15. 1. 1978
Specialia
fibrillary acidic p r o t e i n a n d i n t e r p r e t e d as a s t r o c y t e s s. H o w e v e r , t h e d e m o n s t r a t i o n of capillary origin a n d t h e p r e s e n c e of m a r k e r e n z y m e , alkaline p h o s p h a t a s e , b e i n g c h a r a c t e r i s t i c of t h e e n d o t h e l i a l cells, r e n d e r it possible t h a t t h e original tissue used as a n t i g e n was in p a r t i c o n t a m i n a t e d b y capillaries. A n o t h e r possible i n t e r p r e t a t i o n could b e t h a t t h e cells g r o w i n g in c u l t u r e s w i t h v e r y similar m o r p h o l o g i c a l c h a r a c t e r i s t i c s m a y be c o m p o s e d of 2 t y p e s : one r e a c t i n g w i t h tile a n t i b o d i e s of glial fibrillary acidic p r o t e i n , a n d a n o t h e r s h o w i n g alkaline p h o s p h a t a s e p o s i t i v i t y . Before p e r f o r m i n g b i o c h e m i c a l e x p e r i m e n t s , one has to be c e r t a i n of t h e cell p o p u l a t i o n of cultures. T h e d e m o n s t r a t i o n of alkaline p h o s p h a t a s e in ceils of e n d o t h e l i a l origin m a y be of h e l p ill d e t e r m i n i n g t h e ratios b e t w e e n cells growing in vitro.
97
T h e c u r r e n t s t a t e of k n o w l e d g e of n e u r a l dissociation h a s b e e n claimed 3 to be still far f r o m s a t i s f a c t o r y . Our r e s u l t s s h o w e d t h a t , a f t e r m e c h a n i c a l dissociation of b r a i n tissue, viable cells of c a p i l l a r y origin, w h o s e n a t u r e was e v i d e n c e d as e n d o t h e l i a l cells, were p r e s e n t in t h e cultures. K e e p i n g in m i n d t h e i m p o r t a n t t r a n s p o r t p r o c e s s e s u n d e r l y i n g t h e c o m p l e x r e g u l a t o r y f u n c t i o n of t h e b l o o d b r a i n barrier, a t t e m p t s are m a d e t o o b t a i n c u l t u r e s c o n s i s t i n g m a i n l y of e n d o t h e l i a l cells in t h e h o p e of using t h e m as a n o v e l a p p r o a c h in f u r t h e r studies.
7 A. Bertler, B. Fatck, Ch. 0wman and E. Rosengren, Pharmac. Rev. 18, 369 (1966). 8 B.H. Choi and L. W. Lapham, Exp. mol. Path. 24, 110 (1976).
'Decreased serum responsiveness by'primary monolayer cultures of'preneoplastic and 'neoplastic mammary epithelial cells M. K. F e l d m a n 1
Bruce Lyon Memorial Research Laboratory, Children's Hospital Medical Center, 51st ~ Grove Streets, Oakland (California 94609, USA), 26 April 1977 Summary. M o n o l a y e r c u l t u r e s of n o r m a l , p r e n e o p l a s t i c a n d n e o p l a s t i c m u r i n e m a m m a r y epithelial cells were e x p o s e d to v a r i o u s t y p e s of m a m m a l i a n serum. A p r o g r e s s i v e decline in levels of t h y m i d i n e i n c o r p o r a t i o n t o g e t h e r w i t h a c h a n g e in t h e o r d e r i n g of sera w h i c h s t i m u l a t e s o p t i m a l i n c o r p o r a t i o n was o b s e r v e d in t h e t r a n s f o r m e d cells. M o n o l a y e r c u l t u r e s of n o r m a l m o u s e m a m m a r y epithelial cells (MMEC) r e s p o n d to t h e p r e s e n c e of s e r u m b y increasing a) t h e i r levels of D N A s y n t h e s i s 2-4, b) m i t o t i c r a t e s 5 a n d c) final cell d e n s i t i e s 6. T u m o r cells o b t a i n e d f r o m s p o n t a n e o u s l y arising (MTV-induced) m a m m a r y a d e n o c a r c i n o m a s r e s p o n d in a similar m a n n e r 7-8, H o w ever, c u l t u r e s o r i g i n a t i n g f r o m cells of t h e p r e n e o p l a s t i c ,
