[CANCER RESEARCH 48, 3586-3590, July 1, 1988]

Effect of Testosterone on Imidazole-induced Tyrosinase Expression in B16Melanoma Cell Culture1

Ellis L. Kline,2Karen C. Garland, James T. Warren, Jr., and Terry J. Smith

Department of Microbiology, Clemson University, Clemson, South Carolina 29634-1909 [E. L. K., K. C. C., and J. T. WJ, and Department of Medicine, State Universityof New York at Buffalo and the Veterans Administration Medical Center, Buffalo, New York 14215 [T. J. S.J

ABSTRACT

To assess the effect of androgens on tyrosinase activity in B16/C3melanoma cell cultures, proliferating cultures were treated with testosterone (50 IIM) or one of several other androgenic analogues and metabolites. None of these compounds influenced basal enzyme activity. Imid-azole (10 HIM),however, is a potent inducer of tyrosinase in this cell line.Testosterone blocked induction of tyrosinase by imidazole almost completely. This effect was dose dependent, being maximal at 10 n,\i andhalf-maximal at ~3 IIM,and was rapid, occurring within 15 min. Whencultures treated with both imidazole and testosterone were shifted tomedium containing only imidazole, enzyme activity approximated thatseen in cultures never receiving testosterone within 10 h of the shift. Theother steroids tested failed to influence imidazole induction of the enzyme.This action of testosterone could not be demonstrated in broken cellpreparations. Results of studies involving inhibitors of protein and RNAsynthesis, as well as those quantitating mRNA hybridizable to a synthesized 20-base pair deoxyoligonucleotide tyrosinase probe, suggest thattestosterone is blocking imidazole induction at a pretranslational level.

INTRODUCTION

Tyrosinase (EC 1.14.18.1 ), the rate-limiting enzyme involvedin melanogenesis in mammalian cells (1), is influenced by avariety of hormones in both normal and neoplastically transformed tissue. Perhaps the most extensively studied and bestcharacterized regulator of melanin production is melanocyte-stimulating hormone. The response to MSH3 is associated with

an increased intracellular cyclic AMP content (2) and is mediated through binding to glycoprotein determinants on the cellsurface (3, 4). Disagreement exists concerning the mechanisminvolved in the stimulation of tyrosinase by MSH. Evidencehas been introduced (5) for an activation of preexisting enzymemolecules, while other investigators (6) have suggested that denovo protein synthesis may be involved.

This laboratory (7) reported recently that 3,3',5-L-triiodothy-

ronine, the active thyroid hormone, could inhibit basal andchemically induced tyrosinase activity in B16/C3 murine melanoma cell cultures. The effects were stereospecific and occurred within 2 h of treatment at physiologically relevant concentrations. The effects of 3,3',5-L-triiodothyronine were ap

parently mediated through pretranslational events independentof mRNA destabilization (7). This cell culture line is thus auseful model for studying hormonal regulation of melanogenesis in vitro.

Sex steroids may be important influences of melanoma behavior in vivo, as suggested by the finding of receptors inmelanoma tumors (8) and sex differences in the prognosis and

Received 8/31/87; revised 3/15/88; accepted 3/31/88.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.

1This research was funded in part by the ELK-PAM research fund and by the

Veterans Administration Research Service.1To whom requests for reprints should be addressed.3The abbreviations and trivial names used are: MSH, melanocyte-stimulating

hormone; androstenedione, 4-androstene-3,17-dione; dihydrotestosterone, 5a-androstan-17/3-ol-3-one; epitestosterone, 4-androsten-17«-ol-3-one; testosterone,4-androsten-170-ol-3-one.

clinical course of these tumors (9). Survival time followingmetastasis of the primary tumor is significantly greater infemales than in males and the secondary tumor-doubling timeis slower in female patients (10). These observations promptedus to explore what effect, if any, testosterone and relatedsteroids might have on tyrosinase activity in cultured B16/C3cells. Here we report that the hormone can block induction ofthat enzyme by imidazole.

