stimulation of melanogenesis in a human melanoma cell line by … · stimulation of melanogenesis...

[CANCER RESEARCH 40, 3345-3350, September 1980]0008-5472/80/0040-0000$02.00

Stimulation of Melanogenesis in a Human Melanoma Cell Line by Retinoids1

Reuben Lotan2and Dafna Lotan

Department of Biophysics, The WeizmannInstitute of Science, Rehovot, Israel

ABSTRACT

Retinoic acid was found to be a potent stimulant of pigmentation in human Hs939 melanomacells. Exposure to 1 @sMretinoicacid for longerthanfour dayscausedbotha decreasein the rate of cell proliferationand a concomitantincreaseinmolanogenesis. Those effects of retinoic acid progressed unearly in a time-dependent and a dose-dependent fashion suchthat at the end of a seven-day treatment cell growth wasinhibited by approximately 65%, and both melanin content andtyrosinase activity increased more than three-fold over thecontrol. Interpolation of the dose-response curves indicatedthat 3 nM netinoic acid would cause half-maximal melanogenosisstimulation.No elevationin the levelof cyclic adenosine3':5'-monophosphate could be detected in the melanoma cellsfollowing various periods of exposure to netinoic acid, and thecells were unresponsive to a-melanocyte-stimulating hormone.In the presence of the tyrosinaso inhibitor phenylthiocanbamate, retinoic acid was capableof inhibitingcell proliferationwithout enhancing melanin synthesis. The tumor promoterphorbol mynistate acetate did not affect either the proliferationor the differentiationof the Hs939 melanomacells. However,the enhancement of melanogenosis by 1 @tMrotinoic acid wasinhibited by 66% in the presence of 0.1 @tMphorbol myristateacetate.Thetumor promoterdid not reversethe gnowth-inhibitony effect of retinoic acid. Phorbol, a non-tumor promoter,was ineffective. Other retinoids, such as 13-cis-notinoic acid,retinyl acetate, and the tnimethylmothoxyphenyl analog of retinoicacid,alsoinhibitedtheproliferationandenhancedmelaninproduction in the Hs939 cells. In contrast, retmnylpalmitate, thephenyl analog of netinoic acid, and the pynidyl analog of netinoicacid were ineffective.

INTRODUCTION

In recent studies, we noticed that, when mouse 591 melanoma cells (27, 30) on human Hs939 melanoma cells (28) areexposed to rotinoic acid in culture, their growth is inhibited andtheir cellular melanin content increases over that of the untreated cells. Since melanin synthesis is a specialized functionof pigmentcells which servesas a markerof differentiationinnormal melanocytes (16, 50) and in melanoma cells (17, 24,38, 52), we considered it important to investigate, in greaten

detail, the stimulation of melanogenesis in the human Hs939melanoma cells by retinoic acid and several other rotinoids(46), putting an emphasis on elucidating the possible relation

ship between growth inhibition and stimulation of differentiation.

MATERIALS AND METHODS

Cells and Culture Techniques. The human melanoma cell

line Hs939 (derived from a metastasis to the mediastinum of a24-year-old Caucasian female) was supplied at the fifth passage by the Naval Biosciences Laboratory, School of PublicHealth, University of California, Berkeley, Calif. All tests forMycop!asma (3, 26, 47) were negative. The cells wore culturedin Dulbecco'smodifiedEagle'smedium(GrandIslandBiological Co., Grand Island, N. V.) supplemented with 10% (v/v)heat-inactivated (30 mm, 56Â°)fetal bovine serum (Flow Laboratonios,Inglewood,Calif.),nonessentialaminoacids (Eagle'sbasal medium; Grand Island Biological Co.), and 50 j@ggentamicin per ml (Schening Corp., Kenilworth, N. J.) in tissue culturedishes (Falcon Plastics, Oxnand, Calif.). Cells wore incubatedin the humidified atmosphere of 7% CO2:93% air at 37Â°.Forpassage and at the termination of each experiment, cells weredetached with 2 mM EDTA in a phosphate-buffered NaCI solution containing (pen liter H2O)8.0 g NaCI, 0.2 g KCI, 1.15 gNa2HPO4, and 0.2 g KH2PO4. Cells were counted with anelectronic particle counter (Eloctrozone/Celloscope Model112 ct; Particle Data, Inc. , Elmhurst, Ill.), and cell viability wasdetermined from the proportion of cells excluding 0. 1% trypanblue. In all the experiments, cells of passage level <1 5 (i.e.,<1 0 in our laboratory) were used.

