title: 4-hydroperoxy-cyclophosphamide mediates caspase-independent T-cell apoptosis involving oxidative stress-induced nuclear relocation of mitochondrial apoptogenic factors AIF and EndoG.
abstract: Apoptosis is a major mechanism of treatment-induced T-cell depletion in leukemia and autoimmune diseases. While 'classical' apoptosis is considered to depend on caspase activation, caspase-independent death is increasingly recognized as an alternative pathway. Although the DNA-damaging drug cyclophosphamide (CY) is widely used for therapy of hematological malignancies and autoimmune disorders, the molecular mechanism of apoptosis induction remains largely unknown. Here, we report that treatment of Jurkat, cytotoxic, and primary leukemic T cells with an activated analog of CY, 4-hydroperoxy-cyclophosphamide (4-OOH-CY), induces caspase activation and typical features of apoptosis, although cell death was not prevented by caspase inhibition. Also depletion of murine thymocytes and splenocytes after CY treatment in vivo was not inhibited by Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk). Caspase-8 and receptor-induced protein (RIP) were dispensable for 4-OOH-CY-mediated apoptosis, while overexpression of Bcl-2 was partially protective. 4-OOH-CY treatment induced reactive oxygen species production, upregulation of Bax, and nuclear relocation of the mitochondrial factors apoptosis-inducing factor (AIF) and endonuclease G (EndoG). The antioxidant N-acetyl-L-cysteine substantially inhibited conformational changes of Bax, loss of mitochondrial membrane potential, nuclear relocation of mitochondrial factors, and apoptosis induction in 4-OOH-CY-treated T cells. These results strongly indicate that oxidative damage-induced nuclear translocation of AIF and EndoG in 4-OOH-CY-treated T cells might represent an alternative death pathway in the absence of caspase activity.
authors.affiliation: University Children's Hospital, Ulm, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Strauss
authors.forename: M-A
authors.initials: MA
authors.lastname: Westhoff
authors.forename: P
authors.initials: P
authors.lastname: Fischer-Posovszky
authors.forename: S
authors.initials: S
authors.lastname: Fulda
authors.forename: M
authors.initials: M
authors.lastname: Schanbacher
authors.forename: S M
authors.initials: SM
authors.lastname: Eckhoff
authors.forename: K
authors.initials: K
authors.lastname: Stahnke
authors.forename: N
authors.initials: N
authors.lastname: Vahsen
authors.forename: G
authors.initials: G
authors.lastname: Kroemer
authors.forename: K-M
authors.initials: KM
authors.lastname: Debatin
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D021581:Active Transport, Cell Nucleus; D000818:Animals; D017209:Apoptosis; D051033:Apoptosis Inducing Factor / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D003520:Cyclophosphamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000494:pharmacology; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D019169:Jurkat Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D008928:Mitochondria / Q000378:metabolism; D018384:Oxidative Stress; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects* / Q000502:physiology; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors
chemical_list: D051033:Apoptosis Inducing Factor; D016166:Free Radical Scavengers; D007166:Immunosuppressive Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D003520:Cyclophosphamide; D004706:Endodeoxyribonucleases; C052391:endonuclease G; D020169:Caspases; C011272:perfosfamide; D000111:Acetylcysteine