Download pdf - Cutaneous Photobiology. The Melanocyte vs. the Sun: Who Will Win the Final Round?

Review

Cutaneous Photobiology. The Melanocyte vs. the Sun: Who Will Win the

Final Round?

ANA LUISA KADEKARO1, RENNY J. KAVANAGH

1, KAZUMASA WAKAMATSU

2, SHOSUKE ITO

2,

MICHELLE A. PIPITONE1 and ZALFA A. ABDEL-MALEK1

1Department of Dermatology, University of Cincinnati College of Medicine, Cincinnati, OH, USA; 2Department of Chemistry, Fujita Health

University School of Health Sciences, Toyoake, Aichi, Japan*Address reprint requests to Zalfa A. Abdel-Malek, Department of Dermatology, University of Cincinnati, 231 Albert Sabin Way, PO Box 670592,Cincinnati, OH 45267-0592, USA. E-mail: [email protected]

Received 13 June 2003; in final form 3 July 2003

Solar ultraviolet radiation (UV) is a major environmental

factor that dramatically alters the homeostasis of the skin as an

organ by affecting the survival, proliferation and differentiation

of various cutaneous cell types. The effects of UV on the skin

include direct damage to DNA, apoptosis, growth arrest, and

stimulation of melanogenesis. Long-term effects of UV include

photoaging and photocarcinogenesis. Epidermal melanocytes

synthesize two main types of melanin: eumelanin and pheo-

melanin. Melanin, particularly eumelanin, represents the major

photoprotective mechanism in the skin. Melanin limits the

extent of UV penetration through the epidermal layers, and

scavenges reactive oxygen radicals that may lead to oxidative

DNA damage. The extent of UV-induced DNA damage and

the incidence of skin cancer are inversely correlated with total

melanin content of the skin. Given the importance of the

melanocyte in guarding against the adverse effects of UV and

the fact that the melanocyte has a low self-renewal capacity, it

is critical to maintain its survival and genomic integrity in order

to prevent malignant transformation to melanoma, the most

fatal form of skin cancer. Melanocyte transformation to

melanoma involves the activation of certain oncogenes and the

inactivation of specific tumor suppressor genes. This review

summarizes the current state of knowledge about the role of

melanin and the melanocyte in photoprotection, the responses

of melanocytes to UV, the signaling pathways that mediate the

biological effects of UV on melanocytes, and the most common

genetic alterations that lead to melanoma.

Key words: Human melanocytes, Ultraviolet radiation, Mel-anin, Photoprotection, DNA damage, Apoptosis, Melanoma

SOLAR UV SPECTRA AND THE EFFECTSOF UV ON THE SKIN

The skin is the largest and first line of defense that protectsthe internal organs from various chemical and physicalenvironmental insults. Solar ultraviolet radiation (UV) is a

major environmental factor that influences the function,survival, and proliferation of many cell types. The predom-inant form of solar UV that reaches the earth’s surface is in

the form of long wavelength ultraviolet A (UVA) (320–400 nm) and only a minority (less than 10%) is in the form ofultraviolet B (UVB) (280–320 nm) [Reviewed by Dillman (1)and Gilchrest et al. (2)]. The short wavelength ultraviolet C

(UVC) (200–280 nm) is highly energetic, but very little

reaches the earth’s atmosphere. However, with the depletionof the ozone layer, more UVB and UVC can penetrate theearth’s atmosphere, increasing the risk for UV-induced

mutagenesis and photocarcinogenesis (3). The UVA andUVB spectra differ in their biological effects and in theirdepth of penetration through the skin layers. The shorter

wavelength UVB radiation is more energetic and mutagenicthan UVA. UVB rays are absorbed directly by DNA, andcan cause characteristic dipyrimidine sites in various criticalgenes (4–6). Similar effects have been found experimentally

with UVC (6). UVB also induces the generation of oxygen

Abbreviations – 4-AHP, 4-amino-3-hydroxyphenylalanine; bFGF, basic fibroblast growth factor; BTCA, 6-(2-amino-2-carboxyethyl-2-carboxy-4-hydroxybenzothiazole); CREB, cAMP response element binding protein; ET-1, endothelin-1; MC1R, melanocortin 1 receptor; a-MSH, a-melanocortin;POMC, proopiomelanocortin; PTCA, pyrrole 2,3,5-tricarboxylic acid; RB, retinoblastoma protein; TNF-a, tumor necrosis factor-a; UV, ultravioletradiation

PIGMENT CELL RES 16: 434–447. 2003 Copyright � Blackwell Munksgaard 2003

Printed in UK—all rights reserved ISSN 0893-5785

434 Pigment Cell Res. 16, 2003

radicals, yet the impact of this effect on DNA damage hasnot been well explored. UVA, because of its longer wave-length, penetrates deeper through the epidermis, reaching the

dermis. UVA causes DNA damage primarily by the genera-tion of reactive oxygen species that result in single-strandbreaks in DNA and in DNA-protein crosslinks (7–9).Oxygen radicals also cause lipid peroxidation that can result

in membrane and protein damage.Exposure of the skin to UV affects the survival and

proliferation of epidermal and dermal cells and alters various

cutaneous functions. In general, the effects of UV exposureon the skin are detrimental, an exception being stimulation ofvitamin D synthesis, a hormone that is crucial for normal

skeletal growth and development (10). Lack or inadequateexposure of the skin to UV results in rickets because ofvitamin D insufficiency. Exposure of the skin to UV is critical

for the isomerization of 7-dehydrocholesterol to pre-vitaminD, which is then converted to 1,25(OH)2 vitamin D3, theactive form of the hormone that is most efficient in calciumabsorption. 1, 25 (OH)2 vitamin D3 can be formed in the

skin, as epidermal keratinocytes express the enzymes thatcatalyze the hydroxylation of vitamin D (11). The acuteeffects of UV on the skin are mostly adverse, and include

DNA damage, apoptosis that is exemplified by the genera-tion of sunburn keratinocytes, erythema, immune suppres-sion that is evidenced by reduction in the number of

Langerhans cells, and increased pigmentation (12–17). Thedrastic long-term effects of UV on the skin include photo-aging, characterized histologically by solar elastosis due todegradation of collagen and the accumulation of abnormal

elastin in the dermis, and skin cancers, including melanoma,the most deadly form (18–23). The effects of UV on the skinare direct, as well as indirect. The direct effects of UV are

exemplified by the generation of DNA photoproducts. Theindirect effects of UV, such as cell cycle arrest and melan-ogenesis, are mediated by a variety of UV-induced cytokines

and growth factors that regulate the survival, proliferation,and function of different cell types in the epidermis and thedermis (24–28).

