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Enhancement of spontaneous mammary tumorigenesis at advanced age in mice by temporary stimulation of mammary growth during youth
Life Sciences, Vol. 39, pp. 389-393 Printed in the U.S.A.
Pergamon Journals
ENHANCEMENT OF SPONTANEOUS MAMMARY TUMORIGENESIS AT ADVANCED AGE IN MICE BY TEMPORARY STIMULATION OF MAMMARY GROWTH DURING YOUTH H i r o s h i Nagasawa a n d M a s a h a r u I s h i d a E x p e r i m e n t a l Animal R e s e a r c h L a b o r a t o r y , Meiji U n i v e r s i t y , T a m a - k u , K a w a s a k i , K a n a g a w a 214, J a p a n (Received in final form May i, 1986)
Summary As a p o s s i b l e step to c l a r i f y t h e r e l a t i o n s h i p b e t w e e n mammary g l a n d g r o w t h d u r i n g y o u t h a n d mammary t u m o r i g e n e s i s at a d v a n c e d a g e , t h e l o n g - t e r m e f f e c t s of t h e t e m p o r a r y s t i m u l a t i o n of mammary g l a n d g r o w t h at y o u t h on s p o n t a n e o u s mammary t u m o r i g e n e s i s was s t u d i e d i n a h i g h mammary t u m o r s t r a i n of SHN v i r g i n mice. T h e y r e c e i v e d a s i n g l e p i t u i t a r y g r a f t u n d e r t h e k i d n e y c a p s u l e or a s u b c u t a n e o u s i m p l a n t a t i o n of p r o g e s t e r o n e for 60 d a y s b e t w e e n 30 a n d 90 d a y s of a g e . B o t h t r e a t m e n t s e n h a n c e d mammary g l a n d g r o w t h a n d t h e effect of p i t u i t a r y g r a f t i n g was h i g h e r . S p o n t a n e o u s mammary t u m o r i g e n e s i s p a r a l l e d mammary g l a n d c o n d i t i o n s at y o u t h . T h e r e s u l t s i n d i c a t e t h a t t h e s t i m u l a t i o n of mammary g l a n d g r o w t h d u r i n g y o u t h e n h a n c e s s p o n t a n e o u s mammary t u m o r i g e n e s i s at a d v a n c e d age in mice, which is q u i t e c o n t r a r y in r a t s , a n d t h e y e x p l a i n t h e c a u s e of t h e h i g h e r mammary t u m o r p o t e n t i a l i n b r e e d e r s t h a n i n v i r g i n mice. We p r e v i o u s l y f o u n d i n r a t s t h a t t h e t e m p o r a r y i n h i b i t i o n of mammary g l a n d g r o w t h r e s u l t e d i n a m a r k e d s u p p r e s s i o n of b o t h c a r c i n o g e n - i n d u c e d (1) a n d s p o n t a n e o u s ( 2 , 3 ) mammary t u m o r i g e n e s i s . On t h e c o n t r a r y , mammary t u m o r i g e n e s i s i n mice was e n h a n c e d b y t h e same t r e a t m e n t (4), which may p a r t l y b e d u e to t h e i n e f f e c t i v e n e s s of l o n g - t e r m p r o l a c t i n s u p p r e s s i o n b y CB-154 ( 5 ) . It was also r e p o r t e d t h a t t h e p r e - s t i m u l a t i o n of mammary g l a n d g r o w t h i n h i b i t e d c a r c i n o g e n - i n d u c e d mammary t u m o r i g e n e s i s i n r a t s ( 6 ) . H o w e v e r , no d a t a a r e a v a i l a b l e on t h e l o n g - t e r m e f f e c t s of t h e t e m p o r a r y s t i m u l a t i o n of t h e g r o w t h of mammary g l a n d s ( b o t h n o r m a l a n d p r e n e o p l a s t i c ) on s p o n t a n e o u s mammary t u m o r i g e n e s i s i n mice. T h i s p a p e r d e a l s with t h i s p r o b l e m . Materials a n d Methods A n i m a l s V i r g i n mice of a h i g h mammary t u m o r s t r a i n of SHN ( 7 , 8 ) b r e d in t h e a u t h o r s ' l a b o r a t o r y were u s e d . At 30 d a y s of a g e , t h e y were d i v i d e d i n t o 4 g r o u p s . G r o u p I was g r a f t e d with a s i n g l e i s o l o g o u s a n t e r i o r p i t u i t a r y u n d e r t h e k i d n e y c a p s u l e . G r o u p II was g i v e n a piece of t h e similar size of s u b m a x i l l a r y g l a n d at t h e same site a n d s e r v e d as t h e c o n t r o l of g r o u p I. G r o u p III r e c e i v e d t h e s u b c u t a n e o u s i m p l a n t a t i o n of p e l l e t (30 rag; 5 mm i n d i a m e t e r a n d i . 5 mm i n t h i c k n e s s ) of p r o g e s t e r o n e (Sigma Chem. C o . , St L o u i s , Mo). G r o u p IV was g i v e n t h e c h o l e s t e r o l p e l l e t of t h e same size as t h e c o n t r o l of g r o u p I I I . At 90 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Journals Ltd.
