GENETIC TARGETS OF NITRIC OXIDE IN LEUKEMIA CELLS

白血病细胞中一氧化氮的基因靶点

• 批准号: 6621777
• 负责人: Paul J Shami
• 金额: $ 20.78万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6621777
• 关键词: NOD mouse; SCID mouse; acute myelogenous leukemia; apoptosis; cell differentiation; cell growth regulation; clinical research; complementary DNA; fusion gene; gene expression; gene targeting; green fluorescent proteins; hematopoietic stem cells; human tissue; laboratory rabbit; microarray technology; nitric oxide; nucleic acid sequence; representational difference analysis; tissue /cell culture; transfection /expression vector

DESCRIPTION: (provided by applicant) Nitric oxide (NO) inhibits growth, induces differentiation and apoptosis in acute myeloid leukemia (AML) cells. Using the technique of Representational Difference Analysis of cDNA (RDA), I have identified a novel gene that I have named regulated by nitric oxide or rno. rno is upregulated by NO in AML cells and is expressed exclusively in hematopoietic cells. A cDNA clone of rno was obtained from a normal leukocyte library. The predicted sequence of rno shows that it belongs to the family of leucine rich repeat proteins and has a high degree of homology to the human ribonuclease inhibitor. PCR and sequencing also revealed that there are at least 3 isoforms of rno. Expression of rno in AML cells inhibits growth, and induces differentiation and apoptosis. The HYPOTHESIS for this proposal is that NO affects the growth and differentiation of AML cells by modulating the expression of specific genes. rno could be involved in mediating the effects of NO on hematopoietic cells. The SPECIFIC AIMS are: AIM 1-Clone and sequence the complete cDNA for rno isoforms: This will be done by screening a cDNA library made from RNA obtained from normal human peripheral blood leukocytes. AIM 2-Demonstrate that rno affects the growth and differentiation of hematopoietic cells. This will be accomplished by transfecting rno into HL-60 cells and determining the effect of rno expression on cell growth and differentiation. The subcellular distribution of rno will be determined using a rno Green Fluorescent Protein fusion gene. rno will be expressed in E. coli and rno protein will be purified to study its effects on RNAse function. Antisera to rno will be raised for further functional studies. rno expression will be determined in normal hematopoietic cells at different stages of differentiation. AIM 3-Identify genes that are modulated by rno: rno will be cloned in an expression vector with an inducible promoter. The vector will be transfected into HL-60 cells. By using cDNA array technology, gene expression differences between rno expressing and non-expressing cells will be identified. AIM 4-Determine the effect of rno expression on AML cell growth in vivo. I will use a human AML xenograft model in NOD/SCID mice. The animals will be inoculated with HL-60 cells that have been transfected with an inducible rno expression vector. Induction of rno expression in the leukemia cells in vivo will be done by treatment of the animals with Tetracycline. The effect of rno expression on leukemia cell growth in vivo will be determined. This work will help understand the mechanism by which NO affects hematopoietic cell growth and differentiation and may constitute the basis for the development of novel strategies for the treatment of AML.

描述：(申请人提供)一氧化氮(NO)抑制生长，诱导 急性髓系白血病(AML)细胞的分化与凋亡使用 代表性差异分析技术(RDA)，我有 发现了一种新基因，我将其命名为受一氧化氮或RNO调控。RNO 在急性髓系白血病细胞中被NO上调，并且仅在造血细胞中表达 细胞。从正常白细胞文库中获得了RNO的cDNA克隆。这个 预测的RNO序列表明它属于富含亮氨酸的家族 重复蛋白，与人类核糖核酸酶高度同源 抑制剂。聚合酶链式反应和测序也显示至少有3种异构体 RNO.RNO在AML细胞中的表达抑制生长并诱导 分化和细胞凋亡。这项提议的假设是没有 影响急性髓系白血病细胞的生长和分化 特定基因的表达。RNO可以参与调节 在造血细胞上没有。具体目标是：目标1-克隆和测序 RNO亚型的完整c DNA：这将通过筛选c DNA文库来完成 从正常人外周血白细胞中提取的RNA制成。目标 2-证明RNO影响造血细胞的生长和分化 细胞。这将通过将RNO导入HL-60细胞和 检测RNO表达对细胞生长和分化的影响。 RNO的亚细胞分布将使用RNO绿来确定 荧光蛋白融合基因。RNO将在大肠杆菌和RNO中表达 蛋白质将被提纯以研究其对核糖核酸酶功能的影响。抗血清 RNO将被提出用于进一步的功能研究。RNO表达式将为 正常造血细胞在不同时期的测定 差异化。目标3-识别受RNO调控的基因：RNO将被 克隆到带有可诱导启动子的表达载体中。向量将是 转染HL-60细胞。利用基因芯片技术，基因表达 RNO表达细胞和非表达细胞之间的差异将被识别。 目的4-确定RNO表达对AML细胞体内生长的影响。这就做 在NOD/SCID小鼠中使用人AML异种移植模型。动物们将会是 用导入诱导型RNO的HL-60细胞接种 表达载体。RNO在白血病细胞中的体内诱导表达 将通过用四环素治疗动物来完成。RNO的作用 白血病细胞在体内生长的表达情况将被确定。 这项工作将有助于理解一氧化氮影响造血的机制。 细胞的生长和分化，并可能构成 开发治疗急性髓细胞白血病的新策略。