TANNING, A HALLMARK OF UV EXPOSURE

Epidermal melanocytes play a central role in determining

the responses of the skin to UV exposure. Melanocytesrepresent 8–10% of all epidermal cells, yet they serve acritical function in protecting the skin from UV-induced

photodamage. The skin has developed two main defensemechanisms to guard against the damaging effects of UV:epidermal thickening and hyperkeratosis, and stimulation of

melanin synthesis by epidermal melanocytes. Between thesetwo mechanisms, increased melanogenesis, a hallmark ofUV exposure evident as tanning, is the more photoprotec-

tive. Increased skin pigmentation in response to UVexposure is a two-step process: immediate pigment darken-ing, which occurs within minutes of UV exposure, anddelayed tanning response, which becomes apparent

2–3 days after sun exposure [reviewed in Pathak et al. (29)and Dillman (1)]. Immediate darkening is induced primarilyby UVA, and is due to photooxidation of preexisting

melanin. This immediate effect involves reorganization of

intermediate filaments in melanocytes and keratinocytes, aswell as increased dendrite formation, in order to facilitatethe transfer of melanin-containing melanosomes from mel-

anocytes to keratinocytes. The delayed tanning response isinduced by UVB and UVA, and involves an increase in thenumber of functional melanocytes, stimulation of melano-genesis, increased dendricity of melanocytes, and increased

synthesis and transfer, as well as altered packaging, ofmelanosomes.The pigmentary response of the skin to UV is determined

to a large extent by constitutive pigmentation. The classi-fication of skin phototypes I–VI has been based on theability of individuals with different constitutive pigmenta-

tion to tan in response to sun exposure (29). Skin phototypeI represents individuals with very fair skin who always burnand do not tan when exposed to the sun. Skin type II

individuals tan slightly and often burn, and skin types IIIand IV individuals tan readily and rarely burn. Finally, skinphototypes V and VI represent individuals with very darkskin who never burn upon sun exposure. Melanocytes from

different pigmentary phenotypes differ in their rate ofmelanin synthesis, their capacity to synthesize the brown–black eumelanin and the red–yellow pheomelanin, and in

the rate and manner of melanosome transfer from melano-cytes to keratinocytes. These variables account to a largeextent for the tremendous diversity of human pigmentation.

Individuals with dark skin have a higher total melanincontent, and a higher amount of eumelanin than individualswith light skin color (30–32). Recently, it was reported thatindividuals with �fiery� or �carroty� hair color, who have a

low minimal erythemal dose and a high tendency for actinicdamage, have a characteristic degradation product ofpheomelanin, namely 6-(2-amino-2-carboxyethyl-2-carboxy-

4-hydroxybenzothiazole) (BTCA) in their hair (33, 34).Based on the association of BTCA with this phenotype, itwas proposed that the presence of BTCA may be a useful

marker for skin cancer susceptibility. The size, number, andpackaging of melanosomes is another contributing factor tothe diversity of pigmentation, with larger size and more

melanosomes present in dark skin than in fair skin (35)[reviewed by Pathak et al. (29)]. Ultrastructural studies haverevealed that in dark skin, melanosomes are mostly evidentas single entities, while in lightly pigmented skin, melano-

somes are present as clusters, in keratinocytes in thesuprabasal layers of the epidermis.Individual variations in total melanin and in eumelanin

and pheomelanin contents are not only evident in the skin insitu, but are also detectable in melanocytes cultured fromdifferent pigmentary phenotypes (Table 1). Studies on cul-

tured human melanocytes revealed that those derived fromdark skin consistently had higher total melanin and eumel-anin contents, as well as a higher ratio of eumelanin to

pheomelanin, than those derived from light color skin.Furthermore, the activity of tyrosinase and the protein levelsof tyrosinase, TRP-1 and TRP-2 correlated directly withmelanin content, i.e. melanocytes derived from fair skin with

a low melanin content consistently have lower tyrosinaseactivity and levels of tyrosinase, TRP-1 and TRP-2 thanmelanocytes derived from dark skin with a high melanin

content (31, 36–38).

Pigment Cell Res. 16, 2003 435

COMPARISON OF THE PHOTOPROTECTIVECAPACITIES OF EUMELANIN ANDPHEOMELANIN

Melanin, mainly eumelanin, is best known for its photopro-tective role in the skin. Photoprotection is afforded by theability of melanin to serve as a physical barrier that scattersincident UV, and as a filter that reduces the penetration of

UV through the epidermis (39, 40). These effects are achievedby the localization of melanosomes in the perinuclear area ofepidermal melanocytes and keratinocytes, where they form

supranuclear caps that protect nuclear DNA from impingingUV rays (41).In dark skin, large, heavily-melanized melanosomes,

enriched in eumelanin and resistant to degradation bylysosomal enzymes, persist throughout the epidermal layers,and contribute considerably to photoprotection against

UV-induced damage (35, 42, 43). In contrast, in fair skinthat contains a low eumelanin content, intact melanosomesare rare, or even absent, in the suprabasal layers of theepidermis, which accounts for the increased susceptibility of

this skin type to the photodamaging effects of UV. Animportant property of melanin, particularly eumelanin, is itsability to scavenge free radicals, and to function as a

superoxide dismutase that reduces reactive oxygen to hydro-gen peroxide (44, 45). In contrast to eumelanin, pheomelaninis photolabile and potentially phototoxic (46–48). Studies on

purified eumelanin and pheomelanin showed that irradiation

of pheomelanin results in the generation of hydroxyl radicalsand superoxide anions that might contribute to oxidativeDNA damage. Additionally, pheomelanin increases the

release of histamine, which contributes to the sun-inducederythema and edema in fair skinned individuals (49).Exposure of the skin to UV reduces the levels of glutathionereductase, which is important for pheomelanin synthesis, and

is more prevalent in light-colored than in dark skin (50–52).This observation implies that the response to UV involvesreduction in pheomelanin production, which is expected to

limit the phototoxic effects of pheomelanin.The photoprotective role of melanin is supported by

numerous epidemiological data demonstrating an inverse

correlation between skin pigmentation and the incidence ofsun-induced skin cancers (18, 19) [reviewed in Gilchrest et al.(2)]. The incidence of all forms of skin cancer, basal and

squamous cell carcinomas and melanoma, is by far higher inskin types I and II individuals, with fair skin who burn inresponse to UV exposure, than in skin types III–VI individ-uals that have dark skin with a high eumelanin content and a

good tanning ability. In the USA, the rates of basal andsquamous cell carcinoma are 50 times higher, and theincidence of melanoma is at least 10-fold higher in Cauca-

sians than in African Americans (53–57). Indeed, theincidence of melanoma worldwide is highest in the Celticpopulation of Australia (58). Further evidence for the

photoprotective role of melanin comes from the observationthat albinos living in tropical regions are highly prone tophotoaging and non-melanoma skin cancer (59). The signi-ficance of melanin in general, and of the relative amounts of

eumelanin and pheomelanin in human skin, in determiningthe risk for skin cancer is supported by solid epidemiologicaland clinical evidence. However, more rigorous experimental

evidence is needed to define accurately the comparativeimpact of eumelanin and pheomelanin on photoprotectionin situ, in the context of the melanocyte and the skin.

