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Estrogen and progesterone receptors in transplantable Noble art mammary tumors Yeung, Chuck C. H. 1983

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ESTROGEN AND PROGESTERONE RECEPTORS IN TRANSPLANTABLE NOBLE RAT MAMMARY TUMORS BY CHUCK CH. YEUNG B.Sc, THE UNIVERSITY OF BRITISH COLUMBIA, 1 9 7 7 A Thesis Submitted in Partial Fulfillment of The Requirements For The Degree of Master of Science in THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept this thesis as conforming to the required standard The University of British Columbia. Apr i l 1983 © Chuck C H . Yeung, 1983 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 i t 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 or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Department of \ <Vl 1^ DE-6 (3/81) ABSTRACT The relationship between sex steroid hormone receptors and hormone dependent status of mammary tumors has been studied using an animal model. Hormone-induced Noble rat mammary tumor lines were maintained by serial transplantations. Transplants were carried out by injecting small pieces of viable tissues into the backs of animals. Depending on the hormonal status of the tumors, exogenous estrogen was supplemented. Tumors (5-10 g) were harvested and their cytoplasmic estrogen receptors (ERc), nuclear estrogen receptors (ERn) and cytoplasmic progesterone receptors (PgR) profiles were analyzed. The majority of the tumors studied did not depend on exogenous estrogen for growth i.e. they were hormone autonomous. They arose from transplants of a hormone dependent tumor, an autonomous tumor, or a tumor which had undergone a transition from dependency to autonomy upon removal of the exogenous estrogen source from the host. Histologically they appeared very homogeneous. Characterization of the receptor profiles of the autonomous tumors were intended to answer whether or not (1) the measurements of ERc, or ERn and PgR can indicate the autonomous status of these tumors (2) the levels of ERc, ERn and PgR are influenced by sequential transplantation (3) there is a change in the receptor profiles concurrent to tumor progression from dependency to autonomy. - i i i -Receptors were measured by i s o l a t i n g the cytoplasmic and nuclear fractions prior to an exchange assay with radioactive hormone ligand. Cytoplasmic contamination of the ERn assay was avoided by the development of a procedure to isola t e clean intact nuclei as starting material. Almost a l l of the autonomous tumors reported i n this study (48 out of 52) have measurable amounts of cytoplasmic estrogen receptors (ERc+). This supports the general b e l i e f that ERc alone i s not a s u f f i c i e n t indicator of hormone dependence. The l e v e l of receptors was also found to be quite constant i n transplants over a number of generations of male and female hosts. This i s consistent with the postulate that production of receptors i s "genetically" coded by individual c e l l type and i s not greatly influenced by the hormonal milieu. Noble rat mammary tumors began to progress towards autonomy when the estrogen p e l l e t s were removed. Changes i n receptor p r o f i l e s were studied with respect to tumor progression while under some degree of estrogen stimulation. Transplants were carried out i n either intact female animals or males supplemented with an estrogen p e l l e t , after the tumor had undergone a t r a n s i t i o n from dependency to autonomy. Results show that such tumors seemed to maintain an intact stimulatory pathway i . e . ERc+/ERn+/PgR+ i f transplants were performed shortly after the removal of the estrogen p e l l e t s from the dependent tumors ( <13 weeks). However, i f - i v -duration of progression was extended, receptor profile would become negative and implied a total autonomous state. Therefore, i t is suggested that tumor progression might be attenuated with hormone supplement. This would keep the estrogenic mechanism intact; thus maintaining an avenue for controlling growth via hormone manipulation. Two major groups of autonomous tumors with different receptor profiles were also observed. Group I tumours were shown to have measurable amounts of cytoplasmic estrogen receptors, nuclear estrogen receptors and cytoplasmic progesterone receptors (ERc+/ERn+/PgR+). Further investigation is necessary to elucidate whether these tumors might be hormone responsive thus required the maintenance of their estrogen stimulatory mechanism. In contrast, group II was ERc+/ERn +or- /PgR-. The absence of PgR in this group implies the presence of biochemical lesions or hormone nonresponsiveness and would agree with their autonomous behavior. This suggests that the measurement of PgR, which is believed to be an end product of estrogen- stimulation, might be more informative concerning the condition of the estrogenic stimulatory mechanism than the assays of ERc or ERn. -V-TABLE OF CONTENTS Page Abstract i i Table of Contents v L i s t of Figures v i i i L i s t of Tables x L i s t of Abbreviations x i Acknowledgements x i i Introduction 1 ( i ) H i s t o r i c a l background 1 ( i i ) Postulates f o r ER+ nonresponders 2 ( i i i ) Mechanism of estrogen stimulation 3 ( i v ) Inherent l i m i t a t i o n of cytoplasmic ER as a p r e d i c t i v e 5 index (v) A d d i t i o n a l markers as p r e d i c t i v e indices 5 ( v i ) Estrogen and progesterone receptor p r o f i l e s i n 8 experimental tumor systems ( v i i ) Transplantable Noble rat mammary tumor l i n e s 9 ( v i i i ) Objectives 10 - v i -Page Materials and Methods 11 ( i ) Chemicals and radiochemicals 11 ( i i ) Temperature control 11 ( i i i ) Maintenance of mammary tumor l i n e s i n Noble rats 12 (i v ) Preparation of cytosol 14 (v) Binding assay for cytoplasmic estrogen receptors (ERc) 15 (v i ) Scatchard analysis 17 ( v i i ) Woolf analysis 17 ( v i i i ) Binding assay for progesterone receptors (PgR) 18 (ix) S t a b i l i t y of estrogen receptor (ERc) i n l y o p h i l i z e d cytosol 18 (x) I s o l a t i o n of n u c l e i 19 (xi) Electron microscopic examination of nuclear p e l l e t s 20 ( x i i ) Binding assay for nuclear estrogen receptors 20 ( x i i i ) Protein determination 22 (xiv) Deoxyribonucleic acid determination 22 Results and Discussion 24 ( i ) E f f e c t s of temperature and time.of incubation on cytoplasmic 25 estrogen receptors assay ( i i ) E f f e c t of protein concentration on cytoplasmic estrogen 27 receptors assay ( i i i ) E f f e c t of time of incubation on progesterone receptor assay 29 ( i v ) Estrogen receptor a c t i v i t y i n l y o p h i l i z e d cytosol 29 - v i i -Page (v) Assessment of quality of nuclear preparation 39 (vi) Nuclear estrogen receptor assay 45 (vi i ) Effect of time of incubation on nuclear estrogen receptor U6 assay ( v i i i ) Hormone and receptors status in tumor c e l l lines M9 Conclusion 71 References 73 - v i i i -LIST OF FIGURES Figure Page 1 A generalized model for the interaction of estrogens 4 with target c e l l s . 2 Temperature and time of incubation: effects on cytoplasmic 26 estrogen receptor assay. 3 Effect of protein concentration on cytoplasmic estrogen 28 receptor assay. 4 Effect of incubation period on cytoplasmic progesterone 30 receptor assay. 5 S t a b i l i t y of estrogen receptors i n lyophilized cytosol 31 controls. 6A T i t r a t i o n of estradiol-binding of cytosol of lyo p h i l i z e d 34 Control A. 6B Analysis of estrad i o l binding according to the Woolf plot. 36 6C Analysis of e s t r a d i o l binding according to the Scatchard 38 plot. 7-12 Assessment of nuclear preparation. 41 13,14 Electron micrographs of isolated intact nuclei. 42 15 Electron micrograph of nuclei isolated via employment of a 44 microdismembrator as the i n i t i a l tissue homogenizing t o o l . 16 Elution pattern of e s t r a d i o l binding in nuclear fraction of 47 tumor from a column of Sephadex G 25 (5cm x 1.5 cm diameter). - i x -Figure Page 17 E f f e c t of time of incubation on nuclear estrogen receptor 48 assay. 18 Composite pedigree of Noble rat mammary tumor l i n e 51 15 EMM l 6 ( l ) / 6 . 19 Schematic representation of the hormone receptors p r o f i l e s 52 of Noble rat mammary tumor l i n e 15 EMM l 6 ( l ) / 6 . 20 Estrogen receptors status of tumors a r i s i n g i n male and 54 female hosts of Noble rat mammary tumor l i n e 15 EMM l 6 ( l ) / 6 . 21 Composite pedigree of Noble rat mammary tumor l i n e s 61 31 EMM-(D), 31 EMM-(E), and 31 EMM-(F). 22 Schematic representation of the hormone receptors p r o f i l e s 63 of Noble rat mammary tumor l i n e s 31EMM - (D) 23 Schematic representation of the hormone receptors p r o f i l e s 67 of Noble rat mammary tumor l i n e 31 EMM - ( E ) . 24 Schematic representation of the hormone receptors p r o f i l e s 69 of Noble rat mammary tumor l i n e 31 EMM - (F). -X-LIST OF TABLES Page Table I. Estrogen and progesterone receptor concentrations 55 i n sublines of 15 EMM 16(1)/6. Table I I . Receptor concentrations i n sublines of 31 EMM - ( D ) , 64 (E), and (F). i - x i -LIST OF ABBREVIATIONS DCC - dextran coated charcoal DNA - deoxyribonucleic acid DES - d i e t h y l s t i l b e s t r o l EDTA - ethylenediamine tetraacetate, disodium s a l t ER - estrogen receptor ERc - cytoplasmic estrogen receptor ERn - nuclear estrogen receptor g - gr a v i t a t i o n a l constant HA - hormone autonomous (not requiring estrogen for growth) HD - hormone dependent Kj) - equilibrium dissociation constant mRNA - messenger ribonucleic acid. Nb - Noble rats PgR - progesterone receptor R5020 - dimethyl-19-nor-pregna-4,9-diene -3,20-dione. SB - s p e c i f i c binding SDG - sucrose density gradient Tris - t r i s (hydroxylmethyl) aminomethane - x i i -ACKNOWLEDGEMENT I would like to thank my supervisor, Dr. W.J. Godolphin, for his excellent guidance throughout the course of this thesis. His advice and patience are deeply appreciated. It has also been a gratifying experience to work with the wonderful staff in the Chemistry Department of Vancouver General Hospital. In particular, the members of the routine estrogen receptor laboratory, including Miss Beryl Jacobson, Miss Lulu Garcia, Mrs. Sandy Iscernia, Mrs. Mary McLennan and Mr. Robert Rose have been most accommodating and helpful. Their friendliness and encouragement w i l l always be remembered. I would also like to thank Mrs. Eva Chang for her technical advice on electron microscopy. The fine efforts of Mrs. Ginette Vogel and Mrs. Vivien Roberts in preparing this manuscript are also appreciated. - 1 -INTRODUCTION H i s t o r i c a l Background Breast cancer i s the commonest malignancy among North American women. About 1 i n every 11 females w i l l eventually develop t h i s disease ( 1 ) . Since the breast i s a target organ of female sex hormones, i t i s not su r p r i s i n g that some breast tumors are hormone