MECHANISMS OF PROGRAMMED CELL DEATH IN CHEMOTHERAPY

拟议研究的目标是确定程序化的角色 细胞死亡在化疗药物作用中的作用，从而鉴定生化和 易受或保护细胞免受核酸内切酶影响的代谢因素 DNA损伤后的激活。程序性细胞死亡，或称凋亡，是 通过形态和生化特征可与坏死性区分， 通常发生在胎儿发育、内分泌萎缩和AS 这是细胞免疫的结果。细胞凋亡的标志是 内源性钙离子将染色质切割成寡核小体片段 /Mg++依赖的内切酶。最近的证据表明，某些 化疗药物对间期细胞DNA的损伤可能引发 程序性细胞死亡，以及初步实验使用 2-氯-2‘-脱氧腺苷(CDA)支持这一机制。 淋巴细胞白血病(CLL)细胞。然而，人们对此知之甚少 对钙离子通量、大分子合成或ADP-核糖化的要求 在DNA损伤诱导的靶细胞中发生凋亡 化疗药物。研究计划侧重于两个目标细胞 研究化疗中细胞程序性死亡的模型系统：(I)CLL和 毛细胞白血病是恶性的、未分裂的细胞模型，以及(Ii) 正常单核细胞，不分裂细胞模型吞噬和 分泌功能在自身免疫发病中的作用 精神错乱。靶向CLL和毛细胞白血病细胞和单核细胞 在体外暴露于CDA和其他已知的化疗药物 造成DNA损伤。时间进程测量将由DNA链进行 断裂和寡核小体DNA断裂，后者通过方法 片段释放和电泳法后的可视化。 还将测量NAD新陈代谢的扰动，并具体 钙离子通量的调节、大分子合成和ADP核糖化 将被执行以确定控制 从DNA链断裂到细胞程序性死亡的转变。CDA是 在这个治疗慢性阻塞性肺疾病的研究所进行临床试验 淋巴系统恶性肿瘤和类风湿性关节炎。研究计划 为补充体外生化分析提供了一个独特的机会 用流式细胞术检测同一靶细胞中的细胞凋亡 体内CDA治疗期间的种群。除了临床上的 这一组合方法的相关性，对生化的理解 程序性细胞死亡的机制应提供重要的基础 抗癌和免疫抑制治疗的新策略。

The goal of the proposed research is to define the role of programmed cell death in chemotherapy drug action, so as to identify biochemical and metabolic factors that predispose or protect cells from endonuclease activation after DNA damage. programmed cell death, or apoptosis, is distinguishable from necrosis by morphologic and biochemical features, and typically occurs during fetal development, endocrine atrophy, and as a consequence of cell mediated immunity. The hallmark of apoptosis is chromatin cleavage into oligonucleosomal fragments by an endogenous Ca++ /Mg++dependent endonuclease. Recent evidence suggests that certain chemotherapy drugs at damage the DNA of interphase cells may trigger programmed cell death, and preliminary experiments using 2-chloro-2'-deoxyadenosine (CdA) support such a mechanism in chronic lymphocytic leukemia (CLL) cells. However, little is known about the requirements for Ca++ flux, macromolecular synthesis, or ADP-ribosylation in target cells induced to undergo apoptosis by DNA-damaging chemotherapeutic drugs. The Research Plan focuses on two target cell model systems to study programmed cell death in chemotherapy: (i) CLL and hairy cell leukemia as models of malignant, nondividing cells, and (ii) normal blood monocytes, a model of non-dividing cells with phagocytic and secretory functions contribute to the pathogenesis of autoimmune disorders. Target CLL and hairy cell leukemia cells and monocytes will be exposed in vitro to CdA and to other chemotherapy agents known to cause DNA damage. Time-course measurements will be made of DNA strand breaks and of oligonucleosomal DNA fragmentation, the latter by methods of fragment release and by visualization after electrophoresis. Perturbations in NAD metabolism will also be measured, and specific modulation of Ca++ flux, macromolecular synthesis, and ADP-ribosylation will be performed to determine critical biochemical pathways that govern the transition from DNA strand breaks to programmed cell death. CdA is in clinical trial at this institute for the treatments of chronic lymphoid malignancies and for rheumatoid arthritis. The research plan presents a unique opportunity to complement in vitro biochemical analyses with flow cytometric studies to detect apoptosis in the same target cell populations during CdA treatment in vivo. In addition to the clinical relevance of this combined approach, an understanding of biochemical mechanisms in programmed cell death should provide an important basis for novel strategies for anticancer and immunosuppressive therapy.