig.
4),
indicating that the appearance of the flat
c e l l s was n o t due t o t h e s e l e c t i o n but to d i r e c t activity
had been e x p o s e d f o r more
of depudeein-resistant
cells,
a c t i o n o f d e p u d e e i n on t h e ras/NIHBT3 c e l l s .
o f d e p u d e c i n was r e v e r s i b l e ,
This
s i n c e the f l a t phenotype
o f ras/NIHBTB c e l l s r e v e r s e d t o t h e t r a n s f o r m e d one w i t h more t h a n 24 h i n c u b a t i o n a f t e r
removal o f d e p u d e c i n (Fig.
2).
l n d u e t i o n o f t r a n s f o r m a t i o n t o t h e f l a t morphology by d e p u d e e i n was n o t c a u s e d by i n h i b i t i o n o f t h e s y n t h e s i s o f ras-mRNA (Fig. 6).
A p r e l i m i n a r y e x p e r i m e n t u s i n g a monoclonal a n t i b o d y a g a i n s t
p21 "'~ showed t h a t d e p u d e c i n d i d n o t decrease, t h e amount o f p21' as. These r e s u l t s
indicated that the reversion of the
t r a n s f o r m e d p h e n o t y p e by d e p u d e c i n was n o t c a u s e d by i n h i b i t i o n o f t h e s y n t h e s i s o f r a s - o n e o g e n e macromolecules. Although d e p u d e e i n has a d i f f e r e n t i a t i o n - i n d u c i n g Friend leukemia c e l l s
(unpublished data),
activity
on
the induced phenotype
by d e p u d e c i n i n ras/NIHBTB c e i l s was q u i t e d i f f e r e n t
from t h o s e
i n d u c e d by TSA, DMSO and HMBA, which have a l s o been r e p o r t e d t o have d i f f e r e n t i a t i o n - i n d u c i n g ceils
(15-17).
Our r e s u l t
d e p u d e c i n was d i f f e r e n t
activities
on F r i e n d l e u k e m i a
showed t h a t t h e mechanism o f a c t i o n o f from t h e o t h e r t h r e e compounds. S u p p o r t
f o r t h i s came from t h e f a c t t h a t d e p u d e c i n i n h i b i t e d t h e growth o f T S A - r e s i s t a n t FMBA mouse mammary g l a n d tumor c e i l s
(a g i f t
from Dr. T. Beppu, U n i v e r s i t y o f Tokyo) and t h a t d e p u d e e i n c o u l d induce d i f f e r e n t i a t i o n
o f t h e DMSO-resislant v a r i a n t o f a mouse
e r y t h r o l e u k e m i a 745A c e l l
l i n e (a g i f t
from Dr. M. Oishi,
U n i v e r s i t y o f Tokyo) ( u n p u b l i s h e d d a t a ) . We a l s o examined t h e e f f e c t s of tar-transformed cells
(a g i f t
o f d e p u d e e i n on t h e morphology from Dr. K. Yanagihara, 385
Vol. 182, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Hiroshima U n i v e r s i t y ) ,
a s Lhere h a s b e e n a r e p o r t
product of raf-oncogene (Raf-1), serine/threonine
k i n a s e (18),
and r a s f u n c L i o n s (19).
which h a s been shown t o be a
is a signal
Lransducer afLer src
Our r e s u l L s showed thaL d e p u d e c i n a l s o
r e v e r s e d t h e Lransformed phenoLype o f Lhese c e l l s daLa).
Therefore,
Lhat t h e
(unpublished
Lhe LargeL o f d e p u d e e i n i s LhoughL Lo be
i n v o l v e d in t h e s i g n a l
Lransduekion a f k e r the s i g n a l o f r a f -
oneogene.
ACKNOWLEDGMENTS The a u t h o r s would l i k e t o thank Dr. ft. Matsumoto f o r p r e p a r i n g d e p u d e c i n and TSA, and Dr. T. Yoshida f o r h i s s u p p o r t of t h i s work. They a l s o thank Mr. K. G. Higgins f o r h e l p in preparing t h i s manuscript.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
13. Davis, L. G., Dibner, M. D., and Battey, J. F. (1986) Basic MeLhods in Moleeular Biology. p143-146. Elservier Seienee Publishing. New York. 14. 01den, K., and Yamada, K. M. (1977) Cell ll, 957-969. 15. Yoshida, M., Nomura, S., and Beppe, T. (1987) Cancer Res. 47, 3688-3691. 16. F r i e n d , C., Seher, W., Holland, J . , and ~ L o , T. (1971) Proe. N a t l . Aead. S e i . 68, 378-382. 17. Reuben, R. C., Wife, R. k . , Breslow, R., R i f k i n d , R. A., and Marks, P. A. (1976) l ~ o e . Natl. Aead. S e i . USA 73, 862-866. 18. M o e l l i n g , K., Heimann, B., Beimling, P., Rapp. U. R., and S a n d e r T. (1984) NaLure (London) 312, 558-561. 19. M o ~ i s o n , D. K., Kaplan, D. R., Rapp, U. R., and R o b e r t s T. M. (1988) Proe. N a t l . Aead. S e i . USA 85, 8855-8859.