CONSTITUTIVE MELANIN CONTENT AND THERESPONSE OF CULTURED HUMANMELANOCYTES TO UV

Clinical evidence for the photoprotective role of melanin iscorroborated by experimental data demonstrating that mel-

anin content is an important determinant of the responses ofmelanocytes to UV. The feasibility of culturing humanmelanocytes from different pigmentary phenotypes and the

demonstration that these cells respond in vitro to UV in amanner similar to their responses in the skin in situ havecontributed significantly to the understanding of the photo-

biological effects of UV. The first study comparing theresponses of human melanocyte cultures derived fromdifferent pigmentary phenotypes illustrated that melanocytes

cultured from dark skin were less sensitive to the cytotoxiceffect of UV than their counterparts cultured from lightly-pigmented skin (60). Additionally, highly melanotic melano-cytes, but not melanocytes with a low melanin content,

exhibited an increase in melanin content following UVexposure (60). Another study comparing melanoma cells withdifferent melanin contents also found that melanotic melan-

oma cells were more resistant to the cytotoxic effect of UVB

Table 1. Analysis of eumelanin, pheomelanin, and total melanin con-tents of a panel of human melanocyte cultures derived from dark orlightly-pigmented skin

Eumelanin(lg/106 cells)

Pheomelanin(lg/106 cells)

Ratio of eumelaninto pheomelanin

Total melanin(lg/106 cells)

NHM-D1 14.90 3.64 4.09 33.802 27.04 3.86 7.00 49.303 34.40 2.24 15.36 41.804 25.90 3.05 8.49 41.705 33.28 4.95 6.72 42.706 66.08 4.87 13.57 57.727 63.84 14.93 4.28 N.D.

NHM-L1 1.23 1.29 0.95 14.172 0.66 0.77 0.86 3.303 1.36 1.21 1.12 5.734 5.89 4.85 1.21 8.905 7.71 7.88 0.98 14.246 2.30 2.06 1.11 8.217 1.25 0.97 1.29 7.97

Each human melanocyte culture was derived from a single dark, or lightcolor neonatal foreskin, with the exception of no. 7 in the upper panel,which was derived from a 22-yr old female. NHM-D list representmelanocyte cultures derived from dark skin, and NHM-L list representsmelanocyte cultures derived from light color skin. Melanocytes wereanalyzed for eumelanin and pheomelanin contents using a microassay, asdescribed previously (185, 186). Eumelanin content was calculated bydetermining the amount of pyrrole-2,3,5-tricarboxylic acid (PTCA) andmultiplying it by 160. Pheomelanin content was assessed by measuringthe amount of 4-amino-3-hydroxyphenylalanine (4-AHP) and multiply-ing it by 9. Total melanin was measured spectrophotometrically at anoptical density of 475 nm, as described previously (66). The values foreumelanin, pheomelanin, and total melanin contents are expressed aslg/106 melanocytes, and each represents the mean of two determinationsper culture.


than amelanotic melanoma cells (61). Further comparison ofthe responses of melanoma cells with different melanincontents to UVB showed that the most melanotic cells had

the least amount of photoproducts and were most resistant tocytotoxicity (62). Human melanocytes with a high melanincontent were also found to be more resistant to UVAcytotoxicity than melanocytes with a low melanin content

(63).Compared with keratinocytes, human melanocytes have

lower peroxidase, catalase, glutathione peroxidase, and

superoxide dismutase activities (64). Yet despite the reduc-tion in these antioxidant defenses, cultured melanocytes areless sensitive than keratinocytes to the lethal effect of UVA,

suggesting alternative protective mechanisms including highermelanin content in melanocytes relative to keratinocytes (64).In support of these findings are the results of a study in which

the responses of melanocytes and keratinocytes to UVA,UVB and UVC were compared (65). It was observed thatmelanocytes were less sensitive than keratinocytes to thecytotoxic effects of all three UV spectra, with the greatest

difference being in their responses to UVA.Marked differences in the responses of melanocytes with a

high melanin content and their counterparts with a low

melanin content to UV were detected using changes inproliferation, survival, melanogenesis, and DNA photoprod-uct formation as endpoints (66). The UV source used in this

study consisted of FS20 lamps, with 75% emission in theUVB range, 25% emission in the UVA range that hasnegligible biological effects. The UVC emission of thoselamps is minimal, yet it contributes significantly to the

biological effects of UVB. Only melanocytes derived fromdark skin exhibited an increase in melanin content followinga single irradiation with UV. Exposure of melanocytes to UV

resulted in dose-dependent induction of cyclobutane pyrim-idine dimers, with more dimers generated in melanocyteswith a low, rather than high melanin content at all the UV

doses used, suggesting photoprotection by melanin. Thecorrelation between melanin content and UV-induced pho-toproduct formation was further illustrated in another study

that showed that melanocytes derived from a skin type Idonor encountered more cyclobutane pyrimidine dimers and6,4-photoproducts than melanocytes derived from a skintype VI donor (67). Furthermore, increasing melanin content

in melanocytes by raising the concentration of tyrosine in theculture medium drastically decreased photoproduct forma-tion in response to UV. A third study suggested a link

between increased melanin synthesis and DNA repair (68).Repeated exposure to UV from a solar simulator, whichresulted in a tan, was associated with a faster repair rate of

