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Cultures consisting almost entirely of human melanocytes were obtained from epidermal single-cell suspensions by using phorbol 12-myristate 13-acetate (10 ng/ml) in the culture medium. At this concentration, phorbol ester is toxic to human keratinocytes but not to melanocytes. When the seeding density was optimal (0.8-2 x 104/cm2) and the medium contained both phorbol ester and cholera toxin, melanocytes proliferated extensively. Under these conditions, human melanocytes could be passaged serially in vitro and grown in quantity. This cell culture system can thus be used to answer basic questions related to pigment cell biology and may serve as a control for studies of malignant melanocytes.Melanocytes constitute a minor component of the cell population in normal epidermis, being scattered at relatively low density throughout the entire epidermal sheet. The keratinocytes/ melanocytes ratio is -=35:1 (1). In normal epidermis, melanocytes undergo very little replication because they are rarely shed; on the other hand, keratinocytes undergo replication to replace those that are shed (2-4). In vitro studies ofmelanocytes have therefore had to overcome the following difficulties: (i) the lack of a good source tissue that contains melanocytes as the major component; (ii) the slow division rate ofmelanocytes; and (iii) the presence ofcells that grow faster than melanocytes, such as keratinocytes and fibroblasts.Current cell-culture techniques yield heterogeneous cell populations and lead to eventual overgrowth by keratinocytes and fibroblasts (for review, see ref. 5). Thus, studies of in vitro characteristics of human melanocytes have been complicated by the presence of contaminating populations of rapidly multiplying cells other than melanocytes. This has prevented subculturing and long-term study of melanocytes in culture.This paper presents evidence that, by using phorbol 12-myristate 13-acetate (PMA) (10 ng/ml), selective plating of melanocytes from a mixed cell population can be achieved. Moreover, in addition to suppressing the growth of keratinocytes, PMA also promotes the growth of melanocytes. By using a combination of PMA with cholera toxin, extended growth of melanocytes can be obtained. MATERIALS AND METHODSTissue Culture. Epidermal single-cell suspensions were prepared as described (6). Briefly, facial skin was reduced to split skin thickness (15/1000 in.; 1 in. = 2.54 cm) using a hand dermatome with a disposable blade (DAVOL, Cranston, RI); foreskin samples were freed from fatty tissue and washed with medium containing antibiotics. Each sample was cut into 2 x 5 mm pieces and washed in 0.02% EDTA (Sigma), and 0.5-1.5 g of tissue was placed in 2.5 ml of 0.25% trypsin (diluted 1:250; Difco) at 40C for 12-15 hr. Then, the trypsin was replaced by growth medium [Eagle's minimal essential medium with Earle's salts/0.01 mM nonessential amino acids (GIBCO)/2 mM L-glutamine (GIBCO)/5% fetal calf serum, pH 7.2, containing penicillin at 100 units/ml, streptomycin at 0.1 mg/ml, and Fungizone (GIBCO) at 0.25 ,...

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The chemical basis of melanogenesis is well documented, but the mechanism of melanosome transfer and the regulation of pigmentation by keratinocyte-melanocyte interactions are not well understood. Therefore we examined the effects of serine protease inhibitors on skin pigmentation and found that the protease-activated receptor 2, expressed on keratinocytes, may regulate pigmentation via keratinocyte-melanocyte interactions. Here we show that modulation of protease-activated receptor 2 activation affects melanosome transfer into keratinocytes, resulting in changes in pigment production and deposition. SLIGRL, the protease-activated receptor 2 activating peptide, enhanced melanosome ingestion by keratinocytes, thus increasing pigment deposition. RWJ-50353, a serine protease inhibitor, led to reduced pigment deposition in melanocytes and depigmentation. Electron microscopy studies illustrated an accumulation of immature melanosomes inside melanocytes and abnormal dendrite dynamics in RWJ-50353-treated epidermal equivalents. RWJ-50353 induced a visible and dose-dependent skin lightening effect in the dark-skinned Yucatan swine. Examinations by electron microscopy indicated that the in vivo transfer of melanosomes from melanocytes to keratinocytes was affected. Our data suggest that modulation of keratinocyte-melanocyte interactions via the protease-activated receptor 2 pathway affects melanosome transfer. The use of RWJ-50353 to modulate protease-activated receptor 2 activation could lead to a new class of depigmenting agents.

Knowledge about the surface antigens of malignant melanoma has grown rapidly since the advent of monoclonal antibodies (1)(2)(3)(4)(5). A large number of cell lines derived from melanomas have now been established, and these have facilitated the serological analysis of melanoma surface antigens. Many of the melanoma antigens that have been identified with mouse monoclonal antibodies are not expressed by all melanoma lines but show instead a differential pattern of expression that defines melanoma subsets on the basis of surface antigenic phenotype. It seems likely that this diversity of melanoma phenotype reflects a corresponding diversity in the surface phenotype of normal cells undergoing melanocyte differentiation. To pursue this idea, we analyzed the surface antigens of melanocytes, using a recently described method for growing melanocytes from normal skin (6). Most, but not all, of the antigens initially detected on melanomas were also detected on melanocytes, and the pattern of antigen expression on newborn and adult melanocytes could be distinguished. From these studies of melanocytes and melanomas, we propose a scheme of surface antigenic changes occurring during melanocyte differentiation and a classification of melanoma based on expression of melanocyte differentiation antigens. Materials and MethodsMelanocyte Cultures. Skin from face, trunk, or thigh of 16 adults, foreskin of 14 newborns, and skin from the trunk of a 12-wk fetus served as the source of melanocytes. Cultures of melanocytes were grown in the presence of the phorbol ester, TPA (12-O-tetradecanoyl-phorbol-13-acetate) 10 ng/ml (Consolidated Midland Corp., Brewster, NY), and 1 × 10 -s M cholera toxin (Schwartz/Mann Div., Becton, Dickinson & Co., Orangeburg, NY), according to previously published procedures (6).Melanoma Cultures. For derivation of melanoma cell lines, see ref 7. Cultures were maintained in Eagle's minimum essential medium supplemented with either 10% fetal bovine serum (FBS) or 8% newborn calf serum mixed with 2% FBS, 2 mM glutamine, 1% nonessential amino acids, 100 U/ml penicillin, and 100 #g/ml streptomycin. Cultures were regularly tested for mycoplasma, and contaminated cultures were discarded. Five melanoma cell lines were tested between passage one and three 163,164,165,173), while the remaining cell lines were tested after passage 8, usually between passages 15 and 50. In addition, eight melanoma cell lines 19,28,37,64,127,130, and MeWo) were grown in the presence of TPA (10 ng/ml) and cholera toxin (1 X 10 -s M) for 2-3 wk to determine the effect of these growth factors on surface antigen expression.Serological Procedures. The anti-mouse immunoglobulin (anti-Ig) assay and protein A (PA) *