Biochem. J. (2005) 385
(595–603) (Printed in Great Britain)
Apo2L/TRAIL induction and nuclear translocation of inositol hexakisphosphate kinase 2 during IFN-b-induced apoptosis in ovarian carcinoma
Bei H. MORRISON, Zhuo TANG, Barbara S. JACOBS, Joseph A. BAUER and Daniel J. LINDNER1
Center for Cancer Drug Development and Discovery, Taussig Cancer Center, Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, U.S.A.
Previously, we have reported that overexpression of IHPK2 (inositol hexakisphosphate kinase 2) sensitized NIH-OVCAR-3 ovarian carcinoma cell lines to the growth-suppressive and apoptotic effects of IFN-b (interferon-b) treatment and g-irradiation. In the present study, we demonstrate that Apo2L/TRAIL (Apo2L/tumour-necrosis-factor-related apoptosis-inducing ligand) is a critical mediator of IFN-induced apoptosis in these cells. Compared with IFN-a2, IFN-b is a more potent inducer of Apo2L/TRAIL and IHPK2 activity. Overexpression of IHPK2 converts IFN-a2-resistant cells into cells that readily undergo apoptosis in response to IFN-a2. In untreated cells transfected with IHPK2-eGFP (where eGFP stands for enhanced green fluorescent protein), the fusion protein is localized to the cytoplasm and perinuclear region. After treatment with IFN-b, IHPK2-eGFP translocated to the nucleus. In cells transfected with mutant IHPK2-NLS-eGFP (where NLS stands for nuclear localization sequence), containing point mutations in the NLS, the fusion protein remained trapped in the cytoplasm, even after IFN-b treatment. Cells expressing mutant NLS mutation were more resistant to IFN-b. The IC50 value of IHPK2-expressing cells was 2–3-fold lower than vector control. The IC50 value of NLS-mutant-expressing cells was 3-fold higher than vector control. Blocking antibodies to Apo2L/TRAIL or transfection with a dominant negative Apo2L/TRAIL receptor (DR5D) inhibited the antiproliferative effects of IFN-b. Thus overexpression of IHPK2 enhanced apoptotic effects of IFN-b, and expression of the NLS mutant conferred resistance to IFN-b. Apo2L/TRAIL expression and nuclear localization of IHPK2 are both required for the induction of apoptosis by IFN-b in ovarian carcinoma.
Key words: Apo2L/tumour-necrosis-factor-related apoptosis-inducing ligand (Apo2L/TRAIL), apoptosis, inositol hexakisphosphate kinase 2, interferon, ovarian carcinoma, translocation.
Abbreviations used: Ac-DEVD-AMC, acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin; Ac-DEVD-CHO, acetyl-Asp-Glu-Val-Asp-aldehyde; TNFa, tumour necrosis factor a; Apo2L/TRAIL, Apo2L/TNF-related apoptosis-inducing ligand; DcR, decoy receptor; DR, death receptor; eGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; HEK-293T cells, human embryonic kidney 293T cells; GAP3DH, glyceraldehyde-3-phosphate dehydrogenase; HRP, horseradish peroxidase; IFN, interferon; IHPK2, inositol hexakisphosphate kinase 2; IP6, inositol hexaphosphate; mAb, monoclonal antibody; NLS, nuclear localization sequence; PI, propidium iodide; TdT, terminal deoxynucleotidyl transferase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling; Z-VAD, benzyloxycarbonylvalyl-alanyl-aspartic acid (O-methyl)-fluoromethylketone.
1To whom correspondence should be addressed (email lindned@cc.ccf.org).
Received 8 June 2004/14 September 2004; accepted 24 September 2004
Published as BJ Immediate Publication 24 September 2004, DOI 10.1042/BJ20040971
The Biochemical Society, London ©2005
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