ENHANCING ACTIVITY OF ALKYLATING AGENTS IN NEUROBLASTOMA

描述(根据调查人员摘要改编)：神经母细胞瘤是 是儿童最常见的颅外实体瘤，是一种高危肿瘤， 在大多数情况下会导致死亡。烷基化试剂是主要的 神经母细胞瘤中使用的化疗药物和耐药性有助于 复发死亡率。最近，我们已经证明了1)细胞内 丁硫氨酸亚磺胺(BSO)对谷胱甘肽(GSH)的耗竭具有细胞毒性 与低氧拮抗的烷化剂马法兰(L-PAM)的协同作用 2)维甲酸可提高高危神经母细胞瘤患者的存活率，但可能 拮抗烷化剂活性。我们的长期目标是在 神经母细胞瘤：1)烷化剂抵抗和缺氧的分子决定因素 拮抗作用并确定克服它们的因素；2)确定烷化剂 具有最大的单一药物活性，并为他们量身定做患者治疗；3) 识别烷化剂交叉阻力的模式，并避免这些组合 临床；4)确定具有最高BSO协同作用的烷基化剂；5)与 L-PAM等代理商。明确的目标。1)确定单个代理的活动 在常氧和低氧条件下的临床烷化剂，它们的模式 交叉耐药性，与BSO的协同作用。2)确定对于BSO/L-PAM，是否 低氧对内源性ROS、GSH耗竭、DNA的影响 损伤/P53诱导，以及如果生物还原剂逆转了 缺氧。3)在临床试验中确定其毒性和有效性 清髓性BSO/L-PAM治疗神经母细胞瘤研究设计与方法。我们 将使用我们独特的由140个细胞系组成的小组，它们唯一的药物暴露是 在体内，这包括来自确诊患者的配对细胞系 复发，包括骨髓移植后复发。我们会揭露 选定的品系与临床相关剂量的烷化剂、BSO和 生物还原剂(替拉帕明和咪唑)在常氧和低氧条件下的应用 使用隔离室，并使用我们的习惯进行细胞毒性测试， 半自动DIMSCAN定量成像系统。因为我们的电池面板是 大型的，我们唯一有统计能力来检测烷化剂的模式 这些药物在常氧和低氧条件下的交叉耐药性，首次研究 在任何肿瘤系统中都是如此。对于BSO/L-PAM低氧拮抗作用的研究，我们 将通过谷胱甘肽还原酶-DTNB循环测定总和核GSH 用羧基-DCFDA流式细胞术检测ROS，ROS诱导的8-氧脱氧鸟苷DNA 亲和素结合技术损伤，DNA单链断裂和链间 碱洗脱法交联，免疫印迹法检测p3/p21水平 DIMSCAN的细胞毒作用，DNA梯状凋亡。TDT标记/流式细胞术 和半胱氨酸酶的激活。我们预计这项研究将有助于 了解神经母细胞瘤的烷化剂抵抗并将导致更多 有效的治疗方式。

DESCRIPTION (As Adapted from the Investigator's Abstract): Neuroblastoma is the most common extracranial solid tumor in children and is a high-risk tumor that causes death in the majority of cases. Alkylating agents are the primary chemotherapeutic agents used in neuroblastoma and resistance contributes to relapse mortality. Recently, we have demonstrated that 1) intracellular depletion of glutathione (GSH) by buthionine sulfoximine (BSO) has a cytotoxic synergy with the alkylator melphalan (L-PAM) that is antagonized by hypoxia and 2) that retinoids improve survival in high-risk neuroblastoma but may antagonize alkylator activity. Our long term goals are to define in neuroblastom: 1) the molecular determinants of alkylator resistance and hypoxia antagonism and to identify agents that overcome them; 2) identify alkylators with the greatest single agent activity and tailor patient therapy to them; 3) identify patterns of alkylator cross-resistance and avoid these combinations clinically; 4) identify alkylators with the highest BSO synergism; 5) with L-PAM and other agents. Specific Aims. 1) Determine the single agent activity of clinical alkylators in normoxia and hypoxia, their patterns of cross-resistance, and synergism with BSO. 2) Determine, for BSO/L-PAM, if hypoxia effects endogenous reactive oxygen species (ROS), GSH depletion, DNA damage/p53 induction, and if bioreductive agents reverse the effects of hypoxia. 3) Determine in clinical trials the toxicity and efficacy of myeloablative BSO/L-PAM, for neuroblastoma. Research Design and Methods. We Will use our unique panel of 140 cell lines, whose sole drug exposure has been in vivo, and that includes matched cell line pairs from patients at diagnosis and relapse, including relapse after bone marrow transplant. We will expose selected lines to clinically relevant doses of alkylators, BSO, and bioreductive agents (tirapazamine and misonidazole) in normoxia, and in hypoxia using isolation chambers, and assay cytotoxicity using our custom, semi-automated DIMSCAN quantitative imaging system. Because our cell panel is large, we uniquely have the statistical power to detect patterns of alkylator cross-resistance in both normoxia and hypoxia for these agents, the first study of its kind in any tumor system. For BSO/L-PAM hypoxia antagonism studies, we will measure total and nuclear GSH by glutathione-reductase-DTNB recycling assay, ROS by carboxy-DCFDA flow cytometry, ROS-induced 8-oxodeoxyguanosine DNA damage by avidin binding technique, DAN single strand breaks and interstrand crosslinking by alkaline elution, p3/p21 levels by immunoblotting and cytotoxicity by DIMSCAN, apoptotic DNA laddering. TdT-labeling/flow cytometry and caspase activation. We anticipate that this research will contribute to the understanding of alkylator resistance in neuroblastoma and will result in more effective treatment modalities.