dependent and require a supply of female sex hormones for continued p r o l i f e r a t i o n . Such a re l a t i o n s h i p was confirmed as early as I896 when Beatson was able to demonstrate the regression of some tumors i n young women with advanced breast cancer by removal of the ovaries ( 2 ) . Since then various attempts to block the production of the hormones such as adrenalectomy (3), hypophysectomy (H), administration of androgen ( 5 ) , estrogen ( 6 ) or treatment with antiestrogen (7) have been successful i n inducing tumor regression. Unfortunately not a l l breast tumors respond to hormone manipulation therapy. Markers and guidelines have been sought to d i s t i n g u i s h nonresponders from responders so that the former group can be spared unnecessary morbidity associated with the endocrine therapy treatment. Various markers such as urinary ster o i d s and t h e i r metabolites ( 8 ) serum proteins such as alpha 1 - a n t i t r y p s i n , IgA, beta 1 - t r a n s f e r r i n ( 9 ) , plasminogen a c t i v a t o r ( 1 0 ) and others have been -2-investigated as potential predictive indices. Experiments with t r i t i a t e d hexestrol and estradiol showed that i n target tissues which require estrogen stimulation for growth (11, 12), there i s a spe c i f i c cytoplasmic hormone binding component. In 1967, Jensen et a l (13).demonstrated s p e c i f i c estrogen binding i n hormone dependent 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors, but not by non-respondent tumors. Jensen et a l (14) were also able to show that human breast tumors which have measurable amounts of receptors (ER+) would respond to endocrine therapy at a higher rate than those which have very low or no measurable amounts of receptors (ER-). Similar studies over the past years (15, 16) have now generally agreed that 55—60% of ER+ tumors w i l l respond to hormonal treatment and 90% of ER- tumors w i l l not. Postulates for ER+ nonresponders A question of interest i s : why are a 40 - H5% of ER+ tumors nonresponsive to endocrine therapy? Several hypotheses have been suggested to explain this phenomenon and three popular ones are: 1. Tumor heterogeneity theory Tumors might consist of both hormone dependent c e l l s with high ER content and independent c e l l s with low ER content. Such heterogeneity has been demonstrated i n both mouse and rat mammary tumors (17, 18). In human breast cancer, heterogeneity has also been shown by fluorescent cytochemical staining (19). When such tumors are treated with hormone therapy, only the dependent c e l l s would respond whereas independent ones would continue to p r o l i f e r a t e . -3-2. Additional hormone control pathways Control mechanisms by other hormones such as prolactin or progestin might be important in modulating c e l l growth (20). Prolactin has been shown to have a positive and progestin a negative regulatory effect on the normal pathway of estrogen signals (21). 3. Biochemical lesions There might be defects in the estrogen stimulatory pathway dista l to the i n i t i a l hormone binding step to cytoplasmic ER. Mechanism of estrogen stimulation (Figure 1) The current concept can be summarized as follows:-Estrogen is believed to diffuse in and out of a c e l l quite freely because of i t s lipophilic nature (22). Upon entrance into a target c e l l , i t combines with the cytoplasmic estrogen receptor. Such binding is highly specific with the dissociation constant close to 10-10 _ 10-H m o i / i . The complex then undergoes a transformation into an activated state to be translocated to the nucleus. Such a step has been shown by Jensen et al (23) by autoradiographic technique and believed to be essential for estrogen - i t -F i g . 1. A generalized model for the i n t e r a c t i o n of estrogens with target c e l l s . (Reproduced with permission from reference 111). -5-stimulation (24). Once inside the nucleus, the receptor complex binds to the chromatin acceptor s i t e s (25) to e l i c i t RNA (26), protein (27), DNA synthesis (28) and other physiological events. Inherent l i m i t a t i o n of cytoplasmic ER as a predictive index Measurement of the cytoplasmic ER l e v e l w i l l f a i l to reveal the state of the rest of the estrogen stimulatory pathway. Failure of the receptor complex to undergo transformation into an activated form, or defects i n translocation would render the c e l l s nonresponsive to estrogen stimulation. Sucrose density gradient centrifugation has been employed to q u a l i t a t i v e l y measure the receptor complex. Receptor with an 8S sedimentation c o e f f i c i e n t has been suggested as the required species for transformation into an active form (23). In addition lesions i n the chromatin acceptor s i t e s or transcription or translation defects would also result i n i n s e n s i t i v i t y towards estrogen stimulation. However, measurement of cytoplasmic ER s t i l l plays an important role in the management of breast cancer. Studies by Heuson et a l (29) and more recently by Godolphin et a l (30) have demonstrated the prognostic value of quantitative cytoplasmic ER determination. Additional markers as predictive indices With the understanding of the l i m i t a t i o n s of cytoplasmic ER measurements, alternate or additional markers have been sought to -6-improve the predictability. The two of interest to this project are nuclear estrogen receptors and progesterone receptors. Nuclear estrogen receptors (ERn) Assay of estrogen receptors in both the cytoplasm and the nucleus may indicate a defect in the translocation process. Such a defect might partly explain the failure rate of ERc+ tumors to endocrine therapy. Previous studies have reported that as many as 20 - 30% of ERc+ tumors do not have nuclear ER (31,32) . However, there are some unclear issues concerning the nuclear ER assay i t s e l f . F i r s t l y , i t has been proposed that the amount of unbound nuclear ER i s in equilibrium between cytoplasm and nucleus and partitioned according to the water content in the compartments (33). Therefore, the content of nuclear ER measured might depend on the homogenization procedure and the buffer being used . Secondly, depending on assay conditions, one may be measuring unoccupied or occupied receptors (34, 35). Unoccupied nuclear ER are also believed to e l i c i t a physiological response (36). Patients whose tumors have unoccupied nuclear ER have been suggested to represent a group which might benefit better from antiestrogen therapy (37)-Yet i t i s s t i l l unclear as to whether occupied or unoccupied nuclear estrogen receptors provide a more informative index. Thirdly, assay procedures for nuclear ER generally include either an extraction of nuclear protein with high salt buffer prior to an exchange assay (38) or incubation of isolated nuclei with radioactive tracers -7-(39). The former method has been c r i t i c i z e d because i t measures only the s a l t e x t r a c t a b l e form of r e c e p t o r s . I n a d d i t i o n , under high s a l t c o n d i t i o n , s t r i p p i n g o f bound s t e r o i d s from the receptors might lead to an i n c o r r e c t estimate of the r a t i o o f occupied to unoccupied receptors. Receptors r e s i s t a n t to s a l t e x t r a c t i o n have been suggested to be the required species f o r inducing e s t r o g e n i c response (40). However, the measurement of such species of receptors w i l l r e q u i r e the use of whole n u c l e i and can e a s i l y y i e l d over-estimates due to cytoplasmic contamination or trapping of s t e r o i d s during the washing procedure. Progesterone receptors (PgR) The presence of a product of estrogen s t i m u l a t i o n may be more i n d i c a t i v e o f a complete pathway and hence o f the tumor's responsiveness. The production of progesterone receptors i s s t i m u l a t e d i n human and animal uterus (41, 42), breast tumor c e l l l i n e (35) and r a t mammary tumor (43). I t has been post u l a t e d that simultaneous measurement of ER and PgR might be more in f o r m a t i v e than ER alone (44). Studies have shown that about 35 - 40? of the p o p u l a t i o n of tumors are ER and PgR p o s i t i v e , 15 - 20? are ER+ PgR-, 4 - 5 ? are ER- PgR+, 35 - 40? are ER- PgR - (45, 46). Among those t h a t are p o s i t i v e f o r both receptors, 70 - 80? respond to endocrine therapy (15, 47). -8-Estrogen and progesterone receptor p r o f i l e s i n experimental mammary  tumors A number of experimental systems have been used to investigate various aspects of breast cancer such as tumorigenesis, mechanism of hormone stimulation, tumor progression and therapy. The popular models include 7, 12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumor (48),the MCF-7 human breast c e l l l i n e (49), MXT mouse mammary tumors (50) and MT-W9 rat mammary tumor (18). Studies on hormone receptors have been described i n the hormone dependent and autonomous species of these experimental models. In DMBA-induced rat mammary tumor, the hormone-dependent type possessed ERc whereas the independent tumors were generally ERc- (105). Ovariectomy produced a reduction of ER and a progressive s h i f t towards autonomy. However, ERc l e v e l could be restored by stimulation with prolactin (106). Furthermore, PgR could also be demonstrated i n regressing and autonomous tumors (107). Similar receptors behavior were also shown i n MT-W9 rat mammary tumor (108) and MXT-3590 mouse mammary tumor (109). In the MCF-7 human breast c e l l l i n e , cultured tumor c e l l s were shown to be ERc+ despite estrogen-independent growth (49). Yet enhancement of growth rate could be accomplished by estrogen stimulation (110). While studies using these models (50,106,49,110) generally agree that both ER and PgR levels are higher i n hormone dependent than -9-autonomous tumors and the presence of ERc i n some autonomous growth, changes i n the receptor p r o f i l e s (ERc, ERn, PgR) within a transplantation series has not been reported along with tumor progression from hormone dependency to autonomy. Transplantable Noble rat mammary tumor lines Noble rat mammary tumors and their hormone related characteristics have been described in d e t a i l (51,52, 53, 54). B r i e f l y , tumors arise either spontaneously or after prolonged treatment with estrogen p e l l e t s . These tumors are found i n a variety of organs and include carcinomas of the mammary glands, p i t u i t a r y , ovary, thymus and others. While most of the spontaneous tumors i n females and a l l i n the males are autonomous, most of the tumors a r i s i n g from prolonged hormone treatment require estrogen for growth after transplantation. Most hormone dependent mammary tumors grow slowly and are predominantly p a p i l l a r y or adenocarcinomas. Metastases do occur though not frequently i n l i v e r and lung. Unlike the other i n i t i a l l y hormone dependent tumors such as DMBA-induced rat mammary tumors which tend to progress rapidly to autonomy after a few transplantations, hormone dependence i n Noble rats can be maintained over ten years. In addition, the transplanted c e l l s can remain dormant, but viable i n the hosts and retain hormone dependence when stimulated. -10-A number of transplantable tumor lines have been harvested in our laboratory. The ones studied in this project consisted of mostly autonomous tumors which arose from dependent ones. The lines were maintained by serial transplantation and might differ histologically and in their growth patterns. However, the major difference was that the regression of the original dependent tumor was controlled, thus the degree of progression towards autonomy might differ. Furthermore, growth of some of the transplants were re-stimulated with an estrogen pellet. Objectives The objective of this project was to determine the levels of cytoplasmic estrogen receptors, nuclear estrogen receptors and cytoplasmic progesterone receptors of the Noble rats mammary tumor lines. Such profiles might offer some insights or answers to the following questions:-1. Can the levels of ERc, ERn, PgR (or any combination considered together) indicate the hormonal dependency of the Nb rat mammary tumors? 