COMMENTARY

Colour genes, oncogenes and melanocyte differentiation

DOROTHY C. BENNETT

St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK

Introduction

Pigmentation has long been a favourite and easily usedmarker in the genetics of diverse organisms. Mammalianintegumental pigments are melanins, synthesized bymelanocytes in the epidermis and hair bulbs. Ourunderstanding of mammalian pigmentation genes hasbeen advanced significantly in the last few years (Hearingand Jim6nez, 1989), partly by the advent of methods forthe culture and immortalization of melanocytes. Incultured melanocytes, homozygous recessive germlinemutations can be visibly expressed, complemented or evenrevert spontaneously, as will be discussed. In the mousethere are over 130 mutations that affect coat colour,mapping to well over 50 loci (Silvers, 1979). For the sake ofbrevity, most of this commentary will be concerned withthe recent molecular characterization of mutations at justfour of these loci, two of which encode developmentallycontrolled, melanocyte-specific products while the othertwo play a part in melanocyte development.

The corresponding cancer, melanoma, makes an excel-lent system for the study of cell differentiation and itsrelationship to malignancy, partly again because of theready visibility of melanin (Bennett, 1989). The commen-tary will conclude with discussion of some relationshipsbetween malignancy and cell differentiation in melano-cytes transformed by oncogenes or other agents.

Culture and Immortalization of melanocytes

Although short-term cultures of normal human melano-cytes were obtained by Hu et al. (1957) and others, long-term cultures were first reported by Eisinger and Marko(1982), the key components of their culture medium beingcholera toxin and the phorbol ester tetradecanoyl phorbolacetate (TPA), also known as PMA. Human diploidmelanocytes senesced like other human cell types. How-ever, murine melanocytes grown in similar media withTPA proved capable of spontaneous immortalization, andthree groups of workers soon established murine melano-cyte lines (Sato et al. 1985; Bennett et al. 1987; Tamura etal. 1987). The first such lines were from black mice andwere followed by lines from mice carrying albino (c) andother germline coat-colour mutations (Abe et al. 1986;Halaban et al. 1988; Bennett et al. 1989).

Immortal melanocytes usually retain a high level ofdifferentiated function in culture, synthesizing pigmentJournal of Cell Science 98, 135-139 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

characteristic of the mice of origin. For example, melano-cyte lines from black (nonagouti, a/a) and brown (b/b)mice produce black and brown pigment, respectively (e.g.see Sato et al. 1985; Bennett et al. 1989). Even albinomelanocytes produce abundant premelanosomes, theunpigmented precursors of melanosomes (pigment organ-elles). Immortal melanocytes also tend to retain otherproperties of their normal counterparts including a diploidor near-diploid chromosome number, dependence on TPAfor growth in standard culture media, inability to grow insuspension and lack of tumorigenicity in syngeneic ornude mice (Bennett et al. 1987; Dotto et al. 1989; Dennerand Knowles, personal communication). It is theseproperties that have made such cultures useful for studiesof both pigmentary gene expression and oncogenesis.

The tyrosinase or albino (c) locus

It was long suspected but not proven that albino (pink-eyed white) mutations in a number of organisms affectedthe gene for tyrosinase, the principal enzyme in melaninbiosynthesis (Silvers, 1979). Recently, both mouse andhuman tyrosinase genes were cloned and sequenced, theoriginal cDNA clones having been obtained from culturedmelanocytes (Yamamoto et al. 1987; Kwon et al. 1987).Active cDNAs were identified by the property of transfer-ring tyrosinase activity by transfection to cells that lackedit (Muller et al. 1988; Yamamoto et al. 1989). The mappingof the sequence at or near the mouse albino (c) locus onchromosome 7 (Kwon et al. 1987), its absence in mice withalbino deletions (Ruppert et al. 1988) and especially itsrestoration of pigmentation on transfection to albinomelanocytes (Yamamoto et al. 1989) combined to identifythe c locus as containing the tyrosinase structural gene.Some functional aspects of the protein sequence, to bediscussed, are shown in Fig. 1A (top half).

Kwon et al. (1989) pointed out a single G to C change,resulting in a Cys to Ser substitution, in the tyrosinasesequence derived from albino mice compared with thewild-type sequence (Fig. IB). Although only part of thealbino sequence was described, so that further differenceswere possible, this Cys85 (numbering of Muller et al. 1988)falls in a region highly conserved between tyrosinases andtyrosine-related proteins (Muller et al. 1988; Hearing andJimenez, 1989) (see next section and Fig. IB). Thesubstitution is thus a candidate for the effective mousealbino mutation.

