Relative responsiveness of cultured human epidermal melanocytes and melanoma cells to selected mitogens.

Early cellular events in the malignant transformation of melanocytes to melanoma are virtually unknown. In vitro investigation of this phenomenon has been hampered by the fastidious nature of the human epidermal melanocyte, which has proven difficult to cultivate. The present study compares responsiveness of cultured human epidermal melanocytes and established melanoma cell lines to serum, cholera toxin, and melanocyte growth factor (MGF), three established melanocyte mitogens. Four of four established human melanoma lines were substantially stimulated by fetal bovine serum, as were newborn foreskin-derived epidermal melanocytes. In contrast, none of the four melanoma lines responded to hypothalamic preparations containing MGF that consistently produced an approximately 30-fold increase in newborn melanocyte cell yield over a 2-week period. Cholera toxin, required for successful establishment of primary melanocyte cultures, had small and variable effects on the melanoma lines, with slight stimulation in one case, moderate inhibition in another, and essentially no effect in two others. These data suggest that transformation of epidermal melanocytes to melanoma often involves at least one phenotypic change resulting in escape from MGF regulation and another associated with insensitivity to cyclic AMP modulation; while at least some of the pathways conferring serum dependence are unaltered. Improved culture systems for the human epidermal melanocyte should facilitate further studies into the mechanism of its malignant conversion and may provide useful insights for the prevention and treatment of human melanoma.

Selective cultivation of human melanocytes from newborn and adult epidermis.

Development of adequate culture systems for the human epidermal melanocyte is critical to further advances in pigment cell biology. We now report selective growth and long-term maintenance of melanocytes derived from both newborn and adult skin specimens. Disaggregated epidermal cell suspensions were plated in a hormone-supplemented medium containing cholera toxin and a hypothalamic extract treated to remove keratinocyte growth-promoting activity. After 3-4 weeks, pure melanocyte populations could be harvested and serially passaged up to 6 times over several months for a total of 10 or more cumulative population doublings in vitro. Electron microscopic studies revealed metabolically active cells with abundant melanosomes in various stages of melanization throughout the culture lifespan. Differences in size and number of melanosomes attributable to race of the tissue donor were readily apparent, and pigment content of melanocytes from both black and Caucasian donors appeared to increase with time in culture. Newborn melanocytes proliferated more rapidly and survived longer than did adult melanocytes, but there were no consistent morphologic differences as a function of donor age. Comparison of growth potential for the 3 major skin-derived cell types in this hormone-supplemented medium revealed striking specificity for melanocytes, with total elimination of keratinocytes over 1-2 weeks, and no fibroblast proliferation whatever in the absence of serum supplementation. This system promises to facilitate in vitro investigation of epidermal melanocytes in normal and diseased human skin.

Autocrine and paracrine growth stimulation of cells derived from human skin.

A sensitive serum-free culture system was used to demonstrate that cells derived from normal human skin release soluble mediators which can modulate keratinocyte, melanocyte, and fibroblast growth in vitro. In M199 supplemented with epidermal growth factor, hydrocortisone, insulin, transferrin, triidothyronine, bovine serum albumin, and an extract of bovine hypothalamus, keratinocytes underwent approximately three to five cumulative population doublings (CPD) over a 7-day period. Addition of 20% keratinocyte-conditioned medium more than doubled keratinocyte growth and increased fibroblast growth 25-40% above controls. Dermal fibroblasts maintained in the same serum-free hormone-supplemented medium (SFHSM) alone underwent approximately 4.5-5.5 CPD over a 7-day period; 20% fibroblast-conditioned medium increased fibroblast growth 50-80% and increased keratinocyte growth 30-50%. Melanocytes maintained in the same SFHSM underwent approximately 1 CPD over a 14-day period and 2 CPD if the medium was supplemented with 2% fetal bovine serum (FBS). Addition of 20% melanocyte-conditioned medium more than doubled melanocyte growth in either SFHSM or in medium containing 2% FBS, but decreased both keratinocyte and fibroblast growth up to 30-60%. None of the conditioned media altered cellular morphology. These data provide the first demonstration of mutual growth modulation by cell types normally contiguous in vivo and expand existing evidence for autocrine and paracrine growth regulation by normal human cells.