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d a y s of a g e o r 60 d a y s a f t e r t r e a t m e n t s , some mice in e a c h g r o u p w e r e k i l l e d b y d e c a p i t a t i o n a n d t h e b i l a t e r a l t h i r d t h o r a c i c mammary g l a n d s a n d t h e l e f t i n g u i n a l f at p a d s w e r e u s e d f o r t h e w h o l e m o u n t e v a l u a t i o n a n d DNA d e t e r m i n a t i o n , r e s p e c t i v e l y , as t h e i n d i c e s o f mammary g l a n d g r o w t h . T h e r e m a i n i n g animals w e r e u s e d fo r t h e e x a m i n a t i o n of s p o n t a n e o u s mammary t u m o r i g e n e s i s . K i d n e y s with p i t u i t a r y o r s u b m a x i l l a r y g l a n d g r a f t s w e r e r e m o v e d s u r g i c a l l y in g r o u p I o r II a n d all p e l l e t s w e r e also r e m o v e d in g r o u p s III a n d IV. In g r o u p I, t h e c o m p l e t e r e m o v a l of p i t u i t a r y g r a f t was c o n f i r m e d b y t h e r e c u r r e n c e of e s t r o u s cycles. T h r o u g h o u t t h e e x p e r i m e n t s , mice w e r e k e p t in p l a s t i c c a g e s (16 x 30 x 13 c m ) , 4-6 e a c h , m a i n t a i n e d in an animal room a i r - c o n d i t i o n e d (22-24 °C a n d 55-75 % r e l a t i v e h u m i d i t y ) a n d a r t i f i c i a l l y i l l u m i n a t e d (14 h o u r s of l i g h t from 5:00 AM to 7:00 PM) an d p r o v i d e d with a commercial d i e t ( C E - 2 : CLEA J a p a n I n c . , T o k y o , J a p a n ) a n d t ap w a t e r ad l i b i t u m . Normal a n d p r e n e o p l a s t i c mammary g l a n d g r o w t h in t h e w h o l e m o u n t p r e p a r a t i o n s T h e p r e p a r a t i o n s w e r e e x a m i n e d u n d e r t h e 10-fold m a g n i f i c a t i o n . T h e d e g r e e of e n d - b u d o r l o b u l o - a l v e o l a r f o r m a t i o n was r a t e d from 1 to 7 in i n c r e m e n t s of 1 ( 9 ) . T h e mean of t h e r a t i n g in t h e b i l a t e r a l g l a n d s was e m p l o y e d f o r t h e r e p r e s e n t a t i v e v a l u e f o r e a c h m o u s e . T h e n u m b e r of p r e n e o p l a s t i c mammary h y p e r p l a s t i c a l v e o l a r n o d u l e s (HAN) was c o u n t e d a n d t h e v a l u e f o r e a c h animal was e x p r e s s e d in t e r m s of t h e sum of t h e n u m b e r s in t h e b i l a t e r a l g l a n d s . Mammary DNA c o n t e n t acid and determined by the d e f a t t e d a n d d r i e d with h o t same as d e t a i l e d p r e v i o u s l y
Mammary DNA was e x t r a c t e d with h o t t r i e h l o r o a c e t i c diphenylamine reaction using left inguinal glands a l c o h o l - e t h e r . All p r o c e d u r e s w e r e e s s e n t i a l l y t h e (10).
