Biochemical Strategies to Increase Leukemia Response

描述(由申请人提供)：本提案是CA57629的扩展，该提案侧重于了解核苷类似物的新陈代谢、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，抑制核糖核苷酸还原酶(与较新的类似物)，最后抑制DNA合成。DNA合成的持续抑制会通过细胞凋亡而导致细胞死亡。当在活跃地循环和复制DNA的细胞系中进行测试时，这种情况似乎是适当的。然而，当一个人试图在治疗过程中验证这一过程时，结果是相互矛盾和耐人寻味的。白血病细胞在体内的生物学与培养中的细胞株有很大的不同。外周血中的白血病细胞一般为非周期或慢周期细胞，有很小一部分细胞处于S期(0-5%)。然而，在有效的核苷类似物治疗5天后，有大量的细胞减少(1到3个对数减少)。我们的假说就是围绕这些前提提出的，即除了传统的S期介导的细胞死亡外，可能还有其他途径导致非S期细胞在治疗过程中死亡。为了验证这一假设，我们想通过类比来追求三个特定的目标，这些目标集中在不同的细胞死亡模式上。首先，我们计划确定传统的DNA合成抑制途径在治疗过程中导致细胞死亡的因素。利用奈拉滨和氯法拉滨这两种临床上最成功的新核苷类似物，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。这些参数将与这些疗法的临床反应进行比较。第二，我们计划在治疗过程中识别线粒体诱导的白血病细胞死亡。核苷类似物可能直接和/或间接作用于线粒体，在治疗过程中诱导循环白血病细胞死亡。直接的影响，如线粒体的呼吸功能，包括三磷酸腺苷合成酶、腺苷核苷酸转运体(ANT)，以及线粒体早期膜电位的降低，将被用来阐明线粒体诱导细胞凋亡的作用。间接作用包括细胞色素c的释放，以及对膜电位的迟发作用。最后，我们将研究受体介导的白血病细胞死亡在治疗过程中的作用。根据我们在模拟掺入导致Fas配体诱导以及Fas介导的非S期细胞死亡的细胞系中的领先地位，我们计划在治疗过程中探索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使用核苷类似物的白血病的最佳治疗方法。

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.