Human melanoma progression in skin reconstructs : biological significance of bFGF.

Human skin reconstructs are three-dimensional in vitro models consisting of epidermal keratinocytes plated onto fibroblast-contracted collagen gels. Cells in skin reconstructs more closely recapitulate the in situ phenotype than do cells in monolayer culture. Normal melanocytes in skin reconstructs remained singly distributed at the basement membrane which separated the epidermis from the dermis. Cell lines derived from biologically early primary melanomas of the radial growth phase proliferated in the epidermis and the basement membrane was left intact. Growth and migration of the radial growth phase melanoma cells in the dermal reconstruct and tumorigenicity in vivo were only observed when cells were transduced with the basic fibroblast growth factor gene, a major autocrine growth stimulator for melanomas. Primary melanoma cell lines representing the more advanced stage vertical growth phase invaded the dermis in reconstructs and only an irregular basement membrane was formed. Metastatic melanoma cells rapidly proliferated and aggressively invaded deep into the dermis, with each cell line showing typical invasion and growth characteristics. Our results demonstrate that the growth patterns of melanoma cells in skin reconstructs closely correspond to those in situ and that basic fibroblast growth factor is critical for progression.

Identification and characterization of a melanocyte-specific novel 65-kDa peripheral membrane protein.

In order to study proteins of the melanosome, we developed a panel of antisera against various protein fractions of melanosomes from B16 melanoma cells. An antiserum raised against a Triton X-100 insoluble fraction of melanosomes recognized a 65-kDa protein in melanocytes from mice homozygous for the buff mutation, but not in their wild type counterparts. Further studies were conducted using a specific, second generation antiserum raised against the purified protein. The protein was also detected in melanocytes cultured from albino mice, but absent in cultured mouse cell lines not of melanocyte origin. Density gradient centrifugation of subcellular organelles and indirect immunofluorescent cell staining, indicated that the protein was associated with melanosomes and vesicles. The protein on intact organelles could be made soluble using sodium carbonate, and digested with proteases in the absence of detergent suggesting that it was a peripheral membrane protein localized on the cytosolic face of organelle membranes. Metabolic labelling of cells and N-glycosidase F digestion of cell extracts indicated that the protein was not N-glycosylated. Based on its intracellular localization and biochemical defects in the buff mouse, a potential role has been suggested for the 65-kDa protein in intracellular membrane trafficking.

Ex vivo study of skin phototypes.

We studied skin phototypes ex vivo to validate a model of epidermal reconstruction with melanocytes. We made autologous epidermal reconstructs with keratinocytes and melanocytes of healthy donors of skin phototypes I to VI. Keratinocytes and melanocytes were seeded on a dead de-epidermized dermis (Pruniéras type) at a 1:20 melanocyte/keratinocyte ratio. Reconstructed epidermis was grown for 15 d at the air-liquid interface with or without ultraviolet B irradiation. A macroscopic, chromometric. histologic, and ultrastructural evaluation was performed. Reconstructs reproduced the initial phototype with few modifications. The intensity of melanin transfer correlated with the in vivo situation and was stimulated after ultraviolet B irradiation in reconstructs of all categories of skin phototypes.

EGF receptor expression and growth of psoriatic and normal human keratinocytes are modulated by 1.25 (OH)2-vitamin D3 ex vivo.

Calcitriol or 1.25 (OH)2-vitamin D3 is used in the treatment of psoriasis as an inhibitor of cell proliferation. We studied the action of calcitriol ex vivo on the growth of psoriatic and normal human keratinocytes, and on the expression of the EGF receptor. Third passaged normal and psoriatic keratinocytes were seeded (10(4)/cm2) in 24-well dishes in serum-free medium (MCDB supplemented with amino acids, with either 0.1 or 1.1 mM of calcium) and 10(-9) M calcitriol. When subconfluence was reached, cell counts and 125I-EGF binding studies were performed. Cell counts showed at least a 50% decrease in growth under all conditions studied (normal or psoriatic keratinocytes; 0.1 or 1.1 mM calcium) when calcitriol was added. 125I-EGF binding studies showed a decrease in total receptor numbers in the presence of calcitriol with acceleration of binding at low concentrations of 125I-EGF. Scatchard plot analysis showed only one type of high affinity receptor. Receptor sites were decreased (30% to 40% of controls) in the presence of calcitriol together with a decrease in the dissociation constant. In conclusion, at almost physiological concentrations ex vivo, calcitriol strongly decreased normal and psoriatic keratinocyte growth. This potent antiproliferative effect could in part be explained by the capacity of calcitriol to downregulate EGF receptor expression.

Melanosomal and lysosomal alterations in murine melanocytes following transfection with the v-rasHa oncogene.