387
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 379-387
Januaw 15,1992
A NOVEL COMPOUND, DEPUDECIN, INDUCES PRODUCTION OF TRANSFORMATION TO TIlE FLAT PHENOTYPE OF NIIBT3 CELLS TRANSFOP3/IED BY RAS-ONCOGENE K e n j i SugiLa*, H i r o s h i Yoshida,
MakoLo Matsumoto, and S h i g e r u
Matsutani Division ofOneology,
Department o f M i c r o b i o l o g y , S h i o n o g i
Research Laboratories, Sagisu,
S h i o n o g i & Co., L t d . ,
Fukushima-ku,
4-12,
5-chome,
Osaka 553, J a p a n
Received December 4, 1991
SUMMARY: A n o v e l compound, d e p u d e c i n , i n d u c e d p r o d u c t i o n o f t h e f l a t phenoLype o f K i - r a s - L e a n s f o e m e d NIH3TB c e l l s a t t h e low c o n c e n t r a t i o n o f 1 vg/ml. T h i s e f f e e t was r e v e r s i b l e . A e t i n s t r e s s f i b e r was d e t e c t e d in t h e s e c e l l s a f t e r d e p u d e c i n t r e a t m e n t . Almost c o m p l e t e r e v e r s i o n t o t h e f l a t p h e n o t y p e was observed at 6 h after depudeein addition. The s y n t h e s i s o f Pas-~RNA d i d n o t d e c r e a s e enough w i t h d e p u d e e i n t r e a t m e n t a t t h e c o n c e n t r a t i o n o f 10 #g/ml t o r e v e r s e t h e t r a n s f o r m e d morphology. © 1992 A c a d e m i c
Press,
Inc.
r a s - O n e o g e n e h a s been r e p o r t e d human tumors and tumor c e i l
lines
ko be e x p r e s s e d in many (1).
The p r o d u c t o f r a s -
oncogene i s a k i n d o f GTP-binding p r o / e i n , activity
can be c o r r e l a t e d
GTPase
with /he transforming a c t i v i t y
There have been o n l y a few r e p o r t s r a s - o n e o g e n e (6, 7).
and i t s
on i n h i b i t o e s
(2-5).
against
Oxanosine was found t o i n h i b i t
the function
* To whom c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d . A b b r e v i a t i o n s u s e d : TSA, t r i e h o s t a t i n A; MTT, 3 - ( 4, 5 - d i m e t h y l t h i a z o l - 2 - y l )-2, 5 - d i p h e n y l t e t r a z o l ium bromide; PEG, p h o s p h a t e - b u f f e r e d s a l i n e ( 0 . 1 5 M NaC1); D-MEM, D u l b e c e o ' s m o d i f i e d minimum e s s e n t i a l medium; ICs0, c o n c e n t r a t i o n o f d r u g r e q u i r e d f o r 50% i n h i b i L i o n o f c e l l growth; DMSO, d i m e t h y l s u l f o x i d e ; HMBA, h e x a m e L h y l e n e - b i s - a c e L a m i d e ; Pas/NIH3T3, r a s - t r a n s f o r ~ a e d N I HBT3. 0006-291Xd92 $1.50
379
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
V o l . 182, N o . 1, 1 9 9 2
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
o f r a s - o n c o g e n e by d e c r e a s i n g t h e pool o f g u a n i n e n u c l e o t i d e s i n s i d e t h e c e l l s by i n h i b i t i n g
IMP d e h y d r o g e n a s e (6).
Azatyrosine
was n o t e d Co r e v e r s e t h e p h e n o t y p e o f t h e r a s - t r a n s f o r m e d c e l l s , g i v i n g a f l a L r e v e F t a n L a f L e r long-Lerm culLune (7).
