Activation of hypoxia-inducible factor 1 in human T-cell leukaemia virus type 1-infected cell lines and primary adult T-cell leukaemia cells

Abstract

HTLV-1 (human T-cell leukaemia virus type 1) is the causative agent for ATL (adult T-cell leukaemia). HTLV-1 Tax can activate the PI3K (phosphoinositide 3-kinase)/Akt signalling pathway, which is responsible for survival of HTLV-1-infected T-cells. HIFs (hypoxia-inducible factors) are transcriptional regulators that play a central role in the response to hypoxia. Overexpression of HIF-1alpha in many cancers is associated with a poor response to treatment and increased patient mortality. Our objectives in the present study were to investigate whether HIF-1 was activated in HTLV-1-infected T-cells and to elucidate the molecular mechanisms of HIF-1 activation by focusing on the PI3K/Akt signalling pathway. We detected a potent pathway that activated HIF-1 in the HTLV-1-infected T-cells under a normal oxygen concentration. Enhanced HIF-1alpha protein expression and HIF-1 DNA-binding activity were exhibited in HTLV-1-infected T-cell lines. Knockdown of HIF-1alpha by siRNA (small interfering RNA) suppressed the growth and VEGF (vascular endothelial growth factor) expression of the HTLV-1-infected T-cell line. HIF-1 protein accumulation and transcriptional activity were enhanced by Tax, which was inhibited by dominant-negative Akt. Importantly, mutant forms of Tax that are defective in activation of the PI3K/Akt pathway failed to induce HIF-1 transcriptional activity. The PI3K inhibitor LY294002 suppressed HIF-1alpha protein expression, HIF-1 DNA-binding and HIF-1 transcriptional activity in HTLV-1-infected T-cell lines. In primary ATL cells, HIF-1alpha protein levels were strongly correlated with levels of phosphorylated Akt. The results of the present study suggest that PI3K/Akt activation induced by Tax leads to activation of HIF-1. As HIF-1 plays a major role in tumour progression, it may represent a molecular target for the development of novel ATL therapeutics.

Figure 1. Overexpression of HIF-1α protein in HTLV-1-infected T-cell lines

(A) HIF-1α and Tax protein expression was analysed in HTLV-1-infected T-cell lines [HTLV-1 (+)], HTLV-1-uninfected T-cell lines [HTLV-1 (−)] and CoCl₂ (300 μM, 24 h) treated- [CoCl₂ (+)] and untreated- [CoCl₂ (−)] HeLa cells by Western blotting. The arrow indicates Tax protein and the arrowhead indicates a fusion protein between the envelope and the Tax coding sequence. (B) RT-PCR analysis showing comparable HIF-1α mRNA levels in HTLV-1-infected and -uninfected T-cell lines. Total RNA was extracted and subjected to RT-PCR analysis. β-Actin expression was the control for cDNA synthesis. M represents DNA size markers. (C) Proteasome inhibition causes HIF-1α accumulation in an HTLV-1-infected T-cell line. HTLV-1-infected (HUT-102) and -uninfected (MOLT-4) T-cell lines were treated with 20 μM LLnL for the times indicated before collection. Western blot analysis of total protein extracts from cell lines were probed with an anti-HIF-1α antibody. Actin was analysed as a loading control. (D) The half-life of HIF-1α protein is not extended in HTLV-1-infected T-cell lines. HTLV-1-infected (HUT-102) and -uninfected (MOLT-4) T-cell lines were treated with 12.5 μg/ml cycloheximide (CHX) to block new protein synthesis. Extracts were prepared at the times indicated after cycloheximide block and were then analysed for HIF-1α protein expression by Western blotting (upper panels). Five times more cell extract (100 μg) was used to detect the expression of HIF-1α protein in MOTL-4 cells compared with HUT-102 cells. The kinetics of HIF-1α degradation (lower panels) were analysed using AlphaEase® FC software (Alpha Innotech).

Figure 2. HIF-1 DNA-binding activity is enhanced in HTLV-1-infected T-cell lines

(A) HIF-1 DNA-binding activity of HTLV-1-infected and -uninfected T-cell lines was analysed by EMSA using the W18 oligonucleotide probe containing the HIF-1-binding site from the human EPO gene. The arrow indicates specific DNA–protein complexes. (B) Sequence specificity of HIF-1 DNA-binding activity and competition assays were performed with nuclear extracts from MT-2 cells. Where indicated, 100-fold excess amounts of unlabelled competitor oligonucleotide (lanes 2–4) were added to the reaction mixture with the [³²P]-labelled W18 probe. A supershift assay of HIF-1–DNA complexes in the same nuclear extracts was also performed. An antibody against HIF-1α was added to the reaction mixture before the addition of the [³²P]-labelled probe (lane 5). The arrow indicates specific DNA–protein complexes. The supershifted complexes are indicated by an arrowhead.

