THE USE OF EXPRESSION CLONING TECHNIQUE IN STUDYING HUMAN TUMOR CELLS

这一组使用了一种表达cDNA克隆策略来处理 癌症研究中的当代问题。(1)一个转化株的克隆 丝氨酸激酶癌基因Est是从尤文氏肉瘤基因中分离得到的 代表COT基因的原致癌形式的文库，一个 最近克隆的丝氨酸激酶癌基因可能是由一种C- 终端截断。EST转录本的表达水平极低 被检查的各种细胞类型，但可能被诱导到非常高的水平 欧卡地酸和白介素1。通过原位杂交，EST是 定位于人类10号染色体，带p12-13，其中 图为多发性内分泌腺瘤病II型。(2)开发一种新的 原核表达载体--一种改进的表达载体 在细菌细胞中的克隆被开发出来。一种新的双重专一性 磷酸酶VHR是从本实验室构建的cDNA文库中分离得到的。 向量。VHR介导激活的生长因子去磷酸化 受体以及丝氨酸磷酸化酪蛋白。(3)识别 多药耐药基因，以了解其分子机制 对化疗药物阿霉素的抗药性，cdna文库， 从HL60多药耐药细胞系(HL60R)构建而成 将其导入NIH/3T3细胞，获得1个存活集落。 聚合酶链式反应鉴定了与此有关的cdna。 表型为人类MRP基因，一个可能的离子转运蛋白基因 在阿霉素耐药的肺肿瘤细胞系中扩增。MRP基因 在HL60R和NIH/3T3转染体中也扩增(60倍) 为MRP介导多药耐药提供了强有力的证据 表型。

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.