Actions of oestrogens and antioestrogens on rat mammary gland development: Relevance to breast cancer prevention

J. steroid Biochem.

Vol. 30, No. 1-6, pp. 95-103, 1988 Printed in Great Britain. All rights reserved

0022-4731/K+ $3.00+0.00 Copyright 0 1988 Pergamon Press plc

ACTIONS OF OESTROGENS AND ANTIOESTROGENS ON RAT MAMMARY GLAND DEVELOPMENT: RELEVANCE TO BREAST CANCER PREVENTION R. I. NICHOLSON,K. E. GOTTING, J. GEE and K. J. WALKER Tenovus Institute for Cancer Research, University of Wales College of Medicine, The Heath, Cardiff, CF4 4Xx, Wales. Summary-The proliferative actions of a series of antioestrogens on the development of the second thoracic mammary gland of ovariectomized immature Sprague-Dawley rats have been investigated. Evidence is presented that shows ~~u~~-tamoxifen, LY 1170 IS and LY 13948 1, like oestradiol- 178 and cam-~moxifen, promote full mammary gland ductal development and induce a high rate of cell proliferation in the undifferentiated epithelial cells of the terminal end buds, the main growth region for ductual growth. Conversely, ICI 164,384, a new antioestrogen, is without effect on ductal elongation. In vivaexposure of truns-tamoxifen and LY 1170 18 treated glands in medically castrated animals to the carcinogen DMBA, results in a high rate of mammary tumour development. Indeed, the actions of these so-called antioestrogens are equivalent to those observed in oestradiol-treated rats.

INTRODUCTION

the proposal that tamoxifen should be administered prophylactica~y to women at high risk of developing breast cancer, that is, to healthy women who do not, as yet, have overt disease [3]. However, this population must contain a proportion of women who would not under normal circumstances go on to develop breast cancer and since this has been estimated to represent three out of four women in the proposed treatment group [3] any therapy given must be free of all adverse side-effects [4].

The two major sho~comings associated with the use of antiho~onal drugs in the treatment of established breast cancer are that they are effective only in a minority of patients and are palliative [l]. It is significant, however, that these limitations are not a consequence of the antihormonal drugs being pharmacologic~ly inactive, but result from the aggressive and adaptive nature of the disease. Based on these notions, it may be argued that little further benefit in terms of disease remission is likely to be gained by modifying existing drugs or by developing similar classes of agents. Improvements may, however, accrue through their earlier application either in the preclinical phase of the development of breast cancer, when a more rigorous hormone sensitivity may be envisaged, or through their use to block tumour initiation or early promotional events. Many difficulties are, of course, evident with regard to this approach, notwiths~nding our lack of knowledge of the proliferative actions of drugs on the structures of the normal breast and the consequences of such putative proliferations. In this light, the current study documents our initial approach to these problems and concentrates on resolving the actions of anti~strogens on the growth of the mammal gland of the rat and their modifying influence on the process of carcinogenesis. Special emphasis is placed on the actions of tamoxifen, a drug commonly used in breast cancer therapy and which has a reported low incidence of sideeffects [2]. Indeed, the latter characteristic has led to

EXPERIMENTAL

Oestradiol (3/?,17~-dihydroxy~estra-1,3,5( 1O)-triene), ICI 164,384 (N-n-butyl-N-methyl- 1 l-(3/?, 17/?dihydroxy - oestra - 1,3,5( 10) - triene - 7a - yl)undecamide), trans-tamoxifen (l-(p-dimethylaminoethyoxyphenyl) - 1,2 - diphenylbut - 1 - ene), cis-tamoxifen (isomer of above), LY 117018 (6-hydroxy-2~~hydroxyphenyl~benzo-thien-3-yl, 4-(2-( 1-py~olidinyl~ ethoxy)phenylketone) and LY 139481 (6-hydroxy-2(4-hydroxyphenyl)-benzo-thien-3-yl, 4-(2-( l-piperidinyl)ethoxy)phenylketone) were dissolved in a minimum volume of ethanol and diluted to the required concentration in sesame oil (max. 5% ethanol-oil). A solution of ergobromoc~ptine (CBI 54) was prepared by adding 25 mg of the drug and 25 mg tartaric acid to 3 drops of water and 1 ml of 95W ethanol. The mixture was shaken until the drug was completely dissolved and then made up to the desired concentration with physiological saline (0.9W w/v).

