九州大学学術情報リポジトリ
Kyushu University Institutional Repository
Activation of protein kinase C induces
differentiation in the human T-lymphoblastic cell line MOLT-3
山内, 保生
https://doi.org/10.11501/3052539
出版情報：Kyushu University, 1990, 医学博士, 論文博士 バージョン：
権利関係：
治 V
Br
. J . C a n c e
r( 1 9 8 9 ) 6 0 ， 1 5 ‑ 1 9 。 T h eM a c m i l l a n P r e s s
Ltd . 1 9 8 9
A c t i v a t i o n o f p r o t e i n k i n a s e C i n d u c e s d i f f e r e n t i a t i o n i n t h e human I ・ l y m p h o b l a s t i cc e l l l i n e MOLT ・ 3
Y . Yamauchi ， K. Nagasawa ， T . Mayumi ， T . H o r i u c h i & Y. Niho
F i r s r D e p o r r m e n r o ( J n r e r n a f
，Vle J i c i n e
，F
，αc u f l y o J M e d i c i n e
，K y u s h u U n i v e r s i t y . Muida s h i 3 ‑ 1 ‑ 1
，Higa s h i ‑ k u
，Fuku o kα812
，J O p U
I1S
ummary W e a t t e m p t e d
10d
巴t e r m i n ew h e t h e r o r n o t a c i t v a t i o n o f c a l c i um p h o s p h o l i p i d ‑ d e p e n d e n t p r o t e i n k i n a s e C (PK
C)i s a s s o c i a t e d w i t h t h e i n d u c t i o n o f d i f f e r e n t i a t i o n b y 1 2 ‑ 0 ‑ t
巴t r a d e c a n o y l p h o r b o l ‑ 1 3
・a c e t a t e ( T PA ) i n t h e human T ‑ I y mph o b l a s t i c c e l l l i n e MOL T ‑ 3 . PKC a c t i v i t i e s w e r e a s s a y e d i n MOLT ‑ 3 a n d i t s f i v e s u b c l o n e s r e s i s t a n t
10TPA ‑ i n d u c e d c e l l d i f f e r e n t i a t i o n . T h e c y t o s o l i c PKC a c t i v i l i e s o f TPA ‑ r e s i s t a n t s u b c l o n e s w e r e 3
6‑53% o f t h a t o f t h e
凹r e n t a lMOL T ‑ 3 c e l l s . TPA t r e a t m e n t l e d t o a r a p i d d e c r e a s e i n PK C a c t i v i t i e s i n t h e c y t o s o l t o g e t h e r w i t h a c o n c o m i t a n t i n c r e a s e i n PKC a c t i v i t i e s i n t h e p a r t i c u l a t e f r a c t i o n . i n b o t h MOLT‑3 a n d a TPA
・r e s i s t a n t s u b c l o n e . T h u s . t r a n s l o c a t i o n o f PKC f r o m t h e c y t o s o l t o t h e membr a n e o c c u r r c d f o l l o w i n g t r e a t m e n t w i t h TPA
，i n b o t h c e l l
Iin e s . H o w e v e r . t h e a m o u n t o f PKC t r a n s l o c a t e d f r o m t h e c y t o s o l t o p a r t i c u l a t e f r a c t i o n f o r 6 0 m i n i n a T P A ‑ . e s i s t a n t s u b c l o n e w a s a b o u t 20% o f t h a t o f t h e p a r e n t a l MOL T ‑ 3 c e l l s
.Th e s e f i n d i n g s s u g g e s t t h a t t h e q u a n t i t y o f c y t o s o l i c PKC a c t i v i t y a n d t h e
巴x t e n to f t r a n s l o c a t i o n may r e l a l e t o r e s p o n s e s t o T P A ‑ i n d u c e d c e l l d i f f e r e n t i a t i o n i n t h i s T ‑ c e l l I l n e
TPA . a most p o t e n t tumour p r omo t e r p h o r b o l e s t e . r e x e r t s i t s c f f c c t o n t i s s u e s a nd c u l t u r e d c e l l s ( D i a mond e l a l
叶1 9 8 0 ) Thc i n i t i a l c v c n t i n i t s a c t i o n i n v o l v e s b i n d i n g t o t h e s p e c i f i c r c c c p t o r s on c c l l mcmbrane
、now i d e n t i f i e d t o b e c a l c i u m pho s p h o l i p i d ‑ d c p e n d e n t PKC ( B e r r i d g e
、1 9 8 4 ;N i e d e l e t a l .
，1 9 8 3 ; N i s h i z u k a e l a l .
、1 9 8 4 ; P a r k c r e l a l
べ1 9 8 4 ;Sando &
Youn g 1 9 8 3 : Shoy a b
&Todaro
、1 9 8 0 ) .
We r e p o r t e d TPA‑induced d i f f e r e n t i a t i o n i n human m a l i g n a n t T ‑ c e l l l i n e s MOLT‑3 a nd J u r k a t and a n a l y s e d t h e p r o c e s s e s i n t h e d i f f e r e n t i a t i o n
、u s i n gT ‑ c e l d l i f f e r e n t i a t i o n m a r k e r s s u c h a s c e l l p r o l i f e r a t i o n
司E r o s e t t e f o r m a t i o n . t e r m i n a l d e o x y n u c l e o t i d y l t r a n s f e r a s e a c t i v i t y
，monoclonal OKT a n t i g e n e x p r e s s i o n and m o r p h o l o g i c a l c h a n g e s ( Naga s a wa & Mak
，1 9 8 0
、1 9 8 2
司N a g a s a wae t a 人 1 9 8 1 a
，b ) U s i n g MOL T ‑ 3 a nd i l s s u b c l o n e s r e s i s t a n t t o TPA i n d u c t i o n
守wea l s o f o u n d t h a t t h e r e c e p t o r s f o r p h o r b o l e s t e r p l a y a n i m p o r t a n t r o l e a t t h e i n i t i a l p r o c e s s o f i n d u c t i o n o f c e l l d i f f e r e n t i a t i o n (Mayumi e l a l
司1 9 8 8 ) .Our main i n t e r e s t h a s b c c n w h c t h c r a c t i v a t i o n o f PKC m e d i a t e s t h e s i g n a l s f o r d i f f c r e n t i a t i o n a s i t d o e s i n t h e p r o c e s s o f s t i m u l a t i o n o r p r o l i f c r a t i o n i n v a r i o u s c e l l s y s t e m s ( K a i b u c h i e t αL .
