title: Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1.
abstract: We previously developed an antibody-avidin fusion protein (ch128.1Av) that targets the human transferrin receptor 1 (TfR1) and exhibits direct cytotoxicity against malignant B cells in an iron-dependent manner. ch128.1Av is also a delivery system and its conjugation with biotinylated saporin (b-SO6), a plant ribosome-inactivating toxin, results in a dramatic iron-independent cytotoxicity, both in malignant cells that are sensitive or resistant to ch128.1Av alone, in which the toxin effectively inhibits protein synthesis and triggers caspase activation. We have now found that the ch128.1Av/b-SO6 complex induces a transcriptional response consistent with oxidative stress and DNA damage, a response that is not observed with ch128.1Av alone. Furthermore, we show that the antioxidant N-acetylcysteine partially blocks saporin-induced apoptosis suggesting that oxidative stress contributes to DNA damage and ultimately saporin-induced cell death. Interestingly, the toxin was detected in nuclear extracts by immunoblotting, suggesting the possibility that saporin might induce direct DNA damage. However, confocal microscopy did not show a clear and consistent pattern of intranuclear localization. Finally, using the long-term culture-initiating cell assay we found that ch128.1Av/b-SO6 is not toxic to normal human hematopoietic stem cells suggesting that this critical cell population would be preserved in therapeutic interventions using this immunotoxin.
authors.affiliation: Division of Surgical Oncology, Department of Surgery, David Geffen School of Medicine, University of California, Los Angeles, CA, USA. tdaniels@mednet.ucla.edu
authors.forename: Tracy R
authors.initials: TR
authors.lastname: Daniels-Wells
authors.forename: Gustavo
authors.initials: G
authors.lastname: Helguera
authors.forename: José A
authors.initials: JA
authors.lastname: Rodríguez
authors.forename: Lai Sum
authors.initials: LS
authors.lastname: Leoh
authors.forename: Michael A
authors.initials: MA
authors.lastname: Erb
authors.forename: Graciel
authors.initials: G
authors.lastname: Diamante
authors.forename: David
authors.initials: D
authors.lastname: Casero
authors.forename: Matteo
authors.initials: M
authors.lastname: Pellegrini
authors.forename: Otoniel
authors.initials: O
authors.lastname: Martínez-Maza
authors.forename: Manuel L
authors.initials: ML
authors.lastname: Penichet
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000276:immunology*; D000972:Antineoplastic Agents, Phytogenic / Q000008:administration & dosage*; D017209:Apoptosis / Q000187:drug effects; D001360:Avidin / Q000235:genetics*; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D020869:Gene Expression Profiling; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics*; D051379:Mice; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism; D011990:Receptors, Transferrin / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage*; D054789:Ribosome Inactivating Proteins, Type 1 / Q000008:administration & dosage*; D000076985:Saporins
chemical_list: D015703:Antigens, CD; D000972:Antineoplastic Agents, Phytogenic; C068126:CD71 antigen; D007074:Immunoglobulin G; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D001360:Avidin; D000076985:Saporins