Development of the adenovirus vector containing upstream region of tyrosinase gene

通过使用表达p53家族蛋白之一的重组腺病毒，研究了在黑素瘤细胞系中诱导凋亡。1)以人脑RNA为模板，通过RT-PCR合成p53家族（p53，p51 A，p73β）cDNA，构建了人巨细胞病毒早期启动子（human cytomegalovirus early promoter，HCMV）p53家族蛋白的重组腺病毒（Ad-p53，Ad-p51 A，Ad-p73β）。应用酵母功能分析法对6株人黑色素瘤细胞系的p53进行了分析。其中一个（70 W）被发现含有错义突变的突变型p53。三种细胞系（SK-mel-23、SK-mel-118和70 W）通过表达p53、p51 A和p73β的腺病毒感染显示凋亡性细胞死亡。在p53家族成员中，p51 A比p53或p73β更能诱导黑色素瘤细胞凋亡。感染p51 A的SK-mel-118细胞含有活化的caspase-3。2)酪氨酸酶是黑素生物合成的起始步骤中将酪氨酸转化为多巴的必需酶，在黑素细胞中特异性转录。我们测定了酪氨酸酶基因上游3.6kb启动子序列的核苷酸序列，构建了含有酪氨酸酶基因上游3.6kb启动子的重组质粒（p3.6-p53和p3.6-p51 A）和腺病毒（Ad3.6-p53和Ad3.6-p51 A）。因此，表达p53家族蛋白的重组腺病毒是黑色素瘤基因治疗的良好候选者，但需要进一步研究黑色素瘤特异性表达p53及其相关基因通过酪氨酸酶启动子转录诱导细胞死亡。

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.