Oestrogen receptor activity and endocrine status in DMBA-induced rat mammary tumours

Europ. J. Cancer Vol. 13, pp. 223-228. Pergamon Press 1977. Printed in Great Britain

Oestrogen Receptor Activity and Endocrine Status in DMBA-Induced Rat Mammary Tumours R. A. HAWKINS, A. HILL, B. FREEDMAN, ENID KILLEN, P. BUCHAN, W. R. M I L L E R and A. P. M. F O R R E S T Department of Clinical Surgery, University of Edinburgh, Great Britain Abstract--Oestrogen receptor activity (unfilled sites) has been determined in D MBAinduced rat mammary tumours and at sacrifice, theplasma concentrations of oestradiol-17/3 and prolactin were determined by radioimmunoassay. In intact animals, receptor levels werefound to fluctuate in inverse relationship with the plasma oestrogen concentration, but following ovariectomy, levels were significantly reduced. Administration of oestrogen to ovariectomized animals did not detectably change receptor levels but administration of perphenazine significantly increased receptor levels towards those seen in intact animals. In animals bearing two or more tumours, similar receptor concentrations were detected in tumoursfrom the same animal. It is concluded that the concentration of oestrogen receptor sites in rat mammary tumours is influenced by the circulating levels of both oestrogen and prolactin.

receptor assay. Tumours were excised after exsanguination of the rat under ether anaesthesia. Vaginal smears were taken daily from 45 intact animals and the stage in the oestrous cycle was classified as described by Yoshinaga et al. [11].

INTRODUCTION DMBA-INDUCED rat m a m m a r y tumours, like some h u m a n breast cancers, are hormonesensitive [1]. They contain receptors for oestrogen [2-6], prolactin [7, 8] and progesterone [9]. The oestrogen receptors in these rat tumours have been shown to be similar to those in human breast cancers chemically [10] and in their relation to response to endocrine surgery [4]. Prior to evaluating further the relationship between receptor measurements and the endocrine responsiveness of experimental m a m m a r y tumours, we have investigated the influence of endocrine status on the level of receptor activity.

Endocrine manipulation

Fourteen tumour-bearing rats were injected subcutaneously (s.c.) with perphenazine (5 mg/ kg body weight) for 14 days. A further 57 tumour-bearing rats were ovariectomized under ether anaesthesia and divided into three groups which were treated differently. In one group (OVX) of 11 rats, no further treatment was given until sacrifice 14 days later. In a second group ( O V X + OE2) , 33 animals were left for 9-14 days and then injected s.c. with oestradiol 17-/3 (1-5 mg day in corn oil) for a further 6 days. In the third ( O V X + P e r p h e n ) 13 animals were treated either immediately (9 rats) or after a period of 6-13 days of no treatment (4 rats) by subcutaneous injection of perphenazine (5 mg/kg body weight) for 6-14 days.

MATERIAL AND METHODS Tumours

Female Sprague-Dawley rats were given D M B A (30mg) by intragastric instillation. Tumours induced within 6 months were measured with calipers twice weekly. Size was expressed as the product of measurements in two dimensions. On reaching 1.5 x 1.5 cm tumours were either studied directly or subjected to endocrine manipulation prior to

Multiple tumours in the same rat

In a total of 24 animals in various reproductive states (intact or endocrine manipulated),

Accepted 15 September 1976. 223

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R. A. H a w k i n s et al.

oestrogen receptor activity was determined in two or more tumours from the same animal. Determination of oestrogen receptor concentration

T u m o u r receptor concentrations were determined by a modification of a method described previously [12]. This modification was necessary to improve assay precision since by the original method, the low concentrations of receptor activity encountered in rat tumours lead frequently to non-linearity of the resulting Scatchard [ 13] graph. Tumours were left in Tris buffer solution (0.25 M sucrose, 10 m M Tris and 1 m M ethylene diamine tetra-acetate pH 8.0) containing 0 . 5 m M dithiothreitol (14) for 1 hr at 4°C prior to homogenization at the rate of 300 mg/ ml of Tris buffer and centrifugation of the homogenate at 105,000 g for 50 min (based on 15). The resulting cytosol was subjected to saturation analysis as previously described [ 12]. Determination o f plasma hormone levels

Animals were exsanguinated at death through the abdominal aorta under ether anaesthesia, and the blood was used for the determination of plasma oestradiol-17/? and plasma prolactin concentrations [16]. Since the quantity of blood collected was sometimes limited, assay sensitivity for plasma oestradiol17/? calculated for 4.0 ml plasma, was 0.13 ng/ 100 ml. For plasma prolactin assay, 12 si_iodo_prolactin was prepared by the method of Redshaw and Lynch [17] a procedure which we found more reproducible than that previously employed. The sensitivity for 22 assays varied from 1 to 7.6 ng prolactin/ml with a mean value of 3.3 ng/ml. Inevitable deterioration of prolactin standard occurs on storage ( N I A M D D instructions) and a correction for this has been made using the quality control data, a procedure which results in slightly lower values than those we reported previously.

