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Tirapazamine Cytotoxicity for Neuroblastoma Is p53 Dependent

Authors' Affiliation: ¹ Developmental Therapeutics Program, USC-CHLA Institute for Pediatric Clinical Research, Division of Hematology-Oncology, Children's Hospital Los Angeles and Departments of ² Pediatrics and ³ Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California

Requests for reprints: C. Patrick Reynolds, Developmental Therapeutics Program, USC-CHLA Institute for Pediatric Clinical Research, Children's Hospital, Los Angeles, MS#57, 4620 Sunset Boulevard, Los Angeles, CA 90027. Phone: 323-669-5646; Fax: 323-664-9226 or 9455; E-mail: preynolds{at}chla.usc.edu.

Relapse of neuroblastoma commonly occurs in hypoxic tissues, and is associated with an acquired and sustained high-level drug resistance, often due to p53 loss of function. Abrogating p53 function with HPV 16 E6 transduction in drug-sensitive neuroblastoma cell lines caused high-level drug resistance. Tirapazamine (TPZ) is a bioreductive agent that forms a toxic free radical in hypoxia. We determined in six neuroblastoma cell lines the cytotoxicity of TPZ using DIMSCAN, a digital imaging fluorescence assay, apoptosis and mitochondrial membrane potential (_m) by flow cytometry, and protein expression by immunoblotting. TPZ exhibited high cytotoxicity, especially in hypoxia (2% O₂), for all four p53-functional neuroblastoma cell lines, achieving >3 logs of cell kill (LC₉₉ 0.7 µg/mL). In p53-nonfunctional neuroblastoma cell lines, all TPZ LC₉₉ values were >3.0 µg/mL (average clinically achievable level). TPZ (24 hours) induced apoptosis in >46% of cells in p53-functional cell lines but failed to cause apoptosis in p53 nonfunctional cell lines. Induction of p53 and p21 expression by TPZ was observed in a p53-functional cell line (SMS-SAN) but not in a p53-nonfunctional cell line (CHLA-90). Significant _m loss and glutathione (GSH) depletion in response to TPZ was observed in p53-functional cell lines (SMS-SAN, SMS-SAN EV, and CHLA-15) but not in p53-nonfunctional cell lines (SMS-SAN E6 and CHLA-90). N-Acetylcysteine inhibited TPZ-mediated _m loss and GSH depletion, but neither N-acetylcysteine nor Boc-d-fmk inhibited apoptosis caused by TPZ. In response to TPZ, _m loss preceded apoptosis. Thus, TPZ cytotoxicity for neuroblastoma cell lines in hypoxia occurred via a p53-dependent mitochondrial pathway that caused induction of p53 and p21, _m decrease, GSH depletion, and apoptosis. These data further define the mechanism of action of TPZ and suggest that as a single agent, TPZ would only have clinical activity against p53-functional neuroblastomas.

Key Words: hypoxia • TPZ • p53 • mitochondrial membrane potential • apoptosis

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