，1 9 8 3 ;
K可 i k a w ae l 0 1 .
、1 9 8 3
、M a l a i s s ee l a L .
、1 9 8 3 ;R o z e n g u r t e l 0 1
.、19
8 4 ) 、 i n c l u d i n ghuman mature T ‑ I y m p h o c y t e s
(Is a k o v e t a l
.， 198 7 ; Man g e r e t 0
1.，1 9 8 7 ) .
In
t h e i n d u c t i o n o f d i f f e r e n t i a t i o n i n t h e p r o m y e l o c y t i c l e u k a e m i a c e l l l i n e HL
・6 0
、al i n e most o f t e n u s e d a s a model o f d i f f c r e n t i a t i o n
，some i n v e s t i g a t o r s s u g g e s t e d t h a t TPA c x e r t s a n i n d u c t i o n e f f e c t t h r o u g h PKC a s a s i g n a l m e d i a t o r (Andcr s o n e l a l .
，1 9 8 5 ; V a ndcnbark e r a l .
司1 9 8 4 )
，w h e r e a s o t h e r s r c f u t e d t h i s ( K r e u t t e r e l α i
吋 1985). Vandenbark e tα1 . ( 1 9 8 4 )
日r s t p r o p o s e d t h a t t h e TPA
司i n d u c e d m a t u r a t i o n o f HL
・6 0m i g h t b e m e d i a t e d b y l h e a c t i v a t i o n o f i n t r a c e l l u l a r PKC w h e r e a s K r e u t t e r e t a l . ( 1 9 8 5 ) o b s e r v e d a d i s s o c i a t i o n o f t h e a c t i v a t i o n o f PKC and t h e i n d u c t i o n o f c e l l m a t u r a t i o n
，d e t c r m i n e d u s i n g l ‑ o l c o y
l‑ 2 ‑ a c c l y l g
lyc e r o l
、as y n t h e t i c com‑
pound which a l s o d i r e c t l y a c t i v a t e s PK
C. However、l i t t l ei s known o f t h e r o l e o f PKC i n t h e i n d u c t i o n o f T ‑ I y m p h o c y t e d i f f e r e n t i a t i o n .
A c t i v a t i o n o f PKC i s a s s o c i a t e d w i t h i t s t r a n s l o c a t i o n from t h c c y t o s o l t o t h e membrane ( K r a f t e t a
l.
，1 9 8 2 ; K r a f t
&
Ander s on
，1 9 8 3 ) . K r a f t e l 0 1 . (
198 2 ) and K r a f t and Ander s o n (
198 3 ) d e m o n s t r a t e d t h a t TPA c a u s e d a t r a n s ‑
lo c a t i o n o f PKC from t h e c y t o s o l t o t h e membrane i n i n t a c t c e l l
C o r r e s p o n d e n c e : Y . Y a m a u c h i φ
c l o s e l y r c l a t e d t o t h e TPA
・i n d u c e dd i f f e r e n t i a t i o n i n HL
・6 0 . We h a v e now e x a mined t h e r o l e o f PKC
，i n p a r t i c u l a r i t s a c t i v a t i o n and s u b c e l l u l a r d i s t r i b u t i o n . i n t h e TPA‑induc e d d i f f e r e n t i a t i o n o f MOL T ‑ 3
M a t e r i a l s a n d m
ethodsC h e m i c o l s
H i s t o n e HI ( t y p e I I I ‑ S )
，p h o s p h a t i d y l s e r i n e ( P S )
，TPA a nd A TP were p u r c h a s e d from Sigma C h e m i c a l C o . ( S t L o u i s
，M
0). y̲32P‑A TP ( 3
，0 0 0 C i mmol
‑1)、o b t a i n e d from Amersham Japan L
td was d i l u t e d w i t h n o n ‑ r a d i o a c t i v e ATP t o 1 0 0 c . p . m . pmol
‑Ij u s t b e f o r e u s e . TPA was d i s s o l v e d a t 2mgml‑
1i n d i m e t h y l s u l p h o x i d e ( DMSO) o r 100mgl ‑
1i n a c e t o n e and s t o r e d a t ‑2 0
oC.C e l l c u l t u r e
The human T ‑ I y m p h o b l a s t i c c e l l l i n e MOL T ‑ 3 was o b t a i n e d from E . G e l f a n d ( H o s p i t a l f o r S i c k C h i l d r e n
，Toronto
，C a n a d a ) . MOL T ‑ 3 s u
bclones (RO l.R02
、R03、R04and R05)r e s i s t a n t t o t h e growth i n h i b i t i o n e f f e c t b y TPA w e r e o b t a i n e d from c o l o n i e s formed i n 0.8% m e t h y l c e l l u l o s e c o n t a i n i n g 16nM TPA and 1 5 % f e t a l c a l f serum ( FCS) a s d e s c r i b e d (Mayumi e t α1 .
，1 9 8 8 ) . T h e s e c e l l s w e r e m a i n t a i n e d i n RPMII640 medium s u p p l e m e n t e d w i t h 1 0 % FCS
，1 0 0 mg 1 ‑
Is t r e p t o m y c i n and 1 0 0 U m l
‑1p e n i c i l l i n . The c o n c e n l r a t i o n o f a c e t o n e u s e d i n t h e c e l l c u
ltured i d n o t e x c e e d 0 . 0 1 %
T e r m i n a l d e o x y n u c l e o t i d y l r r a n s J e r a s e a s s a y
The t e r m i n a l d e o x y n u c l e o t i d y l t r a n s f e r a s e (TdT) a c t i v i t i e s i n MOLT‑3 c e l l s and i t s s u b c l o n e s w e r c measured b y b i o c h e m i c a l a s s a y
，a s d e s c r i b e d (Okamura e l 0 1 . ， 1 9 7 8 ) .