RESULTS

For most rat m a m m a r y tumours, the dissociation constant of binding (Kd) was found to be 0.4-0.5 x 10 -1° molar (see Table 1), in agreement with our earlier report [12]. The values found for tumour receptor concentration (Po) by the method described in this paper are, however, 1-67___0.29 (s.c., N -- 11) times lower than those derived by the earlier method. By the modified method, a linear Scatchard plot was obtained for most of the tumours examined, and tumours with apparent receptor concentrations down to 0.05 fmoles/mg tissue clearly showed increasing displacement of (3H) oestradiol-17/? with increasing mass of non-radioactive oestradiol17/? added. Effect of the oestrous cycle on tumour receptor concentration

In 45 cycling rats, tumour receptor concentration varied during the oestrous cycle (Fig. 1 and Table 1) reaching a minimum in proestrous which was significantly lower than the values in all other stages in the cycle (P < 0.01), and 9-

t~m~ourreceptor concn. (f molcslmg wet wt. } ~

plasma ~ t radioM?flconcn. (rig/lOOm[} 54-

21-

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i/

*//

dioestrus

/

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T o

= proestrus

"\,,\ o \

oestrus

tumour

receptors

t p~asm,1. plasma o oestradlol-17jFJ rnetoestrus

stage of oestrus cycle

Fig. 1. The effect of stage in the oestrous cycle upon tumour oestrogen receptor concentration and plasma oestradiol-17~ concentration. Each circle represents the oestrogen receptor concentration in an individual tumour. Lines join the mean receptor concentration ( - - - - ) and mean plasma oestradiol-17~ concentration ( - - - ) . The standard errors for plasma oestradiol- 17B concentration have been omitted for clarity but are to be found in Table 1.

Protein concentration was determined by the method of Lowry, Rosebrough, Farr and Randall [18] using bovine serum albumin as standard.

a m a x i m u m in metoestrus. The changes in receptor concentration were in inverse relation to those in plasma oestradiol-17/? (Fig. 1). The changes in plasma prolactin concentration followed, with a delay, those in oestradiol-17/? but did not reach statistically significant levels.

Statistical analysis

Effect of ovariectomy and oestrogen administration

Except where otherwise stated, statistical evaluation was carried out by the Wilcoxon rank test.

Following ovariectomy, tumour receptor concentrations (Fig. 2) and the plasma levels of both prolactin and oestradiol-17/? (Table 1)

Determination o f cytosol protein concentrations

Oestrogen Receptor Activity and Endocrine status in DMBA-Induced Rat Mammary Tumours gturnout receptor conch. 8(f moles / mg wet wt.)

restoration of prolactin levels to those seen in intact animals (Table i). NS

7-

p=O-Ol

Effect of perphenazine administration

5o o

5,~-

8,

r

3-

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Intact

Fig. 2. The effects of ovariectomy and of ovariectomyfollowed by administration of oestradiol-I 7fl on turnout receptor activity. The values for the turnoutsfrom the intact animals shown in Fig. 1 are reproducedfor comparison. Each circle represents the valuefor an individual tumour P values refer to comparison with the concentrations in ovariectomisedanimals.

were significantly diminished relative to the values found in the tumours of intact animals (P < 0-01). Administration of oestrogen to ovariectomized rats caused an insignificant increase in detectable receptor activity (Fig. 2), and a Table I.

225

T u m o u r concentrations of receptor activity in untreated rats were compared with the values in perphenazine-treated rats for both intact and ovariectomized states. The results are shown in Fig. 3 and Table 1. In the intact animal, neither t h e mean tumour receptor concentration nor the plasma oestradiol-17fl concentration after perphenazinc-treatment was significantly different from the value found in untreated animals in dioestrus. Although perphenazine-treatment significantly increased the plasma prolactin concentration over the value found in untreated rats in dioestrus ( P < 0.01), the increased concentration was not significantly greater than the highest concentration observed in untreated animals at proestrus. In ovariectomized animals, perphenazinetreatment caused a significant elevation in plasma prolactin concentration and in tumour receptor concentration (P < 0.01), whilst dissociation constant of oestrogen binding by the

