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The effect of antisense tyrosinase‐related protein 1 on melanocytes and malignant melanoma cells
Author(s) -
Li CY.,
Gao TW.,
Wang G.,
Han ZY.,
Shen Z.,
Li TH.,
Liu YF.
Publication year - 2004
Publication title -
british journal of dermatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.304
H-Index - 179
eISSN - 1365-2133
pISSN - 0007-0963
DOI - 10.1111/j.1365-2133.2004.05929.x
Subject(s) - tyrosinase , melanoma , cancer research , melanin , medicine , chemistry , microbiology and biotechnology , biology , biochemistry , enzyme
Summary Background Tyrosinase‐related proteins (TRPs) include tyrosinase, TRP‐1 and TRP‐2. The functions of tyrosinase and TRP‐2 have been determined, but the biological role of TRP‐1 is still controversial and is not well known in humans. Objectives To study further the biological role of the human TRP‐1 gene in melanocytes and melanoma cells. Methods TRP‐1 cDNA was subcloned into eukaryotic expression vector pcDNA3.1 in the reverse direction, and antisense recombinant vector was transfected into melanocytes and a melanoma cell line using Lipofectamine 2000. Positive cells were selected by geneticin. TRP‐1 mRNA level was measured by reverse transcription–polymerase chain reaction (RT–PCR), and TRP‐1 protein level by Western blot. Cell cycles were determined by flow cytometry, and the activity of tyrosinase was evaluated by L ‐DOPA reaction. Light microscopy, electron microscopy and flow cytometry were used to observe cell morphology and apoptosis. For in vivo assays, the antitumour activity of antisense TRP‐1 against the malignant melanoma (MM) cell line, Libr, was evaluated in an animal‐tumour model of subcutaneous tumours. Results Positive transfected cells steadily expressed TRP‐1 antisense RNA. RT–PCR and Western blot showed a low level of TRP‐1 mRNA and TRP‐1 protein, respectively. Cell cycles were blocked in the G 1 stage, and the activity of tyrosinase decreased significantly ( P < 0·01). Light and electron microscopy showed abnormal cell morphology, and apoptosis was detected. The neoplasia activity of antisense TRP‐1‐transfected MM cells was significantly lower than that of MM cells ( P < 0·01). Conclusions TRP‐1 plays an important role in the proliferation, morphology and tyrosinase activity of melanocytes and melanoma cells.