GENETIC TARGETS OF NITRIC OXIDE IN LEUKEMIA CELLS

白血病细胞中一氧化氮的基因靶标

• 批准号: 6436675
• 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/6436675
• 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：（由申请人提供）一氧化氮（NO）抑制生长，诱导 急性髓性白血病（AML）细胞的分化和凋亡。使用 cDNA的代表性差异分析（RDA）技术， 我发现了一种新的基因，我命名为受一氧化氮或rno调控的基因。rno 在AML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rno的cDNA克隆。的 预测的rno序列表明，它属于富含亮氨酸的家族， 重复蛋白，并与人核糖核酸酶具有高度同源性 抑制剂. PCR和测序也显示至少有3种异构体 的rno。rno在AML细胞中的表达抑制生长，并诱导 分化和凋亡。该提案的假设是， 影响AML细胞的生长和分化， 特定基因的表达。rno可能参与介导 对造血细胞无影响。具体目标是：AIM 1-克隆和测序 rno亚型的完整cDNA：这将通过筛选cDNA文库来完成 由正常人外周血白细胞中的RNA制成。目的 2-证明rno影响造血干细胞的生长和分化 细胞这将通过将rno转染到HL-60细胞中来实现， 确定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绿色 荧光蛋白融合基因。rno将在E.大肠杆菌和rno 蛋白质将被纯化以研究其对RNA酶功能的影响。抗血清 将提出rno以进行进一步的功能研究。rno表达式将是 在不同阶段的正常造血细胞中测定， 分化目的3-识别由rno调节的基因： 克隆到具有诱导型启动子的表达载体中。矢量将是 转染HL-60细胞。利用cDNA阵列技术， 将鉴定rno表达细胞和非表达细胞之间的差异。 目的4-确定rno表达对体内AML细胞生长的影响。我会 在NOD/SCID小鼠中使用人AML异种移植物模型。将动物 接种已转染诱导型rno的HL-60细胞， 表达载体在体内诱导白血病细胞表达rno 将通过用四环素处理动物完成。rno的作用 将测定对体内白血病细胞生长的表达。 这项工作将有助于了解NO影响造血的机制 细胞的生长和分化，并可能构成的基础， 开发治疗AML的新策略。