Regulation of the catalytic activity of preexisting tyrosinase in black and Caucasian human melanocyte cell cultures

The activity of tyrosinase, the rate-limiting enzyme for melanin synthesis, is higher in Black skin melanocytes than in melanocytes derived from Caucasian skin. This variation in enzyme activity is not due to differences in tyrosinase abundance or tyrosinase gene activity, but, rather, is due to differences in the catalytic activity of preexisting tyrosinase. In melanocytes, tyrosinase is localized to the membrane of melanosomes and in Caucasian melanocytes the melanosome-bound enzyme is largely inactive. Conversely, in melanosomes of Black melanocytes, tyrosinase has high catalytic activity. Treatment of Caucasian melanocytes with the lysosomotropic compound ammonium chloride or with the ionophores nigericin and monensin results in a rapid and pronounced increase in tyrosinase activity. This increase occurs without any change in tyrosinase abundance, indicating that these compounds are increasing the catalytic activity of preexisting enzyme. Inhibition of the vacuolar proton pump V-ATPase by treatment of Caucasian melanocytes with bafilomycin also increases tyrosinase activity. In contrast to the 10-fold increase in tyrosinase observed in Caucasian melanocytes, neither ammonium chloride, monensin, nigericin, nor bafilomycin is able to increase the already high level of tyrosinase activity present in melanosomes of melanocytes derived from Black skin. Finally, staining of Caucasian melanocytes with the fluorescent weak base acridine orange shows that melanosomes of Caucasian, but not Black, melanocytes are acidic organelles. These data support a model for racial pigmentation that is based on differences in melanosome pH in Black and Caucasian skin types. The models suggests that melanosomes of Caucasian melanocytes are acidic, while those of Black individuals are more neutral. Since tyrosinase is inactive in an acid environment, the enzyme is largely inactive in Caucasian melanosomes but fully active in Black melanosomes.     

Melanocytes in Human Embryonic and Fetal Skin: A Review and New Findings

Melanocytes account for approximately 5–10% percent of the cells in adult epidermis. Unlike the ectodermally derived keratinocytes, they originate in the neural crest and migrate into the epidermis early in development. There has been an interest in melanocytes in developing human skin since the late 1800s, when concentrated pigmented cells were identified in the sacro-coccygeal skin of Japanese fetuses. This observation led to speculation and subsequent investigation about the racial nature of the melanocytes in this site (the Mongolian spot), the presence of melanocytes in fetuses of other races, the timing of appearance of these cells in both the dermis and epidermis, and their origin. The early investigators relied primarily on histochemical methods that stained either the premelanosome or the pigmented melanosome, or relied upon the activity of tyrosinase within the melanosome to effect the DOPA reaction. Studies by electron microscopy added further documentation to the presence of melanocytes in the skin by resolving the structure of the melanosome regardless of its state of pigmentation. All of these methods recognized, however, only differentiated melanocytes. The thorough investigations of melanocytes in the skin from a large number of black embryos and fetuses by Zimmerman and colleagues between 1948 and 1955 provided insight into the time of appearance of melanocytes in the dermis (10–11 weeks' menstrual age) and the epidermis (11–12 weeks) and revealed the density of these cells in both zones of the skin of several regions of the body. The precise localization of the melanocytes in the developing hair follicles was contributed by the studies of Mishima and Widlan (J Invest Dermatol 1966; 46:263–277). More recently, monoclonal antibodies have been developed that recognize common oncofetal or oncodifferentiation antigens on the surface or in the cytoplasm of melanoma cells and developing melanocytes (but not normal adult melanocytes). These antibodies recognize the cells irrespective of the presence or absence of melanosomes or their activity in the synthesis of pigment and therefore are valuable tools for re-examining the presence, density, and distribution patterns of melanocytes in developing human skin. Using one of these antibodies (HMB-45), it was found that dendritic melanocytes are present in the epidermis between 40 and 50 days estimated gestational age in a density comparable with that of newborn epidermis and are distributed in relatively non-random patterns. A number of questions about the influx of cells into the epidermis, potential reservoirs of melanoblasts retained within the dermis, division of epidermal melanocytes, and the interaction of melanocytes and keratinocytes during development remain unresolved. The tools now appear to be available, however, to begin to explore many of these questions.     

