Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Morphological effects of estrogen removal on an estrogen-dependent adrenocortical carcinoma in rats Nichols, Thomas Matthew 1970

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1970_A6_7 N53.pdf [ 18.04MB ]
Metadata
JSON: 831-1.0101994.json
JSON-LD: 831-1.0101994-ld.json
RDF/XML (Pretty): 831-1.0101994-rdf.xml
RDF/JSON: 831-1.0101994-rdf.json
Turtle: 831-1.0101994-turtle.txt
N-Triples: 831-1.0101994-rdf-ntriples.txt
Original Record: 831-1.0101994-source.json
Full Text
831-1.0101994-fulltext.txt
Citation
831-1.0101994.ris

Full Text

THE MORPHOLOGICAL EFFECTS OF ESTROGEN REMOVAL ON AN ESTROGEN-DEPENDENT ADRENOCORTICAL CARCINOMA IN RATS ' by Thomas M. N i c h o l s M.D.CiM., M c G i l l U n i v e r s i t y " 1959 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF . THE REQUIREMENTS .FOR THE DEGREE OF M.Sc. i n t h e Department of P a t h o l o g y We accept t h i s t h e s i s as con f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA May, 1970 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver 8, Canada o i i - M. Sc. THESIS - T. M. NICHOLS -THE MORPHOLOGICAL EFFECTS OF ESTROGEN REMOVAL ON  AN ESTROGEN-DEPENDENT ADRENOCORTICAL CARCINOMA IN RATS ABSTRACT The morphologic e f f e c t s of estrogen withdrawal from an estrogen-dependent tumor were investigated using l i g h t and electron microscopy. Twenty-five male hooded rats received interscapular implants of an estrogen-induced, estrogen-dependent adrenocortical tumor as well as subcutan-eous estrone p e l l e t s . The tumors were examined when they had reached one and two centimeters i n s i z e . The p e l l e t s were removed from a second group and the tumors examined when they had decreased to a minimum s i z e . A t h i r d group received a second p e l l e t a f t e r the tumor had regressed to a minimum s i z e f o l l o w i n g removal of the f i r s t p e l l e t . The primary tumors were characterized by plump, active-looking c e l l s growing usually i n a sheet-like pattern. The u l t r a s t r u c t u r a l features of these tumors included large, b i z a r r e mitochondria with tubular c r i s t a e , prominent polyribosomal aggregates and Golgi zones as well as f a i r l y frequent c e n t r i o l e s . The regressing tumors consisted of smaller c e l l s growing i n a trabecular pattern and heavily i n f i l t r a t e d with eosinophils. The f i n e structure of the tumor c e l l s consisted of small mitochondria with a dense matrix and a few i r r e g u l a r c r i s t a e . Myelin figures and r e s i d u a l bodies were present i n s i g n i f i c a n t numbers only i n the regressing tumors while (primary)lysosomes were more frequent i n the growing tumors. The tumors examined af t e r reimplantation of estrone showed features of the primary growth tumors plus r e s i d u a l f o c i of regression. It thus appears that estrogen acts as a trophic factor for t h i s experi-mental tumor much as i t does for the uterus. Tumor c e l l s can survive i n i t s absence, but require i t for growth. The u l t r a s t r u c t u r a l expression of estrogen administration seen i n t h i s study i s the p r o l i f e r a t i o n of mitochondria as an energy source and polyribosomes f o r the production of proteins for endogenous consumption. The removal of estrogen r e s u l t s i n a generalized reduction i n c e l l u l a r a c t i v i t y and an accumulation of the products of autophagy. Further work i s required to t i e i n these r e s u l t s with the studies being done on the basic mechanisms of estrogen action at the l e v e l of molecular biology. i i i TABLE OF CONTENTS T i t l e P a g e i A b s t r a c t i i T a b l e o f C o n t e n t s i i i L i s t o f T a b l e s i v L i s t o f F i g u r e s v T H E S I S I n t r o d u c t i o n 1 M a t e r i a l s a n d M e t h o d s 1 R e s u l t s 3 E l e c t r o n M i c r o s c o p e F i n d i n g s 7 D i s c u s s i o n 11 B i b l i o g r a p h y 21 i v LIST OF TABLES I Construction of Experiment following page 2 II Course of Tumor Growth 3 II I Light Microscopic Appearance of tumors 3 IV Changes i n Organs Due to Presence of Estrogen and/or Tumor 5 V LIST OF FIGURES 1. Primary growth - sheet-like pattern follox<dng page 3 2. Primary growth - trabecular pattern 3 3. Regression - compact c e l l s 3 4. Secondary growth - dimorphic p i c t u r e 4 5. Secondary growth - clear c e l l s 4 6. Regression - cyst formation 4 7. Regression - degenerative f o c i and fibrous septa 4 8. Regression - ir o n deposition 4 9. Regression - eosinophil leukocyte i n f i l t r a t e 4 10. Primary growth c e l l s 7 11. Primary growth - c e n t r i o l e and Golgi apparatus 7 12. Primary growth - dark c e l l s 7 13. Dark c e l l 7 14. Dark c e l l i n mitosis 7 15. Primary growth - mitochondrial morphology 7 16. Primary growth - mitochondrial morphology 7 17. Primary growth - mitochondrial morphology 8 18. Primary growth - mitochondria enfolding cytoplasm 8 19. Primary growth - electron-dense intramitochondrial material 8 2 0. Primary growth - polyribosomes and nuclear pores 8 21. Primary growth - mitochondria and l i p i d droplets 8 22. Primary grox<?th - convoluted m i c r o v i l l i and lysosomes 9 2 3. Primary growth - desmosomes 9 2 4. Primary growth - double basement membrane 9 25. Primary growth - septal collagen f i b r i l s 9 26. Primary growth - collagen f i b r i l s 9 27. Regression - o v e r a l l view 9 2 8. Regression - o v e r a l l view 9 29. Regression - mitochondrial morphology 9 30. Regression - electron dense intracytoplasmic granules 9 v i 31. Regression - eosinophil granules within tumor c e l l s following page 9 32. Regression - intracytoplasmic dense granules 9 33. Regression - collagen f i b r i l s i n tumor c e l l 9 34. Regression - collagen f i b r i l s i n tumor c e l l 9 35. Regression - eosinophil granules i n tumor c e l l 9 36. Regression - l i p o f u s c i n granules 9 37. Regression - intravascular free eosinophil granules 10 3 8. Regression - c i l i a ? or double centriole? 10 39. Regression - l i p o f u s c i n granules 10 40. Regression - myelin f i g u r e 10 41. C r y s t a l l o i d of eosinophil granule 10 42. Secondary growth - a c t i v e c e l l s 10 43. Secondary growth - a c t i v e c e l l s 10 44. Secondary growth - eosinophil i n f i l t r a t e 10 45. Secondary growth - i n a c t i v e area 10 46. Ecdysone 12 47. Regression - eosinophils emigrating from vessel 19 48. Adrenal zona f a s c i c u l a t a - tubulovesicular c t i s t a e mitochondriales 19 49. Adrenal zona f a s c i c u l a t a - v e s i c u l a r c r i s t a e mitochondriales 19 50. Adrenal zona glomerulosa - s h e l f - l i k e c r i s t a e 19 51. Steroid synthetic pathway 19 52. Adrenal zona glomerulosa - c i l i u m 19 M.Sc. THESIS - T.M. NICHOLS THE MORPHOLOGICAL EFFECTS OF ESTROGEN REMOVAL ON  AN ESTROGEN-DEPENDENT ADRENOCORTICAL CARCINOMA IN RATS INTRODUCTION In the 75 years since the association of hormones and tumors was demonstrated by Beatson, a vast amount of l i t e r a t u r e has accumulated, attesting to the complex nature of the problem. Although no f i n a l answers are yet available as to the exact relationship, some points have been established, l ) Hormones can be carcinogenic, anti-carcinogenic, co-carcinogenic, tumor-stimulating, or tumor-inhibiting. 2) They can act as carcinogens on endocrine and non-endocrine tissues. 3) They can 5k affect the induction or course of tumors induced by chemical carcinogens or viruses. h) They do not have to be metabolized to another compound to be e f f e c t i v e . 5) The carcinogenic effects of hormones vary between and . . . . . k7, 5 1 . within species . With two exceptions to d a t e ^ ' ^ only estrogens have induced hi malignant neoplasms. In humans, moreover, there i s no unequivocal evidence of hormonal tumorigenesis Rather there i s only evidence of aggravation or suppression of pre-existing tumors by endocrine manipulation. This s i t u a t i o n has led to a search for suitable animal models of hormone-responsive and hormone-dependent tumors . One such model i s the es:trogen-77 induced adrenocortical carcinoma developed by Noble which under certain conditions i s e n t i r e l y dependent upon the continued presence of estrogens f o r i t s growth. This thesis i s directed toward a morphological evaluation of the regression induced i n this tumor by the removal of estrogen. MATERIALS AND METHODS The present study involves a transplantable adrenal c o r t i c a l carcinoma which arose i n the zone f a s c i c u l a t a as the result of prolonged subcutaneous implantation of an esjtrone p e l l e t i n a female hooded rat. The tumor requires the continued administration of estrogen for i t s continued growth and i n order for transplantation to be successful the host animal must be estrogen treated. I f the p e l l e t i s removed, the transplanted tumor dwindles to a minimum size of approximately 5 mm., and readministration of estrogen produces a regrowth which i s more rapid than the primary growth. -2-T w e n t y - f i v e male hooded r a t s two months o f age were i m p l a n t e d s u b c u t a n e o u s l y i n t h e i n t e r s c a p u l a r r e g i o n w i t h a volume o f tumor a p p r o x i m a t i n g 1.0 m l . and t h e n r e c e i v e d subcutaneous e s t r o n e p e l l e t s . F o u r a n i m a l s r e c e i v e d a p e l l e t b u t no tumor, and a n o t h e r was g i v e n a tumor i m p l a n t b u t no p e l l e t . Of t h o s e t h a t r e c e i v e d b o t h tumor and p e l l e t , two were a a c r i f i e d when t h e tumor had r e a c h e d 0 .5 cm., t h r e e a t 1 cm. and three, a t 2 cm. Twelve had t h e p e l l e t removed when t h e tumor had r e a c h e d 1 cm. and t h r e e were s a c r i f i c e d when t h e tumor had d i m i n i s h e d t o 0 .5 cm. and t h r e e when i t had r e a c h e d a minimum s i z e and had remained s t a b l e f o r f o u r weeks. The r e m a i n i n g a n i m a l s had e s t r o n e p e l l e t s r e i m p l a n t e d when t h e i r tumors had remained a t a minimum s i z e f o r f o u r weeks. A phase o f s e c o n d a r y g r o w t h ensued and t h r e e tumors each were examined when t h e y had regrown t o 1.0 and 2.0 cm. T a b l e I p o r t r a y s t h e e x p e r i m e n t a l d e s i g n and t h e t i m e s o f e x a m i n a t i o n o f t h e tumors. A f u r t h e r f i v e a n i m a l s b e a r i n g tumors a v e r a g i n g 1.0 cm. d i a m e t e r were added as c o n t r o l s f o r t h e r e g r e s s i n g t u m o r s . I n t h e group examined d u r i n g p r i m a r y tumor g r o w t h , one was e l i m i n a t e d because t h e r a t e o f tumor growth f e l l o u t s i d e two s t a n d a r d d e v i a t i o n s f r o m t h e mean. L i k e w i s e , t h e r e s u l t s f r o m two. a n i m a l s w i t h r e g r e s s i n g tumors and two w i t h tumors shewing s e c o n d a r y g r o w t h were d i s c a r d e d on t h e b a s i s o f u n u s u a l l y r a p i d o r s l o w g r o w t h r a t e s . A l l tumors h a v i n g s i m i l a r r a t e s o f g r o w t h , r e g r e s s i o n and r e g r o w t h were examined b y l i g h t m i c r o s c o p y u s i n g h e m a t o x y l i n and e o s i n s t a i n i n g . I n a d d i t i o n two c h a r a c t e r i s t i c tumors f r o m each group were examined u n d e r t h e e l e c t r o n m i c r o s c o p e . T i s s u e s were removed under e t h e r a n e s t h e s i a and f i x e d i n k% g l u t a r a l d e h y d e i n 0 . T r i p h o s p h a t e b u f f e r o r i n Iffd osmium t e t r o x i d e i n 0.1M pho s p h a t e b u f f e r . The g l u t a r a l d e h y d e - f i i x e d t i s s u e s were p o s t - f i x e d i n osmium t e t r o x i d e . S i l v e r t o g r e y s e c t i o n s were s t a i n e d w i t h u r a n y l a c e t a t e and l e a d h y d r o x i d e and examined on a Siemens E l m i s k o p I o r a P h i l i p s EM-200 e l e c t r o n m i c r o s c o p e . A complete a u t o p s y was p e r f o r m e d on each a n i m a l and t h e a d r e n a l s , p i t u i t a r y , k i d n e y s , t e s t e s and l u n g s were examined m i c r o s c o p i c a l l y b y h e m a t o x y l i n and e o s i n s t a i n i n g . y TREATMENT ALLOCATION OF ANIMALS TUMOR SIZE WHEN EXAMINED STAGE Estrone p e l l e t implant Tumor implant Estrone pellet, removed Estrone p e l l e t reimplant PRIMARY 'GROWTH \ REGRESSION Ocif. SECONDARY GROWTH TABLE I.