DNA photoproducts in skin type IV than in skin type IIindividuals. This intriguing association between increasedmelanogenesis, amount of DNA damage and DNA repair

offers an explanation for the variation in the extent ofmutagenesis and skin cancer risk among individuals withdifferent pigmentary phenotype and tanning ability.Most of the above in vitro results are strongly corrobor-

ated by a recent study which examined the relationshipbetween melanin content, on one hand, and the extent ofUV-induced DNA photoproducts and DNA repair rates, on

the other, in normal human skin (69). Volunteers from six

different ethnic groups representing different skin phototypeswere included in this study. The results obtained illustratedan inverse relationship between constitutive pigmentation

and the extent of DNA photoproducts. However, the rate ofDNA repair varied tremendously among the participants,regardless of their ethnic background or pigmentary status.Importantly, it was noted that very low UV exposure caused

measurable DNA photoproducts even in the darkest skin,suggesting that there is no absolute photoprotection in anyskin type. Seven days after exposure to a single dose of UV,

only darker skin types exhibited an increase in melanincontent, as demonstrated previously in vitro (60, 66). It wasconcluded that the difference in the extent of photoproducts

in �white� vs. �black� skin is not solely because of the differencein melanin content. On average, there was a fourfold highermelanin content in African American skin than in white skin,

but the levels of DNA damage were seven- to eightfold lowerin African American skin. These results suggest that inaddition to the absolute amount of melanin, the distributionof melanosomes and the relative amounts of the different

types of melanin contribute to the extent of cutaneousphotoprotection.

SIGNALING PATHWAY OF UV INMELANOCYTES

In many cell types, exposure to UV causes growth arrest andapoptosis because of activation of the p53 and p16 pathways.An early response to DNA damage is the rapid phosphory-lation and activation of the MAP kinases p38 and

JNK-SAPK, which are prominent activators of p53 andinducers of apoptosis (70–74). Irradiation with UV inducesthe accumulation of p53, and the increased expression of

p53-dependent genes, such as the cyclin-cdk inhibitor p21,and the proapoptotic Bax (75–78). Inhibition of cyclin-cdkcomplexes by p21 prevents the phosphorylation and inacti-

vation of the retinoblastoma protein (RB), the G1 gatekeeper(79) [reviewed in Nevins (80) and Levine (81)]. P53 isconsidered a universal sensor of genotoxic stress and is a cell

cycle checkpoint, which arrests cells in G1 phase in order toallow for DNA repair (82–84). P53 also induces apoptosis toremove mutated cells. In this regard, p53 is considered theguardian of the genome. The UV-induced G1 arrest also

results from increased expression of CDKN2K, which codesfor the tumor suppressor protein p16INK4a (p16). The specificfunction of p16 and of p14 is to inhibit cyclin-cdk complexes

by competing with cyclin D1 for binding to cdk4 (85). Thiscompetition inhibits RB phosphorylation by cyclin D1/cdk4complexes, and sustains its activity as the gatekeeper of the

G1 transition (86).Cultured human melanocytes, regardless of their constitu-

tive melanin content, responded to a single irradiation with

increasing doses of UV (UVB + UVC) with dose-dependentG1 arrest and cell killing (66). Yet these effects were morepronounced in melanocytes with a low rather than a highmelanin content. The rapid events that occur immediately

after irradiation of human melanocytes with UV include thephosphorylation of the MAP kinases p38 and JNK-SAPK,known to be activated by various stress stimuli (70–72, 87)

(Fig. 1). Exposure to UV in the absence of mitogens in the


melanocyte culture medium did not phosphorylate or acti-vate the MAP kinases ERK1/2, known to be activated by

many melanocyte-specific mitogens, such as 12-O-tetradeca-noyl phorbol-13-acetate (TPA), basic fibroblast growthfactor (bFGF), endothelin-1 (ET-1), stem cell factor, and

hepatocyte growth factor (87–90). Irradiation of humanmelanocytes with a single dose of UV (UVB + UVC)resulted in the accumulation of the tumor suppressor p53,

increased expression of p21 and p16, and hypo phosphory-lation of RB (66, 91) (Fig. 1). Exposure of human melano-

cytes to UVA (365 nm wavelength) also induced theaccumulation of p53 and caused growth arrest (92). The

observed alterations in p53, p21, p16 and RB in UV-irradiated human melanocytes suggest a role for theseproteins in the response of melanocytes to UV-induced

DNA damage, and in the maintenance of genomic stabilityand prevention of malignant transformation (Fig. 1).Cells with UV-induced DNA damage that surpasses

their DNA repair capacity either die by apoptosis, orsurvive with genetic instability that might lead to malignant

Cell cyclearrest

G2 G1

M

p38

Mitf

CREB

JNK

USF-1

p53 +

p14Akt

p21

HDM2

USF-1 Mitf tyrosinase

MitfTRP-1

Mitf Bcl2

Cyclin-cdk

complexes

RB E2F

p16

p53

p53 Bax

Bad

M

S

M

p38

MitfMitf

CREB

USF-1USF-1USF-1

p53p53 +

p14Akt

p21p21

HDM2

USF-1USF-1USF-1 Mitf

Mitf

Mitf

Cyclin-cdk

complexes

RB E2FRB E2FRB E2F

p53p53p53

p53p53p53

Bad

CREB

p38 JNK p14Akt

p16

DNA repair Apoptosis

p21survival

Fig. 1. Summary of the signaling pathway of UV in human melanocytes. Exposure of human melanocytes to UV results in the activation of a stresssignaling pathway that includes rapid phosphorylation of the MAP kinases JNK and p38, increased expression of CDKN2A gene, which codes for thecyclin-cdk inhibitor p16, and activation of the p53 pathway, resulting in apoptosis, G1 arrest and DNA repair. Few hours after UV exposure,accumulation of p53 in response to DNA damage, increase in p21expression, hypophosphorylation of RB, reduction in Bcl2 level, and increase in Baxlevel are observed. Activation of JNK and p38 contributes to the transcriptional activity of p53. Increased expression of p16 and p21 inhibit cyclin/cdkcomplexes, resulting in G1 arrest. UV activates Akt, which in turn phosphorylates and inhibits the apoptotic effect of Bad. UV also phosphorylatesCREB in a p38-dependent manner and Akt contributes to this effect. Activated CREB is expected to activate Mitf that regulates melanogenesis andpromotes the survival of melanocytes. Solid arrows indicate events known to occur in human melanocytes; dotted arrows point to expected events, basedon findings in other cell types.


transformation. In this context, apoptosis is beneficial, as itrids the skin of mutated cells that otherwise might developinto skin cancer. The best evidence for the apoptotic effect of

UV on the skin is the formation of sunburn cells, orapoptotic keratinocytes. The molecular mechanisms involvedin the formation of sunburn cells as a consequence ofUV-induced DNA damage are well defined (93). UV-induced

apoptosis of keratinocytes occurs because of the formation ofcyclobutane pyrimidine dimers, and enhancement of repairof these photoproducts reduces the formation of sunburn

cells (13, 94). Apoptosis of keratinocytes in UV-irradiatedskin is regulated to a large extent by p53 (14). Activation ofthe intrinsic or mitochondrial apoptotic pathway is import-

ant in the formation of sunburn cells. Irradiation of humanskin or cultured human keratinocytes with UV reduces theexpression of Bcl-2 and enhances the expression of Bax

