G1P3, an IFN-induced survival factor, antagonizes TRAIL-induced apoptosis in human myeloma cells
Venugopalan Cheriyath, … , Mohamad A. Hussein, Ernest C. Borden
Venugopalan Cheriyath, … , Mohamad A. Hussein, Ernest C. Borden
Published October 1, 2007
Citation Information: J Clin Invest. 2007;117(10):3107-3117. https://doi.org/10.1172/JCI31122.
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Research Article Oncology

G1P3, an IFN-induced survival factor, antagonizes TRAIL-induced apoptosis in human myeloma cells

Abstract

The effectiveness of IFN-α2b for human multiple myeloma has been variable. TRAIL has been proposed to mediate IFN-α2b apoptosis in myeloma. In this study we assessed the effects of IFN-α2b signaling on the apoptotic activity of TRAIL and human myeloma cell survival. While TRAIL was one of the most potently induced proapoptotic genes in myeloma cells following IFN-α2b treatment, less than 20% of myeloma cells underwent apoptosis. Thus, we hypothesized that an IFN-stimulated gene (ISG) with prosurvival activity might suppress TRAIL-mediated apoptosis. Consistent with this, IFN-α2b stabilized mitochondria and inhibited caspase-3 activation, which antagonized TRAIL-mediated apoptosis and cytotoxicity after 24 hours of cotreatment in cell lines and in fresh myeloma cells, an effect not evident after 72 hours. Induced expression of G1P3, an ISG with largely unknown function, was correlated with the antiapoptotic activity of IFN-α2b. Ectopically expressed G1P3 localized to mitochondria and antagonized TRAIL-mediated mitochondrial potential loss, cytochrome c release, and apoptosis, suggesting specificity of G1P3 for the intrinsic apoptosis pathway. Furthermore, RNAi-mediated downregulation of G1P3 restored IFN-α2b–induced apoptosis. Our data identify the direct role of a mitochondria-localized prosurvival ISG in antagonizing the effect of TRAIL. Curtailing G1P3-mediated antiapoptotic signals could improve therapies for myeloma or other malignancies.

Authors

Venugopalan Cheriyath, Keith B. Glaser, Jeffrey F. Waring, Rachid Baz, Mohamad A. Hussein, Ernest C. Borden

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Figure 5

Ectopically expressed G1P3 localized into mitochondria.

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Ectopically expressed G1P3 localized into mitochondria. (A) Relative ind...
(A) Relative induction of G1P3 protein in RPMI 8226 cells by IFN-α2b. WCEs from cells left untreated or treated with IFN-α2b (250 IU/ml) for 18, 48, and 72 hours were immunoblotted with an anti-G1P3 antibody. β-Actin was used as a loading control. (B) Schematics of G1P3 expression vector. HAT at N-terminal and putative structural elements derived from primary amino acid sequence analysis with protean software (Lasergene Inc.) are shown. The arrow indicates the cleavage site, which is located following a putative mitochondrial localization signal peptide of 20 amino acids. (C) Ectopic expression of HAT-G1P3 fusion protein in RPMI 8226 cells. Anti-G1P3 antibody specifically recognized a 13-kDa band in the positive control (treated with 250 IU/ml of IFN-α2b) and 13- and 17-kDa bands in HAT-G1P3 lysates. (D) Relative expression of TRAIL receptors in HAT or HAT-G1P3 clones. WCEs (30 μg) from untreated or IFN-α2b–treated (250 IU/ml) RPMI 8226 cells were immunoblotted with DR4, DR5, DcR1, and DcR2 antibodies. β-Actin was used as a loading control. (E) Mitochondrial localization of constitutively expressed G1P3. Immunoblotting of cytoplasmic (Cyto) and mitochondrial (Mito) fractions of pQCXIP, HAT, or HAT-G1P3 cells with anti-G1P3 antibody showed the localization of G1P3 in mitochondrial fractions (top panel). Reprobing with anti–Cox IV (middle panel) and β-actin antibodies (bottom panel) confirmed the purity of subcellular fractions.