Key words: colour genes, oncogenes, melanocytes.

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The spontaneous appearance of black, apparentlyrevertant cells in a line of albino melanocytes (Fig. 2)provided a test for this (Jackson and Bennett, 1990). Theblack cells were cloned to give a black subline. White re-mutant cells were detected in this culture and were alsosubcloned. Amplification and sequencing of part of thetyrosinase gene from these sublines snowed that therevertant line was heterozygous for an exact reversion tothe wild-type codon for Cys85, while the white re-mutantsublines had lost this reversion, by allele loss. Thus thispoint substitution is sufficient to produce the mouse albinomutation (Jackson and Bennett, 1990). A restriction-fragment-length polymorphism revealed the same basechange at this position in 15/15 other albino mousestrains, indicating a common genetic origin for all thesestrains (Jackson and Bennett, 1990).

Yamamoto et al. (1989) and Kwon et al. (1989) havepublished about 2500 and 300 bases, respectively, of thesequence 5' to mouse tyrosinase, which are in agreement.This region contains various notable features includingTATA- and CCAAT-like elements, palindromes, a longregion of GA repeats, and a sequence with 8/10 homologyto the consensus thyroid/retinoid-responsive element.Yamamoto et al. (1989) have constructed a tyrosinase'minigene' capable of inducing pigmentation on expressionin albino melanocytes, by linking their 5' sequence to afull-length cDNA. They have reported tissue-specificexpression of this construct in transgenic mice (Tanaka etal. 1990). The requirements for such expression can now beanalysed, as well as those for the stimulation of tyrosinasegene transcription by melanocyte-stimulating hormone(MSH) or cyclic AMP (Kwon et al. 1988; Hoganson et al.1989).

The murine brown (b) locus encoding tyrosinase-related protein 1

The first tyrosinase-related gene was identified after asequence designated pMT4 was cloned from mouse

melanoma cDNA using monoclonal anti-tyrosinase anti-bodies; it was thought originally to be a tyrosinase cDNA(Shibahara et al. 1986). However, pMT4 could not confertyrosinase activity on transfected cells (Miiller et al. 1988),and was mapped to the murine brown (b) locus (chromo-some 4) rather than the c locus (Jackson, 1988). Thesequence nonetheless showed high homology to authenti-cated tyrosinases (Miiller et al. 1988), and was designatedtyrosinase-related protein-1 or TRP-1 (Jackson, 1988). (Asecond related sequence, TRP-2 (Jackson, 1988) will not bediscussed here.)

The wild-type TRP-1 sequence was inserted into aretroviral vector, which was used to infect cultures ofimmortal melanocytes homozygous for the brown (b)mutation (Bennett et al. 1990). These cells produce mutantbrown pigment in culture. However, many cells with blackto dark brown (wild-type) pigment were observed incultures expressing the exogenous gene. Thus TRP-1complements the brown mutation and is the product of thislocus (Bennett et al. 1990). A human cDNA sequence forTRP-1 has also been reported (Cohen et al. 1990).

The function of TRP-1 is not known, although Hearingand Jimenez (1989) do not dismiss the possibility that it isanother tyrosinase, suggesting that the failure to conferthis activity by transfection could be for trivial reasons.The protein sequence is about 40 % homologous with thatof tyrosinase and shares its known functional character-istics (Fig. 1A), including a leading signal sequence, atrans-membrane region, nearly all cysteine residues(indicating similar secondary structure) and two potentialcopper-binding sites (Muller et al. 1988; Jackson, 1988;Hearing and Jimenez, 1989). TRP-1 may thus be anotherpigmentary enzyme, located with tyrosinase on the innersurface of the membrane of the melanosome. It has alsobeen reported to have catalase activity, which couldprevent bleaching of melanin by the hydrogen peroxidethat is a by-product of melanin synthesis (Halaban andMoellmann, 1990). As with tyrosinase, expression of TRP-1 appears to be specific to pigment-synthesizing cells

Tyrosinase

Signalsequence

Copper1

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86=TyrTRP-1 in brown

Fig. 1. Comparison of features of the amino acid sequences ofmurine tyrosinase and TRP-1. Compiled from Muller et al. (1988),Hearing and Jimenez (1989), Jackson and Bennett (1990) andZdarsky et al. (1990). Gaps have been introduced to maximisealignment. (A) Domains of the two proteins. Copper 1 and 2 denotethe two potential copper-binding sites. Y indicates consensus N-glycosylation sites. (B) Full sequence of a highly conserved regionas indicated in A. Filled blocks represent exact concordance ofamino acids while stippled blocks indicate functional similarity.Conserved cysteine residues are marked by diamonds. The albinoand brown mutations each alter one such cysteine residue, the twomapping close together in the aligned genes.