Effect of bromocriptine on the larval skin of the green toad, Bufo viridis viridis leurenti

In the premetamorphic larval green toad, B. viridis viridis, as in other anurans, the skin is made up of a fibrous dermis and an epidermis of stratified epithelium. The effects of bromocriptine, an antiprolactin drug, on the premetamorphic skin of B. viridis viridis was examined. Bromocriptine, dissolved in rearing water at four different concentrations, induced a number of changes in the skin of treated tadpoles. In rough sequence of appearance, these changes include: retraction ofthe melanocyte dendrites, synchronous burst ofthe apical vesicles of the superficial epithelial cells, gradual disappearance of the melanosomes from the epithelial cells and widening of the intercellular spaces. In addition, macrophages appeared in the superficial dermis amongst the retracted melanocytes. White crystals were observed on the skin surface and similar crystals were ingested by the macrophages. Prolonged treatment with bromocriptine resulted in hypertrophy and extraction of some epidermal cells. Deep melanocytes of the mesenteries were not affected by bromocriptine-treatment indicating that the drug did not penetrate deep into the tadpole tissue. Whether the macrophages observed in the dermis were recruited from deeper tissues or were converted melanocytes is another issue in need of study.     

Pigmentation in intrinsically aged skin of A1 guinea pigs

It is known that skin often shows irregular pigmentation during aging which is frequently associated with hyperpigmentation. Many studies have utilized brownish A1 guinea pigs to investigate the pathogenesis of ultraviolet (UV)-induced skin pigmentation, however, changes associated with intrinsic aging in A1 guinea pig skin have not been documented. To characterize such changes, skin from the dorsal and neck areas of 20-week, 1-, 2-, 3- and 5-yr-old guinea pigs was examined. Skin color was measured using a colorimeter, and biopsy specimens were stained with Masson-Fontana, L-3,4-dihydroxyphenylalanine (DOPA), and antibodies against KIT (ACK-45), gp100 (HMB-45) and S-100 proteins. The L* value of skin color decreased with aging and melanin deposits increased in the epidermis. Further, DOPA+, gp100+ and S-100+ melanocytes increased, indicating that the number of melanocytes had increased with age, whereas KIT+ melanocytes did not increase in dorsal skin and actually decreased in neck skin with aging. Further, rippled pigmented areas appeared in the neck skin of the 3-yr-old animals, and in the dorsal and neck skin of 5-yr-old guinea pigs in the absence of UV irradiation. Melanocytes were distributed uniformly in younger skin, whereas they were clustered in older skin. UV irradiation caused an increase in the number of melanocytes, although they were not clustered. These results are the first to provide evidence that pigmentation is induced in the skin of intrinsically aged A1 guinea pigs in the absence of UV irradiation, a process that differs from that elicited by UV irradiation.     

Histological,Biochemical, and Ultrastructural Studies on Hyperpigmented Human Skin Xenografts

