Estrogen-sensitive cell lines

Copyright All rights

0 1973 by Academic Press, Inc. in any form reserved

of reproduction

Experimental

Cell Research 78 (1973) 41-46

ESTROGEN-SENSITIVE Uptake

of Labeled Sex Steroids

C. SONNENSCHEIN, Department

CELL

of Anatomy,

and Growth

M. POSNER,I S. SAUDUDDINZ Tufts

University

School

LINES

of Medicine,

Properties and M. KRASNAY

Boston,

Mass.

02111,

VSA

SUMMARY Cells of a rat pituitary cell line injected into Wistar/Furth rats produce tumors when inoculated into intact females or estradiol-17/l primed males. No tumors are produced when the same number of cells are injected into spayed females, intact males or castrated males. Experiments carried out in animals and in tissue culture indicate the selective uptake of these cells for physiological amounts of estradiol-17,9 when compared with testosterone and when compared with an undifferentiated cell line.

The effects of sex hormones on the organism in general and on target tissue in particular, may affect cell metabolism in a specific, coordinated way, both in normal and malignant cells. These integrated responsesare a result of a sequence of events that start when the hormone triggers mechanismswhose full expression may not be seen before hours or days have elapsed. Hormones may act directly on the target tissues or through their metabolites and it is possible that the hormone action could be triggered either by (a) a contact between the hormone and a single molecule in the target cell or (b) by contacts of the hormone with multiple molecules [S, 9, 101. The understanding of the mechanism of action of steroids in the target cell is hindered by the circumstance that target organs in 1 Present address: Department of Physics, University of Massachusetts at Boston, Boston, Mass. 02116, USA. 2 Present address: Department of Physiology and Pharmacology, College of Veterinary Medicine, Ohio State University, Columbus, Ohio 43210, USA.

which the effect is measured, usually consist of a mixture of cells with different affinity for the steroid in question. So, the biochemical approach usually represents the summation of results derived from a heterogeneouspopulation of cells in which there is a predominance of the target cells. The isolation of target cells sensitive to estrogens and androgens in a long-term cell culture system is of great importance in order to explore the mechanismsof action of these hormones in a well monitored homogeneous cell system susceptible to modification in different directions without the necessity of having to consider homeostatic mechanisms known to affect expetimental results in animals. A number of tumors that are sensitive to estrogens have been reported [5, 6, 7, 131but no established cell line has been obtained from them. Tumors sensitive to other hormones have been described [3, 161but sensitivity to estrogenshas not been explored. This report deals with attempts to demonstrate Exptl

Cell Res 78 (1973)

42

C. Sonnenschein et al.

the estrogen sensitivity of cells grown in tissue culture condition and transplanted into animals. MATERIALS

AND

METHODS

Cell lines: GH, cells were derived from a radiationinduced pituitary tumor of the rat originally obtained by J. Furth and adapted to grow in long-term tissue culture conditions [4, 121. These cells are epitheliallike, having a modal chromosome number of 69 with two large metacentric chromosomes serving as markers and produce tumors when injected into Wistar/ Furth rats which is the strain of rats in which the tumor was induced and transplanted [l, 2, 41. They also secrete growth hormone and prolactin in tissue culture. The secretion rate of these hormones appears to be influenced by the addition of cortisol to the medium in which the cells are growing 1141. Clone 1D is a mouse undi?feren&ied-established cell line derived from L929 cells. It has the nrooertv of being 5 bromo-deoxyuridine resistant [15] and a modal number of 55 with several big metacentric marker chromosomes. Culture media: Cells were grown in Dulbecco’s modification of Eagle medium with the addition of horse serum 15 %, fetal calf serum 2.5 %, penicillin, streptomycin, fungizone and anti-PPLO agent. Hormones: Estradiol 178-6, 7-SH (spec. act. 4&50 Cij mM) and testosterone 1, 2-SH (spec. act. 3@-50 Ci/mM) were obtained from New England Nuclear Co., Boston, Mass. Rat prolactin radioimmunossay (RZA): The RIA for rat prolactin used in measuring the serum prolactin level listed in table 1 is a homologous rat prolactin radioimmunoassay [22]. The assay measures competitive inhibition of binding of radio-iodinated rat prolactin antibodies by unlabeled antigen, using Is11for the radioiodination and antirabbit gamma globulin to precipitate the antigen-antibody complexes. The materials for the assay were obtained from a kit distributed bv the National Institute of Arthritis and Metabolic Diseases (NIAMD) and has been used and tested recently [22, 231. Serum prolactin levels down to at least the nanogram level can be accurately measured using this radioimmunoassay system.