Treatment of Cells with Retinoids, Tumor Promoter, andPTC.3 Retinoids, the generous gift of Dr. Beverly Pawson(Hoffmann-La Roche Inc., Nutloy, N. J.), were freshly dissolvedin ethyl alcohol and diluted 1:10,000 into the growth mediumimmediately before each experiment. The cultures were usuallyrefod every 3 days.

Phonbol and PMA, purchased from Consolidated MidlandCorp. (Brewster, N. V.), were stoned in ethyl alcohol undernitrogen at â€”20Â°.Immediately before each experiment, aliquots of the stock solutions were diluted into the growthmedium such that alcohol concentration would not exceed0.01 %. PTC (Sigma Chemical Co., St. Louis, Mo.) was dis

solved directly in the growth medium immediately before eachexperiment or refeeding.

All control cultures received growth medium containing the

samefinalalcoholconcentrationasthatof the treatedcultures.Ethyl alcohol concentration did not exceed 0.1 % in theseexperiments.

Assay for Growth Inhibition. At the end of each experiment,the cells were harvested with EDTA and counted. Percentageof inhibitionwas calculatedas: 100 â€”(TIC) x 100, where Tand C are the numbers of cells in treated and control cultures,respectively.

Assay for Tyrosinase Activity. The tyrosine hydroxylaseactivity of tyrosinase (O-diphonyl:O2 oxidoneductase, EC1.10.3. 1) in living cells was estimated from the amount of 3H2Oreleased into the medium during the conversion of L-[ring-3',5'-3H]tyrosine to dihydroxyphenylalanino according to the follow

, Supported by USPHS Grant CA-22823 from the National Cancer Institute.

2 To whom requests for reprints should be addressed.

Received December 17, 1979; accepted May 12, 1980.

3 The abbreviations used are: PTC, phenylthiocarbamate; PMA, phorboi-1 2-myristate-1 3-acetate; cAMP, cyclic adenosine 3' :5'-monophosphate; (I-MSH, nmelanocyte-stimulating hormone.

SEPTEMBER 1980 3345

Research. on September 5, 2021. © 1980 American Association for Cancercancerres.aacrjournals.org Downloaded from

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TreatmentaDuration (mm)cAMPb(pmol/mg protein)MelanogenesisstimulationC

(TIC)Control3015.8

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6017.5 Â±1.5

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acid (10'6 N) +301 4.0 Â±1.01 .9 Â±0.2aMSH(5 X i0-@ M)

R. Lotan and D. Lotan

ing adaptation of previous methods (36, 40): L-[3',5'-3H]tyrosine (specific activity, 56 Ci/mmol; New England Nuclear,Boston, Mass.) was dried twice from distilled H2O under anitrogen stream to remove residual 3H2Oand added to freshgrowth medium to give 0.5 @tCi/mIand a final specific activityof approximately 1 mCi/mmol. After a 24-hr incubation, themedium was removed, and the amount of 3H2Oin duplicate 1-ml medium aliquots was measured after absorption of [3H]-tyrosine onto charcoal by the method of Pomerantz (40). Allvalues were corrected by subtracting the amount of 3H2Oformed spontaneously from [3H]tyrosine in growth mediumwithout cells [this value was usually 490 Â±30 cpm (S.E.)].Enzyme activity was expressed as 3H2O formed in units ofcpm/hr/103 or 106 cells.