- Construction of experiment - 3 -RESULTS Course The time taken f o r the tumor to become palpable ( 1 - 3 mm.) varied from k - 1 1 weeks and the a d d i t i o n a l time to reach 1 . 0 cm. ranged from 3 - 1 2 weeks. Regression and regrowth were also v a r i a b l e , but the secondary growth rate was on the average twice the primary. The r e l a t i o n s h i p of time, tumor s i z e , and s a c r i f i c e are shown i n Tabjke I I . Also demonstrated are degrees of estrogen independence. P a r t i a l independence i s seen i n tumors 5 , 9> 1 9 ? 8 and 2 1 . In these animals the tumor continued to enlarge a f t e r removal of the p e l l e t before shrinking to a minimum s i z e . Greater independence i s seen i n tumors l 6 , l 8 and 2 5 where the tumor kept r i g h t on growing a f t e r p e l l e t removal. In other experiements at the Cancer Research Center, independence (which varied with the s t r a i n ) was more common i n tumors which were allowed to reach a r e l a t i v e l y large s i z e before the p e l l e t was removed. No metastases occurred due to the r e l a t i v e l y short time the tumor was allowed to grow i n the presence of estrogen. Pathologyob-I... Gross: The tumors were lobulated, sometimes co n s i s t i n g of separate nodules. They had a minimal pseudocapsule and a marked tendency to hemorrhage during biopsy. Their consistency was soft and the color lavender. Microscopic: H & E sections of the tumor were evaluated semi-quantitatively f o r pattern of growth, cytology, m i t o t i c a c t i v i t y , necrosis, cyst formation, f i b r o s i s , v a s c u l a r i t y , hemorrhage, i r o n and i n f i l t r a t i o n by eosinophils and other inflammatory c e l l s . The r e s u l t s are tabulated i n Table I I I . The growth pattern was e i t h e r i n the form of sheets ( F i g . l ) or trabeculae ( F i g . 2 ) . The sheets were composed of f a i r l y uniform, plump, a c t i v e l y growing c e l l s which tended to predominate when the tumor was growing i n the presence of the p e l l e t . The c e l l s i n the trabecular areas were again regular but were smaller with more compact n u c l e i . These were found predominantly i n regressing tumors and at the margins of tumors undergoing secondary growth. Concerning the c e l l types i n the regressing tumors, i t was noted that an intermediate c e l l s i z e was seen i n the tumors which had been growing a shorter time i n the absence of estrogen support (Nos. 1 2 , 9 and 2k). Of those that had been allowed to reach a minimum s i z e , a l l had small compact c e l l s with n u c l e i showing pyknosis ( F i g . 3 ) . >-•2 2 3 o - o uJ ? ->) o o ON • x to I-it Ul 5. 3 Vo V) ^ \y) p-a * zz Vo <r Vo <r r-J ro ro Li p- r~ •5 — a*! s £ -— c-J <JN O <>2 -£> 4 - c » io w &o o Vo ol O* — 5 »-UJ O -2. O U) f r> — M d — — ro <5 0^ cO Vo V> <j- <j-U- -J 5 c. 0"^  CO ~ -<? »0 V> CL -J c.1 t^ i'S • -9 . OK> V o -4 - c n ^ ^ o jlt3Q ^ r4 ^ rJ -rst i 1 — - £ ± m - ? J- rJ ^ r l r-J — JL ^ ^ - ^ ^ . ^ <f ^ ^ ^ r J ^ _ ^ ^ r4 o-' — "— _£ O O -O o o ro ^ o o 1" ^ ^ co * ^ ^ ° _> _^ O oi j£ — rs) > I f - J r-i r O i _ — ^ to r o V 1. d ^ O r-i _ C<> \ \ \ \ \ \ ^ • \ \ \ \ \ \ \ \ \ \ \ \ \ - E a 1 O s O N < ~ CA crs ^ 0 > ^ ^ o O oj £ rs, r. Q ^ ^ U Vo W) o o o o ^ ^ 2. 2 o O O v ^ ^ j _ _j_ ^ f x . O ^ o v<,-o o o — — — C-J i — — r-vi (v-s F i g . 3 Regression - compact c e l l s . x250 -k-The tumors e x p e r i e n c i n g s e c o n d a r y growth showed a d i m o r p h i c ( F i g s , h, 5) p i c t u r e w i t h b o t h a c t i v e g r o w t h a r e a s and remnants o f s m a l l c e l l t r a b e c u l a e f r o m t h e r e g r e s s i n g p h a s e . The m i t o t i c ns&te c o r r e l a t e d w e l l w i t h t h e p r e s e n c e o r absence o f the p e l l e t , s howing d e c r e a s i n g numbers as t h e tumor s h r a n k i n t h e absence o f e s t r o g e n i c s t i m u l a t i o n . N e c r o s i s was d i v i d e d i n t o t h r e e t y p e s : one t y p e c o n s i s t e d o f c y s t f o r m a t i o n , a s s o c i a t e d w i t h v e s i c u l a r a r e a s , w h i c h o f t e n c o n t a i n e d b l o o d ( F i g . 6 ) . O t h e r t y p e s o f n e c r o s i s i n c l u d e d m a s s i v e n e c r o s i s r e s e m b l i n g an i n f a r c t , d e g e n e r a t i o n l e a d i n g t o l o s s o f d i s t i n g u i s h a b l e c e l l m a r g i n s , and l y t i c n e c r o s i s o f t h e c e l l s i n t h e c e n t e r s o f l o b u l e s . These l a t t e r t y p e s were seen uncommonly. A d e g e n e r a t i v e c y t o l y s i s , w h i c h c o u l d n o t d e f i n i t e l y be l i n k e d t o a n e c r o t i c p r o c e s s as n u c l e a r i n v o l v e m e n t was n o t r e g u l a r l y o b s e r v e d , was c h a r a c t e r i z e d b y i n d i v i d u a l c e l l s w i t h empty c y t o p l a s m . T h i s p r o c e s s i n v o l v e d s i n g l e c e l l s i n t h e m i d s t o f h e a l t h y c e l l s , began as c y t o -p l a s m i c v a c u o l i z a t i o n , p r o g r e s s e d t o l o s s o f c y t o p l a s m i c s t a i n i n g , and r e s u l t e d i n c l e a r c e l l s . T h i s p r o c e s s was seen f r o m t i m e t o t i m e d u r i n g p r i m a r y g r o w t h , b u t became p a r t i c u l a r l y p r o m i n e n t i n tumors f r o m a n i m a l s w i t h t h e e s t r o g e n p e l l e t i m p l a n t e d f o r t h e second t i m e . C y s t f o r m a t i o n was seen i n r e g r e s s i n g tumors and i n t h o s e showing s e c o n d a r y growth. The f o c i o f s e v e r e a t r o p h y l e a d i n g t o d e g e n e r a t i o n and n e c r o s i s were th e s i t e o f f i b r o b l a s t i c a c t i v i t y and a p p e a r e d t o be t h e p o i n t o f o r i g i n o f t h e f i b r o u s t r a b e c u l a e w h i c h d i v i d e d t h e tumors i n t o v a r y i n g s i z e d l o b u l e s ( F i g . 7 ) . V a s c u l a r i t y was d i f f i c u l t t o a s s e s s as t h e tumors were r e l a t i v e l y b l o o d f r e e on s e c t i o n a p a r t f r o m i n t e r s t i t i a l hemorrhage. However, t h e r e c e n t l y i m p l a n t e d tumors app e a r e d t o have a more p r o m i n e n t v a s c u l a r system. Hemorrhage was a v a r i a b l e phenomenon, b u t was g e n e r a l l y more p r o m i n e n t i n t h e r e g r e s s i n g and s e c o n d a r y g r o w t h tumors. I r o n a c c u m u l a t i o n ( F i g . k, 8 ) , a c u m u l a t i v e i n d e x o f h e m o r r h a g i c e v e n t s , showed a s i m i l a r b u t more d e f i n i t e d i s t r i b u t i o n ; t h e p r e s e n c e o f l a r g e amounts o f i r o n c o r r e l a t e d w e l l w i t h t h e l e n g t h o f t i m e t h e tumor had been g r o w i n g . The p r e s e n c e o f e o s i n o p h i l s was a v e r y s t r i k i n g f i n d i n g ( F i g . 9 ) j i n tumor No. 5 t h e y a c c o u n t e d f o r r o u g h l y 50$ o f t h e c e l l s p r e s e n t i n t h e t i s s u e s e c t i o n . They were se e n i n two s i t u a t i o n s : l ) t h e r e g r e s s i n g tumors, y F i g . 5 Secondary growth - c lear c e l l s . xAOO F i g .-7 R e g r e s s i o n - d e g e n e r a t i v e f o c i and f i b r o u s s e p t a . x l O O F i g . 9 Regression - eosinophil leukocyte i n f i l t r a t e . x275 e x c e p t f o r Wo. 12 w h i c h had been w i t h o u t e s t r o g e n s u p p o r t f o r o n l y one week and had n o t y e t begun t o s h r i n k , a l l c o n t a i n e d v a s t numbers o f e o s i n o p h i l s i n f i l t r a t i n g between t h e t r a b e c u l a e . 2) I n t h e s e c o n d a r y growth tumors, t h e e o s i n o p h i l s were s e e n i n s i g n i f i c a n t numbers among t h e s m a l l c e l l s w h i c h d i d n ot appear t o be t a k i n g p a r t i n t h e renewed growth. O t h e r i n f l a m m a t o r y c e l l s , w h i c h c o n s i s t e d o f s m a l l numbers o f n e u t r o p h i l s o r s m a l l c l u s t e r s o f lym p h o c y t e s were p r e s e n t i n v a r i a b l e b u t i n s i g n i f i c a n t numbers. They were n o t a s s o c i a t e d w i t h n e c r o s i s o r w i t h e o s i n o p h i l i n f i l t r a t e s b u t were s e e n o c c a s i o n a l l y i n t h e t h i n c a p s u l e o r f i b r o u s t r a b e c u l a e . I n d i v i d u a l mast c e l l s were a l s o seen w i t h some r e g u l a r i t y i n t h i s l o c a t i o n . I n summary t h e n , t h e p r i m a r y g r o w t h tumors were c h a r a c t e r i z e d b y l a r g e plump c e l l s o c c u r r i n g g e n e r a l l y i n s h e e t s . They showed a c t i v e m i t o s i s , m i l d s i n g l e c e l l n e c r o s i s , moderate v a s c u l a r i t y and o c c a s i o n a l e o s i n o p h i l s . W i t h t h e pas s a g e o f t i m e a f t e r r e m o v a l o f t h e p e l l e t , t h e s h r i n k i n g tumors showed i n c r e a s i n g prominence o f s m a l l a t r o p h i c c e l l s i n a t r a b e c u l a r p a t t e r n , v i r t u a l absence o f m i t o t i c a c t i v i t y , and c y s t f o r m a t i o n . T h e i r most s a l i e n t f e a t u r e was t h e heavy i n f i l t r a t i o n b y e o s i n o p h i l s . When a p e l l e t was r e i m p l a n t e d , o r when t h e tumor became autonomous, t h e e n l a r g i n g tumor d e m o n s t r a t e d a d i m o r p h i c p i c t u r e w i t h a r e a s r e s e m b l i n g t h e a c t i v i t y o f t h e p r i m a r y g r o w t h group and p e r i p h e r a l a r e a s s t i l l s howing r e g r e s s i v e