Mammary t u m o r i g e n e s i s E a c h mouse of all g r o u p s was c h e c k e d f o r p a l p a b l e mammary t u m o r s e v e r y 7 d a y s u n t i l 3 w e e k s a f t e r t h e f i r s t t u m o r a p p e a r a n c e o r 12 m o n t h s of a g e w h e n all mice of g r o u p III d e v e l o p e d t u m o r s . A f t e r t h e size of t h e f i r s t t u m o r s , e x p r e s s e d as t h e g e o m e t r i c mean of t h e major two d i a m e t e r s , a n d t h e n u m b e r of t u m o r s w e r e r e c o r d e d , t h e t u m o r o u s mice w e r e k i l l e d . S t a t i s t i c s S t a t i s t i c a l s i g n i f i c a n c e of d i f f e r e n c e in mammary t u m o r i g e n e s i s was e v a l u a t e d b y t h e a n a l y s i s of v a r i a n c e ( 1 1 ) . B y t h i s m e t h o d , t h e d i f f e r e n c e between g r o u p s could be checked c o n s i d e r i n g simultaneously both the incidence a n d t h e o n s e t a g e of mammary t u m o r s . T h e d i f f e r e n c e s of o t h e r p a r a m e t e r s w e r e e v a l u a t e d b y D u n c a n ' s multiple r a n g e t e s t . All d a t a in g r o u p s II a n d IV w e r e p o o l e d as t h e c o n t r o l v a l u e s in R e s u l t s e c t i o n , s i n c e no d i f f e r e n c e was s e e n b e t w e e n g r o u p s in a n y p a r a m e t e r . Results T h e r e s u l t s of n o r m a l mammary g l a n d g r o w t h a n d t h e f o r m a t i o n of HAN on 60 d a y s a f t e r t h e t r e a t m e n t s a r e s h o w n in T a b l e 1. Mammary r a t i n g as t h e i n d e x of t h e g r o w t h of e n d - b u d or l o b u l o - a l v e o l a r s y s t e m was s i g n i f i c a n t l y h i g h e r in g r o u p I t h a n in t h e o t h e r g r o u p s . G r o u p III was also s i g n i f i c a n t l y h i g h e r t h a n g r o u p II+IV ( c o n t r o l ) in t h e r a t i n g . Mammary DNA c o n t e n t d i f f e r e d l i t t l e b e t w e e n t h e two e x p e r i m e n t a l g r o u p s , while t h e y w e r e h i g h e r t h a n t h e c o n t r o l . B o t h t h e p e r c e n t of mice with HAN a n d t h e n u m b e r of HAN p e r HAN b e a r i n g mouse w e r e s i g n i f i c a n t l y h i g h e r in g r o u p s I a n d III t h a n in g r o u p II+IV ( c o n t r o l ) . G r o u p I was also h i g h e r t h a n g r o u p III in t h e n u m b e r of HAN. C u m u l a t i v e mammary t u m o r i n c i d e n c e in e a c h g r o u p is i l l u s t r a t e d in F i g u r e 1.
Vol. 39, No. 5, 1986
Mammary Gland Growth and Tumorigenesis
TABLE
391
1
Normal a n d p r e n e o p l a s t i c mammary g l a n d g r o w t h i n e a c h g r o u p 60 d a y s a f t e r t r e a t m e n t s Group and treatment a
No. of mice
Mammary rating
Mammary DNA P e r c e n t of No. of HAN c o n t e n t (~g) mice with p e r HAN HAN b bearing mouse
I.
1 A P
11
4.3 + 0 . 3 c*
323 *_ 19"
100 (11)*
3.0 + 0 . 6 "
II.
Progesterone
12
3.5 + 0 . 1 5
319 +38*
75 (9)*
1.3+0.45
13
2.6 + 0.1 t
196 + 135
0 (0)$
II+IV.
Control
a G r o u p s I a n d II r e c e i v e d a s i n g l e i s o l o g o u s a n t e r i o r p i t u i t a r y (lAP) and a submaxillary gland grafts under the kidney capsule, r e s p e c t i v e l y . G r o u p III or IV r e c e i v e d a s u b c u t a n e o u s i m p l a n t a t i o n of p r o g e s t e r o n e o r c h o l e s t e r o l . All t r e a t m e n t s were g i v e n f o r 60 d a y s b e t w e e n 30 a n d 90 d a y s of a g e . V a l u e s i n g r o u p s II a n d III were pooled as t h e c o n t r o l . b p r e c a n c e r o u s mammary h y p e r p l a s t i c a l v e o l a r n o d u l e s . CMeans -+ SEM. Values with t h e d i f f e r e n t s u p e r s c r i p t s were s i g n i f i c a n t l y d i f f e r e n t from each o t h e r at P< 0.05 o r 0.01. Mammary t u m o r i n c i d e n c e i n c r e a s e d with H o w e v e r , t h e r a t e of i n c r e a s e was m u c h two g r o u p s a n d t h e d i f f e r e n c e s b e t w e e n were s t a t i s t i c a l l y s i g n i f i c a n t . G r o u p III mammary t u m o r i g e n e s i s .