Determination of Relative Melanin Content. Melanin content was measured by the colorimetric method described byWhittaker (50). Cell pellets containing 5 x 106 cells placed induplicate Beckman microfuge polyethylene tubes were suspended, lysed in 0.5 ml of deionized H2O, and subjected to 2cycles of freezing and thawing. Perchloric acid was added toa final concentration of 0.5 N, and the suspension was kept onice for 10 mm and then centrifuged for 5 mm in a BeckmanModel B microfuge. The pellets were extracted twice more with0.5 N HCIO4followed by 2 extractions with a cold mixture ofethyl alcohol:ethyl ether (3:1 , v/v) and a final extraction withethyl ether. The pellets were air dried, 1 ml of 0.85 N KOH wasadded, and the pellets were dissolved by heating to 100Â°for10 mm. After insoluble residue was pelleted the supernatantwas cooled to room temperature and the absorbance at 400nm was measured in a Beckman double-beam spectrophotometer. A standard curve constructed using synthetic melanin(Sigma) dissolved in hot KOH at concentrations ranging from5 to 150 1zgper ml indicated that the absorbance at 400 nmincreases linearly with melanin concentration up to an absorbance of 1.9 (corresponding to a concentration of 115 @igmelanin per ml). The relative melanin content is expressed asthe absorbance at 400 nm per 5 x 106 cells.

cAMP Assay. Intracellularlevelsof cAMP were measuredbya competitive protein-binding assay (10) using the radioimmunoassay kit of Amersham/Searle (Arlington Heights, Ill.). Cellsexposed to various treatments (indicated in Table 1) wereprocessed as follows. The medium was removed by aspiration,and the dishes were placed on ice and rapidly washed twicewith ice-cold phosphate-buffered NaCI solution, pH 7.2. Toeach dish was then added 1 ml of 6% (w/v) ice-cold trichloroacetic acid containing 0.1 pmol of cyclic [8-3H]adenosine-3':5'-monophosphate (28 Ci/mmol) included as an internal tracerto monitor recovery of cAMP. The cell monolayers were thenscraped off the dishes and transferred to an all-glass 1.5-mIhomogenizer. An additional 0.5 ml of cold 6% trichloroaceticacid used to wash the dishes was added to the homogenizer.After homogenization by 20 strokes on ice, the suspensionwas centrifuged. The trichloroacetic acid-insoluble residue wasdissolved in 0.5 ml of 1 N NaOH, and aliquots of it were usedfor protein determination (31). The trichloroacetic acid-solublefractions containing cAMP were extracted 6 times each with 4volumes (6 ml) of H2O-saturated ether, and the residual etherwas evaporated under a nitrogen stream. The remainingaqueous phase (1.5 ml) was lyophilized, and the dried residuewas dissolved in 300 @dof the Amersham kit buffer (Tris-EDTA).Aliquots of 50 @Llwere used for determination of cAMP as

described in the Amersham kit. Phosphodiesterase treatmentof the extracted material according to the procedure describedby Giotta et a!. (11) resulted in the complete loss of competitionfor binding to the cAMP-binding protein, indicating that thecompeting material in the extract is indeed cAMP. The resultsare expressed as pmol cAMP per mg protein.

RESULTS

Effects of Retinoic Acid on Cell Proliferation and Melanogenesis. Hs939 melanomacellsexposedto 1 j@Mretinoicacidfor several days exhibit enhanced pigment production (Chart1). After 4 days in culture, increases were observed in theactivity of tyrosinase [the enzyme involved in melanin biosyn

Table 1Levels of intracellular cAMP and extent of melanogenesis stimulation in cells

exposed to retinoic acid or aMSH

a Cells were plated at 5 x 106/dish in 10-cm dishes, and after 24 hr duplicatecultures received retinoic acid, a-MSH (Calbiochem, San Diego, Calif.), or both.Control cultures received 0. 1% ethanol. The final cell density in all cultures was1.54 Â±0.1 x 10'/sqcm.

b After the indicated periods of treatment, the amount of cAMP was determined as described in@ â€˜Materialsand Methods.' â€˜A value of 385 Â±25 gigproteinper 1 x 106 cells was obtained in protein assays performed on control as well astreated cultures.