(unpublished results). Also, activation of death receptors,

such as Fas (CD95), tumor necrosis factor-a (TNF-a)receptor 1, or increased expression of their ligands, i.e.CD95 ligand and TNF-a, respectively, results in apoptosis ofkeratinocytes (95, 96).Undoubtedly, cultured human melanocytes undergo apop-

tosis when irradiated with UV. This is observed as a dose-dependent increase in the percentage of melanocytes that

stain positively with Annexin V, a marker of early apoptosis(Fig. 2). Exposure to UV results in dose-dependent reductionin Bcl2 levels, accompanied with an increase in the levels of

Bax, suggesting a role for the mitochondrial apoptoticpathway in the UV-induced melanocyte death. Apoptosisof human melanocytes and the alterations in Bcl2 and Bax

levels were induced by UVB, as well as by UVB + UVC (97)(unpublished results). Cultured human melanocytes are moreresistant to UV-induced cell death than either human

keratinocytes or fibroblasts. This can be attributed to severalfactors, including less extent of DNA damage because of thephotoprotective effect of melanin, more efficient DNArepair, and/or higher activity of anti-apoptotic and survival

pathways in melanocytes. It is well documented that highlevels of Bcl2 are constitutively expressed in melanocytes inthe skin (98, 99). Moreover, expression of Bcl2 is crucial for

melanocyte survival, since Bcl2 knockout mice undergoprogressive loss of melanocytes from their hair follicles,resulting in a grey coat color (100). In comparison, the

extrinsic or death receptor apoptotic pathway has not beenamply investigated in melanocytes. Human melanocytesrespond to TNF-a, indicating that they express deathreceptors, particularly TNF-receptors (101). Human melano-

cytes seem to be resistant to Fas ligand- and TNF-relatedapoptosis-including ligand (TRAIL)-induced apoptosis, sug-gesting low abundance, or lack of expression of Fas or

TRAIL receptors 1 and 2 (102).UV activates specific survival pathways that limit

UV-induced apoptosis. Exposure of the skin to UV activates

an important survival pathway, namely the Akt/PKB path-way (103). Akt is a serine-threonine kinase that is a substratefor the lipid kinase IP3kinase, which in turn is activated by

binding of survival factors to their cognate receptors (104–108). In addition to IP3kinase, Akt can also be activated viathe cyclic adenosine monophosphate (cAMP) pathway (109).Activation of Akt by phosphorylation of Thr 308 and Ser 473

directly inhibits apoptosis by suppressing the activity of theproapoptotic Bad, and caspase 9 (108, 110, 111). Addition-ally, Akt contributes to cell survival by activating the

transcription factors NFjB and cAMP response elementbinding protein (CREB) (112, 113).Exposure of cultured human melanocytes to UV activates

the IP3kinase-Akt Pathway, as evidenced by stimulation ofAkt activity and phosphorylation of its substrate Bad (114).Activation of Akt is expected to promote melanocyte survival

by inhibiting the apoptotic effects of Bad and caspase 9.Irradiation of human melanocytes with UV results in thephosphorylation of the CREB on Ser 133, which increases itstranscriptional activity (87). The UV-induced phosphoryla-

tion of CREB is dependent, at least in part, on the MAPkinase p38, and is markedly inhibited by the specific p38inhibitor SB 203580 (87). Phosphorylated CREB is expected

to activate Mitf, a transcription factor that is critical for

0

10

20

30

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UVB 105

UVB 120P

erce

nta

ge

incr

ease

in a

po

pto

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e in

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se in

ap

op

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c ce

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–10

0

10

20

30

40

UV 7UV 14

UV 21

UV 28

A

B

Fig. 2. Dose-dependent induction of apoptosis in human melanocytes byUVB and by UVB + UVC. In (A) and (B), The source of UV was a setof FS20 lamps that has 75% emission in the UVB range, and a minimal,but biologically effective emission in the UVC range. In (A), Kodacelfilter was used to filter out any UVC rays, and melanocytes wereirradiated with 90, 105, and 120 mJ/cm2 UVB. In (B), no filter was used,and melanocytes were irradiated with 7, 14, 21, and 28 mJ/cm2

UVB + UVC. Twenty-four hours thereafter, attached as well asdetached melanocytes were harvested, stained with APC-Annexin andpropidium iodide, and analyzed by flow cytometry.


melanocyte survival (115, 116) [reviewed in Goding (117)].Recently, Mitf was shown to regulate the expression of Bcl2,which is pivotal for preventing melanocyte death (116).

Activation of Akt is expected to contribute to the survivalpathway that involves CREB and Mitf. Maintaining thesurvival of epidermal melanocytes is of particular significancefor photoprotection of the skin, given that they are highly

differentiated cells with a low self-renewal capacity.Melanogenesis that occurs in human melanocytes subse-

quent to UV exposure is regarded by some as a stress

response that is p53-dependent (118). Further evidence forthe possible regulation of melanogenesis by p53 is providedby the finding that the genes for tyrosinase and TRP-1

contain p53-responsive elements in their promoters (119).When these two genes were transfected into heterologouscells, their promoters could be activated in vivo by p53, and

by its homologs p73 and p63. A link between p53 andmelanogenesis was further suggested by the findings that theUV-induced increase in the levels of tyrosinase mRNA inmelanoma cells was dependent on the expression of p53

(120). Such an increase was not evident after UV-irradiationof melanoma cells deficient in p53. If these results areapplicable to normal human melanocytes, then they will

provide an interesting link between the p53 pathway, thesensor of DNA damage and a signal for DNA repair, and themelanogenic pathway, which confers photoprotection. Such

a link would underscore the complex adaptive pathways inthe melanocyte that insure preservation of its genomicintegrity and prevention of photocarcinogenesis.The UV-induced melanogenesis might involve the activa-

tion of the transcription factors Mitf and USF-1. In mousemelanocytes, Mitf is critical for the regulation of melano-genesis, as it increases the expression of the tyrosinase and

TRP-1 genes by binding to a conserved M box core motif intheir promoters (121–124). In human melanocytes, Mitf hasbeen shown to regulate the expression of the TRP-1 gene, as

evidenced by selective down-regulation of TRP-1 as a resultof inhibition of Mitf activity (125). USF-1 is directlyactivated by p38 in UV-irradiated mouse melanocytes and

melanoma cells (126). Activated USF-1, like Mitf, binds tothe M and E boxes in the tyrosinase promoter, and increasestyrosinase expression (126). Whether or not USF-1 isactivated by UV in human melanocytes is not known.

