boc-d-fmk and Neuroblastoma

The F7-26 monoclonal antibody (Mab) has been reported to be specific for single-strand DNA damage (ssDNA) and to also identify cells in apoptosis. We carriedout studies to determine if F7-26 binding measured by flow cytometry was able to specifically identify exogenous ssDNA as opposed to DNA damage from apoptosis. Neuroblastoma cells were treated with melphalan (L-PAM), fenretinide, 4-hydroperoxycyclophosphamide (4-HC)+/-pan-caspase inhibitor BOC-d-fmk, topotecan or with 10Gy gamma radiation+/-hydrogen peroxide (H2O2) and fixed immediately postradiation. Cytotoxicity was measured by DIMSCAN digital imaging fluorescence assay. The degree of ssDNA damage was analyzed by flow cytometry using Mab F7-26, with DNA visualized by propidium iodide counterstaining. Flow cytometry was used to measure apoptosis detected by terminal deoxynucleotidyltransferase (TUNEL) assay and reactive oxygen species (ROS) by carboxy-dichlorofluorescein diacetate. Irradiated and immediately fixed neuroblastoma cells showed increased ssDNA, but not apoptosis by TUNEL (TUNEL-negative). 4-HC or L-PAM+/-BOC-d-fmk increased ssDNA (F7-26-positive), but BOC-d-fmk prevented TUNEL staining. Fenretinide increased apoptosis by TUNEL but not ssDNA damage detected with F7-26. Enhanced ssDNA in neuroblastoma cells treated with radiation+H2O2 was associated with increased ROS. Topotecan increased both ssDNA and cytotoxicity in 4-HC-treated cells. These data demonstrate that Mab F7-26 recognized ssDNA due to exogenous DNA damage, rather than apoptosis. This assay should be useful to characterize the mechanism of action of antineoplastic drugs.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Benzyl Compounds; Cell Line, Tumor; Child; DNA Damage; DNA, Single-Stranded; Enzyme Inhibitors; Fenretinide; Flow Cytometry; Gamma Rays; Humans; Hydrocarbons, Fluorinated; Hydrogen Peroxide; In Situ Nick-End Labeling; Melphalan; Neuroblastoma; Oxidants; Reactive Oxygen Species; Topotecan

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 (DeltaPsim) 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 < or = 0.7 microg/mL). In p53-nonfunctional neuroblastoma cell lines, all TPZ LC99 values were >3.0 microg/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 DeltaPsim 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 DeltaPsim loss and GSH depletion, but neither N-acetylcysteine nor Boc-d-fmk inhibited apoptosis caused by TPZ. In response to TPZ, DeltaPsim 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, DeltaPsim 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.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Benzyl Compounds; Blotting, Western; Caspase Inhibitors; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Flow Cytometry; Glutathione; Humans; Hydrocarbons, Fluorinated; Intracellular Membranes; Membrane Potentials; Mitochondria; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; Tirapazamine; Triazines; Tumor Suppressor Protein p53