Spontaneous and BCR-induced changes in telomerase activity in CLL cases. Purified B cells from 6 U-CLL and 6 M-CLL cases were cultured without or with BCR stimulation. (A) Spontaneous decrease in telomerase activity within residual live cells at day 1, 2, and 3 of culture in U-CLL (left) and M-CLL (right) cases. (B) Telomerase activity assessed in cell extracts prepared from uncultured cells (A1 and A2), cells cultured in medium alone (B1 and B2), or cells cultured with HB57-dex for 3 days (C1 and C2). A1, B1, and C1 represent heat-treated internal controls for A2, B2, and C2, respectively. The representative autoradiograph on the left (U-CLL case) shows induction/retention of telomerase activity, whereas the M-CLL case on the right does not. (C) Change in telomerase activity over time, elicited by anti-IgM stimulation, in 1 representative case each. Stimulation using HB57-dex induces telomerase activity in U-CLL cases, but not in M-CLL cases. (D) Pooled data for fold increase in telomerase activity within HB57-dex cultures, relative to control in 17 U-CLL (left) and 17 M-CLL cases (right). U-CLL showed a significantly higher fold change in telomerase activity than M-CLL cases (P < .001). (E) Pharmacologic inhibition of telomerase activity induced by BCR stimuli: B cells from 10 U-CLL cases were subjected to pretreatment with inhibitors of PI3K/Akt, Erk, or p38MAP kinase and then activated using HB57-dex for 3 days. Cells were harvested and cell lysates subjected to quantification of telomerase activity. Data represent percent inhibition of the HB57-dex-mediated telomerase activity as elicited by the inhibitors. (F) Effect of preincubation with Akt-specific inhibitor on HB57-dex-induced hTERT phosphorylation (upper panel) in 3 U-CLL cases (U1, U2, and U3) and 3 M-CLL cases (M1, M2, and M3). The inhibitor exerts a dose-dependent effect on phosphorylation hTERT. Lower panel: Functional telomerase activity in parallel cultures.