The mechanisms for hyperpigmentation observed in human cutaneous xenografts placed on athymic nude mice was investigated. Histologic, biochemical, histochemical, and ultrastructural examinations were performed on human skin prior to grafting and at various times ranging from 2 weeks to 30 weeks post-grafting (PG). Hyperpigmentation was macroscopically visible on the graft as early as 4–6 weeks. The number of Dopa-positive melanocytes per unit area was increased at 2 weeks PG and remained elevated until 20 weeks PG. The surface area of the melanocytes, a measure of the activity of the cells, also increased significantly and remained above the pre-grafting size throughout the study. Western blot analysis using tyrosinase specific antibody (αTy-SP) revealed the presence of tyrosinase exclusively in the grafted skin from 2 weeks to 12 weeks PG tested. Histological and ultrastructural observations revealed the presence of numerous dendritic melanocytes, indeterminant clear cells suggestive of Langerhans cells, and dermal melanophages. The results of this study suggest that the observed hyperpigmentation in grafted tissue is caused by an increase in the number of Dopa-positive melanocytes and probably from enhanced melanin production. Extracts of proteins from the xenografts exhibited prominent differences in low and high molecular proteins between pre- and post-grafted skin. Among them, the exclusive appearance of a protein doublet with apparent mw ～14 kDa was found in grafted skin, and subsequent studies indicate it has potent effects on melanocyte function.     

Dermal melanocytosis in Japanese monkeys (Macaca fuscata)

The skin of Japanese monkeys (Macaca fuscata) shows diffuse discolorations resembling human dermal melanocytosis. Very few laboratory animals have melanocytes in the dermis. The purpose of this study was to clarify the dermatologic characteristics of Japanese monkeys in terms of gross appearance, skin color, and histopathologic findings. A colorimeter was used to record the skin colors of pigmented and nonpigmented sites. Tissue specimens obtained from both types of sites were examined histopathologically. All animals examined had pigmented sites on their bodies, and the discolorations extended over 25% to 33% of the body surface. The colorimeter could detect differences in skin color due to dermal melanocytosis. All parameters of the colorimetric systems used (Yxy, L*a*b*, and L*C*h* systems) demonstrated significant differences between pigmented and nonpigmented sites. In pigmented sites, the epidermis lacked melanocytes, but the dermis had numerous melanocytes with abundant melanin. Activated melanocytes with well-developed dendrites were distributed throughout the upper part of the dermal layer. Melanocytes were not arranged in clusters, and elastic and collagen fibers in the dermis showed no histological abnormalities. Nonpigmented sites lacked melanin granules in both the epidermis and dermis. This study revealed that gross dermal melanocytosis correlated well with colorimetric results and histopathologic findings. These findings suggest that the pigmentation of Japanese monkeys is equivalent to dermal melanocytosis in humans, to the end that Japanese monkeys may be a useful animal model for investigating dermal melanogenesis.     

Development and validation of a simple method for the extraction of human skin melanocytes

Primary melanocytes in culture are useful models for studying epidermal pigmentation and efficacy of melanogenic compounds, or developing advanced therapy medicinal products. Cell extraction is an inevitable and critical step in the establishment of cell cultures. Many enzymatic methods for extracting and growing cells derived from human skin, such as melanocytes, are described in literature. They are usually based on two enzymatic steps, Trypsin in combination with Dispase, in order to separate dermis from epidermis and subsequently to provide a suspension of epidermal cells. The objective of this work was to develop and validate an extraction method of human skin melanocytes being simple, effective and applicable to smaller skin samples, and avoiding animal reagents. TrypLE? product was tested on very limited size of human skin, equivalent of multiple 3-mm punch biopsies, and was compared to Trypsin/Dispase enzymes. Functionality of extracted cells was evaluated by analysis of viability, morphology and melanin production. In comparison with Trypsin/Dispase incubation method, the main advantages of TrypLE? incubation method were the easier of separation between dermis and epidermis and the higher population of melanocytes after extraction. Both protocols preserved morphological and biological characteristics of melanocytes. The minimum size of skin sample that allowed the extraction of functional cells was 6 × 3-mm punch biopsies (e.g., 42 mm²) whatever the method used. In conclusion, this new procedure based on TrypLE? incubation would be suitable for establishment of optimal primary melanocytes cultures for clinical applications and research.     

