stimulative effects of estrogens on tumor growth and 5a-steroid … · huseby (15) that...

[CANCER RESEARCH 46, 573-576, February 1986]

Stimulative Effects of Estrogens on Tumor Growth and 5a-Steroid Productionin a Mouse Leydig Cell Tumor Line (T 124958-R)1

Shinzaburo Noguchi, Yasuko Nishizawa, Daishiro Takatsuka, Bunzo Sato, and Keishi Matsumoto2

Departments of Pathology [S. N., Y. N., D. T., K. M.] and Internal Medicine [B. S.], Osaka University Medical School, Kita-ku, Osaka 530, Japan

ABSTRACT

The effects of estrogens on the growth and enzyme activitiesfor androgen synthesis in a mouse Leydig cell tumor line (T124958-R) were studied. The s.c. implantation of a diethylstil-

bestrol pellet resulted in a marked enhancement of the tumorgrowth. 5Â«-Reductase activity (nmol/g/h) in tumors rapidly grown

in the presence of diethylstilbestrol pellet was 4 times higherthan that in tumors slowly grown in the absence of diethylstilbestrol, whereas an inverse relation was found for 17/3-hydrox-ysteroid oxidoreductase activity. 17-Hydroxylase activities weresimilar in both tumors. The major C2i- and C19-steroids formed

from progesterone by the tumors grown in the presence ofestrogen were 5Â«-steroids such as 3Â«-or 30-hydroxy-5a-preg-nan-20-one, 3a,17-dihydroxy-5a-pregnan-20-one, androsterone,and 5(*-androstane-3a,17|8-diol, whereas the major steroidsformed by the tumors in the absence of estrogen were 4-ene-3-ketosteroids such as 20a-hydroxy-4-pregnen-3-one, 17-hy-droxy-4-pregnene-3,20-dione, and testosterone. Furthermore,10"8 M of 170-estradiol added in serum-free medium for 10 dayssignificantly enhanced 5a-reductase activities per 106 cells but

significantly inhibited 170-hydroxysteroid oxidoreductase activity

in primary cell culture. These results indicate that estrogensstimulate the growth of T 124958-R in vivo and that estrogensmay directly enhance 5a-reductase activity but inhibit 17/3-hy-droxysteroid oxidoreductase activity in T 124958-R cells.

INTRODUCTION

Previous studies by us and others have shown that testesfrom adult rodents such as rats, golden hamsters, and mice canrapidly convert progesterone to testosterone but not to Ci9-5a-steroids, whereas rapid-growing testes from immature rodentsproduce C19-5Â«-steroidsas major products from progesteroneand that 4-ene-5a-reductase activity is much higher in immaturethan adult rodent testes (1-7). In immature rodent testes, the5<i-reductase is localized primarily in Leydig cells (8, 9) and is

regulated by luteinizing hormone (10,11). However, the patternof conversion of progesterone to 4-ene-3-keto- and 5a-steroids

in Leydig cell tumors of rodents has not been reported.In BALB/c mice, Leydig cell tumors are developed by chronic

treatment with estrogens (12, 13). In our laboratory, one (T124958-R) of the mouse Leydig cell tumor lines supplied by Dr.

R. A. Huseby was found to be estrogen responsive. The stimulative effect of estrogen on the growth of T124958-R was shown

Received 7/8/85; revised 10/4/85; accepted 10/10/85.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by a grant-in-aid for cancer research from the Ministry of

Education, Science, and Culture and by the Ministry of Health and Welfare.2To whom requests for reprints should be addressed.

to be dose dependent (14), supporting the findings reported byHuseby (15) that gonadotrophin had no stimulative effect on thetumor growth. Since C,9-5a-steroids are produced as the majorCi9-steroids in rapid-growing immature mouse testes but not in

adult mouse testes (6,7), and since estrogens markedly enhancethe growth of T 124958-R cells, 5 a-reductase activity relativeto other enzyme activities in T 124958-R was investigated in the

presence or absence of estrogens in the present study.