MATERIALS AND METHODS

Chemicals. Testosterone, epitestosterone, and 5a-dihydrotestoster-one were purchased from Sigma Chemical Co. (St. Louis, MO), andimidazole was purchased from Fisher Scientific (Pittsburgh, PA). Androstenedione was obtained from Steraloids (Wilton, NH). All otherchemicals used were of the highest purity available.

Cell Culture. The B16/C3 mouse melanoma cell line was a generousgift of Drs. J. Kreider and G. Bartlett (Hershey Medical Center,Hershey, PA). Cultures were incubated at 37°Cin 25-cm2 Corning

flasks (Corning Medical, Medfield, MA) with 5 ml antibiotic-freeminimum essential medium supplemented with donor calf serum 10%(vol/vol), as previously described (11). Cells were removed for passagein calcium-free, magnesium-free phosphate-buffered saline containing0.05% EDTA disodium, pH 7.4. Experimental flasks were seeded with1.5 x 10s late exponential cells and allowed to attach for 6 h. Fresh

medium supplemented with the steroid tested, imidazole, or diluent(95% ethanol) was then added, and cultures were allowed to incubate.Unless indicated otherwise, the final hormone concentrations were 50IIM,and that of imidazole was 10 HIM.

Inhibitor Studies. Studies involving inhibitors were conducted asfollows. Actively dividing cultures were treated with imidazole for 18 hwith or without testosterone. Cultures were then shifted to mediumcontaining the same supplements and cycloheximide (10 ng/ml, aconcentration which inhibited >90% of protein synthesis) for 5 h, aperiod of time which allowed maximal inhibition of the enzyme activity(11). After the cultures had been washed extensively, they were shiftedto fresh medium supplemented with only actinomycin D (2 tig/ml, aconcentration inhibiting >90% of RNA synthesis). This time corresponds to time 0 in Figs. 5 and 6.

Tyrosinase Assay. Tyrosinase activity in whole cell sonicates wasdetermined as previously described (12). Briefly, monolayers wererinsed thoroughly with 0.05% disodium EDTA in calcium-free, magnesium-free phosphate-buffered saline (5 ml), suspended in 1.5 mlsodium phosphate buffer (80 mM, pH 6.8), and frozen at —20°Cuntilassayed. Samples were thawed and disrupted at 4°Cwith two 15-s

sonication bursts with a microtip probe (sonicator model W-220F, HeatSystems, Plainview, NY). The assay mixture contained 0.4 (imol L-tyrosine, 0.04 ¿¿mol3,4-dihydroxy-L-phenylalanine in sodium phosphate buffer (26 /¿mol;pH 6.8), 2.5 /¿CiL-[3H]tyrosine (specific activity,

52.5 Ci/mmol; New England Nuclear, Boston, MA), and 0.2 ml cellsonicate (final volume, 0.4 ml). Reactions were carried out at 37°Cfor

l h in a shaking water bath and terminated with the addition of 0.5 mlice-cold trichloroacetic acid (10%, v/v). Unreacted L-tyrosine was extracted by adding 0.7 ml activated charcoal suspension (100 mg/ml;Norit A; Fisher) and centrifuging. An aliquot of supernatant wassubjected to liquid scintillation spectroscopy in a Beckman LS-7000counter (Beckman, Palo Alto, CA), with Aquasol (New England Nuclear) as the scintillant. Protein concentrations were determined by themethod of Lowry et al. (13).

Hybridization Studies. Tyrosinase mRNA abundance was quantitated3586

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TESTOSTERONE REGULATION OF TYROS1NASE EXPRESSION

using the quick blot hybridization method of Gillespie and Bresser (14)using an [«-32P]dCTP-labeled20-base pair probe which corresponds tobase pairs 224-244 of a cloned B16 tyrosinase sequence similar to thatreported previously (15). mRNA was extracted from cultures usingguanidine thiocyanate (14) and equal amounts of total cellular RNAwere loaded onto membranes. Hybridization conditions were similar tothose suggested (14).