As t h e r e a r e
many GTP-binding pFoLeins which acL a s normal s i g n a l LFansduceFs, an i n h i b i t o r
o f r a s - o n c o g e n e mighL modulaLe t h e normal c e l l
funcLion. To a v o i d s e l e c L i n g s u c h an i n h i b i L o r ,
we d e v i s e d a
s c r e e n i n g syshem u s i n g NItBT3 c e l l s d o u b l y h r a n s f o r m e d by r a s and s r c - o n c o g e n e s , b a s e d on Lhe h y p o t h e s i s Lhak Lhere would be a pathway beLween t h e s i g n a l LransducLion a f L e r s i g n a l s of
common
ras
and
src
functions
phenotH3e
of such
iL should
be an
afLer
and
from
ras
depudecin,
phenotype We describe
inhibitor
cells
broth
of
was
can
AfLer
fungi,
isolated
It could
which
induce
of cells
doubly
here
the action
reverses
be detected
in the pathway
functions.
which
brassicicola.
If an agent
transfor~ned
src
a culture
(8).
of signal
screening
we
found
from
the
a novel
Lransformation
Iransduction samples
compound, AILernaria
into
by ras-
of depudecin
by screening,
numerous
fungus
transformed
the
and
against
the
flat
src-oncogenes. Fas/NIH3T3
cells.
MATERI ALS AND METHODS Chemicals. Depudecin (Fig. 1) and TSA (9) were p r e p a r e d i n o u r l a b o r a t o r i e s . DMSO was p u r c h a s e d from Nakarai Tesque Inc. (Kyoto, J a p a n ) , h e x a m e t h l e n e - b i s - a c e t ~ m i d e (HMBA) and MTT from Wako Pure Chemical Co. (Osaka, J a p a n ) , and v - K i - r a s probe from Takara Shuzo Co., LLd. (Kyoto, J a p a n ) . H
.,,\\ 0
,~,.\0
_
R
~ H
Fig.
=-
C
"
H
~
"~.,~ OH
"~
"
OH
I.
SLrucLure
380
of depudecin.
3
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Cells. v - K i - r a s - t r a n s f o r m e d NItBT3 (ras/NIH3TB) c e l l s were a g i f t from Dr. K. g a n a g i h a r a a t t h e R e s e a r c h I n s t i t u t e f o r N u c l e a r M e d i c i n e and B i o l o g y , H i r o s h i m a U n i v e r s i t y . These c e l l s were grown i n D u l b e c e o ' s m o d i f i e d minimum e s s e n t i a l medium (D-MEM) s u p p l e m e n t e d w i t h 10% f e t a l b o v i n e serum. MTT a s s a y I n h i b i t i o n o f c e l l growth by d e p u d e c i n was d e t e r m i n e d a c c o r d i n g t o a method d e s c r i b e d p r e v i o u s l y u s i n g MTT a s a dye (10). Staining of aetin stress fiber with rhodamine-phalloizin. F o r f l u o r e s c e n c e m i c r o s c o p y , t h e a e t i n s t r e s s f i b e r in c e l l s was o b s e r v e d a f t e r s t a i n i n g w i t h r h o d a m i n e - p h a l l o i z i n a s r e p o r t e d p r e v i o u s l y ( 11 ). N o r t h e r n bl o t t i n g . P r e p a r a t i o n o f t o t a l RNA from r a s - t r a n s f o r m e d and normal NIHBTB c e l l s w i t h o r w i t h o u t d e p u d e e i n t r e a t m e n t , e l e c t F o p h o r e t i e s e p a r a t i o n o f RNA and N o r t h e r n b l o t a n a l y s i s were done a s r e p o r t e d p r e v i o u s l y (12, 1B).
RESULTS E f f e c t o f d e p u d e c i n on t h e growth and morphology o f r a s t r a n s f o r m e d NItBTB c e l l s . Depudeein a t more than 1 ~g/ml r e v e r s e d t h e t r a n s f o r m e d p h e n o t y p e o f ras/NIHBTB e e l l s for this aetivity
(Fig.
2).
The e o n c e n t r a t i o n n e e d e d
was mueh l o w e r t h a n t h a t
for inhibition
t h e i r growth ( t h e v a l u e f o r IC50 was 10 gg/ml, assay)
( d a t a n o t shown).
when t h e c e l l after
its
The e f f e e t
removal (Fig.
shape,
4).
The t r a n s f o r m e d c e l l s
(14).
