Development of the adenovirus vector containing upstream region of tyrosinase gene

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By using recombinant adenovirus which express one of the p53 family proteins, induction of apoptosis was studied in melanoma cell lines. 1) p53 family (p53, p51A, p73β) cDNAs were synthesized by RT-PCR from human brain RNA.Then, recombinant adenoviruses (Ad-p53, Ad-p51A and Ad-p73β) which express one of the p53 family proteins by human cytomegalovirus early promoter were constructed. p53 of six human melanoma cell lines were analyzed by yeast functional assay. One of them (70W) was found to contain mutant p53 with a missense mutation. Three cell lines (SK-mel-23, SK-mel-118 and 70W) showed apoptotic cell death by infection of p53-, p51A- and p73β-expressing adenoviruses. Among p53 family members, p51A induced apoptosis most significantly than p53 or p73β in melanoma cells. SK-mel-118 cells infected with p51A contained activated caspase-3. 2) Tyrosinase, an essential enzyme which convert tyrosine to dopa in the initial step of melanin biosynthesis, is transcribed specifically in melanocytes. We determined nucleotides of 3.6 kb promoter sequence upstream of tyrosinase gene, and constructed recombinant plasmid (p3.6-p53 and p3.6-p51A) and adenovirus (Ad3.6-p53 and Ad3.6-p51A) which contain the 3.6 kb tyrosinase promoter upstream of p53 and p51A.p3.6-p53 induced apoptosis in melanoma cells but p3.6-p51A enhanced growth of melanoma cells. From these, it is suggested than recombinant adenoviruses which express p53 family proteins are good candidate for melanoma gene therapy, but further study is required for melanoma-specific expression to induce cell death by p53 and its related genes by transcription from tyrosinase promoter.