f e a t u r e s . I t was i n t h i s s i t u a t i o n t h a t c l e a r c e l l s showing v a c u o l a r d e g e n e r a t i o n were seen. Changes i n o t h e r o r g a n s : A l t h o u g h t h i s s t u d y was n o t d e s i g n e d t o d e f i n i t i v e l y e v a l u a t e t h e s y s t e m i c e f f e c t s o f e s t r o g e n - t u m o r i n t e r a c t i o n , t h e f o l l o w i n g o b s e r v a t i o n s a r e i n c l u d e d f o r c o m p l e t e n e s s and b y way o f s u g g e s t i n g a r e a s o f p o s s i b l e f u t u r e i n v e s t i g a t i o n . The changes a r e shown i n T a b l e I V . A n i m a l w e i g h t and g e n e r a l c o n d i t i o n : The a n i m a l s were two months o l d and weighed 100 - l8o gms. when t h e tumors and p e l l e t s were i m p l a n t e d . The avera g e w e i g h t r e a c h e d w h i l e r e c e i v i n g e s t r o g e n was 215 gms. i n i t i a l l y and 320 gms. d u r i n g s e c o n d a r y growth. When t h e p e l l e t s were removed, t h e a n i m a l s g a i n e d weight, t o an aver a g e o f 3^0 gms. I n g e n e r a l , t h e p e l l e t e d a n i m a l s ( b o t h t h e c o n t r o l s and t h o s e g r o w i n g tumors) seemed l e s s r o b u s t and more p r o n e t o i l l n e s s t h a n t h e u n t r e a t e d a n i m a l s . • • r T a b l e I V . C h a n g e s i n O r g a n s Due t o P r e s e n c e o f E s t r o g e n a n d / o r Tumor GROUP & A M I M A L 4? Normal rirnary WEEKS GfcGWTU OF TUMOR. size. IT p l T U i Tf>R.Y W E I G - H T 2 ; II £ f Yr>m 1 ? ' 4 13 st il 22 J 2- 20 14 14 $0 21 9 10 /• 2 T 10 9 31 20 9 zo - :; 2 — W E E K S S I N C E P E L L E T R E N \ 0 v e ] > ,*, -12 1 11 f : ( ' 9 ,. 12 \% 24 9 It r 20 x r -7 19 17 (9 IS 4 (34) 2. i t J W E E K S S I N C E . (0 ? • IS" r 10 32 2! t 9 3? y t IS" 4 t ( 7 22. 30 3 7 23 M , Indepen dent* W E E W S I N C E P E t u e r R e M o v e D It js* 30 12 !? 20 2<r It W E E K S 17 — 27 2?. V g E .£kS S w c e PELLET <?ehAove& 2.1 — 29 22 t v E t K S S I N C E p e LL E r R e m w . AWTC> — Wf 27 z - I?' tt » 3 " 30 0 10 2? 19 3t W E l Q H T ^0 S"t t4 4t t2 44, 6>4 7 M t t 4 ? J t 2 t 4 J 2? 2t 3t r t 30 S4 + 7.0 *4 4: 9.0 S9 w e i Q t + r <? 500 % 3S0 n t 22V 2 l t 20H-20*f lib 334 35TO 292 33? 33? 31? 298 3 10 332 3 "2-0 112. 342 37t 332-3 1 4 Testes: Tes t i cu la r atrophy and absence of spermatogenesis occurred upon estrogen administrat ion. Recovery of s ize and function followed d iscont in-uation of the treatment and th is phenomenon confirmed complete removal of a' the p e l l e t . . This confirmation was p a r t i c u l a r l y he lpfu l i n the case of the independent tumors. Adrenals: The adrenal weights i n the estrogen treated group were above normal, considering the s ize of the animals. Variat ions between pe l le ted and non-pelleted animals were smal l . However, when the estrogen was discontinued, the r a t i o of adrenal weights to animal weight,-be came smaller, suggesting that the estrogen had a trophic effect on the adrenal glands. In any case, the large adrenal weight gave no ind ica t ion that the tumor was producing.an ACTH-suppressing substance leading to adrenal atrophy. On sect ion, no s ign i f i can t changes were noted i n the zona fasc icu la ta or zona r e t i c u l a r i s . However, upon examination of the zona glomerulosa there was a cor re la t ion between estrogen administrat ion and zone thickness and c e l l morphology. In the animals receiving estrogen, the zona glomerulosa was 3 - 5 c e l l s i n thickness and c e l l s appeared shrunken while i n the animals without the p e l l e t the thickness was 5 - 7 c e l l s and the c e l l s were plumper. This suggested the p o s s i b i l i t y that the a c t i v e l y growing tumor was producing a minera locor t icoid and suppressing s t imulat ion of the zona glomerulosa. The kidney tubule c e l l s were examined for evidence of minera locor t icoid excess but none was found. ^ ' ^ ' P i t u i t a r y : There was a def in i te estrogen effect on the p i t u i t a r y . Pe l le ted animals, whether tumor-bearing or not, had p i t u i t a r y weights 1 . 5 - 2 times those of non-pelleted animals, despite the fact that the pe l l e ted animals were smaller. No cytologic differences were noted on H & E sect ions. Lungs: During the course of the experiment the animals were frequently a f f l i c t e d wi th the " s n i f f l e s " . On several occasions th i s state progressed to a pneumonia wi th labored breathing, weight loss and lethargy. These animals received chloramphenicol and responded promptly. Two animals had to be sac r i f i ced because of ver t igo ; th i s s i tua t ion has been ascribed to distant spread of organisms from a pneumonitis to the middle ear with subsequent d is rupt ion of ves t ibu la r funct ion. Kidneys: While examining the kidneys f o r the p o s s i b i l i t y of nephropathy r e s u l t i n g from excess m i n e r a l o c o r t i c o i d p r o d u c t i o n , p r o m i n e n t accumulations of yellow-brown r e f r a c t i l e vacuoles were seen i n both the proximal and d i s t a l convoluted tubular l i n i n g c e l l s and o c c a s i o n a l l y i n the tubular lumina of animals r e c e i v i n g estrogen. These bodies stained negatively f o r i r o n but took up Schmorl's s t a i n under conditions which caused the lipochrome of the zona r e t i c u l a r i s to s t a i n . I t would thus appear l i k e l y that t h i s pigment was a degenerative lipochrome re l a t e d to estrogen administration. ELECTRON MICROSCOPE FINDINGS  Primary growth tumors C e l l types: The vast majority of the tumor c e l l s are polygonal with a moderately electron-dense cytoplasm ( F i g . 10). A c e n t r a l nucleus containing a small amount of p e r i p h e r a l l y clumped chromatin and one or more u s u a l l y c e n t r a l n u c l e o l i i s present i n the glutaraldehyde-fixed t i s s u e . In the cytoplasm, mitochondria, polysomes, rough and smooth endoplasmic reticulum, f a t droplets, and Golgi apparatus, are seen r e g u l a r l y . Desmosomes, c e n t r i o l e s , i n t r a c e l l u l a r f i b r i l s and lysosomes are o c c a s i o n a l l y present ( F i g . l l ) . A less frequent c e l l type i s a f l a t t e n e d c e l l which i s u s u a l l y found wedged i n between surrounding polygonal c e l l s (Figs. 12, 13)- These c e l l s have organelles i n the same proportion as t h e i r p a l e r counterparts though i n some c e l l s the mitochondria showed a tendency toward, degeneration. The most s t r i k i n g feature of these c e l l s i s the high e l e c t r o n density of both nucleus and cytoplasm. This i s due to an increased density of the nuclear sap, cytoplasmic ground substance, and mitochondrial matrix rather than to any p a r t i c u l a t e or organellar change. One instance of mitosis was noted i n one of these dark c e l l s ( F i g . 1^1-). In addition to the parencb-ymal c e l l s , endothelium, capsular and septal f i b r o b l a s t s , macrophages and the rare eosinophil are i d e n t i f i e d . Parenchymal c e l l organelles: Mitochondria. The most s t r i k i n g u l t r a -s t r u c t u r a l feature of the tumor i s the mitochondria ( F i g s . 15, 16, 17). They are l a r g e , v a r i a b l e i n s i z e and morphology, and as i n the normal adrenal cortex they are the c h i e f cytoplasmic constituent. However, while the F i g . 10 Primary growth c e l l s - low magnificat ion. F i g . 11 Primary growth - centr io le and Golgi apparatus. x20,700 F i g . 13 Dark c e l l . x7,100 F i g . 16 Primary growth - mitochondrial morphology. x8,500 m i t o c h o n d r i a o f t h e i n n e r zones o f t h e a d r e n a l c o r t e x have t u b u l o v e s i c u l a r c r i s t a e , t h e c r i s t a e i n t h i s tumor a r e l i n e a r w i t h a v a r i e t y o f c o n f i g u r a t i o n s . The v a r i o u s p a t t e r n s o f t h e c r i s t a e a r e shown i n t h e a c c o m p a n y i n g f i g u r e s and r ange f r o m r e c t i l i n e a r l a m e l l a e t o c o n c e n t r i c c i r c l e s t o i r r e g u l a r f o r m s . I n a d d i t i o n , b i z a r r e d i s t o r t e d forms i n w h i c h t h e m i t o c h o n d r i o n a p p e a r s t o be f o l d e d o v e r a p o r t i o n o f c y t o p l a s m ( F i g . 18) a r e s e e n i n some t u m o r s . The m a t r i x i s m o d e r a t e l y e l e c t r o n - d e n s e , w h i l e t h e i n t e rmembranous s p a c e i s c l e a r b y c o m p a r i s o n . I n some i n s t a n c e s , amorphous g r a n u l e s a r e p r e s e n t w i t h i n t h e m a t r i x , w h i c h have a n e l e c t r o n d e n s i t y s i m i l a r t o l i p i d ( F i g . 1 8 ) / These a r e i n a d d i t i o n t o t h e s m a l l e r , more d i s c r e t e m i t o c h o n d r i a l dense g r a n u l e s . R i b o s o m e s : A f t e r m i t o c h o n d r i a , t h e most common c y t o p l a s m i c component i s t h e r i b o s o m e s . G e n e r a l l y g r o u p e d i n p o l y r i b o s o m a l a g g r e g a t e s ( F i g . 17, 20'), t h e s e s t r u c t u r e s f i l l mos t o f t h e r e m a i n i n g c y t o p l a s m . The o c c a s i o n a l c e l l has c y t o p l a s m a l m o s t e n t i r e l y composed o f r i b o s o m e s ( F i g . h-2). G o l g i a p p a r a t u s : One o r more G o l g i zones ( F i g . l l ) c a n be s e e n i n t h e m a j o r i t y o f c e l l s s e c t i o n e d i n t h e p l a n e o f t h e n u c l e u s and i n many s e c t i o n e d i n o t h e r p l a n e s as w e l l . The a p p a r a t u s i s as a r u l e w e l l d e v e l o p e d and c o n v e n t i o n a l i n f o r m . F r e q u e n t l y , one o r b o t h p a r t s o f a c e n t r i o l e i s o b s e r v e d i n t h e m i d d l e o f t h e G o l g i z o n e . The l a r g e number o f t h e s e c e n t r i o l a r s t r u c t u r e s , p l u s t h e f a c t t h a t some a r e p r e s e n t i n t h e c e l l p e r i p h e r y and away f r o m t h e G o l g i a p p a r a t u s s u g g e s t two p o s s i b i l i t i e s : t h e tumor c e l l s a r e a c t i v e l y d i v i d i n g and t h e c e n t r i o l e s a r e t h e r e f o r e p r o m i n e n t , o r t h a t some o f them r e p r e s e n t c r o s s - s e c t i o n s o f c i l i a r y b a s a l b o d i e s . No t r u e c i l i a were s e e n w i t h i n v a c u o l e s o r a t t h e c e l l s u r f a c e . F a t d r o p l e t s : V a r i a b l e numbers o f f a t d r o p l e t s , u s u a l l y i n c l u s t e r s and a v e r a g i n g 700 mu i n s i z e a r e p r e s e n t ( F i g . 10). These a r e a t t i m e s o b s e r v e d t o be c l o s e l y a t t a c h e d t o m i t o c h o n d r i a ( F i g . 