r--] Normal Preneoplastic ~Tumor
h y p e r p l a s t i c alveolar n o d u l e ( p r o b a b l y N I V - i n d u c e d ) h a v e n o t y e t b e e n e x a m i n e d . A l t h o u g h it is g e n e r a l l y k n o w n t h a t b o t h n o r m a l a n d a b n o r m a l m a m m a r y cells s y n t h e s i z e g r e a t e r a m o u n t s of D N A in t h e p r e s e n c e of serum, we s o u g h t here to c o m p a r e t h e degrees of r e s p o n s i v e n e s s t o v a r i o u s t y p e s of m a m m a l i a n sera u n d e r o t h e r w i s e i d e n t i c a l cell c u l t u r e conditions. The results of t h e s e e x p e r i m e n t s f o r m t h e s u b j e c t of t h i s r e p o r t . Material and methods. N o r m a l M M E C were o b t a i n e d f r o m t h e g l a n d s of 1 6 - 1 7 - d a y p r e g n a n t B A L B / c f C 3 H mice (Cancer R e s e a r c h L a b o r a t o r y , Berkeley, California). P r e n e o p l a s t i c cells were collected f r o m p r i m a r y h y p e r p l a s t i c o u t g r o w t h (HOG) of a nodule arising s p o n t a n e o u s l y in a n 8 - 1 0 - m o n t h - o l d mouse, w h e r e a s t u m o r cells were o b t a i n e d f r o m a m a m m a r y a d e n o c a r c i n o m a arising f r o m a similar HOG, b o t h f r o m B A L B / c f C 3 H mice. N o r m a l a n d p r e n e o plastic tissues were e n z y m a t i c a l l y d i s s o c i a t e d as p r e v i o u s l y d e s c r i b e d 9. N e o p l a s t i c tissue was m i n c e d a n d d i s s o c i a t e d 1
._g il;iil iilill
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Histogram showing levels of incorporation of aH-thymidine in the presence of various types of mammalian serum on normal, preneoplastic and neoplastic mammary epithelial cells. Each value represents the average of 4 determinations. SEM for each value is indicated by the bars.
Acknowledgments. I wish to thank Mr Donald Wong for excellent technical assistance, Ms ffolyce Hardesty for typing the final manuscript and Dr S. Abraham for reviewing the final manuscript. This work was supported by grant CA 15764 from the National Cancer Institute and grant RR 05467, Biomedical Research Support, National Institutes of Health, DHEW. 2 H.W. Hseuh and F. E. Stockdale, ~. Cell Physiol. 83, 297 (1974). 3 M . K . Feldman and D. L. Wong, Am Zool. 16, 229 (1976). 4 M. K. Feldman and D. L. Wong, In Vitro 13, 275 (1977). 5 M.K. Feldman, Ph. D. Thesis, University of California, Berkeley, California (1972). 6 M.K. Feldman, In Vitro 9, 386 (1974). 7 H.L. Hosiek and S. Nandi, Exp. Cell Res. 84, 419 (1974). 8 L . J . T . Young, R. D. Cardiff and C. M. McGrath, Tissue Cult. Ass. Manual 1, 161 (1975). 9 R.C. Foster and M. K. Feldman, Tissue Cult. Ass. Manual 1, 27 {1975). 10 T.T. Puck, S. J. Cicciura and H. W. Fisher, J. exp. Med. 106, 145 (1957). 11 R. Dulbecco and G. Freeman, Virology 8, 396 (1959).
