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.
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