21) w i t h an a p p a r e n t d i s s o l u t i o n o f t h e o u t e r m i t o c h o n d r i a l membrane a t t h e p o i n t o f c o n t a c t as d e s c r i b e d i n t h e n o r m a l a d r e n a l ^ b y I d e l m a n . O c c a s i o n a l l y a m i t o c h o n d r i o n i s s e e n e n v e l o p i n g a f a t d r o p l e t . E n d o p l a s m i c r e t i c u l u m : S c a t t e r e d s m a l l c o l l e c t i o n s o f r o u g h o r smooth e n d o p l a s m i c r e t i c u l u m a r e s e e n b u t a r e n o t a s t r i k i n g f e a t u r e o f t h e t u m o r . C e l l membranes: The c e l l membranes a r e p r e d o m i n a n t l y s t r a i g h t and w i t h o u t s p e c i a l fo rms o f a t t a c h m e n t l e a v i n g a 200A e l e c t r o n - l u c e n t s p a c e b e t w e e n F i g . 18 Primary growth - mitochondria enfolding cytoplasm. xlA,700 - 9 -a d j o i n i n g c e l l s . A t p o i n t s where more t h a n two c e l l s meet t h e r e i s a t r i a n g u l a r i n t e r c e l l u l a r space w i t h o f t e n a few a d j a c e n t m i c r o v i l l o u s p r o j e c t i o n s . O n l y r a r e l y do t h e s e become c o n v o l u t e d . I n t h e t r a b e c u l a r a r e a s t h e tumor c e l l s a r e prone t o p u l l a p a r t l e a v i n g wide i n t e r c e l l u l a r spaces w i t h d a n g l i n g m i c r o v i l l i ( F i g . 2 2 ) . I n c o n t r a s t t o t h e n o r m a l a d r e n a l , w i d e l y s c a t t e r e d desmosomes a r e seen ( F i g . 2 3 ) . These a r e composed o f s h o r t zones where f i n e e l e c t r o n - d e n s e g r a n u l e s s u r r o u n d a t h r e e - l a y e r e d membrane s h a r e d b y t h e two a d j o i n i n g c e l l s . The c a p i l l a r i e s a r e s u r r o u n d e d by a c o n t i n u o u s basement membrane and i n some i n s t a n c e s an e p i t h e l i a l basement membrane i s p r e s e n t as w e l l ( F i g . 2k), as i s seen n o r m a l l y i n e n d o c r i n e t i s s u e s . S c a t t e r e d s i n g l e g r a n u l e s w i t h an e l e c t r o n - l u c e n t r i m and a g g r a g a t e s o f s i m i l a r g r a n u l e s a r e p r e s e n t w h i c h resemble t h e l i p o f u s c i n pigment d e p o s i t s o f t h e n o r m a l zona r e t i c u l a r i s . S p a r se c o l l a g e n f i b r i l s a r e seen i n t h e s e p t a . ( F i g . 25 , 2 6 ) . R e g r e s s i n g tumors: Low power p h o t o m i c r o g r a p h s ( F i g s . 27 , 28) c o n f i r m t h e i m p r e s s i o n g a i n e d f r o m t h e l i g h t m i c r o s c o p e t h a t t h e number o f p a r e n c h y m a l c e l l s p e r u n i t a r e a i s much l e s s i n r e g r e s s i n g t h a n i n g r o w i n g tumors and t h e c e l l s appear s m a l l e r . Numerous e o s i n o p h i l s a r e p r e s e n t as w e l l as s c a t t e r e d macrophages and p l a s m a c e l l s . Accompanying t h e d e c r e a s e i n c e l l s i z e and number t h e r e i s a d e c r e a s e i n t h e number and c o m p l e x i t y o f o r g a n e l l e s . The m i t o c h o n d r i a a r e s m a l l and compact and have o n l y a few r e c t i l i n e a r o r i r r e g u l a r c r i s t a e ( F i g s . 2 9 , 3 0 ) . The p o l y r i b o s o m a l a g g r e g a t e s and f a t d r o p l e t s a r e g r e a t l y r e d u c e d and t h e G o l g i a p p a r a t u s / c e n t r i o l a r complexes a r e not as w e l l d e v e l o p e d as i n t h e g r o w i n g tumors. O t h e r i n t r a c y t o p l a s m i c m a t e r i a l s i n c l u d e i r r e g u l a r a g g r e g a t e s o f m o d e r a t e l y f i n e , . h l g h - l y l e l e c t r o n -opaque g r a n u l e s ( F i g s . 3 0 , 3 l ) w h i c h a r e a l s o e l e c t r o n - d e n s e i n u n s t a i n e d s e c t i o n s ( F i g . 3 2 ) . They a l s o o c c u r o c c a s i o n a l l y i n l a r g e a g g r e g a t e s s u r r o u n d e d b y m a t e r i a l h a v i n g t h e appearance o f m i t o c h o n d r i a l m a t r i x ( F i g . 3 0 ) . The o c c a s i o n a l tumor c e l l g i v e s t h e appearance o f h a v i n g i n t r a c e l l u l a r f i b r i l s ( F i g s . 33 , 3^ -) r e s e m b l i n g c o l l a g e n and o t h e r s c o n t a i n f r e e e o s i n o p h i l g r a n u l e s ( F i g s . 2 9 , 31 , 35, 3 6 ) . The l a t t e r u s u a l l y had a v i a b l e e o s i n o p h i l g r a n u l o c y t e i n t h e v i c i n i t y . The g r a n u l e s were a l s o seen i n t r a v a s c u l a r l y ( F i g . 37)-Fig. 23 Primary growth - desmosomes. x24,600 F i g . 25 Primary growth - septal collagen f i b r i l s . x6,700 Fig. 29 Regression - mitochondrial morphology. x9,700 F i g . 31 Regression - eosinophil granules within tumor c e l l s . xl0,700 F i g . 32 Regression - intracytoplasmic dense granules. Unstained. xl0,700 F i g . 34 R e g r e s s i o n - c o l l a g e n f i b r i l s i n t u m o r c e l l . x9,000 F i g . 36 Regression - l i p o f u s c i n granules. x8,900 -10-I n one c e l l c r o s s - s e c t i o n s o f two c e n t r i o l e s o r c i l i a r y b a s a l b o d i e s a r e seen i n an a r e a o f m i t o c h o n d r i a and a g r a n u l a r r e t i c u l u m ( F i g . 3 8 ) . These d e m o n s t r a t e d 9 groups o f p e r i p h e r a l f i b e r s and no c e n t r a l f i b e r s . -.: Lysosomes were o b s e r v e d i n s m a l l numbers i n c e l l s f r o m a l l tumor g r o u p s . They were, i n t e r e s t i n g l y , more f r e q u e n t i n t h e g r o w i n g tumors t h a n i n t h e r e g r e s s i n g . The a v e r a g e number p e r c e l l i n p r i m a r y g r o w t h tumors r a n g e d f r o m 1 t o 3 w i t h an o v e r a l l a v e r a g e o f I . 8 5 . I n t h e r e g r e s s i n g tumor c e l l s t h e range was f r o m 0 .03 t o 1 w i t h an a v e r a g e o f 0 A 5 - The s e c o n d a r y g r o w t h tumors had an o v e r a l l a v e r a g e of l.h. The r e g r e s s i n g tumors w h i c h c o n t a i n e d t h e s m a l l e s t numbers o f ( p r i m a r y ) lysosomes a l s o had t h e most p r o m i n e n t and numerous r e s i d u a l b o d i e s and m y e l i n f i g u r e s . Non-parenchymal elements i n t h e tumor i n c l u d e d i n t e r c e l l u l a r c o l l a g e n f i b e r s , macrophages and e o s i n o p h i l s . The l a s t - n a m e d had c h a r a c t e r i s t i c g r a n u l e s ( F i g . ^ l ) . S e c o n d a r y growth: The c e l l s o f tumors u n d e r g o i n g s e c o n d a r y growth had no u n i q u e f e a t u r e s a t t h e u l t r a s t r u c t u r a l l e v e l , b u t r a t h e r showed c h a r a c t e r i s t i c s o f e i t h e r t h e g r o w i n g ( F i g s . k2, k-3, hh) o r r e g r e s s i n g ( F i g . -^5) tumor c e l l s as w o u l d be a n t i c i p a t e d f r o m l i g h t m i c r o s c o p y . Fig. 38 Regression - c i l i a ? or double centriole? x 5 A , 0 0 0 Fig. AO Regression - myelin figure. xll , 0 0 0 F i g . 41 C r y s t a l l o i d of eosinophil granule. xl63,000 F i g . 42 Secondary growth - ac t i v e c e l l s . x8,100 F i g . A3 S e c o n d a r y g r o w t h - a c t i v e c e l l s . x 5 , 0 0 0 F i g . AA S e c o n d a r y g r o w t h - e o s i n o p h i l i n f i l t r a t e . x 3 , 1 0 0 -11-DISCUSSION H i s t o r i c a l : In 1896 Beatson observed that ovariectomy was b e n e f i c i a l i n human breast cancer. In•1905 Lathrop and Loeb reduced the incidence of breast cancer i n a s t r a i n of mice with a high rate of spontaneous mammary carcinoma by ovariectomy p r i o r to s i x months of age. Following this up, i t was l a t e r found that male mice of the same s t r a i n , o r d i n a r i l y immune to the carcinoma, would develop i t with ovarian grafts; and i n 1932 Lacassagr produced breast cancer i n male mice with an extract of germinal f o l l i c l e s . Estrogens were i d e n t i f i e d as the carcinogen by Lacassagne i n mice and by 37 Geshickter i n the rat some t h i r t y years after Lathrop and Loeb's observation. These and subsequent experimental and c l i n i c a l studies have led to the following general pri n c i p l e s of endocrine carcinogenesis: •1) Cancer i s not necessarily autonomous, but may be hormone dependent. 2) Cancer can be maintained by hormones at physiological levels 3) Hormones regulate responses for which c e l l s possess i n t r i n s i c properties rather than i n i t i a t i n g new . . 3h reactions h) Carcinogenesis may be influenced by hormones either d i r e c t l y ( v i a hyperplasia) or by a permissive action (as i n chemical carcinogenesis) Mechanisms of hormonal carcinogenesis: The following immediate mechanisms of action are currently under consideration as possible pathways by which hormones may cause tumors. Hormones may: 1) Give r i s e to neoplasia-induced metabolites. 2) Cause tissues to produce carcinogens. 3) Provide for the growth of tissues upon which other mechanisms a c t . ^ In considering endocrine carcinogenesis, one has an environment, acting to produce a tumor, which manifests varying degrees of hormone - 1 2 -r e s p o n s i v e n e s s . 3 2 2 1 b y F u r t h and Dao as f o l l o w s : These t h r e e a s p e c t s o f t h e p r o b l e m have been s u b d i v i d e d E n v i r o n m e n t I n i t i a t i o n Tumor Degree o f R e s p o n s i v e n e s s i n d u c t i o n M u t a t i o n / N s e l e c t i o n -J> P r o m o t i o n — ^ . P r o g r e s s i o n Hormones Hormone dependent Hormone r e s p o n s i v e Autonomous Dependent - exogenous hormones - endogenous hormones R e s p o n s i v e Non-re spons i v e V i r u s Hormone-induced h y p e r p l a s i a Many e x p e r i m e n t a l models o f t h e s e v a r i o u s c l a s s e s o f ; h o r m o n e - r e s p o n s i v e tumors have been d e v e l o p e d i n t h e p i t u i t a r y , t h y r o i d , o v a r y , u t e r u s , t e s t i c u l a r i n t e r s t i t i a l c e l l s , mammary g l a n d s , and a d r e n a l s . Among t h e v a r i o u s h o rmonal m a n i p u l a t i o n s employed t o p r o d u c e them, a d d i t i o n o f exogenous e s t r o g e n s o r r e m o v a l o f endogenous e s t r o g e n s appear t o be g e n e r a l l y e f f e c t i v e . Thus, one o f t h e b a s i c q u e s t i o n s i n t h i s f i e l d i s : what i s t h e e f f e c t o f e s t r o g e n on tumors s e n s i t i v e t o i t ? I n t h e p r e s e n t s t u d y , t h i s e f f e c t i s i n v e s t i g a t e d u s i n g as a model a r a t a d r e n a l tumor i n d u c e d by e s t r o g e n and dependent upon i t f o r i t s g r o wth. By way o f b a c k g r o u n d I w i l l f i r s t c o n s i d e r t h e p h y s i o l o g i c mechanisms o f e s t r o g e n a c t i o n and s e c o n d l y t h e e n d o c r i n e a s p e c t s o f v a r i o u s a d r e n a l tumors f r o m t h e l i t e r a t u r e . k2 The c u r r e n t h y p o t h e s e s w i t h r e g a r d t o t h e a c t i o n o f e s t r o g e n s a r e : autonomous 1 ) 2 ) 3 ) Hormones may c o m plete some p r o t e i n s t o f o r m an a c t i v e enzyme. 