PIGMENT CONTENT OF CULTURED HUMAN MELANOCYTES DOES NOT CORRELATE WITH TYROSINASE MESSAGE LEVEL
BRITISH JOURNAL OF DERMATOLOGY
Authors: NAEYAERT, JM; ELLER, M; GORDON, PR; PARK, HY; GILCHREST, BA
Abstract
Tyrosinase is considered to be the rate-limiting enzyme for the biosynthesis of melanin in epidermal melanocytes, and thus tyrosinase activity is thought to be a major regulatory step in melanogenesis. To determine whether the rate of pigment production was controlled at the level of tyrosinase gene expression, we developed a culture system capable of generating large populations of pure human melanocytes and then measured both melanin content as determined spectrophotometrically by absorption at 475 nm and mRNA levels as detected by hybridization with cloned cDNA Pmel 34, encoding human tyrosinase. We examined the relationship between pigment content and tyrosinase mRNA levels among human melanoma and melanocyte lines with very different levels of basal pigmentation; between two clones of a single human melanoma line, one pigmented and one amelanotic; and sequentially in melanocytes before and after simulation with isobutylmethylxanthine to increase melanin content per cell. Using Northern blot analysis and in-situ hybridization we found no correlation between tyrosinase message levels and melanin content, suggesting that post-transcriptional regulation of tyrosinase and/or other events determine the rate of pigment synthesis in human melanocytes.
Melanoma-associated antigens as messenger RNA detection markers for melanoma
CANCER RESEARCH
Authors: Sarantou, T; Chi, DDJ; Garrison, DA; Conrad, AJ; Schmid, P; Morton, DL; Hoon, DSB
Abstract
Melanoma is heterogeneous for its biological properties and melanoma-associated antigens (MAAs), This diversity is partially observed in the expression of the MAAs involved with the melanin synthesis pathway, We therefore developed a sensitive multimarker reverse transcription-PCR plus Southern blot assay using five MAAs as molecular markers to detect primary and metastatic melanoma cells, Melanoma cell lines, melanocytes (cultured), primary and metastatic malignant melanoma tissues, and blood from patients with American Joint Committee on Cancer stage I-IV melanoma were assessed for tyrosinase, tyrosinase-related proteins 1 and 2, Pmel 17, and MART-1/melan-A. All of the MAA mRNA markers were expressed in 100% of melanoma cell lines and cultured melanocytes, 74% of primary and metastatic tumors (excluding tumor-draining lymph nodes), 43% of tumor-involved lymph nodes, and 43% of patients' bloods, Hypomelanotic melanoma tissues expressed a lower frequency of individual. mRNA markers, Overall, at least one mRNA marker was expressed in more than 86% of specimens assayed, Normal tissue specimens from patients and blood from normal volunteer donors were negative for MAA mRNA expression, The multimarker MAA reverse transcription-PCR plus Southern blot analysis was more reliable and sensitive than a single-molecular marker assay for the detection of melanoma cells. This molecular assay can also provide information on MAA mRNA expression of metastatic melanoma cells that may assist in monitoring the therapeutic efficacy of active specific immunotherapy toward specific MAA-bearing melanomas.
COA
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