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-hydroperoxycyclophosphamide (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-CYtreated 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.

T-cell clones persisting in the circulation after autologous hematopoietic SCT are undetectable in the peripheral CD34+ selected graft

“…CY is a potent alkylating agent that induces DNA fragmentation and apoptosis in lymphocytes, which have low levels of aldehyde dehydrogenase, the enzyme that converts the active alkylating compound phospharamide mustard into the inert compound carboxyphosphamide. 14,15 Common protocols for the evaluation of hematopoietic graft composition include flow cytometric enumeration of CD3 þ cells but do not assess their apoptotic status. We hypothesize that T cells contained in HSC grafts obtained with mobilization schemes not using CY or other lymphotoxic agents may have greater engraftment potential, and it would be important to test this hypothesis in future studies.…”

Endoplasmic Reticulum Stress-induced Apoptosis in Leishmania through Ca2+-dependent and Caspase-independent Mechanism

“…Endo G is a mitochondrial nuclease that has been suggested to play a role in mitochondrial DNA replication (38), and its role in nuclear DNA fragmentation during apoptosis has been evidenced from the studies on Caenorhabditis elegans (39,40). The release of Endo G from mitochondria and its translocation to nuclei have been documented in different cell lines during apoptotic stimuli (41)(42)(43). In Leishmania, both Cyt c and Endo G have been reported to play important roles in apoptosis (6).…”

TRAIL–Receptor Costimulation Inhibits Proximal TCR Signaling and Suppresses Human T Cell Activation and Proliferation

“…Western blot analysis was carried out as described previously (22). In brief, 20 mg cell lysates was separated on 8.5 or 10% SDS-PAGES and electroblotted onto Hybond ECL nitrocellulose membrane (GE Healthcare Life Sciences).…”