CELL DEATH INDUCED BY ANTICANCER AGENTS

抗癌剂引起的细胞死亡

• 批准号: 2093662
• 负责人: ALAN R EASTMAN
• 金额: $ 24.09万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-30 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2093662
• 关键词: DNA replication; DNA topoisomerases; acidity /alkalinity; antibody formation; antineoplastics; apoptosis; biological signal transduction; cell cycle; cell cycle proteins; cell death; cis platinum compound; complementary DNA; cytotoxicity; deoxyribonuclease I; drug adverse effect; drug metabolism; fluorescent dye /probe; gel electrophoresis; hyperthermia; laboratory rabbit; molecular cloning; molecular genetics; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; protein kinase C

Cell death induced by anticancer agents involves many targets but in each case, a subsequent step involves endonuclease-mediated digestion of cellular DNA. This DNA digestion is characteristic of "programmed cell death" or "apoptosis" and is considered to represent an essential step in the process of cell death. The timing of apoptotic DNA degradation after equitoxic treatment varies with agent occurring within 30 min after hyperthermia but 3 days after cisplatin. In the latter case, death occurs at the G2/M phase of the cell cycle. The first goal of this proposal is to purify and characterize an acidic endonuclease, identified as deoxyribonuclease II, that has been implicated in cell death after hyperthermia. The purification has been achieved and a partial amino acid sequence obtained. Based on this sequence, oligonucleotides will be synthesized and used to isolate the cDNA. Antibodies will also be raised. The cDNA and antibodies will be used as probes to investigate the normal function of this endonuclease and its contribution to cell death: one approach will involve suppression or enhancement of endonuclease activity. The role of intracellular acidification after lethal insults will be investigated using a pH sensitive fluorescent dye. The structure of the cut ends of DNA will be investigated as a potential diagnosis of the activity of this endonuclease. Another goal is to establish a cause or effect relationship between cell death and the p34cdc2 kinase that regulates passage into mitosis. Assays will be performed to confirm that the kinase activity is suppressed during G2 arrest and to determine whether activity increases again as cells progress toward death. A cell line temperature sensitive for p34cdc2 kinase will be used to resolve the cause or effect relationship. Other experiments will investigate the role of the endonuclease and p34cdc2 in cell death following other insults including aphidicolin, and conditions considered as physiologic cell death (e.g. glucorticoid-mediated death of thymocytes). Subsequent experiments will investigate the regulation of the endonuclease and kinase. The outcome of these experiments may be the development of new strategies for drug development, for synergistic therapy, and for overcoming drug resistance.

抗癌剂诱导的细胞死亡涉及许多靶标，但在每个目标中 案例，随后的步骤涉及核酸内切酶介导的消化 细胞DNA。 该DNA消化是“编程细胞的特征” 死亡”或“凋亡”，被认为是重要的一步 细胞死亡的过程。 凋亡DNA降解后的时机 均毒性治疗随着后30分钟的发生而变化 高温，但在顺铂后3天。 在后一种情况下，死亡发生 在细胞周期的G2/m期。 该提议的第一个目标是 纯化和表征酸性内切核酸酶，被确定为 脱氧核糖核酸酶II，与细胞死亡有关 热疗。 已经实现了纯化和部分氨基酸 获得的序列。 基于此序列，寡核苷酸将是 合成并用于分离cDNA。 抗体也将升高。 cDNA和抗体将用作研究正常的探针 该核酸内切酶的功能及其对细胞死亡的贡献：一个 方法将涉及抑制或增强核酸内切酶活性。 致命损伤后细胞内酸化的作用将是 使用pH敏感荧光染料进行了研究。 结构 DNA的切割末端将作为潜在的诊断 这种核酸内切酶的活性。 另一个目标是建立原因或 细胞死亡与p34CDC2激酶之间的效应关系 调节导致有丝分裂。 将进行测定以确认 激酶活性在G2逮捕过程中被抑制，并确定是否是否 随着细胞向死亡的发展，活动再次增加。 细胞系 对p34CDC2激酶的温度敏感性将用于解决原因 或效果关系。 其他实验将研究 核酸内切酶和p34CDC2在其他侮辱之后的细胞死亡中 蚜虫蛋白和被视为生理细胞死亡的疾病（例如 糖苷介导的胸腺细胞死亡）。 随后的实验将 研究核酸内切酶和激酶的调节。 结果 这些实验可能是制定药物新策略的发展 开发，协同疗法以及克服耐药性。