| TRP-1 (tyrosinase related protein 1,TRP-1) or glycoprotein 75 is the most abundant glycoprotein in melanocytes. It is well know that TRP-1 has an important role in the maturation and stability of melanomsomes, site organelles of the mealanin synthesis and deposition. TRP-1 mutation affects the expression of brown color by functioning as 5,6-dihydroxyindole-2-carboxylic acid (DHICA) oxidase The human homologue of TRP-1 of the mouse brown gene maps to the short arm of chromosome 9 to the known region of homology with mouse chromosome 4. However, it has been shown that DHICA oxidase activity is limited only to the murine TRP-1, and not to humans . The biological role of TRP-1 is still controversial and not well known in humans.In order to identify the biological function of the TRP-1 gene and encoding product in human melanogenesis, Hara exposed human melanocytes and melanoma cells to UVB. Those cells with high tyrosinase activity and melanin sythesis revealed increased signals for mRNA and protein expression of tyrosinase and TRP-1. In another study, they found that the single transfectants of human tyrosinase gene revealed early cellthat the single transfectants of human tyrosinase gene revealed early cell death during melanogensis and this programmed cell death was inhibited by the cotransfection of human TRP-1 gene . TRP-1 has been suggested to alter the activity of tyrosinae by stabilization that can be attributed either to direct protection of tyrosinase itself from degeneration by toxic melanin intermediates or to the involvment of TRP-1 in the glycosylation and trafficking of tyrosinase. Within the multimeric complex formed by TRP-1 enzymes in melanosomal membrane, TRP-1 play an important role in stabilization of tyrosinase, thus indirect controlling the melanin production . There are extensive genetic studies regarding gene changes of TRP-1 in brown mutant in mice providing basic information and explanation of allelism and phenotypes of mouse TRP-1 mutation . Some recessive TRP-1 mutations involve single amino acid substitutions. Changing an arginine residue to the signal sequence cysteine resulting in either complete loss function, partial loss of function or temperature sensitive function . Dominant mutations affecting the TRP-1 gene have also arisen as a result of a base pair mutation, destabilizing the melanosomal membrane and allowing the intermediates of melanogenesis to interfere with normal melanocyte function. Another type of dominant mutation may arise due to the TRP-1 gene having undergone some rearrangment; as a result, TRP-1 is not properly transcribed .Leading to melanocyte desfunction or death. It has therefore been suggested that mutant TRP-1 protein is involved in cell degeneration or death of melanocytes, which is associated with a fault in scavenging inherently toxic products of the melanogenesis process.It can not be excluded that TRP-1 protein play an important role in the preliferation and function of melanocytes. But the question should be answer whether the TRP-1 directly effect the preliferation and function of melanocytes? In order to answer such a question, in this study we attempted to look at the possible role of TRP-1 in melanocytes and melanoma cells with antisence technology and RNAi technology. The main methods and results are:1.Construction of TRP-1 antisense eukaryotic expression vectors and the confirmation on the transfected cells.The antisense eukaryotic expression vectors pcDNA3.1/TRP-1(-) was constructed by subcloning the target gene into eukaryotic expression vector pcDNA3.1(+). The direction was confirmed by endonuclease digestion. Using liposome mediated method, the TRP-1 highly exprssed human melanocytes and huamn malignant melanoma cells(Libr) were transfected with the antisense recominant vector and control plasmid. Stable clones Libr -AS and Libr -P were obtained after G418 screening. Western blot studies and RT-PCR assay testified that the protein and mRNA level were both down-regulated in the transfected Libr -AS cell...
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