Positive regulation of melanin pigmentation by two key substrates of the

melanogenic pathway, L-tyrosine and L-dopa

ANDRZEJ SLOMINSKl'1*, GISELA MOELLMANN1, ELIZABETH KUKLINSKA1,

ANDRZEJ BOMIRSKI2 and JOHN PAWELEK1

' Department of Dermatology, Yale University School of Medicine, Xetc Haven, CT 06510, USA2 Department of Biology, Gdansk Medical School, Gdansk, Poland

•Author for correspondence

Summary

We describe results demonstrating the positiveregulation of melanogenesis by two substrates ofthe melanogenic pathway. We have found thatL-tyrosine and L-dihydroxyphenylalanine(L-dopa), 'whose metabolic fates are affected bythe activity of that pathway, can also act as itsregulators. In living pigment cells, tyrosinase (EC1.14.18.1), a crucial and rate-limiting enzyme ofmelanogenesis, acts in subcellular organellesknown as melanosomes. Melanin is laid downonly in these organelles. We demonstrate thatsupplementing Ham's F-10 medium with ad-ditional L-tyrosine or L-dopa during the culture ofamelanotic Bomirski hamster melanoma cellsresults in a rapid increase in melanin formation,which is not simply due to greater availability of

substrate. There is a rapid increase in tyrosinaseactivity and a large scale synthesis of melano-somes. The effects of L-tyrosine and L-dopa areprevented by the addition of cycloheximide. Theactions of L-tyrosine and L-dopa are specificin that under similar conditions D-tyrosine,D-dopa, N-acetyl-L-tyrosine, L-phenylalanine,L-tryptophan and L-valine have little or no effect.The two substrates, L-tyrosine and L-dopa, ap-pear to act through related but distinct mechan-isms. Our findings provide an example of alittle-known phenomenon: regulation of a differ-entiated eukaryotic phenotype through positivecontrol by substrates in the pathway.

Key words: melanogenesis, tyrosine, dopa, melanoma, cellculture.

Introduction

The biosynthesis of melanin is initiated by the enzy-matic oxidation of L-tyrosine to L-dihydroxy-phenylalanine (L-dopa) by tyrosinase (monophenoldihydroxyphenylalanine: oxygen oxidoreductase EC1.14.18.1) in a reaction that uses L-dopa as a cofactor(Lerner et al. 1949). In the animal kingdom, thisprocess takes place in subcellular organelles, the mela-nosomes (Seiji, 1967). In mice, control of melano-genesis is known to be a highly complex processinvolving precise regulation of more than 50 geneticloci that influence both foetal and neonatal develop-ment of the pigmentary system as well as variousaspects of melanogenesis in the mature animal (Silvers,1979).

Journal of Cell Science 89, 287-296 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

It has been known for almost two decades that ifmelanoma cells are cultured in media relatively low intyrosine (e.g. 1 X 1 0 ~ 5 M ) , increasing the concentrationof tyrosine causes increased production of melanin(Ulrich et al. 1968). It has been assumed that thiseffect occurred because tyrosine was limiting as asubstrate. We supply here a full description of ourobservation, published previously in the form of anabstract (Slominski et al. 1987) that with certainhamster melanoma lines L-tyrosine and L-dopa eachhave regulatory roles in melanogenesis in addition totheir known functions as substrate and cofactor. Theimportance of this finding as a general regulatory modelin pigment cell biology has been confirmed by twoindependent reports in which it was shown thatL-tyrosine in human melanomas stimulates tyrosinase

287

activity and that L-dopa regulates the type of melano-gcnesis, either eu- or pheomelanogenesis, in a mousemelanoma (McEwan & Parsons, 1987; Sato et al.1987).