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w i t h 0.25% t r y p s i n in P u c k ' s saline A 1~ for 30 rain at 37 ~ in a g y r a t o r y w a t e r b a t h shaker. I n all cases, tile cells were initially plated in a Dulbecco's M E M m e d i u m 11 c o n t a i n i n g 4500 m g glucose/1 and supp l e m e n t e d w i t h 20% fetal bovine serum (FBS) and gentam y c i n (0.05 mg/ml, Shering) at 37 ~ in an a t m o s p h e r e of 5% CO2:95% air. After 3 days the a m o u n t of F B S was reduced to 5% and a d a y later the m e d i u m was changed to one containing 5% of the m a m m a l i a n serum to be tested. The final cell density of each culture well at this p o i n t was 5.0-6.0 • 104 cells/cm ~ of surface area as det e r m i n e d b y s t a n d a r d h e m o c y t o m e t r i c methods. Sera selected for testing included the following: bovine (Grand Island, S a n t a Clara, Calif.), fetal bovine (Grand Island), horse (Grand Island), h u m a n (Microbiological Associates, Los Angeles, Calif.), l a m b (Grand Island), porcine (Grand Island), and r a b b i t (Microbiological Associates). T h y m i d i n e incorporation was measured as follows: After: t h e ceils had been exposed to the selected s e r u m for 20 h~ ~H-thymidine (45 Ci/mmole, Research P r o d u c t s Corp., E l k Grove Village, Ill.) was added to the m e d i u m at a c o n c e n t r a t i o n of 1 [zCi/ml media for the final 2 h of incubation. The ceils were washed once in unlabeled media, twice w i t h cold 5% trichloroacetic acid (TCA) and t h e n placed a t 4 ~ for t h e n e x t 2 days w i t h fresh TCA to rem o v e a n y u n i n c o r p o r a t e d 8H-thymidine. Ceils were finally rinsed w i t h distilled w a t e r and dissolved in 88% formic acid. An aliquot of this solution was assayed for radioa c t i v i t y and the results were expressed as c p m incorporated into TCA-insoluble m a t e r i a l over the 2-h labelling period. Results and discussion. N o r m a l MMEC responded m a r k edly to selected sera b y incorporating more 3H-thymidine
EXPERIENTIA34/1
t h a n serum-free controls. R a b b i t serum was the m o s t p o t e n t (3.5 times the control), followed by porcine (2.8 times), calf (2.7 times), and h u m a n sera (2.1 times) (figure). Preneoplastic cells, on the o t h e r hand, incorporated somew h a t less a H - t h y m i d i n e in response to s e r u m (only 2fold a b o v e serum-free controls in the presence of porcine serum) (figure). Regardless of the t y p e of serum used, the H O G cultures always incorporated less 3H-thymidine t h a n similarly s u p p l e m e n t e d n o r m a l cultures. A further observ a t i o n is t h a t b o t h n o r m a l and preneoplastic cells respond almost identically in serum-free medium. Thus, it appears t h a t cultures of preneoplastic cells, while displaying a decreased sensitivity to serum, retain as high a rate of incorporation in the absence of serum as do identically cultured n o r m a l cells. T u m o r cells incorporated 3H-thymidine at a rate m a r k e d l y lower t h a n cells from n o r m a l glands and hyperplastic outgrowths (figure). F B S yielded the m a x i m u m stimulus of 1.8fold above serum-free controls. H o w e v e r , unlike n o r m a l and preneoplastic cells, t u m o r cells exposed to serum-free m e d i u m incorporated c o m p a r a t i v e l y low levels of 8Ht h y m i d i n e . Thus, we m a y conclude t h a t these cultured t u m o r cells h a v e a decreased sensitivity to serum and possess a s u b s t a n t i a l l y reduced ability to incorporate t h y m i d i n e in m e d i u m free of serum. The significance of these d a t a lies in the area of cellular recognition. Morphologically and biochemically, monolayer cultures of normal, preneoplastic and neoplastic MMEC often closely resemble one another. Thus, the differential responsiveness of n o r m a l and a b n o r m a l ceils observed in these studies m a y p r o v e i m p o r t a n t as an additional m e a n s of identifying these cells in vitro.
