[CANCER RESEARCH 44,1128-1134, March 1984]

Characterization of Melanogenesis and Morphogenesis of Melanosomes by

Physicochemical Properties of Melanin and Melanosomes inMalignant Melanoma1

Kowichi Jimbow,2 Yasuhiro Miyake, Koichi Homma, Katsunari Yasuda, Yoshinobu Izumi, Akihiro Tsutsumi, and

Shosuke Ito

Department of Dermatology, Sapporo Medical College, Sapporo [K. J., K. H.]; Department of Polymer Science, Faculty of Science, Hokkaido University, Sapporo [Y. M.,K. Y., Y. I., A. T.]; and Institute for Comprehensive Medical Science, School of Medicine, Fujita-Gakuen University, Toyoake [S. I.], Japan

ABSTRACT

This study elucidates the nature of melanogenesis in B16 andHarding-Passey (HP) mouse melanomas producing melanin andmelanosomes of different color and fine structure, i.e., brown-black eumelanosome-like B16 granules and reddish brown pheo-melanosome-like HP granules, and compares them with "typical"

3,4-dihydroxyphenylalanine (DOPA) and sepia eumelanins and

sepia eumelanosomes. The melanin content of B16 melanosomes was more than three times higher than that of HPmelanosomes. The content of free and protein-bound DOPA and5-S-cysteinyldopa varied greatly in B16, HP, and sepia melano

somes and was unrelated to melanin content. Chemical analysisof the eumelanin:pheomelanin ratio in melanosomes and elemental analysis of isolated melanin showed that B16 and HP melaninsare primarily eumelanic, with a higher ratio of pheomelanic component in HP melanin. The spectra of electron spin resonanceand IR and X-ray small-angle scattering of B16 and HP melanins

were basically similar to those of sepia and DOPA melanins.B16, HP, and DOPA melanins were dissolved in aqueous NH3,while sepia melanin was dissolved to a far lesser extent. It wasconcluded that both B16 and HP melanomas are primarily involved in eumelanogenesis, although the fine structure of theirmelanosomes is entirely different, and that the marked colordifference in the two melanosomes is related to a difference inthe absolute content of eumelanin, the presence of a smallamount of pheomelanin, and the mode of chemical bindings ofmelanin to structural proteins. In contrast to normal skin andhair, melanosome morphogenesis may not directly correspondto melanogenesis type in malignant melanoma.

INTRODUCTION

Because of its unique biological property, melanogenesis, I.e.,synthesis of melanin within melanosomes, has been used eitherexperimentally or clinically to provide rational approaches forlaboratory diagnosis and treatment of malignant melanoma. Melanin pigments are classified into 2 major types, i.e., brown-blackeumelanin pigment composed of indole units derived from DOPA3

and yellow to reddish brown pheomelanin pigment composed of

1Supported by Grants-in-Aid No. 58010030, No. 58390016, and No. 58480265from the Ministry of Education, Science, and Culture, and No. 58-42 from theMinistry of Health and Welfare, Japan, by the Japan O'Learly Foundation, the

Kurata Foundation, and the Alfred-Marchionini Foundation.2To whom requests for reprints should be addressed, at Department of Der

matology, Sapporo Medical College, Minami 1, Nishi 16, Sapporo 060, Japan.3The abbrevations used are: DOPA, 3,4-dihydroxyphenylalanine; HP, Harding-

Passey; ESR, electron spin resonance; AHP, aminohydroxy phenylalanine; HPLC,high-performance liquid chromatography.

Received July 7,1983; accepted November 23,1983.

benzothiazine units derived from cysteinyldopas (34). In normalskin, 2 types of melanin synthesis strictly correspond to the finestructure of melanosomes, i.e., ellipsoidal-lamellar (filamentous)eumelanosomes and spherical-granular (microvesicular) pheo-

melanosomes (21, 31 ). In malignant melanoma, the fine structureof melanosomes can also be grouped into these 2 basic forms(9, 12). It is, however, unknown which types of melanin pigmentare synthesized in the 2 morphologically different melanosomesand to what extent the fine structure of melanosomes reflectsthe types of melanin pigment within melanosomes.