Studies done in animals One million cells were injected into the left hind leg of 13 Wistar/Furth males weighing 150-170 g and 12 females weighing 130-150 g (Microbiological Associates, Bethesda, Md). They were approx. 8 weeks old. Of the 13 males, 3 were castrated 72 h prior to the injection of the cells, 5 were kept intact and 5 were injected S.C. with 4 mg of esttadiol valerate (Delestrogen; E. R. Squibb & Sons, Inc.). Of the 12 females, 4 were spayed and the rest were kept intact. In a previous experiment, animals carrying tumors Exptl

Cell Res 78 (1973)

as a result of inoculation of GH, cells were injected with radioactive estradiol-17,K Three females carrying the GH, tumors were spayed and 72 h later, a single S.C. injection of SH-estradiol (0.1 pLg/lOO g body wt) was given. Two hours later the animals were sacrificed and tumor slices, the uterus and diaphragm muscle were processed to measure the radioactivitv retained by them. The SH-estradiol-17B extraction procedure 1171consisted of homogenization of the tisue in 2 ml distilled water; contents were transferred to 20 ml screw can vials and the tubes were rinsed with 10 ml of toluene/isoamyl alcohol (19: 1) mixture. The vials were shaken mechanically for 1 h. After settling down, 5 ml of the organic phase was transferred to scintillation vials and 10 ml of toluene scintillator was added and each sample counted for 10 min.

Studies done in tissue culture Experiments were designed to test the ability of the GH, cells to uptake and retain sex steroids and compare this uptake with a putative undifferentiated mouse fibroblast line, Clone 1D [15]. $H-estradiol178 was added to fully grown small Falcon Plastic flasks (30 ml capacity) in which GH, cells and Clone 1D cells were growing separately and the experiment was terminated at different time intervals. Once the cells were harvested by short trypsin treatment, the pellet was washed 3 times with balanced salt solution in an effort to eliminate all the loosely bound (unspecifically) labeled sex steroid. Comparable amounts of cells were taken in order to determine the amount of counts retained by the two cell lines investigated. Radioactivity was counted using the same procedure as described above. In order to test for the specificity of this uptake and retention, experiments were conducted simultaneously using SH-testosterone as an alternate sexual steroid.

RESULTS

Experiments done in animals The results of the injection of GH, cells growing in tissue culture conditions in the left rear leg are summarized in table 1. In summary, only intact females and estrogen primed malesdeveloped tumors after a period of 60 days of observation. The tumors became sizeable enough to be palpable (pea size) after 30 days of inoculation in the estradiol primed males. The tumors became apparent between 30 and 45 days of inoculation in intact females. At the termination of the experiment (75 days) the tumor reached a size of a small nugget (3 x2 x2 cm). As can be seen in table 1, the weight of animals with tumors