Fibroblasts play a regulatory role in the control of pigmentation in reconstructed human skin from skin types I and II

Human melanocytes in monolayer culture are extremely dependent on a wide range of soluble signals for their proliferation and melanogenesis. The advent of three-dimensional models of reconstructed skin allows one to ask questions of how these cells are regulated within a setting which more closely approximates normal skin. The purpose of this study was to investigate to what extent melanocytes within a reconstructed skin model are sensitive to regulation by dermal fibroblasts, basement membrane (BM) proteins and the addition of alpha-melanocyte-stimulating hormone (alpha-MSH). Sterilized acellular de-epidermized dermis (prepared to retain BM proteins or deliberately denuded of BM by enzymatic treatment) from skin type I or II was reconstituted with fibroblasts, melanocytes and keratinocytes. In all but one case (9/10), cell donors were skin type I or II. The presence of BM antigens was found to be necessary for positional orientation of the melanocytes; in the absence of BM, melanocytes moved into the upper keratinocyte layer pigmenting spontaneously. Addition of fibroblasts suppressed the extent of spontaneous pigmentation of melanocytes within this model. Neither alpha-MSH nor cholera toxin induced pigmentation in this model despite the fact that melanocytes clearly had the ability to synthesize pigment.     

Changes in the proliferation and differentiation of neonatal mouse pink-eyed dilution melanocytes in the presence of excess tyrosine

Changes in the proliferation and differentiation of epidermal melanocytes derived from newborn mice wild-type at the pink-eyed dilution (p) locus (P/P) and from congenic mice mutant at that locus (p/p) were investigated in serum-free primary culture, with or without the addition of L-Tyr. Incubation with added L-Tyr inhibited the proliferation of P/P melanocytes in a concentration-dependent manner and inhibition was gradually augmented as the donor mice aged. In contrast, L-Tyr stimulated the proliferation of p/p melanoblasts-melanocytes derived from 0.5-day-old mice, but inhibited their proliferation when derived from 3.5- or 7.5-day-old mice. L-Tyr stimulated the differentiation of P/P melanocytes. However, almost all cells were undifferentiated melanoblasts in control cultures derived from 0.5-, 3.5- and 7.5-day-old p/p mice, but L-Tyr induced their differentiation as the age of the donor mice advanced. The content of the eumelanin marker, pyrrole-2,3,5-tricarboxylic acid as well as the pheomelanin marker, 4-amino-3-hydroxyphenylalanine in p/p melanocytes was greatly reduced compared with P/P melanocytes. However, the contents of eumelanin and its precursor, 5,6-dihydroxyindole-2-carboxylic acid, as well as the contents of pheomelanin and its precursor, 5-S-cysteinyldopa in culture media from p/p melanocytes were similar to those of P/P melanocytes at all ages tested. L-Tyr increased the content of eumelanin and pheomelanin two- to threefold in cultured cells and media derived from 0.5-, 3.5- and 7.5-day-old mice. These results suggest that the proliferation of p/p melanoblasts-melanocytes is stimulated by L-Tyr, and that the differentiation of melanocytes is induced by L-Tyr as the age of the donor mice advanced, although eumelanin and pheomelanin fail to accumulate in p/p melanocytes and are released from them at all ages of skin development.     

ELECTRON MICROSCOPE STUDIES OF EPIDERMAL MELANOCYTES, AND THE FINE STRUCTURE OF MELANIN GRANULES

Melanocytes and melanin granules have been studied by electron microscopy in normal human and cat skin, and in hyperplastic human skin lesions. The melanocytes have always been found as free cells within the epidermis,i.e., on the epidermal side of the dermal membrane. Melanocytes frequently rest on the dermal membrane or bulge towards the dermis. In such cases the uninterrupted dermal membrane is uniformly thin and smooth in appearance, in contrast with the regions alongside Malpighian cells, where it appears appreciably thicker and seemingly anchored to the basal cell layer. Two types of melanin granules have been distinguished according to their location in the melanocytes and to morphological characteristics which may only express different stages in the maturation of the granules: (a) light melanin granules in which a structure resembling a fine network is apparent; (b) dense melanin granules which, in osmium-fixed preparations, appear as uniformly dense masses surrounded by a coarsely granular, intensely osmiophilic shell. Treatment of sections of osmium-fixed tissues with potassium permanganate has revealed within the dense granules the existence of an organized framework in the form of a regular, crystalline-like lattice. It is suggested that this basic structure is protein in nature and may include the enzymatic system capable of producing melanin. The existence is reported of fine filaments located in the cytoplasm of melanocytes and morphologically distinct from the tonofilaments found in Malpighian cells.     