t h e a d v a n c e of age i n e a c h g r o u p . more m a r k e d i n g r o u p I t h a n i n t h e o t h e r g r o u p I a n d g r o u p III o r II+IV ( c o n t r o l ) was also h i g h e r t h a n t h e c o n t r o l i n
At a u t o p s y 3 w e e k s a f t e r t h e f i r s t t u m o r a p p e a r a n c e , t h e r e was l i t t l e d i f f e r e n c e b e t w e e n g r o u p s i n e i t h e r t h e g r o w t h r a t e of t u m o r s or t h e n u m b e r of t u m o r s p e r t u m o r b e a r i n g mouse ( d a t a n o t s h o w n ) . T h r e e o u t of 69 mice (4.3%) died i n t h e c o n t r o l g r o u p b e f o r e a n y t u m o r s were e v i d e n t , while all a n i m a l s i n t h e e x p e r i m e n t a l g r o u p s u r v i v e d u n t i l t h e e n d of t h e e x p e r i m e n t (12 m o n t h s of a g e ) . U n i l a t e r a l k i d n e y r e m o v a l showed n o d e l e t e r i o u s e f f e c t s i n g r o u p s I a n d I I . Discussion T h i s s t u d y shows t h a t t h e t e m p o r a r y s t i m u l a t i o n of mammary g l a n d g r o w t h d u r i n g y o u t h e n h a n c e d s p o n t a n e o u s mammary t u m o r i g e n e s i s i n mice, while t h e same t r e a t m e n t s s u p p r e s s e d c a r c i n o g e n - i n d u c e d mammary t u m o r i g e n e s i s i n r a t s ( 6 ) . This d i s c r e p a n c y between species can be explained b y the species-difference in t h e f r e q u e n c y of mammary cell d i v i s i o n o r t h e r a t e of mammary DNA s y n t h e s i s , which is a l i m i t i n g f a c t o r for mammary t u m o r i g e n e s i s ; if t h e r a t e is h i g h at t h e time w h e n c a r c i n o g e n i c a g e n t s act o n t h e g l a n d s , mammary t u m o r i g e n e s i s is e n h a n c e d a n d vice v e r s a (12). T h e r a t e of DNA s y n t h e s i s of mammary g l a n d s differentiated by p i t u i t a r y g r a f t i n g or p r e g n a n c y decreases in rats (13-15), b u t it e l e v a t e s i n mice a s s o c i a t e d with t h e i n c r e a s e d DNA c o n t e n t ( 1 5 - 1 7 ) . Both a n i m a l s a n d h u m a n s , i n c l u d i n g mice u s e d i n t h i s e x p e r i m e n t , a r e e x p o s e d
392
Mammary Gland Growth and Tumorigenesis
Vol.
39. No. 5, 1986
100I.
I A P
l~
III.
U
II + I V .
4
5
(30}
Progesterone
(29J
C o n t r o l (69)
LU
~9 Z LU a J ~9 Z f~
o 50
I< <
f'
6
i
iiii 8
7
A
G
E
9
10
11
12
(months)
FIGURE 1 C u m u l a t i v e i n c i d e n c e of mammary t u m o r s i n each g r o u p . See T a b l e 1 for d e t a i l s of t r e a t m e n t s . I n i t i a l n u m b e r of mice e x a m i n e d is i n t h e p a r e n t h e s e s . S t a t i s t i c a l s i g n i f i c a n c e of d i f f e r e n c e b e t w e e n g r o u p s i n mammary t u m o r i g e n e s i s w a s e v a l u a t e d b y t h e a n a l y s i s of v a r i a n c e . I
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Mammary Gland Growth and Tumorigenesis
393
References 1. 2. 3. 4. 5.
