FUNCTIONAL ANALYSIS OF THE HUMAN LEUKEMIA GENE E2A-PBX1

人类白血病基因E2A-PBX1的功能分析

• 批准号: 3201291
• 负责人: MARK P KAMPS
• 金额: $ 18.72万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-10 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3201291
• 关键词: 3T3 cells; B lymphocyte; DNA binding protein; athymic mouse; bone marrow; carcinogenesis; cell differentiation; cell growth regulation; chimeric proteins; colony stimulating factor; gene expression; genetic regulatory element; genetic transcription; hematopoiesis; homeobox genes; human genetic material tag; leukemia; molecular cloning; molecular genetics; myelogenous leukemia; neoplasm /cancer genetics; neoplastic transformation; neutrophil; nucleic acid hybridization; nucleic acid sequence; oncogenes; polymerase chain reaction; protein purification; protein structure function; tissue /cell culture; transcription factor

The objective of this proposal is to understand how expression of an altered form of PBX1, the prototype of a new homebox gene family, alters hematopoiesis and induces leukemia in man. I have shown that expression of a fusion transcript between the transcription factor gene, E2A, and the homebox gene, PBX1, is the genetic consequence of the t(1;19) translocation of childhood pre-B cell acute lymphocytic leukemia, and that the presumptive chimeric transcription factor encoded by this transcript produces myeloid leukemia in mice. Discovery of this new human oncogene prompts three distinct questions: first, what biochemical activities are required to make E2A-Pbx1 a transforming gene (Aims 1 and 2); second, what specific cellular events does E2A-Pbx1 alter to produce leukemia (Aim 3); and third, do other PBX-related genes control elements of hematopoetic differentiation (Aim 4). Aim 1: Determine the structural domains of E2A-Pbx1 required for transformation. Whereas E2A-pbx1 cDNA's are in hand, a full-length pbx1 cDNA must be isolated. The leukemic potential of normal Pbx1, an aminoterminal truncation of Pbx1 at the E2A- Pbx1 junction, and Pbx1 fusion proteins with other transactivation domains will then be examined in mice. Analysis of a shorter spliced variant of E2A-Pbx1 that lacks 7 Kda's of Pbx1 will permit further resolution of the functional requirements of Pbx1. Aim 2: Determine the transcriptional activity of Pbx1 and E2A-Pbx1. Recombinant Pbx1 proteins will be expressed in bacteria, purified, and then used to select a Pbx1 DNA-binding sequence from degenerate oligonucleotides or genomic DNA fragments. Transcriptional activities of Pbx1 and E2A-Pbx1 will then be assessed by cotransfecting a reporter construct (CAT), transcriptionally regulated by the Pbx1-responsive DNA sequence, with an expression plasmid, producing forms of Pbx1 or E2A-Pbx1. Transcriptional properties will then be correlated with transforming ability to determine a biochemical basis for transformation. Aim 3: Examine the cellular effects of E2A-Pbx1. The ability of E2A-Pbx1 to alter the growth and/or differentiation of granulocyte and granulocyte-macrophage, colony- forming cells from mouse marrow will be examined in vitro. The ability of E2A-Pbx1 to inhibit inducible differentiation of cell lines in vitro will also be tested. These results will reveal whether E2A-Pbx1 alone can mediate, the factor-dependent, differentiation-inhibited, in vivo phenotype of E2A-Pbx1-induced leukemias. Aim 4: Initiate studies to determine whether other PBX1 homologues are expressed during hematopoesis. The possibility that other Pbx1 homologs are expressed during hematopoetic differentiation and bind the Pbx recognition sequences will be addressed by cloning PBX homologues, using the known sequence identity between PBX1 and PBX2 to develop identification strategies. New cloned PBX CDNA'S will provide the basis to initiate studies on the role of PBX gene expression during hematopoeisis, as well as provide a basis for determining whether other PBX genes are involved in other types of human malignancy.

该提议的目的是了解如何表达 PBX1的改变形式，新的Homebox基因家族的原型，变化 造血和诱导人的白血病。我已经表明了表达 转录因子基因，E2a和 Homebox基因PBX1是t（1; 19）的遗传结果 儿童期前B细胞急性淋巴细胞性白血病和 由此编码的推定嵌合转录因子 转录本在小鼠中产生髓样白血病。发现这个新的 人类癌基因提示三个不同的问题：首先，什么生化 需要活动才能使E2A-PBX1成为转化基因（目标1和 2）;其次，E2A-PBX1改变了哪些特定的细胞事件以产生 白血病（目标3）；第三，其他与PBX相关的基因控制元件 造血分化（目标4）。目标1：确定结构 转换所需的E2A-PBX1域。而E2A-PBX1 cDNA 在手中，必须隔离全长PBX1 cDNA。白血病 正常PBX1的潜力，PBX1在PBX1处的截断 E2A-PBX1结和与其他反式激活的PBX1融合蛋白 然后将在小鼠中检查域。较短的剪接分析 缺少7 kDa的pBx1的E2A-PBX1变体将允许进一​​步 PBX1功能要求的分辨率。目标2：确定 PBX1和E2A-PBX1的转录活性。重组PBX1蛋白 将在细菌中表达，纯化，然后用于选择PBX1 来自退化寡核苷酸或基因组DNA的DNA结合序列 碎片。然后，PBX1和E2A-PBX1的转录活动将是 通过共转移记者结构（CAT）评估，转录 由PBX1反应性DNA序列调节，表达式 质粒，产生PBX1或E2A-PBX1的形式。转录特性 然后将与确定A的转换能力相关 转化的生化基础。目标3：检查细胞 E2A-PBX1的效果。 E2A-PBX1改变增长和/或 粒细胞和粒细胞巨噬细胞的分化，菌落 - 将在体外检查由小鼠骨髓形成细胞。能力 E2A-PBX1的抑制细胞系的诱导分化 也将进行测试。这些结果将揭示是否仅E2A-PBX1 可以在体内介导，依赖因子依赖性，分化抑制 E2A-PBX1诱导的白血病的表型。目标4：开始学习 确定是否在 血小化。表达其他PBX1同源物的可能性 在造血分化过程中并结合PBX识别 将使用已知的PBX同源物克隆来解决序列 PBX1和PBX2之间的序列身份以开发鉴定 策略。新的克隆PBX cDNA将提供启动的基础 关于PBX基因表达在造血中的作用的研究， 作为确定是否涉及其他PBX基因的基础 在其他类型的人类恶性肿瘤中。