FUNCTION OF 13-CIS-RETINOIC ACID IN HL-60 CELLS

13-顺式-维甲酸在 HL-60 细胞中的功能

• 批准号: 3458545
• 负责人: MALFORD E CULLUM
• 金额: $ 9.18万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-06 至 1993-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3458545
• 关键词: acute leukemia; affinity chromatography; antineoplastics; cell bank /registry; cell differentiation; gene expression; karyotype; laboratory mouse; laboratory rabbit; monoclonal antibody; mutagens; myelogenous leukemia; neoplasm /cancer genetics; protein sequence; retinoate

The object of this proposal is to identify the control mechanisms responsible for retinoic acid induced differentiation and reversal of malignancy, a central unsolved problem in retinoid biology and biochemistry. Recently novel approaches have developed in treating patients with leukemia using agents that modify differentiation and growth properties of preleukemic cells; 13-cis- and all-trans-retinoic acids have been promising agents. Beneficial clinical effects of 13-cis-retinoic acid have been described for a subgroup of patients with acute promyelocytic leukemia. Retinoids have a practical importance in clinical medicine if toxicity and resistance problems can be overcome. Since 13-cis-retinoic acid has a relatively low toxicity in humans compared to all-trans-retinoic acid but is equipotent in differentiation of HL-60 cells, the mechanisms that control the biologic activity these retinoids should be defined. The specific aims of this proposal are 1) to develop a stable 13cisretinoic acid resistant HL60 (differentiation defective, HL60D-) cell line from the original differentiation positive HL60D+ that is characterized by proliferative capacity, alteration in nitroblue tetrazolium reduction capacity, by karyotypic and two dimensional electrophoretic analysis and compare it to HL-60D+; 2) to create revertants from HL60D+ and HL60D- that have been mutagenized with ethyl methane sulfonate (EMS) that will not differentiate, HL60EMSD-, or differentiate, HL60EMSD+ in presence of 13cis-retinoic acid; 3) to identify the cellular localization of 13cisretinoic acid in HL- 60-, HL60D+, HL-60EMSD- and HL60EMSD+ after treatment with 13-cis-retinoic acid using polyclonal and monoclonal anti(13cisretinoic acidhemocyanin) antibody; 4) to characterize the proteins from HL60D+, D-, EMSD+, and EMSD- that interact with retinoic acid, by affinity chromatography and amino acid sequence analysis. The role of 13cis-retinoic acid in gene expression will be examined. Gene expression will be evaluated at 1) the chromosome level by karyotypic analysis, 2) the phenotypic level by developing a 13cisretinoic acid resistant cell line, 3) the genotypic level by mutagenizing HL60 with a selective transition (point mutation) type mutagenic agent, EMS and 4) at the protein level by monitoring expression of transglutaminase in a specific response for retinoic acidinduced differentiation. Gene expression will be assessed at the cellular level by correlating the subcellular localization of 13cisretinoic acid and the ability to differentiate in HL60 and in constructed HL60 variants.

该提案的目的是确定控制机制 负责视黄酸诱导的分化和逆转 恶性肿瘤是类维生素A生物学中一个未解决的核心问题 生物化学。 最近开发了新方法 使用修饰药物治疗白血病患者 白血病前期细胞的分化和生长特性； 13-顺式- 和全反式视黄酸一直是有前途的药物。 13-顺式视黄酸的有益临床效果已被证实 描述了一组患有急性早幼粒细胞疾病的患者 白血病。 类维生素A在临床上具有重要的实际意义 如果可以克服毒性和耐药性问题。 由于13-顺式视黄酸对人体的毒性相对较低 与全反式视黄酸相比，但在以下方面是等效的 HL-60细胞的分化，控制机制 应定义这些类维生素A的生物活性。 该提案的具体目标是 1) 开发稳定的 13顺视黄酸抗性HL60（分化缺陷， HL60D-)细胞系由原始分化阳性 HL60D+的特点是增殖能力， 硝基蓝四唑还原能力的改变，通过 核型和二维电泳分析和 与HL-60D+比较； 2) 从 HL60D+ 创建回复体 和 HL60D- 已用乙基甲烷诱变 不会区分的磺酸盐 (EMS)、HL60EMSD-，或 区分 HL60EMSD+ 在 13cis-视黄酸存在下； 3） 鉴定 HL- 中 13 顺视黄酸的细胞定位 60-、HL60D+、HL-60EMSD- 和 HL60EMSD+ 治疗后 13-顺式视黄酸使用多克隆和单克隆 抗(13顺视黄酸血蓝蛋白)抗体； 4）到 表征 HL60D+、D-、EMSD+ 和 EMSD- 的蛋白质 通过亲和色谱法与视黄酸相互作用 氨基酸序列分析。 13cis-视黄酸在基因表达中的作用将是 检查了。 基因表达将在 1) 处进行评估 通过核型分析进行染色体水平，2）表型水平 通过开发13顺视黄酸抗性细胞系，3) 通过选择性转变诱变 HL60 来提高基因型水平 (点突变)型诱变剂、EMS和4)处的蛋白质 通过监测特定区域中转谷氨酰胺酶的表达水平 视黄酸诱导分化的反应。 基因 表达将在细胞水平上通过关联 13顺视黄酸的亚细胞定位及其能力 HL60 和构建的 HL60 变体之间存在差异。