Les cellules m a m m o t r o p e s et s o m a t o t r o p e s de l ' a n t e h y p o p h y s e c h e z la souris C3H porteuse de t u m e u r m a m m a i r e spontan@e. Etude quantitative au m i c r o s c o p e @lectronique 1, 2 M a m m o t r o p i c and s o m a t o t r o p i c pituitary cells in s p o n t a n e o u s m a m m a r y t u m o r bearing C3H f e m a l e m i c e . A quantitative electron m i c r o s c o p e s t u d y K. H. H o l l m a n n et J. M. Verley
Laboratoire de cytopathologie et de microscopie dlectronique, H6pital Broussais, 96, rue Didot, F-7567d Paris Cddex 14 (France), 12 mai 5977 Summary. Pituitaries of m a m m a r y t u m o r - b e a r i n g mice (C3H) were e x a m i n e d b y q u a n t i t a t i v e electron microscopy. The results indicate t h a t considerable modifications occur in m a m m o t r o p i c and somatotropic cells. B o t h types show an increase of the surface of the endoplasmic r e t i c u l u m and a decrease of tile v o l u m e of secretory granules (in percent of cytoplasmic volume), suggesting a heightened secretory a c t i v i t y of these cells during m a m m a r y carcinogenesis. I1 existe de n o m b r e u x a r g u m e n t s en f a v e u r du r61e de l ' h y p o p h y s e dans la g6n~se et la croissance des t u m e u r s m a m m a i r e s t a n t chez l ' a n i m a l a-~ que chez l ' h o m m e 1~ P a r m i les h o r m o n e s hypophysaires, la prolactine a une action m a j e u r e et sa p r o d u c t i o n sembte augment@e au cours de la carcinog6n@se m a m m a i r e . Ainsi la concentration ell prolactine de l ' h y p o p h y s e est plus 61ev6e chez la chienne a t t e i n t e de cancer m a m m a i r e 14 et chez la f e m m e a t t e i n t e de cancer du sein ~ un stade avanc615-17 que chez les sujets t6moins. De m~me la c o n c e n t r a t i o n dans l ' h y p o p h y s e est g@n6ralement plus @lev@e chez la souris de souche 5~ h a u t e incidence canc@reuse is, et le t a u x p l a s m a t i q u e de prolactine semble a u g m e n t 6 chez les f e m m e s ~ h a u l risque ~. A l'oppos@ les d6riv6s de l'ergot de seigle, qui i n h i b e n t la s@cr@tion de prolactine, r6duisent l'incidence e t l a croissance des t u m e u r s m a m m a i l e s chez le rat et chez
la souris ~0. L'influence de l ' h o r m o n e de croissance est moins bien pr~cis6e mais semble 6galement i m p o r t a n t e : chez la souris hypophysectomis@e, ovariectomis6e et surr@nalectomis6e l'injection d ' h o r m o n e de croissance est n@cessaire g l ' i n d u c t i o n de 16sions pr6n6oplasiques et A l e u r t r a n s f o r m a t i o n canc6reuse, au moins dans certaines souches ~, 5. L ' o b j e t de ce t r a v a i l est d'@tudier les modifications morphologiques des cellules m a m m o t r o p e s et somatotropes chez la souris porteuse de t u m e u r rnammaire spontan@e et d'appr@cier le degr6 d'activit6 de ces 2 t y p e s cellulaires de fa~on aussi pr6cise que possible par Yapplication de m6thodes m o r p h o m 6 t r i q u e s au microscope 61ectronique. Matdriel et mdlhodes. Le mat@riel p r o v i e n t de 8 souris C3H ~ (MTV + ) porteuses de t u m e u r m a m m a i r e spontan@e et de 4 souris C3H ~? (MTV + ) t6moins non
Specialia
