Vol. 155, No. 2, 1988

September 15, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 773-778

DIFFERENT SUSCEPTIBILITIES OF MELANOMA CELLS TO RETINOIC ACID-INDUCED CHANGES IN MELANOTIC EXPRESSION

Michael Edward , J ean A. Gold a n d Rona M. MaeKie

D e p a r t m e n t of Dermato logy , U n i v e r s i t y of Glasgow, A n d e r s o n Col lege, 58 D u m b a r t o n R d . , Glasgow, G l l 6NU, Scot land

Received June 27, 1988

Summary : The e f fec t of r e t i n o i c acid on m u r i n e B16 melanoma cell g r o w t h , t y r o s i n a s e a c t i v i t y a n d melan in s y n t h e s i s was i n v e s t i g a t e d . Ret ino ic acid i n h i b i t e d the g rowth of B16F1, B16F10 a nd B16BL6 melanoma ce l l s , b u t e n h a n c e d me lan in s y n t h e s i s on ly in the B16F1 ce l l s . The B16F10 a n d B16BL6 cells e x h i b i t e d r e t i no i e a c i d - i n d u c e d s u p p r e s s i o n of t y r o s i n a s e a c t i v i t y and melan in s y n t h e s i s , which was most a p p a r e n t i n the B16F10 cell v a r i a n t . For c o m p a r i s o n , Cloudman $91 melanoma cel ls p r o v e d to be p a r t i c u l a r l y s e n s i t i v e to r e t i no i c a c i d - i n d u c e d g rowth i n h i b i t i o n a n d s t im- u l a t i on of the e x p r e s s i o n of t h e i r melanot ie p h e n o t y p e . The se r e s u l t s s u g g e s t c o n s i d e r a b l e h e t e r o g e n e i t y in the B16 melanoma with r e s p e c t to t h e i r r e s p o n s e to r e t i no i c ac id . © 1988 Academic Press, Inc.

Re t ino ids e x e r t a wide r a n g e of b io logica l e f f ec t s on norma l a n d

t u m o u r cells i n c u l t u r e and in v ivo , the most i m p o r t a n t b e i n g the

modula t ion of cell g rowth and d i f f e r e n t i a t i o n ( 1 - 3 ) . However , t he u n d e r -

l y i n g mechan i sms w h e r e b y r e t i n o i d s ach ieve t h e i r b io logica l f u n c t i o n is

e s s e n t i a l l y u n k n o w n , b u t may be b y modif ica t ion of gene e x p r e s s i o n . The

r e c e n t l y d i s c o v e r e d human r e t i no i e acid r e c e p t o r , which b e l o n g s to the

family of n u c l e a r r e c e p t o r s (4 ) , is p r o b a b l y the t r a n s c r i p t i o n a l media tor

of at l eas t some of the biological e f fec t s of r e t i n o i d s . The r e s p o n s e of

s e v e r a l t u m o u r cell l i nes to r e t ino ic a c i d - i n d u c e d g rowth i n h i b i t i o n is

v a r i a b l e (5) a n d t h e r e can be v a r i a b i l i t y b e t w e e n cell l i ne s d e r i v e d from

the same t u m o u r (8) . Th i s u n d o u b t e d l y r e l a t e s to p h e n o t y p i c h e t e r o -

g e n e i t y of t u m o u r cell s u b p o p u l a t i o n s r e s u l t i n g from the e lonal e m e r g e n c e

of v a r i a n t s u b p o p u l a t i o n s d u r i n g solid p r i m a r y t u m o u r g r o w t h . Melanin

s y n t h e s i s has b e e n u s e d as a r e a d i l y m e a s u r a b l e i n d i c a t o r of melanoma

cell d i f f e r e n t i a t i o n , a n d r e t i no i c acid has b e e n shown to s t imula te melano-

genes i s while i n h i b i t i n g ce l lu l a r g rowth ( 7 , 9 ) , b u t in a d d i t i o n , t h e r e has

b e e n a r e p o r t of r e t ino ie a c i d - i n d u c e d s u p p r e s s i o n of me lan in s y n t h e s i s

(10).

Here we d e m o n s t r a t e v a r i a t i o n s in the r e s p o n s e to r e t i no i e ac id -

i n d u c e d c h a n g e s i n m e l a n o g e n e s i s b y B16 melanoma cell v a r i a n t s se lec ted

for t h e i r ab i l i t y to m e t a s t a s i s e and i n v a d e (11) , a n d u s e d for compar i son

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, lnc.

773 All rights of reproduction in any form reserved.

Vol. 155, No. 2, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

t he C loudman $91 melanoma cel l l ine which is k n o w n to be p a r t i c u l a r l y

s e n s i t i v e to r e t i n o i c a c i d - i n d u c e d g r o w t h i n h i b i t i o n a n d s t i m u l a t i o n of

m e l a n o g e n e s i s (12) .

