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BackgroundMelanocytic proliferations are common in horses but the diagnosis of malignancy is not always straightforward. To improve diagnosis and prognosis, markers of malignancy are needed. Receptor for activated C kinase 1 (RACK1) protein may be such a marker. RACK1 was originally found to characterize malignant melanocytic lesions in the Melanoblastoma-bearing Libechov minipig (MeLiM) and, later, in human patients. Our purpose was to investigate the value of RACK1 in the classification of cutaneous melanocytic proliferations in horses.ResultsUsing immunofluorescence, we report here that both MITF (Microphthalmia-associated transcription factor) and PAX3 (Paired box 3) allow the identification of melanocytic cells in horse skin samples. Importantly, RACK1 was detected in melanocytic lesions but not in healthy skin melanocytes. Finally, we found that RACK1 labeling can be used in horses to distinguish benign melanocytic tumors from melanomas. Indeed, RACK1 labeling appeared more informative to assess malignancy than individual histomorphological features.ConclusionsThis study confirms that horses provide an interesting model for melanoma genesis studies. It establishes MITF and PAX3 as markers of horse melanocytic cells. RACK1 emerges as an important marker of malignancy which may contribute to progress in the diagnosis of melanomas in both human and veterinary medicine.
Background: Constitutive activation of the ERK pathway, occurring in the vast majority of melanocytic neoplasms, has a pivotal role in melanoma development. Different mechanisms underlie this activation in different tumour settings. The Grey phenotype in horses, caused by a 4.6 kb duplication in intron 6 of Syntaxin 17 (STX17), is associated with a very high incidence of cutaneous melanoma, but the molecular mechanism behind the melanomagenesis remains unknown. Here, we investigated the involvement of the ERK pathway in melanoma development in Grey horses. Methods: Grey horse melanoma tumours, cell lines and normal skin melanocytes were analyzed with help of indirect immunofluorescence and immunoblotting for the expression of phospho-ERK1/2 in comparison to that in non-grey horse and human counterparts. The mutational status of BRAF, RAS, GNAQ, GNA11 and KIT genes in Grey horse melanomas was determined by direct sequencing. The effect of RAS, RAF and PI3K/AKT pathways on the activation of the ERK signaling in Grey horse melanoma cells was investigated with help of specific inhibitors and immunoblotting. Individual roles of RAF and RAS kinases on the ERK activation were examined using si-RNA based approach and immunoblotting.
Summary
The paired box gene 3 (Pax3) is expressed during pigment cell development. We tested whether the targeted allele Pax3GFP can be used as a reporter gene for pigment cells in the mouse. We found that enhanced green fluorescent protein (GFP) can be seen readily in every melanoblast and melanocyte in the epidermis and hair follicles of Pax3GFP/+ heterozygotes. The GFP was detected at all differentiation stages, including melanocyte stem cells. In the dermis, Schwann cells and nestin‐positive cells of the piloneural collars resembling the nestin‐positive hair follicle multipotent stem cells exhibited a weaker GFP signal. Pigment cells could be purified by fluorescent activated cell sorting and grown in vitro without feeder cells, giving pure cultures of melanocytes. The Schwann cells and nestin‐positive cells of the piloneural collars were FACS‐isolated based on their weak expression of GFP. Thus Pax3GFP can discriminate distinct populations of cells in the skin.
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