HTLV-I–associated up-regulation of hTERT mRNA expression. (A) Expression of hTERT mRNA was investigated by real-time RT-PCR at time 0 and at 3, 5, 7, and 12 weeks after coculture of PHA-stimulated PBMCs with HTLV-I–transformed lethally irradiated MT-2 cells (100 Gy). Human β-2-microglobulin was used as endogenous control. Reactions were in triplicate for each sample. Experimental variations are indicated by error bars. The expression level in nonactivated PBMCs was defined as 1.0 and the expression levels were expressed as fold induction relative to the PBMCs. Expression of hTERT mRNA in MT-2 is shown for comparison. (B) Western blot analysis of viral protein expression in MT-2–derived cocultured MU04 cell line and MT-2 control. (C) Telomeric amplification protocol (TRAP) assay was performed as described in “Materials and methods” using 125 ng protein lysate in CHAPS buffer. IC indicates internal 36-bp control. Quantification of telomerase activity, telomerase generated product (TGP) was calculated according to the standard formula³² : TGP (units) = {[(X – X₀)/C]/[R – X₀/C_R]} ×100. X indicates telomerase amplification signal of sample; X₀, background; C, internal control signal of sample; R, telomerase amplification signal of TSR8 positive control (1 μL = 0.1 amole); and C_R, internal control signal of TSR8. It is normal that the intensity of the internal control (IC) is very low in the TSR8 reaction, however, this was previously demonstrated not to affect quantification as linear range of this assay is 0.001 amole to 10 amole.³²  In our assay, 0.1 amole TSR8 was used and corresponds to 100% of TGP.