MATERIALS AND METHODS

Cells: Cloudman $91 melanoma cells, Clone M3, were obtained from Flow Laboratories, Rickmansworth, England, while the B16F1, B16F10 and B16BL6 melanoma cells were kindly supplied by Dr. I.R. Hart, Imperial Cancer Research Fund Laboratories, London, England.

Cell Culture: Cell~ were seeded in 90mm Nunc petri dishes (BI6F1, F10 and BL6 cells at 10 ~ cells/dish; Cloudman $91 cells at 5 x 10 ~ cells/ dish), and incubated in Eagle's minimal essential medium supplemented with 10% foetal calf serum, 100u/ml penicillin and 100~g/ml streptomycin (Gibeo Ltd., Paisley, Scotland), with medium changes on days 3 and 6. For dose response studies, triplicate dishes were treated with retinoic acid (Sigma Chemical Co., Poole, England) in a range of concentrations up to 10-UM for 6 days, while those dishes used for time dependent respo_r~se to retinoic acid were treated with 10-6M (B16F1, F10 and BL6) or 10-'M (Cloudman $91) retinoic acid or without retinoic acid, and triplicate dishes taken for analysis daily from day 3. The cells were harvested with a. rubber policeman and a single cell suspension obtained by gentle pipetting in PBS, and 10% of the suspension taken for tyro- sinase estimation and cell counts, while the remainder was used for melanin estimation.

T y r o s i n a s e A s s a y : T h e me thod is e s s e n t i a l l y t h a t d e s c r i b e d b y Ha l aban et a l . (13) . B r i e f l y , t h e cel l s u s p e n s i o n was c e n t r i f u g e d at 400g fo r 3 rain a n d .the ce l l p e l l e t r e s u s p e n d e d in 0.2ml o f PBS c o n t a i n - i n g 1% N o n i d e t , v o r t e x e d , a n d c e n t r i f u g e d at 2000g fo r 5 min. To t h e s u p e r n a t ~ n t was a d d e d 0 .2ml of a4so lu t ion o f PBS c o n t a i n i n g L - d o p a (2 x 10- M), L - t y r o s i n e (4 x 10- M) a n d 2 .0~Ci o f [ 3 , 5 - 3 H ] t y r o s i n e (52 Ci /m mol; A m e r s h a m I n t e r n a t i o n a l p l c , A m e r s h a m , E n g l a n d ) . The m i x t u r e was i n c u b a t e d fo r l h at 37°C with o c c a s i o n a l s h a k i n g , a f t e r which t h e r e a c t i o n was t e r m i n a t e d b y the a d d i t i o n o f 0 .5ml of 10% TCA. The m i x t u r e was t h e n c e n t r i f u g e d at 2000g for 5 min, a n d t h e s u p e r - n a t a n t t r a n s f e r r e d to t u b e s c o n t a i n i n g a c t i v a t e d c h a r c o a l , m i x e d , a n d le f t fo r 30 rain. The m i x t u r e was a g a i n c e n t r i f u g e d at 2000g for 5 rain, and 0.3ml o f t h e s u p e r n a t a n t t a k e n a n d mixed with 5ml o f Ecosc in t s c i n t i l l a t i o n f l u id ( N a t i o n a l D i a g n o s t i c s , A y l e s b u r y , E n g l a n d ) a n d c o u n t e d .

Melanin A s s a y : Melanin c o n t e n t was m e a s u r e d b y t h e c o l o r i m e t r i c me thod d e s c r i b e d b y W h i t t a k e r (14) . Cell s u s p e n s i o n s were c e n t r i f u g e d (400g, 3 min) a n d t h e cel l p e l l e t s r e s u s p e n d e d in 0.5ml d i s t i l l e d w a t e r , a n d t h e n s u b j e c t e d to two c y c l e s of f r e e z e / t h a w i n g . P e r c h l o r i c a c id (0 .5ml , 1M) was t h e n a d d e d a n d t h e m i x t u r e k e p t on ice for 10 min b e f o r e c e n t r i f u g i n g (3000g, 5 min ) , a f t e r which t h e p e l l e t s we re e x - t r a c t e d twice each wi th 0.5M p e r c h l o r i c a c id a n d a 3:1 V / v m i x t u r e of e t h a n o l : e t h e r , a n d f i na l l y wi th e t h e r . Pe l l e t s were d r i e d , i ml o f 0.85M KOH a d d e d , a n d t u b e s h e a t e d to 100°C for 10 min, a f t e r which t h e a b s o r b a n c e was m e a s u r e d at 400nm.