C Melanin content was measured in cultures exposed to the various treatments

for 7 days (with a medium change on Day 3). The values are the ratios of melanincontent in treated (7) and control (C) cultures. The control values were 0.31 Â±0.03 A.a,@,,/5 x 106cells.

d Average Â± SE. of duplicate cultures (each assayed in duplicate). Similar

results were obtained in 2 independent experiments.

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Chart 1. Time course of retinoic acid-induced melanogenesis stimulation.Cells were plated in 10-cm dishes at 0.2 x 106 cells/dish in control medium(0)orinmediumcontaining1 @at.iretinoicacid(â€¢).ThemediumwaschangedonDays 3 and 5. L-(3',5'-3H]Tyrosine was added to the cultures 24 hr before theend of each treatment period, and at the indicated times duplicate cultures wereremoved from the incubator, the medium was collected for determination oftyrosinase activity, the cells were harvested, and their melanin content wasdetermined. Inset, growth inhibition measured as described in â€˜â€˜MaterialsandMethods.â€˜â€˜The values are the averages of duplicate cultures. Bars, SE. Similarresults were obtained in 2 additional independent experiments.

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3346 CANCERRESEARCHVOL. 40



Enhancement of Melanogenesis by Retinoids

thesis (25, 35)] and in the melanin content of the treated cellsover the untreated cells. The retinoic acid-induced melanogenesis stimulation progressed linearly concurrently with the inhibition of cell proliferation. It should be noted that both untreated and treated cultures grew exponentially throughout the7-day incubation, although the latter had a slower proliferationrate (28). There was a positive correlation between the enhancement of tyrosinase activity and the increase in melanincontent up to Day 6. At the end of a 7-day exposure to retinoicacid, cell growth was inhibited by 65%, and both melanincontent and tyrosinase activity increased more than 3-fold overuntreated cells. The apparent discrepancy between the lack ofincrease In tyrosinase activity between Days 5 to 6 and 7 to 8and the simultaneous increase in melanin content during thistime may be due to the previously reported inhibition of tyrosinase in intensely melanized melanosomes (43).

Both inhibition of cell proliferation and stimulation of melaninproduction were dependent on the concentration of retinoicacid (Chart 2). A linear dose dependence was observed between 0.1 flM and 0.1 @LM.Interpolation of the dose-responsecurves (Chart 2) indicates that 3 ni@iretinoic acid could causea half-maximal effect after a 6-day treatment.

Lack of Changes in the Levels of cAMP in Cells Treatedwith Retlnolc Acid or a-MSH. Numerous studies have suggested a role for cAMP in the stimulation of melanogenesis inmelanoma cells exposed to aMSH (38, 52, 54), cholera toxin(37), N@,O@'-dibutyrylcyclic adenosine 3':5'-monophosphate(20), or inhibitors of cAMP phosphodiesterase (23, 24). Inaddition, cAMP has been implicated in the control of cellproliferation (1, 23). It was therefore important to determinewhether retinoic acid, which affects both growth and melanogenesis in the Hs939 cells, causes an elevation in cAMP levels.Similar to our previous observations with the murine melanoma

Bi 6 and 591 (27, 30), exposure of the human melanoma cellsto retinoic acid failed to alter the intracellular level of cAMPafter 30 or 60 mm of treatment (Table 1). Further treatment forup to 6 hr also had no effect (data not shown). The Hs939melanoma cells failed to respond to aMSH. The hormoneneither induced an elevation in cAMP levels nor stimulatedmelanin production in these cells (Table 1). It is noteworthythat the same aMSH preparation effectively augmented thelevel of cAMP and enhanced pigmentation in murine S91melanoma cells (27). For a reason which is still obscure, theconcurrent treatment of the Hs939 cells with both retinoic acidand aMSH, while not affecting cAMP levels, resulted in aconsiderably lower stimulation of melanogenesis than that observed in cultures treated with retinoic acid alone. The lack ofresponsiveness to aMSH found with the Hs939 cells is notunique. For example, a recent study revealed that only 2 of 8human melanoma cell lines were responsive to aMSH stimulation of melanin synthesis (8).

Effects of PTC on Retinoic Acid-induced Growth Inhibitionand Tyrosinase Stimulation. Actively melanizing melanomacells can produce toxic melanin precursors capable of inhibiting the growth of various cells including the melanoma cellsthat generate them (12, 14, 39). It was therefore plausible toconsider a possible relationship between the ability of retinoicacid to enhance tyrosinase activity and its ability to inhibit cellproliferation. To explore this possibility, we attempted to dissociate the growth-inhibitory effect of retinoic acid from thestimulation of tyrosinase. To this end, we used PTC, a compound which forms a weakly dissociable complex with copperand thereby inhibits the copper-requiring tyrosinase (25). PTChas been shown to inhibit tyrosinase activity in living cells andto provide protection to melanoma cells from the toxic effectsof melanin precursors (38). Initial experiments using 0.5 mMPTC, a concentration previously shown to inhibit tyrosinaseactivity in mouse S91 melanoma cells without affecting cellproliferation (38, 52), resulted in growth inhibition of the humanHs939 melanoma cells (55 Â±5% inhibition by the end of an 8-day treatment). However, after the concentration and treatmentschedule were varied, conditions were found under which 0.1mM PTC inhibited tyrosinase activity without affecting cell proliferation in otherwise untreated cells (Table 2). Under theseconditions, PTC also prevented the elevation of tyrosinaseactivity occurring between Days 3 and 5 and between Days 5and 7 in cells treated with retinoic acid (compare tyrosinaselevels in Table 2 and in Chart 1). PTC, however, did not alterthe growth-inhibitory effect of retinoic acid on the melanomacells. These results suggest that the growth-inhibitory effect ofretinoic acid in the Hs939 melanoma cells is not the indirectresult of tyrosinase stimulation.

Effects of PMA on Retinoic Acid-induced MelanogenesisStimulation. PMA and several other phorbol diesters, whichare tumor promoters in the 2-stage mouse skin carcinogenesis(2), have been found to inhibit cellular differentiation in certaincell types (7) and to stimulate or induce differentiation in othercells (18). In murine B16 melanoma cells, PMA delayed theexpression of melanogenesis without affecting cell proliferation(33). In previous studies, we found that PMA both inhibitedgrowth and suppressed melanin production in mouse S91melanoma cells (27). Furthermore, PMA inhibited retinoic acidinduced melanogenesis stimulation in S91 melanoma cells(27). In the present investigation, we exposed the Hs939

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stimulation. Cells were plated in 10-cm dishes at 0.3 x 106 cells/dish in controlmedium or in medium containing the indicated concentrations of retinoic acid.The cultures were refed with fresh medium on Day 3, and L-(3',5'-3Hjtyrosine(0.5 @iCi/ml)was added on Day 5. On Day 6, the experiment was terminated, andthe medium was removed for determination of tyrosinase activity. The cells wereharvested and counted, and their melanin content was determined. Percentageof growth inhibition was determined as described in â€˜â€˜Materialsand Methods.â€•The values are the averages of duplicate cultures. Bars, SE. Similar results wereobtained in 2 additional independent experiments.

SEPTEMBER1980 3347



Tyrosinase activity (3H20RelativemelaninTreatmentNo.

of cells (x106) on Day 8formed,

cpm/hr/ 106 cells)content (A4U),,,,,/5 x 106cells)

on Day8Day5 Day8Control6.35

Â±0,186310 Â±22 599 Â±380.19 Â±0.03PTC(10@M)'6.02Â±0.17162Â±17202Â±310.12Â±0.01All-trans-retinoic

acid (106M)2.43 Â±0.06897 Â±63 2509 Â±1850.98 Â±0.11AlI-trans-retinoicacid (106 M) +