fibrillary acidic p r o t e i n a n d i n t e r p r e t e d as a s t r o c y t e s s. H o w e v e r , t h e d e m o n s t r a t i o n of capillary origin a n d t h e p r e s e n c e of m a r k e r e n z y m e , alkaline p h o s p h a t a s e , b e i n g c h a r a c t e r i s t i c of t h e e n d o t h e l i a l cells, r e n d e r it possible t h a t t h e original tissue used as a n t i g e n was in p a r t i c o n t a m i n a t e d b y capillaries. A n o t h e r possible i n t e r p r e t a t i o n could b e t h a t t h e cells g r o w i n g in c u l t u r e s w i t h v e r y similar m o r p h o l o g i c a l c h a r a c t e r i s t i c s m a y be c o m p o s e d of 2 t y p e s : one r e a c t i n g w i t h tile a n t i b o d i e s of glial fibrillary acidic p r o t e i n , a n d a n o t h e r s h o w i n g alkaline p h o s p h a t a s e p o s i t i v i t y . Before p e r f o r m i n g b i o c h e m i c a l e x p e r i m e n t s , one has to be c e r t a i n of t h e cell p o p u l a t i o n of cultures. T h e d e m o n s t r a t i o n of alkaline p h o s p h a t a s e in ceils of e n d o t h e l i a l origin m a y be of h e l p ill d e t e r m i n i n g t h e ratios b e t w e e n cells growing in vitro.
97
T h e c u r r e n t s t a t e of k n o w l e d g e of n e u r a l dissociation h a s b e e n claimed 3 to be still far f r o m s a t i s f a c t o r y . Our r e s u l t s s h o w e d t h a t , a f t e r m e c h a n i c a l dissociation of b r a i n tissue, viable cells of c a p i l l a r y origin, w h o s e n a t u r e was e v i d e n c e d as e n d o t h e l i a l cells, were p r e s e n t in t h e cultures. K e e p i n g in m i n d t h e i m p o r t a n t t r a n s p o r t p r o c e s s e s u n d e r l y i n g t h e c o m p l e x r e g u l a t o r y f u n c t i o n of t h e b l o o d b r a i n barrier, a t t e m p t s are m a d e t o o b t a i n c u l t u r e s c o n s i s t i n g m a i n l y of e n d o t h e l i a l cells in t h e h o p e of using t h e m as a n o v e l a p p r o a c h in f u r t h e r studies.
7 A. Bertler, B. Fatck, Ch. 0wman and E. Rosengren, Pharmac. Rev. 18, 369 (1966). 8 B.H. Choi and L. W. Lapham, Exp. mol. Path. 24, 110 (1976).
'Decreased serum responsiveness by'primary monolayer cultures of'preneoplastic and 'neoplastic mammary epithelial cells M. K. F e l d m a n 1
Bruce Lyon Memorial Research Laboratory, Children's Hospital Medical Center, 51st ~ Grove Streets, Oakland (California 94609, USA), 26 April 1977 Summary. M o n o l a y e r c u l t u r e s of n o r m a l , p r e n e o p l a s t i c a n d n e o p l a s t i c m u r i n e m a m m a r y epithelial cells were e x p o s e d to v a r i o u s t y p e s of m a m m a l i a n serum. A p r o g r e s s i v e decline in levels of t h y m i d i n e i n c o r p o r a t i o n t o g e t h e r w i t h a c h a n g e in t h e o r d e r i n g of sera w h i c h s t i m u l a t e s o p t i m a l i n c o r p o r a t i o n was o b s e r v e d in t h e t r a n s f o r m e d cells. M o n o l a y e r c u l t u r e s of n o r m a l m o u s e m a m m a r y epithelial cells (MMEC) r e s p o n d to t h e p r e s e n c e of s e r u m b y increasing a) t h e i r levels of D N A s y n t h e s i s 2-4, b) m i t o t i c r a t e s 5 a n d c) final cell d e n s i t i e s 6. T u m o r cells o b t a i n e d f r o m s p o n t a n e o u s l y arising (MTV-induced) m a m m a r y a d e n o c a r c i n o m a s r e s p o n d in a similar m a n n e r 7-8, H o w ever, c u l t u r e s o r i g i n a t i n g f r o m cells of t h e p r e n e o p l a s t i c ,