B16 and HP mouse melanomas are useful animal models forclarifying the relation between melanosomes morphogenesis andmelanogenesis in malignant melanoma (20, 23), inasmuch asB16 melanosomes are ellipsoidal-lamellar and brown-black, whileHP melanosomes are spherical-granular and reddish brown. This

study characterizes the nature of melanogenesis in B16 and HPmelanomas by comparing the contents of melanin, DOPA, and5-S-cysteinyldopa within melanosomes, and the chemical com

position and solubility as well as spectrum patterns of IR, ESR,and X-ray small-angle scattering of melanin with those of sepia

melanosomes, and sepia and DOPA melanins, which are knownas "typical" eumelanosomes and eumelanins. Elemental analysis

and solubility tests of melanin were carried out because, atpresent, the major criteria for differentiation of pheomelanin fromeumelanin are high sulfur content and solubility in dilute alkali(28, 37, 38). The content of eumelanin and pheomelanin withinmelanosomes was also estimated by our recently developedtechnique after chemical degradation (16).

MATERIALS AND METHODS

Preparation of Melanosomes

B16 and HP melanomas are grown in C57BL/6 and ICR mice, respectively. The details of melanoma preparation and melanosome isolationfollowed our previously described methods with a minor modification (19,39). Isolated melanosomes were treated with Brij-35 in 4 MIMphosphate

buffer in an ACE tissue homogenizer (Nihon Seiki Kaisha, Ltd., Japan)at 15,000 rpm for 3 min in ice and then underwent sucrose densitygradient ultracentrifugation (1.0 to 2.0 M sucrose) to remove externalcontaminants as reported previously (23). The purity of melanosomeswas checked under electron microscopy after fixing melanosome pelletswith 1% osmium tetroxide (20).

Preparation of Melanin

The purified melanosomes were washed extensively with distilledwater and were lyophilized. Then the lyophilized melanosomes werehydrolyzed to extract melanin by heating with 6 N HCI under reflux forabout 30 hr. Sepia melanosomes were collected from the ink sac of a

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Melanogenesis in Melanoma

cephalopod: Sepia (Acanthosepion) sebaculecta Sasaki kindly suppliedby Dr. Y. Fujinuma (Shiseido Cosmetic Co., Yokohama, Japan), and their

melanin was extracted by a method similar to that for B16 and HPmelanosomes. DOPA melanin was prepared from L-dopa by autoxidation

in phosphate buffer (pH 8.1) by passing oxygen into a DOPA solution forabout 50 hr at room temperature, after which it was washed repeatedlywith diluted HCI solution and water, dialyzed, and lyophilized (30).

Analytical Methods

Content of Catechols in Melanosomes. The contents of free andprotein-bound forms of DOPA and 5^S-cysteinyldopa in B16, HP, and

sepia melanosomes were analyzed by HPLC following our methoddescribed previously (14). Briefly, lyophilized melanosomes were homogenized in 0.4 M HCIO4 solution and were centrifuged at 10,000 x g for 5min. The supernatant was analyzed for free DOPA and 5-S-cysteinyldopa

by direct injection into HPLC. The precipitate was heated under reflux in10 ml of 6 N HCI containing 50 mg of cysteamine hydrochloride for 24hr. Insoluble black melanin was collected by centrifugation, washedsuccessively with 0.1 N HCI (twice) and acetone (twice), and dried(Method A). Alternatively, the precipitate was heated under reflux in 10ml of 6 N HCI containing 50 mg of cysteamine hydrochloride for 24 hr.The catechols liberated from proteins were analyzed by HPLC as described above. Insoluble melanin was washed and dried (Method B). Thetyrosine concentration in the hydrolysate solution was also analyzed byHPLC (14).