Subc e
l/u l a r j
トa c t
lOn a t
lOn
MOLT‑3 o r T P A ‑ r e s i s t a n t c e l l s ( 3
‑6 x 1 0
7c e l l s ) m a i n t a i n e d w i t h o u t TPA f o r a t l e a s t a month w e r e u s e d i n t h e f o l l o w i n g s t u d y
.A I I s u b s e q
uent st e p s w e r e done a t 4
0C. Thcc c l l s w e r e washed t w i c e w i t h d i v a l e n t c a t i o n ‑ f r e e PBS
司r e s u s p e n d e di n 3 ml h o m o g e n i s i n g b u f f e r ( 2 0 mM T r i s pH 7 . 5
、2 mM EDTA
，0 . 5 mM EGT A . 0 . 3 3 M s u c r o s e
，2 mM p h e n y l m e t h y l s u l p h o n y l
f1
u o r i d e ( PMSF) a nd 5 0 mM 2 ‑ m e r c a p t o e t h a n o l ) a nd s o n i c a t e d w i t h a Branson Model S o n i f i e r f o r 4 5 s a t 2 0 W.
The homogenates w e r e c e n t r i f u g e d f o r 6 0 min a t 1 0 0 βOOg
and t h e s u p e r n a t a n t s e r v e d a s t h e c y t o s o l f r a c t i o n . The
p e l l e t s w e r e wa s h e d w i t h homo g e n i s i n g b u f f e r a nd r e ‑
c c n t r i f u g e d f o r 6 0 min a t 1 0 0 . 0 0 0 g . The p e l l e t was u s e d f o r
1 7 o T ‑ L YMPHOBLAST
a 3
一
F的二
ωU
∞ OF
TC
‑E
仏 ト
︽
‑o EC )
﹀H E
H υ
のω凶
m wc ‑
t c‑ 2
﹄0
仏
03 0
2
a
2
060 30
o
5 b O3
01
三
c
0 . . ...
の'‑
03 C
B
c o u
02 U
《司
Z
01
O b
E
D.. υ
×
("1
0
こ 1
0
ー
〉.〉....
仁J f百 むω
司 C
‑""
C
‑
ω O 』a...
5 O
1
0F
10
2
0F r a c t i o n number
F i g u r e 2 PKC a c t i v i t i e s o n D E A E ‑ s e p h a r o s e c h r o m a l o g r a p h y o f MOLT‑3 ( a ) a n d T P A ‑ r e s i s t a n t s u b
c10 n e ( R O I ) ( b )
.C y t o s o l f r o m 1 0
8c e l l s (MOL T ‑ 3 . 1 0
.0 m g ; RO I . 1 0 . 1 m g ) w e r e a p p l i e d t o a n d e l u t e d f r o m t h e c o l u m n
、a s d e s c r i b e d i n M a t e r i a l s a n d
methods.PKC a c t i v i t y w a s d e t e r m i n e d a s d e s c r i b e d w i t h 5 0 μ l a l i q u o t s f r o m t h e i n d i c a t e d e r n u e n t i n t h e p r e s e n c e
(・一一一一・)o r a b s e n c e (0
一 一 一0)of 1 μ g m l
‑ITPA
、w i t hO.lmM C a z
‑ra n d 80μgml
‑IP S a n d e x p r e s s e d a s c . p . m . J
2p
"ir i c o r p o r a t e d i n t o
・ ‑h i s t o n e H I f o r 5 m i n p e r 5 0 μ I s a m p l e .
一 一」 一、N a C I c o n c e n t r a t l o n
30 O
O~
1
0
5 O3 5
Time ( d a y s )
F i g u r e 1 G r o w l h c u r v e s o f MOL T ‑ 3 ( a ) a n d T P A ‑ r e s i s t a n t s u b c l o n e ( R O I ) ( b ) i n t h e p r e s e n c e
(・一一一一・)o r a b s e n c e (
0一一一一0)o f 1 6 n M TP
A.E a c h p o i n t i s t h
巴mean
土s
.e . o f
vi a b l e c e l l n u m b e r o f t w o s e p a r a t e e x p e r i m e n t s . R 0 2 R 0 3 . R 0 4
an d R05 p r o l i f e r a t e d e q u a l l
yw e l
l.w i t h o r w l t h o u t TPA
、a sRO 1 .
a 106
1 0
5t h e p a r t i c u l a t e f r a c t i o n . PKC i n t h e p a r t i c u l a t e f r a c t i o n was
e x t r a c t e d w i t h 0
.5% Nonidet P ‑ 4 0
、o v e r n i g h ton i c e . PKC from t h e c y t o s o l f r a c t i o n s was p a r t i a l l y p u r i f i e d b y
ap p l y i n g c e l l e x t r a c t s from
恥10LT
・3and t h e T P A ‑ r e s i s t a n t s u b c l o n e ROI t o a s m a l l DEAE‑sepharose column ( 0
.9
x2
.4cm) e q u i l i b r a t e d w i t h b u f f e r A
(2 0 mM T r i s
、pH7
.5
，2mM EDTA. O.Sm
'V¥EGTA and 2mM PMSF). A f t e r s a m p l e a p p l i c a t i o n . t h e column was washed w i t h 3 0 ml o f b u f f e r A and t h e enzyme was e l u t e d w i t h a i I n e a r g r a d i e n t o f
~、~aCI (0‑0.3M.t o t a l
volumc o f 3 0 m ¥ . now r a t e 60mlh
1)
.F r
ac t i o n s o f 1 . 0 ml w e r e c o l ¥ e c t e d and 5 0 μ ¥ o f e a c h f r a c t i o n was u s c d t o m c a s u r e t h e a c t i v i t y o f PK C . On t h e b a s i s o f t h e c l u t i o n p r o f i l e o b t a i n e d
、t h e c y t o s o l and p a r t i c u l a t e p r e p a r a t i o n s w e r e f r a c t i o n a t e d on a DEAE‑sepharose column
、u s i n ga one s t e p w i t h 0 . 1 5 M Na
Cl in 4 ml o f b u f f e r
A
、andPKC a c t i v i t i e s w e r e d e t e r m i n e d
. E的
@
し)1
0
6Subcellular disrribution of PKC
f n t a c t MOLT‑3 c e l l s and t h e T P A ‑ r e s i s t a n t s u b c l o n e RO 1 ( 3
X1 0
7c e l l s ) w e r e p r e i n c u b a t e d a t
37~Cf o r 3 0 min
，t h e n TPA was added a t a f i n a l c o n c e n t r a t i o n o f 1 6 nM. The c e l l s w e r e i n c u b a t e d f o r v a r i o u s t i m e p e r i o d s up t o 6 0 min and washcd t w i c e w i t h i c e ‑ c o l d PBS
.PKC i n t h e c y t o s o l and t h e p a r t i c u l a t e f r a c t i o n was p a r t i a l l y p u r i f i e d on a DEAE
・s e p h a r o s e column
、u s i n ga one s t e p w i t h 0 . 1 5 M NaCJ
，and PKC a c t i v i t y was d e t e r m i n e d .