2. How are the levels of ERc, ERn and PgR affected by sequential transplantation? 3. In the progression of hormone dependency to autonomy, is there an accompanying change in the ERc, ERn or PgR pattern which might indicate the presence of a biochemical lesion? MATERIALS AND METHODS Chemicals and radiochemicals 1 , 3 ,5 (10) - estratriene - 3 , 1 7 ^ - d i o l , (2 , 4 , 6,7 - 3H(N ) ) , (17<3 - e s t r a d i o l ) , 90-115 Ci/mmol, Dimethyl - 19-nor-pregna-4,9-diene -3,20 dione,(l7°t - methyl - 3H}(R5020), 70-87 Ci/mmol and nonradioactive R5020 were purchased from New England Nuclear (Boston, MA 02118). Radioactive steroids shipped in absolute ethanol were adjusted to 1 umol/1 concentration with 100? ethanol and stored at 4°C (refrigerator temperature). Working solutions were prepared freshly by appropriate d i l u t i o n with buffer to required concentration. D i e t h y l s t i l b e s t r o l was from Sigma Chemical Co. (St. Louis, MO 63178). Sephadex G-25M prepacked columns and dextran T70, MW -v 70,000 were obtained from Pharmacia (Pharmacia Fine Chemicals, AB, Uppsala, Sweden). Activated charcoal, radioimmunoassay grade came from Schwarz/Mann (Orangeburg, NY 10962). Unogel emulsifier was from Becton Dickinson Co. (Orangeburg, NY 10962). A l l other chemicals used were reagent grade Temperature control The different temperatures mentioned throughout this thesis were achieved as follows unless otherwise specified: 1. 0-4oc - ice water bath. 2. 4oc - refrige r a t o r . - 1 2 -3. 10°C, 12°C - c i r c u l a t i n g bath - model TE 9 equipped with a temperature controlled bath cooler - model PBC-4 (Neslab Inst. Inc. Portsmount, NH 03801). 4. 25°C - room temperature 5. 30°C, 37°C - Blue M constant temperature bath (Blue M E l e c t r i c Co., Blue Island, I l l i n o i s ) . Maintenance of mammary tumor li n e s i n Noble rats Transplantable tumor l i n e s were maintained i n Dr. G.L. Slemmer's laboratory i n the Department of Pathology at the University of B r i t i s h Columbia with the valuable cooperation of Dr. R.L. Noble (present address: Cancer Research Centre, 601 West 10th Avenue, Vancouver, B.C.) Male and female rats were used for transplantation. Small pieces of tumors were injected subcutaneously via a trochar into the backs of groups of two or three animals. The tissue was prepared by excision of viable growing areas of tumors which were then scissored into small pieces i n Hank's medium containing a n t i b i o t i c . Remaining tissues were quickly frozen i n l i q u i d nitrogen and stored at -80°c. Depending on the hormone dependency of the tumor, an estrogen p e l l e t (.90% estrone + 10% cholesterol) was inserted at the time of tumor implant. Four major classes of tumors with different growth rates can be c l a s s i f i e d according to t h e i r growth response to estrogen -13-suppleraent. They were harvested when t h e i r c h a r a c t e r i s t i c s were f u l l y manifested and usually weighed from 5-10 grams. However, si z e s up to 20 grams or bigger were also sampled. 1. Hormone Dependent Type (HD) - these tumors required adequate doses of estrogen f or growth and could only be maintained by s e r i a l transplantation. I f the estrogen p e l l e t was removed, the tumor would regress but remained dormant. Yet upon reexposure to estrogen, they had the capacity to resume growth and stayed hormone dependent. 2. T r a n s i t i o n from hormone dependent to autonomous type (HD/HA) -these tumors started growing as dependent tumors. Upon challenge by removal of estrogen p e l l e t , they regressed just as HD tumors would. However, a f t e r a va r i a b l e period of remission (3-15 months), spontaneous regrowth occurred and did not require estrogen supplement. 3. Hormone stimulated Type (HA') - t h i s type of tumors was observed only i n male rats which required an estrogen p e l l e t for the i n i t i a t i o n of tumor growth. Removal of the estrogen source a f t e r 2 days to 10 weeks of implant did not seem to a f f e c t growth. Neither was the growth pattern affected i f the p e l l e t was l e f t i n the animal. 4. Hormone Autonomous Type (HA) - these tumors would grow i n the absence of an exogenous estrogen supply. They were more aggressive and fa s t e r growing than the HD c l a s s and often exhibited hemorrhagic and necrotic centres. -14-Four tumor l i n e s were mainta ined over a number o f passages. Al though each t r a n s p l a n t passage was c a r r i e d out i n 3 or more an imals , on ly tumors w i t h t h e i r cy top lasmic recep tors (ERc+) measured are repor ted i n t h i s s tudy . These were most ly autonomous tumors which de r ived from dependent t ypes . H i s t o l o g i c a l l y , they appeared to be very homogeneous. Each tumor was coded to i d e n t i f y which tumor l i n e i t belonged to . For example, 15EMM l 6 ( l ) / 8 s i g n i f i e s tha t the tumor belonged to l i n e 15EMM 16(1) and the l a s t number "8" that i t arose i n the e i g h t h t r a n s p l a n t . P r e p a r a t i o n o f C y t o s o l A l l procedures were c a r r i e d out a t 0-4°C un less o therwise s t a t e d . Tumors were f i r s t thawed i n a 4°C c o l d room. Uninvo lved t i s s u e s and fa t were trimmed and t i s s u e was d i ced i n t o 2 mm cubes and q u i c k l y f rozen i n l i q u i d n i t r o g e n . On the day o f assay, samples were thawed i n 4 volumes o f Buf f e r A (Tr is -HC1 10 mmol/1, EDTA 1.5 mmol/1, d i t h i o t h r e i t o l 0.5 mmol/1, sodium molybdate 10 mmol/1, sucrose 0.25 m o l / 1 , pH 7 . 5 ) . They were then homogenized w i t h a P o l y t r o n PT-10 homogenizer (Brinkman Instrument I n c . , Rexdale , Ont. M9W 4Y5) a t a s e t t i n g o f 4.2 f o r 3 x 15s w i t h 1 min c o o l i n g p e r i o d i n an i c e - b a t h between b u r s t s . The homogenate was f i l t e r e d through Brunswick gauze ( K e n d a l l , Toronto , Ont, M4B 1X2) and fu r the r homogenized w i t h a Dounce, a l l - g l a s s t i s s u e homogenizer (Ca t . no. -15-357546 Wheaton, M i l v i l l e , NJ 08332): 10 strokes with a loose pestle and 7 strokes with a tight pestle. The homogenate was centrifuged at 800 xg for 10 min, the supernatant was removed and the pe l l e t was washed once with 2 volumes of buffer. The combined supernatants were centrifuged at 105,000 xg for 30 min with a Sorvall OTD-2 ultracentrifuge i n an AH 650 swinging bucket rotor (Dupont Inst., Newtown, CO 06470). The protein concentration of the soluble cytosol fraction was estimated by UV spectrophotometry and the following calculation (55). C1•55 A 2 g 0 - 0.74 A 2 6 o ) x Diluti o n Factor Binding Assay for Cytoplasmic Estrogen Receptors (ERc) Incubations were carried out i n 1.5 ml volume Eppendorf polypropylene tubes (Brinkman Instrument Inc.) i n duplicate with a f i n a l adjusted volume of 0.5 ml. The cytosol f r a c t i o n (350 j i l ) was incubated with 100 i l l of 3H-estradiol solution (0.21, 0.32, 0.54, and 0.84 nmol/1 f i n a l concentration) with or without 50 j i l of competitor ( d i e t h y l s t i l b e s t r o l , 0.4 jjmol/1 f i n a l concentration). A buffer blank was prepared by incubating 400 ,ul of buffer with 100 j j l of ^H-estradiol solution (concentrations same as above). After 2 hours of incubation at 4°C, 0.5 ml dextran-coated charcoal (DCC, 5 g Norit A activated charcoal, 0.5 g Dextran 70 per l i t r e T r i s buffer, pH 7.5) was added to remove unbound or loosely bound steroids. The suspension was thoroughly mixed and incubated for 30 minutes at 4orj. The charcoal was separated by centrifugation for -16-4 min at 10,000 xg i n an Eppendorf 5412 centrifuge (Brinkman Instrument Inc.). The supernatant (500 JJI) was counted for t r i t i u m i n 10 ml Unogel emulsifier with a Beckman LS 9000 s c i n t i l l a t i o n counter (Beckman Instrument, Palo Alto, CA 94304) at H2% counting e f f i c i e n c y . Doses were determined by counting 100 jul of ^H-estradiol solutions used i n the assay and correction for buffer blank. Specific binding (SB) at each dose was calculated as follows: 1. Total binding = (Amount of ^H-estradiol bound by cytosol i n the absence of DES) - (background binding) 2. Nonspecific binding = (Amount of ^H-estradiol bound by cytosol i n the presence of DES) - (background binding) 3. S p e c i f i c binding = Total binding - Nonspecific binding 4. Free unbound hormone (F) = dose - 3H-estradiol s p e c i f i c a l l y bound. Receptor concentrations and dissociation constants were estimated by Woolf and Scatchard analysis (56,57) with the assumption that there are only two types of binding s i t e s i n the system. One i s a saturable s p e c i f i c binding with high a f f i n i t y and the other i s a nonsaturable low a f f i n i t y nonspecific binding. -17-Scatchard analysis (56) The Michaelis-Menten equation can be rewritten as SB / F = Bmax / + SB / Where SB = number of s p e c i f i c binding s i t e s occupied at a parti c u l a r dose Bmax = maximum available number of spe c i f i c binding s i t e s F = free unbound hormone = dissociation constant In this analysis, SB/F i s plotted against SB. A straight l i n e i s f i t t e d to the data points by least square regression. The dissociation constant i s estimated by :-l/slope x 1/total assay volume. The receptor concentration can thus be calculated as follow: x-intercept/amount of protein i n the assay Woolf analysis (57) Transformation of the Michaelis-Menten equation y i e l d s F /SB = KJJ / Bmax + F /Bmax Analysis i s done by pl o t t i n g F/SB against F followed by a simple li n e a r regression analysis. The dissociation constant i s estimated by : x-intercept/total assay volume. The receptor concentration can be calculated as : 1/slope x 1/amount of protein i n the assay. Nonlinearity i n Woolf and Scatchard plots or inaccurate protein measurement could induce s i g n i f i c a n t errors i n receptor concentration estimation. -18-Binding Assay for Progesterone Receptors (PgR) Cytosol was prepared i n the same fashion as i n the estrogen receptor assay except 1.5 mmol/1 glycerol was substituted for 0.25 mol/1 sucrose i n the buffer. The incubation conditions and other assay procedures were i d e n t i c a l with the following exceptions: 1) The period of incubation was 1.5 hour. 2) The radioactive ligand used was ^H-R5020 at 0.4, 0.7, 1.0 and 1.4 nmol/1 ( f i n a l concentration). 