RESULTS

Testosterone, 5a-dihydrotestosterone, and epitestosteronefailed to influence tyrosinase activity in proliferating B16/C3cells in culture (Fig. IA). Compounds were present for a totalof 19 h and none influenced the rate of proliferation. Imidazole(10 HIM)induced the activity ofthat enzyme 2-3-fold (Fig. IB),consistent with earlier reports (11). We have previously reported (10) that maximal induction of tyrosinase occurs by 19h in proliferating cultures. When testosterone was added to theculture medium in addition to imidazole, the androgen blockedthe increase in activity (Fig. IB). In contrast, none of the othersteroids influenced the imidazole induction. Androstenedionealso failed to influence both basal and imidazole-induced activity (data not shown).

The inhibition by testosterone of imidazole induction was

8 -

Com Testos- 5a dihydno-terone testos-

terone

Epitestos-

terone

14-

\2~

V c IO-

B

I

Cont I Testos- 5a ditiydro-testos-

Im

Epitestosterone

ImIm

Fig. 1. Effects of testosterone, dihydrotestosterone, and epitestosterone ontyrosinase activity in B16/C3 cell cultures. Proliferating cultures were treatedwith diluent (control) or one of the steroids (SOnM) for 19 h without ( -I) or with(B) imidazole (Im) (10 niM). Cultures were then assayed for tyrosinase activity.Columns, means ±range (bars) of duplicate culture plates from a representativeexperiment.

dose dependent. As Fig. 2 demonstrates, the threshold of effectoccurred at a concentration of 1 nM, was half-maximal at ~3HM, and was maximal at 10 nM. The addition of up to 10-foldhigher concentrations of the hormone failed to inhibit enzymeactivity any further.

To determine how rapidly testosterone could affect chemicalinduction, proliferating cultures which had received imidazoleat time 0 were treated with the steroid at 18 or 23 h and assessedfor tyrosinase activity. As Fig. 3 demonstrates, there was a rapiddecline in enzyme activity regardless of when testosterone wasadded. Within 3-5 h of testosterone addition, activities fell tolevels observed in noninduced cultures. In contrast, platestreated with imidazole alone exhibited increasing activity until30 h (the end of the study), when levels were almost 4-fold

Ixlo'm 5xK>9mIxK) m

TESTOSTERONE CONCENTRATION

Fig. 2. Dose-response curve of the effect of testosterone on imidazole-inducedtyrosinase activity. Cultures were incubated for 19 h with imidazole (10 nist) plusthe concentrations of testosterone indicated along the abscissa. Points, means ±range (bars) of duplicate culture plates.

* IMIDAZOLEi IMIDAZOLE +

TESTOSTERONE

So Cvj

L/Ai12 14 16 18 20 22 24 26 28 30

Time (hours)

Fig. 3. Time-course of the effect of testosterone on imidazole-induced tyrosinase activity. Cultures were treated with imidazole (10 HIM)for 18 h. At 18 or 23h (arrows), some plates received testosterone (SO nM) as well. Cultures wereharvested at the times indicated along the abscissa and assayed for tyrosinaseactivity. Points, means ±range (bars) of triplicate cultures.

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TESTOSTERONE REGULATION OF TYROSINASE EXPRESSION

above basal levels. This effect was rapidly reversible. Culturesreceiving both imidazole and testosterone which were shiftedto medium containing only imidazole manifested increasingenzyme activity within l h of the shift and levels which werenear maximal within 6 h (Fig. 4).

An intact cell is apparently necessary to demonstrate imidazole stimulation of tyrosinase activity and the testosteroneblockade thereof. Whole cell sonicates incubated with eitherimidazole or the steroid and incubated for up to 4 h failed toalter activity (Table 1). In another set of experiments, cells wereharvested and sonicated in the presence of sodium molybdate(10 HIM),under conditions which stabilize the androgen receptor (16). Results obtained were similar to those contained inTable 1 (data not shown).