Demonstrating activation of USF-1 and its ability to increaseexpression of tyrosinase gene in UV-irradiated humanmelanocytes would suggest the participation of this tran-

scription factor in the tanning response. Collectively, thesefindings suggest a connection between the survival andmelanogenic pathways in melanocytes. The ultimate outcome

of activating these pathways is increased photoprotection byinsuring the survival of melanocytes and stimulating melaninsynthesis.

REGULATION OF THE RESPONSE OFMELANOCYTES TO UV BY PARACRINEFACTORS

There is overwhelming evidence for the presence of aparacrine network in the epidermis that regulates human

melanocyte survival, proliferation, and function. The first

evidence for the regulation of melanocytes by epidermalfactors came from the observation that conditioned mediafrom human keratinocyte cultures enhanced the growth,

dendricity, and melanogenesis of cultured human melano-cytes (127). This paracrine network is upregulated by UV,and mediates many of the responses of melanocytes to UVexposure (24–27, 128–130). The contribution of keratinocytes

to the response of melanocytes to UV is exemplified by themarked increase in melanogenesis induced by irradiationwith UVB of melanocytes co-cultured with keratinocytes,

compared with the same melanocytes grown as mono-culture(131). Keratinocytes seemed to contribute specifically to themelanogenic response to UVB, but not UVA. The paracrine

factors that are up regulated by UV include growth factorsand cytokines that inhibit proliferation and melanogenesisand activate apoptotic pathways, as well as other factors that

are mitogenic, melanogenic, and promoters of melanocytesurvival. Interleukin 1-a and -b and TNF-a are threecytokines that are upregulated by UV and have inhibitoryeffects on melanocyte proliferation and melanogenesis

(28, 101). Examples of paracrine factors that promotemelanocyte survival and proliferation are bFGF, the mel-anocortins a-melanocyte-stimulating hormone (a-MSH) and

adrenocorticotropic hormone (ACTH), and ET-1 (24, 25, 27,129, 132). Increases in the expression of ET-1 and proopi-omelanocortin (POMC), the precursor of melanocortins, and

in the levels of a-MSH and ACTH derivatives were depictedin the epidermis of UV-irradiated skin (24–26). The increasedproduction of factors with opposing effects on melanocytesmight be a mechanism by which UV balances apoptosis and

growth arrest with survival and proliferation, and limits theextent of melanogenesis to prevent the build up of certaincytotoxic melanin intermediates (133, 134).

The paracrine factors a-MSH, ACTH, and ET-1 that aresynthesized by epidermal keratinocytes are mitogenic andmelanogenic for melanocytes. The effects of ET-1 on

melanocytes are mediated by binding the ET-B receptor,and activating the MAP kinases ERK1/2 (87, 90) (Fig. 3). Inturn, ERK1/2 phosphorylate p90rsk, which phosphorylates

CREB. Activated CREB is expected to lead to the activationof Mitf (115, 116). The effects of a-MSH are mediated bybinding to the melanocortin 1 receptor (MC1R), andactivating adenylate cyclase (135) (Fig. 3). Increasing cAMP

levels is known to be a principal mechanism for stimulatingmelanogenesis and enhancing the proliferation of melano-cytes (136). a-MSH also contributes to the ET-1 induced

activation of ERK1/2 and CREB (87). As a result of themitogenic effects of a-MSH, ET-1, and bFGF, they havebeen utilized as major constituents of a culture medium used

for establishing and maintaining primary cultures of humanmelanocytes (137). These three factors interact synergistical-ly, and the crosstalk of their signaling pathways (calcium

mobilization, increased formation of inositol phosphates,and activation of PKC by ET-1, activation of the cAMPpathway by a-MSH, and of tyrosine kinase receptor andPKC by bFGF) promote melanocyte survival and prolifer-

ation (Fig. 3).a-MSH and ET-1 also stimulate melanogenesis in human

melanocytes by increasing tyrosinase activity and the protein

levels of tyrosinase, TRP-1 and TRP-2 (97, 138). Treatment


of human melanocytes with a-MSH increases the ratio ofeumelanin to pheomelanin, and concomitant treatment with

ET-1 further augments this effect (A.L. Kadekaro et al.,unpubl. results). Beside the role of a-MSH in up regulatingeumelanin synthesis and proliferation of human melanocytes,

it might also act as an antioxidant (139). Irradiation ofmelanocytes with UV results in dose-dependent generation ofhydrogen peroxide, and this effect is markedly inhibited by a-MSH (A.L. Kadekaro et al., unpubl. results). a-MSH and

ET-1 enable human melanocytes to overcome the UV-induced G1 arrest and mediate the melanogenic effect of

UV (97, 140). In vitro, an increase in tyrosinase activity andin the protein levels of tyrosinase and TRP-1 in humanmelanocytes irradiated with UVB + UVC was only detected

when a-MSH, or an alternative cAMP inducer, was presentin the culture medium (140). An increase in the expression ofPOMC was observed 2 days after exposure of the skin in situ

to UV, and was followed 3 days thereafter by an increase in

MitfMitf

CREBCREB

AktAkt

Cell survival

ETBR MC1R Tyrosine

kinase receptor;

PKC

ET-1 α -MSH b-FGF

PKC; calcium mobilization

non-receptor tyrosine kinases

ERK

p90p90

PKA

MelanogenesisProliferation

Bad

Apoptosis

ERK 1/2

Fig. 3. The role of the paracrine factors a-MSH, ET-1 and bFGF in modulating the UV-signaling pathway in melanocytes. Exposure to UV upregulates the synthesis by keratinocytes of an array of growth factors and cytokines, which include a-MSH, ET-1 and bFGF. a-MSH binds the MC1R,which activates adenylate cyclase and the cAMP-dependent PKA. ET-1 binds to the ETBR, activates PKC and tyrosine kinases, and increases calciummobilization. Basic FGF binds and activates its specific tyrosine kinase receptor and PKC. The crosstalk of these signaling pathways results in activationof the MAP kinases ERK1/2, which leads to the phosphorylation of CREB. a-MSH and ET-1 promote melanocyte survival by activating the Aktsurvival pathway. Active Akt is expected to contribute to the activation of CREB, which in turn is expected to stimulate Mitf activity, thus inhibitingapoptosis, promoting melanocyte survival and stimulating melanogenesis. As in the legend for Fig. 2, solid arrows indicate events known to happen inhuman melanocytes, and dotted arrows point to expected events shown to occur in other cell types.


tyrosinase, TRP-1 and TRP-2 mRNA levels (26). Theseresults suggest a role for melanocortins in the delayedmelanogenic response to UV. Furthermore, the observation

that stimulation of melanogenesis in human melanocytes byconditioned medium from UV-irradiated keratinocytes wasabrogated by antibody neutralization of ET-1 stronglysuggests a role for ET-1 in UV-induced melanogenesis (141).