98 Hormones may a c t as a l l o s t e r i c e f f e c t o r s o f enzymes. E s t r o g e n s i n c r e a s e t r a n s h y d r o g e n a s e a c t i v i t y p r o v i d i n g n£ 1 0 1 f o r b i o s y n t h e t i c r e a c t i o n s ( s i n c e abandoned) TPKH^o d r ATP Hormones a c t as gene a c t i v a t o r s Hormonal gene a c t i v a t i o n c a n a c t u a l l y be v i s u a l i z e d i n i n s e c t s E c d y s o n e , t h e p u p a t i n g hormone, w h i c h has t h e b a s i c s t e r o i d n u c l e u s , p r o d u c e s a v i s i b l e p u f f - l i k e e n l a r g e m e n t o f g i a n t chromosomes and t h i s e f f e c t i s f o l l o w e d b y an i n c r e a s e i n messenger RNA l e v e l s ( F i g . U 6 ) . 5 ) Membrane p e r m e a b i l i t y ^ 3 H e c h t e r p o s t u l a t e s t h a t s t e r o i d hormones o p e r a t e by i n s e r t i n g t h e m s e l v e s as a s e r i e s o f h e x a g o n a l d i s c s i n t o r e c e p t o r s i t e s on c e l l 56. -13-membranes, thus a l t e r i n g i t s conformation and permeabil i ty . This would account for the estrogen-induced increase i n trans-membrane movement of 87 amino-acids noted by Segal. A variant of th i s theory proposes that RNA inducifcionu i s a resul t of changes i n the Na/K ra t io which i n turn are the resu l t of hormone-induced permeabil i ty changes. I t has been recent ly demonstrated that l abe l l ed estrogen i s bound p r e f e r e n t i a l l y to c e l l s of the uterus, vagina, anterior p i t u i t a r y , hamster 92 53 59 72 29 85 kidney, DMBA-induced mammary tumors, ' and human breast tumors. The reason for th i s preference has been demonstrated i n cer ta in instances to 53 be an estrogen-specific p ro te in receptor. In addi t ion , those breast tumors which had a good response to oophorectomy had'a greater estrogen uptake hi 85 than those wi th a poor response. ' Both of these groups had a greater uptake than the estrogen-independent tumors of the same type. 71 23 Autoradiographic and biochemical studies indicate that the s ign i f i can t binding s i t e for estrogen i s i n the nucleus. No biochemical 52 transformation occurs. The subsequent events fol low two routes: l ) ac t iva t ion of p re -ex i s t ing enzyme machinery - th i s i s unaffected by actinomycin D. 2) an increase i n RNA polymerase leading to new ribosome, 97 new enzyme and then new pro te in production. Recent experiments suggest that th i s l a t t e r effect i s the resul t of ac t iva t ion of RNA polymerase from an inac t ive s tate . Much current in teres t centers around the p o s s i b i l i t y that g estrogens may interact wi th histones with resultant derepression of the histone-associated, DNA-dependent, RNA polymerase. In a survey of adrenal c o r t i c a l tumors of animals, W i l l i s states that c o r t i c a l adenomas and carcinomas are among the commonest v i s c e r a l tumors of ca t t l e and that they also occur i n the hen and wart-hog. Among mouse s t r a ins , frequent spontaneous tumors occur i n the NH and Bagg a lb ino . In te res t ing ly , i n the former there i s an ear ly menopause. In ra t s , spontaneous tumors occur with frequency only i n the Osborne Mendel s t r a i n . Experimentally induced mouse adrenal tumors were f i r s t produced by 27 Woolley i n 19^1 employing neonatal ovariectomy. This act ion was blocked by 105 s t i l b e s t r o l r a i s ing the question whether tumorigenesis occurred v i a the 11 12 p i t u i t a r y . Browning reported s imi l a r f ind ings . ' S i m i l a r l y , Houssay and -Ik-i l l 14.14. 1 4 V G a r d e z a ' ' f o u n d t h a t s t i l b e s t r o l i n h i b i t e d t h e development o f 2 8 a d r e n a l c a r c i n o m a s i n c a s t r a t e d a l b i n o r a t s and F l a k s o b t a i n e d a h i g h l y m a l i g n a n t ' a d r e n o c o r t i c a l c a r c i n o m a by ACTH t r e a t m e n t o f gonadectomized m i c e . R o l e s f o r ACTH, GTH and an u n d e t e r m i n e d p i t u i t a r y hormone have been i n d i c a t e d . Most a d r e n a l tumors p r o d u c e d by t h i s means, i f f u n c t i o n a l , ;ro£ 1 5 A d r e n a l tumors have a l s o been p r o d u c e d b y t h e i n j e c t i o n o f e s t r o g e n i c )mmc 5 5 s e c r e t e e s t r o g e n s o r m i x t u r e s o f e s t g e n s and a n d r o g e n s ^ ^ r a t h e r t h a n g l u c o - o r m i n e r a l o - c o r t i c o i d s . ' s u b s t a n c e s i n t o m i c e , ^ b u t more commonly i n r a t s ^ (where t h e r e i s a s t r a i n d i f f e r e n c e ) and i n hamsters.' P r o g e s t e r o n e , on t h e o t h e r hand, i s n o t t u m o r - i n d u c i n g , b u t may be c o - c a r c i n o g e n i c , s h o r t e n i n g t h e l a t e n t p e r i o d and i n c r e a s i n g t h e y i e l d i n e s t r o g e n and c h e m i c a l c a r c i n o g e n e s i s - 5 1 + J 7 6 , 9 ^ j n Qther s i t u a t i o n s , s u c h as 1 8 an e s t r o n e - i n d u c e d mammary c a r c i n o m a o f r a t s , p r o g e s t e r o n e p r o l o n g e d t h e l a t e n t p e r i o d and c a u s e d t e m p o r a r y r e g r e s s i o n o f e s t a b l i s h e d tumors. D e t e r m i n i n g t h e degree o f hormone dependency and t e s t i n g t h e r e l a t i o n s h i p o f t r o p h i c hormones t o hormone p r o d u c t i o n by t h e .tumor a r e most r e a d i l y c a r r i e d out i n t r a n s p l a n t a b l e c a r c i n o m a s . Such tumors have a r i s e n s p o n t a n e o u s l y and a f t e r c a s t r a t i o n o r i r r a d i a t i o n . They v a r y i n t h e i r hormone p r o d u c t i o n and e s t r o g e n i c , a n d r o g e n i c , g l u c o c o r t i c o i d o r m i n e r a l o c o r t i c o i d 2 6 9 a c t i v i t y . Host c a s t r a t i o n o r hypophysectomy i s r e q u i r e d f o r t h e p r o l i f -e r a t i o n o f t r a n s p l a n t a b l e mouse tumors p r o d u c e d b y gonadectomy. O t h e r s u c h tumors r e p o r t e d t o have gene.ralikyrbeen autonomous . Whether t h e e f f e c t o f e s t r o g e n on t h e tumor under s t u d y i s v i a t h e p i t u i t a r y r a t h e r t h a n d i r e c t i s p r o b l e m a t i c a l i n t h e absence o f a u t o r a d i o g r a p h i c s t u d i e s o r e x p e r i m e n t s e m p l o y i n g hypophysectomy. The r o l e o f t h e p i t u i t a r y has been v a r i a b l e i n p r e v i o u s l y r e p o r t e d h o r m o n e - r e s p o n s i v e 9 9 tumors o f t h e b r e a s t and a d r e n a l . Hypophysectomy p r e v e n t e d gonadectomy-p r o d u c e d o r spontaneous a d r e n a l tumors o f m i c e . ^ ' ^ L i k e w i s e i t p r e v e n t e d 5 7 e s t r o g e n s t i m u l a t i o n o f mammary c a r c i n o m a and c a u s e d r e g r e s s i o n o f 1 9 2 0 e s t a b l i s h e d tumors. ' However, t h e absence o f t h e p i t u i t a r y was w i t h o u t J+7 e f f e c t on o t h e r e s t r o g e n - i n d u c e d mammary tumors o r on t h e i n t e r a c t i o n 6 6 5 8 between b r e a s t t i s s u e and androgens. Kim ~ f o u n d t h a t e s t r o g e n , p r o g e s -- 1 5 -terone, and testosterone had both a d i r e c t and an i n d i r e c t e f f e c t v i a the U7 p i t u i t a r y on DMBA-induced breast tumors. Huseby reported s i m i l a r r e s u l t s . Adrenal secretory a c t i v i t y and ACTH a c t i v i t y have been both i n c r e a s e d ^ ^ ' ^ 1 ' ^ 2 and decreased^'"'"^ by e s t r a d i o l under d i f f e r e n t circumstances. Gompertz^ has presented evidence i n d i c a t i n g that e s t r a d i o l i i n c r e a s e s the e f f e c t of ACTH. A l l these d i f f e r e n t e f f e c t s are not s u r p r i s i n g , considering the number of l e v e l s at which the adrenal axis and the gonadal axis can i n t e r a c t . Thus, on the basis of existent information i t i s not possible to p r e d i c t the route of estrogen e f f e c t on the adrenal tumor or to i n t e r p r e t the enlarged p i t u i t a r y , the borderline adrenal enlargement, or the hypoplasia of the zona glomerulosa. The question of the r e l a t i o n s h i p of ;.tissue i n t e g r i t y to tumor autonomy has been raised by the work of Algard"1" which demonstrated tumor dependence i n vivo, autonomy i n t i s s u e c u l t u r e , and an intermediate p i c t u r e i n organ cult u r e . Quantitation of steroids and trophic hormones i n plasma and i n the appropriate organs would shed a good deal of l i g h t on these problems as w e l l as help sort out the changes taking place i n those tumors that become autonomous. Light microscope f i n d i n g s . The h i s t o l o g i c p i c t u r e of t h i s tumor when a c t i v e l y growing i s one of sheets of regular polyhedral c e l l s with moderate m i t o t i c a c t i v i t y and sparse, small f o c i of necrosis. When the tumor i s shrinking a f t e r the removal of estrogen support, the necrosis i s a c t u a l l y diminished. The processes accounting f o r the decrease i n bulk are atrophy and a markedly decreased m i t o t i c a c t i v i t y , together with a pronounced reduction i n c e l l s i z e and nuclear p.yknosis. The occurrence of the trabecular pattern may be a r t e f a c t u a l as suggested by the work of Bohman et a l " ^ , which shows that the separation of cells seen i n e l ectron microscope preparations may be prevented by i n f u s i n g a f i x a t i v e s o l u t i o n of r e l a t i v e l y high osmolarity. The preponderance of the trabecular, pattern i n the regressing tumors would indicate an a c t u a l a l t e r a t i o n i n c e l l (surface) properties as a background f o r the trabeculation even i f a r t e f a c t u a l . -16-79 The d i f f e r e n t c e l l t y p e s seen i n t h e tumors, namely t h e l i g h t c e l l s , t h e d a r k c e l l s and t h e c l e a r ( c y t o l y t i c ) c e l l s a l l w o u l d appear t o have t h e i r c o u n t e r p a r t s i n t h e n o r m a l a d r e n a l r e s p o n d i n g t o v a r i o u s p h y s i o l o g i c s t i m u l i . The p r e d o m i n a n t l i g h t c e l l c o r r e s p o n d s t o t h e e u t r o p h i c c e l l w h i l e t h e d a r k c e l l s c o r r e s p o n d t o t h o s e c e l l s w i t h dense c y t o p l a s m seen most commonly i n t h e zona r e t i c u l a r i s . ^ ^ These w o u l d appear t o "be r e s t i n g o r e l a b o r a t i n g c e l l s o f some s o r t and a r e a b s e n t i n r e g e n e r a t i n g a d r e n a l t i s s u e 88 The c l e a r c e l l s were d e s c r i b e d b y S e l y e and Stone i n t h e n o r m a l a d r e n a l where 88 t h e y o c c u r r e d w i t h i n t e n s e c o r t i c o t r o p h i c s t i m u l a t i o n . They and C o t t e 17 and C o t t e f e l t t h a t t h i s was a s s o c i a t e d w i t h h o l o c r i n e s e c r e t i o n b u t t h i s w o u l d not f i t w e l l w i t h t h e c o n c e p t t h a t a d r e n a l s t e r o i d s a r e n o t s t o r e d i n t e r c e l l u l a r l y . The c l e a r c e l l s seen i n t h e g r o w i n g tumors a r e a l s o s i m i l a r t o t h e f i n d i n g s i n t h e e a r l y s t a g e s o f t u b u l a r n e c r o s i s d e s c r i b e d by R i c h ^ and W i l b u r and R i c t u ^ ' T h i s f o r m o f n e c r o s i s was p r o d u c e d b y f u l m i n a t i n g i n f e