title: Upon drug-induced apoptosis in lymphoma cells X-linked inhibitor of apoptosis (XIAP) translocates from the cytosol to the nucleus.
abstract: The X-linked inhibitor of apoptosis (XIAP) and cellular inhibitor of apoptosis-1 (cIAP-1) are emerging as versatile proteins in programmed cell death with a scope of possible functions reaching far beyond their well known inhibitory effects on caspases. We previously demonstrated that the ability of drugs to modify expression and cleavage of the IAPs are crucial for the synergistic effects achieved by the combinations of different cytotoxic drugs employed to treat malignant lymphomas. In order to more clearly assess the underlying molecular mechanisms, we here evaluated the consequences of drug-induced apoptosis on the localization and aggregation of XIAP and cIAP-1. The influence of drug-induced apoptosis on localization of IAPs was investigated using immunofluorescence microscopy as well as western blot analysis. Apoptosis was induced by chemotherapeutic drugs with different modes of action (bendamustine, cladribine, fludarabine, doxorubicin and mitoxantrone) and assessed by flow-cytometry using Annexin V. We demonstrate that XIAP and cIAP-1 are downregulated and/or cleaved in a dose-dependent manner upon treatment with a variety of anti-cancer drugs. Moreover we provide evidence that in the context of drug-induced apoptosis XIAP, its BIR3-RING cleavage product and cIAP-1 undergo an extensive change of subcellular localization. Immunofluorescence microscopy reveals that XIAP, in contrast to cIAP-1, is located in discrete cytosolic protein aggregates and-upon induction of apoptosis with cytotoxic drugs--redistributes into large nuclear inclusions. This translocation of XIAP and its BIR3-RING cleavage product from the cytosol into the nucleus is confirmed by cell fractionation and western blot analyses. Of note, in this experimental setting putative interaction partners of XIAP-such as Apaf-1, caspase-3 and -7--do not co-localize with XIAP. These results imply a new unknown function of XIAP and its BIR3-RING fragment in the nucleus in the context of drug-induced apoptosis. The localization of cIAP-1 in mitochondria and its liberation from these indicate a profoundly different function of this protein despite its similar modular structure to XIAP.
authors.affiliation: Department of Internal Medicine III, Hematology and Oncology, University Hospital Frankfurt, Theodor-SternKai 7, 60590 Frankfurt am Main, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Nowak
authors.forename: Simone
authors.initials: S
authors.lastname: Boehrer
authors.forename: Angela
authors.initials: A
authors.lastname: Brieger
authors.forename: Soo-Zin
authors.initials: SZ
authors.lastname: Kim
authors.forename: Simone
authors.initials: S
authors.lastname: Schaaf
authors.forename: Dieter
authors.initials: D
authors.lastname: Hoelzer
authors.forename: Paris S
authors.initials: PS
authors.lastname: Mitrou
authors.forename: Eckhart
authors.initials: E
authors.lastname: Weidmann
authors.forename: Kai Uwe
authors.initials: KU
authors.lastname: Chow
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001402:B-Lymphocytes / Q000378:metabolism / Q000473:pathology; D002451:Cell Compartmentation; D045744:Cell Line, Tumor / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D045586:Intranuclear Inclusion Bodies / Q000378:metabolism; D016393:Lymphoma, B-Cell / Q000473:pathology*; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism*; D021381:Protein Transport; D011506:Proteins / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D051636:X-Linked Inhibitor of Apoptosis Protein
chemical_list: D000970:Antineoplastic Agents; D051034:Inhibitor of Apoptosis Proteins; D009363:Neoplasm Proteins; D011506:Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; C494833:BIRC2 protein, human; D044767:Ubiquitin-Protein Ligases