Animals Female Sprague-Dawley rats were bred either at the Tenovus Institute or obtained from Charles River (Margate, U.K.). Animals were housed in groups of

Proceedings of the 8th International Symposium of The Journal of Steroid Biochemistry “Recent Advances in Steroid Biochemistry” (Paris, 24-27 May 1987). 95

R. I. NICHOLSON et al.

96

five and allowed food and water ad ~~b~t~rn.Two series of experiments were undertaken. (a) At 2X days of age, the rats were ovariectomized and injected intramuscularly ( 100 ~1) with the above compounds at the doses indicated in the text, beginning on day 30. Treatments were continued daily until day 50. (b) Intact animals were administered a 0.5 mg depot of a sustained-release formulation of the luteinizing hormone-releasing hormone (LH-RH) agonist ICI 118,630 (D-Ser (B~‘)~Azgly~O,LH-RH) on day 26. On day 30 oestradiol (1 @day) and antioestrogen (1Opg trans-tamoxifen or LY 117018/day) therapy was initiated and daily injections of these compounds were maintains for 20 days. The animals were intubated with graded doses of dimethylbenzanthracene (DMBA) totalling 20 mg on days 35,40,45 and 50. The animals in (b) were palpated for tumours at weekly intervals. When tumours reached an approximate size of 2 cm mean diameter a tumour biopsy was removed for histological examination and the animals were ovariectomized to establish the hormone sensitivity of the induced neoplasms. Whole-mount preparations

Animals in (a) were killed by cervical dislocation at the times indicated in the text. The pelts were removed and immersed in freshly prepared Bouin’s fixative (75 ml saturated aqueous picric acid solution, 25 ml neutralized formaldehyde (40%) and 5 ml glacial acetic acid) for approx 16 h and subsequently washed in running tap water for 24 h. The epidermal layer was excised [ 51, placed in Mayer’s haemalum for 24 h, rewashed and differentiated with acid alcohol (5% hydrochloric acid in 70% ethanol). After differentiation, the mammary glands were washed in tap water until ‘blued’ and dissected from the preparations. They were then dehydrated in increasing concentrations of ethanol (70-1OOW) and placed in xylene. Preparations of the second thoracic mammal gland were mounted in Pertex on acid-washed slides. The second thoracic gland is virtually two-dimensional (Fig. la) and was therefore deemed most suitable for the assessment of alterations in mammary gland length and area. Measurements were taken from the first bifurcation of the ductal system to the furthest point at the perimeter of the gland, Area was assessed by tracing around the gland onto cellophane and weighing the traced area in comparison with standards. Individual structures of the mammary glands were carefully dissected from the whole-mount preparations following an overnight incubation of the slides in xylene. The structures were processed manuaily for histology [S], wax embedded and 5-pm sections stained with Ehrlich’s haematoxylin and 5% eosin (e.g. Fig. lb). Cell kinetic measurements (a) Metaphase-arrest index.