O 5 30
Time ( m i n u t e s )
F i g u r e 3 T i m e c o u r s e o f PKC a c t i v i t i e s i n t h e c y
lOs o l i c ( a ) a n d p a r t i c u l a t e ( b ) f r a c t i o n
.MOL T ‑ 3
(・一一一一・)a n d R O l (
0一一一一0) w e r e i n c u b a t e d f o r t h e i n d i c a t e d t i m e s a t 3 7 C w i t h 16nM TPA i n 0 . 0 0 0 5 % DMSO. E a c h p o i n t i s t h e m e a n : t s . e . o f t h r e e s e p a r a t e e x p e r i m e n t s . PKC a c t i v i t y i s e x p r e s s e d a s n m o l J Z p i n c o r p o r a t e d i n t o h i s t o n e H 1 p e r 1 0
8c e l l s
60
s t i m u l a t i o n . However
，t h e amount o f d e c r e a s e f o r 6 0 min was 0 . 5 7 nmol min
‑1 p e r 1 0
8c e l l s i n RO 1
、24% o f 2
.40nmolmin
‑1 p e r 1 0
8c e l l s i n MOLT‑3 ( F i g u r e 3 a ) and t h e amount o f i n c r e a s e f o r 6 0 min was 0 . 5 1 nmol min
‑1 p e r 1 0
8c e l l s i n RO
1，20% o f 2 . 6 0 nmol min
‑1 p e r 1 0
8c e l l s i n MOL T ‑ 3 ( F i g u r e 3 b ) . T h e r e was no d e t e c t a b l e PKC a c t i v i t y i n t h e c y t o s o l and p a r t i c u l a t e f r a c t i o n s o f b o t h MOLT‑3 and ROI a t 2 4
，7 2 and 120h a f t e r a d d i t i o n o f 16nM TPA ( d a t a n o t s h o w n ) . DMSO ( 0 . 0 0 0 5 % ) had no a p p a r e n t e f f e c t on t r a n s l o c a t i o n i n MOLT‑3 o r t h e T P A ‑ r e s i s t a n t s u b c l o n e s ( d a t a n o t s h o w n ) .
D i s c u s s i o n
To i n v c s t i g a t e t h e r o l e o f p h o r b o l e s t e r r e c e p t o r s o r PKC i n c a s e 0
'1TPA‑induced d i f f e r e n t i a t i o n
，a c o m p a r i s o n b e t w e e n T P A ‑ s e n s i t i v e and r e s i s t a n t s u b c l o n e s s h o u ¥ d b e o f g r e a t u s e The T P A ‑ r e s i s t a n t s u b c l o n e s from MOL T ‑ 3 p r o l i f e r a t e d i n e i t h e r t h e p r e s e n c e o r a b s e n c e o f TPA
.The T P A ‑ r e s i s t a n c e was a l s o c o n f i r m e d b y t h e a s s a y o f TdT a c t i v i t i e s
.TdT a c t i v i t i e s i n t h c s c s u b c l o n e s r e m a i n e d h i g h e v c n i n t h e p a r e n t a l MOL T ‑ 3 we a s s a y e d t h e c y t o s o l i c PKC a c t i v i t i e s i n
MOL T ‑ 3 a nd T P A ‑ r e s i s t a n t s u b c l o n e s b y p a r t i a J p u r i t i c a t i o n o
nDEAE‑sepharose columns w i t h a one s t e p w i t h 0
.1 5 M
a C I i n 4
mlo f b u f f e r A . The amount o f c y t o s o l i c PKC a c t
ivity o f f i v e T P A ‑ r e s i s t a n t s u b c l o n e s ROI
，R02
，R03
、R04 a
nd R05 was I
、1 7 1 : t 2 6 4 ( 3 6
%).