RESULTS AND DISCUSSION

The dose response relationship of retinoic acid-induced growth

inhibition is shown in Figure I(A). The BI6F1 and Cloudman $91 cell

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181 15

× 12 "o

i A ~ '9,0 36 × 3(3 ~ 24

~ O

6

_ i ' i , i 0

Retinoic acid (M) C

i i i i i

-10 -9 -8 -7 -6 -5 10 10 10 10 10 10 Retinaic acid (M)

Retinoic acid (M)

Figure 1

Dose response relationship of retinoic acid-induced changes in growth inhibition (A), tyrosinase activity (B) and melanin synthesis (C) of BI6FI (O o), BI6FI0 (~------4), BI6BL6 ('- -') and Cloudman $91 (D D) melanoma cells. Data points are the mean of t r ipl icate cu l tures ± s tandard deviations.

l ines p r o v e d 1:o be p a r t i c u l a r l y s e n s i t i v e to r e t i n o i c a c i d , b e i n g i n h i b i t e d

b y 91.2% a n d 54.9% r e s p e c t i v e l y at i0-6M r e t i n o i c a c i d , while t h e B16BL6

a n d B16F10 ce l l l i ne s were more r e s i s t a n t to g r o w t h i n h i b i t i o n , b e i n g i n -

h i b i t e d b y 43.4% a n d 39.9% r e s p e c t i v e l y at 10-6M r e t i n o i c a c i d . T h e dose

r e s p o n s e r e l a t i o n s h i p s of r e t i n o i c a c i d - i n d u c e d c h a n g e s in t y r o s i n a s e

a c t i v i t y a n d me lan in s y n t h e s i s a r e shown in F i g u r e s I ( B ) a n d 1 ( C ) .

The B16F1 a n d Cl0udman $91 cel l l i ne s b o t h e x h i b i t e d r e t i n o i e a c i d - i n d u c e d

e n h a n c e m e n t of t y r o s i n a s e a c t i v i t y a n d melan in s y n t h e s i s , while t h e t y r o -

s i n a s e a c t i v i t y a n d me lan in s y n t h e s i s d e c r e a s e d wi th i n c r e a s i n g r e t i n o i c

ac id c o n c e n t r a t i o n in t h e B16BL6 a n d B16F10 cel l l i n e s . The B16BL6 cel l

l ine d i d h o w e v e r show a s l i g h t i n c r e a s e in t y r o s i n a s e a c t i v i t y a n d melan in

s y n t h e s i s a t v e r y low r e t i n o i c ac id c o n c e n t r a t i o n s . T h e t ime c o u r s e of

r e t i n o i c a c i d - i n d u c e d c h a n g e s a r e shown in F i g u r e s 2 ( g r o w t h ) , 3 ( t y r o -

s i n a s e ) a n d 4 (me lan in s y n t h e s i s ) . Here a g a i n t h e r e t i n o i c a c i d i n h i b i t e d

t h e g r o w t h s i g n i f i c a n t l y of t h e B16F1 a n d Cloudman $91 ce l l s whi le o n l y

s l i g h t l y r e d u c i n g t h e g r o w t h r a t e of t h e B16F10 a n d B16BL6 c e l l s . How-

e v e r , whi le r e t i n o i c ac id e n h a n c e d t h e t y r o s i n a s e a c t i v i t y a n d me lan in

s y n t h e s i s of t h e Cloudman $91 ce l l s , wh ich i n c r e a s e d g r a d u a l l y o v e r t h e

7 d a y i n c u b a t i o n p e r i o d , t h e B16F1 ce l l s s h o w e d a r e d u c e d t y r o s i n a s e

a c t i v i t y b u t e n h a n c e d melan in s y n t h e s i s in t h e p r e s e n c e of r e t i n o i c a c i d .

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201

× I~

"(3

201 A

16'

i T2

..,~ . . . . . . "~ 4

3 4 5 6 7 0

B

i 3 4 5 6 7

days in c u l t u r e

F i g u r e 2

Time c o u r s e o f g r o w t h i n h i b i t i o n b y r e t i n o i c a c i d on (A) B16F1 (o) a n d C l o u d m a n $91 ( o ) , a n d (B) B16FI0 (o) a n d B16BL6 (1) me_l~noma ce l l s g r o w n i n t h e p r e s e n % e ( . . . . ) o r a b s e n c e ( ) o f 10 M ( B 1 6 F 1 , F10 a n d BL6) o r 1 0 - ' M ( C l o u d m a n $91) r e t i n o i c a c i d . D a t a p o i n t s a r e t h e m e a n o f t r i p l i c a t e c u l t u r e s _+ s t a n d a r d d e v i a t i o n s .