Testosterone appears to block imidazole-induced tyrosinaseexpression at a pretranslational level (Fig. 5). Cultures wereincubated with imidazole (10 HIM),imidazole plus testosterone(SO DM),or diluent for 18 h. They were then shifted to mediumcontaining cycloheximide (10 pg/ml) and the respective supplements for an additional 5 h (pretreatment). All cultures werethen washed and shifted to medium containing actinomycin D(2 Mg/ml) alone at the time designated "0" in Fig. 5. As thatfigure demonstrates, enzyme activity increases 3-4-fold in the4 h following the shift to actinomycin D in cultures which hadpreviously been treated with imidazole. In contrast, tyrosinaseactivity in cultures which had received imidazole and testosterone increased less than 2-fold and more closely resembledcultures not pretreated with imidazole. Under these treatmentconditions, tyrosinase mRNA accumulated presumably duringthe pretreatment period, when translation was effectively

blocked. Any accumulated message should be available fortranslational and posttranslational processing during the actinomycin D treatment. These results suggest that imidazole-pretreated cultures accumulated more translatable tyrosinasemRNA than did those which had received imidazole and testosterone or neither compound.

Fig. 6 demonstrates the results of an experiment designed toassess the effect of imidazole and testosterone on preformed,accumulated tyrosinase mRNA. Cultures were treated withimidazole or diluent for 18 h and allowed to proliferate actively.Fresh medium containing cycloheximide with or without imidazole was added for 5 h and then at the time designated 0 inFig. 6, fresh medium containing actinomycin D and imidazole,testosterone, or diluent was added and cultures were harvestedat the times indicated along the abscissa. Those cultures whichhad received imidazole manifested increasing tyrosinase activityregardless of whether testosterone was present, suggesting thatthe hormone failed to block the translation or posttranslationalprocessing of preformed mRNA.

The increase in tyrosinase activity following imidazole exposure and the blockade of that induction by testosterone wereaccompanied by changes in the abundance of cellular RNAwhich hybridized to a 20-base pair tyrosinase probe. As Fig. 7clearly demonstrates, imidazole markedly increased the abundance of hybridizable RNA compared to levels in cells receivingtestosterone. The hybridization pattern in the testosterone-treated cultures was identical to that in untreated controls (datanot shown). When testosterone was added to cultures receivingimidazole, the steroid completely blocked the increase in tyrosinase mRNA abundance.

iMDUOUi IMIDAZOLE +

TESTOSTERONE

O 12 14 16 18 2O 22 24 26 28 JO

Tim«(hours)Fig. 4. Reversibility of testosterone effects on imidazole-induced tyrosinase

activity. Cultures were treated with testosterone (SO nM) and imidazole (10 HIM)for 18 h and then one-half of the plates were shifted to medium containing onlyimidazole while the others continued to be treated with both compounds. Cultureswere harvested at the times indicated along the abscissa. Points, means ±range(harx) of duplicate culture plates.

DISCUSSION

These results demonstrate that testosterone can block imidazole induction of tyrosinase rapidly at concentrations whichhave physiological relevance. This inhibitory effect apparentlyinvolves de novo protein synthesis.

There exists a priori evidence to suspect steroid hormones assignificant regulators of melanoma behavior and metabolism.High affinity binding for androgens, estrogens, progesterone,and glucocorticoids has been demonstrated in human melanoma (8,17-1 9), yet there is generally a lack of clinical responseto endocrine therapy (8). Thus, the binding activities mayrepresent an association with inactive receptors or with unrelated proteins.

We found that testosterone but none of the other relatedsteroids influences the induction of tyrosinase activity in B16cells in vitro. The lack of response to 5a-dihydrotestosteronesuggests that a 5«reductive pathway is not involved in themediation of this effect, consistent with androgen action innormal dermal tissue not derived from the genital anläge(20).Whether any alternative biotransformation of testosterone (3areduction or aromatizaron to estradici, as examples) occurs in

Table 1 Effects of testosterone and imidazole on tyrosinase activity in cell sonicatesCell sonicates from imidazole-induced cultures were preincubated at 37°Cfor the times indicated in the presence of diluent, imidazole (10 HIM),or testosterone

(SOnM) and then assayed for tyrosinase activity. Means ±SD of 3 determinations (nmol 3H2O/mg protein/h).