c t i o n o r h i g h ACTH l e v e l s . The r e l a t i o n s h i p o f t h i s c y t o l y s i s t o t h e v a c u o l i z a t i o n a s s o c i a t e d w i t h m e t h y l a n d r o s t e n e d i o l a d m i n i s t r a t i o n ^ ' ^ i s p r o b l e m a t i c a l . These c l e a r c e l l s c o u l d n o t be i d e n t i f i e d i n e l e c t r o n m i c r o s c o p e p r e p a r a t i o n s . Three p o s s i b l e e x p l a n a t i o n s a r e s u g g e s t e d : l ) t h e r e were no c l e a r c e l l s i n t h e t i s s u e t a k e n f o r e l e c t r o n m i c r o s c o p y , 2 ) t h e v a c u o l a r c o n t e n t s do n o t s t a i n w i t h h e m a t o x y l i n and e o s i n b u t a r e e l e c t r o n - d e n s e a f t e r l e a d and u r a n i u m s t a i n i n g , 3 ) t h e l i g h t m i c r o s c o p i c appearance i s an a r t i f a c t o f f o r m a l i n f i x a t i o n . W i t h r e g a r d t o t h e d a r k c e l l s , i n DMBA-induced b r e a s t tumors under-2 g o i n g e s t r o g e n t r e a t m e n t , A r c h e r f o u n d t h a t t h e d a r k c e l l s i n t h a t s i t u a t i o n had many e l e c t r o n - d e n s e d r o p l e t s w i t h i n c i s t e r n a e i n d i c a t i v e o f p r o t e i n p r o d u c t i o n . The p a l e c e l l s , on t h e o t h e r hand, c o n t a i n e d l a r g e l i p i d - f i l l e d v a c u o l e s as a p a r t o f a d e g e n e r a t i v e p r o c e s s . T h i s w o u l d n o t ap p e a r t o be what i s h a p p e n i n g i n t h e a d r e n a l tumors under s t u d y . The f i n d i n g s i n t h e r e g r e s s i n g tumors a r e s i m i l a r t o t h o s e o f S c o t t 86 85 e t a l and o f Sander and AttramLadel who o b s e r v e d mouse mammary tumors f o l l o w i n g oophorectomy. They d e s c r i b e d i n s i g n i f i c a n t n e c r o s i s and r e d u c t i o n o f c e l l s i z e b y 50%. S c o t t e t a l . s u g g e s t e d t h a t t h e a t r o p h y t o o k p l a c e b y autophago< an immunologic p r o c e s s . 93 c y t o s i s and t h a t i t c o u l d r e p r e s e n t t h e r e s u l t o f - 1 7 -O t h e r p r e v i o u s s t u d i e s o f r e g r e s s i n g , h o r m o n e - r e s p o n s i v e tumors 2 0 i n c l u d e t h o s e o f C u t t s who o b s e r v e d c e l l s h r i n k a g e t o 13$>5 n u c l e a r p y k n o s i s , a d e c r e a s e i n number o f d i v i d i n g c e l l s , c y s t i c s p a c e s , and l e u k o c y t i c i n f i l t r a t i o n a f t e r r e m o v a l o f e s t r o n e f r o m an e s t r o g e n - d e p e n d e n t b r e a s t t u m o r . 1 0 7 Young e t a l . o n t h e o t h e r hand o b s e r v e d no h i s t o l o g i c change b e t w e e n g r o w i n g and s p o n t a n e o u s l y r e g r e s s i n g mammary t u m o r s . E l - B o l k a i n y 2 5 e t a l . d e s c r i b e d t h e r e g r e s s i v e e f f e c t s o f e s t r o g e n a d m i n i s t r a t i o n o n a s p o n t a n e o u s a d r e n a l t u m o r . T h e r e was m i t o t i c a r r e s t and l i p i d a c c u m u l a t i o n i n i n t a c t a n d h y p o p h y s e c t o m i z e d a n i m a l s c o n s i s t e n t w i t h a d i r e c t t o x i c e f f e c t i n t h i s i n s t a n c e . These f i n d i n g s w o u l d f i t i n w e l l w i t h t h e t h e o r y t h a t s m a l l doses o f e s t r o g e n s a c t i n g v i a t h e p i t u i t a r y a r e s t i m u l a t o r y t o 5 2 c e r t a i n t i s s u e s w h i l e l a r g e dose s c a n e x e r t a d i r e c t i n h i b i t o r y e f f e c t . A h i s t o l o g i c f e a t u r e n o t p r e v i o u s l y d e s c r i b e d i n e x p e r i m e n t a l w o r k o n r e g r e s s i n g tumors i s t h e m a s s i v e i n f i l t r a t i o n o f e o s i n o p h i l s s e e n i n t h a t s i t u a t i o n . The s i g n i f i c a n c e o f t h e e o s i n o p h i l s i s n o t c l e a r a l t h o u g h t h e y c o u l d be a r e f l e c t i o n o f an i m m u n o l o g i c p r o c e s s . I t has b e e n d e m o n s t r a t e d 81+ t h a t e o s i n o p h i l s a r e a t t r a c t e d b y a n t i g e n - a n t i b o d y c o m p l e x e s . On t h e o t h e r h a n d , t h e number o f e o s i n o p h i l s i n t h e r a t u t e r u s has b e e n n o t e d t o i n c r e a s e 7 8 2 m a r k e d l y d u r i n g e s t r u s ' s u g g e s t i n g t h e r e l e a s e o f a c h e m o t a c t i c s u b s t a n c e f r o m e s t r o g e n - s t i m u l a t e d t i s s u e s . A t one t i m e i t was f e l t t h a t t h e p r e s e n c e o f an e o s i n o p h i l i c i n f i l t r a t e , p a r t i c u l a r l y i n c a r c i n o m a o f t h e c e r v i x , 6 3 3 8 a l l o w e d a b e t t e r p r o g n o s i s , b u t t h i s i d e a has s i n c e been a b a n d o n e d . E l e c t r o h T M i c r o s c o p e f i n d i n g s To a l a r g e e x t e n t , what t h e EM s t u d i e s a c c o m p l i s h i s t o c o n f i r m t h e r e s u l t s o f t h e l i g h t m i c r o s c o p i c o b s e r v a t i o n s . The f i n d i n g s o f d e c r e a s e d c e l l and n u c l e a r s i z e i n t h e r e g r e s s i n g tumors t o g e t h e r w i t h a d e c r e a s e i n a l l o r g a n e l l a r sys t ems b e s p e a k s a g e n e r a l i z e d a t r o p h i c p r o c e s s . T h i s a f f e c t s b o t h s y s t e m s o r i e n t e d t o w a r d t h e e x p o r t o f m a t e r i a l s f r o m t h e c e l l ( r o u g h and smooth e n d o p l a s m i c r e t i c u l u m , G o l g i a p p a r a t u s ) and t h o s e o r g a n e l l e s a s s o c i a t e d ^ , w i t h c e l l g r o w t h and r e p l i c a t i o n ( n u c l e u s , n u c l e o l i , and r i b o s o m e s ) . A n a l o g i c a l l y , ACTH has a t w o - f o l d e f f e c t o n t h e n o r m a l a d r e n a l and i t has b e e n shown t h a t ACTH w i l l s t i m u l a t e g r o w t h and a c t i v i t y i n e s t r o g e n - d e p r i v e d - 1 8 -tumors. These f i n d i n g s i m p l i c a t e p i t u i t a r y i n v o l v e m e n t and s u g g e s t t h a t a t t h i s s t a g e e s t r o g e n i s a c t i n g as a c o n t r o l l e r o f c a n c e r g r o w t h r a t h e r t h a n as a c a n c e r - c a u s i n g a g e n t . Dark c e l l s s i m i l a r t o t h o s e seen i n t h e r e g r e s s i n g tumors have been d e s c r i b e d b y o t h e r s . R e c e n t l y i n t h e German l i t e r a t u r e s u s p i c i o n has been c a s t on t h e s i g n i f i c a n c e o f t h e s e c e l l s . I t was s u g g e s t e d t h a t t h e d a r k appearance i s t h e r e s u l t o f w a t e r l o s s d u r i n g f i x a t i o n . As i n t h e c a s e o f th e t r a b e c u l a r p a t t e r n , i t w o u l d seem r e a s o n a b l e t h a t o n l y a r e a l a l t e r a t i o n i n c e l l s c o u l d g i v e r i s e t o t h e a r t e f a c t u a l i n v o l v e m e n t o f s i n g l e c e l l s i n a f i e l d o f u n i n v o l v e d c e l l s . O t h e r i n t e r e s t i n g f e a t u r e s o f t h e r e g r e s s i n g t u mors, namely t h e appearance o f c o l l a g e n f i b e r s and f r e e e o s i n o p h i l g r a n u l e s w i t h i n tumor c e l l s , a r e n o t r e a d i l y e x p l a i n e d . T h e i r i n f r e q u e n t o c c u r r e n c e w o u l d be more i n k e e p i n g w i t h t h e i r b e i n g p a r t o f a c l e a n - u p o p e r a t i o n r a t h e r t h a n t h e e f f e c t o r mechanism o f tumor r e g r e s s i o n . The p r e s e n c e o f e o s i n o p h i l g r a n u l e s i n tumor c e l l s has n o t p r e v i o u s l y been d e s c r i b e d t o t h e a u t h o r ' s knowledge. The u s u a l mode o f o p e r a t i o n f o r e o s i n o p h i l s i s i n g e s t i o n o f f o r e i g n p a r t i c l e s i n d i g e s t i v e v a c u o l e s . S u b s e q u e n t l y t h e d i g e s t i v e v a c u o l e f u s e s w i t h a s p e c i f i c g r a n u l e w h i c h a l l o w s 3 t h e r e l e a s e o f l y t i c enzymes i n t o t h e p h a g o c y t i c v a c u o l e . However, Ross 82 and K l e b a n o f f have r e p o r t e d t h e p r e s e n c e o f e o s i n o p h i l g r a n u l e s w i t h i n t h e 16 macrophages o f t h e e s t r o g e n - s t i m u l a t e d mouse u t e r u s , and C l i n e e t a l . have o b s e r v e d e o s i n o p h i l s t o l o s e t h e i r g r a n u l e s d u r i n g y e a s t p h a g o c y t o s i s . Thus, e o s i n o p h i l e g r a n u l e s i n tumor c e l l s c o u l d r e p r e s e n t an u n u s u a l mechanism o f e o s i n o p h i l a c t i v i t y o r i t c o u l d mean t h a t t h e tumor c e l l s had a c t i v e l y p h a g o c y t i z e d them. That e o s i n o p h i l g r a n u l e s a r e r e l e a s e d i n t o t h e tumor m i l i e u i s shown i n F i g . 3 7 where t h e g r a n u l e s c a n be seen i n t r a v a s c u l a r l y . E m i g r a t i o n o f e o s i n o p h i l s f r o m t h e b l o o d s t r e a m i s d e m o n s t r a t e d i n F i g . h7• The g r e a t e r number o f l y s o s o m e - l i k e s t r u c t u r e s i n t h e g r o w i n g tumors and t h e g r e a t e r number o f m y e l i n f i g u r e s and r e s i d u a l b o d i e s d u r i n g r e g r e s s i o n s u g g e s t two p o s s i b i l i t i e s , 1) t h e d e c r e a s e i n lysosomes p a r a l l e l s t h e g e n e r a l d e c r e a s e i n c e l l u l a r a c t i v i t y f o l l o w i n g e s t r o g e n r e m o v a l , 2 ) t h e lysosomes p l a y e d a r o l e i n t h e c e l l s h r i n k a g e and t h e r e s i d u a l b o d i e s a r e a - 1 9 -r e s u l t o f t h i s p r o c e s s . The second a l t e r n a t i v e has a p a r a l l e l i n t h e m e n s t r u a l c y c l e where i t has been o b s e r v e d t h a t e s t r o g e n and p r o g e s t e r o n e cause a p r o l i f e r a t i o n o f lysosomes and t h e i r w i t h d r a w a l r e s u l t s i n f i n d i n g s 8 9 , 9 0 i n d i c a t i n g t h e s e s t r u c t u r e s had been i n v o l v e d i n autophagy. i n t e r e s t i n g l y , ACTH has been f o u n d t o p r o d u c e an i n c r e a s e i n t h e number o f 9 5 a d r e n a l l i p o f u s c i n r e s i d u a l b o d i e s . A l t h o u g h the m i t o c h o n d r i a , p a r t i c u l a r l y t h o s e o f t h e g r o w i n g t u m o r s , have a h i g h l y b i z a r r e a p p e a r a n c e , i t i s n o t p o s s i b l e t o draw any f u n c t i o n a l i n f e r e n c e s a p a r t f r o m c o n j e c t u r e s as t o t h e i r r e l a t i o n s h i p t o t h e a n a e r o b i c r e s p i r a t i o n o f tumors o b s e r v e d b y Warburg o v e r t h i r t y y e a r s ago. ' hi Recent i n v e s t i g a t i o n s b y Green e t a l . have shown a r e l a t i o n s h i p between • m i t o c h o n d r i a l m e t a b o l i c a c t i v i t y and morphology, b u t t h e changes o b s e r v e d i n t h e tumors under s t u d y h e r e w o u l d n o t appear t o be o f t h a t t y p e . I t i s i n t e r e s t i n g t o note t h a t t h e m i t o c h o n d r i a had s h e l f - l i k e c r i s t a e whereas t h e p a r e n t zona f a s c i c u l a t a c e l l s have t u b u l o v a s i c u l a r c r i s t a e , ( F i g s . hQ, h$). 