Chen Y T, Stockert E, Tsang S, Coplan K A, Old L J
Ludwig Institute for Cancer Research, New York Unit, Memorial Sloan-Kettering Cancer Center, NY 10105, USA.
Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8125-9. doi: 10.1073/pnas.92.18.8125.
Tyrosinase (EC 1.14.18.1), the key enzyme in melanin synthesis, has been shown to be one of the targets for cytotoxic T-cell recognition in melanoma patients. To develop serological reagents useful for immunophenotyping melanoma for tyrosinase, human tyrosinase cDNA was expressed in an Escherichia coli expression vector. The purified recombinant tyrosinase was used to generate mouse monoclonal and rabbit polyclonal antibodies. The prototype monoclonal antibody, T311, recognized a cluster of protein moieties ranging from 70 to 80 kDa in tyrosinase mRNA-positive melanoma cell lines and melanoma specimens as well as in L cells transfected with tyrosinase cDNA. Untransfected L cells and L cells transfected with tyrosinase-related protein 1, TRP-1(gp75), were nonreactive. Immunohistochemical analysis of melanomas with T311 showed tyrosinase in melanotic and amelanotic variants, and tyrosinase expression correlated with the presence of tyrosinase mRNA. Melanocytes in skin stained with T311, whereas other normal tissues tested were negative. The expression pattern of three melanosome-associated proteins--tyrosinase, TRP-1(gp75), and gp100--in melanoma was also compared. Tyrosinase and gp100 are expressed in a higher percentage of melanomas than TRP-1(gp75), and the expression of these three antigens was discordant. Tyrosinase expression within individual tumor specimen is usually homogenous, distinctly different from the commonly observed heterogeneous pattern of gp100 expression.
酪氨酸酶（EC 1.14.18.1）是黑色素合成中的关键酶，已被证明是黑色素瘤患者中细胞毒性T细胞识别的靶标之一。为了开发用于黑色素瘤酪氨酸酶免疫表型分析的血清学试剂，人酪氨酸酶cDNA在大肠杆菌表达载体中表达。纯化的重组酪氨酸酶用于制备小鼠单克隆抗体和兔多克隆抗体。原型单克隆抗体T311在酪氨酸酶mRNA阳性的黑色素瘤细胞系、黑色素瘤标本以及转染了酪氨酸酶cDNA的L细胞中识别出一组分子量在70至80 kDa之间的蛋白质部分。未转染的L细胞和转染了酪氨酸酶相关蛋白1（TRP-1，gp75）的L细胞无反应。用T311对黑色素瘤进行免疫组织化学分析显示，黑色素瘤的色素沉着型和无色素型变体中均有酪氨酸酶，且酪氨酸酶表达与酪氨酸酶mRNA的存在相关。皮肤中的黑素细胞用T311染色呈阳性，而其他检测的正常组织为阴性。还比较了黑色素瘤中三种黑素小体相关蛋白——酪氨酸酶、TRP-1（gp75）和gp100——的表达模式。与TRP-1（gp75）相比，酪氨酸酶和gp100在更高比例的黑色素瘤中表达，且这三种抗原的表达不一致。单个肿瘤标本中酪氨酸酶表达通常是均匀的，与gp100表达常见的异质性模式明显不同。