r--] Normal Preneoplastic ~Tumor
h y p e r p l a s t i c alveolar n o d u l e ( p r o b a b l y N I V - i n d u c e d ) h a v e n o t y e t b e e n e x a m i n e d . A l t h o u g h it is g e n e r a l l y k n o w n t h a t b o t h n o r m a l a n d a b n o r m a l m a m m a r y cells s y n t h e s i z e g r e a t e r a m o u n t s of D N A in t h e p r e s e n c e of serum, we s o u g h t here to c o m p a r e t h e degrees of r e s p o n s i v e n e s s t o v a r i o u s t y p e s of m a m m a l i a n sera u n d e r o t h e r w i s e i d e n t i c a l cell c u l t u r e conditions. The results of t h e s e e x p e r i m e n t s f o r m t h e s u b j e c t of t h i s r e p o r t . Material and methods. N o r m a l M M E C were o b t a i n e d f r o m t h e g l a n d s of 1 6 - 1 7 - d a y p r e g n a n t B A L B / c f C 3 H mice (Cancer R e s e a r c h L a b o r a t o r y , Berkeley, California). P r e n e o p l a s t i c cells were collected f r o m p r i m a r y h y p e r p l a s t i c o u t g r o w t h (HOG) of a nodule arising s p o n t a n e o u s l y in a n 8 - 1 0 - m o n t h - o l d mouse, w h e r e a s t u m o r cells were o b t a i n e d f r o m a m a m m a r y a d e n o c a r c i n o m a arising f r o m a similar HOG, b o t h f r o m B A L B / c f C 3 H mice. N o r m a l a n d p r e n e o plastic tissues were e n z y m a t i c a l l y d i s s o c i a t e d as p r e v i o u s l y d e s c r i b e d 9. N e o p l a s t i c tissue was m i n c e d a n d d i s s o c i a t e d 1
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Fetal bovine Porcine Human
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Serum supplement
Histogram showing levels of incorporation of aH-thymidine in the presence of various types of mammalian serum on normal, preneoplastic and neoplastic mammary epithelial cells. Each value represents the average of 4 determinations. SEM for each value is indicated by the bars.
Acknowledgments. I wish to thank Mr Donald Wong for excellent technical assistance, Ms ffolyce Hardesty for typing the final manuscript and Dr S. Abraham for reviewing the final manuscript. This work was supported by grant CA 15764 from the National Cancer Institute and grant RR 05467, Biomedical Research Support, National Institutes of Health, DHEW. 2 H.W. Hseuh and F. E. Stockdale, ~. Cell Physiol. 83, 297 (1974). 3 M . K . Feldman and D. L. Wong, Am Zool. 16, 229 (1976). 4 M. K. Feldman and D. L. Wong, In Vitro 13, 275 (1977). 5 M.K. Feldman, Ph. D. Thesis, University of California, Berkeley, California (1972). 6 M.K. Feldman, In Vitro 9, 386 (1974). 7 H.L. Hosiek and S. Nandi, Exp. Cell Res. 84, 419 (1974). 8 L . J . T . Young, R. D. Cardiff and C. M. McGrath, Tissue Cult. Ass. Manual 1, 161 (1975). 9 R.C. Foster and M. K. Feldman, Tissue Cult. Ass. Manual 1, 27 {1975). 10 T.T. Puck, S. J. Cicciura and H. W. Fisher, J. exp. Med. 106, 145 (1957). 11 R. Dulbecco and G. Freeman, Virology 8, 396 (1959).