6. 7. 8'. 9. i0. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
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"
Pergamon Journals
ENHANCEMENT OF SPONTANEOUS MAMMARY TUMORIGENESIS AT ADVANCED AGE IN MICE BY TEMPORARY STIMULATION OF MAMMARY GROWTH DURING YOUTH H i r o s h i Nagasawa a n d M a s a h a r u I s h i d a E x p e r i m e n t a l Animal R e s e a r c h L a b o r a t o r y , Meiji U n i v e r s i t y , T a m a - k u , K a w a s a k i , K a n a g a w a 214, J a p a n (Received in final form May i, 1986)
Summary As a p o s s i b l e step to c l a r i f y t h e r e l a t i o n s h i p b e t w e e n mammary g l a n d g r o w t h d u r i n g y o u t h a n d mammary t u m o r i g e n e s i s at a d v a n c e d a g e , t h e l o n g - t e r m e f f e c t s of t h e t e m p o r a r y s t i m u l a t i o n of mammary g l a n d g r o w t h at y o u t h on s p o n t a n e o u s mammary t u m o r i g e n e s i s was s t u d i e d i n a h i g h mammary t u m o r s t r a i n of SHN v i r g i n mice. T h e y r e c e i v e d a s i n g l e p i t u i t a r y g r a f t u n d e r t h e k i d n e y c a p s u l e or a s u b c u t a n e o u s i m p l a n t a t i o n of p r o g e s t e r o n e for 60 d a y s b e t w e e n 30 a n d 90 d a y s of a g e . B o t h t r e a t m e n t s e n h a n c e d mammary g l a n d g r o w t h a n d t h e effect of p i t u i t a r y g r a f t i n g was h i g h e r . S p o n t a n e o u s mammary t u m o r i g e n e s i s p a r a l l e d mammary g l a n d c o n d i t i o n s at y o u t h . T h e r e s u l t s i n d i c a t e t h a t t h e s t i m u l a t i o n of mammary g l a n d g r o w t h d u r i n g y o u t h e n h a n c e s s p o n t a n e o u s mammary t u m o r i g e n e s i s at a d v a n c e d age in mice, which is q u i t e c o n t r a r y in r a t s , a n d t h e y e x p l a i n t h e c a u s e of t h e h i g h e r mammary t u m o r p o t e n t i a l i n b r e e d e r s t h a n i n v i r g i n mice. We p r e v i o u s l y f o u n d i n r a t s t h a t t h e t e m p o r a r y i n h i b i t i o n of mammary g l a n d g r o w t h r e s u l t e d i n a m a r k e d s u p p r e s s i o n of b o t h c a r c i n o g e n - i n d u c e d (1) a n d s p o n t a n e o u s ( 2 , 3 ) mammary t u m o r i g e n e s i s . On t h e c o n t r a r y , mammary t u m o r i g e n e s i s i n mice was e n h a n c e d b y t h e same t r e a t m e n t (4), which may p a r t l y b e d u e to t h e i n e f f e c t i v e n e s s of l o n g - t e r m p r o l a c t i n s u p p r e s s i o n b y CB-154 ( 5 ) . It was also r e p o r t e d t h a t t h e p r e - s t i m u l a t i o n of mammary g l a n d g r o w t h i n h i b i t e d c a r c i n o g e n - i n d u c e d mammary t u m o r i g e n e s i s i n r a t s ( 6 ) . H o w e v e r , no d a t a a r e a v a i l a b l e on t h e l o n g - t e r m e f f e c t s of t h e t e m p o r a r y s t i m u l a t i o n of t h e g r o w t h of mammary g l a n d s ( b o t h n o r m a l a n d p r e n e o p l a s t i c ) on s p o n t a n e o u s mammary t u m o r i g e n e s i s i n mice. T h i s p a p e r d e a l s with t h i s p r o b l e m . Materials a n d Methods A n i m a l s V i r g i n mice of a h i g h mammary t u m o r s t r a i n of SHN ( 7 , 8 ) b r e d in t h e a u t h o r s ' l a b o r a t o r y were u s e d . At 30 d a y s of a g e , t h e y were d i v i d e d i n t o 4 g r o u p s . G r o u p I was g r a f t e d with a s i n g l e i s o l o g o u s a n t e r i o r p i t u i t a r y u n d e r t h e k i d n e y c a p s u l e . G r o u p II was g i v e n a piece of t h e similar size of s u b m a x i l l a r y g l a n d at t h e same site a n d s e r v e d as t h e c o n t r o l of g r o u p I. G r o u p III r e c e i v e d t h e s u b c u t a n e o u s i m p l a n t a t i o n of p e l l e t (30 rag; 5 mm i n d i a m e t e r a n d i . 5 mm i n t h i c k n e s s ) of p r o g e s t e r o n e (Sigma Chem. C o . , St L o u i s , Mo). G r o u p IV was g i v e n t h e c h o l e s t e r o l p e l l e t of t h e same size as t h e c o n t r o l of g r o u p I I I . At 90 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Journals Ltd.
