Abstract | BACKGROUND: OBJECTIVES: 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.
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Authors | C-Y Li, T-W Gao, G Wang, Z-Y Han, Z Shen, T-H Li, Y-F Liu |
Journal | The British journal of dermatology
(Br J Dermatol)
Vol. 150
Issue 6
Pg. 1081-90
(Jun 2004)
ISSN: 0007-0963 [Print] England |
PMID | 15214892
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
- Oligonucleotides, Antisense
- Recombinant Proteins
- Oxidoreductases
- tyrosinase-related protein-1
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Topics |
- Animals
- Apoptosis
- Blotting, Western
(methods)
- Cell Cycle
- Cell Division
- Cell Line, Tumor
- Cells, Cultured
- Flow Cytometry
- Humans
- Melanocytes
(metabolism, pathology)
- Melanoma
(metabolism, pathology)
- Mice
- Mice, Nude
- Neoplasm Transplantation
- Oligonucleotides, Antisense
(pharmacology)
- Oxidoreductases
(analysis, genetics)
- Recombinant Proteins
(genetics)
- Reverse Transcriptase Polymerase Chain Reaction
- Skin Neoplasms
(metabolism, pathology)
- Transfection
(methods)
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