PTC (10@ M)b2.51Â±0.13185 Â±22 210 Â±290.14 Â±0.03

Effects of PMA and phorbol on all-trans-retinoic acid-inducedmelanogenesisstimulationCells

were plated at 0.3 x 106/dish in 10-cm dishes in growth mediumaloneorsupplemented with the indicated compounds. The cultures were refed withtheappropriate

medium on Day 3, and L-(3',5'-3Hltyrosine was added on Day 5.OnDay6, the mediumwas removed for determination of tyrosinase activity; thecellswereharvested and counted; and their melanin content wasdetermined.Tyrosinase

ac- Relative melaNo. of cells/dish tivity (3H20 nmcontentafter

6 days formed, cpm/@ xTreatment(x 1O_6) hr/i 06 cells) 1 @6cells)Control

6.93 Â±0.186 464 Â±30 0.27 Â±0.OiPMA(10w M) 6.12 Â±0.06 393 Â±26 0.24 Â±0.04PMA(108 M) 6.59 Â±0.27 471 Â±15 0.28 Â±0.03PMA(10@ M) 7.1 Â±0.26 428 Â±18 0.27 Â±0.04Phorbol(i07M)

7.12Â±0.11 441 Â±45 0.26Â±0.02AII-trans-retinoicacid 3.i 5 Â±0.07 3090 Â±200 0.97 Â±0.05(106

M)AlI-trans-retinoicacid 3.31 Â±0.14 1060 Â±160 0.56 Â±0.02(10.6

M) + PMA(10@M)All-trans-retinoic

acid 3.26 Â±0.21 1970 Â±93 0.73 Â±0.09(106M) + PMA(108PA)All-trans-retinoic

acid 3.46 Â±0.15 2710 Â±244 0.92 Â±0.17(106M) + PMA(10@M)All-trans-retinoic

acid 3.08 Â±0.03 2970 Â±155 1.08 Â±0.13(106N) +phorbol(10@N)Similar

results were obtained in 2


Table 2Effect of PTC on all-trans-retinoic acid-induced growth inhibition and tyrosinase stimulation

Cells were plated in 6-cm dishes at 0.1 x 106/dish in growth medium alone or supplemented withretinoic acid. The cultures were refed on Days 3 and 5 with fresh growth medium alone (control) or withmedium containing all-trans-retinoic acid alone, PTC alone, or both compounds. On Day 4, L-(3',5'-3Hjtyrosine was added to one-half of the cultures; on Day 7, it was added to the rest of the cultures. Themedium was removed for determination of 3H2Ocontent on Days 5 and 8 from the former and the lattercultures, respectively, and the cells were detached and counted. The melanin content of cells cultured for8 days was determined.

aAverageÂ±SE. ofduplicatecultures.Similarresultswereobtainedin2 independentexperiments.b PTC was added to untreated and retinoic acid-treated cultures on Days 3 and 5.

human melanoma cells to PMA and found that it has no effecton either the proliferation or the production of melanin byotherwise untreated cells (Table 3). The unresponsiveness ofthe Hs939 cells to PMA differs from the behavior of human HOmelanoma cells in which PMA has recently been shown toinduce melanin synthesis and to inhibit cell proliferation (18).Unlike untreated Hs939 cells, the retinoic acid-treated melanoma cells were affected by PMA. In cells treated with bothretinoic acid and PMA, the tumor promoter caused a dosedependent suppression of melanin production (Table 3). However, PMA had no effect on the ability of retinoic acid to inhibitcell proliferation. Phorbol, which is inactive in tumor promotion,had no effect in this system (Table 3). These results demonstrate that, in human Hs939 melanoma, as in mouse 591melanoma cells, PMA counteracts the stimulatory effect ofretinoic acid on melanin synthesis. The 2 systems differ, however, in 2 respects: (a) PMA inhibited cell proliferation in themouse melanoma but not in the human melanoma; and (b) inthe mouse cells PMA completely inhibited melanin productionin retinoic acid-treated cultures (27), whereas in the human

melanoma cells PMA suppressed melanogenesis by no morethan 66% (Table 3).