In an attempt to develop a suitable experimentalmodel for studying this process, we have used Bomirskihamster melanoma lines (Bomirski, 1977; Bomirski etal. 1987). One of them, Bomirski Ab amelanoticmelanoma, shows a unique property: it remains highlymalignant and anaplastic in vivo but expresses thedifferentiated melanocytic phenotype in cell culture(Stominski, 1983, 1985; Slominski et al. 1983;Bomirski & Slominski, 1986). In this paper we describethat L-tyrosine, when added to the culture medium, ofamelanotic Bomirski hamster melanoma cells, caused adramatic rise in tyrosinase activity and melanin con-tent, accompanied by the appearance of large numbersof melanosomes. L-dopa had similar effects but ap-peared to act through a pathway different from that ofL-tyrosine.

Materials and methods

Cell lines

The hamster melanoma cells used in this study were derivedfrom Bomirski Ma melanotic, MI hypomelanotic, and Abamelanotic melanomas (Bomirski, 1977; Bomirski et al.1987). In addition, a subline of the Ab amelanotic tumourline, designated AbCl, was selected through cloning in softagar. Cloudman mouse melanoma lines were PSl-wt and asubline derived from PSl-wt, designated M1B, which wasselected for its ability to grow under clonal conditions in thepresence of melanotropin (Pawelek et al. 1975).

Culture conditionsCells were cultured in Corning Tissue Culture Flasks in anatmosphere of 5 % CO2 in air, at a humidity of 80% and atemperature of 37°C (Pawelek, 1978). The culture mediumwas Ham's F-10 (Gibco Labs) containing horse serum (10%)streptomycin ( l l ^ g m l " ' ) , and penicillin (lOOumtsml"1).Cells (1SX 10°) were inoculated into 25-cm2 culture flasks in5 ml medium to initiate the experiments. Where noted,primary cultures were started from solid tumours with cellsderived by a non-enzymatic method (Stominski, 1983). Theseparate experimental cultures were performed over a periodof 2 years (1985 and 1986).

Tymsinase activityMelanoma cells were cultured as described above. Followingthe various experimental treatments, they were harvested inTyrode's balanced saline solution containing EDTA (1 mM),and pelleted by centrifugation. The pellets were then lysed insodium phosphate buffer (01 M, pH68) containing TritonX-100 (0-5%) or stored at -70°C and lysed later. Resultswere the same whether or not the cells had been frozen. Boththe tyrosine hydroxylase (conversion of tyrosine to dopa) anddopa o.xidase (conversion of dopa to dopa quinone) activities

of the enzyme were measured as described previously(Pawelek, 1978). Dopa oxidase was measured spectrophoto-metrically at 475 nm, with 1 mM-L-dopa (Hoffmann-LaRoche) as substrate in 0 1 M-phosphate buffer, p H 6 8 .Results are expressed as nmoles dopachrome produced by1 X106 cells h"1. Tyrosine hydroxylase activity was measuredas the amount of [3H]water released during the conversion ofL-[3H]tyrosine to L-dopa, with lS^Ciml"1 of [3H]-3-,5-L-tyrosine (55-7 Cimmole"1, New England Nuclear Corpor-ation) as substrate and 0 1 mM-L-dopa as cofactor in 0 1 M-phosphate buffer, pH 6-8. Results are presented as ctsmin"'from [3H]water formed by IX 106cellsh~'.

Acid phosphatase and fi-glucuronidase activitiesLysates of melanoma cells were prepared as described abovewith the exception that sodium acetate buffer (50mM,pH 5-0) was used in place of sodium phosphate buffer. Therewas no difference in the results whether or not the cells hadbeen frozen prior to lysis. Lysates were assayed for acidphosphatase activity according to the method of Trouet(1974) by measuring the formation of inorganic phosphatefrom jS-glycerophosphate (Sigma Chemicals). /3-Glucuroni-dase activity was assayed according to the method of Stahl &Touster (1972) by measuring the formation of phenol-phthalein from phenolphtalein glucuronide (Sigma Chemi-cals).