3) Unlabelled R5020 (0.7 jumol/1, f i n a l concentration) was used as a competitor (rather than d i e t h y l s t i l b e s t r o l ) to estimate non-specific binding. S t a b i l i t y of estrogen receptor (ERc) i n lyophilized cytosol Cytoplasmic fractions from a number of estrogen receptor positive tumors were pooled, frozen and lyophilized overnight i n a Labconco refrigerated freeze dryer I I I (Labconco Corporation, Kansas City, M0 64132). After thorough mixing, the dry powder was aliquoted into Nunc cryotubes (Gibco Canada, Calgary, Alta T2G 4B7) and stored at 4orj. Samples were assayed for ER after various intervals of storage to test for s t a b i l i t y and also served as quality control specimens . Two batches (control A and B) with different receptor concentrations were tested. -19-I s o l a t i o n of Nuclei A l l steps were carried out at 0-4°C unless otherwise specified. Trimmed tissues were thawed i n 4 volumes of Buffer C (Tris 10 mmol/1, calcium chloride 3 mmol/1, d i t h i o t h r e i t o l 0.5 mmol/1, sucrose 0.25 mol/1, pH 7.5), homogenized with a Polytron PT-10 homogenizer, setting at 4.2, for 3 x 15 s. The homogenate was f i l t e r e d through Brunswick gauze and further homogenized i n a Dounce homogenizer (10 strokes with a loose pestle and 7 strokes with a ti g h t one). I t was centrifuged at 800 xg for 10 min and the p e l l e t was washed twice with 4 volumes of buffer. The p e l l e t was resuspended i n equal volume of buffer (5 strokes with a tight pestle in a Dounce homogenizer) and f i l t e r e d through Nitex nylon cloth with 150 jam openings (Cat. no. HC 3-150 Thompson Co. Ltd., St. Laurent, Montreal 379, Quebec). The f i l t r a t e was then centrifuged for 10 minutes at 800 xg . The p e l l e t was resuspended i n buffer, sonicated gently with a Biosonik I I I sonicator (Bronwill S c i e n t i f i c , Rochester, NY 14603) for 2 x 12 s with 50 watts output. The suspension was f i l t e r e d through Nitex nylon cloth with 48 .um pores (Cat. no. HC-48) and was washed once more followed by a f i n a l f i l t r a t i o n through a Nitex nylon cloth with 30 jum pores (Cat. no. HC-30). Degree of contamination and the intactness of the nuclear membrane were assessed by l i g h t and electron microscopic examinations. Pulverization of tissues i n a precooled teflon mortar with a stainless steel b a l l (1 cm diameter) and a Microdismembrator -20-(B Braun Melsungen AG, W. Germany) was also tested as a substitute i n the homogenization step. Electron Miorosoopio Examination of Nuclear P e l l e t s Samples were fixed with 25 ml glutar-aldehyde (Ladd Industries Inc., Burlington, VT 05401) per l i t r e of 0.1 mol/1 sodium dimethylarsonate buffer pH 7.3. A f t e r f i x a t i o n for 30 minutes, they were washed once i n buffer and post-fixed for 1-1.5 hours at 0-4° i n osmium tetroxide (10 g / l ) i n veronal buffer (sodium b a r b i t a l 35 mmol/1, sodium acetate 35 mmol/1, pH 7.4). Specimens were washed 3 times with 0.1 mol/1 sodium dimethylarsonate buffer and dehydrated for 5 minutes each i n 50%, 15%, 95% ethanol followed by 15 minutes each i n absolute ethanol and propylene oxide. Samples were embedded i n Effapoxy r e s i n (Fullam Inc., NY 12301) and allowed to polymerize i n a 37°C oven for 1 day followed by a 60° incubation f o r 2 days. S e r i a l sections of 0.1 jum thickness were cut using an LKB Ultramicrotome (LKB Inst. Ltd., Rockville, MD 20852), stained with aqueous uranyl acetate (50g/l) and lead c i t r a t e (58) and examined with a P h i l i p s EM 400 electron microscope ( P h i l i p s Industries, Eindhoven, The Netherlands). Binding assay for nuclear estrogen receptors A l l procedures were ca r r i e d out at 0-4oc unless otherwise stated. Clean n u c l e i free from contaminating debris were suspended i n 1.5 ml of Buffer D ( T r i s 10 mmol/1, EDTA 1.5 mmol/1, - 2 1 -d i t h i o t h r e i t o l 0.5 mmol/1, sodium molybdate 10 mmol/1, sucrose 0.25 mol/1, potassium chloride 0.4 mol/1, pH 8.5) and sonicated at 300 watts output for 3 x 10 s with 1 min cooling i n ice bath between. The extraction was carried out for 1 hour with vortex mixing at 10 min i n t e r v a l s . The soluble nuclear protein was separated from other nuclear debris by ultracentrifugation at 105,000 x g for 30 min i n an AH 650 swinging bucket rotor. The nuclear extracts (200 j j l ) were incubated with 100 u l of JH-estradiol solution (0.57, 1.43, 2.14 nmol/1 f i n a l concentration) with or without 50 J J I of d i e t h y l s t i l b e s t e r o l (0.57 pmol/1 f i n a l concentration) at 4° for 1.5 hour. The free estradi o l was separated by gel f i l t r a t i o n column chromatography. Sephadex G-25 prepacked column (9 ml volume) from Pharmacia were conditioned with 30 ml of water followed by 20 ml of Buffer A. The incubated mixture (300 JJ1) was layered on the column and the receptor complex were eluted with buffer A pumped at a flow rate of 1.4 ml/min with a Technicon 2-speed proportioning pump and 0.06 inch (internal diameter) tubing (Technicon Instruments Corporation, Tarrytown, NY 10591). Fractions of the eluant (450 jul) were collected, mixed with 10 ml of Unogel emulsifier and counted i n the l i q u i d s c i n t i l l a t i o n counter. Specific binding was calculated by substracting nonspecific binding (incubation with DES) from t o t a l binding (incubation without DES). Receptor concentrations and dissociation constant were estimated by Woolf and Scatchard analysis -22-(56, 57). Columns were regenerated with 50 ml water followed by 20 ml Buffer D and reused up to a maximum of three times. Fractions were collected for counting and columns were considered properly reconditioned when eluted radioactivity equalled background. Protein determination Protein concentrations were measured by a modification of the Lowry procedure (59). Appropriate volumes (5-20 jul) of the samples in t r i p l i c a t e were mixed with 1 ml reagent (1 volume CuS04-5H20 (5 g/1) and 1 volume sodium potassium tartrate (10 g/1) freshly mixed with 50 volumes Na 2C0 3 (20 g/1 in 0.1 mol/1 NaOH)) and allowed to stand for 10 min or longer at room temperature. To these were added, with rapid vortex mixing, 100 jjl 1 mol/1 Folin-Ciocalteau phenol reagent (Fisher Scientific Co., NJ 07410). Absorbance was measured after 30 min at 500 nm on a Cary Model 118C dual-beam spectrophotometer (Varian Inst., Monrovia, CA 91016) with buffer blank as reference. A standard curve was constructed with bovine serum albumin (Cat. no. A-4378, Sigma Chemical Co., St. Louis, MO 63178) up to 100 g/1 concentration. Deoxyribonucleic acid (DNA) determination The indole method was used (60). Nuclear pellets were washed with 2 ml ice cold trichloroacetic acid (100 mg/1) to remove free nucleotides. They were resuspended in 1.5 ml water with an equal -23-volume of 0.66 mol/1 perchloric acid added and hydrolyzed for 20 min at 80°C. The extraction was repeated once and supernatants were combined. Aliquots of these hydrolyzed sample supernatants were adjusted to a f i n a l volume of 1.5 ml with water, then 750 jul indole reagent (6.83 mmol/1 indole, 60 jjmol/1 cupric sulphate) and 750 u l of concentrated hydrochloric acid (12 mol/1) were added. The samples were heated at 100°C i n a water bath for 10 min. After cooling to room temperature, the samples were extracted 3 times with 2 ml volumes of chloroform. Absorbance of the aqueous phase was measured at 490 nm. Hydrolyzed deoxyribonucleic acid (Sigma Chemical Co., St. Louis, MO 63178) (concentration up to 25 g/1) was used as standard. _24-RESULTS AND DISCUSSION Optimal assay conditions were f i r s t sought. A number of systems using different buffers, length of incubation and incubation temperatures have been reported. Yet due to the difference in types of tissue used and their proteolytic enzyme content, assay parameters might require adjustment. In the buffering system for cytoplasmic receptors assays, Tris-HCl was used to control the pH. EDTA was included to chelate divalent cations which have been believed to be important cofactors in receptor activation (63). Also included is molybdate, a potent phosphatase inhibitor, which has also been reported to stabilize ER and PgR by preventing their activation and inhibiting protease activity during the homogenization step (64, 65, 66). Sucrose was also added to adjust the buffer osmolarity to simulate the in vivo environment thus stabilizing the nuclei and lysosomes. Sulfhydryl reagent (dithiothreitol) supplement to prevent receptor loss has also been recommended (67, 68) and was included in the system. Glycerol was used in place of sucrose for the PgR assay to prevent dissociation of the hormone complex which happens much faster than is the case for cytoplasmic estrogen receptors (69). Dextran coated charcoal was used to separate bound from free ligand in both the cytoplasmic ER and PgR assays. This is not the -25-ideal method to employ since i t w i l l alter the equilibrium between hormone-receptor complex and hormone, and thus is unable to measure the true dissociation constant. However, i t is simple, reproducible and economical. Equilibrium dialysis would be the most suitable approach but is not practical due to the time required especially when a large number of specimens are being assayed. Nevertheless, for a receptor having a high a f f i n i t y for the ligand, the shift in equilibrium from bound to free would not be significant within a short period of perturbation. Effects of temperature and time of incubation on cytoplasmic estrogen  receptors assay. Because of- the reported heat l a b i l i t y of estrogen receptors (61) assays were set up to measure their activity at 4°, 10°, 12°, 25°, 30° and 37°C for various lengths of time to select the optimal assay conditions. Cytosols were incubated with a 1.0 nmol/1 ^H-estradiol ( f i n a l concentration) with or without 0.4 jumol/l ( f i n a l concentration) of DES. Unbound steroids were removed by DCC treatment as described in the materials and methods. Results are summarized in Figure 2. At 30° and 37°C, loss of receptor activity became very significant even after a short 20 min incubation. This observation correlates quite well with the literature (61). At room temperature, activity reached a maximum in 40 min and - 2 6 -Time (hour) Fig. 2. Temperature and time of incubation: effects on cytoplasmic estrogen receptor assay. Cytosol ( 3 5 0 jjl) was incubated at specified temperature for various lengths of time with 100 nl of 3n-estradiol ( 1 . 