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d a y s of a g e o r 60 d a y s a f t e r t r e a t m e n t s , some mice in e a c h g r o u p w e r e k i l l e d b y d e c a p i t a t i o n a n d t h e b i l a t e r a l t h i r d t h o r a c i c mammary g l a n d s a n d t h e l e f t i n g u i n a l f at p a d s w e r e u s e d f o r t h e w h o l e m o u n t e v a l u a t i o n a n d DNA d e t e r m i n a t i o n , r e s p e c t i v e l y , as t h e i n d i c e s o f mammary g l a n d g r o w t h . T h e r e m a i n i n g animals w e r e u s e d fo r t h e e x a m i n a t i o n of s p o n t a n e o u s mammary t u m o r i g e n e s i s . K i d n e y s with p i t u i t a r y o r s u b m a x i l l a r y g l a n d g r a f t s w e r e r e m o v e d s u r g i c a l l y in g r o u p I o r II a n d all p e l l e t s w e r e also r e m o v e d in g r o u p s III a n d IV. In g r o u p I, t h e c o m p l e t e r e m o v a l of p i t u i t a r y g r a f t was c o n f i r m e d b y t h e r e c u r r e n c e of e s t r o u s cycles. T h r o u g h o u t t h e e x p e r i m e n t s , mice w e r e k e p t in p l a s t i c c a g e s (16 x 30 x 13 c m ) , 4-6 e a c h , m a i n t a i n e d in an animal room a i r - c o n d i t i o n e d (22-24 °C a n d 55-75 % r e l a t i v e h u m i d i t y ) a n d a r t i f i c i a l l y i l l u m i n a t e d (14 h o u r s of l i g h t from 5:00 AM to 7:00 PM) an d p r o v i d e d with a commercial d i e t ( C E - 2 : CLEA J a p a n I n c . , T o k y o , J a p a n ) a n d t ap w a t e r ad l i b i t u m . Normal a n d p r e n e o p l a s t i c mammary g l a n d g r o w t h in t h e w h o l e m o u n t p r e p a r a t i o n s T h e p r e p a r a t i o n s w e r e e x a m i n e d u n d e r t h e 10-fold m a g n i f i c a t i o n . T h e d e g r e e of e n d - b u d o r l o b u l o - a l v e o l a r f o r m a t i o n was r a t e d from 1 to 7 in i n c r e m e n t s of 1 ( 9 ) . T h e mean of t h e r a t i n g in t h e b i l a t e r a l g l a n d s was e m p l o y e d f o r t h e r e p r e s e n t a t i v e v a l u e f o r e a c h m o u s e . T h e n u m b e r of p r e n e o p l a s t i c mammary h y p e r p l a s t i c a l v e o l a r n o d u l e s (HAN) was c o u n t e d a n d t h e v a l u e f o r e a c h animal was e x p r e s s e d in t e r m s of t h e sum of t h e n u m b e r s in t h e b i l a t e r a l g l a n d s . Mammary DNA c o n t e n t acid and determined by the d e f a t t e d a n d d r i e d with h o t same as d e t a i l e d p r e v i o u s l y
Mammary DNA was e x t r a c t e d with h o t t r i e h l o r o a c e t i c diphenylamine reaction using left inguinal glands a l c o h o l - e t h e r . All p r o c e d u r e s w e r e e s s e n t i a l l y t h e (10).
Mammary t u m o r i g e n e s i s E a c h mouse of all g r o u p s was c h e c k e d f o r p a l p a b l e mammary t u m o r s e v e r y 7 d a y s u n t i l 3 w e e k s a f t e r t h e f i r s t t u m o r a p p e a r a n c e o r 12 m o n t h s of a g e w h e n all mice of g r o u p III d e v e l o p e d t u m o r s . A f t e r t h e size of t h e f i r s t t u m o r s , e x p r e s s e d as t h e g e o m e t r i c mean of t h e major two d i a m e t e r s , a n d t h e n u m b e r of t u m o r s w e r e r e c o r d e d , t h e t u m o r o u s mice w e r e k i l l e d . S t a t i s t i c s S t a t i s t i c a l s i g n i f i c a n c e of d i f f e r e n c e in mammary t u m o r i g e n e s i s was e v a l u a t e d b y t h e a n a l y s i s of v a r i a n c e ( 1 1 ) . B y t h i s m e t h o d , t h e d i f f e r e n c e between g r o u p s could be checked c o n s i d e r i n g simultaneously both the incidence a n d t h e o n s e t a g e of mammary t u m o r s . T h e d i f f e r e n c e s of o t h e r p a r a m e t e r s w e r e e v a l u a t e d b y D u n c a n ' s multiple r a n g e t e s t . All d a t a in g r o u p s II a n d IV w e r e p o o l e d as t h e c o n t r o l v a l u e s in R e s u l t s e c t i o n , s i n c e no d i f f e r e n c e was s e e n b e t w e e n g r o u p s in a n y p a r a m e t e r . Results T h e r e s u l t s of n o r m a l mammary g l a n d g r o w t h a n d t h e f o r m a t i o n of HAN on 60 d a y s a f t e r t h e t r e a t m e n t s a r e s h o w n in T a b l e 1. Mammary r a t i n g as t h e i n d e x of t h e g r o w t h of e n d - b u d or l o b u l o - a l v e o l a r s y s t e m was s i g n i f i c a n t l y h i g h e r in g r o u p I t h a n in t h e o t h e r g r o u p s . G r o u p III was also s i g n i f i c a n t l y h i g h e r t h a n g r o u p II+IV ( c o n t r o l ) in t h e r a t i n g . Mammary DNA c o n t e n t d i f f e r e d l i t t l e b e t w e e n t h e two e x p e r i m e n t a l g r o u p s , while t h e y w e r e h i g h e r t h a n t h e c o n t r o l . B o t h t h e p e r c e n t of mice with HAN a n d t h e n u m b e r of HAN p e r HAN b e a r i n g mouse w e r e s i g n i f i c a n t l y h i g h e r in g r o u p s I a n d III t h a n in g r o u p II+IV ( c o n t r o l ) . G r o u p I was also h i g h e r t h a n g r o u p III in t h e n u m b e r of HAN. C u m u l a t i v e mammary t u m o r i n c i d e n c e in e a c h g r o u p is i l l u s t r a t e d in F i g u r e 1.