Therefore,
the morphologieal
a d d i t i o n o f d e p u d e e i n t o ras/NIHBTB
T h i s ehange was o b s e r v e d a f t e r
bundles of actin stress cells
o f d e p u d e e i n was r e v e r s i b l e
B). To f i n d t h e l e n g t h o f d e p u d e e i n
change was f o l l o w e d a f t e r
(Fig.
d e t e r m i n e d by MTT
c u l t u r e was i n e u b a t e d f o r more t h a n 24 h a t B7°C
e x p o s u r e n e e d e d go i n d u e e t h e f l a t
cells.
of
fiber,
e x p o s u r e f o r more t h a n 6 h
have been r e p o r t e d t o l o s e whieh a r e o b s e r v e d i n t h e normal
we examined t h e a p p e a r a n c e o f t h e a e k i n 381
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. 2. E f f e c t o f d e p u d e e i n on t h e morphology o f ras/NIHBT3 c e l l s , ras/NIH3T3 c e l l s were i n o e u l a L e d i n t o p e L r i d i s h e s ( 6 6 em, F a l c o n ) a t 2 . 5 x 10" e e I I s / m l in % mI o f D-MEM (10% FBS) and c u l t u r e d o v e r n i g h t in a 57; C02 i n c u b a t o r . The c e i l s were i n c u b a t e d f o r a f u r t h e r 24 h i n the a b s e n c e (a) o r t h e p r e s e n c e (b) o f d e p u d e e i n a t 5 ~g/ml, and t h e n s t a i n e d w i t h h e m a g o x y l i n and e o s i n , and p h o t o g r a p h e d .
Fig. 3. Reversible effects of depudeein on the morphological change o f ras/NIH3T3 c e l l s , ras/NIH3T3 c e l l s were i n c u b a t e d in t h e a b s e n c e (a) o r t h e p r e s e n c e (b) o f d e p u d e c i n a t 5 ~g/ml. A f t e r d e p u d e e i n t r e a t m e n t , t h e c u l t u r e was washed and i n c u b a t e d f o r a f u r t h e r 48 h in t h e a b s e n c e o f d e p u d e c i n (e), and photographed.
382
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Fig. 4. KineLies o f act.ion o f depudeein in r e v e r s i n g the phenot.ype o f Pas/NIH3T3 e e l l s . The procedure was the same as t h a t in Fig. 2, exeepL Lhat t.he morphology o f the e e l l s was observed at. 0 h (a), 2 h (b), 4 h (e), 6 h (d), 8 h (e), and 10 h (f) a f t e r the a d d i t i o n o f depudeein, and photographed. stress
fiber
i n ras/NIHBT3 c e l l s
with depudecin treatment.
Clear
b a n d s were o b s e r v e d by f l u o r o s c e n e e m i c r o s c o p y i n ras/NIHBTB ceils
after
depudeein treatment
f o r 24 h ( F i g .
whether the morphological reversion o f ras-mRNA s y n t h e s i s , Northern blotting
To d e t e r m i n e
was c a u s e d by t h e i n h i b i t i o n
t h e amount o f ras-mRNA was m e a s u r e d by
u s i n g a r a s - o n e o g e n e p r o b e i n Pas/NIH3TB c e l l s
with or withouL depudeein treatment. significantly
5).
affect
the synthesis
Depudecin d i d n o t
o f ras-nRNA a t
10 ~g/ml,
a
c o n e e n L r a t i o n a t which t h e p h e n o t y p e t r a n s f o r m a h i o n was c l e a r l y rever~oed ( F i g .
6). DISCUSSION
We h a v e d e s c r i b e d reverse
the phenotype transformation
v e r y low c o n c e n t r a t i o n ~g/ml).
how t h e n o v e l compound d e p u d e c i n c a n o f ras/NIHBTB c e l I s
(1 v g / m l ) compared t o a z a C y r o s i n e
Depudeein indueed the transformation 383
at a (500
of ras/NItBTB cells
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. 5. R e s t o r a t i o n o f a c t i n s t r e s s f i b e r in ras/NIHBT3 c e l l s wiLh d e p u d e c i n ~ e a t m e n L . AcLin s t r e s s f i b e r was s t a i n e d w i t h r h o d a m i n e - p h a l l o i z i n . (a) UnLreated c e l l s , (b) d e p u d e c i n - t r e a t e d cells.