0 nmol/1 f i n a l concentration) with or without 50 u l diethylstilbestrol (0.U jjmol/1 f i n a l concentration). Unbound steroids were removed by dextran coated charcoal treatment. Specific binding was obtained by subtracting nonspecific binding from total. Two hours incubation at 4 o c was selected as the control condition. -27-began to f a l l to 60% after 4 hours. Incubations carried out at 10°C or 12°C did not d i f f e r much i n t h e i r affect on receptor a c t i v i t y . Maximum s p e c i f i c binding capacity was comparable to that measured at 4°C. At 4°C, 1.5 -2.0 hours of incubation were necessary to reach maximum binding. In addition, the le v e l was equal to or higher than that attained at other temperatures. Incubation at lower temperature might slow down the rate of protease degradation but prolonged assay such as overnight incubation resulted i n a loss of 20% of the binding capacity. Thus the conditions selected for cytoplasmic ER assays were 2 hours of incubation at 4°C. Effect of protein concentration on cytoplasmic estrogen receptor  assay Cytosols of increasing concentration of 0.72 - 5.0 mg/ml of protein were assayed for cytoplasmic ER. Results are summarized i n Figure 3> Within the protein concentration tested, there was l i t t l e difference i n the receptor concentrations measured. Panko et a l i n their recent review (70) recommended a minimum concentration of 1.5 - 2.0 mg/ml, an optimum of 3.0 - 4.0 mg/ml and no maximum l i m i t for DCC exchange assay for cytoplasmic estrogen receptors. In addition, Poulsen (71) has emphasized the importance of having a s u f f i c i e n t amount of protein i n order to obtain r e l i a b l e ER data. Markland et a l (72) have demonstrated a - 2 8 -protein concentration (mg/ml) Fig. 3- Effect of protein concentration on cytoplasmic estrogen receptor assay. Cytosol ; ( 3 5 0 jjl) with protein concentrations of 0 . 7 2 to 5 . 0 mg/ml were incubated with 1 0 0 ial of 3n-estradiol ( 1 . 0 nmol/1 f i n a l concentration) with or without 50 jjl diethylstilbestrol ( 0 . 4 jjmol/1 f i n a l concentration) for 2 hours at 4°C. Free ligand was removed by dextran coated charcoal treatment. Estradiol specifically bound was obtained by subtracting nonspecific binding (in presence of diethylstilbestrol) from total binding. Maximum binding sites available (Bmax) were estimated by Woolf and Scatchard analyses ( 5 6 , 5 7 ) . Results shown were obtained using three different tumors. -29-si m i l a r p r o f i l e i n PgR analysis showing a linear relationship between concentration of ligand bound and protein concentration i n the assay. I f enough tissue i s available, protein concentration of 1.5 mg/ml i s recommended. P a r t i c u l a r l y when a low sp e c i f i c binding tumor i s encountered, the signal to noise r a t i o for binding would be increased for better d i f f e r e n t i a t i o n from background, thus less error would be incurred i n calculating s p e c i f i c bound. Otherwise, the error could be as large as 50%. Effect of time of incubation on progesterone receptor assay. Progesterone receptors have been reported to be even more heat l a b i l e than ER (62), thus 4°C was chosen as the incubation temperature. Figure 4 i l l u s t r a t e d an increase of nonspecific binding with respect to time of incubation, but the maximum le v e l of sp e c i f i c binding was reached i n 1.5 - 2.0 hours. S i m i l a r l y to the ERc assay, overnight incubation resulted i n a 20% loss of s p e c i f i c binding capacity. The conditions thus selected for routine PgR assay were incubation for 1.5 hours at 4°C. Estrogen receptor a c t i v i t y i n lyoph i l i z e d cytosol. Estrogen receptors measured i n two different batches of lyophilized cytosol kept at 4°c showed no s i g n i f i c a n t decrease i n binding a c t i v i t y up to 50 days of storage (Figure 5). For Control A, the mean concentration was 38.9 + 1.3 (standard deviation) -30-Fig. 4. Effect of incubation period on cytoplasmic progesterone receptor assay. Cytosol (350 .ul) were incubated with 100 jjl of 3H-R5020 (1.4 nmol/1 fi n a l concentration) with or without 50 ul of cold R5020 (0.7 jjmol/1 f i n a l concentration) at 4oc. Unbound steroids were removed by dextran coated charcoal treatment. -31-ControlAmean 38.86 S.D. 1.3 n=ll I I I I I I I I I i ' 0 10 20 30 40 50 Time (days) Fig. 5. Stability of estrogen receptors in lyophilized cytosol controls. Isolated cytoplasmic fractions of Nb rats mammary tumors were pooled, lyophilized and stored at 4oc in a dessicator. After various periods of storage, aliquots were assayed for estrogen receptors by performing a four dose binding study. Results were analyzed by the Woolf (57) and Scatchard plots (56). -32-fmol/mg protein. Control B had a lower concentration of 21.6 + 2.0 fmol/mg protein. Both pools demonstrated high a f f i n i t y binding with -11 -11 mean dissociation constants of 3.9 x 10 mol/1 and 5.3 x 10 mol/1 respectively. A t i t r a t i o n curve showed saturated s p e c i f i c binding (Figure 6A). Binding sit e s and dissociation constants were similar using either the Scatchard or the Woolf method (Figure 6 B,C). Despite the large number of methods available for data analysis from graphic representation (84, 85) l i n e a r plots of Woolf (57), Lineweaver Burk (86), Scatchard (56) or the non-linear and weighed analysis (87), to the more complex approaches employing computer programmes (88), the l i n e a r Scatchard plot has remained the most popular one i n the hormone receptor f i e l d . I f one has a large number of good data points over a wide concentration range, then i t r e a l l y does not pose a problem as to which i s the appropriate method to select, for the end result w i l l not d i f f e r much. Yet due to the limited amount of tissue available, a multi-dose binding assay cannot always be performed. I f the small number of data also include one or two e r r a t i c points, they must be interpreted with extreme caution. For cytoplasmic ER and PgR assays i n this study, l i n e a r Scatchard and Woolf plots were employed to analyse the data. Four ligand concentrations were selected to provide good d i s t r i b u t i o n of points along the plots. P a r a l l e l incubations with competitor at each dose avoids a) the dependence on the accuracy of 1 or 2 -33-Addendum t o F i g u r e 6A I n t e r p r e t a t i o n o f raw d a t a Raw d a t a o b t a i n e d from s c i n t i l l a t i o n c o u n t i n g : 1. Amount o f - e s t r a d i o l bound by c y t o s o l i n t h e absence o f DES. 3 2. Amount o f K - e s t r a d i o l bound by c y t o s o l i n t h e p r e s e n c e o f DES. 3. Dose. 4. R e s i d u a l background count f o r each dose a f t e r d e x t r a n - c o a t e d c h a r c o a l t r e a t m e n t . 5. B u f f e r b l a n k . C a l c u l a t i o n : 1. D i l u t i o n f a c t o r = 2, s i n c e o n l y 0.5 ml was c o u n t e d o u t o f a t o t a l volume o f 1.0 ml (0.5 ml i n c u b a t i o n m i x t u r e + 0.5 ml DCC). 2. T o t a l b i n d i n g = ((Amount o f 3 H _ e s t r a d i o l b o u n d b y c y t o s o l i n t h e absence o f DES) - ( R e s i d u a l background count f o r t h e r e s p e c t i v e dose )) x D i l u t i o n f a c t o r . 3. N o n s p e c i f i c B i n d i n g = ((Amount o f 3 H - e s t r a d i o l bound by c y t o s o l i n t h e presence o f DES) - ( R e s i d u a l background count f o r t h e r e s p e c t i v e d o s e ) ) x D i l u t i o n f a c t o r . 4. S p e c i f i c B i n d i n g = T o t a l B i n d i n g - N o n s p e c i f i c B i n d i n g . 5. F r e e unbound l i g a n d s = Dose - S p e c i f i c B i n d i n g . -34-80 h 60 o E — 40 ~D c 3 o CQ "5 o L-LU r? 20 Total: without DES ^ ' Specific - Total —nonspecific Nonspecific: with DES 0.2 0.4 0-6 -Estradiol dose (nmol/l) 0.8 Fig. 6A. Titration of estradiol-binding of cytosol of lyophilized Control A. Cytosol (350 ju.1) with 2.0 mg protein was incubated with 100 u l of 3H-estradiol (0.2 - 1.0 nmol/l f i n a l concentration) for 2 hours at Hoc with or without 50 u l of 500 fold excess DES. Duplicates were done with a final adjusted volume of 0.5 ml. After incubation, 0.5 ml dextran coated charcoal (5 g charcoal, 0.5 g dextran per l i t r e pH 7.5 Tris buffer) was added to remove unbound steroids. -35-Addendum to Figure 6B Analysis of e s t r a d i o l binding by Woolf pl o t (57): The Michaelis-Menten equation can be rewritten as F/SB = K n / B + F / B D max max where SB = number of s p e c i f i c binding s i t e s occupied at a p a r t i c u l a r dose Bmax = maximum av a i l a b l e number of s p e c i f i c binding s i t e s F = free unbound ligand = d i s s o c i a t i o n constant In t h i s analysis, F/SB i s plotted as a function of Free ligand. A s t r a i g h t l i n e i s f i t t e d to the data points by least square regression The d i s s o c i a t i o n constant (K^) estimation = (y-intercept / slope )( 1 / t o t a l volume of assay mixture ) Maximum a v a i l a b l e number of binding s i t e s (Bmax) = ( 1 / slope )( 1 / amount of protein i n assay) -36-Fig. 6B Analysis of estradiol binding according to the Woolf plot (57). Dissociation constant (K D) estimation = ( y-intercept / slope )( 1 /total volume of assay mixture ) = 21.1 x lO -^ m o i / Q.5 ml = 4.2 x 10_n Maximum available number of binding sites ( Bmax ) = ( 1 / slope )( 1 / amount of protein in assay ) = 81.95 x 10-15 m o l / 2.01 mg = 40.2 x 10-15 mol / mg protein. -37-Addendum to Figure 6C Analysis of e s t r a d i o l binding by Soatchard p l o t (56) Transformation of the Michaelis-Menten equation y i e l d s S B / F = Bmax / + SB / Kp Analysis i s performed by p l o t t i n g SB / F against SB followed by a simple regression analysis. The d i s s o c i a t i o n constant (K^) estimation = -( 1 / slope )( 1 / t o t a l volume of assay mixture ) Maximum a v a i l a b l e number of binding s i t e s (Bmax) = -( y-intercept / slope )( 1 / amount of protein i n assay) -38-r C Scatchard Plot r 2 0.96 Specific Bound (fmole) Fig. 6C. Analysis of estradiol binding according to the Scatchard plot (56). Dissociation constant (Kp) estimation = ( 1 / slope )( 1 / total volume of assay mixture ) = 24.4 x 10 _ 15 m o i / 0 . 5 ml = 4.9 x 10-11 m o i / i . Maximum available number of binding sites ( Bmax ) = ( y-intercept / slope )( 1 /amount of protein in assay ) = 8 3 . 