Tumour receptor concentrations and plasma hormone levels in tumour-bearing rats in various endocrine states Plasma

T u m o u r receptor activity

Reproductive state

No. of rats

No. of tumours

Sensitivity* Intact proestrus

/Ca ( X 10- z OM)

P0 (fmoles/mg) (tissue)

--

0.05

0.64 4-0.10 0"53 4-0-04 0.40 § 4- 0.03 0.49 4-0.05

Po, protein (fmoles/mg) (protein)

Oestradiol17fl (ng/100ml)

Prolactin (ng/ml)

1

0" 13

3

1"21 +0.20 2"42t +0.23 3.77 t _+0.60 2"70 t 4-0.27

34 4-7 58++ _+8 94++ 4- 25 70 4- I1

4.70 4-0.69 0.41 t 4-0.15 0"32t _+0.07 0"70 t _+0.17

54 4- 16 51 4-7 35 4- 6 26 _+6

10

13

9

10

metoestrus

11

11

dloestrus

13

17

Intact + perphenazine (dioestrus)

14

19

0.39 4- 0.03

3-11 4- 0.30

72 + 11

0.43 4- 0.08

77 t 4- 16

Ovariectomised OVX O V X + OE2

11

15

33

36

13

16

0"72 4-0.11 0.62 +0.05 0"42t 4- 0.005

0.94 4-0-17 1-00 +0.11 1-86 4- 0-19

23 4-5 22 4-3 44 4- 6

0.13 _+0"02 0"26 +0.05 0-16 4- 0-03

12 +2 40t 4-6 59t 4- 11

oestrus

OVX+perphenazine

Results are the mean values _ 1 standard error. *Sensitivity defined as described under "Methods and Materials" and first section of results. t P < 0.01, ++P < 0.02, §P < 0-05 when compared with appropriate reference value: during oestrous cycle, values compared with that in proestrus; " I n t a c t q- perphenazine" values compared with "intact-dioestrus" value; " O V X - k OE2" and " O V X + p e r p h e n a z i n e " values compared with " O V X " value.

226

R. A. Hawkins et al.

tumour was significantly reduced (P < 0.01) following perphenazine-treatment. No differences were noted between the values found for rats treated immediately after ovariectomy and those in which the start of treatment was delayed: accordingly the data for these two, slightly different modes of treatment have not been separated. 7-

(a!

[b)

• tumour

I~:eptor conch.

p <0"01

6-

NS

( f m0tes/mg wetwt ) 5o eo

4-

o o 8' o

o o o o

3-

8

o cp

2o

o o

o o

o +

controls

+

perphcnozin¢

controls

Ovx.

Intact

perphcnazin¢

( dioestrus )

Fig.

3. The effect of perphenazine-administration in vivo upon tumour oestrogen receptor concentration in (a) ovariectomised and (b) intact rats. Each czrcle represents the value from an individual tumour. P values refer to the effect of perphenazine within each endocrine state.

5re¢~ot or eoncn, in lurnour A [

0

4-

0

0

0

fmoles ling wctwt) 3-

0

0

0

0

0

2-

O O' O ~ . O

[ R o n k torte[oriOn te~t ]

1-

o I

i

i

i

I

1

2

3

~

5

receptor cohen,in t urnour B ( f moles I mg wet wt )

Fig. 4. Tumour receptor concentrations in rats bearing 2 tumours : receptor concentration for one tumour, A is plotted against that for the second tumour, B from the same rat. The rats were sacrificed in various reproductive states. Each circle represents the value from a single rat.

Receptor concentrations in multiple tumours from the same rat When two or more tumours from the same animal were examined, the receptor concentration found for one tumour ("A") was more or less identical to the value for a second tumour ("B") from the same animal (Fig. 4). For animals with more than two tumours, only two values, selected by random selection (tables of random numbers), have been plotted. In most cases, tumours from the same animal contained very similar concentrations of oestro-

gen receptors. By the R a n k Correlation test, there was a highly significant ( P < 0.01) correlation between the receptor levels in tumours from the same animal.