Reconstruction of a tissue-engineered skin containing melanocytes

The objective of this study was to establish a new method for reconstruction of a tissue-engineered skin containing melanocytes by employing tissue engineering. The keratinocytes, melanocytes and dermal fibroblasts were isolated and purified from human foreskin biopsies. Then the cells were used to construct a tissue-engineered skin containing melanocytes. The localization of melanocytes in the tissue-engineered skin was detected by DOPA staining, S-100 immunohistochemical staining and transmission electron microscope (TEM). The results showed that the melanocytes could be detected in the basal layer of the constructed skin and the melanocytes showed dendritic morphology. Moreover the constructed skins were used to repair the athymic mice skin defects. Animal experiment results indicated that the skin equivalents could successfully repair full thickness skin defects in athymic mice and generated black skins by 6weeks after grafting. Melanocytes located in the basal layer of the athymic mice skin could also be detected by using the S-100 immunohistochemical staining. Our established method is useful to repair the full-thickness skin defects.     

Bimodal effect of retinoic acid on melanocyte differentiation identified by time-dependent analysis

Retinoic acid (RA) is considered to control melanocytes; however, its precise mechanism remains unclear because of a bimodal effect, which promotes or inhibits melanin synthesis depending on the cell type, culture condition of melanocytes and skin conditions. In this study, we examined the effects of RA throughout each stage of differentiation of melanocytes using a mouse embryonic stem cell culture system to induce melanocytes. The results showed that RA has significantly different effects depending on the stage of differentiation of melanocytes. More specifically, RA promoted differentiation in earlier stages, wherein embryonic stem cells became melanoblasts via neural crest cells, and inhibited differentiation in later stages, wherein melanoblasts became melanocytes. It was revealed for the first time that melanocytes show markedly different reactions to RA depending on the stage of differentiation.     

Cellular and hormonal regulation of pigmentation in human ocular melanocytes

The purpose of this study was to examine some of the factors that may be relevant to regulating pigmentation in the human eye, specifically whether choroidal and iridial melanocytes are sensitive to regulation by epithelial and stromal cells and alpha-melanocyte stimulating hormone (alpha-MSH). Human choroidal and iridial melanocytes were established in culture and co-cultured with epithelial cells and stromal cells derived both from skin and from eye in order to determine their influence on choroidal and iridial melanocyte dopa oxidase activity. In all cases, co-culture of melanocytes with either epithelial cells or fibroblasts led to an increase in dopa oxidase activity during 5 days of co-culture. The extent of the increase ranged from 60% (non-significant) to as much as 185% when both fibroblasts and keratinocytes were present. The optimal ratio of fibroblasts to melanocytes was 1:10 (for dermal fibroblasts) or 1:2 (for iridial fibroblasts) and 1:1 for all epithelial cells to melanocytes. Both choroidal (three out of three cultures) and iridial (two out of three cultures) melanocytes showed increases in dopa oxidase activity to alpha-MSH when cultured in Green's media but the same cells cultured in MCDB153 were unresponsive to alpha-MSH. These in vitro studies suggest that ocular melanocytes have the capacity to be influenced by adjacent epithelial and stromal cells with respect to pigmentation.     