agent vincristine

sulphate

The stathmokinetic (Eli Lilly, Basingstoke,

U.K.) was used to arrest cells in the metaphase stage of division. The drug was injected intravenously (1 mglkg body wt) in 500 ~1 of a sterilized solution of methyl and propyl parabens with mannitol BP as a preservative. The collection of metaphases was only allowed to continue to 2 h to avoid any degeneration of blocked metaphases, a phenomenon which has been noted in serial blockade studies [6]. Metaphasearrested cells are characterized by condensed chromatin and a transluscent cytoplasm (Fig. lc). These features readily distinguish them from non-dividing cells. The numbers of metaphase arrested cells in the individual structures of the mammary gland were expressed as a propo~ion of the total epithelial cells counted. A correction factor was, however, applied to the index since metaphase arrested cells were observed to be larger in diameter than the interphase nuclei and therefore appear in a proportionally higher number of histological sections. The factor varied between treatments and was found to be 0.76 (ratio of section thickness plus diameter of interphase nuclei to section thickness plus diameter of whole metaphase arrested cell) for the tamoxifen, LY 1170 18, LY 13948 1 and oestradiol treated groups and 0.66 for the ICI 164,384 treated and ovariectomized control groups. (b) ‘S’ phase labe~~~ngindex. The autoradiographic demonstration of tritiated thymidine ([3H]TdR) incorporation into nuclei was used to determine the proportion of cells in the DNA synthetic phase during the period of exposure to the nucleotide. A dose of 1 @i/g body weight [3H]TdR (methyQ3H]thymidine, sp. act. 52 Ci/mmol, Amersham International, Buckinghamshire, U.K.) was injected peritoneally 1 h prior to the animals being killed. The mammary glands were carefully removed as previously described and embedded in resin [5]. Histological sections (1 pm) were cut, mounted and dipped in Ilford K5 emulsion [0.2pm crystal size, emission sensitive @ford Ltd, Cheshire, U.K.)] at 43°C in a water-bath under darkroom safelight conditions. They were then air dried, placed in lightproof boxes, sealed in aluminium foil and exposed for 30 days at 4°C. After this period the autoradiographs were brought to room temperature and opened in the darkroom. At a temperature of 20°C the slides were immersed in Kodak D 19 developer (3.5 min), a 1% acetic acid stop bath (1 min) and finally a Kodak Unifix solution (diffusion A, 7.5 min). They were then washed in freshly distilled water (10 min) and air dried. The sections were stained with a poiychrome stain at 100°C for 5-8 s. This was followed rapidly by a brief wash in 70% ethanol to remove the background stain in the gelatin layer. Nuclei with more than three grains overlying them were taken as positively labelled (see Fig. Id). This was determined from counts of background grains in control slides [ 51. The labelling index of the epithelial cells was expressed as a proportion of the total epithelial cells counted.

Actions

of oestrogens

and antioestrogens

on rat mammary

gland development

91

Fig. 1. Whole-mount and histological preparations of the second thoracic mammary gland. (a) Wholemount preparation from a 30-day-old intact animal. (b) TEB (-) from a 50-day-old intact rat ( x 200). (c) Metaphase arrested cells (-+) in a tamoxifen treated TEB (x 1000). (d) Autoradiograph of a tamoxifen treated TEB (x 2000, -_), labelled cell).

RESULTS

Normal mammary gland ductal development

The mammary glands of the Sprague-Dawley rat begin to develop under ovarian control at 30-35 days

of age [7]. At this time tree which originates branching into the fat ducts are capped by

the glands consist of a ductal at the nipple and spreads by pad (Fig. la). The ends of the club shaped structures called

R. I. NICHOLSINef al.

98

terminal end buds (TEB). These are the main growth regions of the gland concerned with ductal elongation and branching [8]. Histological sections through the TEB reveals the presence of multilayered epithelial cells which are continuous with the ductal epithelia (plate lb). The TEB have a narrow central lumen which may show invaginations into the main body of the epithelial cells. They are characterized by high rates of cell proliferation and mitotic figures are often seen within the epithelial cell population (Fig. lb). The number of dividing cells observed in TEB may be amplified by the administration of the stathmokinetic agent vincristine prior to the sacrifice of the animals [5]. After correction for anomalies in the relative size of metaphase-arrested and non-dividing cells, the percentage of arrested cells in TEB from 30-day-old animals was calculated to be approx 2.5, a value higher than that recorded in either ductal or alveolar epithelium (Fig. 2). 25

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Fig. 2. Metaphase-arrest index in the structures of the mammary gland. Histological sections of the individual structures of the second thoracic mammary gland were prepared from 30-, 40- and 50-day-old intact (Cl) and ovariectomized (Q animals administered 1 mg vincristinel kg body wt 2 h before being killed. The number of metaphase arrested cells in the total epithelial cell population was determined in individual structures. Counting was continued until a minimum of 2000 cells had been assessed. Sections were derived from five animals per group and the results are expressed as the mean+SD for each treatment group.