Content of Eumelanin and Pheomelanin in Melanosomes. Themelanin content of melanosomes was estimated by our HPLC methodafter chemical degradation (16). Briefly, eumelanin content in melanosomes was estimated by multiplying the amount of pyrrole-2,3,5-tricar-

boxylic acid a major pyrrolic product of eumelanin, by a factor of 50 afterpermanganate oxidation of melanin. A melanosome sample, approximately 30 mg, was homogenized in 5 ml of K2CO3, and to this, 3%KMnO4 was added until the purple color of KMn04 persisted. SolidNa2SO3 was added to decompose residual KMnO4, and the mixture wascentrifuged. The supernatant was acidified to pH 1 with 6 N HCI andextracted 5 times with ether. The combined ether extract was dried overMgSO4 and evaporated, and the residue was dissolved in water andcentrifuged. The supernatant injected into the HPLC for analysis ofpyrrole-2,3,5-tricarboxylic acid. Pheomelanin content was estimated by

multiplying the amount of AHP, a specific indicator of pheomelanin, by afactor of 5 after haemagglutination inhibition hydrolysis of melanin. Asample (approximately 30 mg) was suspended in 5 ml of 57% hydriodicacid containing 100 mg of H3PO4 and was heated under reflux. Thehydrolysate was evaporated to dryness, and the residue was dissolvedby 0.1 N HCI and was centrifuged. The supernatant was injected into theHPLC for analysis of AHP.

Elemental Analysis of Melanin. Elemental analysis of melanin wascarried out at the Central Instrument Analysis Laboratory of HokkaidoUniversity and at the Elemental Analysis Laboratory, Faculty of Science,Osaka University, Japan.

Solubility of Melanin. Solubility tests of melanin were carried out bypreparing mixtures of melanin and solvents at reagent grade after whichthe mixtures were put in glass tubes and sealed with a flame. All the testmixtures had less than a 3% weight concentration with the exception ofsepia melanin, which had from a 3 to 6% weight concentration.

The mixtures were then shaken, heated, and cooled until they showedno further change and were left standing at room temperature for 1week. Solubilities were checked by the naked eye and divided into 4classes: G, good solvent; F, fairly good solvent; P, poor solvent; and N,nonsolvent for samples.

IR, ESR, and X-Ray Small-Angle Scattering of Melanin. IR absorp

tion spectra were taken with a JASCO A102 spectrometer using a pelletof sample mixed in KBr. ESR absorption spectra were obtained by aJEOL JES-1XG spectrometer. Measurements at the X band (9.4 GHz)

with a modulation frequency of 100 KHz were made at room temperatureon lyophilized melanin contained in quartz tubes (1-mm internal diameter),

degassed, and sealed off with a flame. X-ray small-angle scattering

measurements were carried out by the same method as described in

our previous report (18).

RESULTS

Fine Structure of Melanosomes. The fine structure of thepurified B16 and HP melanosomes before characterization oftheir physicochemical properties is shown in Fig. 1. As in ourprevious report (20), the purified melanosomes lacked an outermembrane and were devoid of external contaminants, but theircharacteristic internal structure was well preserved.

Contents of Melanin and Catechol. The contents of melaninand free and protein-bound forms of catechol in the purified

melanosomes are given in Tables 1 and 2. Treatment of themelanosomes in 6 N HCI under reflux revealed a marked difference in the content of melanin, with 30 to 31% in B16, 3 to 10%in HP, and 59 to 64% in sepia melanosomes. There was littledifference in the content of melanin prepared in the absence orpresence of cysteamine, except for the case of the HP No. 1preparation (Table 1).