MATERIALS AND METHODS

Animals and Tumors. The original Leydig cell tumors, which hadbeen developed in testes of BALB/c mice by s.c. implantation of a DES3

pellet [fused 10-mg pellet of 10% DES in cholesterol (12)], were seriallytransplanted s.c. in BALB/c mice. These tumor-bearing mice and host

mice were kindly supplied by Dr. R. A. Huseby and have been maintainedin our laboratory. One tumor line (T 124958-R) used in the present studywas recently established in vivo from a slightly estrogen-responsive

tumor line (T 124958) under the selection pressure of estrogen (14). Theadministration of estrogen to host mice bearing T 124958-R resulted ina marked enhancement of the tumor growth (14). T 124958-R cells

developed in castrated mice in the presence and absence of a DES pelletwere used 3-4 and 7-9 wk after transplantation of seed tumors, respec

tively.Chemicals. [1,2,6,7-3H]Progesterone (112 Ci/mmol), [1,2,6,7-3H]tes-

tosterone (99 Ci/mmol), [1,2-3H]androsterone (41 Ci/mmol), and 4-[4-14C]androstene-3,17-dione (52 mCi/mmol) obtained from New England

Nuclear Corporation (Boston, MA) were purified by paper chromatogra-phy using the hexane:formamide or Hexane:benzene (1:1, vol/vol) form-

amide system (16) just before use. The purified radioactive steroidscontained very small amounts (<0.3%) of contaminating steroids (forexample, see Table 1). Nonradioactive steroids were obtained fromSteraloids, Inc. (Wilton, NH) and Ikapharm, Israel. Other reagents wereof analytical grade.

Incubation Procedure. Tumors without necrotic tissues were homogenized, and the homogenates (5-90 mg tissue) were incubated withpurified [3H]progesterone (specific activity, 6.3 ^M; 2.5 jiCi/5 nmol) or 4-[14C]androstene-3,17-dione (specific activity, 9.6 /Â¿M;0.4 ^Ci/7.7 nmol)and cofactor in air at 34Â°Cfor 30 min in 0.8 ml incubation mixture, as

previously described (5). The incubation mixture consisted of 0.15 Mpotassium phosphate buffer (pH 7.4), 0.13 M sucrose, 0.03 M nicotina-

mide, 1 mw MgCI2, 0.5 CTIMEDTA, and 1.5 mM NADPH.Metabolism of Progesterone. The method used was the same as

previously described (17,18).Estimation of Activities of 17-Hydroxylase, 170-Hydroxysteroid

Oxidoreductase, and 5a-Reductase. Methods for the estimation ofthese enzyme activities were previously described (17-19). These assays

met quantitative criteria when at least 50% of the substrate remainedand the incubation time did not exceed 30 min. Under the assay conditions used, the rate of production of 17-OH-C2i-products plus CÂ«-products, 17/3-hydroxy-, or 5a-products was proportional to the amount

of tissue and the incubation time, when a sufficient amount (1.5 mM) ofcofactor was added. The enzyme activities were expressed as nmol of

3The abbreviations used are: DES, diethylstilbestrol; HCG, human chorkxiic

gonadotrophin.

CANCER RESEARCH VOL. 46 FEBRUARY 1986

573

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5a-ANDROGEN PRODUCTION IN MOUSE LEYDIG CELL TUMOR

Table 1Formation of tritium-labeled 5a- and 4-ene-3-ketosteroids from [3H]progesterone

by T 124958-R in castrated mice with or without a DES pellet, expressed aspercentage of substrate

Homogenates of tumors were incubated with [3H]progesterone (5 nmol; 2.5(iCi/tube) and NADPH at 34Â°Cfor 1 h in 0.8 ml incubation mixture. Values wereobtained after recrystallizationto constant specific activity. Tumors were obtained4 and 9 wk after transplantation of seed tumors in the presence and absence ofDES, respectively.

