Reduction of Melanogenic Activity and Responsiveness to α-Melanocyte-Stimulating Hormone during Serial Passage of Melanoma Cells

Abstract

Background: Murine melanoma cells such as Cloudman S91 or B16 mouse melanoma cells have been used extensively to study mechanisms involved in pigmentation because these cells have tyrosinase, the key enzyme in pigmentation, and produce pigment. We have observed that serial passaged S91 cells tend to decrease their basal pigment content and to lose their responsiveness to α-melanocyte-stimulating hormone (α-MSH). Objective: Because this reduction of melanogenic capacity is a widely acknowledged but virtually unstudied characteristic of both human and murine melanoma cell lines in culture, we wished to document and quantify the phenomenon. Methods: Commercially attained S91 melanoma cells were serially passaged. Basal pigmentation as well as α-MSH responsiveness and expression of protein kinase C-beta (PKC-β) were assessed. Results: S91 cells progressively lost their basal pigmentation under standardized conventional conditions of culture, from an initial melanin content of 20 ± 4 pg/cell content to 12 ± 5 pg/cell within 70 population doublings (16 passages) and to 4.5 ± 6 pg/cell, a level at or below the detectable level of our melanin assay, by 110 population doublings (28 passages). When responsiveness to α-MSH was assessed, a 6-day treatment with 10−6M α-MSH initially induced the pigment content four-fold from 20 ± 4 to 82 ± 2 pg/cell. In contrast, after 110 population doublings, identical treatment with α-MSH induced pigment content less than two-fold from 4.5 ± 6 to 7.5 ± 2 pg/cell. PKC-β expression was readily detected by immunofluorescence in early passage pigmented cells, but not in late passage nonpigmented cells. Conclusion: These results confirm that while murine melanoma cells are a useful model system for pigmentation studies, it is important to monitor changes in the cells' basal abilities to pigment and to respond to exogenous pigment-inducing factors. They further suggest that factors in the culture environment or the internal milieu of melanoma cells exposed to continuous mitogenic stimulation inhibit melanogenesis. One candidate mechanism is down regulation of PKC-β.