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


OBJECTIVE
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.
Li
C-Y
CY
Department of Dermatology, Xijing Hospital, Fourth Military Medical University. Xi'an 710033, China.
Gao
T-W
TW
Wang
G
G
Han
Z-Y
ZY
Shen
Z
Z
Li
T-H
TH
Liu
Y-F
YF
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western / Q000379:methods; D002453:Cell Cycle; D002455:Cell Division; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D008544:Melanocytes / Q000378:metabolism* / Q000473:pathology; D008545:Melanoma / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010088:Oxidoreductases / Q000032:analysis / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012878:Skin Neoplasms / Q000378:metabolism* / Q000473:pathology; D014162:Transfection / Q000379:methods
D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D010088:Oxidoreductases; C064056:tyrosinase-related protein-1