Resistance to Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and constitutive expression of Apo2L/TRAIL in human T-cell leukemia virus type 1-infected T-cell lines

Abstract

Adult T-cell leukemia (ATL), a CD4+-T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1), is difficult to cure, and novel treatments are urgently needed. Apo2 ligand (Apo2L; also tumor necrosis factor-related apoptosis-inducing ligand [TRAIL]) has been implicated in antitumor therapy. We found that HTLV-1-infected T-cell lines and primary ATL cells were more resistant to Apo2L-induced apoptosis than uninfected cells. Interestingly, HTLV-1-infected T-cell lines and primary ATL cells constitutively expressed Apo2L mRNA. Inducible expression of the viral oncoprotein Tax in a T-cell line up-regulated Apo2L mRNA. Analysis of the Apo2L promoter revealed that this gene is activated by Tax via the activation of NF-kappaB. The sensitivity to Apo2L was not correlated with expression levels of Apo2L receptors, intracellular regulators of apoptosis (FLICE-inhibitory protein and active Akt). NF-kappaB plays a crucial role in the pathogenesis and survival of ATL cells. The resistance to Apo2L-induced apoptosis was reversed by N-acetyl-L-leucinyl-L-leucinyl-lLnorleucinal (LLnL), an NF-kappaB inhibitor. LLnL significantly induced the Apo2L receptors DR4 and DR5. Our results suggest that the constitutive activation of NF-kappaB is essential for Apo2L gene induction and protection against Apo2L-induced apoptosis and that suppression of NF-kappaB may be a useful adjunct in clinical use of Apo2L against ATL.

FIG. 1.

Growth-inhibitory responses of T-cell lines and primary ATL cells following exposure to Apo2L. (A) Sensitivity of T-cell lines and primary ATL cells (left) and PBLs of healthy donors (right) to Apo2L. The cells were incubated for 24 h in the absence (open bars) or presence (solid bars) of Apo2L (100 ng/ml). Cell survival was determined using the WST-8 assay. A, acute type; C, chronic type. The bars represent the mean plus standard deviation. (B) Dose-response survival curves based on WST-8 assay. Cells were incubated with increasing concentrations of Apo2L for 24 h. Cell survival was determined using the WST-8 assay and is expressed as a percentage of the control (untreated cells). (C) Effects of Apo2L on apoptosis of T-cell lines. Cells were incubated for 24 h in the absence (open bars) or presence (solid bars) of Apo2L (100 ng/ml). The cells were harvested and then labeled with phycoerythrin-conjugated Apo2.7 and analyzed by flow cytometry.

FIG. 2.

Apo2L is expressed in HTLV-1-infected T-cell lines and primary ATL cells and induced by Tax. (A) RT-PCR analysis of Apo2L mRNA levels in HTLV-1-infected and uninfected T-cell lines. Total RNA was prepared from the indicated T-cell lines. β-Actin expression served as a control. (B) Expression of Apo2L mRNA in ATL cells. ATL cells and normal lymphocytes were collected for RNA preparation. RT-PCR was performed to amplify both Apo2L and β-actin mRNAs. (C) Induction kinetics of the Apo2L gene in JPX-9 cells treated with CdCl₂. Total-RNA samples were prepared from CdCl₂ (20 μM)-treated JPX-9 cells at the indicated time points (0 to 72 h). The expression of Tax and Apo2L in the extracted RNA was analyzed by Northern blotting and RT-PCR analysis, respectively. GAPDH served as a loading control for Northern analysis, whereas β-actin served as an internal control in the RT-PCR procedure.

FIG. 3.

Transactivation of Apo2L promoter by Tax is dependent on NF-κB signaling. (A) Jurkat cells were transfected with HTLV-1 Tax (Tax WT), Tax M22, Tax 703, or pHβAPr-neo vector (5 μg) and a luciferase reporter construct containing the −1056 Apo2L promoter (ApoP1056; 5 μg). phRL-TK (2 μg) was also cotransfected as an internal control plasmid. Luciferase activity was measured 48 h following transfection and normalized based on the Renilla luciferase activity from phRL-TK. The results are expressed as induction (n-fold) relative to the basal level measured in cells transfected with the empty vector (pHβAPr-neo). (B) Functional effects of IκBα and IκBβ dominant-interfering mutants and kinase-deficient IKKα, IKKβ, and NIK mutants on Tax-induced activation of the Apo2L promoter. The indicated effector plasmids (5 μg) were cotransfected with ApoP1056. Open bar, luciferase activity of empty vector (pCMV4) without Tax; solid bars, luciferase activities of IκBα and IκBβ mutants and kinase-deficient IKKα, IKKβ, and NIK mutants in the presence of Tax. The activities are expressed relative to that of empty vector (pCMV4) without Tax, which was defined as 1. (C) Localization of the Tax response region in the Apo2L 5′ flanking sequences. Constructs containing deletions of the Apo2L gene 5′ flanking region were transfected into Jurkat cells, together with Tax (solid bars) or empty vector (pHβAPr-neo) (open bars). The activities are expressed relative to that of cells transfected with the empty vector and ApoP/1056, which was defined as 1. The data are the mean plus standard deviation of three independent experiments.