Eumelanin content, obtained by HPLC analysis of permanganate oxidation products, agreed fairly well with the melanincontent after acid hydrolysis. However, in HP melanosomes, theeumelanin content was approximately one-half of the melanin

content. It is not clear at present whether the difference shouldbe ascribed to artificial production of insoluble melanin-like ma

terial during acid hydrolysis, or to an underestimation of theeumelanin content. Pheomelanin content, obtained by HPLCanalysis of hydriodic acid hydrolysis products, was much lowerthan the eumelanin content in the 3 types of melanosomes, thusindicating that the 3 melanins were primarily eumelanic. However,the highest pheomelanin content was found in HP melanosomeswhich contained the lowest level of eumelanin (Table 1).

The content of free DOPA in the 3 types of melanosomes wasnot parallel to that of the melanin. Free 5-S-cysteinyldopa wasfound at a similar level in the 3 types of melanosomes. Protein-bound DOPA was found in large quantities, with ratios of DOPAto tyrosine in the hydrolysates of B16, HP, and sepia melanosomes of 1:21 to 25, 1:25 to 40, and 1:10, respectively. It isnoteworthy that sepia melanosomes did not contain 5-S-cys-teinyldopa in the protein-bound form (Table 2).

Elemental Analysis. Table 3 demonstrates an elemental analysis of B16, HP, and sepia melanins and a comparison with thatreported for feather pheomelanin (27). Theoretically, pure eumelanin should have a C:N:S ratio of 8 to 9:1:0, while purepheomelanin should have one of 5 to 6:1:0.5. Little difference

Table 1

Content of melanin in B16, HP, and sepiamelanosomesMelanosomeB1

6melanomaHP

melanomaSepia

inkPrepara

tion1

21

2Melanin

(%,w/w)Method

A"31

317.69.560Method

B30

313.0

8.559,64Eumelanin

(mean %,w/w)32

(2)"

28(2)2.3

(2)5.2(2)57

(3)Pheo

melanin(%,w/w)0.07

0.050.20

0.200.01

a Method A, hydrolysis in 6 N HCI; Method B, hydrolysis in 6 N HCI containing

0.5% cysteamine hydrochloride.0 Numbers in parentheses, number of determinations.

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Table 2

Content of catechols in 816, HP. and sepia melanosomes

Free catechols (^g/g) Bound catechols

MelanosomeB16

melanomaHP

melanomaSepia

inkPrepa

ration1212DOPA7423171314MeanS^S-CD"4.43.1

(2)°4.43.5

(2)3.1

(2)DOPA10307805506101705-S-CD1208812075ND(2)DOPA(mmol/mol

tyrosine)647402540975-S-CD(mmol/moltyrosine)3.52.83.43.0ND(2)

8 5-S-CD, 5-S-cysteinyldopa; ND, not detectable." mmol of catechol per mol of tyrosine in hydrolysate.c Numbers in parentheses, number of determinations

TablesElemental analysis of B16, HP, and sepia melanina and comparison with pheomelanin

Elemental composition(%)MelaninB16Method

A0MethodB8HP

Method AMethodB8Sepia

Method AMethod B8C53.17

55.1651.59

53.5154.15

55.04H4.55

4.434.55

4.633.57

3.83N7.13

7.847.25

8.208.00

8.42S0.74

2.131.80

2.630.48

1.46(Sf(0.24)

(0.67)(1.32)

(1.17)(0.00)

(0.00)(Of(34.48)

(30.44)(34.81)

(31.03)(33.80)

(31.25)Empirical

formula8Cs.7Ha.7NO4.2So.045

Cs.2H7.9NO3.4So. 12C7.sH7.aN03.3So.14C"Hs2N037Soo2.

