MOLECULAR MECHANISMS OF ARA C THERAPY IN MAN

我们以前已经证明，阿糖胞苷纳入白血病细胞 DNA，并且这种掺入的程度与DNA的丢失相关。 克隆存活率 我们还证明， 阿拉伯糖基残基用作不良引物末端，从而导致 抑制DNA合成。 （ara-C）DNA结构异常， 在暴露于碱时发生断链。 此外，抑制 通过掺入阿糖胞苷导致DNA合成的增加， DNA链断裂后进行修复和复制合成。 最后， （ara-C）DNA的形成导致了末端的诱导， 分化和致死细胞事件。 这些发现提供了重要的新见解的作用机制， 是治疗急性髓细胞白血病最有效的药物。 拟议的研究将扩展我们在2010年期间纳入ara-C的工作。 通过监测DNA修复过程中ara-C的掺入来进行复制合成。 在复制过程中，（ara-C）DNA形成的生物化学效应 修复合成将被监测，作为一个结果， 3-氨基苯甲酰胺和咖啡因。 分子参数将被关联 具有通过诱变和克隆形成丧失确定的生物效应 生存 最后，我们将采用一种新的体外后标记试验， 监测临床样品中（ara-C）DNA的形成。 这种方法将是 适用于从接受低剂量连续给药的患者中获得的药物 输注区-C作为我们生物和生化 问题研究 这些研究将在HL-60前髓母细胞和 密度停滞的平台期二倍体成纤维细胞。 掺入 在复制和修复DNA合成过程中的ara-C将被监测， 平衡离心和DNA损伤将使用 碱洗提技术 这种方法应该提供分子 阿糖胞苷诱导致死性的基础 细胞事件 这项工作是重要的，在提供实验 阿糖胞苷生化合理临床试验设计的基础 以及对人类白血病的分子控制的理解。

We have previously demonstrated that ara-C incorporates in leukemic cell DNA and that the extent of this incorporation correlates with loss of clonogenic survival. We have also demonstrated that the incorporated arabinosyl residue serves as a poor primer terminus and thereby results in inhibition of DNA synthesis. The (ara-C)DNA is structurally abnormal and undergoes strand scission upon exposure to alkali. Furthermore, inhibition of DNA synthesis by the incorporation of ara-C causes the accumulation of strand breaks in DNA undergoing repair and replicative synthesis. Finally, the formation of (ara-C)DNA results in both the induction of terminal differentiation and lethal cellular events. These findings provide major new insights into the mechanism of action of the most effective agent in the treatment of acute myelogenous leukemia. The proposed studies will extend our work on incorporation of ara-C during replicative synthesis by monitoring ara-C incorporation during DNA repair. The biochemical effects of (ara-C)DNA formation during both replicative and repair synthesis will then be monitored as a result of exposue to 3-aminobenzamide and caffeine. The molecular parameters will be correlated with biologic effect as determined by mutagenesis and loss of clonogenic survival. Finally, we will employ a new in vitro post-labeling assay to monitor formation of (ara-C)DNA in clinical samples. This approach will be applied to speciments obtained from patients receiving low dose continuous infusion area-C as a clinical correlate of our biologic and biochemical studies. These studies will be performed on HL-60 promyeloblasts and density-arrested, plateau phase diploid fibroblasts. The incorporation of ara-C during replicative and repair DNA synthesis will be monitored by equilibrium centrifugation and DNA damage will be monitored using the alkaline elution technique. This approach should provide the molecular basis for understanding the effects of ara-C on the induction of lethal cellular events. This work is important in providing the experimental basis for the design of biochemically rational clinical trials with ara-C and for gaining an understanding of the molecular control of human leukemia.