The effects of single gene substitution on the mammalian melanocyte system—a qualitative and quantitative histological study in the C57BL and DBL mice

About 160 skin samples from the tail, sole, palm, ear and scrotum of DBL and C57BL/St mice were “split” with NaBr and treated with DOPA. A quantitative and qualitative microscopic analysis showed that: (a) the two strains did not differ consistently in the frequency of epidermal or dermal melanocytes; nor did the sexes differ from each other in this respect; (b) the melanocytes of the two strains differed morphologically. The DBL melanocytes were generally larger, with melanin-congested perikarya. They had fewer dendrites than the C57BL melanocytes and their dendrites were shorter; and (c) the melanocytes of the DBL and C57BL strains differed in activity, the DBL melanocytes donating less melanin to Malpighian cells than did the C57BL melanocytes. It was concluded that the morphology of DBL and C57BL melanocytes is largely autonomously determined, although regional differences in melanocyte morphology suggest that the cellular environment also plays some part in influencing melanoblast differentiation.     

The melanocytorrhagic hypothesis of vitiligo tested on pigmented, stressed, reconstructed epidermis

Common generalized vitiligo is an acquired depigmenting disorder characterized by a chronic and progressive loss of melanocytes from the epidermis and hair follicles. We previously proposed a new theory that vitiligo involves the chronic detachment and transepidermal loss of melanocytes caused by autoimmune, neural and impaired redox mechanisms associated with mechanical trauma. In this study, we reconstructed epidermis on dead de-epidermized dermis with normal and/or non-segmental non-lesional vitiligo (NSV) cells and tested catecholamines or sera or hydrogen peroxide. Under unstressed conditions, the number of melanocytes located in the basal layer was significantly lower in reconstructs made with melanocytes from non-lesional NSV skin and normal keratinocytes compared with controls made with autologous normal melanocytes. The number of non-lesional NSV melanocytes was even lower in reconstructs made with keratinocytes from non-lesional NSV skin. Epinephrine and H(2)O(2) could trigger the transepidermal loss of normal and vitiligo melanocytes. Some sera induced melanocyte detachment but without any clear correlation with disease activity in the donors. In conclusion, our results are the first step to obtaining a reproducible melanocytorrhagic model in vitro with some of the stressors investigated. They support the hypothesis that NSV melanocytes have an intrinsic defect, which limits their adhesion in a reconstructed epidermis, with an enhancer effect of the vitiligo keratinocyte milieu.     

Timeline and distribution of melanocyte precursors in the mouse heart

Apart from the well-studied melanocytes of the skin, eye and inner ear, another population has recently been described in the heart. In this study, we tracked cardiac melanoblasts using in situ hybridization with a dopachrome tautomerase (Dct) probe and Dct-LacZ transgenic mice. Large numbers of melanoblasts were found in the atrioventricular (AV) endocardial cushions at embryonic day (E) 14.5 and persisted in the AV valves into adulthood. The earliest time Dct-LacZ-positive cells were observed in the AV endocardial cushions was E12.5. Prior to that, between E10.5 and E11.5, small numbers of melanoblasts traveled between the post-otic area and third somite along the anterior and common cardinal veins and branchial arch arteries with other neural crest cells expressing CRABPI. Cardiac melanocytes were not found in the spotting mutants Ednrb s-l/s-l and Kit w-v/w-v, while large numbers were observed in transgenic mice that overexpress endothelin 3. These results indicate that cardiac melanocytes depend on the same signaling molecules known to be required for proper skin melanocyte development and may originate from the same precursor population. Cardiac melanocytes were not found in zebrafish or frog but were present in quail suggesting an association between cardiac melanocytes and four-chambered hearts.     

Maintenance of distinct melanocyte populations in the interfollicular epidermis

Hair follicles and sweat glands are recognized as reservoirs of melanocyte stem cells (MSCs). Unlike differentiated melanocytes, undifferentiated MSCs do not produce melanin. They serve as a source of differentiated melanocytes for the hair follicle and contribute to the interfollicular epidermis upon wounding, exposure to ultraviolet irradiation or in remission from vitiligo, where repigmentation often spreads outwards from the hair follicles. It is unknown whether these observations reflect the normal homoeostatic mechanism of melanocyte renewal or whether unperturbed interfollicular epidermis can maintain a melanocyte population that is independent of the skin's appendages. Here, we show that mouse tail skin lacking appendages does maintain a stable melanocyte number, including a low frequency of amelanotic melanocytes, into adult life. Furthermore, we show that actively cycling differentiated melanocytes are present in postnatal skin, indicating that amelanotic melanocytes are not uniquely relied on for melanocyte homoeostasis.     