Melanomas exhibiting mutated ras genes are frequently invasive and amelanotic. Transfecting melanocytes with ras oncogenes causes transformation and a loss of visible pigmentation. We analyzed murine melanocytes rendered amelanotic by transfection with the v-rasHa oncogene. Consistent with previous reports, tyrosinase and tyrosinase-related protein-1 (TRP-1) were not expressed by transformed cells. In addition, lack of expression of TRP-2 and the product of the silver locus was documented. Levels of melanosomal matrix antigens, the pink-eyed dilution locus protein and lysosome-associated membrane protein-1 were markedly reduced. Residual matrix antigens were localized by immunofluorescence to large vacuoles distributed peri-nuclearly in transfected cells. Electron microscopy demonstrated the absence of typical melanosomes and the presence of large vacuolar structures, also in a peri-nuclear distribution. Although levels of lysosomal hydrolases, such as beta-glucuronidase and cathepsin D, were diminished, marked elevations were observed in the expression of cathepsins B and L, 2 thiol proteases implicated in the acquisition of invasiveness. Our data demonstrate that transfection of melanocytes with v-rasHa is sufficient to disrupt the biogenesis of melanosomes and to up-regulate thiol protease synthesis, providing insights into the amelanotic and invasive nature of melanomas exhibiting mutations in ras genes.

Interaction of melanosomal proteins with melanin.

Melanin is deposited in melanosomes upon a proteinaceous matrix enveloped by a melanosomal membrane. Since melanin is highly detergent insoluble, we hypothesized that the detergent solubility of proteins of the melanosomal matrix might be inversely related to the state of melanosomal melanization. Immunoblotting analyses were performed on extracts of albino and black melanocytes to test this hypothesis. The protein products of the silver (si) and the pink-eyed-dilution (p) loci as well as other matrix constituents were present at twofold higher levels in extracts of albino cells. When black cells were rendered amelanotic by growing cultures in the presence of the tyrosinase inhibitor phenylthiourea, the apparent levels of these proteins were also increased. To obviate the potential role of different levels of synthesis in contributing to these differences, we developed a cell-free melanosomal melanization assay. Upon incubation of a melanosome-rich fraction with the melanin precursor L-3,4-dihydroxyphenylalanine (Dopa) followed by immunoblot analysis, the si locus protein, the p locus protein, and other putative matrix constituents became rapidly insoluble in SDS when compared with the members of the tyrosinase-related family of melanosomal membrane proteins. Our results suggest that melanosomal proteins that interact with melanin may be identified by their relative insolubility in SDS under conditions of increasing melanization. In addition to the si locus protein and other putative melanosomal matrix proteins, the membrane-bound p locus protein may also interact closely with melanin.

Cultured human melanocytes respond to MSH peptides and ACTH.

Although the administration of melanocyte-stimulating hormone (MSH) peptides results in skin darkening in man, cultured human melanocytes have been reported to be unresponsive to these peptides. This may be a consequence of the conditions under which the cells were maintained in vitro, particularly the use of phorbol esters and cholera toxin as melanocyte mitogens. By culturing the cells in the absence of these additives, we demonstrate that alpha-MSH and its synthetic analogue Nle4DPhe7 alpha-MSH (NDP-MSH) induce dose-related increases in melanin content and tyrosinase activity and affect cell morphology in the majority of human melanocyte cultures. In addition, NDP-MSH induces increases in tyrosinase mRNA and tyrosinase-related protein-1 (TRP-1) mRNA. The dose-response curves for the MSH peptides are sigmoidal and the two peptides are equipotent in their effects on human melanocytes. Adrenocorticotropic hormone (ACTH) also affects morphology and stimulates melanogenesis and tyrosinase activity in human melanocytes. However, the dose-response curves for ACTH are biphasic, and the melanocytes respond to lower concentrations of ACTH than MSH peptides, similar to those normally present in human plasma. These findings may be important in understanding the role of these pro-opiomelanocortin peptides in human skin pigmentation.

Identification of a melanosomal matrix protein encoded by the murine si (silver) locus using "organelle scanning".

To identify a broad spectrum of melanosomal proteins, antisera were raised in rabbits against melanosomal protein fractions separated on the basis of their solubility in the nonionic detergent Triton X-114. Antisera against the different fractions each recognized a distinct set of bands when used for immunoblotting analysis with extracts of melanocytes cultured from wild-type black mice. Immunoblotting with antisera to whole melanosomes or to Triton X-114-soluble melanosomal proteins that segregated with the detergent phase gave identical patterns with protein extracts from melanocytes from wild-type mice and from mice homozygous for the si (silver) coat color mutation. By contrast, an antiserum against Triton X-114 soluble melanosomal proteins that segregated in the aqueous phase recognized an 85-kDa protein that was present in extracts from wild-type melanocytes but was absent from si melanocytes. This suggested that the protein was encoded at the si (silver) locus. This was confirmed by employing an antiserum directed against the carboxyl terminus of the predicted murine silver protein sequence. The detergent solubility, biochemical characteristics, and immunologic properties of the 85-kDa protein and of the authentic si gene product were identical. Further analysis demonstrated that this protein corresponds to a melanosomal matrix glycoprotein that we recently described. Our results suggest that employing polyclonal antisera to fractionated organelles such as melanosomes, to screen tissues from mutant mice, a technique that we call "organelle scanning", can serve as a powerful means of identifying new organellar proteins and their respective genes.