DES (+) DES (-)

30 90 30 90mg mg mg mgof of of of

tumor tumor tumor tumor Buffer

6.3 65.2 66.5 97.50.4 7.1 6.0 <0.1

11.8 3.8 6.4 0.2

Progesterone(unchanged) 4.220Â«-Hydroxy-4-pregnen-3-one 0.75rt-Pregnane-3n,20tt-diol 1.25a-Pregnane-3,20-dione 9.23Â«-or 3fi-Hydroxy-5a-pregnan-20-one 62.6 55.7 6.7 3.517-Hydroxy-4-pregnene-3,20-dione 0.3 0.2 0.6 0.817-Hydroxy-5Â«-pregnane-3,20-dione 0.1 0.4 <0.1 <0.13a,17-Dihydroxy-5a-pregnan-20-one 2.0 3.1 0.6 0.530,17-Dihydroxy-5a-pregnan-20-one <0.1 <0.1 0.2 0.24-Androstene-3,17-dione 0.1 0.1 0.2 0.4Testosterone 0.5 0.7 0.2 1.05tt-Androstane-3,17-dione <0.1 <0.1 <0.1 <0.117/?-Hydroxy-5Â«-androstan-3-one <0.1 0.2 0.1 0.1Androsterone 0.1 0.5 <0.1 0.13tÃ-Hydroxy-5a-androstan-17-one <0.1 0.1 <0.1 <0.15u-Androstane-3a,17/J-diol 0.2 0.4 <0.1 0.15Â«-Androstane-3/3,17/3-diol <0.1 0.1 <0.1 <0.1

Total CiÂ»-4-ene-3-ketosteroids

Total C,9-5o-steroids

0.6 0.8 0.4 1.4 0.0

0.3 1.3 0.1 0.3 0.0

steroids formed per g wet tissue per h. Because the concentrations ofsubstrates (5.0 or 7.7 nmol/5-80 mg tissue) were supraphysiological,

the assumption was made that endogenous levels of the steroid intermediates can be disregarded when estimating enzyme activities (forexample, endogenous levels of progesterone and testosterone estimatedby radioimmunoassay in Leydig cell tumors were less than 0.08 nmol/10 mg tissue and 0.01 nmol/10 mg tissue, respectively).

Assay of Progesterone and Androgens. Methods for extraction andthe competitive protein binding assay of progesterone were previouslydescribed (20). Methods for extraction and radioimmunoassay of testosterone or 5Â«-androgens (androsterone plus 5a-androstane-3a,17/3-diol)were also previously described (21). Testosterone anitbody cross-reacted with 17/3-hydroxy-5<*-androstan-3-one (70%), 5Â«-androstane-3a,17/3-diol (9%), and 5a-androstane-3/3,17/3-diol (4%) but not with 170-estradiol, progesterone, or coritsol. Androsterone antibody cross-reactedsubstantially with 5a-androstane-3a,17/3-diol (56%), and, to much lesserdegree, with 3Â«-hydroxy-5<*-pregnan-20-one (5%), 3Â«-hydroxy-5/3-an-drostan-17-one (2%), and 5Â«-androstane-3,17-dione (1%). The antibodywas not found to cross-react with various C2i- and Ci9-5a-steroids,various C2r and d9-5/3-steroids, various C2t- and Ci9-4-ene-3-ketoste-roids, dehydroepiandrosterone, or 17/3-estradiol (less than 1% cross-

reaction for these steroids). Since androsterone was measured withoutchromatography, androsterone levels used in the present study mustrepresent androsterone plus 5Â«-androstane-3a,17/3-diol levels. The intra-

and interassay coefficients of variation obtained from 10 assays of tumorsamples were 7.7-8.8 and 9.4-12.2%, respectively.

Estimation of Enzyme Activities in Cell Culture. Tumor-bearing

castrated mice with DES pellet were sacrificed by cervical dislocation,and the tumors were excised under sterile conditions. In order to obtainthe dispersed cells, the minced tumor was digested with Hanks' balanced

salt solution (Ca2+and Mg2+ free, 1 g/10 ml) containing trypsin (Worthing-

ton; 0.1 mg/ml), collangenase (Sigma; type IV, 0.5 mg/ml), bovine serumalbumin (1 mg/ml), and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic

acid (5 nriM)for 30 min at 37Â°C,as previously reported (22). The mixture

was then centrifuged at 300 x g for 5 min, and the pellet was washed

once with 10% fetal calf serum: Eagle's minimal essential medium to

inhibit the activity of trypsin. The pellet was then washed 3 times withHanks' balanced salt solution. These dispersed cells in the pellet werefinally resuspended in 2 ml of serum-free medium [Ham's F-10: Eagle's

minimal essential medium (1:1, vol/vol) supplemented with insulin (1 ^g/ml), human transferrin (20 Mg/ml), and 0.1% bovine serum albumin] withor without 10~8 M 170-estradiol [in a few experiments, 10-50 ng of HCG

(Teizo, Tokyo, Japan) per ml were added] and inoculated onto 35-mmplastic dishes (1 x 106 cells/dish). The serum-free media were changed

every 2 days. 17/3-Estradiol dissolved in ethanol solution was added to

the medium, in which final concentrations of ethanol were less than0.1%.

