The effect of antisense tyrosinase-related protein 1 on melanocytes and malignant melanoma cells.

Abstract

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.

• 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)

Chemical References

• Oligonucleotides, Antisense
• Recombinant Proteins
• Oxidoreductases
• tyrosinase-related protein-1

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)