136 D. C. Bennett

(Jackson, 1988). Its genomic and flanking sequencesremain to be described.

The sequences of cDNAs for the murine brown mutantTRP-1 gene and an induced revertant to wild-type (partialsequence) have been reported (Zdarsky et al. 1990). Thereare several differences between the brown and (original)wild-type sequences, but only one of these is restored inthe revertant, showing that it is the critical mutation. Thisis in the codon for a conserved cysteine residue, Cys86which, interestingly, falls very close to the Cys85 of thealbino mutation of tyrosinase when the two proteinsequences are aligned by homology (Zdarsky et al. 1990;Fig. IB). Both cysteine residues lie within a regionparticularly highly conserved between the two proteins,and presumably vital for protein function, althoughwithout any identified external homology (Fig. IB). Thereare several other such regions (not shown; Zdarsky et al.1990).

The S/and c-kit/ W loci: a growth/maturationfactor and its receptor

An interesting link between malignancy and developmentwas found in the identification of the murine dominantwhite spotting (W) locus with the proto-oncogene c-kit, theoncogenic homologue of which is carried by a felinesarcoma virus. Mutant alleles of W, even when hetero-zygous, reduce the numbers of melanocytes developing inthe skin, in some cases severely. Germ cells and somehaemopoietic stem cells are similarly affected, from earlyembryonic to adult stages (Silvers, 1979). After c-kit wasmapped close to the W locus (mouse chromosome 5), c-kitgenomic or cDNA sequences from a number of W mutantstrains were analysed by Southern blotting or sequencing.Some restriction fragment patterns and all the sequenceswere found to differ from those of normal c-kit (Geissler etal. 1988; Nocka et al. 1990). This constituted convincingevidence for the identity of the two genes, c-kit transcriptsare detectable in cell types including melanocytes, mastcells and the other known targets of W mutations (Nockaet al. 1989). Now, the c-kit product has tyrosine kinase

Fig. 2. A group, probably a clone, of phenotypic revertant cellsseen in a culture of melan-c albino melanocytes. To illustratethe prominent visibility of living pigmented cells amongunpigmented, even by phase-contrast optics as here. Thisprominence is invaluable for the somatic-cell genetics ofpigmentation. Bar, 100 fim.

activity, and from its sequence is a trans-membranereceptor, with homology to a family of growth factorreceptors (Nocka et al. 1990; Stenman et al. 1989),Interestingly a different receptor in the same family, theplatelet-derived growth factor A (PDGF-A) receptor, isencoded close to the human c-kit gene (Stenman et al.1989).

Several W mutations are associated with impairedtyrosine kinase activity in immunoprecipitable c-kit(Nocka et al. 1990), and impaired or absent responses ofmast cells and pluripotent haemopoietic stem cells to agrowth- and differentiation-inducing factor produced bycultured cells of several types (Witte, 1990). Theseresponses have now been used by several groups to purifymouse and rat ligands, and sequence the mouse, rat andhuman genes, as reported in a striking set of eight articles(Martin et al. 1990, and adjacent papers, reviewed byWitte, 1990). The ligands appear to be soluble ormembrane-bound products of the same novel gene, despitereceiving various names including MGF (mast cell growthfactor), KL (kit ligand), SLF (Steel factor) and SCF (stemcell factor) (Witte, 1990). This author prefers SCF, as theother terms each describe only one aspect of the factor —and MGF already denotes a melanocyte growth factorfrom brain. It is satisfying that the SCF gene maps to theSteel (SI) locus (mouse chromosome 10) and is deleted inmutant alleles of SI, while its product corrects haemopoi-etic deficiencies in SI mutant mice (Witte, 1990). This isbecause the SI locus was widely speculated to encode a Wligand as its mutations have similar phenotypes to Wmutations, yet SI melanocytes and other target cells arenormal if transplanted into normal mice (Silvers, 1979). Insummary, SCF is encoded at the SI locus, its receptor atthe W/c-kit locus, and both are required for the normaldevelopment of germ cells, various haemopoietic stemcells and melanocytes.