Estrogen-sensitive

cell lines

43

Table 1 Average Weight Tumors

Initial

Final

Prolactin w/ml serum

5

0

3 5

0 515

160+10 16OklO 160&10

328 + 20 313125 350*55

30 45 670

140+10 140*10

254 k 40 232+10

76 50

Animals Injected Males

Normal Castrated El Primed Females

Normal Spayed

8

8/8

4

0

was not significantly higher than those withments run for 24 h the level of 3H-estradiolout them. The high serum prolactin levels 178 retained in the GH, cells did not decline. found in estrogenized male rats are in agree- We assume that estradiol-17/3 in the culture ment with the results reported by other re- medium is not metabolized or conjugated searchers which show that estrogens can be and always remains in the growth medium potent stimulators of pituitary prolactin secre- being constantly incorporated by the cells, tion [28, 291. It is not clear at this time whe- released and reincorporated again. Evidence ther the high prolactin levels we found reflect supporting the fact that estradiol-171 is enhancement of prolactin secretion by the rat metabolized at a minimum in target organs pituitary or by the transplanted functional is available [8,9]. On the other hand, we GH, tumor cells. cannot assume that the same is true with When these tumors were studied for their testosterone, due to evidence indicating that ability of uptaking labeled estradiol, the re- testosterone is metabolized and that 5crsults shown in table 2 indicate that these dihydrotestosterone is the more active androtumors behave as other ‘target tissues’ for genie compound in prostate tissue of different estradiol behave. Diaphragm muscle was species [9, 24, 251. Furthermore, we think included in this experiment as an example of that if the labeled testosterone is indeed mea ‘non-target tissue’ for estradiol. tabolized into other androgenic metabolites Experiments done in tissue culture Experiments measuring the uptake of labeled estradiol-17/3 by the GH, and Clone 1D cells are summarized in fig. 1 for two different concentrations of labeled estradiol in the culture medium (estradiol-178 at 1O-8 M and 1O-g M), spec. act. 40 Ci/mM. The data in fig. 1 show that under the culture conditions employed the GH, cells selectively uptake estradiol-17/3 when compared with undifferentiated fibroblasts (Clone 1D). In experi-

Table 2

Rat 1 Rat 2 Rat 3

GH, Tumor mass

Uterus

Diaphragm

130a 130 130

100 140 170

11 10 15

a Values represent cpm/mg of wet tissue weight in castrated rats (2 days before tritiated estradiol was injected). Animals were sacrificed 2 h after S.C.estradiol injection. Exptl

Cell Res 78 (1973)

44

C. Sonnenschein et al.

0

8

6

0

GH3(10-*

WE,1

1 15’

60’

120’

240’

Fig. 1. Abscissa: time; ordinate: 6, 7, aH-estradiol lOa cpm/mg protein. Uptake of tritiated estradiol by the GH, and Clone 1D cells versus time. Each experimental point represents the average uptake for 3 replicate flasks of cells at the time indicated after the labeled estradiol was added to the culture medium. Total cell protein in each culture flask was determined by the Lowry method. The concentrations listed for each curve are the unlabeled carrier estradiol concentrations. Spec. act. of 3H-estradiol-17 8: 40 Ci/mM.

it is very likely that such sex steroids would bind to high affinity binding sites should they be present in the cells growing in tissue culture conditions or in cells growing as tumors in the animals. Although experiments directed towards identification of probable androgenic metabolites derived from testosterone are in order, experiments designed to clarify other aspectsof this subject indicate that these GH, cells do not have a testosterone binding protein [26]. In order to test the specificity of the uptake of estradiol-17/3 by the GH, cells, experiments were done comparing the relative uptake of tritiated estradiol and testosterone by the GH3 cells. The results of these experiments are shown in fig. 2. The experiments were done by adding similar amounts of labeled estradioL17P and testosterone to the culture medium of replicate flasks of GH, cells to achieve a final concentration of 1OP M, spec. act. 40 Ci/mM. The difference in uptake and/ or retention between the two hormones is Exptl Cell Res 78 (1973)

evident and indicates that the uptake of estradiol-17b by the GH, cells is specific for that steroid and a comparable uptake and/or retention of testosterone is not observed. In order to make sure that the small uptake of testosterone by the GH, cells in fig. 2 is a nonspecific effect, an experiment comparing the uptake of tritiated testosterone by the GH, and Clone 1D cells was performed and the results are shown in fig. 3. In this experiment replicate flasks of GHB and Clone ID cells were exposed to tritiated testosterone at 10egM, spec.act. 40 Ci/mM. Since, as shown in fig. 3, Clone 1D cells uptake testosterone to a comparable or somewhat greater degree than the GH, cells, we can conclude that the small uptake of testosterone by the GH, cells is a nonspecific effect. In conclusion, we can state that the uptake of estradiol-17/I by the clonal rat pituitary cell line, GH,, seemsto be a specialized function of these cells when compared with their uptake of testosterone and when compared with the uptake of estradiol-17/I and testosterone by undifferentiated fibroblasts (Clone