Growth and differentiation of normal human melanocytes in a TPA-free, cholera toxin-free, low-serum medium and influence of keratinocytes.

Melanocyte cultures were obtained from a modification of the keratinocyte culture system MCDB153. Either promelanocytes or mature melanocytes were selected from epidermal cell primary cultures. Pure subcultures of actively dividing melanocytes of both types were grown in a low-serum medium totally deprived of TPA and cholera toxin called melanocyte growth medium (MGM). Early passaged cells from MGM primary cocultures were similar to normal adult human melanocytes in vivo, exhibiting numerous melanosomes, strong dopa positivity and a high dendricity. The ability of MGM to support melanocyte growth was mainly a consequence of its basic composition, combined with a low serum concentration. Bovine pituitary extract significantly enhanced melanocyte growth. Using complete MGM, in the absence of mitogens and keratinocytes, cell growth was maintained, but the differentiation of melanocytes decreased. The presence of keratinocytes was found to promote melanocyte growth. The coculture system used strongly suggests the action of soluble keratinocyte-derived factors. Keratinocyte contact was necessary to sustain melanocyte dendricity and melanization. Melanization and dendricity behaved mostly as independent features when keratinocyte influence was withheld. Our results underline the essential role of keratinocytes in the regulation of melanocyte growth and differentiation in a physiological culture system.

Production of epidermal sheets in a serum free culture system: a further appraisal of the role of extracellular calcium.

Rhenwald and Green's technique is currently the standard method for growing stratifying epidermal cell cultures. The serum free system developed in Ham's laboratory (MCDB 153) was designed to grow keratinocyte monolayers in clonogenic conditions. Our aim was to optimize conditions in serum-free MCDB 153 for culturing epidermal sheets from adult normal skin, and to assess the effect of extracellular calcium and temperature on proliferation and differentiation of cultured keratinocytes. Sixteen strains derived from plastic surgery specimens (mean age of donors 37 years; range 5-89) were used. Primary cultures were seeded at an optimal density of 8 x 10(4) cells/cm2 in primary cultures and 10(4) cells/cm2 in secondary cultures in complete medium including EGF, insulin, hydrocortisone and bovine pituitary extract, supplemented with isoleucine, tyrosine, methionine, phenylalanine, tryptophane and histidine. Amino acid (AA) supplementation allows a 5.8-fold increase in cell counts at confluency and monolayers with densely packed cells are obtained. In AA supplemented cultures, confluency is obtained in 16 +/- 3 days in primary cultures and in 13 +/- 0.5 days at first passages. Switches to 1.1 mM calcium at first or second passages resulted in a significant increase in cell counts (P less than 0.001), when compared with AA supplemented low calcium cultures. Low temperature/low calcium cultures resulted in a 50% decrease in cell counts. Low temperature/high calcium cultures gave similar cell counts as the 37 degrees C controls. AA and calcium supplemented cultures were evaluated for differentiation markers: involucrin expression was increased, keratins 5, 6, 14, 17 were expressed, and the sheets were 6-10 layers thick by electron microscopy, with keratohyalin granules and cornified envelopes appearing at layers 3-6 (from basal layer). Dispase treatment allowed an easy detachment of these sheets. These results show that the culture medium MCDB 153 can be adapted without serum supplementation to batch culture of human adult keratinocytes to produce epidermal sheets suitable for grafting. They also indicate that extracellular calcium in physiological range of concentration is not a sufficient signal for growth arrest when other growth conditions are optimized.

The expression of functional MSH receptors on cultured human melanocytes.

Although alpha-MSH increases skin darkening in humans, there are several reports that it fails to have melanogenic effects on human melanocytes in vitro. The purpose of this study was to see whether cultured human melanocytes express MSH receptors. Human melanocytes were grown in the absence of artificial mitogens such as 12-O-tetradecanoyl phorbol-13-acetate (TPA) and cholera toxin (CT) and incubated for 2 h at room temperature with increasing amounts of 125I-labelled Nle4DPhe7-alpha-MSH with and without excess cold peptide. Binding was saturable and specific: Scatchard analysis gave a Kd of 4.9 x 10(-11) M and approximately 700 binding sites/cell. Human keratinocytes and fibroblasts showed no specific binding. The addition of 1 mM dibutyryl cAMP to the culture medium caused a 62% increase in MSH binding to human melanocytes. A smaller increase (25%) was seen with 10(-9) M CT while 25 mM TPA caused a 24% decrease. These results show that human melanocytes in culture express MSH receptors and that this expression can be modulated by mitogens.

Treatment of leg ulcers with an allogeneic cultured-keratinocyte-collagen dressing.

A living cellular allogeneic dressing made up of cultured keratinocytes adhering to a collagen film was used to treat 20 leg ulcers of various aetiologies in 16 patients. A reduction in pain was noted in 80% of cases, and promotion of granulation tissue in the ulcer bed in 70% of cases. In 10 patients, epithelialization of 71 +/- 29% of the ulcer was noted at Day 30.