THE USE OF EXPRESSION CLONING TECHNIQUE IN STUDYING HUMAN TUMOR CELLS

表达克隆技术在人类肿瘤细胞研究中的应用

• 批准号: 3774869
• 负责人: S A AARONSON
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3774869
• 关键词: 3T3 cells; Ewing's tumor; complementary DNA; doxorubicin; gene expression; genetic library; genetic mapping; growth factor receptors; human tissue; in situ hybridization; interleukin 1; lung neoplasms; molecular cloning; multidrug resistance; neoplasm /cancer genetics; neoplastic cell; okadaic acid; oncogenes; phenotype; phosphoprotein phosphatase; phosphorylation; polymerase chain reaction; protein kinase; protooncogene; transfection /expression vector

This group utilizes an expression cDNA cloning strategy in addressing contemporary issues in cancer research. (1) Cloning of a transforming serine kinase oncogene, est, was isolated from Ewing's sarcoma cDNA library that represents the proto-oncogenic form of the cot gene, a recently cloned serine kinase oncogene activated presumably by a C- terminal truncation. Expression of est transcripts are extremely low in various cell types examined but could be induced to very high levels by okadeic acid and interleukin-1. by in situ hybridization, est is localized to human chromosome 10, band p12-13, in which the locus for multiple endocrine neoplasia type II is mapped. (2) Development of a new prokaryotic expression vector, an improved cDNA vector for expression cloning in bacterial cells, was developed. A novel dual specificity phosphatase, VHR, was isolated from a cDNA library generated in this vector. VHR mediates the dephosphorylation of activated growth factor receptors as well as serine-phosphorylated casein. (3) Identification of a multidrug resistant gene, to understand the molecular mechanism of resistance to the chemotherapeutic drug adriamycin, a cDNA library, constructed from an HL60 multidrug resistant cell line (HL60R), was introduced into NIH/3T3 cells and one survival colony was isolated. Polymerase chain reaction identified the cDNA responsible for this phenotype as the human mrp gene, a putative ion-transporter gene amplified in an adriamycin-resistant lung tumor cell line. The mrp gene is also amplified (sixtyfold) in HL60R and the NIH/3T3 transfectant provided strong evidence that mrp mediates the multidrug resistant phenotypes.

该小组利用表达 cDNA 克隆策略来解决 癌症研究的当代问题。 (1)转化体的克隆 丝氨酸激酶癌基因 est，从尤文氏肉瘤 cDNA 中分离出来 代表 cot 基因原癌形式的文库， 最近克隆的丝氨酸激酶癌基因可能被 C-激活 终端截断。 est 转录本的表达极低 研究了各种细胞类型，但可以通过以下方法将其诱导至非常高的水平： 冈胶酸和白细胞介素-1。 通过原位杂交，est为 定位于人类 10 号染色体，带 p12-13，其中基因座 绘制了 II 型多发性内分泌肿瘤的图谱。 (2) 开发新的 原核表达载体，一种改进的cDNA表达载体 开发了细菌细胞克隆技术。 新颖的双重特异性 磷酸酶，VHR，是从在此生成的 cDNA 文库中分离出来的。 向量。 VHR 介导活化生长因子的去磷酸化 受体以及丝氨酸磷酸化酪蛋白。 (3) 身份识别 多重耐药基因的研究，了解其分子机制 对化疗药物阿霉素的耐药性，一个 cDNA 文库， 由 HL60 多重耐药细胞系 (HL60R) 构建， 导入NIH/3T3细胞并分离出一个存活集落。 聚合酶链式反应鉴定了造成这种情况的 cDNA 表型为人类 mrp 基因，一种假定的离子转运基因 在阿霉素耐药肺肿瘤细胞系中扩增。 mrp基因 在 HL60R 和 NIH/3T3 转染子中也被扩增（60 倍） 提供了强有力的证据证明mrp介导多重耐药性 表型。