Ret ino ic ac id r e d u c e d the t y r o s i n a s e a c t i v i t y and melan in s y n t h e s i s in

bo th the B16F10 and BI6BL6 cel ls and was most m a r k e d a f t e r 7 d a y s

i n c u b a t i o n , b y which time the ce l ls had become c o n f l u e n t . An i n t e r e s t i n g

f e a t u r e of t he t y r o s i n a s e a c t i v i t y of t he BI6F1 and B16BL6 ce l l s g r o w n in

the a b s e n c e o f r e t i n o i c ac id is the time d e p e n d e n t d e c r e a s e in a c t i v i t y ,

while melanin s y n t h e s i s i n c r e a s e s . The mechan i sms i n v o l v e d in t h e

modula t ion of m e l a n o g e n e s i s by r e t i n o i c acid a r e u n k n o w n , b u t may i n -

v o l v e the de n o v o s y n t h e s i s of t y r o s i n a s e , a c r i t i ca l r e g u l a t o r y c o n t r o l

po in t in m e l a n o g e n e s i s , or t he s y n t h e s i s of a t y r o s i n a s e i n h i b i t o r . In

a d d i t i o n , r e t i n o i c acid has b e e n shown to a f f ec t g l y c o s y l a t i o n (15) ,

t h e r e f o r e any c h a n g e s in t h e g l y c o s y l a t i o n of t y r o s i n a s e may a f f ec t i t s

a c t i v i t y or h a l f - l i f e . The ma t r ix on which t h e melanoma cel ls a re c u l -

2~ x

"Io ' 6

0

Figure 3

30 A

24

.t • .-J 12

3 4 5 6 7 C

B

/ / " ,.

days in cu l ture

Time course of tyrosinase activity of (A) B16FI (o) and Cloud- man $91 (a) and (B) B16FI0 ( . ) and B16BL6 (u) melanom~a cells grown in the presence ( . . . . ) or absence ( ) of 10-bM (B16FI, F10 and BL6) or 10-'M (Cloudman $91) retinoic acid. Data points are the mean of triplicate cultures + s tandard deviations.

776

Vol . 155, No. 2, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

10

--~ 8 o

~ 6 .~_

E

0 3 4 5 6 7

days in culture

Figure 4_

Time course of melanin synthesis by (A) B16F1 (o) and Cloud- man $91 (o) and (B) B16F10 ($) and B16BL6 (m) melanoma cells grown in the presence ( . . . . ) or absence ( ) of 10-UM (B16F1, F10 and BL6) or 10-°M (Cloudman $91) retinoic acid. Data points are the mean of triplicate cul tures + s tandard deviations.

t u r e d may also af fec t t he i r s t a t e of d i f f e r e n t i a t i o n a nd melan in s y n t h e s i s .

Kle inman et al. (16) have shown tha t melanoma cel ls g rown on a b a s e m e n t

membrane gel d i sp l ay a more e x t e n s i v e p i g m e n t a t i o n a n d m a r k e d l y d i f f e r -

en t cell morpho logy , a n d as r e t i no i c acid is k n o w n to affect the s y n t h e s i s

of e x t r a c e l l u l a r ma t r ix molecules such as f i b r o n e c t i n (17) , co l lagen (18)

and g l y c o s a m i n o g l y c a n s (19) , such modif ica t ion of the de pos i t e d ma t r ix

may c o n t r i b u t e to the o b s e r v e d c h a n g e s in morpho logy a nd m e l a n o g e n e s i s .

The i n t r a c e l l u l a r r e t i n o i d b i n d i n g p r o t e i n s (20) may also be i n v o l v e d in

the d i f f e r e n t i a l e f f ec t s of r e t i no i c acid b y a f f e c t i n g the t r a n s l o c a t i o n of

r e t ino ic acid to the n u c l e u s . However , i t i s u n c l e a r j u s t how melano-

g e n e s i s is s t imu la t ed in one cell l ine a nd i n h i b i t e d in a n o t h e r , b u t t hese

cell l ines may p r o v i d e an idea l sys tem for e x a m i n i n g the mechanisms

i n v o l v e d .

ACKNOWLEDGEMENT

This work was f u n d e d in p a r t b y the C a n c e r R e s e a r c h Campa ign .

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(1987) N a t u r e 330, 444-450. 5. Lo tan , R. (1979) C a n c e r Res . 39, 1014-1019.

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and Lerner , A.B. (1983) J. Cell Biol. 97, 480-488. 14. Whittaker, J . R . (1963) Dev. Biol. 8, 99-127. 15. Deluca, L.M. (1977) Vitam. Horm. 35, 1-57. 16. Kleinman, H .K . , McGarvey, M.L., Hassell, J . R . , Star , V .L . ,

Cannon, F . B . , Lawrie, G.W. and Martin, G.R. (1986) Biochemistry 25, 312-318.

17. Bolmer, S.D. and Wolf, G. (1983) Modulation and Mediation of Cancer by Vitamins, pp.204-210. Karger : Basel.

18. Hein, R . , Mensing, H. , Muller, P .K . , Braun-Falco , O. and Kreig, T. (1984) Br. J. Dermatol. 111, 37-44.

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