During the period 30-50 days of age substantial ductal development of the mammary gland occurs. This results in a two-fold increase in ductal length (Fig. 3b), extensive ductal branching [7] and maintained high levels of cell proliferation in the TEB (Fig. 2). These events are dependent on the presence of functional ovaries, since either the administration of a sustained-release formulation of the LH-RH agonist ICI 118,630 on day 26, a procedure that induces a complete suppression of ovarian hormone production for 4 weeks [5], or surgical ovariectomy on day 28, substantially reduced the increase in

ductal length and TEB proliferative activity in intact animals on day 50 (Ref. [5] and Fig. 3). The major ovarian hormone involved in these events is oestradiol and its daily administration (1 or 50 &day) to ovariectomized animals for 20 days reverses the effects of castration (Fig. 3). The stimulatory actions of oestradiol on the mammary gland of ovariectomized animals are not a consequence of the ability of this steroid to raise circulating prolactin levels, since the concurrent administration of CB154 at a dose that abolishes the oestradiol stimulated rise in prolactin levels [7], was unable to block the tissue actions of oestradiol (Fig. 3). The administration of progesterone to ovariectomized animals did not induce an increase in either the percentage of metaphase arrested cells in TEB, or increases in ductal length or area (Fig. 3). Injluence of antioestrogens on ductal development The data presented in Fig. 4(a) demonstrate that doses of trans-tamoxifena5 &day stimulate a large increase in mammary gland ductal length in ovariectomized animals. Indeed, the increases in size of the mammary gland promoted by the drug are equivalent to those observed in either 50-day-old intact control animals or in ovariectomized animals administered either the oestrogenic cis-isomer of tamoxifen (67.5 &day) or oestradiol(1 or 50&day). Administration of trans-tamoxifen (67.5 &day) concurrently with oestradiol (1 @day) showed no antagonism between the compounds. These data are in marked contrast to the actions of trans-tamoxifen on the uteri of the same animals where the drug showed little agonistic activity and antagonized the actions of oestradiol (Fig. 4b). Administration of CB 154 was unable to modify the stimulatory effects of tamoxifen on mammary gland ductal growth. Examination of TEB from trans-tamoxifen treated rats administered vincristine 2 h before being killed, showed the presence of large numbers of metaphasearrested cells (Fig. lc). The distribution of dividing cells was similar to that observed in TEB from either 50-day intact controls or ovariectomized animals administered oestradiol (not illustrated) with arrested cells being observed in the cap region of the TEB, in the main body of the epithelial cells and also in the luminal cells. The percentage metaphase arrested cells in TEB from trans-tamoxifen treated animals remained relatively constant at doses of tamoxifen between 5 and 67.5&day (Fig. 5a). The levels achieved were higher than those recorded in TEB from either 50-day intact rats or ovariectomized animals administered vehicle (Fig. 5a) or oestradiol (Fig. 4a). High rates of cell proliferation following the administration of tamoxifen were also indirectly demonstrated by monitoring the incorporation of [3H]TdR into replicating S-phase DNA. Autoradiography of histological sections of TEB removed from trans-tamoxifen treated animals injected with [‘H]TdR 1 h before being killed, showed over 20% of

99

Actions of oestrogens and antioestrogens on rat mammary gland development fbl

Fig. 3. Influence of oestradiol on the growth of the rat mammary gland. Groups of animals (n = S/group) were ovariectomized on day 28 and administered the compounds listed in the figure at the doses indicated. Treatments began on day 30 and were continued for 20 days. On day SOthe animals were administered vincristine 2 h before being killed. Mammary gland whole mounts were prepared and ductal length and mammary gland areas measured. The metaphase-arrest index in TEB was determined as outlined in the legend to Fig. 1. The results are compared with those obtained in 50-day intact animals and are expressed as the mean + SD for each treatment group.