Recently, a new role for a-MSH and ET-1 as survivalfactors for human melanocytes was revealed (114). Theincrease in the survival of UV-irradiated melanocytes in

response to ET-1 and a-MSH treatment is a consequence ofactivation of the Akt survival pathway and inhibition ofapoptosis. The UV-induced activation of Akt in human

melanocytes was markedly increased by a-MSH and ET-1(114) (Fig. 3). Given that ET-1 alone, or in combination witha-MSH, activates ERK1/2, and phosphorylates CREB, it is

expected that ET-1 and a-MSH will activate Mitf, thuscontributing further to melanocyte survival (87). Based onthe effects of ET-1 and a-MSH on melanogenesis andsurvival, it can be concluded that these two factors protect

human melanocytes from UV-induced death, first, by imme-diately activating survival pathways, and second, by stimu-lating delayed tanning and increasing eumelanin synthesis

that protects against DNA damage induced by subsequentUV exposures. A third possible mechanism for the survivaleffects of a-MSH and ET-1 is enhancement of repair of

UV-induced DNA damage. Such a mechanism has beendemonstrated for the survival effects of interleukin-12 andinsulin-like growth factor, which promote the survival ofUV-irradiated keratinocytes by enhancing the removal

of DNA photoproducts (142, 143). Promoting the survivalof melanocytes by ET-1 and a-MSH is critical for optimalphotoprotection, but should be accompanied with genomic

stability to prevent mutations that might lead to melanoma.

UV AND MELANOCYTE TRANSFORMATIONTO MELANOMA

The most drastic effect of UV on melanocytes is malignant

transformation to melanoma, the most fatal form of skincancer. Melanoma tumors seem to be the delayed effect ofintense intermittent sun exposure and periodic sunburn,unlike non-melanoma skin cancers that are mostly the

outcome of chronic sun exposure (144–146). Melanoma ismainly a disease of indoor workers, and is common inyoung and middle aged professionals who indulge in

outdoor recreational activities, while non-melanoma skincancers occur mostly in outdoor workers during or afterthe fifth decade of life. The risk for melanoma is thought

to be already determined around the age of 20 yr, andappears to correlate directly with the number of sunburnsencountered during these early years of life. A history of

five or more severe sunburns during adolescence mightdouble the risk for melanoma (147). Thus, it seems thatthe outcome of UV on melanocytes is determined by thedose per exposure, rather than the sum of individual

exposures over the years (2).The biology of melanoma is challenging, and the exact

mechanism of melanocyte transformation is not well

defined. The underlying carcinogenic mechanism in melan-

oma seems to be different from that of other forms of skincancer. Typical �UVB signature� mutations, particularlythose found in the tumor suppressor gene p53, are early

markers for keratinocyte transformation, and are prevalentin squamous and basal cell carcinoma, but are rare inmelanoma (148–152). However, the observation that themutations in CDKN2A found in sporadic melanoma are

UVB-induced mutations suggest that UVB plays a role inthe etiology of melanoma (153–155). The possibilitythat certain mutations in melanocytes arise because of

UV-induced oxidative DNA damage, rather than DNAphotoproducts, has not been explored, and thus needs to beinvestigated.

There is convincing evidence that environmental exposureto long wavelength UVA is a risk factor for melanoma (156,157). Evidence for the involvement of UVA in melanoma

genesis came from two different animal models, the Xipho-phorus fish, and the South American opossum Mondolephisdomestica, in which melanoma tumors can be induced byirradiation with UVA (158, 159). Additionally, UVA can

enhance melanoma tumor formation in mice injected withmelanoma tumor cells, and can act as a tumor promoter,enhancing melanoma formation in mice initiated with

DMBA (20, 160). Although UVB is more mutagenic thanUVA, the amount of UVA that reaches the earth’s atmo-sphere is by far greater than the amount of UVB. Addition-

ally, UVA results in oxidative DNA damage, which ispotentially mutagenic (7, 9). The significance of the oxidativeDNA damaging and mutagenic potential of UVA is under-scored by the finding that the extent of oxidative DNA

damage induced by sunlight in fibroblasts in the skin is atleast equal to the amount of cyclobutane pyrimidine dimers(9). Given that long wavelength UVA penetrates deeper than

UVB through the epidermal layers of human skin to thebasal layer and the underlying dermis, it can readily reach themelanocytes that reside on the epidermal-dermal junction,

and possibly lead to oxidative DNA damage and mutagen-esis (40, 161). These data question the safety of acute UVAexposure, such as prolonged recreational outdoor activities,

tanning booths and PUVA treatment.

GENETIC ALTERATIONS THAT LEAD TOMALIGNANT TRANSFORMATION OFMELANOCYTES

Malignant transformation generally occurs because of

inactivation of tumor suppressor genes and/or activationof oncogenes. The result of either event is deregulation ofcell cycle progression and proliferation, which has a

negative impact on DNA repair. Inactivation of tumorsuppressors, such as p53 or RB, results in escape from cellcycle arrest because of disruption of the G1/S restriction

point (80, 81). The outcome is premature cell cycleprogression and incomplete repair of DNA damage thatresults in genomic instability and mutations. The best prooffor a direct link between DNA repair and prevention of

carcinogenesis is provided by the tremendous increase in therisk for UV-induced carcinogenesis in xeroderma pigmento-sum patients who are deficient in excision repair of

UV-induced photoproducts (162, 163). As discussed below,


melanoma oncogenesis involves both, inactivation of tumorsuppressors and activation of oncogenes in human melano-cytes.

