ENHANCING ACTIVITY OF ALKYLATING AGENTS IN NEUROBLASTOMA

描述（改编自研究者摘要）：神经母细胞瘤是 最常见的儿童颅外实体瘤，是一种高危肿瘤， 在大多数情况下会导致死亡。烷基化剂是主要的 用于神经母细胞瘤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1）细胞内 谷胱甘肽（GSH）被丁硫酰亚砜（BSO）耗尽具有细胞毒性， 与缺氧拮抗的烷化剂美法仑（L-PAM）协同作用， 2)类维生素A能提高高危神经母细胞瘤的存活率， 拮抗烷化剂活性。我们的长期目标是， 神经母细胞瘤：1）烷化剂抗性和缺氧的分子决定因素 拮抗作用，并确定克服它们的药物; 2）确定烷化剂 具有最大的单药活性，并为其定制患者治疗; 3） 识别烷基化剂交叉抗性模式并避免这些组合 临床上; 4）鉴定具有最高BSO协同作用的烷化剂; 5）与 L-PAM和其他试剂。具体目标。1)确定单个代理活动 临床烷化剂在常氧和缺氧，他们的模式， 交叉抗性，并与BSO具有协同作用。2)对于BSO/L-PAM，确定是否 缺氧影响内源性活性氧（ROS）、GSH耗竭、DNA 损伤/p53诱导，并且如果生物还原剂逆转 缺氧3)在临床试验中确定 清髓性BSO/L-PAM，用于神经母细胞瘤。研究设计和方法。我们 将使用我们独特的140种细胞系，其唯一的药物暴露是 在体内，包括来自诊断时患者的匹配细胞系对 和复发，包括骨髓移植后复发。我们将揭露 临床相关剂量的烷化剂、BSO和 常氧和缺氧时的生物还原剂（替拉扎明和米索硝唑） 使用隔离室，并使用我们的习惯测定细胞毒性， 半自动DIMSCAN定量成像系统。因为我们的电池板 大，我们独特的统计能力，以检测模式的烷基化 这些药物在常氧和缺氧条件下的交叉耐药性， 在任何肿瘤系统中都是如此。对于BSO/L-PAM缺氧拮抗作用研究，我们 将通过谷胱甘肽还原酶-DTNB再循环测量总GSH和细胞核GSH 活性氧测定，羧基-DCFDA流式细胞术，活性氧诱导的8-氧代脱氧鸟苷DNA 抗生物素蛋白结合技术损伤，DAN单链断裂和链间断裂 通过碱性洗脱进行交联，通过免疫印迹进行p3/p21水平测定， DIMSCAN细胞毒性，凋亡DNA梯状。TdT标记/流式细胞术 和半胱天冬酶激活。我们预计，这项研究将有助于 了解成神经细胞瘤中的烷化剂耐药性，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.