Vol. 39, No. 5, 1986
Mammary Gland Growth and Tumorigenesis
TABLE
391
1
Normal a n d p r e n e o p l a s t i c mammary g l a n d g r o w t h i n e a c h g r o u p 60 d a y s a f t e r t r e a t m e n t s Group and treatment a
No. of mice
Mammary rating
Mammary DNA P e r c e n t of No. of HAN c o n t e n t (~g) mice with p e r HAN HAN b bearing mouse
I.
1 A P
11
4.3 + 0 . 3 c*
323 *_ 19"
100 (11)*
3.0 + 0 . 6 "
II.
Progesterone
12
3.5 + 0 . 1 5
319 +38*
75 (9)*
1.3+0.45
13
2.6 + 0.1 t
196 + 135
0 (0)$
II+IV.
Control
a G r o u p s I a n d II r e c e i v e d a s i n g l e i s o l o g o u s a n t e r i o r p i t u i t a r y (lAP) and a submaxillary gland grafts under the kidney capsule, r e s p e c t i v e l y . G r o u p III or IV r e c e i v e d a s u b c u t a n e o u s i m p l a n t a t i o n of p r o g e s t e r o n e o r c h o l e s t e r o l . All t r e a t m e n t s were g i v e n f o r 60 d a y s b e t w e e n 30 a n d 90 d a y s of a g e . V a l u e s i n g r o u p s II a n d III were pooled as t h e c o n t r o l . b p r e c a n c e r o u s mammary h y p e r p l a s t i c a l v e o l a r n o d u l e s . CMeans -+ SEM. Values with t h e d i f f e r e n t s u p e r s c r i p t s were s i g n i f i c a n t l y d i f f e r e n t from each o t h e r at P< 0.05 o r 0.01. Mammary t u m o r i n c i d e n c e i n c r e a s e d with H o w e v e r , t h e r a t e of i n c r e a s e was m u c h two g r o u p s a n d t h e d i f f e r e n c e s b e t w e e n were s t a t i s t i c a l l y s i g n i f i c a n t . G r o u p III mammary t u m o r i g e n e s i s .
t h e a d v a n c e of age i n e a c h g r o u p . more m a r k e d i n g r o u p I t h a n i n t h e o t h e r g r o u p I a n d g r o u p III o r II+IV ( c o n t r o l ) was also h i g h e r t h a n t h e c o n t r o l i n
At a u t o p s y 3 w e e k s a f t e r t h e f i r s t t u m o r a p p e a r a n c e , t h e r e was l i t t l e d i f f e r e n c e b e t w e e n g r o u p s i n e i t h e r t h e g r o w t h r a t e of t u m o r s or t h e n u m b e r of t u m o r s p e r t u m o r b e a r i n g mouse ( d a t a n o t s h o w n ) . T h r e e o u t of 69 mice (4.3%) died i n t h e c o n t r o l g r o u p b e f o r e a n y t u m o r s were e v i d e n t , while all a n i m a l s i n t h e e x p e r i m e n t a l g r o u p s u r v i v e d u n t i l t h e e n d of t h e e x p e r i m e n t (12 m o n t h s of a g e ) . U n i l a t e r a l k i d n e y r e m o v a l showed n o d e l e t e r i o u s e f f e c t s i n g r o u p s I a n d I I . Discussion T h i s s t u d y shows t h a t t h e t e m p o r a r y s t i m u l a t i o n of mammary g l a n d g r o w t h d u r i n g y o u t h e n h a n c e d s p o n t a n e o u s mammary t u m o r i g e n e s i s i n mice, while t h e same t r e a t m e n t s s u p p r e s s e d c a r c i n o g e n - i n d u c e d mammary t u m o r i g e n e s i s i n r a t s ( 6 ) . This d i s c r e p a n c y between species can be explained b y the species-difference in t h e f r e q u e n c y of mammary cell d i v i s i o n o r t h e r a t e of mammary DNA s y n t h e s i s , which is a l i m i t i n g f a c t o r for mammary t u m o r i g e n e s i s ; if t h e r a t e is h i g h at t h e time w h e n c a r c i n o g e n i c a g e n t s act o n t h e g l a n d s , mammary t u m o r i g e n e s i s is e n h a n c e d a n d vice v e r s a (12). T h e r a t e of DNA s y n t h e s i s of mammary g l a n d s differentiated by p i t u i t a r y g r a f t i n g or p r e g n a n c y decreases in rats (13-15), b u t it e l e v a t e s i n mice a s s o c i a t e d with t h e i n c r e a s e d DNA c o n t e n t ( 1 5 - 1 7 ) . Both a n i m a l s a n d h u m a n s , i n c l u d i n g mice u s e d i n t h i s e x p e r i m e n t , a r e e x p o s e d
392
Mammary Gland Growth and Tumorigenesis
Vol.