Ten days after plating, activities of 5Â«-reductase and 17/3-hydroxyste-roid oxidoreductase were estimated. The serum-free media werechanged with 2 ml of the fresh serum-free medium without 10~e M 17/8-

estradiol just before the addition of 4-[14C]androstene-3,17-dione (0.4fiCi/7.7 nmol/dish) or [3H]progesterone (2.5 fiCi/5 nmol/dish) dissolvedin 20 ti\ ethanol. Cells were incubated with 14C- or 3H-labeled substrate(3.85 or 2.5 ^M) in a humidified incubator in 5% CO2 at 37Â°Cfor 15 or

30 min. The incubated cells were harvested by a rubber policeman atthe indicated times, and the cell suspension was immediately transferredinto the tubes on ice to be mixed with etherchloroform (4:1, vol/vol)solution in order to stop the reaction. Cell numbers were determined byhemocytometer, and cell viability was assessed by the trypan blue dyeexclusion test. A significant difference in cell number (1.5-1.9 x 106

cells/dish) or viability (60-70%) was not found among the dishes. The

cell suspension was extracted 3 times with 10 ml of etherchloroform(4:1, vol/vol) solution. The methods for the estimation of 5Â«-reductaseand 17/3-hydroxysteroid oxidoreductase activities are already describedin this paper. 5a-Reductase activity for progesterone was expressed asthe sum of all 5a-C2,-17-deoxysteroids (4 steroids shown in Table 1)formed from progesterone, since C2,-17-OH- and d9-steroids were not

formed significantly under the present conditions in culture. Under theassay conditions used, the rate of production of 5Â«-or 17/3-hydroxy

products was proportional to the amount of cells and the incubationtime. The enzyme activities were expressed as nmol of steroids formedper 106 cells per h.

RESULTS

Stimulation of Tumor Growth by Estrogen. In castrated mice,the s.c. implantation of one DES pellet (10 mg), which consistedof 90% cholesterol and 10% DES, resulted in a marked enhancement of the growth of T 124958-R. Although more than 50% of

the host mice with DES pellet died due to the growth of T124958-R within 35 days after the transplantation of seed tu

mors, no tumor mass was detectable until 40 days after thetransplantation in the absence of estrogen.

Metabolism of Progesterone in Tumor Homogenates. Homogenates of 30 and 90 mg of tumors were incubated with [3H]

progesterone and NADPH for 1 h. The results are given in Table1 as the percentage of total steroid introduced and are notrepresentative of the percentage of the activity of different enzymes. Under the incubation conditions used, the rate of production of the sum of Cig-steroids was roughly proportional to

the weight of tumors used.When homogenates of tumors grown in the presence and

absence of estrogen were incubated with [3H]progesterone,

similar amounts of substrate were converted to C19-steroids inboth tumors. However, the formation of C2i-5a-steroids relativeto C2i-4-ene-3-ketosteroids and that of Ci9-5a-steroids relativeto Ci9-4-ene-3-ketosteroids were much higher in tumors rapidlygrown in the presence of estrogen than in tumors slowly grown


574



5Â«-ANDROGEN PRODUCTION IN MOUSE LEYDIG CELL TUMOR

in the absence of estrogen (Table 1). These results suggest thatestrogenization of host mice stimulates 5o-reductase activity inT124958-R.

Activities of 5a-Reductase, 170-Hydroxysteroid Oxidore-ductase, and 17-Hydroxylase in Tumor Homogenates. Specific 5Â«-reductase activity (nmol/g/h) in tumors rapidly grown in

the presence of estrogen was 4 times higher than that in tumorsslowly grown in the absence of estrogen. However, specif ic 17/3-

hydroxysteroid oxidoreductase activity was significantly higherin the tumors grown in the absence of estrogen than in thetumors grown in the presence of estrogen (Table 2). Specific 17-

hydroxylase activities and degenerative changes by morphologywere similar in both tumors.

