Retroviral Infection with Human Tyrosinase-Related Protein-1 (TRP-1) cDNA Upregulates Tyrosinase Activity and Melanin Synthesis in a TRP-1-Deficient Melanoma Cell Line

A human tyrosinase-related protein-1 (TRP-1) cDNA was inserted into the retroviral vector, pBabe-puro. Sense and anti-sense constructs were identified and transfected, as well as vector-alone, into a retrovirus packaging cell line by a liposome-mediated technique and used in turn to infect a human melanoma line deficient in TRY-1 protein/transcript. Polymerase chain reaction (PCR) amplification of genomic DNA from these infectants, using TRY-1 cDNA-specific primers, demonstrate that PCR products were only identified from the sense- and anti-sense-infected clones, not from the parental cells or vector-alone infectants. Northern analysis demonstrated that TRP-1 sense and antisense infectants produced TRY-1 cDNA-related transcripts. Immunoblotting analysis with TA99 (a monoclonal antibody for TRP-1) demonstrated a single band of nonilal molecular weight from melanoma cells infected with sense cDNA, not from cells infected with anti-sense or vector-alone, or from the uninfected-parental melanoma cells. The quantitative and qualitative analysis of melanin in the sense and anti-sense infectant cells demonstrated an increase and decrease in pigmentation, respectively, compared with vector alone. Tyrosine hydroxylase and DOPA oxidase activities of tyrosinase were both increased in sense cDNA infected cells plus unaltered or slightly decreased, respectively, in anti-sense cDNA-infected cells compared with control cells. Immunoblotting analysis with anti-tyrosinase antibody (αTy-SP) demonstrated that the amount of tyrosinase was slightly increased in TRP-1 overexpressing cells but slightly decreased in anti-sense infectant cells. We have demonstrated that the expression of exogenous TRP-1 cDNA in melanoma cells stimulated the activity of tyrosinase and promoted melanogenesis, indicating that TRP-1 plays a role in regulating tyrosinase activity.