Pheomelanin' 49.0 3.8 9.0 9.9 (28.3)a Relative to nitrogen (= 1).'' Obtained by subtracting sulfur contents of sepia melanin.c Obtained by subtracting values for C, H, N, and S from 100%.d Method A, hydrolysis in 6 N HCI; Method B, hydrolysis in 6 NHCI containing 0.5% cysteamine hydrochtoride.8 Values, corrected for ash that remained after combustion (3 to 5%).' Gallopheomelanin 1 (27).

was found in the percentage of composition of C, H, and Ncontents among B16, HP, and sepia melanins. The C:N ratio ofB16 and HP melanins was closer to that of sepia melanin thanto that of pheomelanin. Although B16, HP, and even sepiamelanins contained small quantities of sulfur, the values weremuch lower than those of pheomelanin (27). Hydrolysis in thepresence of cysteamine (Method B) resulted in higher sulfurcontent than did hydrolysis in its absence (Method A), the difference between the 2 methods being 0.8 to 1.4%. Since sepiaappears to be a pure eumelanin, based on analysis of pheomelanin content (Table 1), the sulfur content of B16 and HPmelanins must have compensated for that of sepia melanin. Theaverage sulfur content of HP melanin (1.8%) was more thantwice that of B16 melanin (0.7%), as measured by Method A.

Solubility of Melanin. The solubility of B16, HP, sepia, anddopa melanins is summarized in Table 4. The solutions solubi-

lized very successfully in F were all black, and only a smallamount of unsolvable material was dispersed within them. Incontrast, although the poorly soluble solutions in P were alsoblack, a large amount of unsolved material was noted. Solubility,obtained 6 months after the first check, is presented in thesecond column of each type of melanin. Both HP and B16melanins were easily solubilized in aqueous NH3 after standingfor 3 to 5 months. Sepia melanin was poorly solubilized inaqueous NH3 after standing for 3 to 5 months, but could be

Table4Solubility of HP, B16, sepia, and DOPA melanins

HP, B16, and sepia melanins were extracted by hydrolysis with 6 N HCI underreflux.

SolventNH4OH

aqueous solution (7 N)NH4OH aqueous solution (1 N)EDAEDA aqueous solution (10%)DEADEA aqueous solution (10%)DOPAF8

GNGNGF"

QPGNGSepiap

PNPNPP"P

PPNPHPF

FNFNPF*

GP

FB16F

FNPNPF"

G

8 G, good solvent; F, fairly good solvent; P, poor solvent; N, non-solvent; EDA,

ethylenediamine; DEA, diethylamine.6 Solubility obtained 6 months after the first check in these columns.

dissolved when the melanin concentration was low. Unexpectedly, DOPA melanin was easily solubilized in dilute aqueous NH3.Since distinction between eumelanin and pheomelanin has beenmade by their solubility in alkaline (27, 37, 38), DOPA melanin("typical" eumelanin) was further characterized for solubility (Ta

ble 5). Excellent solubility of DOPA melanin was noted in from0.1 to 0.01 N NH3. DOPA melanin also dissolved successfully in0.1 N LiOH-water, and in NaOH-glycine at pH above 12. It

dissolved very little in H2S04 and H2P03 water solutions. Interestingly, DOPA melanin did not dissolve in pure diethylamine andethylenediamine, while it easily dissolved in dilute water solutions(10%) of diethylamine and ethylenediamine (Table 4). These

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Melanogenesisin Melanoma

findings indicate that water is necessary to dissolve melanin andthat the solubility of synthetic DOPA melanin and of natural B16and HP melanins is different from that of sepia melanin.

IR Absorption Spectra of Melanin. The IB absorption spectraof HP, B16, and sepia melanins are shown in Chart 1. All ofthese melanins had basically similar spectra and possessedidentical absorption bands in the regions around 3400 crrr1 (orthe OH and NH regions) and 1700 to 1650 cm"1 (or the carbonyl

and carboxyl regions). HP and B16 melanins showed an absorption band at 2850 cm"1 which, however, might be related to the

presence of lipid moieties in the sample. On the other hand,sepia and DOPA melanins showed no such absorption band at2850 cm~1. The IR spectrum of sepia melanin corresponded well

with one reported previously by Blois ef al. (5). All of the spectrawere evidently different from those of pheomelanin, which havebeen reported to possess rugged absorption patterns in theregions between 500 and 1500 crrr1 (28).