Mutations in BRAF

Identification of mutations in the Ras pathway in melanomasuggests the involvement of oncogene activation in themalignant transformation of melanocytes. The mitogenic

response to growth factors is mediated mainly by the Ras-RAF-MEK-MAP kinase pathway (164). In melanocytes, thispathway is activated by many melanocyte-specific mitogens

(87, 88, 90). Recently, mutations in BRAF, one of the threemembers of the RAF family of genes that code for Ras-regulated cytoplasmic serine/threonine kinases, were foundin 66% of melanoma tumors tested (165). The BRAF

mutations identified in melanoma tumors were somatic, andresulted in elevated kinase activity, as compared with thewild type gene. In particular, one mutation, namely V599E,

which seems to be specific to melanoma, results in 11-foldincrease in BRAF kinase activity (166). However, BRAFV559E mutation was also expressed at a high frequency of

about 89% in all forms of nevi (167). These findingsimplicate mutations in BRAF as an early and critical eventin the initiation of melanocyte transformation, and UV in

the promotion and progression, rather than in the initiationphase of melanoma.

Alteration in CDKN2A

Loss of tumor suppressor function in melanoma is exem-

plified by mutations in the CDKN2A locus in melanoma.Missense mutations or deletions of CDKN2A are found inthe germline of 40% of melanoma kindreds (168). Import-antly, deletion mutations in CDKN2A that are prevalent in

sporadic melanoma tumors are typical UVB signaturemutations (153–155). The unique characteristic of theCDKN2A locus is that it encodes for two different proteins,

the tumor suppressor p16 and p14 alternative reading frame(ARF), by utilizing two separate reading frames (169, 170).Mutations in p16 result in loss of its function as an inhibitor

of cylcin D1/cdk4 complexes, which leads to escape of thecells from G1 arrest and incomplete DNA repair (171, 172).P14 ARF affects the cell cycle by a different mechanism

than p16, as it normally activates p53 by binding andinactivating HDM2, which targets p53 to degradation bythe ubiquitin pathway (173–175) (Fig. 3). Mutations in p14ARF prevent the accumulation of p53, thus disrupting the

G1 restriction point, and hindering the completion of DNArepair.

The MC1R as a Melanoma Susceptibility Gene

A candidate melanoma susceptibility gene is theMC1R gene,which codes for the MC1R expressed on melanocytes. TheMC1R gene is one of more than 70 genes that are involved inmelanocyte regulation. This gene is of particular significance

for human pigmentation because of its critical role indetermining constitutive pigmentation and the ability to tanupon sun exposure (176, 177). The MC1R gene is highly

polymorphic, and to date, at least 35 allelic variants of thisgene have been identified (178, 179). The wild type gene ispredominantly expressed in African populations with very

dark, skin phototypes V and VI, while allelic variants areexpressed in populations with light skin color (176, 177, 180).SpecificMC1R variants, particularly Arg142His, Arg160Trp,Asp294His, and Arg151Cys variants are highly associated

with red hair phenotype, poor tanning ability, and increasedrisk for melanoma (177, 181). Expression of those alleles isnecessary, but not sufficient for red hair phenotype. Inter-

estingly, the expression of these MC1R variant alleles inindividuals with olive skin color compromises photopro-tection of the skin, and increases the susceptibility to

melanoma (181). These observations imply that the associ-ation of the above alleles with melanoma is independentof skin or hair color, and that melanoma susceptibility can

not be always predicted based solely on pigmentation.Supporting evidence for the function of the MC1R gene asa tumor susceptibility gene for melanoma was provided bytwo studies showing that the MC1R genotype modifies the

risk for melanoma in families with CDKN2A mutations(182, 183). A significant increase in penetrance of CDKN2Amutations, and a reduction in the age of onset of melan-

oma were observed in carriers of both, CDKN2A muta-tions and the MC1R alleles Arg160Trp, Asp294His, orArg151Cys.

The impact of most MC1R allelic variants on melanocytefunction is still unknown. In one study in which the effects ofMC1R genotype on MC1R function, constitutive pigmenta-tion, and the response of human melanocytes to UV were

investigated, the following seminal results were obtained.Expression of Arg151Cys, Arg160Trp, and Asp294His allelicvariants, in the homozygous or compound heterozygous

state, resulted in loss of function of the MC1R. Culturedhuman melanocytes that naturally express these variantswere refractory to a-MSH, and had increased sensitivity to

UV-induced apoptosis (114, 184). This increase in apoptosismight be attributed to the inability of these melanocytes tocope with the UV-induced DNA damage. Expression of

Val92Met substitution in the homozygous state did not seemto alter the function of MC1R or the response to UV,suggesting that not every polymorphism will necessarilyresult in loss of function of the receptor (184). Recently, it

was shown that a-MSH functions as a survival factor thatrescues human melanocytes from UV-induced apoptosis.Melanocytes with loss of function MC1R are at a disadvan-

tage, as they cannot benefit from the protection againstapoptosis that is afforded by activation of the MC1R. Thatpromotion of melanocyte survival by activation of the

MC1R is because of enhancement of DNA repair is anattractive possibility that might link the MC1R, a criticalregulator of melanogenesis and survival, to the DNA repair

machinery of melanocytes. Investigating this possibility isimportant for elucidating how functional MC1R protectsagainst photocarcinogenesis, and how dysfunction of thisreceptor increases the risk for melanoma. Further studies are

urgently needed to understand how the remaining MC1Ralleles affect human melanocytes. Such studies will determinethe usefulness of the MC1R genotype as a predictive marker

for melanoma susceptibility.


SUMMARY AND FUTURE DIRECTIONS

Investigation of the signaling pathways of UV in melanocyteshas revealed the complex interrelationship between thepathways that regulate survival, proliferation and melano-

genesis. Future studies might unravel the potential link ofthese pathways to DNA repair, a key process for preventionof mutagenesis and carcinogenesis. While the contribution of

paracrine factors to the UV responses of melanocytes is wellrecognized, it is not known whether in different skin types,epidermal cytokines and growth factors are differentiallyexpressed constitutively or up regulated to different extents.

The regulation of expression of growth factor receptors andthe sensitivity of melanocytes from different pigmentaryphenotypes to epidermal factors have not been amply

investigated. These issues remain open questions, and theanswers should provide important clues about the variationin the responses of melanocytes from different pigmentary

phenotypes to UV, and its impact on the susceptibility tosun-induced skin cancers. Given the difficulty in curingmelanoma, there has been tremendous emphasis on melan-

oma prevention. Despite the strong correlation betweenconstitutive pigmentation and risk for melanoma, thesusceptibility to this disease cannot be accurately ascertainedbased only on pigmentary phenotype. Therefore, identifying

genetic markers of the disease will allow for more precisescreening of individuals with a high risk for melanoma, andhence will lead to a reduction in its incidence.

Acknowledgements – Supported in part by NIH Grant R01 ES009110 andby a grant from the Ohio Cancer Research Associates (to ZAM), and bySkin Research Grant from the Johnson & Johnson Skin Research Center(to ZAM and ALK).

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