Electron microscopy

Cultures were fixed in situ at room temperature in a mixtureof glutaraldehyde (2-5%) plus formaldehyde (2%) (Kar-novsky, 1965), buffered to pH7-2 with sodium cacodylatebuffer ( 0 1 M ) . Immediately, while in the fixative solution,the cells were scraped off the culture dish with a rubberpoliceman and pelleted by centrifugation. The cells wereexposed to fixative for a total of 30 min. The pellets were thenwashed in sodium cacodylate buffer ( 0 2 M , pH 72) andimmersed for 1 h at room temperature in the same buffercontaining a mixture of osmium tetroxide (1 %) and potass-ium ferrocyanide (15%) (Karnovsky, 1971). They werefurther immersed in an aqueous solution of uranyl acetate(05%) for 10min. Finally, the pellets were dehydrated inethanol and embedded in Spurr's epoxy mixture (Spurr,1969). Ultrathin sections were cut on a diamond knife andcounterstained with lead citrate (Reynolds, 1963).

Cytochenucal assays for dopa oxidase and acid

phosphatase activities

For ultrastructural localization of dopa oxidase activity,cultures were fixed in situ, scraped from the culture flasks,and pelleted as described above. After 30 min in fixative, thepellets were washed extensively with sodium cacodylatebuffer ( 0 1 M , pH7-2) and incubated at 4°C overnight(approx. 15 h) in that buffer alone (controls), or in buffercontaining L-dopa (5 mM). The solutions were discarded andfresh sodium cacodylate buffer and L-dopa were added for anadditional 5 h at 37°C. The reacted pellets were washed withsodium cacodylate buffer without L-dopa and prepared forelectron microscopy as described above.

For ultrastructural localization of acid phosphatase ac-tivity, the cells were fixed for 30 min at 4°C in a dilutedmixture of glutaraldehyde (1-25 %) and formaldehyde (1 %) .

288 A. Slominski et al.

J0 10 20 30 40 50Time in culture (h)

Fig. 1. Kinetics of stimulation of tyrosinase activity by L-tyrosine and L-dopa. Cells of clone AbCl, originating fromBomirski's amelanotic hamster melanoma, were grown asmonolayer cultures in Ham's F-10 medium with thefollowing additions: ( • ) none (control); ( • ) L-tyrosine(200HM); (O) L-dopa (50jtM). At the times indicated, cellswere harvested and lysed and the cell extracts were used toassay tyrosinase activity. A, tyrosine hydroxylase activityexpressed as production (ctsmin"1) of [3H]water from[3H]tyrosine; B, dopa oxidase activity expressed asproduction (nmoles) of dopachrome from L-dopa. Theresults represent a mean from three separate cellcultures ± S.E.M.

After centrifugation, the cell pellets were washed two timeswith sodium cacodylate buffer (0-2M, pH7-2), followed bysodium acetate buffer (0-1 M, pH5). The cells were incu-bated for 30 min at 37°C in Gomori's medium as modified byDejong el al. (1979). The substrate was /J-glycerophosphateand the capture reagent lead nitrate. After incubation, thepellets were washed consecutively in acetate and cacodylatebuffers, fixed as above in osmium tetroxide/potassium ferro-cyanide, and processed for electron microscopy.

Histocliemical assay for dopa oxidase activity inmonolayer cultures

Cultures were fixed in silu as described above. They werethen rinsed three times with sodium cacodylate buffer (01 M,pH 7-2) and stored overnight as above with or without L-dopa(5mM). The cultures were incubated an additional 3 h at37°C in fresh L-dopa solution. After incubation the sampleswere washed with sodium cacodylate buffer, immersed 1 h in

J=

oles

Ec

cell

8.

