Biochemical Strategies to Increase Leukemia Response

描述（由申请人提供）：该提案是CA 57629的扩展，其重点是了解核苷类似物的代谢、作用机制和相互作用。随着类似物在白血病中的成功率，包括我们在内的几个实验室已经研究了这些药物导致细胞死亡的机制。这些步骤包括形成类似物的三磷酸，掺入复制的DNA中，抑制核糖核苷酸还原酶（使用更新的类似物），最后抑制DNA合成。DNA合成的持续抑制通过细胞凋亡导致细胞死亡。当在活跃地循环和复制DNA的细胞系中进行测试时，这种情况似乎已经存在。然而，当一个人试图在治疗过程中验证这个过程时，结果是矛盾和有趣的。体内白血病细胞的生物学与培养中的细胞系非常不同。外周血中的白血病细胞通常是非循环或慢循环的，并且具有非常小百分比的细胞处于S期（0-5%）。尽管如此，在有效的核苷类似物治疗5天后，有大量的细胞减少（1至3个对数减少）。我们的假设是围绕这些前提建立的，以表明除了传统的S期介导的途径之外，可能还有其他途径导致治疗期间的非S期细胞死亡。为了验证这一假设，我们想追求三个特定的目标，这些目标集中在类似物引起的不同细胞死亡模式上。首先，我们计划定义在治疗期间由常规DNA合成抑制途径引起的细胞死亡的要素。使用奈拉滨和氯法拉滨，在临床上最成功的两个新的核苷类似物，我们计划研究细胞药代动力学和细胞药效学在细胞死亡中的作用。将这些参数与这些治疗的临床应答进行比较。其次，我们计划鉴定治疗过程中线粒体诱导的白血病细胞死亡。核苷类似物可以直接和/或间接影响线粒体，以在治疗期间诱导循环白血病细胞中的细胞死亡。直接影响，如线粒体呼吸功能涉及ATP合成酶，腺苷核苷酸转运（ANT），和线粒体膜电位的早期下降将被评估，以阐明线粒体诱导细胞凋亡的作用。间接效应包括细胞色素c的释放和对膜电位的晚期效应。最后，我们将研究白血病细胞在治疗过程中受体介导的细胞死亡的作用。在我们的细胞系中，类似物掺入导致诱导Fas配体，随后是Fas介导的非S期细胞死亡，我们计划在治疗期间追求Fas在细胞死亡中的作用。我们认为通过这些目标获得的知识将有助于我们设计核苷类似物治疗白血病的最佳方案。

DESCRIPTION (provided by applicant): This proposal is an extension of CA57629 that has been focused on understanding the metabolism, mechanism of action, and interaction of nucleoside analogs. With the success rate of analogs in leukemias, several laboratories including ours have investigated the mechanisms of cell death by these agents. The steps include formation of triphosphate of the analog, incorporation into replicating DNA, inhibition of ribonucleotide reductase (with newer analogs) and finally inhibition of DNA synthesis. Continued inhibition of DNA synthesis proceeds to cell death through apoptosis. When tested in cell lines, which are actively cycling and replicating DNA, such scenario seems to be in place. However, when one tries to validate this process during therapy, the outcome is conflicting and intriguing. The biology of leukemia cells in the body is very different from cell lines in culture. Leukemia cells in peripheral blood are generally non- or slow- cycling and with a very small percent of cells in S-phase (0-5%). Nonetheless after 5-days of effective nucleoside analog therapy, there is a massive cytoreduction (1 to 3-log decrease). Our hypothesis is built around these premises to suggest that in addition to conventional S-phase mediated pathway, there may he additional pathways that result in non-S-phase cell death during therapy. To test this hypothesis, we want to pursue three specific aims that are focused toward different mode of cell death by analogs. First, we plan to define the elements of cell death caused by conventional DNA synthesis inhibition pathway during therapy. Using nelarabine and clofarabine, two of the most successful new nucleoside analogs in the clinic, we plan to investigate the role of cellular pharmacokinetics and cellular pharmacodynamics in cell death. These parameters will be compared with clinical response to these therapies. Second, we plan to identify mitochondria induced cell death of leukemia cells during therapy. Nucleoside analogs may affect mitochondria directly and/or indirectly to induce cell death in circulating leukemia cells during therapy. Direct effect such as mitochondrial respiratory function involving ATP synthase, adenosine nucleotide translocator (ANT), and early decrease in mitochondnal membrane potential will be accessed to elucidate the role of mitochondria induced apoptosis. Indirect effect will include release of cytochrome c, and late effect on membrane potential. Finally, we will investigate the role of receptor-mediated cell death of leukemia cells during therapy. Following our lead in cell lines that analog incorporation results in induction of fas ligand followed by fas-mediated cell death of non-Sphase population, we plan to pursue the role of fas in cell death during therapy. We feel that knowledge gained through these aims will assist us in designing optimal therapy of leukemia with nucleoside analogs.