3 x 10-15 mol / 2.04 mg = 41.1 x 10-15 mol/mg protein. -39-nonspecific binding data to extrapolate the nonspecific binding at other doses by assuming i t is a linear phenomenon b) the p i t f a l l of nonlinearity in nonspecific binding which has been suggested in a number of situations (89). Binding curves similar to Figure 6A were plotted to enable detection of any anomaly. Overall, the results obtained from the two plots seemed to agree very well. Assessment of quality of nuclear preparation Edwards et a l (73) have demonstrated the potential error in nuclear estrogen receptors assay due to cytoplasmic contamination. A number of reports have used a crude nuclei preparation and claimed there was no interference (74, 39). However, isolation of clean intact nuclei as the starting material would certainly avoid the controversy. A number of procedures for nuclei isolation have been reported (75, 76). Some of them employ systems incompatible with receptor analysis like organic solvents or strong detergent and the majority would perform ideally only with tissues having l i t t l e stromal material. Nuclei were isolated from tumor 31 EMM - (D) according to the procedure described in materials and methods. The isolation steps were monitored with light microscopy (Figure 7 - 12). Nuclei free of cellular organelles attached were obtained and electron microscopic examination showed intact nuclear membranes (Figure 13, 14) - 4 0 -Fig. 7-12. Assessment of nuclear preparation. Photomicrographs represent preparation along the isolation procedure. A l l sections, x100 or x 160. Fig. 7. Trimmed and diced tumors were homogenized with a Polytron homogenizer in 4 volumes of buffer. Fig. 8. Specimen was filtered through gauze and further homogenized with a Donnce homogenizer. Crude nuclei were collected by centrifugation at 800 x g for 10 min followed by two washes with buffer. Fig. 9. Crude nuclei were resuspended in buffer, homogenized with a Donnce homogenizer and fil t e r e d through nylon cloth with 150 jum openings. Fig. 10. Pellet collected after centrifugation at 800 x g for 10 min was sonicated gently for 2 x 12s with 50 watts output. Fig. 11. Specimen was filtered through nylon cloth with 48.jjm openings. Fig. 12. Nuclei was washed once more with buffer and filtered through nylon cloth with 30 um openings. -41--42-Fig. 13, 14. Electron micrographs of isolated intact nuclei were i n i t i a l l y fixed with glutaraldehyde and post-fixed with osmium tetroxide. Following dehydration procedure with alcoholic washes, specimen was embedded i n Effapoxy resin. After polymerization, 0.1 jum sections were cut using an LKB ultramicrotome, stained with uranyl acetate and lead c i t r a t e and examined with a P h i l i p s EM 400 electron microscope. Magnification for F i g . 13 and 14 are 15,200 x and 56,000 x respectively. -43-Like the scirrhous type of tumor in human, rat mammary tumors have a high content of connective tissue, in particular when the tumors progress towards autonomy. Isolation of clean intact nuclei from such fibrous type of tissue is very d i f f i c u l t . Tissue pulverization as recommended by the E.O.R.T.C. Breast Cancer Group may be performed with (77) a dismembrator (Braun, Melsungen, Germany). This not only broke up the cells and inevitably some nuclei but also the fibrous connective tissue into small pieces and strands which tended to cling onto nuclear membrane very tightly. Nuclei contaminated with cytoplasmic debris might interfere with ERn assay (Figure 15). The isolation procedure described employed gentler homogenization and l e f t the stroma less extensively shattered. Then i t can be removed more easily by f i l t r a t i o n through nylon cloth with discriminating pores. Inclusion of Ca + + in the buffer inhibits receptor release into the wash or soluble fraction from the nuclei. It helps maintain an intact nuclear membrane and does not seem to affect the af f i n i t y of estradiol to receptors (39). Sonication of the nuclear preparation at low amplitude loosens the cytoplasmic debris attaching to the nuclear membrane and faci l i t a t e s i t s removal. Excessive breakage of nuclei resulting in DNA spillage into the cytoplasmic fraction during isolation was -44-Fig. 15. Electron micrograph of nuclei isolated via employment of a microdismembrator as the i n i t i a l tissue homogenizing tool. -45-also checked. The DNA concentrations i n the cytoplasmic f r a c t i o n and in the nuclei were compared. The mean (+ S.D.) DNA i n cytosol was 3-0% (+ 0.4) of that i n the nuclei (analyses done on 300, 400, 500 mg tissue from two different tumors). However, the major l i m i t a t i o n of t h i s procedure i s that a l o t of tissue i s required to generate a good y i e l d of nuclei for multi-dose receptor analysis. Approximately two grams of tissue were required to y i e l d three milligrams of protein thus only three ligand concentrations could be used for this assay. Nuclear estrogen receptor assay Various methods of assaying nuclear estrogen receptors using protamine sulfate p r e c i p i t a t i o n (78), direct nuclear exchange (79) and hydroxylapatite (38) have been reported. Protamine sulfate f a i l s to function i n the presence of high s a l t buffer (0.4 mol/1) which i s used to extract nuclear receptor protein, nor can i t eliminate nuclear protease a c t i v i t y (80). Although adsorption of receptors to hydroxylapatite can prevent proteolytic degradation, r e l a t i v e l y high nonspecific background i s observed as i n the case with the direct nuclear exchange technique (79). The conventional DCC method used i n cytosol estrogen receptors assay i s not applicable with high s a l t buffer. The method would underestimate the receptor quantity due to "stripping" of radioactive ligand from the receptor complex by charcoal under high ionic strength conditions (81). -1J6-The use of Sephadex column chromatography avoids the interference by s a l t (82, 83), but had been unpopular for being impractical for handling large numbers of fractions. However, the miniaturisation of columns f a c i l i t a t e s fast e f f i c i e n t separation between bound and free ligands. The fast separation time also minimizes the length of exposure to non-equilibrium conditions by the receptor complex. A representative Sephadex G-25 elution p r o f i l e i s shown i n Figure 16. A s p e c i f i c binding component was excluded by the column and was absent i n p a r a l l e l incubations with excess DES. The free es t r a d i o l being retarded by the column resin was eluted at a much slower rate. Binding i n the absence of excess DES under the f i r s t elution peak was considered as t o t a l ligand bound. P a r a l l e l incubation with excess DES at f i r s t peak would represent nonspecific binding. Effect of time of incubation on nuclear estrogen receptor assay With the establishment of the gel f i l t r a t i o n p r o f i l e s , studies were carried out to select the optimal length of incubation. After 1.5 hour of incubation, s p e c i f i c binding reached a maximum and was stable up to 20 hours (Figure 17). The conditions thus selected for the assay were 1.5 hour of incubation at 4°C. - 4 7 -Fraction F i g . 16. E l u t i o n p a t t e r n o f e s t r a d i o l b i n d i n g i n nuc lea r f r a c t i o n o f tumor from a column o f Sephadex G25 (5 cm x 1.5 cm d iame te r ) . The 300 ul sample o f incubated mixture c o n t a i n i n g nuc lea r e x t r a c t , 3H-estradiol , w i t h or wi thout excess d i e t h y l s t i l b e s t r o l was a p p l i e d to the column and e l u t e d a t a f low ra te o f 1.4 m l / m i n . The arrow i n d i c a t e s e l u t i o n o f an ovalbumin marker. -48-1.0 2.0 Time (hour) 20 Fig. 17. Effect of time of incubation on nuclear estrogen receptor assay. Nuclear extract (200 jul) was incubated with 100 u l of 3H-estradiol (2.14 nmol/l fi n a l concentration with or without 50 jjl of diethylstilbestrol (0.57 pmol/1 fi n a l concentration). After various periods of incubation, 300 jul of the incubated mixture was separated on a Sephadex G25 column. -49-Hormone and receptors status in tumor cell lines Line 15 EMM l6(l)/6 This tumor line had been maintained for six passages and was hormone dependent throughout. At the seventh passage, three major sublines arose stemming from the original donor demonstrating the heterogeneity of tumor cell types that could propagate in a single animal. Rats A, B and C bore dependent tumors, but upon removal of the estrogen pellet after 5-6 months of implant, tumors continued to grow and no longer required exogenous estrogen. They were thus classified as HD/HA type. Almost all tumors from subsequent transplants were completely autonomous (HA) (Figure 18). Histologically, these tumors were quite different. Tumor C was a highly fibrous type of tumor whereas tumor B was of a ductal type. Two classes of tumor also arose in rat A, a glandular carcinoma (Aa) and a sarcoma (Ab). Transplants from rat A were carried out with tumor Aa. Subsequent passages from Aa, B and C were histologically identical to their predecessors. Two tumors (Ag> B^) harvested in this line were regarded as poor specimens which showed a lot of hemorrhagic infiltration and the receptor status must be interpreted with caution. All the tumors in this line possessed cytoplasmic estrogen receptors i.e. ERc+ (Figure 19). The ERc levels of transplants originated from donors A and B (group I) and donor C (group II) were 24.9 + 12.2 fmoles/mg protein (mean + standard error of mean, n=17) -50-Fig. 18. Composite pedigree of Noble rats mammary tumor l i n e 15 EMM l 6 ( l ) / 6 . Tumor transplants were carried out by i n j e c t i n g subcutaneously small pieces of viable tumor into the backs of 2 or 3 animals. Estrogen pe l l e t (90? estrone + 10? cholesterol) was inserted at the time of implant i f necessary depending on the hormonal status. Only tumors with receptor assays performed are included i n the p r o f i l e . HD = hormone dependent, HD/HA = tr a n s i t i o n from dependent to autonomous after estrogen p e l l e t removal, HA' = hormone stimulated, HA = hormone autonomous. 15 EMM 16(1 )/6 2 2 K I 6 - 3)B86 - 4 ( S - 4 ) 28 E p 19 119 8(6-5) S O *| l2-4(7-4) 12 125 - 4 (62 ) 5-6 ^ 1 3 5 - 0 ( 7 8 ) 3 0 (2)18-4(10-6) (T)5-2(76) V > 1 8 ^ 2 8 8 Y fT124 1(56) rri25-9(3-9) f~122-6(3-6) rT|43-3(l32) /^38-9(2-9) T~\39-t LLlITi L£J~~4^9'"" LU 142 LU 30 v3> 3-e VL1~3-, | 7 9 ( 6 5 ) O 2 1 3 9 ( 0 ) O I H1'3-2(I2 O) fi\6-6(70) O O 39-6(0) 3-7 |V | lO -8( l3 -4) O 6-0(2 I) \ 3 0 ( 7 0(5-4) 0HD/HA6* • HAC/ O h a 9 @ U n k n o w n E R C ( f m o l / m g p r o t e i n ) ( E R n ) r ^ | ! 4 ^ 5 j r^tfLiip*) [3'±3iHi P g R 16-6(9) Q j ) 6 0 ( l 2 - I ) -52-C O CD E "o E WO <D lo CO CO 15 EMM A and B £ 40 20 - t • • 15 EMM G 0 • mmn .23 ERC ER n PgR ERC ER n PgR F i g . 