The concentration of progesterone or testosterone was significantly higher in tumors grown in the absence of estrogen thanin tumors grown in the presence of estrogen. A slightly higherconcentration of 5a-androgens in DES (-) as compared with

DES (+) tumors did not agree with the metabolic data. Thisfinding may be related to the uncertainties of the radioimmu-noassay cross-reactivities. However, the ratio of 5a-androgens

to testosterone was slightly but significantly (P < 0.05) higher inDES (+) than DES (-) tumors (Table 3). A biological evidencefor the formation of androgens in T 124958-R is also clearly

shown in Table 3, since significant increases in weights of theseminal vesicles of castrated male recipients were produced bythe tumors grown in the presence or absence of estrogen.

Effect of 170-Estradiol on Activities of 5a-Reductase and17n'-Hydroxysteroid Oxidoreductase in Tumor Cells in Cul

ture. In order to examine whether the stimulative effect ofestrogen in vivo on 5Â«-reductase activity in T 124958-R is director indirect via estrogen-induced factors secreted from othertissues such as the pitutiary, the effect of 17/3-estradiol added in

Table2Effects of estrogenizationof castrated mice with DESpellet on activities of 5a-

reductase, 17ÃŸ-hydroxysteroidoxidoreductase,and 17-hydroxylaseinT 124958-R

Tumor homogenates (5-80 mg) were incubated with 4-[uC]androstene-3,17-dione (9.6 MM)or [3H]progesterone(6.3 Â»M)and NADPHat 34Â°Cfor 30 min in 0.8

ml incubation mixture.

5a-ReductaseDES (nmol/gtissue/h)+

218 Â±18a-6

55 Â±717/3-Hydroxyste-

roid oxidoreductase (nmol/g

tissue/h)29Â±7Â°

108 Â±1717-Hydroxylase

(nmol/gtissue/h)6.5

Â±0.85.8 Â±0.6

8 Mean Â±SE of 4 separate determinations.6P<0.01.c P < 0.05, when compared to the values of DES (-).

Table 3Weightof the seminal vesicles and concentrations of progesterone, testosterone,and 5a-androgens (androsteroneplus 5a-androstane-3a,Ufl-diol) in tumor found

in castrated mice bearing T 124958-Rin the presence or absenceof DESMice bearing T 124958-R were sacrificed 3-4 and 7-9 wk after transplantaton

of seed tumors in the presenceand absenceof DES. respectively.

Concentration in tumor (ng/10 mg)

T Wt of seminal124958-R DES vesicles (mg) Progesterone Testosterone

5n-Andro-gens

12Â±2"63 Â±2"

153 Â±2164.0 Â±0.6C

23.9 Â±2.7

0.3Â±0.1C

1.0 Â±0.12.0 Â±0.22.9 Â±0.4

medium on 5Â«-reductase activity in T 124958-R cells in culturewas examined. As shown in Table 4, 10~8 M of 170-estradiol

significantly enhanced 5Â«-reductase activities per 106 cells in

primary culture but significantly inhibited specific 170-hydroxy-

steroid oxidoreductase activity. Although the stimulative effecton 5Â«-reductase activity for progesterone was much strongerthan that for 4-androstene-3,17-dione, these 2 experiments werecarried out separately at an interval of 4 mo. The addition of 10-

50 ng of HCG per ml to culture medium for 10 days, however,showed no significant effects on 5a-reductase and 170-hydrox-

ysteroid oxidoreductase activities (data not shown). In contrastwith T 124958-R tumors grown in vivo (Tables 1 and 2), T124958-R cells maintained for 10 days in culture did not formsignificant amounts of C2i-17-OH- and Cie-steroids.