Preincubation time

CompoundDiluentImidazoleTestosterone0min10.87

±0.2310.87

±0.2310.87

±0.2330

min10.89

±0.1710.43

±0.1210.53

±0.14Ih9.48

±0.169.69

±0.149.52

±0.232h7.87

±0.177.66

±0.247.39

±0.103h6.27

±0.126.40

±0.056.18

±0.164

h5.02

±0.075.11

±0.235.21

±0.193588

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TESTOSTERONE REGULATION OF TYROSINASE EXPRESSION

TYR-PROBE

0123456

I Actinomycin D 1

TIME (Hours)

Fig. 5. Protein and RNA synthesis inhibitor effects on imidazole and testosterone regulation of tyrosinase activity. Proliferating cultures were incubated for18 h with imidazole (10 HIM;A), imidazole plus testosterone (50 DM; A), ordiluent (control; •).They were then shifted to medium with these respectivesupplements as well as cycloheximide (10 fig/ml) for 5 h. All plates were thenshifted to medium to which only actinomycin D (2 Mg/ml) was added (time 0)and harvested at the times indicated along the abscissa. These concentrations ofinhibitors blocked >90% of protein and RNA syntheses, respectively. Points,means ±range (hars) of tyrosinase activity in duplicate cultures.

8" 6M •

01 234567

l Actinomycin D 1

Time (hours)

Fig. 6. Effect of testosterone on the translation of preformed tyrosinasemRNA. Cultures were treated for 18 h with imidazole (10 HIM;•,A) or diluent(A) and then shifted to medium containing these respective supplements andcycloheximide (10 >ig/ml) as well for 5 h (pretreatment). At time 0, cultures wereshifted to medium containing actinomycin D plus either imidazole (•),testosterone (A), or diluent (A) and were harvested at the times indicated along theabscissa. Points, means ±range (oars) of duplicate cultures.

these cells and precedes the blockade of tyrosinase induction isnot known. Our recent finding (21) that estradiol can also blockthe induction by imidazole raises the possibility that theseeffects might be mediated through the estrogen receptor.

Imidazole induction of tyrosinase in B16/C3 cells in vitro isindependent of increased intracellular cyclic AMP content (11),and unlike the action of MSH on these cells (22) it involves denovo synthesis of enzyme. This compound can alter gene transcription at specific promoter sites in prokaryotic cells as a"metabolite gene regulator" (23-25). From the data presented

ImTestosterone

Im + Testosterone

Fig. 7. Effect of imidazole (Im) and testosterone on the abundance of RNAhybridizable to a tyrosinase (TYR) probe. Equivalent quantities of RNA wereimmobilized and allowed to hybridize to a 20-base fragment of the tyrosinasegene as described in "Materials and Methods."

here and those recently reported (7, 21), it would appear that3,3',5-L-triiodothyronine, estradiol, and testosterone can influ

ence significantly the chemical induction of tyrosinase. Whilethe effects of all three classes of hormone appear to be pre-translational and independent of niRNA destabilization,whether transcription is affected is uncertain. Preliminary hybridization studies4 with the 20-base pair deoxyoligonucleotide

probe confirm that imidazole increases the cellular content ofmRNA for tyrosinase and that both 3,3',5-L-triiodothyronine

and estradiol block this induction, similar to the effect oftestosterone reported here.

Chemical-induced melanogenesis in B16/C3 cells in vitro isthus apparently under multihormonal control, consistent witha variety of other cellular events including a2„-globulingeneexpression (26, 27), growth hormone synthesis (28), and hyalu-ronate synthesis (29). This culture model may be ideal forstudying the nature of hormone-hormone interactions at thelevel of gene expression and for examining the mechanismsinvolved in hormonal conditioning of the cellular response tochemicals, as has been described in the liver (30, 31).

ACKNOWLEDGMENTS

The authors thank June I luff and Ruth Harvey for their help in thepreparation of this manuscript.

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1988;48:3586-3590. Cancer Res   Ellis L. Kline, Kren C. Carland, James T. Warren, Jr., et al.   Expression in B16 Melanoma Cell CultureEffect of Testosterone on Imidazole-induced Tyrosinase

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