ESR Spectra of Melanin. The ESR spectra of synthetic DOPAmelanin and natural melanin of sepia, HP, and B16 melanosomesare shown in Chart 2. All of the melanins revealed a similarspectrum having a single, structureless absorption pattern. Thisspectrum had neither the side components of the central part inthe low and high fields, although radical concentrations mightnot have been the same in all samples. All of the melanins alsorevealed a basically similar g value, i.e., 2.002 in dopa melanin,2.005 in sepia melanin, 2.003 in HP melanin, and 2.002 in B16melanin.

X-Ray Small-Angle Scattering of Melanin. The intensity profiles of X-ray small-angle scattering in DOPA melanin, and B16

and HP melanins and melanosomes are shown in Chart 3. BothB16 and HP melanins possessed one peak, the spacing size of

TablesSolubility of DOPA melanin

Synthesis of DOPA melanin follows the method described in Ref. 25

IR SPECTRA OF MELANIN

SolventNH4OH

aqueoussolutionNaOH

aqueoussolutionLiOH

aqueoussolutionBuffer

solution (NaOH,glycin)H2SO4

aqueoussolutionH2PO3

aqueous solutionConcentration0.1

N0.01

N0.001

N0.1

N0.01

N0.001

N0.1

N0.01

N0.001

NpH

8.5pH9.3pH10.1pH10.4pH11.0pH11.9pH12.2pH12.6pH12.7100%95%86%76%67%57-17.3%85%80-14%SolubilityGaGFGPNGPNNNPFFFGGGNNFPPNPNG"GFGPNGPNNNPFFFGGGNPFPPNFN

4000 3000 2000 1500 KX30wave number (cm"1 )

Chart 1. IR spectra of HP, B16, sepia, and DOPA melanins.

DORA-Melanin

Sepia-Melanin

BI6-Melanin

H P-Melanin

100

3G, good solvent; F, fairly good solvent; P, poor solvent; N, nonsolvent.

Second solubility value obtained 6 months after the first check.

Chart 2. ESR spectra of HP, B16, sepia, and DOPA melanins

which was 3.7 A in B16 and 4.3 A in HP melanin. While thespacing size of B16 melanin was almost identical to that ofDOPA melanin (3.5 A), the size of HP melanin was higher thanthat of B16 and DOPA melanins. The difference in this peakbetween B16 and HP melanins appears to correspond to thedifference in the stacking profiles of the indole ring and/or itsanalogous ring substances. In contrast, the unfractionated melanosomes of both B16 and HP possessed 2 to 3 peaks in X-raysmall-angle scattering profiles. Of these, 4.1 A in the B16 mela

nosomes and 4.5 A in the HP melanosomes corresponded tothe scattering profiles of the indole ring and/or its analogoussubstances such as seen in the melanin examined. Two othersmall peaks, i.e., 8.1 and 2.3 A in the B16 melanosomes and10.1 and 2.9 A in the HP melanosomes, were quite distinctive ofeach type of melanosome.

DISCUSSION

Melanogenesis is a process which, if more completely understood, might give rise to tools useful in laboratory diagnosis andperhaps even for treatment of malignant melanoma; e.g., inmeasurement of melanin precursors or metabolites in urine forestimation of melanoma stages (2,11,26,33) and in modificationof melanogenesis precursors to be selectively incorporated into

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K. Jimbow et al.

