Blackwell Publishing Journal Backfiles 1879-2005
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 G1 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.
Type of Medium: