Figure 2.

Higher TCL1 levels predict the degree of BCR-induced AKT phospho-activation in CLL short-term cultures. (A) After BCR crosslinking, responsive CLL cases showed rapid ERK1/2 phospho-activation followed by variable and usually prolonged pS473-AKT induction. pAKT activation could be detected in some cases by 15 minutes of BCR stimulation and was most variable between cases at 2 hours, peaked between 24 to 48 hours, and declined thereafter. Such responsive cases frequently showed parallel changes in TCL1 levels (see also Table 1). (B) Using 3 different α-IgM plate concentrations for each of the 70 CLL cultures, the degree of AKT phospho-activation after 2 hours of BCR engagement (graded as none, moderate, or strong) was highly correlated with TCL1 levels (P = .005, graded as in Figure 1). Among the strong pAKT responder group (red text), moderate to high TCL1 levels were seen in 24 of 25 cases, whereas 75% of the TCL1-dim/negative cases were in the nonresponsive group (black). A third group of cases showed moderate levels of pAKT induction, even at high α-IgM concentrations (blue, middle panel). (C) Full-length TCL1, introduced into the TCL1-negative DoHH2 B-cell line, showed both cytoplasmic and nuclear localization with most pAKT phospho-activation in the cytoplasmic fraction (left panel). TCL1 introduction led to mildly increased (1.26-fold) basal pAKT levels, compared with the TCL1⁻ baseline (“*” in right panel) but more prominent phospho-activation after BCR (**1.72-fold after TCL1⁺ baseline was set as 1.0) or LPS stimulation, but not after other B-cell cytokine stimuli. Numbers indicate normalized densitometric quantitation of blot, with 1.0 corresponding to the level seen in the unstimulated control condition (blue indicates TCL1-expressing transfectants).