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.