39. No. 5, 1986
100I.
I A P
l~
III.
U
II + I V .
4
5
(30}
Progesterone
(29J
C o n t r o l (69)
LU
~9 Z LU a J ~9 Z f~
o 50
I< <
f'
6
i
iiii 8
7
A
G
E
9
10
11
12
(months)
FIGURE 1 C u m u l a t i v e i n c i d e n c e of mammary t u m o r s i n each g r o u p . See T a b l e 1 for d e t a i l s of t r e a t m e n t s . I n i t i a l n u m b e r of mice e x a m i n e d is i n t h e p a r e n t h e s e s . S t a t i s t i c a l s i g n i f i c a n c e of d i f f e r e n c e b e t w e e n g r o u p s i n mammary t u m o r i g e n e s i s w a s e v a l u a t e d b y t h e a n a l y s i s of v a r i a n c e . I
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Mammary Gland Growth and Tumorigenesis
393
References 1. 2. 3. 4. 5.
6. 7. 8'. 9. i0. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
H. NAGASAWA, R° YANAI a n d H. T A N I G U C H I , C a n c e r R e s . 36. 2223-2226 (1976) H. NAGASAWA a n d S. MORII, C a n c e r R e s . 4 1 : 1 9 3 5 - 1 9 3 7 (1981) H. NAGASAWA a n d S. MORII, A c t a e n d o c r i n o l . 1 0 1 : 5 1 - 5 5 (1982) H. NAGASAWA a n d T . MORI, P r o e . Soc. E x p . Biol. Med. 1 7 1 : 1 6 4 - 1 6 7 (1982) T . MORI, K. N I K I , H. NAMIKI a n d S. S A T O H , A c t a e n d o c r i n o l . 1 0 9 : 2 0 8 - 2 1 3 (1985) C.W. WELSCH, J.A. CLEMENS and J. MEITES, J. Natl. Cancer Inst. 41: 465471 (1968) H. NAGASAWA, R. YANAI, H. TANIGUCHI, R. TOKUZEN and W. NAKAHARA, J. Natl. Cancer Inst. 57:425-430 (1976) J. STAATS, Cancer Re--s. 45:945-977 (1985) H. NAGASAWA, R. YANAITY. NAKAJIMA, H. NAMIKI, S. KIKUYAMAand K. SHIOTA, Europ. J. Cancer 16:473-480 (1980) H. NAGASAWAand R. YANAT, J. Natl. Cancer Inst. 52:1219-1222 (1974) C.W. SNEDECOR and W.G. COCHRAN, Statistical Methods, 7th ed., Iowa State Univ. Press, Ames (1980) H. NAGASAWA, IRCS Med. Sci, 5:405-408 (1977) H. NAGASAWA and R. YANAI, J_Natl. Cancer Inst. 52:609-610 (1974) H. NAGASAWAand H. VORHERR, Amer. J. Obstet. Gynecol. 127:590-593 (1977) H. NAGASAWA, Med. Hypoth. 5:499-510 (1979) H. NAGASAWAand R. YANAI,J. Endocr. 70:389-395 (1976) H. NAGASAWA, R. YANAIand H. TANIGUCHI, Europ. J. Cancer 12: 10171019 (1976) H. VORHERR, Breast Cancer. Urban &Schwarzenberg, Baltimore (1980) M. ALDERSON. Br. Med. J. 283:9-10 (1981) K.L. WOODS, S.R, SMITH and J.M. MORRISON, Br. Med. J. 281:419-421 (1980) H. NAGASAWA, R. YANAI, H. IWAHASHI, M. FUJIMOTO and K. KURETANI, Endocrinol. Japon. 14:351-356 (1967) ,
21.
"