Oncogenes in melanocyte malignancy anddifferentiation

There is a consistent proliferative difference betweenmelanocytes and melanoma cells in culture, which isproving useful although it is not understood. Melanocytes(mouse or human) will not grow in a standard culturemedium supplemented with serum, but they will grow ifTPA is also added. However, many melanomas can grow insuch a standard medium, and in general the growth ofmetastatic melanomas is inhibited by TPA. Several groupshave 'transformed' immortal murine melanocytes withexogenous oncogenes or chemical carcinogens, and havedescribed properties of the resulting cells including theirresponses to TPA. The results fell into four patterns:(1) there was no detectable effect on the melanocytes,reported only for an activated p53 gene (Dotto et al. 1989).(2) Cells expressing oncogenes v-Ha-ros, x-neu or adeno-virus gene Ela, and cells transformed by two chemicalcarcinogens, all showed an alteration from TPA depen-dence to independence (or to inhibition of growth in thecase of Ha-ras), combined with a loss of pigment and theacquisition of the ability to grow in suspension or (wheretested) to form tumours in mice (Wilson et al. 1989; Dottoet al. 1989; Denner and Knowles, personal communi-cation). (3) Clones expressing \-myc or polyoma middle Tantigen initially showed a mitogenic response to TPA, andwere pigmented. However, growth soon accelerated in theabsence of TPA, while a loss of pigmentation of the

Pigmentation genes 137

cultures was observed (Dooley et al. 1988; Dotto et al.1989). These changes were observed in each of fourinitially pigmented clones expressing middle T antigen (B.Nester and DCB, unpublished data). All these types ofunpigmented cultures were tumorigenic (Dooley et al.1988; Dotto et al. 1989). Pattern (4) was reported only formelanocytes ectopically expressing basic fibroblast growthfactor (bFGF) rather than an oncogene. These cells becameindependent of TPA and unpigmented, but not tumori-genic (Dotto et al. 1989).

Two aspects of this work will be emphasized. The first isthe loss of pigmentation, which in 6/7 treatments wasassociated with tumorigenicity. To put it another way, allcultures that became malignant also became unpig-mented. One interesting question about this is that ofwhether the melanocytes dedifferentiated. While the cellsdescribed by Dotto et al. (1989) were uncloned, the othergroups used the cloned line melan-a (Bennett et al. 1987),or a subline of it (mel-ab; Dooley et al. 1988), so all cellswere progeny of one black melanocyte. Denner andKnowles (personal communication) tested for the ex-pression of the melanocytic mRNAs for tyrosinase andTRP-1. They detected both gene products in melan-a cells,but neither was found in depigmented cells transformedby chemicals, v-Ha-ras or Ela. This indicates somededifferentiation at the level of transcription, which wouldnot be unduly surprising in view of the evidence forinstability of differentiation in melanoma cells (Bennett,1989).

The second point to consider is a comparison betweenpatterns (1) and (2) above. In pattern (2), cells seem toundergo at least two steps of alteration, with unpigmentedcells emerging from and overgrowing initially pigmentedtransfected clones. Thus, although melanocytes express-ing these oncogenes become malignant, some extraspontaneous cellular change may play a part. However, inpattern (1), malignancy may have arisen in one step withintegration of v-ros or v-neu genes, although this has notbeen proven. It should be remembered however that themelanocytes used were already immortal, i.e. not entirelynormal. Thus it cannot be inferred that melanoma can becaused by any dominant oncogene, although up to 25 % ofhuman melanomas are reported to have one or moreactivated ras genes (Shukla et al. 1989). Indeed, obser-vations including the high frequencies of breakpoints inchromosomes 1 and 6 in melanoma, the existence of afamilial form of the disease (Dracopoli and Bale, 1988;Trent et al. 1989) and the loss of malignancy of humanmelanoma cells following the reintroduction of a normalhuman chromosome 6 (Trent et al. 1990) provide evidencefor involvement of the loss of a normal gene.

Perspective

The genetics of pigmentation and of pigment-cell malig-nancy have both entered a phase of analysis at themolecular level. Moreover, a region of overlap has beenindicated, in which exogenous oncogenes can affect theexpression of two coat colour genes, while another coatcolour gene actually is a proto-oncogene. This area ofoverlap has yielded some fascinating results recently and,as indicated by a surge of related research, promises to bea fruitful field in the near future.

The author is grateful to Joachim Denner, Ian Hart and IanJackson for communication of results prior to publication, and toIan Jackson for valuable comments on the manuscript. Research

in the author's laboratory is supported by the Cancer ResearchCampaign and the Wellcome Trust.

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