MYELOID CELL EXPRESSED ONCOGENE

骨髓细胞表达癌基因

• 批准号: 2443059
• 负责人: DENIZ TOKSOZ-EXLEY
• 金额: $ 11.92万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2443059
• 关键词: 3T3 cells; G protein; athymic mouse; biological signal transduction; chromosomes; complementary DNA; gene expression; genetic mapping; human genetic material tag; in situ hybridization; laboratory rabbit; mutant; myeloid stem cell; neoplastic transformation; northern blottings; nucleic acid sequence; oncogenes; open reading frames; phosphorylation; polymerase chain reaction; protooncogene; recombinant proteins; site directed mutagenesis; southern blotting; transfection

The utilization of the NIH-3T3 cell transfection method has enabled the isolation of several novel oncogenes. The goal of this project is to investigate the mechanism of activation and function of the novel human lbc transforming gene obtained by NIH-3T3 transfection with human leukemic DNA and isolated by its ability to form tumors in nude mice. The lbc cDNA sequence shows no identity to known sequences, and encodes a predicted hydrophilic protein of approximately 47 kD. Lbc is expressed in human myeloid and lymphoid cells and muscle, lung and heart tissue. Transfection of NIH-3T3 cells with lbc cDNA results in morphologically transformed foci in culture, demonstrating the biological activity of the lbc cDNA, and presenting a functional in vitro assay for the analysis of lbc transforming activity. The following aims are proposed. A. Preliminary studies indicate that the lbc oncogene represents a novel transcription unit derived from fusion of a truncated lbc proto-oncogene located on chromosome 15 with heterologous sequence encoded on chromosome 7. In order to fully elucidate the mechanism of activation, it is proposed to obtain complete lbc proto-oncogene cDNA sequence from normal hematopoietic tissues, to analyze the transforming activity of the proto-oncogene and its mutant forms, and to precisely define the breakpoint by genomic sequence analysis. B. Human samples/cell lines from hematopoietic malignancies and disorders will be screened for activated lbc genes, based on lbc expression. The exact location of the lbc gene on human chromosome 15, and in the mouse will be pinpointed and compared with the position of genetic loci known to be involved in hematologic and other diseases in human and mouse. C. The transforming activity of lbc will be analyzed by focusing on two functionally implicated domains: 1. A dbl domain associated with regulatory activity for the ras superfamily of small G proteins which control cell proliferation and cytoskeletal organization; this domain is also encoded in the dbl and vav oncogenes, the bcr gene and the yeast cell cycle gene CDC24. 2. A PH (pleckstrin homology) domain found in several intracellular signal transducing molecules, thought to define a novel protein interaction domain. These domains will be subjected to site-directed mutagenesis, and mutants assayed using the 3T3 focus forming assay. D. In order to study lbc function, antibodies will be derived to identify the lbc protein, to determine its cellular localization and to analyze lbc phosphorylation. Based on the activity associated with the dbl domain encoded in other oncogenes, it is proposed to test already available recombinant lbc protein for regulatory activity against a panel of ras-like small G proteins. Furthermore, the role of lbc in the p2lras signal transduction pathway will be analyzed using a well- characterized dominant negative ras mutant Nl7. These proposed experiments will elucidate the transforming activity and function of the novel lbc oncogene.

NIH-3T3细胞转染法的应用使 分离出几个新的癌基因。这个项目的目标是 研究新人的激活和功能机制 NIH-3T3转染人白血病细胞获得LBC转化基因 DNA，并因其在裸鼠体内形成肿瘤的能力而被分离。LBC基因的cDNA 序列与已知序列没有相同之处，并编码预测的 亲水性蛋白约为47kD。LBC在人类体内表达 髓系细胞和淋巴样细胞以及肌肉、肺和心脏组织。转染法 携带LBC基因的NIH-3T3细胞出现形态转化灶 在培养中，展示了LBC cDNA的生物学活性，以及 一种用于LBC分析的功能性体外分析方法 转型活动。提出了以下目标。A.初步报告 研究表明，LBC癌基因代表了一种新的转录 来自截短的LBC原癌基因融合的单位 15号染色体和7号染色体上编码的异源序列。按顺序 为了充分阐明激活的机制，建议获得 正常人外周血淋巴细胞原癌基因全序列分析 组织中，分析原癌基因的转化活性和 它的突变形式，并通过基因组准确地定义断裂点 序列分析。B.来自造血系的人体样本/细胞系 恶性肿瘤和疾病将筛查激活的LBC基因，基于 关于LBC表达式。LBC基因在人类染色体上的准确定位 15，并将鼠标中的位置与所指位置进行比较 已知与中国血液病和其他疾病有关的遗传基因 人和老鼠。C.对LBC的转化活性进行分析 关注两个功能相关的域：1.DBL域 与小G基因ras超家族的调节活动相关 控制细胞增殖和细胞骨架组织的蛋白质； 该结构域也编码在Dbl和VaV癌基因中，bcr基因和 酵母细胞周期基因CDC24。2.Ph(Pleckstrin Homology)结构域 在几个细胞内信号转导分子中发现，被认为是 定义了一个新的蛋白质相互作用结构域。这些域名将受到 到定点突变，并使用3T3焦点检测突变体 形成化验。D.为了研究淋巴细胞的功能，抗体将被 衍生来鉴定LBC蛋白，以确定其细胞 定位并分析LBC的磷酸化。基于活动 与其他癌基因中编码的DBL结构域相关，提出了 测试已有的重组LBC蛋白的调节活性 对抗一组ras样的小G蛋白。此外，LBC的作用 在p2lras信号转导通路中，我们将使用Well- 具有显性负ras突变体n17的特征。这些拟议的实验 将阐明新的LBC的转化活性和功能 致癌基因。