FIG. 4.

Tax response region is located at −74/-65. (A) The Apo2L sequences between −75 and −65 share potential binding sites for SP1 and NF-κB. The SP1 and NF-κB sites are underlined and boxed, respectively. For EMSA, a probe was used that corresponded to the Apo2L-specific sequence between −81 and −51. Lowercase letters indicate the positions (−67, −68, −73, and −74) where mutations were made. (B) Deletional analysis of the cis element required for Tax-induced Apo2L promoter activity. Luciferase reporter constructs containing the wild-type and deleted −1056 Apo2L promoter region (5 μg) were cotransfected with Tax or empty vector (5 μg) into Jurkat cells. The activities are expressed relative to that of cells transfected with ApoP/1056 and empty vector, which was defined as 1. The data are the mean plus standard deviation of three independent experiments. (C) Tax-dependent binding of NF-κB family proteins on the Apo2L sequence between −81 and −51. Nuclear extracts from JPX-9 cells, treated with (+) or without (−) CdCl₂ (20 μM) for 72 h, were mixed with α-³²P-labeled probe. Where indicated, 100-fold excess amounts of each specific competitor oligonucleotide (lanes 3 to 7) were added to the reaction mixture with labeled probe. A supershift assay of NF-κB DNA binding complexes in the same nuclear extracts was also performed. Where indicated, appropriate antibodies were added to the reaction mixture before the addition of α-³²P-labeled probe (lanes 8 to 13). The arrow and arrowheads indicate the locations of NF-κB binding complex and supershifted bands with antibodies, respectively.

FIG. 5.

Binding of nuclear proteins from HTLV-1-infected T-cell lines to the Apo2L −81/−51 oligonucleotide probe. (A) Nuclear extracts were prepared from HTLV-1-infected (lanes 4 and 5) and uninfected (lanes 1 to 3) T-cell lines and incubated with α-³²P-labeled probe. (B) Nuclear extracts from SLB-1 (top) and C5/MJ cells (bottom) were subjected to an EMSA using the Apo2L −81/-51 probe. The NF-κB-DNA complex formation was competed using a 100-fold excess of unlabeled Apo2L −81/-51 (lane 2) or IL-2R κB (lane 5) probe. The same nuclear extracts were subjected to supershift analysis using appropriate antibodies (compare lanes 7 to 12 with control lane 1). The arrows and arrowheads indicate the locations of NF-κB binding complexes and supershifted bands with antibodies, respectively.

FIG. 6.

Expression of Apo2L receptors, FLIP mRNA, and active Akt in T-cell lines. (A) RT-PCR analysis of human T-cell lines for expression of Apo2L receptors (DR4, DR5, DcR1, and DcR2) and FLIP. Total RNA was prepared from the indicated T-cell lines. β-Actin served as an internal control in the RT-PCR procedure. NB4 cells were used as a positive control for DR4, DR5, DcR1, and DcR2. (B) Active Akt in T-cell lines. Western blot analyses were performed with anti-phospho-Akt, anti-Akt, or anti-actin antibody. A schematic of the four Apo2L receptors is shown at the top. (C) Cell surface expression of Apo2L receptors on T-cell lines. T-cell lines were stained with control mouse immunoglobulin G1 or anti-human DR4, DR5, DcR1, and DcR2 monoclonal antibodies and analyzed by flow cytometry. Shaded and unshaded peaks correspond to specific and control stainings, respectively.

FIG. 7.

Inhibition of NF-κB reverses Apo2L resistance of HTLV-1-infected T cells. (A) EMSA of NF-κB and AP-1 activation status in C5/MJ cells before and after exposure to the NF-κB inhibitor LLnL at 25 μM. Constitutive activation of NF-κB, but not AP-1, was reduced in the presence of LLnL. (B) WST-8 assays indicate that pretreatment with LLnL overcomes the Apo2L resistance of HTLV-1-infected T-cell lines. Cells were either not treated or treated with LLnL (25 μM in C5/MJ and 1 μM in the residual cell lines) for 2 h prior to the addition of Apo2L (100 ng/ml). After a further 24 h, cell survival was assessed by WST-8 assays. The data are the mean plus standard deviation of three independent experiments. (C) Expression of DR4 and DR5 increases following LLnL treatment (+). (D) Expression of intracellular apoptosis regulator proteins in C5/MJ cells treated with LLnL. Immunoblot analysis in C5/MJ cells treated with (+) and without (−) LLnL for 24 h. Levels of actin are shown for confirmation of equal protein loading.