1.7 2 5 : t 2 6 4 ( 5 3
%).
l.S 2 2 : t 1 1 9 ( 4 7
%). 1 . 6 8 9 士 2 6 9( 5 2
%)and 1 . 6 3 4 : t 3 6 7 ( 5 0
%)nmol 3
Zp i n c o r p o r a t c d i n t o h i s t o n e H 1 p e r min p c r 1 0
8c e l l s
司r e s p c c t i v c l y a l l b e i n g s i g n i f i c a n t l y ¥ o w compared t o 3 . 2 4 7
士4 6 7( 1 0 0
%)i n t h e p a r c n t a l MOLT‑3. The d a t a a r e e x p r e s s e d a s mean : t s . d . n=7. 7
、4
令4
匂4and 4 ; MOL T ‑ 3
，RO
l . R02
、R03
、R04and ROS
、r e s p e c t i v c l y
.T h c r e f o r e . PKC a c t i v i t i e s i n T P A ‑ r e s i s t a n t s u b c l o n e s w e r e l e s s t h a n t h a t i n MOLT‑3
.T a b l e 1 T
er m i n a
l de o x
yn u c l e o l l d
yl t r a n s f e r a s e
(TdT
)a c t i
vi t i e s o f MOL T
‑3 an d T P A ‑ r e s i s t a n t
u b c l o n e s
3 days after C!ones
D
ayo
TPバsrimu!αriρnMOLT‑3 6 0 . 6
+ 7.8 7
.1
+2
.9 R O I 1 9 9
+3
.2 1 2 5
+9
.4R 0 2 1 0 6 + 9
.7 1 2 5 +
13
.4R 0 3 1 1 0 + 7
.7 1 0 6 + 1 4
.9 R04 1 1 8 + 1 5
.7 9 6
.7+ 7
.3 R 0 5 8 8 . 7 + 1 4
.47 0
.0+ 8
.7
PKC assavPKC a c t i v i t y was d e t e r m i n e d by m e a s u r i n g t h e i n c o r p o r a t i o n o f 3 Z p from i ̲3ZP‑ATP i n t o h i s t o n e HJ
，a s d e s c r i b e d (Kikkawa
et a!.、1 9 8 2 ) .The r e a c t i o n
.m i x t u r e
，i n a f i n a l volume o f 2 5 0 μ 1
，c o n s i s t e d o f 80μg ml
‑1 PS
，2 0 mM T r i
s(pH 7
.5 )
司0
.1 mM CaCl
z5 mM magnesium a c e t a t e
句2
.5 nmol
~，_32P‑ATP ( 1 0 0 c . p . m . pmol
‑1 ) 50μg 0
1'h i s t o n e H I and 5 0 μ I o f s a m p l e i n t h e a b s e n c e o r p r e s e n c e o f Iμgml
一l TPA. I n c u b a t i o n was c a r r i e d o u t f o r 5 min a t 3 0 C . The r e a c t i o n was t e r m i n a t e d by t h e a d d i t i o n o f 2 5
% tr i c h l o r o ‑ a c e t i c a c i d . The a c i d
‑prec i p i t a t e d m a t e r i a l s w e r e c o l l e c t e d on a m
巴mbrane f i l t e r and c o u n t e d f o r r a d i o a c t i v i t y
.PKC a c t i v i t y was d e t e r m i n e d b y s u b t r a c t i n g t h e a c t i v i t y measured i n t h e a b s e n c e o f TPA from t h a t measured i n i t s p r e s e n c e and e x p r e s s e d a s nmol o f 3
Zp t r a n s f e r r e d t o h i s t o n e Hl p e r min a t 3 0 C p e r 1 0
8c e l l s . P r o t e i n was e s t i m a t e d b y t h e method o f B r a d f o r d ( 1 9 7 6 )
.B o v i n e serum albumin was u s e d a s t h e s t a n d a r d
E
. ! f
ect4
TPA on川 bcellulardislribulion of PKCTo i n v e s t i g a t e t h c a c t i v a t i o n o f PKC
、t h ee f f e c t o f TPA on t h e s u b c c l l u
la r d i s t r i b u t i o n o f PKC i n MOL T ‑ 3 and t h e T P A ‑ r e s i s t a n t s u b c l o n e ROI was examined ( F i g u r e 3 ) . Most ( 9 8
‑9 9
%)o f t h e PKC a c t i v i t y was found i n t h e c y t o s o l and t h c r c was l i t t l e PKC a c t
ivit y i n t h e p a r t i c u l a t e f r a c t i o n
、10b o t h c e l l l i n e s
.The s
timula t i o n w i t h 1 6 nM TPA i n t h e p a r e n t a l MOL T ‑ 3 r e s u l t e d i n a 50% d e c r e a s e i n c y t o s o
ilc PKC a c t i v i t y w i t h i n 5 m
川、f o l l o w e db y a g r a d u a l d e c l i n e t o
10% o f t h c i n i t i a l I c v e l a t 6 0 min ( F i g u r e 3 a ) and a c o n c o m l t a n t
lOc r e a s e i n PKC a c t i v i t y i n t h e p a r t i c u l a t e f r a c t i o n o c c u r r e d ( F i g u r e 3 b ) . A s i m i l a r c h a n g e o f s u b ‑ c e l l u l a r PKC d i s t r i b u t i o n a l s o o c c u r r e d i n RO I . Thus
司t h e PKC a c t i v i t i e s i n t h e c y t o s o l and p a r t i c u l a t e f r a c t i o n c h a n g c d i n v e r s e l y
、i nb o t h c c l l l i n e s and i n a t i m e d e p e n d e n t manner
、i n d i c a t i n gt h a t t h e t r a n s J o c a t i o n o f PKC f r o m ' t h e c y t o s o
lt o t h e p a r t i c u l a t e f r a c t i o n was c a u s e d b y TPA TdT a c t i v i t y
:u n i t p c r 1 0
8c e l l
.sOne u n
il o f
e n z y m e a c t l v l t y
IflCO中o r a t e s i n 1 h I n m o l o f dGMP i n t o a c i d ‑ p r e c i p i t a b l e m a t e r i a l a t 3 7 C
，u s i n g 0
iIg o ( d A ) ，
2̲，
8a s a p r i m e
r.T h e v a l u e s
，a r e e x p r e s s e d a s mean
1:: S. e . o f t h r e e
se p a r a t e e x p e n π l e n t s
Elution projile ofり'fosolicPKC of MOLT‑3 and TPA‑ resislanl subclone ROI