32 A f u n c t i o n i n g human a d r e n a l tumor d e s c r i b e d by F r a s e r e t a l . had s a c c u l a r c r i s t a e . The m i t o c h o n d r i a o f t h e zona g l o m e r u l o s a , on t h e o t h e r hand, do have s h e l f - l i k e c r i s t a e ( F i g . 5 0 ) and s i n c e t h e zona f a s c u l u l a t a can be r e p o p u l a t e d b y c e l l s a r i s i n g f r o m t h e zona g l o m e r u l o s a , i t i s i n t e r e s t i n g t o s p e c u l a t e w h e t h e r t h e m i t o c h o n d r i a l m orphology seen i n t h e tumor r e p r e s e n t s a r e v e r s i o n 7 9 t o a p r e c u r s o r f o r m . However, Penny e t a l . d e s c r i b e d c o n v e r s i o n o f c r i s t a e f r o m t u b u l o v e s i c u l a r t o l a m e l l a r i n r e g e n e r a t i n g a d r e n a l t r a n s p l a n t s , so i t w o u l d appear more l i k e l y t h a t when t h e a d r e n a l i s r a p i d l y g r o w i n g and t h e r e i s a s h i f t away f r o m s t e r o i d o g e n e s i s , a m i t o c h o n d r i a l s t r u c t u r a l change o c c u r s . S i n c e a d r e n o c o r t i c a l m i t o c h o n d r i a a r e a s s o c i a t e d w i t h s t e r o i d s y n t h e t i c s t e p s o f c h o l e s t e r o l s i d e c h a i n d e g r a d a t i o n and 1 1 - b e t a and 1 8 h y d r o x y l a t i o n ^ " ' " ( F i g . 5 l ) 5 i t has been s u g g e s t e d t h a t t h e i r p e c u l i a r c r i s t a l m o r p h o l o g y i s r e l a t e d t o s t e r o i d p r o d u c t i o n . I f t h i s were s o , t h e n l o s s o f t h e t u b u l o v e s i c u l a r c r i s t a e w o u l d p r o b a b l y i n t e r f e r e w i t h s t e r o i d o g e n e s i s . I n f a v o r o f t h i s h y p o t h e s i s i s t h e f a c t t h a t a l t h o u g h t h e s e tumors do p r o d u c e hormones, t h e y a r e abnormal. A g a i n s t t h e p r o p o s i t i o n F i g . 47 Regression - eosinophils emigrating from v e s s e l . x3,200 F i g . 49 Adrenal zona f a s c i c u l a t a - v e s i c u l a r c r i s t a e mitochondriales xl6,100 • F i g . 50 Adrenal zona glomerulosa - s h e l f - l i k e c r i s t a e . x20,000 pregnenolone. Coyh'Sof .MtrocttoNWLifit st'de cUafr)> deg radabb?) ft, I ? hydroxylak'oo F i g . 51 S t e r o i d s y n t h e t i c p a t h w a y 9 9 Mfc/coscMes -j> 0ji£z\9n 3fl dehydrogenase. \ I \ \ \ \ / \ S0LU6L£ FR. ACTION I / 11,2.1 hydroxylakon If deoxycork'sol II dzoxy COY kcosfe rone. -20-k i s t h e f a c t t h a t some n o n - e n d o c r i n e t i s s u e s have t u b u l o v e s i c u l a r c r i s t a e . A n o t h e r d i f f e r e n c e between t h e tumor m i t o c h o n d r i a and n o r m a l a d r e n a l m i t o c h o n d r i a i s t h e p r e s e n c e o f m i t o c h o n d r i a l dense g r a n u l e s i n t h e f o r m e r . The c y t o p l a s m i c and l i p i d i n c l u s i o n s seen w i t h i n m i t o c h o n d r i a r e s e m b l e s i m i l a r (and s i m i l a r l y u n e x p l a i n e d ) s t r u c t u r e s o b s e r v e d b y I d e l m a n ^ and 22 D e R o b e r t i s and S a b a t i n i . Thus, t h e a c c u m u l a t i o n o f e l e c t r o n - d e n s e , l i p i d - l i k e m a t e r i a l i n tumor c e l l m i t o c h o n d r i a c o u l d be a m a n i f e s t a t i o n o f i n c r e a s e d o r d i s o r d e r e d s t e r o i d m e t a b o l i s m , b u t a d e f i n i t e i n t e r p r e t a t i o n o f t h e s i g n i f i c a n c e o f t h i s f i n d i n g i s n o t p o s s i b l e . I n a l l l i k e l i h o o d , t h e m i t o c h o n d r i a l d e v i a t i o n s f r o m t h e p a r e n t t i s s u e 78 r e p r e s e n t n o n - s p e c i f i c changes a s s o c i a t e d w i t h m a l i g n a n c y . The p r e s e n c e o f der a n g e d c e n t r i o l e s and/or c i l i a and t h e e c t o p i c o c c u r r e n c e o f desmosomes w o u l d a l s o be i n k e e p i n g w i t h n e o p l a s t i c d e r e p r e s s i o n phenomena. C i l i a a r e seen n o r m a l l y i n t h e zona g l o m e r u l o s a o f t h e r a t a d r e n a l ( F i g . 52), l e n d i n g f u r t h e r s u p p o r t t o t h e i d e a t h a t t h e tumor c e l l s , t hough t h e y a r o s e i n t h e zona f a s c i c u l a t a have r e v e r t e d t o h a b i t s o f t h e i r a l l e g e d p r e c u r s o r s i n t h e zona g l o m e r u l o s a . A more l i k e l y e x p l a n a t i o n i s t h a t t h e s t r u c t u r e s a r e c e n t r i o l e s and t h a t t h e y a r e i n c r e a s e d i n p r o p o r t i o n t o t h e amount o f c e l l d i v i s i o n o c c u r r i n g . R ecent a r t i c l e s on t h e s u b j e c t o f hormonal and a d r e n a l c a r c i n o g e n e s i s i+8 65 a r e t h o s e o f Huvos e t a l . and Lemon. I n t h e f o r m e r t h e r e i s t h e i n t e r e s t i n g c l i n i c a l f i n d i n g t h a t p r e m e n o p a u s a l women a r e more p r o n e t o a d r e n o c o r t i c a l c a r c i n o m a t h a n p o s t m e n o p a u s a l women o r men. Lemon p r o p o s e s a r e l a t i v e e s t r i o l d e f i c i e n c y as a s i g n i f i c a n t f a c t o r i n e s t r o g e n c a r c i n o g e n e s i s . 21 BIBLIOGRAPHY 1. Algard, F. T. Action of sex hormones on dependent tumors i n c e l l and organ-culture, systems. Nat. Cancer Inst. Monograph 11:215, 1963 2. Archer, F. L. Fine structure of spontaneous andestrogen-induced secretion i n breast tumors i n the ra t induced by DMBA. J. Nat. Cancer Inst. 42:347, 1969 3. Banerjee, M. R. Hormonal con t r o l of DNA synthesis. Altered responsiveness of hyperplastic a l v e o l a r nodules of mouse mammary gland. J . Nat. Cancer Inst. 42:227, 1969 4. B e l t , W. D. & D. C. Pease Mitochondrial structure i n s i t e s of st e r o i d secretion. J . Biophys. Biochem. Cyto l . 2(suppl) :369, 1956 5 . Bernhard, W. Some problems of f i n e structure i n tumor c e l l s . Progr. Exp. Tumor Res. 3:1, 1963 6. Bielschowsky, F & E. S. Horning Aspects of endocrine carcinogenesis. Br. Med. B u l l . 14:106, 1958 7. Bjersing, L. & N. E. Borglin E f f e c t of hormones on incidence of uterine e o s i n o p h i l i a i n r a t s . Acta Pat. M i c r o b i o l . Scand. 60:27, 1964 8. Block, M. M. & H. R. Ansley Histone-DNA i n t e r a c t i o n i n r e l a t i o n to c e l l function. Fed. Proc. 27:670, 1968 9. Bloch, E., A. Cohen, & J . Furth Steroid production by normal and adrenal tumor-bearing male mice. J . Nat. Cancer Inst. 24:97, 1960 10. Bohman, 0., & A. B. Maunsbach E f f e c t s on tissue Fine Structure of Variations i n C o l l o i d Osmotic Pressure of Glutaraldehyde F i x a t i v e s . J . U l t r a s t r . Res. 10:195, 1970 11. Browning, H. C. Hormonal dependence of adrenocortical tumors of CE and Balb/C mice i n s e r i a l i n t r a o c u l a r t r a n s f e r . Cancer Res. 18:781, 1958 12. Browning, H. C , W. D. White, & W. A. Sadler I n h i b i t i o n of growth of a transplantable adrenocortical tumor by sex st e r o i d s . Cancer Res. 19:819, 1959 13. Bulbrook, R. D. Hormonal factors i n the etiology and treatment of breast cancer. Canad. Cancer Conf. 6:36, 1966 14. Cardeza, A. F. H i s t o l o g i c des tumeurs surrenales des rats castres. Comp. Rend. Soc. B i o l . 148:2116, 1954 15. C l i f t o n , K. H. Problems i n experimental tumorigenesis of the p i t u i t a r y gland, gonads, adrenal c o r t i c e s and mammary glands: a review. Cancer Res. 19:2, 1959 22 16. C l l n e , J . J . , J . Hanefln, & R. I. Lehrer Phagocytosis by human eosinophils. Blood 32:922, 1968 17. Cotte, G. & Cotte, N. Holocrini e dans l a cort i c o s u r r e n a l e . Z e i t s c h r . Z e l l f o r s c h . 54:182, 1961 18. Cutts, J . H. Estrone-induced mammary tumors i n the r a t . II. E f f e c t of a l t e r a t i o n s i n the hormonal environment on tumor induction, behaviour, and growth. Cancer Res. 24:1124, 1964 19. Cutts, J . H. Estrogen-induced breast cancer i n the r a t . Canad. Cancer Conf. 6:50, 1966 2 0, Cutts, J . H. & G. C. Froude. Regression of estrone-induced mammary cancers i n the r a t . Cancer Res. 28:2413, 1968 21. Dao. T. L. The dual function of hormones i n mammary carcinogenesis. A working hypothesis. On Cancer & the Hormones p. 231, U. of Chicago Press, 1962 2 2. DeRobertis, E. & D. Sabatini. Mitochondrial changes i n the adrenal cortex of normal hamsters. J . Biophys. Biochem. C y t o l . 4:667, 1958 23. De Sombre, E. R., T. Suzuki, T. Kawashime, P. W. Jungblut, & E. V. Jensen. A two-step mechamism for the i n t e r a c t i o n of estrogen with the rat uterus. Fed. Proc. 27:497, 1968 24. Dunning, W. F., M. R. C u r t i s , & A. Segaloff S t r a i n differences i n the response to estrone and the induction of mammary gland, adrenal, and bladder cancers i n r a t s . Cancer Res. 13:147, 1953 25. El-Bolkainy, M., G. B. Pierce, & A. J . French Regression of and adrenocortical carcinoma by e s t r a d i o l treatment. Cancer Res. 27:1846, 1967 26. Engel, L. L. & J . F. Scott. E f f e c t s of s t e r o i d hormones upon DPN-mediated enzymatic reactions. Rec. Progr. Hormone Res. 16:79, 1960 27. Fekete, E., G. W. Woolley, & C. C. L i t t l e . H i s t o l o g i c a l changes following ovariectomy i n mice. J . Exp. Med. 74:1, 1941 28. Flaks, J . Adrenocortical carcinoma with metastases i n an ovariectomized Strong A mouse. J. Path. Bact. 61:266, 1949 2 9. Folca, • P. J . , R. F. Clascock, & W. T. Irvine. Comparison of ti s s u e accumulation of hexestrol with response to b i l a t e r a l adrenalectomy and ovariectomy i n advanced breast cancer. Lancet 2:796, 1961 3 0. Fonzo, D., P.. B. Mims, & D. H. Nelson. Estrogen influence on p i t u i t a r y and adrenal function i n the r a t . Endocrinol. 81:29, 1967 31. Franks, L. M. & F. C. Chesterton Experimental production of morphological changes resembling Conn's syndrome. Lancet 2:1193, 1956 24 32. Fraser, R. et a l . (7 authors) C l i n i c a l and biochemical studies of a patient with a corticosterone-secreting adrenocortical tumor. Lancet 2:1116, 1968 33. Furth, J . Thoughts on the evolution and nature of neoplasms. Cancer Res. 19:241, 1959 34. Gardner, W. U. Hormonal aspects of experimental tumorigenesis. Adv. Cancer Res. 1:173, 1953 35. Gardner. W. U. Summary of discussion of f i r t day. Symposium: Role of hormones i n the o r i g i n and control of abnormal neoplastic growth. Cancer Res, 17:481, 1957 36. Gardner, W. U. Studies on s t e r o i d hormones i n experimental carcinogenesis. Recent Adv. Hormone Res. 1:217, 1947 37. Geschickter, C. F. Mammary carcinoma i n the rat with metastases induced by estrogen. Science 89:35, 1939 3 8. G i l l , A. J . Local e o s i n o p h i l i a i n malignant neoplasms. J. Lab. C l i n . Med. 29;820, 1944 39. Goldzieher, J . W. The i n t e r p l a y of adrenocortical and ovarian function. Mack, H. C , The Ovary, p. 106, Academic Press, 1968 40. Gompertz, D. The e f f e c t of sex hormones on the adrenal gland of the male r a t . J . Endocrinol. 