DISCUSSION

The present findings demonstrate for the first time the stimulative effect of estrogens on specific 5a-reductase activity in

Leydig cells. The effect of estrogens seems to be induced by adirect effect of estrogens on T 124958-R cells, since 170-estradiol (10~8 M) added in medium for 10 days significantlyenhanced 5a-reductase activities per 106 cells in primary cell

culture. Although 5a-reductase activity is regulated by luteinizing

hormone in normal Leydig cells of immature rat and hamstertestes (10, 11), HCG had no effect on 5a-reductase activity in T124958-R cells. Previous studies reporting the direct effect of

estrogens on testicular enzymes involved in androgen synthesisare as follows. In hypophysectomized mice treated with constantdoses of gonadotrophin, treatment with estrogens led to amarked reduction in testicular 17-hydroxylase, 17,20-lyase, and17/3-hydroxysteroid oxidoreductase activities (23). Treatment ofhypophysectomized rats with 170-estradiol or incubation of dispersed rat Leydig cells with 17/3-estradiol for 24 h caused decreases of 17-hydroxylase and 17,20-lyase activities, but not of3/8-ol-dehydrogenase or 17/3-hydroxysteroid oxidoreductase ac

tivities (24, 25). In the present study, estrogens directly inhibited170-hydroxysteroid oxidoreductase activity in T 124958-R cellsbut had no effect on 17-hydroxylase activity.

In T124958-R cells, estrogens stimulate 5a-reductase activity.The estrogen-induced change of enzyme activity in rapid-growing

Table4Effects of 17ÃŸ-estradiol(W u) added in medium on activities of 5a-reductase

and 170-hydroxysteroidoxidoreductasein T 124958-Rcells in cultureTumor cells (1.5-1.9 x 10" cells/dish) cultured for 10 days in the presence or

absenceof 10"" M170-estradiolwere incubated with 4-["C]androstene-3,17-dione(3.85 Â¿IM)or [3H]progesterone(2.5 ^M)for 15 or 30 min. Radioactiveproducts wereidentified by recrystallization to constant specific activity. No significant amountsof 3HC21-17-OH-and 3HCtÂ»-steroidswere formed from [3H[progesteroneunder theconditions used.

4-[14C]Androstene-3,17-dtone"170-

Estra-diol+5a-Reductase

(nmol/10*cells/h)0.46

Â±0.046 c

0.31 Â±0.02170-Hydroxy-

steroid oxidoreductase(nmol/IO*

cells/h)0.94Â±0.04Â°

1.33 Â±0.12[3H]Progesteronea

(5a-Reductase;nmol/10"cells/h)0.53

Â±0.04d

0.14 Â±0.01

a Mean Â±SE of 4 determinations."p < 0.01. when compared to the value of T 124958-R (-).CP< 0.01, when compared to the value of DES (-).

a Radioactivesteroid used as substrate.6 Mean Â±SE of 4 separate determinations.CP<0.05." P < 0.01, when compared to the valuesof 17tf-estradiol(-).


575



5a-ANDROGEN PRODUCTION IN MOUSE LEYDIG CELL TUMOR

T 124958-R results in the production of an immature type oftesticular steroids formed in rapid-growing immature mousetestes, in which the major C2i- and Ci9-steroids produced are5a-steroids (6, 7, 26). These observations suggest that theestrogen-induced increase in 5Â«-reductase activity in T 124958-

R cells may be related to the stimulative effect of estrogen onthe tumor growth. The present results demonstrate for the firsttime the hormone-induced production of 5Â«-steroids in Leydig

cell tumor lines. In connection with the direct effect of estrogenon steroidogenesis in Leydig cells, the present findings are notinconsistent with the finding by Samuels ef al. (23) describedbefore.

Recent studies have shown that the stimulative effect of sexsteroids on cell proliferation may be mediated by specific peptidegrowth factors (27, 28). However, the mechanism of mitogenicaction of estrogens is not clearly known. In order to study themolecular mechanism of estrogen-dependent growth using T124958-R cells as a model, the demonstration of estrogen-

induced marker proteins of the tumor cells and the establishmentof a cell line showning estrogen-responsive growth in culturewere desirable. Although an estrogen-induced secretory protein

with a molecular weight of 34,000 was demonstrated (29), wewere unable to demonstrate estrogen-induced intracellular proteins in T124958-R cells until very recently. Estrogen-responsive5Â«-reductase activity in the tumor cells seems to be a goodestrogen-induced intracellular marker in T 124958-R cells.

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1986;46:573-576. Cancer Res Shinzaburo Noguchi, Yasuko Nishizawa, Daishiro Takatsuka, et al. 124958-R)-Steroid Production in a Mouse Leydig Cell Tumor Line (T

αStimulative Effects of Estrogens on Tumor Growth and 5

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