C y t o s o l p r e p a r a t i o n s o f MOL T ‑ 3 w c r e f r a c t i o n a t e d on DEAE‑sepharose columns l l s i n g a l i n e a r g r a d i e n t and column f r a c t i o n s w e r e a s s a y e d f o r PKC a c t i v i t y
.Thc PKC a c t i v i t y e
¥ute d a t a c o n c e n t r a t i o n o f 0 . 0 6 0
.1 6 M w i t h a p e a k a t 0
.1 0 M Na C l ( F i g u r e 2 a ) . C y t o s o l p r e p a r a t i o n s o f RO 1 showed a n e ¥ u t i o n p r o f i l e o f PKC
、i na
si m i l a r f a s h i o n and w i t h a pcak a t 0
.1 0 M
r、~aCJ( F i g u r e 2 b )
.However
，t h c amount o f PKC a c t i v i t y i n RO 1 was 4 0
% of t h a L o f t h e p a r e n t a l MOLT‑3. The b a s e l i n e o f PKC a c t i v i t y i n ROI was l o w e r t h a n t h a t o f MOL T ‑ 3
，t h e r e b y i n d i c a L i n g t h a t PKC
inROI was n o t a l r e a d y a c t i v a t e d
.To d e t e r m i n e w h e t h c r PKC a c t i v i t i e s o f TPA
・r c s i s t a n ts u b c
lo n e s w c r c l e s s t h a n t h o s e o f
Results
Char町田lerisricsof TPA‑resistant subclones
The p r o l i f e r a t i o n o f MOL T ‑ 3 i n t h e p r e s e n c e o f 1 6 nM TPA was r e d u c e d w i t h 3 d a y s o f c u l t u r e
，a f t e r which t h e c e l l s grew s l o w l y up t o day 9
，w i t h a v i a b i l i t y e x c e e d i n g 90%
，a s d e t e r m i n e d b y t r y p a n b J u e dye e x c l u s i o n
.Thus
，t h e TPA was n o t t o x i c t o t h e s e MOLT‑3 c e l l s ( F i g u r e l a ) . The f i v e TPA‑
r c s i s t a n t s u b c l o n c s o f MOL T
・3o b t a i n e d i n m c t h y l c e l l u l o s e c o n l a i n i n g TPA grew e q u a l l y w e l l i n s u s p e n s i o n c u l t u r e s
、w i t h o r w i t h o u t 1 6 nM TPA ( F i g u r e 1 b )
.The form and s i z e
of t h e s e TPA
・r e s i s t a n ts u b c l o n e s d i d n o t d i f f e r from t h o s e o f t h c p a r e n l a J MOLT‑3. The r e s i s t a n c e o f t h o s c c J o n c s t o TPA wa
sn o t l o s t f o r up t o s e v e r a l monlhs
，e v e n i n c o n t i n u o u s c u l t u r e w i l h o u t TPA
.The p r e s e n c e o f TdT i s c h a r a c t e r i s t i c o f p r o t h y m o c y t e s
and i
sa b s e n t i n mature T ‑ I y m p h o c y t e s ( B o l l u m
，1 9 7 9 ) . The
Icv e l o f t h e e n z
yme o f MOL T ‑ 3 was r e d u c e d d r a m a t i c a l l y i n
t h e p r e s c n c e o f 16nM TPA
，r e a c h i n g a l e v e l o f 1 2
% of t h e
c o n t r o l c u l t u r c a t 3 d a y s
，w h e r e a s t h e l e v e l o f t h e enzymc o f
T P A ‑ r e s i s t a n t s u b c l o n e s r e m a i n e d h i g h a f t e r TPA
st i m u l a t i o n f o r 3 d a y s ( T a b l e
1).T h e s e r e s u l t s i n d i c a t e t h a t
t h e p a r e n t a l MOL T ‑ 3 r e a c h e s a more d i f f e r e n t i a t e d s t a t e
w h e r e a s t h e T P A ‑ r e s l s t a n t s u b c l o n e s r e m a i n immature i n t h e
p r e s e n c e o f TPA
18 Y. Y AMAUCHI el al.
presence of TPA、whereasthey were reduced in the sensitive parental M O L T‑3 cells in 3 days司 culturewith TPA. These results indicated that the TPA・rcsistantsubc10nes were not induced to differentiate by TPA and remained Immature
In previous work. we found that the amount of phorbol estcr binding to TPA‑resIsLant subc10nes from MOLT‑3 was aboul half thaL of lhe parental M O L T‑3、prcsumablydue to a low conccnlralion of rcccptors for phorbol eSLers as assaycd by Scatchard analysis (Mayumi et 01.、1988).Thus、 we conc1uded LhaL Lhe number of reccpLors for phorbol eSlerS played an Important role in the Indllction 0 '1differ‑ cntialion in M O L T‑3 cells by TPA. Several studIes have
;hown Lhat the receptors for phorbol estcrs are copurified with PKC (Ashendel ef 01.、1983、Niedelel al.、1983).The enzyme is now thought to be a major receptor for phorbol esters (Sando & YOllng司 1983;Sharkey白川、 1984)and also one of the major sIgnal mediators from the cell membrane to Lhc nucleus (Nishizuka 1984 Bcrridgc、1984).This prompted
lIS to investigate whelher PKC was relaled lo thc TPA‑ Induccd diffcrentIaLIon in MOLT‑3. Thc levels of PKC in lhe cytosol in five TPA‑resistant subclones were about half lhose in the parcntal MOLT‑3. A similar result is shown in Figure 2、whcre the baseline of PKC activity without TPA is also lower in the TPA‑resislant subc10ne RO 1 than in the parenlal M O L T‑3. These results may renect a decrease in number of phorbol ester receptors in TPA‑resistanl subc10nes of M O L T‑3. as already noted (Mayumi et al.. 1988).