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w i t h 0.25% t r y p s i n in P u c k ' s saline A 1~ for 30 rain at 37 ~ in a g y r a t o r y w a t e r b a t h shaker. I n all cases, tile cells were initially plated in a Dulbecco's M E M m e d i u m 11 c o n t a i n i n g 4500 m g glucose/1 and supp l e m e n t e d w i t h 20% fetal bovine serum (FBS) and gentam y c i n (0.05 mg/ml, Shering) at 37 ~ in an a t m o s p h e r e of 5% CO2:95% air. After 3 days the a m o u n t of F B S was reduced to 5% and a d a y later the m e d i u m was changed to one containing 5% of the m a m m a l i a n serum to be tested. The final cell density of each culture well at this p o i n t was 5.0-6.0 • 104 cells/cm ~ of surface area as det e r m i n e d b y s t a n d a r d h e m o c y t o m e t r i c methods. Sera selected for testing included the following: bovine (Grand Island, S a n t a Clara, Calif.), fetal bovine (Grand Island), horse (Grand Island), h u m a n (Microbiological Associates, Los Angeles, Calif.), l a m b (Grand Island), porcine (Grand Island), and r a b b i t (Microbiological Associates). T h y m i d i n e incorporation was measured as follows: After: t h e ceils had been exposed to the selected s e r u m for 20 h~ ~H-thymidine (45 Ci/mmole, Research P r o d u c t s Corp., E l k Grove Village, Ill.) was added to the m e d i u m at a c o n c e n t r a t i o n of 1 [zCi/ml media for the final 2 h of incubation. The ceils were washed once in unlabeled media, twice w i t h cold 5% trichloroacetic acid (TCA) and t h e n placed a t 4 ~ for t h e n e x t 2 days w i t h fresh TCA to rem o v e a n y u n i n c o r p o r a t e d 8H-thymidine. Ceils were finally rinsed w i t h distilled w a t e r and dissolved in 88% formic acid. An aliquot of this solution was assayed for radioa c t i v i t y and the results were expressed as c p m incorporated into TCA-insoluble m a t e r i a l over the 2-h labelling period. Results and discussion. N o r m a l MMEC responded m a r k edly to selected sera b y incorporating more 3H-thymidine
EXPERIENTIA34/1
t h a n serum-free controls. R a b b i t serum was the m o s t p o t e n t (3.5 times the control), followed by porcine (2.8 times), calf (2.7 times), and h u m a n sera (2.1 times) (figure). Preneoplastic cells, on the o t h e r hand, incorporated somew h a t less a H - t h y m i d i n e in response to s e r u m (only 2fold a b o v e serum-free controls in the presence of porcine serum) (figure). Regardless of the t y p e of serum used, the H O G cultures always incorporated less 3H-thymidine t h a n similarly s u p p l e m e n t e d n o r m a l cultures. A further observ a t i o n is t h a t b o t h n o r m a l and preneoplastic cells respond almost identically in serum-free medium. Thus, it appears t h a t cultures of preneoplastic cells, while displaying a decreased sensitivity to serum, retain as high a rate of incorporation in the absence of serum as do identically cultured n o r m a l cells. T u m o r cells incorporated 3H-thymidine at a rate m a r k e d l y lower t h a n cells from n o r m a l glands and hyperplastic outgrowths (figure). F B S yielded the m a x i m u m stimulus of 1.8fold above serum-free controls. H o w e v e r , unlike n o r m a l and preneoplastic cells, t u m o r cells exposed to serum-free m e d i u m incorporated c o m p a r a t i v e l y low levels of 8Ht h y m i d i n e . Thus, we m a y conclude t h a t these cultured t u m o r cells h a v e a decreased sensitivity to serum and possess a s u b s t a n t i a l l y reduced ability to incorporate t h y m i d i n e in m e d i u m free of serum. The significance of these d a t a lies in the area of cellular recognition. Morphologically and biochemically, monolayer cultures of normal, preneoplastic and neoplastic MMEC often closely resemble one another. Thus, the differential responsiveness of n o r m a l and a b n o r m a l ceils observed in these studies m a y p r o v e i m p o r t a n t as an additional m e a n s of identifying these cells in vitro.