19. Schematic r e p r e s e n t a t i o n of the hormone receptors p r o f i l e s of Noble r a t mammary tumor l i n e 15 EMM 16 ( l ) / 6 . -53-and 11.9 +8.3 fmoles/mg protein (n=26) respectively. Dissociation constants of the two groups were quite comparable (Group I = 7.67 x 10~ 1 1 mol/1, Group I I = 1.1 x 10' 1 0 mol/1). Although the difference of the mean ERc levels between the two groups was s t a t i s t i c a l l y s i g n i f i c a n t (p < 0.01, Student's t - t e s t ) , the ERc levels between tumors a r i s i n g i n female and male recipients within each group (Figure 20) and i n the whole l i n e showed no s i g n i f i c a n t difference (p> 0.05, Student's t - t e s t ) . This implies that the ERc levels of these autonomous tumors were not greatly influenced by the sex or the hormonal environment of the animals they had grown i n . With regard to nuclear ER (ERn), a l l the group I tumors possessed measurable amounts whereas only some of the group I I tumors did. A steady l e v e l of ERn was also observed i n group I tumors carried over several passages . However, the difference i n levels between the two groups was not s i g n i f i c a n t (p> 0.05, Student's t - t e s t ) . A common observation among some of the sublines i s the constant trend of receptor l e v e l (ERc, ERn, PgR) possessed by the tumors harvested over a number of passages into animals of either sex (Table I ) . This phenomenon suggests that 1) production of receptor i s "genetically" coded by individual c e l l type 2) i f there i s a signal for receptor production, the degree of expression of the gene(s) coded for i t s synthesis i s i n turn regulated i.e. the l e v e l of production i s closely monitored 3) the -54-C '55 o O) E A and B ERC15 EMM 40 r ERC 15 EMM o E ^ 20 to <D co CO CO •Ir > l a' Fig. 20. Estrogen receptors status of tumors arising in male and female hosts of Noble rat mammary tumor line 15 EMM l6(l)/6. - 5 5 -Table I . Estrogen and progesterone receptor concentrations i n sublines of 15 EMM l 6 ( l ) / 6 . Receptor Tumors Analyzed Receptor Concentration:(fmol/mg protein) Assay Mean + SD a Aa b, A l , A2 20.6 + 1.9 B, B l , B8, B9, BIO 32.5 + 9.6 B, B1-B7 24.2 + 10.2 C l , C2, C3, C7 6.7 + 0.8 Cl - C6 7.8 + 3.7 C, C8 - C12, C22 1 3 . 1 + 2.1 C13 - C20 10.4 + 7.1 Aa, A l , A2 12.8 + 3.3 B, B1-B7 4.7 + 2.0 B, B l , B8 - BIO 7.7 + 3.8 Aa, A l , A2 1.9 + 0.9 B, B l , B8 - BIO 4.1 + 1.1 B8 - BIO 3.3 + 0.5 B, B l - B7 7.0 + 4.5 a - standard deviation b - the second symbol denotes the tumor number in the subline coded by the f i r s t symbol as the originating tumor e.g. B4 represents tumor #4 i n the subline with tumor B as the o r i g i n a l tumor. -56-l e v e l of expression i s not influenced by the hormonal environment of the tumor except with minor modification. An interesting point i n this tumor l i n e was that group I tumors a l l had PgR whereas only three specimens of the group I I tumors did. Although the presence of PgR seemed to associate with a higher l e v e l of ERc as observed i n group I tumors, no s i g n i f i c a n t correlation was found between them (p> 0.05). Furthermore, ERc was not correlated to ERn nor ERn correlated with PgR (p> 0.05). Similar finding has also been reported by Stojkavic in human breast tumors (73). Therefore with respect to the receptor content, the HA tumors i n t h i s l i n e can be further c l a s s i f i e d into two major groups. The group I tumors were of ductal or glandular type' and possessed the f u l l spectrum of receptors assayed (ERc, ERn, PgR). In contrast the group I I tumors were of a fibrous type and were ERc+, ERn+ or - , and PgR-. The group I HA tumors (transplants from A and B) demonstrated that despite a seemingly intact estrogen stimulatory pathway (ERc+, ERn+, PgR+), they did not depend on exogenous estrogen for c e l l growth. Further investigations to elucidate whether these animals require an intact estrogen stimulation pathway for growth or receptors measured were nonfunctional gene products might better explain the receptors behavior in this type of autonomy. -57-The maintenance of an intact pathway was essential i f the transition from HD to HA behaviour in this group was due to an increase in sensitivity of the cells to estrogen. Such alteration would imply that endogenous level of estrogen in female animals or conversion of testosterone to estrogen in the male hosts was sufficient to sustain growth. The increase in estrogen sensitivity might represent genome adaptation at a variety of sites. Three such alterations might be: First, instead of on a one-to-one production basis, there was a multi-fold increase in mRNA transcription upon binding at a single acceptor site. Changes in the frequency of i n i t i a t i o n of RNA synthesis have been postulated in hormonal control of target tissues (90, 91). Second, permanent retention of receptor due to altered nuclear acceptor sites. Clark and Peck (57) have shown that more than 6 hours of retention is necessary for the hormone-receptor complex to e l i c i t true physiological response. Third, unmasking of "active" acceptor sites upon challenge by hormone removal. Spelsberg et al (92) have shown the presence of masked acceptor sites in nontarget chromatin. However, the a b i l i t y of this group of tumors to grow without exogenous estrogen supplement cannot be interpreted as complete nonresponsiveness to estrogen . Cytoplasmic ER and PgR have also been detected in MCF-7 human breast tumor c e l l culture which can grow in the absence of estrogen (49). Upon estrogen stimulation, these cells have the potential to grow at a faster rate. -58-In summary, the speculation that autonomous growth might always be associated with lack of a b i l i t y to make or translocate ER or PgR could not be demonstrated in this group of tumors. Yet the possession of an intact estrogen stimulatory machinery might be useful for controlling growth by hormone manipulation in this group. Unlike the group I tumors, the group II tumor transplants from tumor C did not have some of the receptors being assayed for. While a l l of them had measurable amounts of ERc, only some possessed ERn. The major difference from group I as mentioned earlier was that almost a l l of them had no PgR. This absence indicated that there may be a potential transcriptional defect resulting in non-production of PgR; or the problem might also be due to failures in translocation or transformation of cytoplasmic ER into an activated form; or a combination of these. A variety of receptor activation mechanisms such as simple conformation change (93), dimerization (94) or addition of other proteins (95) have been reported to be necessary for normal nuclear translocation. Although a translocation step is required, i t is not sufficient to e l i c i t an estrogenic response by i t s e l f . Antiestrogens such as tamoxifen or nafoxidine though capable of being translocated, f a i l to trigger biological response probably due to a transcriptional defect (9,6). Therefore, the lack of an intact estrogen receptor mechanism would predict that these tumors would be nonresponsive to estrogen. It is also unlikely that their growth could be controlled by hormone - 5 9 -manipulation. To further complicate the scheme of estrogenic stimulation, the effect of estrogen on c e l l growth and PgR synthesis might depend on hormone receptor binding at discreetly different acceptor sit e s or the degree of occupancy of a common one. Alteration at the acceptor s i t e for PgR synthesis or a suboptimal degree of occupancy of acceptor s i t e s might result i n nonproduction of PgR. Therefore, the absence of PgR does not preclude the p o s s i b i l i t y that these tumors might be hormone sensitive. Genome adaptations l i k e those suggested for group I tumors may occur or c e l l growth might require fewer occupied acceptor s i t e s . Leake has postulated that only a small number of acceptor sit e s need to be occupied to e l i c i t f u l l physiological response (97). Yet regardless of whether there were difference i n si t e s or occupancy or both, normal transformation and translocation of ERc complex was necessary. Absence of ERn i n some of the group I I tumors apparently indicated a lesion with either transformation or translocation thus contradicting the p o s s i b i l i t y that this group of tumors might be hormone sensitive. Yet t h i s discrepancy might revolve around the controversy of ERn assay. Nuclear estrogen receptors resistant to salt-extraction have been postulated to be the true active species required for stimulation of growth (40, 98). Since only salt-extractable ERn were assayed for, an intact stimulatory pathway would s t i l l be possible i f the sa l t - r e s i s t a n t ERn was the functional -60-form. Compounding the confusion associated with the ERn"assay i s the question whether occupied or unoccupied ERn should be measured. Occupied ERn have been demonstrated i n systems where assay conditions were claimed to be only capable of detecting unoccupied species (39, 79). However, unoccupied ERn have been shown to induce tumor growth i n the absence of estrogen by binding to the acceptor s i t e s (3^,99). Furthermore, ERn levels might fluctuate according to the cycles of estrogen stimulation p a r t i c u l a r l y i n female animals (100). Line 31 EMM - (D) This tumor l i n e represented a l i n e going through t r a n s i t i o n from dependent to autonomous status. However, the progression to f u l l autonomy was attenuated by transplanting the tumors to either female hosts which provided estrogen stimulation via endogenous supply or male receipients supplemented with estrogen p e l l e t s . Transplant of tumor D was carried out on 2 normal female rats and growth was autonomous (Figure 21). Subsequent transplants from tumor #1 were performed on 1 normal female rat and 2 male recipients which were supplemented with estrogen p e l l e t s . Tumor #4 i n one of the males was allowed to grow with the p e l l e t being l e f t i n the animal for the duration whereas the p e l l e t was removed i n rat #3 af t e r 4 weeks of implantation. - 6 1 -31 E M M — ( D ) K - J 2 0 8 /iK 7 -1 (155 ) f?