The PI3K and AKT pathways are associated with increased telomerase activity in HTLV-I–immortalized and –transformed cells. (A,B) IL-2–independent (MT4 and C8166) and –dependent (LAF and 1185) cells were treated with LY29 (LY294002, 10 μM) or control, LY30 (LY303511, 10 μM), for 48 hours, followed by TRAP analysis for telomerase activity. (B) Cells were treated with AKT Inhibitor II (20 μM) or DMSO control (solvent control). TPGs are calculated as the percentage activity compared with cells grown without inhibitor (100%). Results are representative of at least 2 independent experiments, and SD is indicated. To verify inhibitor efficacy, total cell extracts were immunoblotted with anti-phosphorylated AKT (P-AKT). Anti-AKT served as a loading control. The percentage of decreased expression of P-AKT, as stated in the text, was calculated by spot densitometry with cells grown in either LY30 or DMSO considered to be 100%. (C) HTLV-I cell lines, MT4 and LAF, were treated for 48 hours with either LY29 (10 μM), AG490 (50 μM), or control DMSO. Cells were subsequently stained for annexin V and propidium iodine (PI), and analyzed for apoptosis by FACS analysis. The percentage of dead cells for each treatment is indicated. (D) 1185 and LAF cells were cultivated without IL-2 and in the presence of LY29 (10 μM) for 48 hours. After the 48-hour incubation period, the cultures were split and IL-2 was added in the absence or presence of LY29 (10 μM) for an additional 24 hours. TRAP analysis was performed and TPGs were calculated as the percentage of activity compared with cells grown without treatment (100%). As a control, cells were grown for 48 hours without IL-2, then treated an additional 24 hours without IL-2, with or without LY29 (10 μM). Cells grown continuously in IL-2 served as a positive control.

IL-2R/IL-2 signaling regulates hTERT promoter expression. (A) IL-2 was withdrawn from 1185 and LAF cells for 4 or 24 (overnight [O/N]) hours, followed by hTERT RNA expression by RT-PCR. (B) LAF cells were cultured in the absence of IL-2 for 4 hours, and subsequently pulsed with IL-2 for 4 and 24 hours before hTERT RT-PCR analysis. Cells grown continuously in IL-2 media served as a control (first lane). (C) 1185 cells were treated with actinomycin D (5 μg/mL) in the presence or absence of IL-2 for 0, 1, 2, and 4 hours. RNA was extracted and analyzed for hTERT expression by RT-PCR. (A-C) GAPDH was used as an amplification control.

IL-2–dependent WT1 binding to the hTERT promoter. (A) The hTERT-3915 promoter was biotin labeled and bound to streptavidin-agarose beads, followed by incubation with 1185 cellular extracts from cells cultivated overnight in the presence or absence of IL-2. Bound proteins were visualized using anti-WT1 or anti–c-Myc. The percentage of increased binding of WT1 to the hTERT promoter was calculated by spot densitometry as indicated, with 1185 cells grown in the presence of IL-2 considered to be 100%. (B) 293T cells were transfected with wild-type WT1 (SS) or mutant WT1 (FF), along with the hTERT-Luc promoter, or wild-type WT1 (SS) or mutant WT1 (FF), along with the hTERT-Mut-Luc promoter (defective for WT1 binding). Extracts were assayed 48 hours after transfection for luciferase activity. Cell extracts were probed with anti-WT1 and antiactin to verify expression. Average luciferase values were as follows: hTERT-Luc promoter (150 518), + WT1 (SS) (79 578), + WT1 (FF) (241 727) (SD measured from at least 2 independent experiments) and for the hTERT-Mut Luc promoter (1 166 792), + WT1 (SS) (1 468 345), + WT1 (FF) (1 736 347) (SD measured from multiple readings from the same experiment).

Removal of IL-2 from HTLV-I–immortalized cells decreases telomerase activity. (A) Total cell extracts from IL-2–independent (MT2 and MT4) and IL-2–dependent (LAF and 1185) cell lines were immunoblotted with anti-Tax. Actin served as a loading control. (B) LAF and 1185 cells were cultured in the presence (+) or absence (−) of IL-2 for 24 hours, followed by TRAP analysis for telomerase activity. TPGs are calculated as the percentage of activity compared with cells grown with IL-2 (100%), as previously reported.⁹ Results are representative of at least 2 independent experiments, and standard deviation (SD) is indicated. (C) LAF cells were analyzed at either 4 hours or 24 hours after growth in either IL-2–containing or IL-2–deprived media. For each time point, cells were collected and analyzed by FACS for cell-cycle and TRAP for telomerase activity. TPGs were calculated as the percentage of activity compared with cells grown with IL-2 (100%).

Telomerase activity is regulated at the level of transcription by a PI3K-dependent pathway in HTLV-I cell lines. (A) 1185 cells were treated with LY29 (10 μM), AKT II (20 μM), LY29 (10 μM)/AKT II (20 μM), or DMSO (solvent control) for 4 hours, followed by analysis of hTERT RNA expression by quantitative real-time PCR. GAPDH expression was used as a control. The expression level in 1185 cells treated with DMSO was defined as 1.0. Results are representative of 3 independent experiments and standard deviations are indicated by error bars. (B-D) 1185 cells were treated with LY29 (10 μM), AKT II (20 μM), LY29/AKT II (10 μM/20 μM), or DMSO (solvent control) for 48 hours, followed by TRAP analysis for telomerase activity (B), FACS analysis for cell cycle (C), or FACS analysis for apoptosis (D). TPGs were calculated as the percent-age of activity compared with DMSO control (100%). (E) 1185 cells were grown with or without IL-2 for 4 hours followed by Western blot analysis. Nuclear extracts were probed with anti–NF-κB RelA/p65, and cytoplasmic extracts with anti–IκB-α.

Cellular localization of WT1 dictates repression of hTERT expression. (A) MT4 and 1185 cells were treated with LY29 (10 μM), AKT II (20 μM), or DMSO (solvent control) for 48 hours followed by immunostaining with anti-WT1 (green). Nuclear staining was visualized by DAPI (blue). (B) 1185 cells were cultured overnight with or without IL-2 and stained with anti-WT1, as described in panel A. (C) The HTLV-I–transformed cells, MT2 and MT4, or the HTLV-I–immortalized cells, 1185, were electroporated with the hTERT-Luc-Promoter using the Amaxa transfection kit (Amaxa Biosystems). Twelve hours after transfection, MT2 and MT4 cells were treated with either LY29 (10 μM) or DMSO (solvent control) for 8 hours. 1185 cells were electroporated and cultured in media with and without IL-2 for 24 hours. Cell extracts were normalized and assayed for luciferase activity.