Phorbol cstcrs with tumour‑promoting activity cause a translocalion of PKC from the cytosol to the membrane fraction in various cell systems (Kraft et al.. 1982; Kraft &
Anderson， 1983、Shojiet al.， 1987) including hllman mature T‑cells (Isakov et al、1987;Manger et a!.， 1987). In cell diffcrcntiation systems、howcve人therole of PKC is not well understood. [n the human promyelocytic leukaemia cell line H L・60 Vandenbarket α1. (1984) found that the TPA‑
induced differentiation was mediated by PKC whereas other investigators demonstraled that mediator(s) other than or in addition to the activalion of PKC may be required for the induction of diffcrentiation (Kreutter et al.， 1985). 1n conlrast、however、therole of PKC旧 TPA‑induceddiffer‑ cntiation in T‑Iymphocytes is less well understood. We found lhal a translocation of PKC occurred in both TPA・reslstant cells
References
ANDERSON守NL..GEMMELL、M.A叶 COUSSENS、P.M.MURAO，S. &
HUBERMAN， E. (1985). Specific protcin phosphorylation in human promyclocytic HL・60 leukemia cells susceptible or resistant to induction of cell differentiation by phorbol‑12‑ myristate‑13・acetate.Cαncer Res.， 45
，
4955.ASHENDEL， c.L.. STALLER， J.M. & BOUTWELL. R.K. (1983). Protein kinase activity associated with a phorbol ester receptor purifi巴d from mouse brain. Cαncer Res.， 43
，
4333.BALLESTER. R. & ROSEN、O.M.(1985). Fate of immuno‑
preclpitatable protein kinase C in G H J cells treated with phorbol 12‑myristate l3‑acetate. J. Biol. Chem.
，
260，
15194BERRIDGE， 'YU. (1984). InosiLol trisphosphale and diacylglycerol as second messengers. Biochem. J.， 220， 345
BOLLUM. F.J. (1979). Terminal deoxynucleotidyl transferase as a hemalopoietlc C巴1marke1 r. Blood， 54
，
1203BRADFORD. M.M. (1976). A rapid and sensiLive method for the quantitation of microgram qu辻nlilies of proLein utilizing the principle of prot巴in‑dyebmding. Anal. Biochem.， 72
，
248 DIAMOND， L.， 0・BRIEN.TG. & BAIRD、W M. (1980). TumorpromoLers and the mechanism of tumor promotion.ペdνCαncer Res.， 32， 1
HOMMA， Y， HENI吋ING‑CHUBB.C.B & HUBERMAN. E. (1986) TranslocaiLon of protein kinase C in human 1巴ukemiacells suscepLible or resistant to differentiaLion induced by phorbol 12・
myristaLe 13‑acetate. Proc. Narl Acad. Sci. US，ペ83，7316
was only 20% of that in the MOLT‑3 cells. The amount of cytosolic PKC and of its translocation may be a main factor associated with lhe induction of cell differentiation
These results differ from those shown by Homma ef (11. (1986)、inthat the level of cytosolic PKC in TPA‑resistant H L・60variant cells was as high as thal in TPA‑sensitive HL‑
60 cells and that translocalion of PKC to the membrane did not occur in the resistant cells. In contrasl、theresul ts obtained by Shoji er al. (1987) who used lhe acutc myclo・
blastic leukaemia cell line KG‑I， are close to our observations. They showed that cytosolic PKC aClivity in the TPA・resistantsubclone KG‑Ia was about one lhird of lhat in the parental TPA‑sensitive KG‑l cclJs， despite similar patlerns of translocation of PKC司 in bOlh lincs. These discrepancies may be related to differenl cell syslcms. With respect to the phorbol cster rcccptors， thcre is also a difference between M O L T‑3 and HL‑60. The down regulation of phorbol estcr reccplors by TPA was seen bOlh in TPA・resistantcells and in the parental M O L T‑3 (Mayumi el al門 1988) whereas it was not observcd in TPA‑resistanl cells from H L・60(Solanki el al.、1981).The down regulation of phorbol ester receptors was observed wilhin 15 min after stimulation with TPA (Mayumi el α1.， 1988). Thc prccisc relationship between the down regulalion of phorbol csler binding and PKC translocation is unclear、butboth cvcnt may be related
The role of PKC after the onset of cell differentiation is also unclear. Thcre was no detectable PKC activity in lhc cytosol and particulate fractions for up 10 120 h atter treatm巴nt with 16nM TPA， in both MOLT‑3 and ROI.
These results may reflect the continued translocation of PKC from the cytosol to the membrane and the degradation of membrane‑associated PKC (Ballester & Rosen 1985).
While this study provides no direct evidence that PKC translocation from the cytosol to the membrane fraction causes differentiation in MOLT‑3司 neverlheless、theexistence of differences in the amount of cytosolic PKC， the amount of PKC translocation and thc numbcr of phorbol cslcr receptors between TPA‑resistant subclones and Lhe parenlal M O L T‑3 cells do suggest that PKC plays an important role in the induction of differentiation by TPA in lhis T・
Iymphoblastic cell line M O L T‑3.
This work was supported by a Grant‑in‑Aid for Scientific Research from the Ministry of Education Science and Culture of Japan (no 62570291). We thank M. Ohara for pertinent commenls
ISAKOV， N.. MALL Y司 M.1.、SCHOLZ，W. & ALTMAN. A. (1987)， T‑
Iymphocyte activation: the role of protein kinase C and Ihc bifurcating inositol phospholipid signal Iransdluct!On palhway fmmunol. Rev.， 95， 89.
KAIBUCHI. K. .TAKAl， Y， SAWAMURA. M. . HOSHIJIMA， M .. FUJIKURA， T. & NlSHIZUKA. Y (1983). Synergistic functions of protein phosphorylation and calcium mobilization in platelet activation. J. Biol. Chem.， 258. 6701
KAJIKAWA. N.. KAIBUCHI， K.、MATSUBARA，T喝 KIKKAWA.U.， TAKAI. .Y. & NISHIZUKA. Y. (1983). A possiblte role of protein kinase C in signal‑induced Iysosomal enzyme release. 8iochem Biophys. Res. Commun.. 116. 743
KIKKAWA. U ..TAKAI， Y.， MTNAKUCHI， R、INOHARA，S， &
ISHIZUKA， Y. (1982). Calcium‑activated、 phospholipid dep巴ndent protein kinase from rat brain. 1. Biol. Chem.， 257
，
13341
KRAFT， AS， ANDERSON、W.B，COOPER. H し &SANDO. J.J. (1982) Decrease in cytosolic calciumjphospholipid‑dependent prot巴tn kinase activity following phorbol ester treatmcnt of EL4 thymoma cells. 1. Bio .lChem.， 257. 13193
KRAFT， A.S. & ANDERSON， W.B. (1983). Phorbol cstcrs incrcasc the amount of Ca2 +守 phospholipid‑depend巴nt protein kinase associated with plasma membrane. Nalure， 301， 621
KR
モ
UTTER司 D.、 CALDWELL. A.B. & MORfN， M.J. (1985). Dissociation of protein kinase C activation from phorbol este~induced maturation of HL‑60 leukemia cells. 1. Biol. Chem.， 260. 5979.