\\A-3 ( N E ) ^ ~ ¥ 9 Y 1 6 0 (V) 1 2 - 2 ( 3 2 - 2 ) rnil4(60) ^ 2 i - 9 LlJ o— LSI 5 - K N E ) 2 6 - 6 31 E M M ( F ) IF ^ 3 ( 0 ) 5 3 1 V9-K9 -9) 8 4 - 2 0 10(0) 1 0 9 ( 5 0 ) 4 6 - 4 \ I 0 - 6 ( I 3 9 ) 1 2 7 - 9 ^ 1 0 - 2 ( 1 7 9 ) 3 3 5 - 5 ^ ~ | 4 - 4 ( 5 S - 4 ) L U - J 4 5 l ^ H D C C P^HD/HAd" |~1HAC/ O H A O N E Insufficient t issue ' • 4 ( 0 ? o Fig. 21. Composite pedigree of Noble rat mammary tumor lines 31 EMM-(D), 31EMM-(E), and 31 EMM-(F). Tumor transplants were carried out by injecting subcutaneously small pieces of viable tumor into the backs of 2 or 3 animals. Estrogen pellet (90? estrone + 10? cholesterol) was inserted at the time of implant i f necessary depending on the hormonal status. Only tumors with receptor assays performed are included in the profile. HD = hormone dependent, HD/HA = transition from dependent to autonomous after estrogen pellet removal, HA' = hormone stimulated, HA = hormone autonomous. -62-A f a i r l y constant level of ERc was also observed in this line (Table II). Tumors #1-4 had a ERc level of 10.6 + 4.0 fmol/mg protein (mean + standard deviation) (Figure 22). Nuclear ER results were not available for tumor #1 and #4 because of insufficient amount of tissue harvested. Otherwise, a l l tumors had measurable amount of this receptor. Cytoplasmic PgR was also detected in a l l tumors except #3. In contrast to the "high PgR, high ERc" observation discussed earlier in 15EMM 16(1) group I tumors, the PgR levels in this line were comparatively higher than the group I tumors though the ERc levels were lower. With the exception of PgR in tumor #3 and incomplete data, a l l three receptors (ERc, ERn, PgR) were detected in the tumors signifying the absence of any translocation, transcription or translation defect. Continual stimulation of tumors with exogenous and endogenous estrogen might slow down the progression to f u l l autonomy and help maintain an intact estrogen stimulatory pathway. Conversely, an intact pathway might represent a slower degree of progression to autonomy by the tumor ce l l s . As an analogy in the human spectrum, a recurrence of ER+ cancer after a long remission period would be most likely to have ER and be responsive to hormone treatment. The long remission period would signify a slowly progressing tumor with better prognosis (101). When tumors have reached the f u l l autonomous stage, growth is independent of hormone and the maintenance of an intact stimulatory - 6 3 -"a o Q . Ui E "o E to lo to 31 EMM — (D) 30 r 20 r ioh ERC ER n PgR Fig. 2 2 . Schematic representation of the hormone receptors profiles of Noble rat mammary tumor line 3 1 EMM-(D). -64-Table II Receptor concentrations in sublines of 31 EMM-(D), (E), and (F). Receptor Tumors Analyzed Receptor Concentration (fmol/mg protein) Assay Mean + SDa ERc D, D1-D5 10.0 + 3.4 E1-E4 1.5 + 2.2 F1-F4 9.0 + 3.6 ERn D, D2, D3, D5 14.6 + 12.6 PgR D, D1-D5 15.7 + 15.7 a - standard deviation b - the second symbol denotes the tumor number in the subline coded by the f i r s t symbol as the originating tumor e.g. B4 represents tumor #4 in the subline with tumor B as the original tumor. -65-machinery would be redundant. The result of the i n i t i a l change of receptor status as the tumor progressed might be the loss of PgR as shown i n tumor #3- This observation seems consistent with the b e l i e f that PgR i s the more informative index than ERc concerning the potential hormone responsiveness or the biology of the tumor. In human breast cancer, PgR has been reported to be a stronger index for predicting hormone dependency by i t s e l f or i n combination with ERc. Horwitz and McGuire (35) f i r s t suggested that PgR might improve the p r e d i c t a b i l i t y of tumor response to hormone therapy. A number of studies since then have reported that response rate to endocrine therapy i s s i g n i f i c a n t l y higher (70 - 80?) i f tumors possess both ER and PgR (102,103). Progesterone receptor by i t s e l f has also been shown to be a better predictor of response than ER (104,46). In summary, th i s l i n e represents a t r a n s i t i o n from dependent to autonomous tumor and i t s autonmous decendents which were maintained i n the presence of estrogen stimulation. Cytoplasmic ER (ERc), ERn and PgR were detected i n these tumors with the exception that PgR was absent i n tumor #3. Interestingly, the estrogen p e l l e t was removed i n t h i s c a r r i e r after an i n i t i a l implantation of 4 weeks. -66-Line 31 EMM - (E) This line demonstrated quite clearly a transition from hormone dependent (HD) to dependent/autonomous (HD/HA), and finally autonomous growth (HA)(Figure 21, 23). Furthermore, this line could be considered as a "control" to 31EMM-(D) concerning tumor progression and receptor status. Both tumors #1 and #3 were classified as HD/HA tumors. Subsequent transplants of tumor #1 and #3 to two male hosts were supplemented with estrogen pellets whereas tumor #2 was a fully autonomous type. The growth period of 20 weeks before harvest for tumor #4 was similar to what of tumor #2. With regard to receptor status, when tumors #1 and #3 in this line were allowed to grow for a longer period than tumor D in 31 EMM - (D) after the removal of estrogen pellet, basal levels of ERc and ERn were detected. However, the high PgR levels detected in the absence of ERc seemed contradictory. Whether the production of PgR was constitutive because of constantly activated ERc or genome alteration requires further investigation. Tumors #3 and #4 were both ERc+, ERn- but the PgR dropped from a high positive (16 fmol/mg protein) in #3 to nondetectable level in #4. On the other hand, tumor #1 and #2 had no ERc, no ERn and PgR also dropped from 11.5 fmol/mg protein in #1 to 4.6 fmol/mg protein in #2. This phenomenon of PgR decline was similar to tumor #3 in 31EMM-(D) described earlier. -67-c o Q L E B 20 o E 10 oo CO CO CO ERC c u n ^R, PgR Fig. 23. Schematic representation of the hormone receptors p r o f i l e s of Noble rat mammary tumor l i n e 31 EMM - (E). -68-Human breast tumors with ER-/PgR+ characteristic have been reported and the response rate to endocrine therapy i s about k-5% (46,104). Such receptor p r o f i l e (ER-/PgR+) as demonstrated i n t h i s l i n e would be associated with rapid tumor progression which predicts that these tumors would l i k e l y be poor hormone responders i f not altogether resistant. Line 31 EMM - (F) The transplant picture of th i s l i n e was very si m i l a r to l i n e 31 EMM-(D) (Figure 21, 24). The f i r s t passage from tumor F was carried out i n 3 normal female rats (#1,5,9). Intact estrogen stimulatory pathway was found i n a l l three tumors as they were found to have very high l e v e l of PgR i n addition to the presence or ERc and ERn. This supported e a r l i e r suggestion that estrogen responsiveness could be maintained by slowing tumor progression v i a suitable l e v e l of hormone stimulation i n female and male hosts. Transplants from tumor #1 were subsequently carried on with 1 normal female (#2) followed by 2 males (#3, #4) supplemented with estrogen p e l l e t s . Tumor #3 grew for three months af t e r p e l l e t removal whereas #4 was harvested after an equivalent period of growth under continuous estrogen stimulation. A l l three transplants (#2, #3 #4) s t i l l maintained a complete receptor p r o f i l e . Transplants from, tumor #5 were also carried for 3 passages i n male recipients (#6,#7,#8). Both rat 6 and 7 were supplied with -69-_ c n 60 31 EMM—(F) ••••>60 40 20 • • ••• • -•-• • ER„ PgR Fig. 24. Schematic representation of the hormone receptors profiles of Noble mammary tumor line 31 EMM - (F). -70-estrogen pellets at the time of transplant. However, the highlight of this subline of transplants was that tumor #6 was harvested 7 months after the estrogen pellet removed in contrast to a shorter period for tumor #3. Interestingly, a l l three tumors (#6, #7, #8) lost their three receptors. This observation suggests that tumor growth shortly after the removal of estrogen stimulation, which represents the early stage of progression, would maintain an intact pathway for hormone responsiveness. Yet i f progression towards autonomy was permitted after a lengthier period of tumor propagation, estrogen and progesterone receptors would not be detected. In addition, progression could not be reversed by re-exposure to estrogen stimulation as shown in tumor #7. Besides the lack of receptors detected, the tumor was aggressive and fast growing which could be another indication of i t s autonomous status. Transplants into a normal male animal (tumor #8) also grew autonomously.. Similar finding has been demonstrated by Noble in estrogen-dependent prostate carcinoma in Nb rat (106). Transplants from dependent tumors which had undergone slow regression was found to maintain dependent status. In addition, these tumors would regress upon antiestrogen treatment despite continued estrogen stimulation. Furthermore, transplants from the regrowth after tamoxifen treatment s t i l l remained estrogen dependent. CONCLUSION This project is the preliminary portion of an investigation to better understand the relationship between hormone dependence of mammary tumors and their steroid hormone profiles. An animal model, estrogen-induced mammary tumors in Noble rats, i s used and shown to be a valuable system because of i t s distinctive characteristics:-1. This model arose as a cpmseqiemce of hormone manipulation and thus is expected to be more f i t t i n g for the investigation than some popular models which are chemically induced (dimethylbenz(a)antracene or N-nitrosomethylurea). 2. The model displayed a close resemblance to the human spectrum with regard to growth dependence on hormone. This was demonstrated by tumor regression upon estrogen removal i n dependent tumors or the failure to do so in autonomous class. 3. Apart from the dependent and autonomous groups which could be maintained by serial transplantation of small pieces of tumor ( 200 mg tissue), tumors representing a transition group from dependency to autonomy were also harvested for study. 4. Characterization of the tumors with respect to estrogen receptor status and hormone dependency had revealed a subclass - 7 2 -which can be used as an excellent model for receptor positive, hormone autonomous human breast cancer. The receptor profiles of this subclass of estrogen receptor positive, hormone independent tumors were the focus of this report. Two major groups of autonomous growth with different receptor characteristics among the tumor lines studied were observed. The f i r s t group showed an ERc+/ERn +or- /PgR- profile after the transition from hormone dependency to autonomy. The second group was shown to be ERc+/ERn+/PgR+. The absence of PgR in the f i r s t group seemed to indicate a non intact estrogen stimulatory pathway which would be consistent with the fact that these tumors did not require estrogen supplement for growth. The detection of a l l three receptors in the second group despite i t s autonomous growth suggests these tumors might be hormone sensitive. Further investigation is required to test this hypothesis. Examination of ERc, ERn, PgR or any combination failed to predict whether tumors were hormone dependent. Whether a hormone dependent tumor progresses into either a sensitive or independent type upon challenge by removal of estrogen supplement is speculated to be genetically coded according to individual c e l l type. In addition, the level of receptor production is also closely regulated and did not seem to be greatly influenced by sequential transplantations nor the hormonal milieu of the ce l l s . 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