HUMAN HEPATOMA CELL SURFACE PROTEIN P50

我们最近用单抗鉴定了一个细胞表面 蛋白(P50)在乙肝病毒相关蛋白中持续存在 原发性肝细胞癌。P50在正常人群中检测不到 成人和胎儿组织，肾上腺除外。一个非常好的 类似的蛋白质也已被鉴定和部分纯化。 来自牛的肾上腺。我们希望研究它的结构和细胞 该蛋白的功能(S)，并评价其在 恶变。在这份提案中，我们将重点关注三个方面 P50的主要方面如下：1)初级结构：在这个 关于我们计划从人肝癌细胞和牛身上提纯p50 用亲和层析法将肾上腺转化为均一。我们会 通过N-末端测序确定其一级结构。我们会 还通过产生新的单克隆来分析免疫优势表位 纯化蛋白的抗体。2)分子生物学： 我们已经从肝癌细胞中构建了c DNA文库 原核和真核表达载体中的Polya()RNA。 我们计划将我们的特异性单抗用于 P50基因的免疫选择。我们将测定基因组 用双脱氧核苷酸组织目的基因插入片段 测序。3)功能分析：我们将研究p50 正常和再生组织中mRNA和蛋白水平的表达 与肾上腺相比，肝脏和人类肿瘤也是如此。我们会 将克隆基因S导入哺乳动物细胞建立动物模型 P50的瞬时和本构表达系统。为了测试 P50的过度生产是否会改变细胞的生物学特性 已建立的细胞，有代表性的细胞系将在 细胞形状的改变，接触抑制的丧失， 免疫缺陷裸鼠的永生化和致瘤性。 这些调查将使我们能够更好地了解 肝细胞癌相关基因表达的生物学意义 P50和肝细胞癌AS p50的发病机制 也在迄今为止研究的所有人类肿瘤细胞系中发现，如 调查可能对恶性犯罪具有更广泛的意义 哺乳动物细胞的一般转化。

We have recently identified by monoclonal antibodies a cell surface protein (p50) consistently found in hepatitis B virus - related primary hepatocellular carcinomas. p50 was undetectable in normal adult and fetal tissues except in the adrenal glands. A very similar protein has also been identified and partially purified from bovine adrenals. We wish to study the structure and cellular function(s) of this protein and evaluate its possible role(s) in malignant transformation. In this proposal we will focus on three major aspects of p50 as follows: 1) Primary structure: In this regard we plan to purify p50 from human hepatoma cells and bovine adrenals to homogeneity by affinity chromatography. We will determine its primary structure by N-terminal sequencing. We will also analyze immunodominant epitopes by generating new monoclonal antibodies to the purified protein. 2) Molecular biology: In this regard we have constructed cDNA libraries from hepatoma cell polyA(+) RNAs in procaryotic and eucaryotic expression vectors. We plan to use our specific monoclonal antibodies for immunoselection of p50 cDNA's. We will determine genomic organization of cDNA inserts of interest by dideoxynucleotide sequencing. 3) Functional analysis: We will investigate p50 expression at mRNA and protein level in normal and regenerative liver as well as human tumors compared to adrenal glands. We will transfect cDNA clone(s) into mammalian cells to obtain model systems for transient and constitutive expression of p50. To test whether p50 overproduction could alter biological properties of established cells, representative cell lines will be studied in terms of changes of cell shape, loss of contact inhibition, immortalization and tumorigenicity in immunodeficient nude mice. These investigations will permit a better understanding of the biological significance of the hepatoma-associated expression of p50 and the pathogenesis of hepatocellular carcinoma As p50 was also found in all human tumor cell lines thus far studied, such investigations may have a broader significance to the malignant transformation of mammalian cells in general.