1800

1200

600

A / \

• / \

• J \

r *i i • i i

a i =

Q

c(A

5=

8 240

180

120

- B ^ < * v

• /

/

1 1 i 1 1

u0 200 400 600"L-Tyrosine concentration (/JM)

Fig. 2. Stimulation of tyrosinase activity as a function ofconcentration of L-tyrosine in the culture medium. Cellsfrom three variants of the Bomirski hamster melanomawere grown as monolayer cultures for two days in Ham'sF-10 medium in the presence of different concentrations ofL-tyrosine. The cells were harvested and lysed and theextracts were used to assay tyrosinase activity as tyrosinehydroxylase activity (O) expressed as production(ctsmin"') [3H]\vater from [3H]tyrosine, or dopa oxidaseactivity expressed as production (nmoles) of dopachromefrom L-dopa ( • ) . A. MI hypomelanotic melanoma; B, Mamelanotic melanoma; C, Ab amelanotic melanoma. Theresults were taken from representative experiments done induplicate .for each line tested.

the osmium tetroxide/potassium ferrocyanide mixture, dehy-drated in ethanol and embedded ;;/ situ in a thin layer ofEpon.

Results

Effects of L-tyrosine and L-dopa on tyrosinase activityand melanin content

Each amino acid caused a marked stimulation of bothtyrosine hydroxylase (Fig. 1A) and dopa oxidase(Fig. IB) activities, but the kinetics of stimulation atoptimal concentrations (Figs 2, 3) differed. L-dopaacted rapidly, its effect being readily detectable within3h and reaching a plateau by 24 h. The effects ofL-tyrosine appeared more gradually and had notreached a plateau when the experiments were termi-nated at 48 h. Since L-dopa is unstable in culturemedium, the plateau at 24 h could have been due tonon-enzymatic oxidative degradation of the substrate;however, this is unlikely since the media were changed

Regulation of melanin pigmentation 289

0 20 40 60 80 100Dopa concentration (//M)

Fig. 3. Stimulation of tyrosinase activity as a function ofconcentration of dopa in the culture medium. Cells ofAbCl amelanotic melanoma were grown as monolayercultures for 24 h in Ham's F-10 medium in the presence ofdifferent concentrations of L-dopa (O) or D-dopa (A). Thecells were harvested and lysed and the extracts were usedto assay tyrosinase activity as tyrosine hydroxylase activity(A) or dopa oxidase activity (B). The results represent amean from three separate cell cultures ± S.E.M.

after 24 h and fresh L-tyrosine and L-dopa were addedat this point.

The stimulatory effects of L-tyrosine and L-dopawere dose-dependent (Figs 2, 3). For three separateBomirski hamster melanoma cell lines, the stimulationof dopa oxidase activity (Fig. 2A-C), and in one case

tyrosine hydroxylase activity (Fig. 2C), was optimal atan L-tyrosine concentration of approximately 200/AM.With the two cell lines of highest basal tyrosinaseactivity concentrations higher than 200fiM-L-tyrosineresulted in reduced stimulation of tyrosinase activity(Fig. 2A,B). The effects of L-dopa were maximal atapproximately 50/IM (Fig. 3A,B) and were readilydetected at a concentration as low as 5jtiM.

D-dopa was far less effective than L-dopa. Thespecificity of stimulation was investigated further(Table 1). The effects of L-dopa and L-tyrosine ontyrosinase activity were highly specific in that theirD-stereoisomers, as well as L-phenylalanine, L-tryp-tophan, L-valine and N-acetyl-L-tyrosine had little orno stimulator^' activity (Fig. 2, Table 1).

The stimulatory effects of L-tyrosine and L-dopawere dependent on protein synthesis since concomitantaddition of cycloheximide (10/UM) to the culture me-dium prevented the increases of tyrosinase activity(Table 2). This concentration of cycloheximide in-hibits general protein synthesis by more than 90 %(Wong & Pawelek, 1973). The tyrosinase activity ofcells cultured in the presence of cycloheximide only,measured at 24h, did not differ significantly from theuntreated cells. Cultures of 24 h in the presence ofcycloheximide only was done twice with a mean dopaoxidase activity of 2-0nmolesh~' per lXlO6 cells.