8-

-

6-

4-

2-

I:

xI

“, 15’

60’

120’

240’

Fig. 2. Abscissa: time; ordinate: lOa cpm/mg prot GH, cells. 0, 6,7, 3H-estradiol-17,8; x , 1,2, 3H-testosterono 1 x 1O-9 M, spec. act. Wi/mM. Comparison of tritiated estradiol and testosterone uptake in culture by the GH, cells. The conditions and methods of the experiment are the same as described in fig. 1.

Estrogen-sensitive 4

3

1 L

/

15’

,

I

60’

120’

I

240’

Fig. 3. Abscissa: time; ordinate: 1, 2 3H-testosterone [1O-9 M] uptake (lo2 cpm/mg protein). O-O, clone lD, o-0 GH,. Comparison of tritiated testosterone uptake in culture by GH, cells and Clone 1D cells. The conditions and methods of the experiment are the same as described in fig. 1. The concentration of 1,2-SHtestosterone in the culture medium was 1O-s M, spec. act. 40 Ci/mM.

1D). Saturability of uptake has not been explored at this time [8]. This selective uptake of estradiol-17/3 by the GH, cells is presumed to be the first step involved in the stimulation of tumor growth in estrogen-primed Wistar/Furth rats in which GH, cells have been injected. DISCUSSION Data obtained from experiments reported above suggest that cells from the rat cell line GH, derived from a pituitary tumor are able to produce tumors in normal females and estradiol-17/Lprimed malesafter a period of about 45 days. It follows then that estrogens or estrogen-related mechanisms operating in these animals are responsible for the selective growth of these tumor cells. In addition, when these tumor cells are challenged in their ability to incorporate labeled estradiol-17b they show values similar to those of target cells like those of uteri. When uptake experiments were done with the cells growing in tissue culture condition they showed that

cell lines

45

these GH, cells take up estradiol-17p selectively when compared with undifferentiated fibroblast-like cells as well as when compared with another sex steroid, namely testosterone. Pituitary cells in intact animals have been shown to be target cells for a variety of sex steroids including estradiol-i7p [8, 17, 181. These target cells have the ability of taking up and retaining estradiol-17/I and it has also been shown that the pituitary cells contain estradiol binding proteins or receptors in them [19]. Preliminary experiments have indicated the presence of such binding proteins and the receptor sites both in the cytosol fraction as well as in the nucleus of the GH, cells. The concentration of such receptor sites are comparable to that present in uteri cytosol

t271. The availability of a system like the one we are describing involving solid tumors and the ability of selective growth in a defined endocrine environment in tissue culture will facilitate the analysis of factors related to the mechanism of action of hormones on these tumor cells. It is conceivable that differences will be shown between the two experimental conditions. An added value of such cell lines is the possibility of studying this system on genetic terms when mutants that lack steps in the translation and transcription mechanisms induced by estrogen hormones will be available [20, 211. We wish to express our recognition to Dr L. M. Sherwood for the prolactin radioimmunoassays and Dr E. E. Baulieu for encouragement and support. This contribution was supported by NIH Grant No. lRO1 CA 12338, American Cancer Society (Massachusetts Division) and the Tufts Cancer Research Center. C. S. is a’ recipient of a Research Career Development Award (lK4 CA 47, 410) from the National Cancer Institute.

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46

C. Sonnenschein et al.

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Cell Res 78 (1973)

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