CLONING THE GENE FOR A NOVEL TPA-INDUCED PROTEIN

克隆新型 TPA 诱导蛋白质的基因

• 批准号: 3195521
• 负责人: JUDITH K CHRISTMAN
• 金额: $ 23.05万
• 依托单位: BARBARA ANN KARMANOS CANCER INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 1992-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3195521
• 关键词: antisense nucleic acid; chemical carcinogenesis; complementary DNA; gene expression; genetic mapping; hematopoiesis; hematopoietic stem cells; laboratory mouse; molecular cloning; molecular oncology; monoclonal antibody; platelets; transfection; tumor promoters

We have recently characterized and partially purified what appears to be a unique plasma membrane protein, "HEMP". HEMP is found on the surface of normal human eosinophils, megakaryocytes and platelets, but is not detectable on other normal hematopoietic cells or cells in frozen sections of normal adult tissues. HEMP is a 19 kD protein that is post- translationally modified by covalent linkage of lipid to yield a mature form with an apparent MW of 21 kd. Binding of a HEMP-specific monoclonal antibody to HEMP on platelets causes aggregation and ATP release, suggesting that HEMP may be an accessory protein in signal transduction from surface receptors. Initial studies also suggest that a careful characterization of HEMP and the regulation of its synthesis has the potential to enhance understanding of tumorigenesis and the interactions between transformed cells and tumor promoters. We have found that exposure, in culture, to the tumor promoter, 12-0- tetradecanoyl phorbol 13-acetate (TPA), induces expression of HEMP by fetal marrow cells, blast cells of patients with acute monocytic leukemia and several human tumor cell lines. TPA does not induce HEMP expression by normal circulating leukocytes. This indicates that regulation of the HEMP gene is different in fetal and adult hematopoietic cells and is altered in neoplastic cells. Expression of HEMP may also be associated with the ability of some human tumor cells to grow in nude mice, i.e. cells cultured from tumors formed in nude mice by human cell lines that were either inducible for HEMP synthesis or contained a HEMP (+) subpopulation gave rise to lines that express HEMP constitutively. Our immediate goals are: 1. To purify HEMP for peptide analysis and preparation of monospecific and monoclonal antibodies. Peptide sequences will be used either for preparation of nucleotide probes and/or confirmation of cDNA clone identity. 2. To clone the cDNA and gene(s) for HEMP, DNA sequence analysis will allow determination of HEMP's amino acid sequence, the organization of its gene and identification of putative promoter and regulatory sites. It may also give clues as to HEMP's function. 3. To functionally map regulatory sites in the HEMP gene(s). Information derived from characterization of HEMP and the gene(s) that encode it will be the basis for future studies aimed at elucidating the role of tumor promoters in regulating the HEMP gene and determining HEMP's function in normal and transformed cells.

我们最近已经鉴定并部分纯化了一种 独特的质膜蛋白，“HEMP”。HEMP是在 正常人嗜酸性粒细胞，巨核细胞和血小板，但不是 在其他正常造血细胞或冷冻切片细胞上可检测到 正常的成人组织。HEMP是一种19 kD的蛋白质， 通过脂质的共价连接进行修饰，以产生成熟的 表观分子量为21 kd。HEMP特异性单克隆抗体的结合 血小板上的HEMP抗体引起聚集和ATP释放， 提示HEMP可能是信号转导的辅助蛋白 从表面受体。初步研究还表明， HEMP的特性及其合成的调节具有 提高对肿瘤发生及其相互作用的理解的潜力 转化细胞和肿瘤促进剂之间的关系。 我们发现，在培养物中暴露于肿瘤促进剂12-0- 十四酰基佛波醇13-乙酸酯（TPA）诱导胎儿HEMP表达 急性单核细胞白血病患者的骨髓细胞、母细胞， 几种人类肿瘤细胞系。TPA不诱导HEMP表达， 正常的循环白细胞这表明HEMP的调节 基因在胎儿和成人造血细胞中是不同的， 肿瘤细胞HEMP的表达也可能与 某些人肿瘤细胞在裸鼠中生长的能力，即培养的细胞 由人细胞系在裸鼠中形成的肿瘤， 可诱导HEMP合成或含有HEMP（+）亚群的细胞， 上升到组成型表达HEMP的细胞系。 我们的近期目标是：1。纯化HEMP用于肽分析， 单特异性和单克隆抗体的制备。肽序列 将用于制备核苷酸探针和/或 cDNA克隆同一性的确认。 2.为了克隆cDNA和基因， HEMP，DNA序列分析将允许确定HEMP的氨基酸 序列，其基因的组织和鉴定推定的 启动子和调控位点。它也可能提供线索， 功能 3.对HEMP基因的调控位点进行功能定位。 来自HEMP表征的信息和 编码它将是未来研究的基础，旨在阐明 肿瘤促进剂在调节HEMP基因和决定HEMP的 在正常和转化细胞中发挥作用。