Increased tyrosinase activity was reflected inincreased melanin production by the cells (Fig. 4,Table 1). L-tyrosine was always more stimulatory thanL-dopa (Fig. 4). Stimulation of melanogenesis could beseen in cell pellets (Fig. 4, Table 1) and in individualcells growing in monolayer (Fig. 5A,B). Stimulation of

Table 1. Tyrosinase activity and melanization in primary cell cultures of the Bomirski Ab amelanoticmelanoma

Day ofculture

0

2

Addition tomedium

(;IM)

noneL-tryptophan (50)L-phenylalaninc (200)L-valinc (390)D-tyrosinc (200)A'-acetyl-L-tyrosinc (200)L-tyrosinc (200)

Dopa oxidaseactivity

(nmolesh"' per1 X 10* cells)

3-3 ±0-5

3-3 ±0-62-8 ±0-33-1 ±0-22-8 ±0-33-3 ±0-32-8

47-5 ±3-9

Tyrosinehydroxylasc

activity(ctsmin"' h~' per

IX 10° cells)

2808 ±387

4008±5192 433 ±5254383 ±13715 088 ±4713 084 ±3213600

36 642 ± 10101

Colour ofcell

pellets

White

WhiteWhiteWhiteWhiteWhiteWhiteBlack

The cells were grown for 2 days in F10 medium with the different ammo acids added at the concentrations indicated. The cells wereharvested and lyscd, and the extracts were used to assay tyrosine hydroxylase as production (ctsmin"') of [3H]\vater from [""HJtyrosinc,and dopa oxidase as production of nmoles of dopachromc from L-dopa during 1 h of incubation. The data represent the mean from at leastthree separate primary cell cultures ± S.E.M. or two cultures in the case of .V-acetyl-L-tyrosinc.

290 A. Slominski et al.

Table 2. The effect of cycloheximide on tymsmase stimulation in the AbCl clone of the Boinirski amelanoticmelanoma

Time inculture

(n)

6

12

24

Addition to medium (fim)

NoneL-dopa (50)L-dopa (50) + cycloheximide (10)

NoneL-dopa (50)L-dopa (50) + cycloheximide (10)

NoneL-dopa (50)L-dopa (50) + cvcloheximidc (10)L-tyrosine (200)L-tyrosine (200) + cycloheximide (10)

Dopa oxidaseactivity

(nmoles h~' per1X106 cells)

l-8± 0-510-313-03-3 ±0-2

2-5 ±0-718-811-04-210-5

2-210-335-5 1 12-23-81 1-4

14-71 1-04-110-5

Tvrosinchydroxylasc

activity(ctsmin"' h~' per

IX 10° cells)

117125096713 896

114638041216746

ND96 38710 57563 91215 063

AbCl cells were grown for different time periods in L-dopa, or L-tyrosine, 1 cycloheximide. The cells were harvested and lyscd, and theextracts were used to assay tyrosine hydroxylase as production (ctsmin"') of [3H]\vater from [3H]tyrosine, and dopa oxidase activity asproduction of nmoles of dopachromc from L-dopa during 1 h of incubation. The data for dopa oxidase represent the mean from threeseparate cell cultures 1 S.E.M. The data for tvrosinc hydroxylase represent the results from two separate cell cultures with a variation of lessthan 1 1 5 % . ND, not done.

Fig. 4. Relative cellular melanin content as a function ofL-tyrosine or L-dopa. Cells of clone A b C l , originating fromBomirski's amelanotic hamster melanoma, were grown asmonolayer cultures for 2 days in Ham's F-10 medium withthe following additions: A, none (control); B, L-tryptophan(200fiM); C, L-dopa (50ftM); D, L-tyrosine (200 jUM). Thecultures were then harvested and centnfuged into pellets.

dopa oxidase activity by L-tyrosine was detected histo-chemically by incubating aldehyde-fixed cultures withL-dopa (Fig. 5C,D).