DISCUSSION Oestrogen receptor activity has been determined in m a m m a r y tumours of the rat in various endocrine states. It was found necessary to modify the method previously employed [12] in three respects (based on 14 and 15) to permit accurate determination of the relatively low concentration of receptor binding sites present in these tissues. By this modified method, oestrogen receptor activity was detected in 98.5% of the 136 DMBA-induced m a m m a r y tumours examined. The receptor activity in these tumours was characterized by a dissociation constant of binding ranging from 0.16-1.80 x 10 - l ° molar, and in most tumours dissociation constant was in the range 0.40"5 x 10 -1° molar. The range of oestrogen receptor concentrations encountered in rat mammary tumours is approximately ten times lower than that found in human breast cancers [12]. A similar difference is seen between the concentrations of plasma oestradiol-17fl in the two species [16, 19]. Although most methods for determining oestrogen receptor concentrations detect only empty receptor sites, in general workers do not report the stage in the cycle at which tumours have been harvested for receptor assay or estimate plasma oestrogens at the time the tumour is harvested. We have shown that receptor concentrations fluctuate in inverse relationship with the prevailing oestradiol-17~ concentration, and that the tumour receptor concentration is a function of the stage in the oestrous cycle at which the tumour was excised. This finding is in contrast to the recent work of Jordan and Jasper [20] who conducted receptor assays at 30°C, a temperature at which exchange between (3H) oestradiol-17/~ and endogenous oestrogen at filled receptor sites might occur. However, our results agree with those of Lee and Jacobson [21] and of Trams, Engel, Lehmann and Maass [22] in the rat and human uterus respectively, and of Maass, Engel, Nowakowski, Stolzenbach and Trams [23] in human breast cancer. It is known that the effects ofoestrogen upon the oestrogen receptor concentration within a tissue are complex: cytoplasmic receptor levels can be altered by increases in circulating oestrogen in two ways. In addition to (i)

Oestrogen Receptor Activity and Endocrine Status in DMBA-Induced Rat Mammary Tumours filling empty receptors, which are then translocated to the cell nucleus, oestrogen m a y also (ii) stimulate the synthesis of new receptors [24, 25]. Our observations that tumour receptor levels firstly vary during the oestrous cycle and secondly, fall after ovariectomy, are, respectively, in keeping with these two effects. In the rat, changes in circulating oestrogen level can also lead to alterations in the concentration of plasma prolactin (see e.g. [26].) This hormone has also been reported to influence oestrogen receptor levels in the rat m a m m a r y tumour [27-29]. In view of this, it is difficult to ascribe changes in receptor level to the effect of a single hormone. Nevertheless, in intact cycling animals, the tumour levels of detectable receptor activity found in this study were inversely related to changes in circulating oestradiol,17fl concentration and apparently unrelated to changes in plasma prolactin concentration. In the ovariectomised rats treated with perphenazine, tumour receptor levels were clearly elevated in association with a significant increase in prolactin secretion, but in the absence of detectable oestrogen. This effect of prolactin on oestrogen receptor levels is in agreement with the findings of Leung and Sasaki [27, 28] and of Vignon and Rochefort [29]. Although perphenazine-treatment in ovariectomised rats also increased the affinity of the receptors for oestrogen (see Table 1), this apparent difference probably reflects the less accurate determination of K d at low receptor levels rather than a specific effect of prolactin. In ovariectomised rats treated with oestrogen, tumour receptor levels were not detectably changed relative to untreated controls, possibly due to the filling of empty sites by the injected oestrogen. The failure to detect any effect of perphenazine-treatment upon tumour receptor

levels in intact animals m a y either be due to the fact that the plasma prolactin concentrations found after treatment were not significantly greater than those found in untreated animals at proestrus and oestrus, or may indicate that the latter concentrations are already sufficient to maximally stimulate tumour receptor formation. In animals bearing two or more tumours, despite the existence of a different clone of cells in each tumour, receptor levels were strongly correlated between tumours from the same animal, indicating that the endocrine milieu m a y be the major determinant of tumour receptor level. These results support the view that endocrine status markedly influences tumour receptor level. In particular, we have indicated that oestrogen receptor levels may be increased by prolactin or reduced by the filling of empty receptor sites with oestrogen. If a similar situation obtains in women with breast cancer, then endocrine status at biopsy for receptor assay would influence the receptor levels detected and hence the predictive value of this parameter. The adoption of an exchange assay for measuring total cellular receptors should help by reducing the effect of circulating oestrogens on detectable receptor level for (i) clinical, predictive purposes and (ii) separating the effects of oestrogen and prolactin on receptor levels in the rat m a m m a r y tumour model.

Acknowledgements--We thank Mrs. Dorothy Gray for her expert assistance and the Cancer Research Campaign for Grant No. SP1256. We also thank Miss Sheila Gore, Medical Computing and Statistics Group for statistical advice. We are grateful to the N I A M D D rat pituitary hormone distribution program as the sole source of our prolactin reagents and to Drs. A. E. Bolton and W. Hunter of the Radioimmunoassay Team, Forrest Road, Edinburgh for anti-oestradiol-17fl serum.

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