15°

29(MoKt)

20"

Charts. X-ray small-angle scattering patterns of B16 and HP melanins andmelanosomes,which are expressed as arbitrary units.

the melanin pathway for melanoma chemotherapy and radiotherapy (3, 13, 24, 41). However, estimation of melanogenesistype in tissues has largely been dependent on the fine structureof melanosomes, i.e., either ellipsoidal-lamellar or spherical-gran

ular (22). Furthermore, the majority of previous analytical studies(3, 5, 8, 27, 40) have been based on crude pigments extractedfrom entire tissues, not on the purified melanosomes and melaninextracted from these melanosomes, as was the case in thepresent study. In this study, a marked difference was notedbetween B16 and HP melanomas in the content of melanin withinmelanosomes. The melanin content of these 2 melanosomeswas quite different from and opposite to that reported recentlyby Watts ef a/., who estimated melanin content from wholemelanoma tissues (40). In addition, chemical analysis of theeumelanin:pheomelanin ratio in melanosomes and elementalanalysis of isolated melanin showed that B16 and HP melaninsare primarily eumelanic, with a higher ratio of pheomelanic component existing in HP melanin. These findings clearly suggestthat the type of melanogenesis in B16 and HP melanomas isessentially the same, although the fine structure of their melanosomes is entirely different, and that the marked color differencebetween the 2 melanosomes is largely dependent on absoluteeumelanin content.

In this study, the average sulfur content of HP melanin wasmore than twice that of B16 melanin, this also being grossly inparallel to the content of AHP (pheomelanin indicator). Muchnatural melanin has been shown to contain a fairly large amountof sulfur (32, 34). A small amount of the sulfur content ineumelanin has been attributed to bindings of protein to themelanin polymer (25). The sulfur content of natural eumelanin,which has been differentiated from pheomelanin by solubility toalkali solutions, has been found to range from 0.2% (octopusink) to 5.8% (human melanoma) (34). Since the sulfur content ofmuch natural melanin has been reported to be intermediatebetween that of synthetic DOPA melanin and 5-S-cysteinyldopamelanin, it has been suggested that natural melanin is a copoly-mer (mixed-type melanin) of eumelanin and pheomelanin, and

that "pure" eumelanin and pheomelanin seldom, if ever, exist in

malignant melanoma (32, 38). Four possibilities may, however,be raised for the origin of sulfur atoms in eumelanic pigments:(a) copolymerization of DOPA and cysteine residue during melanogenesis; (b) binding of melanin to protein through cysteineresidue during melanogenesis; (c) nucleophilic addition of cysteine to the 5,6-indolequinone moiety in melanin during acidhydrolysis; and (d) electrophilic reaction of cysteine with the 5,6-dihydroxyindole moiety in melanin during acid hydrolysis. Ourprevious study (17) and another study (29) have shown that thelatter 2 types of reactions are chemically possible. The last ofthe 4 reactions mentioned is known to be prevented by theaddition of sulfhydryl compound,4 which is one of the reasons

why cysteamine was added to the hydrolysis medium in thepresent study. Among the 4 reactions (a to d), only the first cangive rise to "pheomelanin" by our HPLC method. The higher

content of sulfur in melanin isolated by Method B of the presentstudy may result from a reaction similar to (c). However, evensepia melanin contained 0.5% sulfur after hydrolysis in the absence of cysteamine. Which one of the reactions, (b) and (of), isthe major source of the sulfur is not clear at present.

Recently, Sealy ef a/. (37, 38) demonstrated that the ESRspectra of eumelanin and pheomelanin of synthetic or naturalorigin are distinctive from each other. Eumelanin showed a singlelinear ESR spectrum, whereas pheomelanin revealed a complexspectrum with 3 distinctive features consisting of a central component and low and high field components. They proposed thatthe ratio of the central component to either the low or high fieldcomponent can be used to characterize the type of melanin. Inour study, B16 and HP melanins revealed, similar to syntheticDOPA and natural sepia melanins, single structureless ESRspectra with a line width of 4 to 6 G. The g values of HP, B16,DOPA, and sepia melanins were similar and close to 2.004, thevalue of which was essentially the same as that of melanin ofsquid and HP and human melanomas reported by Blois andKallman (4). The IR absorption spectra of B16 and HP melaninswere also similar to each other and were apparently differentfrom those of pheomelanin reported previously (27). B16 and HPmelanins further showed one major peak on X-ray small-angle