The effects of L-tyrosine on tyrosinase activity shownabove for the Bomirski hamster melanomas were notseen in Cloudman S91 mouse melanoma cell lines(Table 3). However, L-tyrosine (180jUM) stimulatedmelanin production in Cloudman cells. Higher concen-trations of L-tyrosine (360 /IM) also stimulated melaninproduction but caused a 30-50% reduction of tyrosin-ase activity. The increase of melanin production byCloudman melanoma cells was estimated macroscopi-cally on the basis of the colour of the cell pellet.

Table 3. Dopa oxidase activity in three Cloudmanmelanoma cell lines

Melanomaline

M1B

PSl-wt

Original line

Primary cellculture of PSl-wt

Addition ofL-tyrosinc tomedium (fiM)

None180

None180

None360

None360

Dopa oxidaseactivity

(nmoles h ' perIX 10" cells)

3-32 1

4-03-4

29-422-4

104-255-6

Cloudman melanoma cells were cultured for 2 days with orwithout addition of L-tyrosine. Lines M1B and PSl-wt were clonallines maintained for several generations in monolayer culture inour laboratory. The primary cell culture of PSl-wt was adapted tomonolayer culture from a PSl-wt tumour in a DBA/2J mouse afew days prior to its use in this experiment. Original line refers toa line of Cloudman S91 melanoma cells purchased from theAmerican Type Culture Collection. Cells from the original linewere not purified by cloning in our laboratory. The cells wereharvested and lysed, and the extracts were used to assay dopaoxidase activity as production of nmoles dopachrome from L-dopaduring 1 h of incubation. The data represent the mean from 2—3separate cell cultures. The variation for each point was less than115%.

Regulation of melanin pigmentation 291

:*•

5A * G

Fig. S. Cytological aspects of the stimulation of melanogenesis by L-tyrosine. Cells were isolated from a solid Abaniclanotic tumour and grown for 2 days as primary monolayer cultures in Ham's F-10 medium with the followingadditions: A,C, none (control); B,D, L-tyrosine (200 ;/M). The cells were fixed /;; situ as for electron microscopy,dehydrated, and embedded in Epon. A,B. No further staining. C,D. The aldehyde-fixed cultures were stainedhistochemically for dopa oxidase activity before treatment with osmium tetroxide (see Materials and methods). X275.

Effects of L-tymsine and L-dopa on the fonnation ofmelanin granules

We examined the Ab amelanotic line with the electronmicroscope for ultrastructural differences during pri-mary cell culture in the presence or absence ofL-tyrosine or L-dopa. In plain Ham's F-10 medium,when the cells were completely amelanotic and tyrosin-ase activity was low, there was a general absenceof identifiable melanosomes and premelanosomes(Fig. 6A). After having been cultured in the presenceof L-tyrosine, however, when the cells were highlymelanized and had high tyrosinase activity, the cyto-plasm was packed with melanized, granular melano-somes. The melanosomes were lysosome-like, irregularin shape, with a homogeneous or finely granular matrixof medium electron-opacity plus disorderly material ofhigh electron-opacity resembling melanin (Fig. 6B).The granules are identical in appearance to those in thehighly melanized parental Bomirski Ma melanoma(Bomirski, 1977; Bomirski et al. 1973). In cells cul-tured in the presence of L-dopa, a few melanosomes

were present in some cells (not illustrated). These weremorphologically similar to the ones synthesized in thepresence of L-tyrosine, but they contained less melanin.Unmelanized granules with a finely granular matrixwere seen near the cell centre (Fig. 6C). These arepresumed to be atypical premelanosomes, precursorsto granular melanosomes.

Subcellular distribution of dopa oxidase activityUltrastructural localizations of the dopa oxidase reac-tion revealed striking differences in the abundance anddistribution of enzyme activity under the three con-ditions of cell culture. In cells cultured in F-10 mediumthe dopa reaction product was abundant and localizedin smooth-surfaced cisternae and related vesicles on thetrans side of the Golgi complex (Fig. 6D). In cellscultured in the presence of L-tyrosine, the distributionof the dopa reaction had shifted from the above sites tomelanosomes, i.e. dopa reaction product was scarce inthe trans-Golgi vesicles and cisternae and was localizedpredominantly in melanosomes (Fig. 6E).

