STUDIES ON HUMAN HEPATOMA CELL SURFACE PROTEIN P50

我们最近通过单克隆抗体鉴定了一种细胞表面 在B型肝炎病毒相关的 原发性肝癌 p50在正常人中检测不到， 成人和胎儿的组织，肾上腺除外。 一个非常 类似蛋白质也已被鉴定和部分纯化 牛的肾上腺 我们希望研究它的结构和细胞 该蛋白的功能，并评估其可能的作用， 恶性转化 在本建议中，我们将重点关注三个方面 p50的主要方面如下：1）一级结构：在这个 因此，我们计划从人肝癌细胞和牛肝癌细胞中纯化p50。 通过亲和色谱法将肾上腺纯化至均一。 我们将 通过N-末端测序确定其一级结构。 我们将 还通过产生新的单克隆抗体来分析免疫显性表位， 纯化蛋白的抗体。 2)分子生物学：在这 鉴于我们已经构建了肝癌细胞cDNA文库， 原核和真核表达载体中的polyA（+）RNA。 我们计划使用我们的特异性单克隆抗体， p50 cDNA的免疫选择。 我们将确定基因组 通过双脱氧核苷酸组织感兴趣的cDNA插入片段 测序 3)功能分析：我们将研究p50 在正常和再生组织中mRNA和蛋白水平的表达 与肾上腺相比，肝脏和人类肿瘤。 我们将 将cDNA克隆转染到哺乳动物细胞中以获得模型 用于瞬时和组成型表达p50的系统。 测试 p50的过度产生是否会改变 建立的细胞，代表性的细胞系将在 细胞形状的变化，接触抑制的丧失， 免疫缺陷裸鼠的永生化和致瘤性。 这些调查将有助于更好地了解 肝癌相关基因表达的生物学意义 p50与肝细胞癌的发病机制 在迄今为止研究的所有人类肿瘤细胞系中也发现， 调查可能对恶性肿瘤有更广泛的意义。 哺乳动物细胞的转化。

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.