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Fig. 4. Influence of tamoxifen on the growth of the rat mammary gland and uterus. Groups of animals (n=S/group) were ovariectomized on day 28 and administered the compounds listed in the figure at the doses indicated. Treatments began on day 30 and were continued for 20 days. On day 50 the animals were administered vincristine 2 h before being killed. Mammary glands and uteri were removed and the latter weighed. Whole-mount preparations of the mammary glands were assessed for alterations in ductal length. The results are compared with those obtained in ovariectomized animals killed on day 30 and 50-day-old intact rats and are expressed as the mean+SD.

the epithelial cells of the TEB to be labelled (Fig. Id). Indeed, the percentage of S-phase labelled cells was higher than that observed in TEB from either 50-day ovariectomized animals or intact controls (Fig. 5b). The stimulatory actions of tamoxifen on ductal development were also mimicked by LY 1170 18 and LY 13948 1 (Fig. 6a), antioestrogens reported to possess lower agonistic activity than trans-tamoxifen in the rat [9-l 11. Thus, both compounds, at doses

> 5 &day, stimulated increases in mammary gland ductal length equivalent to those observed in either 50-day intact rats or ovariectomized animals administered either trans-tamoxifen (67.5 @day) or oestradiol(1 ,@day). In the presence of such agonistic activity, it was not possible to demonstrate any antagonistic activity of LY 117018 and LY 139481 towards oestradiol. The metaphase-arrest index in TEB from LY 117018 and LY 13948 1 treated rats exceeded the values obtained in ovariectomized and

R. I.

NICHOLXIN

(bl

ductal system of the rat mammary gland (Fig. 7a). Moreover, when administered concurrently with tamoxifen, ICI 164,384 antagonized the actions of tamoxifen. ICI 164,384 was however, unable to prevent the increase in the size of the mammary gland which occurs in ovariectomized animals between 30 and 50 days. These data are in contrast to the actions of ICI 164,384 on the uteri of the same animals, where the drug prevented the increase in uterine weights in ovariectomized animals during the same interval (Fig. 7b).

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Influence of oestradiol, trans-tamoxifen and L Y 117018 on carcinogenesis The data shown in Fig. 8(a) shows that intact animals exposed to DMBA begin to develop mammary tumours after approx 7 weeks. Tumours continued to arise throughout the study period and by week 20 70% of the animals have at least one tumour. These tumours are predominantly (~85%) hormonedependent adenocarcinomas and will regress following the surgical removal of the ovaries. Administration of the sustained-release formulation of the LH-RH agonist ICI 118,630 during DMBA administration significantly reduced the number of tumours produced. Indeed, this agent which produces a reversible castration-like response lasting 4 weeks [ 51,was as effective as ovariectomy at reducing tumour formation (Fig. 8a). The inhibitory effects of ICI 118,630 were reversed by the concurrent administration of either oestradiol (1 @day), truns-tamoxifen or LY 117018 (100 &day) (Fig. 8b). The majority of tumours formed in oestrogen and antioestrogen

Fig. 5. Cell proliferation in the TEB from tamoxifen treated animals. Groups of animals (n = S/group) were ovariectomized on day 28 and administered various dose levels of trans-tamoxifen. Treatments began on day 30 and were continued for 20 days. On day 50 the animals were administered either (a) vincristine (2 h before being killed) or (b) [3H]TdR (1 h before being killed). Mammary gland whole mounts were prepared and the TEB processed for histology. The metaphase-arrest and S-phase labelling indices were calculated as described earlier. The results are compared with those obtained in TEB removed from the mammary glands of ovariectomized and intact animals on day 50 and are the mean+SD for each treatment group.

intact controls and was equivalent to that seen in tamoxifen treated animals (Fig. 6b). Administration of ICI 164,384, an antioestrogen reported to possess no agonistic activity in rat uterine weight gain tests [ 121, similarly showed no agonistic properties with regard to the development of the

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Fig. 6. Influence of LY 117018 (I) and LY 13948 1 (II) on the growth of the rat mammary gland. Groups of animals (n= S/group), were ovarlectomized on day 28 and administered the compounds listed in the figure at the dose levels indicated. Treatments began on day 30 and were continued for 20 days. On day 50 the animals were administered vincristine 2 h before being killed. Mammary gland whole-mounts were prepared and ductal length assessed. The metaphase-arrest index in TEB was determined as outlined in the legend to Fig. 1. The results are compared with those obtained in ovariectomized animals killed on day 30 and 50-day-old intact rats and are expressed as the mean + SD.