Les cellules m a m m o t r o p e s et s o m a t o t r o p e s de l ' a n t e h y p o p h y s e c h e z la souris C3H porteuse de t u m e u r m a m m a i r e spontan@e. Etude quantitative au m i c r o s c o p e @lectronique 1, 2 M a m m o t r o p i c and s o m a t o t r o p i c pituitary cells in s p o n t a n e o u s m a m m a r y t u m o r bearing C3H f e m a l e m i c e . A quantitative electron m i c r o s c o p e s t u d y K. H. H o l l m a n n et J. M. Verley
Laboratoire de cytopathologie et de microscopie dlectronique, H6pital Broussais, 96, rue Didot, F-7567d Paris Cddex 14 (France), 12 mai 5977 Summary. Pituitaries of m a m m a r y t u m o r - b e a r i n g mice (C3H) were e x a m i n e d b y q u a n t i t a t i v e electron microscopy. The results indicate t h a t considerable modifications occur in m a m m o t r o p i c and somatotropic cells. B o t h types show an increase of the surface of the endoplasmic r e t i c u l u m and a decrease of tile v o l u m e of secretory granules (in percent of cytoplasmic volume), suggesting a heightened secretory a c t i v i t y of these cells during m a m m a r y carcinogenesis. I1 existe de n o m b r e u x a r g u m e n t s en f a v e u r du r61e de l ' h y p o p h y s e dans la g6n~se et la croissance des t u m e u r s m a m m a i r e s t a n t chez l ' a n i m a l a-~ que chez l ' h o m m e 1~ P a r m i les h o r m o n e s hypophysaires, la prolactine a une action m a j e u r e et sa p r o d u c t i o n sembte augment@e au cours de la carcinog6n@se m a m m a i r e . Ainsi la concentration ell prolactine de l ' h y p o p h y s e est plus 61ev6e chez la chienne a t t e i n t e de cancer m a m m a i r e 14 et chez la f e m m e a t t e i n t e de cancer du sein ~ un stade avanc615-17 que chez les sujets t6moins. De m~me la c o n c e n t r a t i o n dans l ' h y p o p h y s e est g@n6ralement plus @lev@e chez la souris de souche 5~ h a u t e incidence canc@reuse is, et le t a u x p l a s m a t i q u e de prolactine semble a u g m e n t 6 chez les f e m m e s ~ h a u l risque ~. A l'oppos@ les d6riv6s de l'ergot de seigle, qui i n h i b e n t la s@cr@tion de prolactine, r6duisent l'incidence e t l a croissance des t u m e u r s m a m m a i l e s chez le rat et chez
la souris ~0. L'influence de l ' h o r m o n e de croissance est moins bien pr~cis6e mais semble 6galement i m p o r t a n t e : chez la souris hypophysectomis@e, ovariectomis6e et surr@nalectomis6e l'injection d ' h o r m o n e de croissance est n@cessaire g l ' i n d u c t i o n de 16sions pr6n6oplasiques et A l e u r t r a n s f o r m a t i o n canc6reuse, au moins dans certaines souches ~, 5. L ' o b j e t de ce t r a v a i l est d'@tudier les modifications morphologiques des cellules m a m m o t r o p e s et somatotropes chez la souris porteuse de t u m e u r rnammaire spontan@e et d'appr@cier le degr6 d'activit6 de ces 2 t y p e s cellulaires de fa~on aussi pr6cise que possible par Yapplication de m6thodes m o r p h o m 6 t r i q u e s au microscope 61ectronique. Matdriel et mdlhodes. Le mat@riel p r o v i e n t de 8 souris C3H ~ (MTV + ) porteuses de t u m e u r m a m m a i r e spontan@e et de 4 souris C3H ~? (MTV + ) t6moins non
