PURPOSE: To establish methodology for the culture of human choroidal melanocytes to compare their responsiveness to melanocyte stimulating hormone (MSH) with that of their transformed melanoma counterparts and with that of the retinal epithelial cell. METHODS: Choroidal melanocytes from the choroid of eyes enucleated for the presence of malignant melanoma were cultured in MCDB 153 medium supplemented with insulin, transferrin, hydrocortisone, glutamine, nystatin, vitamin E, phorbol myristate acetate, bovine hypothalamic extract, cholera toxin, and chelexed fetal calf serum. RESULTS: High yields of pure spindle-shaped bipolar melanocytes were obtained with a doubling time of 3 to 4 days in nine consecutive eyes. Cells continued to divide after 4 months in culture. In contrast, uveal malignant melanoma cells grew rapidly in a relatively simple medium of Ham's F12:DMEM (1:1) supplemented with fetal calf serum, insulin, transferrin and glutamine. This medium was unable to support choroidal melanocytes. Choroidal melanocytes were DOPA-positive with appreciable tyrosinase activity that significantly increased with treatment with MSH. MSH also significantly altered the size, local density, and distribution of primary and mature melanosomes of ocular melanocytes. In contrast, uveal melanoma cells had a low level of tyrosinase activity and failed to respond to MSH with either an increase in enzyme activity or melanosome size. Retinal epithelial cells failed to show significant tyrosinase activity under the conditions studied or any increase in melanosome size in response to MSH. CONCLUSION: Ocular melanocytes show evidence of regulation by MSH and a range of mitogenic stimuli unlike the transformed melanoma cells, implying a loss of regulatory control in the latter.
PURPOSE: To establish methodology for the culture of human choroidal melanocytes to compare their responsiveness to melanocyte stimulating hormone (MSH) with that of their transformed melanoma counterparts and with that of the retinal epithelial cell. METHODS: Choroidal melanocytes from the choroid of eyes enucleated for the presence of malignant melanoma were cultured in MCDB 153 medium supplemented with insulin, transferrin, hydrocortisone, glutamine, nystatin, vitamin E, phorbol myristate acetate, bovine hypothalamic extract, cholera toxin, and chelexed fetal calf serum. RESULTS: High yields of pure spindle-shaped bipolar melanocytes were obtained with a doubling time of 3 to 4 days in nine consecutive eyes. Cells continued to divide after 4 months in culture. In contrast, uveal malignant melanoma cells grew rapidly in a relatively simple medium of Ham's F12:DMEM (1:1) supplemented with fetal calf serum, insulin, transferrin and glutamine. This medium was unable to support choroidal melanocytes. Choroidal melanocytes were DOPA-positive with appreciable tyrosinase activity that significantly increased with treatment with MSH. MSH also significantly altered the size, local density, and distribution of primary and mature melanosomes of ocular melanocytes. In contrast, uveal melanoma cells had a low level of tyrosinase activity and failed to respond to MSH with either an increase in enzyme activity or melanosome size. Retinal epithelial cells failed to show significant tyrosinase activity under the conditions studied or any increase in melanosome size in response to MSH. CONCLUSION: Ocular melanocytes show evidence of regulation by MSH and a range of mitogenic stimuli unlike the transformed melanoma cells, implying a loss of regulatory control in the latter.