MALAISSE. W.J. LEBRUN. P. HERCHUELZ、 A.SENER. A. &
;¥1ALAfSSE‑LAGAE. F. (1983). Synergistic effcct of a tumor‑
promoting rhorbol ester and a hypoglycemic sulfonylurea upon insulin release. Elldocril1()I()~.\\ 113， 1870
MANGER. B ..WEfSS. A日 IMBODEN.J. LAfNG. T & STOBO. 1.D ( 1 9 8 7 ) T 11 E r o l e O
「
p r印ot臼tem凶引em川nk七m附 e C in 川 mby the T c印ellは川ln礼山l1genreceptor じOTηmplcx. Effects of 5幻刊山tii口IIIηlU凶laLion with soluble or i川mmob以ilized CD3 antibodics. 1. fmmul1ol.ー 139
，
2755.
MAYUM. IT ..NAGASAWA. K， HORIUCH .IT. & KUSABA. T (1988) Phorbol ester receptors and the induction of differentialion in the human T Iymphoblastic cell line MOLT‑3. Exp. Cell Biol. .56
，
12.
AGASAWA. K. & MAK， T W. (1980). Phorbol esters induce differ‑ entl辻llOnI日humanmalignant T Iymphoblasts. Proc. Nall Acad Sci. USA， 77， 2964.
NAGASAWA. K ..CHECHIK. B.E ..GELFAND. E.W. .SENGUPTA. S. LETARTE. M. & MAK. T.W. (198Ia). Modulation of human T‑cell differentlation ma rkers by 12‑0‑tetradecanoylphorbol‑13‑acetate Tln'mus， 3， 307
N'¥GASAWA. K ..HOWATSON. A. & MAK. T.W. (19816). Induction of human malignant T‑Iymphoblastic cell lines MOL T‑3 and Jurkat by 12‑0ぺetradecanoylphorbol‑13・acetate:biochemical、physical， and morphological characLcrization. J. Cell. Physiol守 109，181 NAGASAWA. K. & MAK， T.W. (1982). Induclion of differentiation in
human T‑Iymphoblastic Icukemia cell lines by 12‑0‑telra‑ decanoylphorbol 13・acetate (TPA): studies with monoclonal antibodies to T cells. Cell. Irnmunol ..71
，
396NIEDEL. J.E.. KUHN. L.J. & VANDENBARK、GR. (1983). Phorbol dicstcr receptor copurifies with protein kinase C. Proc. Nall A
，cad. Sci じSA，80. 36
NISHIZUKA. Y. (1984). The role of protein kinase C in cell surface ignal transduction and tumour promotion. ，Valure. 308， 693
PROTEIN KINASE C AND T‑L YMPHOBLAST 19
OKAMURA， S ..CRANE. F.， MESSNER. H.A. & MAK. T. w. (1978). Purification of terminal deoxynucleotidyltransferase by oligo・
nucleotide afiinity chromatography. J. Bio .lChem.， 253: 3765 PARKER. Pl， STABEL， S. & WATERFIELD守M.D.(1984). Puritication
to homogencity of protein kinase C from bovine brain‑identity with the phorbol ester receptor. EM BO 1.， 3守953.
ROZENGURT. E.. RODRIGUEZ‑PENA守A COOMBS句 M.& SINNETT‑ SMITH司 J.(1984). Diacylglycerol stimulatcs DNA synthcslS and cell division in mouse 3T3 cells: role of Ca1‑‑sensit(ve phospho‑
lipid‑dependent protein kinase. Proc. .Vall Acad. 50. U5A: 81， 5748，
SANDO. J.J. & YOUNG. M.C. (1983) ldcntification of high‑affinity phorbol estcr receptor in cylOsol of EL4 Ihymo~ma cell~
requirement for calcium、magnesJUm.and phospholipids. Proc. Nall Acad. 5c .iUSA， 80， 2642.
SHARKEY司 N.A. LEACH.K.L. & BLUMBERG. P刈 (1984)
c … 。
petitiveinhibition by出acylglycerolof specific phorbol ester binding. Proc. Nall Acad. 5ci. U5A， 81， 607SHOJ I.M. .GIRARD. PR. CHARP. P.A ..KOEFFLER. HP. VOGLER. W.R. & KUO. 1.F. (1987). Effects of phorbol ester on trans‑ location and down‑regulation of protein kinase C and phosphorylation of endogEnous proteins In human acute myeloid leukemia cell line KG‑l and its phorbol， ester‑resistant s~bline KG‑Ia. Cαncer Res.， 47， 6363
SHOYA
早
M.& TODARO、G，1.(1980). Specific high affinily cell membrane receptors for biologically active phorbol and i~genol esters. Nalure， 288，
451SOLANK. IV SLAGA， T.J. CALLAHAM. M. & HUBERMAN.巳 (1981). Down regulation of specific binding of [20‑JH)phorbol
12，13一dibulyrate and phorbol ester‑induced differentia'tion of human promyelocytic leukemia cells. Proc. Narl Acad. Sci， U5A， 78， 1722.
VANDENBARK、G.R，KUHN唱L.J.& NIEDEL， 1.E， (1984). Possible mechanism of phorbol diester司induced maturation of human promy巴locyticleukemia cells. Activation of prolein kinase C. J. Clin. Jnvesl" 73， 448.
