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: 3458546
• 负责人: MALFORD E CULLUM
• 金额: $ 9.33万
• 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 1992-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3458546
• 关键词: 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.

这项建议的目的是确定控制机制 负责维甲酸诱导的分化和逆转 恶性肿瘤是维甲类生物学中尚未解决的中心问题， 生物化学。最近，新的方法在 使用改良剂治疗白血病患者 白血病前期细胞的分化和生长特性；13-顺式- 而全反式维甲酸一直是很有前途的药物。 13-顺式维甲酸的有益临床疗效已被证实 描述了一组急性早幼粒细胞白血病患者 白血病。类维A酸在临床上具有重要的实际意义。 药物，如果毒性和耐药性问题可以克服的话。 由于13-顺式维甲酸对人体的毒性相对较低 与全反式维甲酸相比，但在 HL-60细胞分化的调控机制 这些维甲酸的生物活性应该被定义。 这项提议的具体目标是：1)发展稳定的 13顺式维甲酸耐药HL60(分化缺陷， HL60D-)细胞系从原代分化阳性 以增殖能力为特征的HL60D+， 硝基蓝四氮唑还原能力的改变，由 核型和双向电泳法分析 将其与HL-60D+进行比较；2)从HL60D+创建回复变种 和HL60D-已被乙基甲烷诱变 不会区分的磺酸盐(EMS)、HL60EMSD-或 在13顺-维甲酸存在下分化，HL60EMSD+；3) 鉴定13顺式维甲酸在HL-1细胞中的定位 60-、HL60D+、HL-60EMSD-和HL60EMSD+ 13-顺式维甲酸的多克隆和单克隆应用 抗(13顺式维甲酸血蓝蛋白)抗体； HL60D+、D-、EMSD+和EMSD-蛋白质的特性 与维甲酸相互作用，通过亲和层析和 氨基酸序列分析。 13顺式维甲酸在基因表达中的作用将是 检查过了。基因表达的评估将在1) 核型分析的染色体水平，2)表型水平 通过建立13顺式维甲酸抗性细胞系，3) 选择性转化诱变HL60的基因水平 (点突变)型诱变剂EMS和4)在蛋白质上 通过监测转谷氨酰胺酶在特定组织中的表达水平 对维甲酸诱导分化的反应。基因 表达将在细胞水平上评估，通过关联 13顺式维甲酸的亚细胞定位及其作用能力 区分HL60和构建的HL60变异体。