Effects of Various Retinoids on Cell Proliferation and Melanin Production. To determine whether other vitamin A analogsshare with retinoic acid the ability to inhibit Hs939 melanomacell proliferation and/or augment melanin synthesis, the cellswere cultured for 8 days in the presence of several retinoids.Table 4 shows that 13-cis-retinoic acid was as active as wasretinoic acid, whereas retinyl acetate and the trimethylmethoxyphenyl analog of retinoic acid were less effective in bothgrowth inhibition and melanogenesis stimulation. Retinyl palmitate and the phenyl and the pyridyl analogs of retinoic acidexhibited marginal or no effect (Table 4). These results indicatethat both growth inhibition and melanogenesis stimulation impose similar structural requirements on retinoids, since noneof the tested compounds was significantly effective in augmentation of melanin synthesis if it did not also inhibit cell proliferation.

DISCUSSION

We found that the growth of human Hs939 melanoma cellsin the presence of certain retinoids results in a reduction in cellproliferation rate and a concomitant increase in melanin pro

Table 3

aAverageÂ±S.E.of duplicatecultures.independent experiments.

duction. The augmented pigmentation was first detected incells exposed to retinoic acid for 4 days. The progressiveenhancement of melanin synthesis with continued treatmentwas demonstrated by measuring both tyrosinase activity andcellular melanin content.

There are several possible mechanisms which may accountfor the enhancement of tyrosinase activity. These include: (a)stimulation or induction of de novo enzyme synthesis (9); (b)inactivation or suppression of the production of an enzymeinhibitor (22, 55); (c) activation of preexisting tyrosinase molecules (53); (d) decreased degradation rate (increased halflife) of the enzyme; and (e) increased availability of tyrosine,e.g. , due to changes in the permeability of the melanosomalmembrane. At this stage, we do not know which of the abovepossibilities is responsible for our observations; however, several recognized properties of retinoids are compatible withmost of these suggestions. For example, it has been suggested




Cells were plated in 10-cm dishes at 0.3 x 106/dish in control medium andat various initial densities from 0.3 to 0.7 x 106/dish, which were chosen inorder to allow all cultures to reach a similar final density (1.4 Â±0.1 x io@cells/sq cm) at the end of an 8-day Incubation. The cultures were refed on Days 3 and6, and the cells were harvested and counted on Day 8.Melanogene

Growth inhi- sis stimulabition6 tionC

Retinold Structure (%)(TIC)i3-cis-Retinoic

acid @â€˜@â€˜@@COOH 61 Â±6c 46 Â±0.3@-AII-trans-retinoic

@ 62 Â±5 4.5 Â±0.2Retinyl

acetate @@L,.L,..CM,OCOCH, Â±3 2.9 Â±0.3Trimethylmethoxy-

COOH 34@@ 1.8 Â±.2phenyl analog of

RARetinylpalmitate

LJL 14 Â±3 1.0 Â±0.iCOON

PhenylanalogofRA I <10 i.0 Â±0.1Pyridyl

analog of RA@ <10 i .0 Â±o.i

Enhancement of Melanogenesis by Retinoids

Table 4Effects of various retinoids on growth and melanogenesis in Hs939 melanoma

cells

without an increase in cAMP (30) suggests that retinoic acidcould activate tyrosinase via a similar pathway as aMSH byacting at a step following the activation of adenylate cyclase.It remains to be determined whether retinoic acid also activatesprotein kinase in Hs939 melanoma cells in which the responseto aMSH is abrogated.

The results also exclude nonspecific, detergent-like effectson the melanosomal membranes as the cause for enhancedtyrosinase activity (32), since retinoids such as the phenyl andthe pyridyl analogs of retinoic acid, which probably possessmembrane-labilizing capacity similar to that of retinoic acid,failed to stimulate melanin synthesis.

In the Hs939 melanoma cells, the increased melanin synthesis seems to be coordinated with the reduction in cell proliferation rate. Although by the concurrent treatment of the cellswith PTC and retinoic acid we excluded the enhanced melanogenesis as the cause of growth inhibition, we could notexclude the possibility that growth inhibition could somehowinduce melanogenesis.