292 A. Slominski et al.

Fig. 6. Ultrastructural aspects of the regulation of themelanogenic pathway for L-tyrosine and L-dopa. Cells wereisolated from solid tumours and grown as primary monolayersin Hani's F-10 medium for 2 days with the followingadditions: A,D, none (control); B,E, L-tyrosine (200|<M);C,F,G, L-dopa (50^(M). The cells were fixed in situ withaldehyde, scraped off the plastic surface and centrifuged intopellets. A-C. No dopa-oxidase histochemistry wasperformed. D-G. Before treatment with osmium tetroxide,the aldehyde-fixed cell pellets were stained histochemically fordopa oxidase activity (see Materials and methods). C, Golgicomplex; large asterisks, melanosomes; small asterisks,presumptive premelanosomes stage 11; arrowheads, dopa-oxidase positive vesicles; arrows, dopa-oxidase positive trans-Golgi cisternae. Bar,

*&<{••••5 ? •••*'<* \^

Regulation of melanin pigmentation 293

Table 4. Acid phosplialase and fi-glucuronidaseactivities in primary cell cultures of the Bomirski Ab

amelanotic melanoma

Day ofculture

0

1

2

Additionto

medium

None

NoneTyrosine

NoneTvrosinc

Acidphosphatase

(nmolch ' per1X106 cells)

60-5 ±7-0

52-0 ±2-353-3 ±10-0

51-1 ±6-150-2 ±9-5

/5-glucuronidase(nmolch""1 per

1 X 106 cells)

6-8 ± 1-3

9-0 ± 1-77-5 ± 1-2

8-3 ±1-47-S± 1-5

The cells were harvested and lyscd, and the extracts were usedto assay acid phosphatase as the formation of nmoles inorganic acidfrom /3-glyccrophusphatc, and /?-glucuronidase as the formation ofnmoles phcnolphthalcin from phcnolphthalcin glucuronide. Thepoints represent the mean from 3—5 experiments ± S.E.M. Tyrosinewas added to a final concentration of 200/(M.

Reaction product was most abundant in cells thathad been cultured in the presence of L-dopa. It waslocalized predominantly at the same sites as in cellsincubated in plain F-10 medium, but these sites weregreatly expanded (Fig. 6F), sometimes to the point ofbeing grotesquely swollen (Fig. 6G). Some reactionproduct also appeared in melanosomes, but to a lesserextent than when the cells were cultured withL-tyrosine.

Lysosomal marker enzyme activities andlocalizationsBecause the newly synthesized pigment granules wereof lysosome-like morphology, we performed biochemi-cal and cytochemical determinations of acid phospha-tase and /J-glucuronidase activities. Quantitativelythere were no differences in the activities of eitherenzyme, whether or not the cells had been culturedwith additional L-tyrosine (Table 4). Ultrastructurallocalizations of acid phosphatase, however, revealedthat following the addition of L-tyrosine to the culturemedium, the enzyme had shifted from smooth-sur-faced cisternae and vesicles (Fig. 7A) to the newlyformed pigment granules (Fig. 7B).

Discussion

We have demonstrated positive regulation of melano-genesis by two substrates in the melanogenic pathway,L-tyrosine and L-dopa. The regulation is specific (seeTable 1, Fig. 3) and highly complex, involving syn-thesis of melanosomes (see Fig. 6), increased activity oftyrosinase (see Figs 1-3), and changes in the subcellu-lar distribution of tyrosinase and acid phosphatase (seeFigs6D-G, 7). Ultrastructural findings suggest that

L-dopa primarily increased tyrosinase activity, whereasL-tyrosine stimulated melanosome synthesis, increasedtyrosinase activity and changed the subcellular distri-bution of the enzyme (see Fig. 6A-E). Even thoughthe two amino acids differed in the mechanism bywhich they exerted their effects on melanogenesis, theyboth required active protein synthesis in order to beeffective (see Table 2).

