Dexamethasone inhibits cell growth in the early, but not the late stage of HTLV-I-induced transformation. IL-2-dependent HTLV-I-infected T cells (D-ED40515, D-ATL43, D-ATL2T cells), representing the early transformation, and their IL-2-independent derivatives (I-ED40515, I-ATL43, I-ATL2T cells), representing the late stage of transformation, were treated with Dex or ethyl alcohol. (a) Cell proliferations of D-ED40515/I-ED40515T cells were examined by SF cell count reagent at 72 h after treatments. Data were shown as mean±s.d. (n=6). ^*P<0.05; ^**P<0.01; ^***P<0.001. (b) At 48 h after Dex treatment, D-ED40515T cells were collected and used for immunoblotting to detect cleaved caspase-3 and PARP. β-Actin served as a loading control. Uncl, uncleaved; cl, cleaved. (c) At 48 h of culture, D-ED40515 (upper panel) and I-ED40515 (lower panel) T cells were collected and used for flow cytometry to assess the proportion of early apoptosis. Data are expressed as mean±s.d. (n=3). (d and e) At 48 h after Dex treatment, I-ED40515T cells were collected and immunoblotting and flow cytometry were carried out, respectively, as in (b) and (c). (f) Cell proliferations of D-ATL43/I-ATL43T cells were as in (a). (g–j) D-ATL43/I-ATL43 cells after Dex treatment were used for immunoblotting and flow cytometry analysis as in (b–d). (k) Cell proliferations of D-ATL2/I-ATL2T cells were as in (a). (l–o) D-ATL2/I-ATL2 cells after Dex treatment were used for immunoblotting and flow cytometry analysis as in (b–d).

GR mediates TBP-2 induction, apoptosis and caspase activation in the early stage of HTLV-I-induced transformation. (a) At 48 h after treatment with doses of Dex, D-ED40515T cells were collected. TBP-2 mRNA levels were measured by quantitative RT-PCR (upper panel). Data are shown as mean±s.d. (n=4). ^***P<0.001. Immunoblotting was also performed to detect TBP-2 protein (lower panel). β-Actin served as a loading control. (b) At 48 h after treatment with doses of Dex, I-ED40515T cells were collected. TBP-2 mRNA (upper panel) and protein (lower panel) were measured as in (a). The ‘0' indicated no detection within the PCR cycles (n=40). (c–f) D-ED40515T cells were treated, respectively, with either 10 n Dex or 100 n RU486, or both, for 48 h. Samples treated with ethanol vehicle (0.004%, v/v) were used as a negative control. (c) Cell growth was examined using SF cell count reagent. (d) Early apoptosis was determined by flow cytometry. (e) Immunoblotting was performed to detect cleaved caspase-3 and PARP. Uncl, uncleaved; cl, cleaved. (f) TBP-2 mRNA (upper panel) and protein (lower panel) were measured by real-time quantitative RT-PCR and immunoblotting, respectively. ^**P<0.01. Representative results of three independent experiments were shown. (g–l) ATL43T cells (D-ATL43/I-ATL43) were used as in (a–f).

Forced expression of GR and treatment with SAHA in the late stage of HTLV-I-induced transformation restores GC sensitivity. I-ED40515T cells were transfected with 5 μg of pCMV-tag3B-GRα or pCMV-tag3B control plasmids. (a) At 24 h after transfection, immunoblotting was performed to verify the expression of GRα. (b–d) Then, ethanol vehicle (0.1%, v/v), dexamethasone (1 μ), SAHA (2.5 μ) or dexamethasone (1 μ)/SAHA (2.5 μ) combinations were added, respectively, into cells. (b) Immunoblotting for PARP was performed. β-Actin was used as a loading control. Each lane number was labeled below the blot. (c) Cell pellets were collected and flow cytometry analysis was performed. Each column number was labeled below the graph. (d) Cell pellets were collected to measure TBP-2 mRNA expression by real-time quantitative RT-PCR. Data of relative TBP-2 expression normalized to the glyceraldehyde 3-phosphate dehydrogenase expression are shown. ^**P<0.01; ^***P<0.001. Each column number was labeled below the graph. (e and f) I-ED40515T cells were transfected with 5 μg of pCMV-tag3B-GRα plus TBP-2 antisense oligonucleotides (RNAi no. 2) or control oligonucleotides. At 24 h after transfection, dexamethasone (1 μ)/SAHA (2.5 μ) combinations were added into cells. (e) Immunoblotting for myc-tagged GRα and TBP-2 was performed to verify the transfection and knockdown efficiency. ‘+', RNAi no. 2 added; ‘−‘, control RNAi added. Densitometric ratios of TBP-2/β-actin (T/β) are listed below. Representative data of two independent experiments were shown. (f) Cell pellets were collected and flow cytometry analysis was performed. Data were shown as mean±s.d. in triplicate.

Expression of GR and TBP-2 was diminished during HTLV-I-induced transformation. The amounts of GRα (a) and TBP-2 mRNA (b) were determined by real-time quantitative RT-PCR. The relative mRNA expressions normalized to the glyceraldehyde 3-phosphate dehydrogenase expressions are shown and expressed as mean±s.d. (n=3). ^*P<0.05; ^**P<0.01. (c) Immunoblotting was performed to detect GRα and TBP-2 proteins. β-Actin served as a loading control. Representative results of three independent experiments were shown.

Knockdown of TBP-2 in the early stage of HTLV-I-induced transformation abolishes GC-induced growth inhibition and apoptosis. (a and b) D-ED40515T cells were transfected with control oligonucleotides (Control RNAi) or two sets of TBP-2 antisense oligonucleotides (RNAi no. 2, RNAi no. 3) as described in the ‘Materials and methods' section. At 24 h after the transfection, cells were treated with 1 μ Dex or the ethanol (0.1%, v/v) for another 24 h. (a) Immunoblotting was performed to detect TBP-2 protein. β-Actin was used as a loading control. Densitometric ratios of TBP-2/β-actin (T/β) are listed below. ‘+', Dex added; ‘−', ethanol added. (b) Flow cytometry assay for detecting the early apoptotic proportion was performed. Data from three independent experiments are shown as mean±s.d., ^*P<0.05. (c and d) D-ATL43T cells were used for the TBP-2 knockdown assays as in (a and b).

Ectopic expression of TBP-2 in the late stage of HTLV-I-transformed T cells inhibits cell growth and causes apoptosis. I-ED40515T cells (a) and I-ATL43T cells (b) were transfected with 2 μg pCMV-tag2A-TBP2 or empty vector pCMV-tag2A, respectively. After 48 h, immunoblotting was performed using an anti-Flag antibody to detect Flag-tagged TBP-2 expression (upper panel) and other corresponding antibodies to detect cleaved caspase-3, PARP, β-actin, respectively (middle panel). Flow cytometry assay was performed to detect early apoptotic proportion (lower panel). Data are shown as mean±s.d. (n=3). ^***P<0.001.