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: 3458540
• 负责人: MALFORD E CULLUM
• 金额: $ 8.26万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 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/3458540
• 关键词: affinity chromatography; antineoplastics; cell bank /registry; cell differentiation; gene expression; karyotype; monoclonal antibody; mutagens; myelogenous leukemia; neoplasm /cancer genetics; 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.

本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，一个中心未解决的问题，在维甲酸生物学和 生物化学 最近开发了新的方法， 使用修饰剂治疗白血病患者 白血病前期细胞的分化和生长特性; 13-顺- 和全反式视黄酸是有前途的药剂。 13-顺式维甲酸的有益临床效果已被证实， 描述了急性早幼粒细胞白血病患者亚组 白血病 维甲酸类药物在临床上具有重要的应用价值 如果药物的毒性和耐药性问题可以克服。 由于13-顺式-视黄酸对人体的毒性相对较低， 与全反式维甲酸相比， HL-60细胞的分化，控制机制， 这些类维生素A的生物活性应该被定义。 该提案的具体目标是：（1）建立一个稳定的 13顺维甲酸抗性HL 60（分化缺陷， HL60D-）细胞系从原始分化阳性 HL 60 D+，其特征在于增殖能力， 硝基蓝四唑还原能力的改变， 核型和二维电泳分析， 将其与HL-60 D+进行比较; 2）从HL-60 D+产生回复突变体 和HL60D-已经用乙基甲烷诱变 不会区分的磺酸盐（EMS），HL60EMSD-，或 分化，HL60 EMSD+存在13顺式维甲酸; 3） 确定13顺维甲酸在HL-60细胞中的定位。 HL-60 D+、HL-60 EMSD-和HL 60 EMSD+治疗后 13-顺式维甲酸，使用多克隆和单克隆 抗（13顺维甲酸血蓝蛋白）抗体; 4） 表征来自HL60 D+、D-、EMSD+和EMSD-的蛋白质 与视黄酸相互作用， 氨基酸序列分析 13cis-维甲酸在基因表达中的作用将是 考察 基因表达将在1） 染色体水平核型分析，2）表型水平 通过开发13顺维甲酸抗性细胞系，3） 通过选择性转化诱变HL 60的基因型水平 （点突变）型诱变剂，EMS和4）在蛋白质 通过监测特定细胞中转氨酶的表达， 对维甲酸诱导分化的反应。 基因 将通过将细胞水平上的表达与细胞水平上的表达相关联来评估表达。 13顺视黄酸的亚细胞定位和 在HL 60和构建的HL 60变体中的差异。