L-tyrosine and L-dopa are consecutive substrates forthe same multifunctional enzyme, tyrosinase (Korner& Pawelek, 1982). Consequently, exogenous additionof L-tyrosine to cultured pigment cells was likely tohave resulted in the generation of some L-dopa. There-fore, the effects of tyrosine on tyrosinase could havebeen due to L-dopa that was generated from L-tyrosine.Consistent with this possibility is the kinetic analysisshown in Fig. 1. Exogenous L-dopa increased tyrosin-ase activity more rapidly than exogenous L-tyrosine,even though the concentration was only 25 % of that ofL-tyrosine. The lag in the tyrosine effect could havebeen due to a prerequisite generation of L-dopa.

Bomirski Ab amelanotic melanoma cells, whengrown as tumours in hamsters, are completely amela-notic and have no melanosomes (Bomirski et al. 1973).When transferred to monolayer culture in Eagle'sMinimal Essential Medium there is a rapid appearanceof melanosomes and increases in tyrosinase activity andmelanin content (Slominski et al. 1983; Slominski,1985; Bomirski & Slominski, 1986). These events areprevented by actinomycin D and cycloheximide (Slo-minski et al. 1984). The tyrosine concentration inEagle's medium is approximately 200 f.iM, whereas inHam's F-10 medium, used in the experiments pre-sented here, the tyrosine concentration is lOjtM. Ourresults make it likely that L-tyrosine is the factor inEagle's medium that caused the differentiation ofBomirski Ab amelanotic cells into active, pigment-producing cells.

The effect of L-tyrosine on tyrosinase described forBomirski hamster melanoma cells did not occur in theCloudman mouse melanoma lines that we tested(Table 3). These lines may serve as an example ofanother type of regulation of melanogenesis in whichL-tyrosine serves only as a substrate of the melanogenicpathway.

Studies have been reported from other laboratoriesthat potentially implicate tyrosine (Aubert et al. 1985),and in one case tryptophan (Chakraborty et al. 1986),as regulators of melanogenesis in melanoma cells.However, we believe that McEwan & Parsons (1987)and we are the first to document a regulatory effect ofindividual melanin precursors on tyrosinase in vivoand, especially, melanosome formation. It will be ofvalue to see whether these effects apply to otherpigmentary systems or whether they are restricted toparticular melanomas. These studies taken together

294 A. Slominski et al.

Fig. 7. Subcellular localization of acidphosphatase as a function of L-tyrosine.Cells were isolated from solid Ab tumoursand grown as primary monolayer cultures inHam's F-10 medium for 2 days with thefollowing additions: A, none (control);B, L-tyrosine (200 /<M). The cells were fixedin situ with aldehyde, scraped off the plasticsurface, centrifuged into pellets and acidphosphatase histochemistry was performed.Arrows, acid phosphatase positive cisternaeor vesicles; large asterisks, acid phosphatasepositive melanosomes. Bar, 0-5//in.

show that precursors of the melanogenic pathway canbe positive regulators of the subcellular apparatus ofmelanogenesis in living melanocytes. Another potentialexample of similar regulation also involves L-tyrosine,which serves as a substrate and possible regulator forcatecholamine biosynthesis (Conlay el al. 1985; VVurt-manel al. 1981).

In conclusion, we suggest that the tyrosine and dopaeffects can serve as excellent models for the study of themechanism of positive regulation of mammalian differ-entiated pathways by substrates of those pathways.

We are indebted to Dr A. B. Lerner for support. This workwas financed by the Lawrence M. Gelb Foundation (Clairol)and by USPHS grants 5 R01 CA04679 and 2 T32 AM07016.

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(Received 25 September 1987 - Accepted17 Sovember 1987)

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