Fig. 6. E f f e c t s o f d e p u d e c i n on t h e s y n t h e s i s o f ras-mRNA. Normal (a, b) and s i s - t r a n s f o r m e d (c, d) NIHBTB c e l l s were i n o c u l a t e d i n t o a 75 cm= b o t t l e ( F a l c o n ) a t 5 x 10" c e l l s / m l i n 15 ml o f D-MEM (10% FBS) and i n c u b a t e d f o r 24 h i n t h e a b s e n c e (a, f) o r t h e p r e s e n c e o f d e p u d e c i n a t 1.0 gg/ml (b, g), 2 . 5 gg/ml (e, h), 5 . 0 ~g/ml (d, i ) , and 10.0 ug/ml (e, j ) . (A) v - K - r a s prx3be, (B) a c t i n probe. 384
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
t o t h e f l a t p h e n o t y p e when t h e c e l l s t h a n 6 h (Fig.
4),
indicating that the appearance of the flat
c e l l s was n o t due t o t h e s e l e c t i o n but to d i r e c t activity
had been e x p o s e d f o r more
of depudeein-resistant
cells,
a c t i o n o f d e p u d e e i n on t h e ras/NIHBT3 c e l l s .
o f d e p u d e c i n was r e v e r s i b l e ,
This
s i n c e the f l a t phenotype
o f ras/NIHBTB c e l l s r e v e r s e d t o t h e t r a n s f o r m e d one w i t h more t h a n 24 h i n c u b a t i o n a f t e r
removal o f d e p u d e c i n (Fig.
2).
l n d u e t i o n o f t r a n s f o r m a t i o n t o t h e f l a t morphology by d e p u d e e i n was n o t c a u s e d by i n h i b i t i o n o f t h e s y n t h e s i s o f ras-mRNA (Fig. 6).
A p r e l i m i n a r y e x p e r i m e n t u s i n g a monoclonal a n t i b o d y a g a i n s t
p21 "'~ showed t h a t d e p u d e c i n d i d n o t decrease, t h e amount o f p21' as. These r e s u l t s
indicated that the reversion of the
t r a n s f o r m e d p h e n o t y p e by d e p u d e c i n was n o t c a u s e d by i n h i b i t i o n o f t h e s y n t h e s i s o f r a s - o n e o g e n e macromolecules. Although d e p u d e e i n has a d i f f e r e n t i a t i o n - i n d u c i n g Friend leukemia c e l l s
(unpublished data),
activity
on
the induced phenotype
by d e p u d e c i n i n ras/NIHBTB c e i l s was q u i t e d i f f e r e n t
from t h o s e
i n d u c e d by TSA, DMSO and HMBA, which have a l s o been r e p o r t e d t o have d i f f e r e n t i a t i o n - i n d u c i n g ceils
(15-17).
Our r e s u l t
d e p u d e c i n was d i f f e r e n t
activities
on F r i e n d l e u k e m i a
showed t h a t t h e mechanism o f a c t i o n o f from t h e o t h e r t h r e e compounds. S u p p o r t
f o r t h i s came from t h e f a c t t h a t d e p u d e c i n i n h i b i t e d t h e growth o f T S A - r e s i s t a n t FMBA mouse mammary g l a n d tumor c e i l s
(a g i f t
from Dr. T. Beppu, U n i v e r s i t y o f Tokyo) and t h a t d e p u d e e i n c o u l d induce d i f f e r e n t i a t i o n
o f t h e DMSO-resislant v a r i a n t o f a mouse
e r y t h r o l e u k e m i a 745A c e l l
l i n e (a g i f t
from Dr. M. Oishi,
U n i v e r s i t y o f Tokyo) ( u n p u b l i s h e d d a t a ) . We a l s o examined t h e e f f e c t s of tar-transformed cells
(a g i f t
o f d e p u d e e i n on t h e morphology from Dr. K. Yanagihara, 385
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Hiroshima U n i v e r s i t y ) ,
a s Lhere h a s b e e n a r e p o r t
product of raf-oncogene (Raf-1), serine/threonine
k i n a s e (18),
and r a s f u n c L i o n s (19).
which h a s been shown t o be a
is a signal
Lransducer afLer src
Our r e s u l L s showed thaL d e p u d e c i n a l s o
r e v e r s e d t h e Lransformed phenoLype o f Lhese c e l l s daLa).
Therefore,
Lhat t h e
(unpublished
Lhe LargeL o f d e p u d e e i n i s LhoughL Lo be
i n v o l v e d in t h e s i g n a l
Lransduekion a f k e r the s i g n a l o f r a f -
oneogene.
ACKNOWLEDGMENTS The a u t h o r s would l i k e t o thank Dr. ft. Matsumoto f o r p r e p a r i n g d e p u d e c i n and TSA, and Dr. T. Yoshida f o r h i s s u p p o r t of t h i s work. They a l s o thank Mr. K. G. Higgins f o r h e l p in preparing t h i s manuscript.
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