BRAF, HRAS, KRAS, NRAS and CDKN2A genes analysis in cultured melanocytes used for vitiligo treatment

Abstract

Cultured pigment cells are increasingly being used in the treatment of stable vitiligo. The melanocyte growth media contain synthetic and human recombinant mitogenic factors. High concentration of growth factors, increased melanin biosynthesis, and the rapid cell cycle progression may lead to the genetic material damage and the initiation of melanocyte malignant transformation in cell culture conditions. Mutations of genes of the RAS/RAF/MEK/ERK signaling pathway and CDKN2A gene are often found in the early stages of melanoma development. 12-O-Tetradecanoyl-phorbol-13-acetate (TPA)/phorbol 12-myristate 13-acetate (PMA) is considered to be an oncogenic factor, but there is no evidence that it is responsible for melanomagenesis initiation. The goal of this research was to assess the risk of the development of mutations in selected genes of the RAS/RAF/MEK/ERK signaling pathway and CDKN2A gene during the culturing of pigment cells in various growth media. Three-hundred melanocyte cultures were established in 10 various growth media. The population doubling time of cultured cells was calculated for all the tested growth media. Cytogenetic analysis was carried out on the HRAS (exon 1 and 2), KRAS (exon 1 and 2), NRAS (exon 1 and 2), BRAF (exon 11 and 15), and CDKN2A (exon 1) genes. Our study revealed that TPA and high concentrations of other growth factors intensify the proliferation of pigment cells, without the risk of damage to the analyzed genes. It is necessary to carry out further similar studies on other signaling pathways to confirm cultured melanocytes transplantation safety.