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: 6763232
• 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 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6763232
• 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 急性髓系白血病（AML）细胞的分化和凋亡。使用 cDNA 代表性差异分析 (RDA) 技术，我有 发现了一个新的基因，我将其命名为受一氧化氮或 rno 调节的基因。诺 在 AML 细胞中被 NO 上调，并且仅在造血细胞中表达 细胞。 rno的cDNA克隆是从正常白细胞文库中获得的。这 rno的预测序列表明它属于富含亮氨酸的家族 重复蛋白质，与人类核糖核酸酶具有高度同源性 抑制剂。 PCR和测序还显示至少有3种亚型 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：否 通过调节 AML 细胞的生长和分化 特定基因的表达。 rno 可能参与调节影响 对造血细胞无NO。具体目标是： AIM 1-克隆并测序 rno 同工型的完整 cDNA：这将通过筛选 cDNA 文库来完成 由从正常人外周血白细胞获得的RNA制成。目的 2-证明rno影响造血细胞的生长和分化 细胞。这将通过将 rno 转染到 HL-60 细胞中并 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 Green 来确定 荧光蛋白融合基因。 rno将在大肠杆菌和rno中表达 蛋白质将被纯化以研究其对 RNAse 功能的影响。抗血清 rno 将被筹集用于进一步的功能研究。 rno 表达式将是 正常造血细胞不同阶段的测定 差异化。 AIM 3-识别受rno调节的基因：rno将是 克隆到具有诱导型启动子的表达载体中。该向量将是 转染至HL-60细胞中。利用cDNA阵列技术，基因表达 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 小鼠中使用人类 AML 异种移植模型。动物们将是 接种已用诱导型 rno 转染的 HL-60 细胞 表达载体。体内白血病细胞rno表达的诱导 将通过用四环素治疗动物来完成。 rno的作用 将确定对体内白血病细胞生长的表达。 这项工作将有助于理解NO影响造血的机制 细胞生长和分化，可能构成细胞生长和分化的基础 开发治疗 AML 的新策略。

项目成果

Gene expression profiling for nitric oxide prodrug JS-K to kill HL-60 myeloid leukemia cells.

• DOI: 10.1016/j.ygeno.2009.03.005
• 发表时间: 2009-07
• 期刊: Genomics
• 影响因子: 4.4
• 作者: Liu J;Malavya S;Wang X;Saavedra JE;Keefer LK;Tokar E;Qu W;Waalkes MP;Shami PJ
• 通讯作者: Shami PJ

JS-K has potent anti-angiogenic activity in vitro and inhibits tumour angiogenesis in a multiple myeloma model in vivo.

• DOI: 10.1211/jpp.62.01.0017
• 发表时间: 2010-01
• 期刊: The Journal of pharmacy and pharmacology
• 作者: Kiziltepe T;Anderson KC;Kutok JL;Jia L;Boucher KM;Saavedra JE;Keefer LK;Shami PJ
• 通讯作者: Shami PJ