17:107, 1958. 41. Green, D. E., J . A s a i , R. A. H a r r i s , & J . T. Pennington Conformational basis of energy changes i n membrane systems. I l l Conformational changes i n the mitochondrial inner membrane induced by changes i n fu n c t i o n a l states Arch. Biochem. Biophys. 125:684,1968 42. Hechter, 0., & I. D. K. Halkerston E f f e c t s of s t e r o i d hormones on gene regulation and c e l l metabolism Ann. Rev. P h y s i o l . 27:133,1965 4 3. Hechter, 0. Hormone action at the c e l l membrane Karlson, P. Mechanisms of Hormone Action, p. 61, 1965 44. Houssay, A. B., A. F. Cardeza, B. A. Houssay, & R. M. Pinto Facteurs susceptibles de modifier 1'apparition et l a fonction des tumeurs surrenales chez l e s rats castres Comp. Rend. Soc. B i o l . 148:2123,1954 45. Houssay, B. A., A. F. Cardeza, & R. M. Pinto Tumeurs surrenales oestrogeniques et tumeurs hypophysaires chez les animaux castres Schweiz. Med. Wchnschr. 85:291,1955 4 6. Huggins, C. Steroids, growth, and Cancer Pincus, G. P. & E. P. Vollmer B i o l o g i c a l A c t i v i t i e s of Steroids i n Relation to Cancer, p. 1, Academic Press, 1960 47. Huseby, R. A. Steroids and tumorigenesis i n experimental animals Dorfman, R. I., Methods in Hormone Research, v. 4, p. 123-164, Academic Press, 196 4 8. Huvos, A. G., S. I. Hajdu, R. D. B r a s f i e l d , & F. W. Foote Adrenal C o r t i c a l carcinoma. C l i n i c o p a t h o l o g i c study of 34 cases Cancer 25:354,1970 25 49. Idelman, S. Mitochondries et liposomes. J . Mlcroscopie 3:437, 1964 50. Idelman, S. Contribution a l a cytophysiologie i n f r a s t r u o t u r a l e de l a c o r t i c o -surrenale chez l e ra t albino. Ann. des S c i . Nat., Zool. 8:205-362, 1966 51. I g l e s i a s , R. Hormones & Tumors. Proc. 2nd Internat. Congr. Endocrinol., p. 1072. 52. Jensen, E. V. Estrogen receptors i n target t i s s u e s . Pincus, G., T. Nakao, & J . F. T a i t , Steroid Dynamics, p. 133, 1965 5 3. Jensen, E. V., E. R. DeSombre, & P. W. Jungblut. Interaction of estrogens with receptor s i t e s i n vivo and i n v i t r o . 2nd Internat. Congr. on Hormonal Steroids, p. 44, 1966 54. J u l l , J . W. E f f e c t s of estrogens and progesterone on the chemical induction of mammary cancer i n mice of the IF s t r a i n . J . Path. Bact. 68:547, 1954 55. J u l l , J . W. Hormonal mechanisms i n carcinogenesis. Canad. Cancer Conf. 6:109, 1966 56. Karlson, P. & C. E. Sekeris Biochemical Mechanisms of hormone acti o n . Acta Endocrinol. 53:505, 1966 57. Kim. U., J . Furth, & K. Yannopoulos Observations on hormonal control of mammary carcinoma. I. Estrogens and mammotropes. J. Nat. Cancer Inst. 31:233, 1963 58. Kim, U. P i t u i t a r y function and hormonal therapy, of experimental breast cancer. Cancer Res. 25:1146, 1965 59. King, R. J . B. & J . Gordqn The a s s o c i a t i o n of (6,7 H) e s t r a d i o l with a nuclear p r o t e i n . J . Endocrinol. 39:533, 1967 6 0. Kirschbaum, A. Role of hormones i n cancer: laboratory animals. Cancer Res. 17:432, 1957 61. Kitay, J . I. Enhancement of steroidogenesis by rat adrenal s l i c e s i n v i t r o with e s t r a d i o l 17 beta. Nature 192:358, 1961 62. Kitay, J . I. E f f e c t s of e s t r a d i o l on p i t u i t a r y and adrenal function i n male and female r a t s . Endocrinol. 72:947, 1963 63. Klebanoff, S. J . Ina c t i v a t i o n of estrogen by rat uterine preparations. Endocrinol. 76:301, 1965 64. Lacassagne, A. Ap a r r i t i b n de cancers de l a mamelle chez l a souris male, soumis a des i n j e c t i o n s de f o l l i c u l i n e . Comp. Rend. Acad. S c i . 195:630, 1932 26 65. Lemon, H. M. Abnormal Estrogen Metabolism and Tissue Estrogen Receptor Proteins i n Breast Cancer. Cancer 25:423, 1970 66. Lerner, L. J . S R . J . H i l f B i o l o g i c a l a c t i v i t i e s of steroids and t h e i r r e l a t i o n s h i p to breast cancer therapy. Segaloff, A., K. Meyer, & S. Debakey, Current Concepts i n Breast. Cancer, p. 80, Williams & Wilkins, 1967 67. Levine, A. J . & F. P. Skelton A l i g h t and electron microscopic study of hyaline droplet and vacuole formation i n the adrenal glands of rat s treated with methylandrostenediol. Am. J . Path. 51:831, 1967 6 8. Lipschutz, A., R. I g l e s i a s , & S. Salinas Ovarian tumo«rs induced by a s t e r i l i z i n g s t e r o i d . Nature 196:946, 1962 69. Lipschutz, A., R. I g l e s i a s , V. I. Panasevich, & S. Salinas Ovarian tumours and other ovarian changes induced i n mice by two 19-nor contraceptives. Br. J . Cancer 21:153, 1967 70. Lipschutz, A., R. I g l e s i a s , V. I. Panasevich, & S. Salinas Pathological changes induced i n the uterus of mice with prolonged administration of progesterone and 19-nor contraceptives. Br. J . Cancer 21:161 / 1967 71. Mobbs, B. G. The uptake and autoradiographic l o c a l i z a t i o n of t r i t i a t e d e s t r a d i o l i n the rat uterus a f t e r l o c a l a p p l i c a t i o n . J . Endocrinol. 41:69, 1968 72. Mobbs, B. G. 3 1 4 The uptake of simultaneously administered H e s t r a d i o l and C progesterone by DMBA-induced rat mammary tumors. J . Endocrinol.'41:339, 1968 73. Motlik, K. & M. Janouskova Hyaline droplet formation and some other adrenocortical changes following methylandrostenediol treatment i n the r a t . Virchows Arch. Path. Anat. 336:427, 1967 7 4. Mueller, G. C. The actions of estrogens at the c e l l u l a r l e v e l . Mack, H. C. The Ovary, p. 188, Academic Press. 1968 7 5. N e v i l l e , A. M., J . M. Anderson, M. H. McCormack, & J . L. Webb Steroid biosynthesis i n v i t r o and i n tissue culture by adrenal tumors of mice of the CE s t r a i n . J . Endocrinol. 41:547, 1968 76. Nishikawa, M., I. Murone, & T. Sato E l e c t r o n Microscopic observations of the adrenal cortex. Endocrinol. 72:197, 1963 77. Noble, R. L. Induced, transplantable, estrogen?dependent carcinomas of the adrenal cortex i n r a t s . Proc. Am. Assoc. Cancer Res. 8:51, 1967 7 8. Oberline, C. &. W. Bernhard. The morphology of cancer c e l l s . Brachet & Mirsky, The C e l l , v. 5 Ch. 7, Academic Press, 1961 27 79. Penney, D. P., I. I. Pra t t , & W. C. Dixon The f i n e structure of regenerating adrenocortical transplants In the r a t . Anat. Rec. 146:319, 1963 80. Puca, G. A. & F. Bre s c i a n i Receptor molecule for estrogens from the rat uterus. Nature 218, 967, 1968 81. Rich, A. R. A pec u l i a r type of adrenal c o r t i c a l damage associated with acute i n f e c t i o n s and i t s possible r e l a t i o n to c i r c u l a t o r y collapse. B u l l . Johns Hopkins Hosp. 74:1, 1944 8 2. Ross, R. & S. J . Klebanoff The eosinophil leukocyte. Fine s t r u c t u r a l studies of changes i n the uterus during the estrus c y c l e . J . Exp. Med.' 124:653, 1966 8 3. R u s s f i e l d , A. B. Tumors of endocrine glands and secondary sex organs. U. S. Dept. Health & Welfare, pp. 25-38, 19.66 84. Sabesin, S. M. A function of the eosinop h i l : phagycytosis of antigen-antibody complexes. Proc. Soc. Exp. B i o l . Med. 112:667, 1963 85. Sander, S. & A. Attramadel The i n v i t r o uptake of e s t r a d i o l 17 beta by hormone responsive and unresponsive breast tumors of the r a t . Acta Path, et M i c r o b i o l . Scand. 74:169, 1968, 74:301, 1968 8 6. Scott, G. B.,H. J . C h r i s t i a n , & A. R. Currie The Huggins rat mammary tumors: c e l l u l a r changes induced with regression. Wissler, R. W., T. L. Dao, & S. Wood, Endogenous Factors Influencing Host-Tumor Balance, p. 99, U. of Chicago Press, 1967 87. Segal S. Hormones, Amino-acid transport, and protein synthesis. Nature 203:17, 1964 .88. Selye, H. &. H. Stone On the experimental morphology of the adrenal cortex. C. C. Thomas, 1950. 89. Smith, R. E. Phosphohydrolases i n C e l l Organelles: Electron Microscopy. Ann. N. Y. Acad. S c i . 166:525, 1969 90. Smith, R. E. and M. R. Henzl. Role of Mucopolysaccharides and lysosomal Hydrolases i n Endometrial Regression Following Withdrawal of E s t r a d i o l and Chlormadinone Acetate. I. Epithelium and Stroma. Endocrinol. 85:50, 1969 91. S n e l l , K. D. & H. L. Stewart. Variations i n h i s t o l o g i c pattern and functional e f f e c t s of a transplantable adrenocortical carcinoma i n i n t a c t , hypophysectomized and newborn r a t s . J . Nat. Cancer Inst. 22:1119, 1959 92. Steggles, A. W. & E. J . B. King. The uptake of 6,7 H e s t r a d i o l by estrogen dependent and independent tumors of hamster kidney. Europ. J . Cancer 4:395, 1968 9 3. Swift, H. & Z. Hruban. Focal degradation as a b i o l o g i c a l process. Fed. Proc. 23:1026, 1964 28 94. Sydnor, K. L., & B. C o c k r e l l . Influence of e s t r a d i o l , progesterone, and hydrocortisone on 3-methylcholanthrene-induced mammary cancer. Endocrinol. 73:427, 1963 9 5. Szabo, D., E. Stark, & B. Varga. The l o c a l i z a t i o n of acid phosphatase a c t i v i t y changes i n lysosomes i n the adrenal zona f a s c i c u l a t a of int a c t and hypophysectomized rats following ACTH administration. Histochemie 10:321, 1967 96. Talal a y , P., & H. G. Williams-Ashman. P a r t i c i p a t i o n of st e r o i d hormones i n the enzymatic t r a n s f e r of hydrogen Ions. Recent Progr. Hormone Res. 16:1, 1960 97. Teng, C. S., & T. H. Hamilton Regulation of polyribosome formation and protein synthesis i n the uterus. Bioch. J . 105:1101, 1967 9 8. Tomkins, G. M. B i o l o g i c a l regulation by steroid hormones. Canad. Cancer Conf. 6:163, 1966 99. T u l l o s , H. S., A. Kirschbaum, & J . J . Trentin Role of gonadotrophic hormone i n the i n h i b i t i o n and progression of adrenocortical tumors i n ovariectomized mice. Cancer Res. 21:730, 1961 100. Van der Vies, J . The e f f e c t s of steroids on adrenocortical function. Acta P h y s i o l . Pharmacol. Neerl. 9:122, 1960 101. V i l l e e , C. A., D. A. Hagerman, & P. B. Joe l An enzymatic basis f o r the physiologic action of estrogens. Recent Progr. Hormone Res. 16:49, 1960 102. Warburg, 0. Uber die entstehung der krebszellen. Naturwissenschaften 42:401, 1955 103. Wilbur, 0. M. & A. R. Rich. A study of the r o l e of ACTH i n the pathogenesis of tubular degeneration of the adrenal. B u l l . Johns Hopkins Hosp. 93:321, 1953 104. Wilson, R. A. The roles of estrogen and progesterone i n breast and g e n i t a l cancer. J . Amer. Med. Ass. 182:327, 1962 105. Woolley, G. W. & C. C. L i t t l e Prevention of adrenocortical carcinoma by d i e t h y l s t i l g e s t r o l . Proc. Nat. Acad. S c i . 32:239, 1946 106. Yamori, T., S. Matsuura, & S. Sakamoto An electronmicroscopic study of the normal and stimulated adrenal cortex i n the r a t . Ze i t s c h r . fur Z e l l f o r s c h . 55:179, 1961 107. Young, S. & D. M. Cowan Spontaneous regression of induced mammary tumors i n r a t s . Br. J . Cancer 17:85, 1963 108. Young, S.,rD. M. Cowan, & L. E. Sutherland Induced mammary tumors i n r a t s . J . Path. Bact. 85:331, 1963 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0101994/manifest

Comment

Related Items