The patchwork mouse phenotype: implication for melanocyte replacement in the hair follicle.

Mice homozygous for the recessive patchwork (pwk) mutation are characterized by a variegated pigment pattern with a mixture of unpigmented and normally pigmented hairs. The pigmented hair bulbs contain functional melanocytes. By contrast, the unpigmented hair bulbs contain no melanocytes. This lack results from the death of melanoblasts in the hair follicle at the end of embryogenesis. Here, we report that melanoblasts and melanocytes are found in the epidermis of pwk/pwk mice. Furthermore, these epidermal pigment cells are able to colonize new hair follicles after skin wounding. Despite the presence of epidermal pigment cells with a colonization potential, a follicle that had produced an unpigmented hair produces a new unpigmented hair during the successive hair growth cycles. This hair color continuity is also true for the pigmented hair follicles. Thus, in normal conditions, the hair acts as an independent functional unit as regards its pigment cells population.     

PIG3V, an immortalized human vitiligo melanocyte cell line, expresses dilated endoplasmic reticulum

Summary Vitiligo is an enigmatic pigmentary disorder of the skin. Factors potentially involved in the progressive loss of melanocytes from the basal layer of the epidermis include genetically determined aberrancies of the vitiligo melanocyte. It follows that analysis of melanocytes cultured from vitiligo donors can contribute to a further understanding of the etiopathomechanism. A setback for vitiligo research has been the limited availability of vitiligo-derived melanocytes. To overcome this limitation, we have generated a vitiligo melanocyte cell line according to a protocol established previously for the immortalization of normal human melanocytes. Vitiligo melanocytes Ma9308P4 were transfected with HPV16 E6 and E7 genes using the retroviral construct LXSN16E6E7. Successful transformants were selected using geneticin and subsequently cloned to ensure genetic homogeneity. The resulting cell line PIG3V has undergone more than 100 cell population doublings ince its establishment as a confluent primary culture, whereas untransfected melanocytes derived from adult skin senesce after a maximum of 50 population doublings. Cells immortalized by this transfection procedure retain lineage-specific characteristics and proliferate significantly faster than parental cells. In this study, the phenotype of PIG3V resembled melanocytes rather than melanoma cells in culture. Tyrosinase was processed properly and melanosomes remained pigmented. Importantly, ultrastructural characterization of PIG3V cells revealed dilated endoplasmic reticulum profiles characteristic of vitiligo melanocytes. An explanation for this dilation may be found in the retention of proteins with molecular weight of 37.5, 47.5, and 56.5 kDa, as determined by gel electrophoresis of microsomal proteins isolated from radiolabeled cells. Presented in part at the Annual Meeting of the Panamerican Society for Pigment Cell Research, Aspen, Colorado, 1998.     

The location of heart melanocytes is specified and the level of pigmentation in the heart may correlate with coat color

Melanocytes are mainly found in the skin and more rarely in other parts of the body, including the heart. We analyzed the localization of heart melanocytes and their levels of pigmentation in a series of mutant mice presenting different numbers of melanocytes and pigmentation in the skin. We found that melanocytes were localized in the valves (mitral, tricuspid, and aortic) and septa (ventricular and atrial). Moreover, the numbers of melanocytes in the heart appears to reflect that of the skin. Mice having a high or low level of pigmented cells and/or melanin in valves and septa have similar lifespan. In this respect, melanocytes found in the valves and septa of the heart are probably not essential in a healthy and non-stressful environment.