101

Actions of oestrogens and antioestrogens on rat mammary gland development

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Fig. 7. Influence of ICI 164,384 on the growth of the rat mammary gland and uterus. Groups of animals (n=S/group) were ovariectomized on day 28 and administered various doses of ICI 164,384 or transtamoxifen. Treatments began on day 30 and were continued for 20 days. On day 50 the animals were killed and their mammary glands and uteri removed. Whole-mount preparations of the mammary glands were assessed for alterations in ductal length. The results are compared with those obtained in 30- and X)-day-old ovariectomized animals and are expressed as mean+SD. lb1

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Fig. 8. Influence of oestrogens and antioestrogens on the development of mammary tumours. Groups of animals (n=25/group) were administered a sustained-release formulation of the LH-RH agonist on day 26. Drug treatments began on day 30 and were continued for 20 days. DMBA was administered by intubation on days 35, 40,45 and 50 (total dose 20 mg DMBA). Animals were palpated for tumours at weekly intervals and the results obtained were expressed as the percentage cumulative number of tumours relative to the value obtained in intact animals after 1 yr of DMBA treatment. Over 90% of animals in the intact group produced at least 1 tumour/animal with a mean of 2.1 tumours/rat. The results are compared to those obtained in animals ovariectomized on day 28.

groups were hormone-dependent adenocarcinomas. The incidence of hormone independent turnout-s did not vary significantly between the groups. treated

DISCUSSION It is widely recognized that the actions of antioestrogens on the growth of normal and neoplastic oestrogen-dependent tissues are predominantly inhibitory[2]. This useful property is, however, often accompanied by stimulatory actions of the drugs. Thus, while tamoxifen inhibits oestradiol-induced cell proliferation in the rat uterus, it also promotes extensive cellular hypertrophy in the luminal and glandular epithelial cells [ 131. Significantly, the stimu-

latory actions of antioestrogens

are not always re-

stricted to cell work and increases in the growth rate of oestrogen-sensitive T-47D [ 141 and ZR-75-1 [ 151

mammary tumour cells in culture have been induced using low concentrations of tamoxifen in charcoal stripped or defined media. The data presented in the current study also demonstrate stimulatory actions of antioestrogens on oestrogen-sensitive tissue growth and show that following the administration of trunstamoxifen, LY 117018 and LY 13948 to ovariectomized immature female rats extensive ductal development of the mammary gland occurs. Moreover, the data are paralleled by antioestrogen-induced increases in the rate of proliferation in the epithelial

102

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cells of the TEB, the main growth regions for ductal elongation and branching in the developing mammary gland. In fact, such are the stimulatory actions of these compounds that it is not possible to demonstrate any antagonism towards oestradiol. These data are in marked contrast to the effects of tamoxifen on the uterus, where the drug displays little agonistic activity and inhibits the tissue growth response to the steroid. Although it is not clear why these differences exist, it is possible that since the present results were obtained during an early developmental phase of mammary gland growth, when antioestrogens are acting on a large undifferentiated cell population, the inhibitory pathways associated with antioestrogenic action [2] may not, as yet, have been gained. Significantly, it has been reported that tamoxifen reduces the DNA content of mammary glands removed from mature animals [ 161 at a time when the glands have achieved a more differentiated state. This may coincide with the development of prolactin sensitivity, a function which is not obligatory to the stimulatory effects of oestradiol or tamoxifen on the growth and proliferative activity of TEBs. Indeed, the observation that the administration of prolactin can override the inhibitory actions of tamoxifen on the growth of well-differentiated mammary adenocarcinomas [ 171, tentatively associates the inhibitory properties of the drug with the requirement of the mammary gland cells for this hormone. In the absence of such a requirement antioestrogens might behave as full oestrogens. It is noteworthy that the proliferating cells of the TEB are believed to be the main site of action of the carcinogenic polycyclic hydrocarbons [ 181. Our current data clearly show that the cell proliferations induced by oestradiol, truns-tamoxifen and LY 117018 retain this sensitivity. This result contrasts with the actions of the antioestrogens on the growth of established carcinogen-induced mammary tumours where both of the compounds examined have been shown to possess significant antitumour activity [7, 19-211. Interestingly, the tumours produced in the present study following the administration of antioestrogens to DMBA treated animals were predominantly hormone dependent and regressed following ovariectomy. These data imply that the acquisition of strict hormone dependency does not occur immediately following tumour initiation, since if this were the case then tamoxifen should inhibit the early lesions. Alternatively, the concentration of antioestrogens used may stimulate sufficient pituitary prolactin release to mask the inhibitory actions of the drugs on early promotional events. In this light, it is significant that the administration of CB 154 concurrently with tamoxifen, despite failing to block the antioestrogen-induced increase in ductal length, significantly inhibits mammary tumour induction by DMBA in LH-RH agonist treated rats (Gotting and Nicholson, unpublished data). Are these observations of any significance to the