The relationship between the rate of growth of melanomacells and the expression of their pigmented phenotype is quitecomplex. Some studies suggested that growth rate and differentiation are inversely related (13), whereas other investigations reported the presence of pigmented cells in all stages ofmitosis (44) and that melanin-producing cells were activelysynthesizing DNA, RNA, and protein at the same rate as werenonpigmented melanoma cells (21). Thus, there seems to beno support for the view of a fundamental antagonism betweencell division and differentiation (pigment production). Nonetheless, there are reports that melanoma cells produce moremelanin in the stationary phase than in the exponential growthphase (41 , 49). It has been proposed that this may be due tothe active synthesis of a tyrosinase inhibitor during rapid growthwhich ceases when growth in slowed (55). It should be notedthat, in the Hs939 melanoma cells, melanin content per cell incultures maintained for 3 weeks at saturation density, wherecell proliferation is greatly reduced, was no greater than onehalf of the melanin content in cells which were allowed to reachsaturation density during an 8-day exponential growth in thepresence of 1 @Mretinoic acid. Thus the stimulatory effect onmelanin synthesis in the Hs939 cells cannot be explained onthe basis of growth inhibition alone and may represent frankdifferentiation. This may be brought about through modifiedgene expression or through direct activation of the tyrosinaseas outlined above.

Previous studies demonstrated the ability of retinoids toaffect differentiation in cultures of normal epithelial (6, 45, 51)and mesenchymal (15) cells and in malignant embryonal carcinoma cells (19, 48). Our observations with the murine 591melanoma (27) and the present results with Hs939 humanmelanoma suggest that retinoids may also stimulate differentiation in melanoma cells.

ACKNOWLEDGMENTS

The major part of this work was performed while the authors were in thelaboratory of Professor Garth L. Nicolson at the University of California in Irvine,Calif. We thank Dr. Nicolson for his support and encouragement, George Neumann for assistance with the cAMP assays, and Adele Brodginski for help inmanuscript preparation.

REFERENCES

1. Abell, C. W., and Monahan, T. M. The role of adenosine 3',5'-cyclic mono

a The percentages of growth inhibition were calculated from the followingequation:

I (Fr/lr) 1I 1 â€” I x 100L (Fc/lc)j

where Fr/lr and Fc/lc are the ratios of the final (Day 8) to that initial cell numbersin retinoid-treated and control cultures, respectively.

b Values are the ratios of the relative melanin content in treated (T) andcontrol (C) cultures. The value measured in the control cultures was 0.24 Â±0.01A4@,,,,,/5 x 106 cells. Similar results were obtained in 2 independent experiments.

C Average Â± SE. of duplicate cultures. Similar results were obtained in

independent experiments where the same initial cell number (0.3 x 106/dish)was plated in both untreated and treated cultures.

that retinoids, like steroid hormones, can form a complex withspecific cellular retinoid-binding proteins [we have detected aretinoic acid-binding protein in the Hs939 cells (29)] and thatthe complex can be translocated to the cell nucleus and modifygene expression (4, 29). Such an effect could account forMechanisms a and b presented above. In addition, retinoidshave been shown to participate in the glycosylation of membrane glycoproteins (5, 6, 29). They could, therefore, increasethe glycosylation of tyrosinase which is a glycoprotein (25)localized in the melanosomal membrane (32, 42). This couldeither activate the enzyme (Mechanism c) or decrease the rateof its degradation (Mechanism d).

Elucidation of the mechanism responsible for retinoid effectson melanogenesis in the Hs939 melanoma cells will be attempted in subsequent investigations. However, we have aready excluded the possibility that retinoic acid acts like aMSH,namely, via activation of adenylate cyclase and a transientelevation in the level of cAMP (38). In fact, the Hs939 failed torespond to aMSH due to either lack of cell surface receptorsfor this hormone or the presence of a defective adenylatecyclase. Although most of the agents that enhance tyrosinaseactivity cause an increase in cAMP levels (20, 23, 24, 37, 38),there have been previous demonstrations of melanogenesisstimulation without an increase in cAMP (11, 49). The recentreport on the ability of retinoic acid to enhance cAMP-dependent protein kinase activity in Bi 6 mouse melanoma cells (34)

SEPTEMBER1980 3349




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1980;40:3345-3350. Cancer Res Reuben Lotan and Dafna Lotan by RetinoidsStimulation of Melanogenesis in a Human Melanoma Cell Line

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