scattering, the spacing size of which was basically similar between B16 and DOPA melanins, and slightly different in HPmelanin. In addition, the melanosomes of B16 and HP showed 2other small peaks which appeared unique to either HP or B16melanosomes. Our previous studies (23, 24) and studies byother researchers (6, 10) have indicated that B16 and HP melanosomes consist of polypeptide components basically similar innumber and molecular weight, although the relative amount ofcommon proteins was quite different between the 2 melanosomes. Thus, it is suggested that the difference in color betweenthe 2 melanosomes may also be controlled by the difference inthe mode of chemical bindings of the melanin to structural matrixprotein within the melanosomes, and that, unlike normal tissues(7, 21, 31, 36), melanosome morphogenesis may not directlyreflect melanogenesis type in malignant melanoma.

Previously, we found no apparent difference in the level of freeDOPA and 5-S-cysteinyldopa in subcellular fractions of B16 and

HP melanomas (14). We also found large amounts of free andprotein-bound forms of DOPA and 5-S-cysteinyldopa in the sol-

4K. Jimbow, Y. Miyake, K. Homma, K. Yasuda, Y. Izumi, A. Tsutsumi, and S.Ito. unpublisheddata.

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uble fraction of the 2 melanomas. In this study, the content offree and bound forms of these catechols varied greatly in the 2melanosomes and was unrelated to melanin content. Our presentand previous findings may suggest that the urinary excretion of5-S-cysteinyldopa in patients and animals bearing melanomas,which has been recently used to estimate the degree of melanoma invasion (1, 2), does not directly relate to the melano-

genesis type occuring in the melanosomes, but rather to thesynthesis of 5-S-cysteinyldopa, which appears to take place

freely in the cytoplasm. Our suggestion may also correspond tothe previous report of Rorsman ef al. (35), indicating that cys-

teinyldopas are excreted in melanoma patients irrespective ofthe types of melanin pigment synthesized in skin and hair.

Our solubility test indicated that it may not be suitable to usethe solubility of melanin in dilute alkali as an absolute criterionfor differentiation between eumelanin and pheomelanin as hasbeen done previously (34, 38). Both B16 and HP melaninsbecame soluble in aqueous NH3, although they were found to beprimarily eumelanic on chemical and elemental analysis as wellas in the spectra of IR and ESR. Solubility has no relation to thefact that the melanoma melanin may be of a mixed type ofmelanin because, in our study, DOPA melanin was also solublein aqueous NH3. We found that the solubility of DOPA melaninin alkali solutions appears to depend on the degree of basicityand melanin concentration, although our method for examiningsolubility is still semiquantitative. In addition, it was possible todissolve sepia melanin in aqueous NH3 when the melanin concentration is low. Inasmuch as we have now found a method tosolubilize natural melanin of melanomas, although the melaninmay not be completely dissolved, the method can be applied tofurther characterization of the physicochemical properties ofmelanin and, thus, provide a basis not only for melanin biologybut also for the development of rational approaches for laboratory diagnosis and treatment of malignant melanoma.

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Fig. 1. Ultrastructure of melanosomes purified from B16 and HP mouse melanomas. The melanosomeswere purified by sucrose density gradient ultracentrifu-gation, and their outer member was leached off by treatment with Brij-35. a, B16melanosomes.x 15,000. b, HP melanosomes.x 16,000.

1134 CANCER RESEARCH VOL. 44

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1984;44:1128-1134. Cancer Res   Kowichi Jimbow, Yasuhiro Miyake, Koichi Homma, et al.   Melanosomes in Malignant MelanomaMelanosomes by Physicochemical Properties of Melanin and Characterization of Melanogenesis and Morphogenesis of

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