Figure 3. Knockdown of HIF-1α suppresses cell growth of a HTLV-1-infected T-cell line

(A) HUT-102 cells were transfected with siRNA for HIF-1α or with a non-target siRNA. The effect of siRNA on cell growth was examined by counting the viable cell number in triplicate using the Trypan Blue dye-exclusion method. (B) RT-PCR analysis showing repression of HIF-1α and VEGF mRNA (one of the target genes of HIF-1α) in HUT-102 cells transfected with HIF-1α siRNA (+) compared with that in cells transfected with non-targeting siRNA (−) 24 and 48 h after transfection. β-Actin expression was used as the cDNA loading control.

Figure 4. Tax induces HRE reporter activity through the PI3K/Akt signalling pathway

(A) HeLa cells were transfected with the following expression vectors: increasing amounts (0, 0.5, 1 or 2 μg) of Tax WT, HIF-1α or a combination of 0.5 μg of Tax WT and increasing amounts (0, 0.5, 1 or 2 μg) of HIF-1α, together with 0.5 μg of luciferase reporter plasmid containing the HRE (pGL3-6×HRE-Luc) as indicated. After 48 h, cells were collected for the luciferase assay. (B) Tax induced HIF-1α expression in HeLa cells. HeLa cells were transfected with increasing amounts (0, 1, 2 or 4 μg) of Tax WT. Cells were harvested 48 h after transfection and analysed for HIF-1α and Tax expression by Western blotting. (C) A combination of Tax and HIF-1α transfection caused a more pronounced accumulation of HIF-1α. HeLa cells were transfected with 0.5 μg of Tax WT and 2 μg of HIF-1α as indicated in the Figure. Cells were harvested 48 h after transfection and analysed for HIF-1α and Tax expression by Western blotting. (D) HeLa cells were transfected with the following expression vectors: increasing amounts (0, 0.5, 1 or 2 μg) of Tax WT or Tax mutants (M22, 703 or K88A), together with 0.5 μg of luciferase reporter plasmid containing the HRE (pGL3-6×HRE-Luc) as indicated. After 48 h, cells were collected for the luciferase assay. (E) Tax-enhanced HRE reporter activity was inhibited by a PI3K inhibitor. HeLa cells were transfected with 2 μg of HIF-1α and 0.5 μg of Tax WT or mutant (M22, 703 or K88A) expression plasmids, together with 0.5 μg of pGL3-6×HRE-Luc as indicated in the Figure. After 24 h, cells were treated with increasing amounts of LY294002 (0, 5, 10 or 20 μM) for a further 24 h. (F) Tax-enhanced HIF-1 reporter activity was inhibited by AKT-DN. HeLa cells were transfected with 2 μg of Tax WT, or a combination of 0.5 μg of Tax WT and 2 μg of HIF-1α as indicated in the Figure, together with increasing amounts (0, 0.05, 0.1 or 0.5 μg) of AKT-DN and 0.5 μg of pGL3-6×HRE-Luc. After 48 h, cells were collected, and transcriptional activity was determined using the luciferase assay. Relative luciferase activities were measured in cell extracts normalized to the Renilla luciferase activity. Luciferase activity is presented as a fold induction relative to the basal level measured in cells transfected with the reporter plasmid alone. Values are the means±S.D. from three separate experiments.

Figure 5. A PI3K inhibitor represses HIF-1α protein expression and HIF-1 DNA-binding activity in HTLV-I-infected T-cell lines

(A) HUT-102 cells were treated with (+) or without (−) 20 μM LY294002 for 24 h. Western blots of total protein extracts from the cells were probed with anti-HIF-1α, anti-phosphorylated Akt (Ser⁴⁷³), anti-Akt and anti-actin antibodies. (B) A PI3K inhibitor prevents HIF-1 DNA-binding activity in HTLV-1-infected T-cell lines. Cells were treated with (+) or without (−) 20 μM LY294002 for 24 h. The inhibition of HIF-1 DNA-binding activity was assessed by EMSA using the oligonucleotide probe W18. An oligonucleotide probe for Oct-1 was used as a control.

Figure 6. HIF-1α and phosphorylated Akt are highly expressed in primary ATL cells

Cell lysates of PBMCs from eight ATL patients (ATL numbers 1–8) and two healthy donors (Normal numbers 1 and 2) were resolved by SDS/PAGE and analysed by Western blotting with antibodies against anti-HIF-1α, anti-phosphorylated Akt (Ser⁴⁷³), anti-Akt and anti-actin antibodies. MT-2 was used as a positive control.