prophylactic use of tamoxifen in women who are at high risk of developing breast cancer? Obviously, in the absence of any published information on the effects of tamoxifen on the normal structures of the breast, this question cannot be answered directly. Evidence does, however, exist to support the view that tamoxifen is not biologically inert and will produce some oestrogen-like responses in treated women (see Ref. [2]). Whether these stimulatory actions impinge on the normal breast and whether they induce cell proliferation remains to be investigated. It will, however, be essential to monitor the responses of individual cell types, since we have no reason to expect a uniform pattern of cell response to antioestrogens. Our data suggest special caution should be directed to the long-term use of antioestrogens in postmenopausal women who have an inherently low rate of cell proliferation and where any proliferative effects caused by antioestrogens would be at their most significant. It is unfortunate that this is the very group proposed for prophylactic trials. Finally, it is evident that a number of the above concerns would be overcome by the availability of antioestrogens with no biological activity. ICI 164,384, appears to fulfil this requirement and studies examining its properties in the DMBAinduced mammary tumour model are currently under way. Acknowledgements-The authors wish to thank the Tenovus Organization for its generous financial support and Drs A. E. Wakeling and B. J. A. Furr (ICI Pharmaceuticals Division, Cheshire, U.K.) for the gift of cis- and trans-tamoxifen, ICI 164,384 and ICI 118,630. ICI 164,384 was synthesized by J. Bowler. Thanks are also given to the Eli Lilly Company, Indianapolis, U.S.A. for the sift of LY 117018 and LY 139481 and to Sandoz (Leeds, U.K.) for the gift of CB 154. MS J. Gee is a recipient of an M.R.C. scholarship. REFERENCES 1. Nicholson R. I., Walker K. J. and Davies P.: Hormone agonists and antagonists in the treatment of hormone sensitive breast and prostate cancer. Cancer Surv. 5 (1986) 463-486. 2. Nicholson R. I.: Antioestrogens and breast cancer therapy. In Pharmacology and Clinical Uses of Inhibitors of Hormone Secretion and Action (Edited by B. J. A. Furr and A. E. Wakeling). Ball&e Tindall (1987) pp. 60-86. 3. Cuzick J., Wang D. Y. and Bulbrook R. D.: The prevention of breast cancer. Lancet January 11 (1986) 83-86. 4. Diver J. M. J., Jackson I. M. and Fitzgerald J. D.: Tamoxifen and non-malignant indications. Lancet March 29 (1986) 733. 5. Gotting K. E.: DMBA tumours in the mammary glands of rats. Ph.D. thesis, University of Wales College of Medicine, Cardiff (1987). studies in rat prostate. 6. Evans G. S.: Cell proliferation Ph.D. thesis, University of Wales College of Medicine, Cardiff (1985). 7. Nicholson R. I. and Gotting K. E.: Comparative biological and antitumour effects of antioestrogens in the rat. In Reviews on Endocrine-Related Cancer (Edited by V. C. Jordan). ICI Publications, U.K. (1986) Suppl. 19, pp. 49-57.

Actions of oestrogens and antioestrogens on rat mammary gland development

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Jordan). Academic Press, New York (1981) pp. 143-162. 14. Reddel R. R. and Sutherland R. L. Tamoxifen stimulation of human breast cancer cell proliferation in vitro: a possible model for tumour flare. Eur. J. Cancer clin. Oncol. 20 (1984) 1419-1424.

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