Lenalidomide increases RNA stability and transcription of CD154 in CLL cells. (A) CD154 mRNA level was measured by real-time reverse-transcription–PCR after 48 hours of treatment with lenalidomide 0.5μM or vehicle control (n = 20, P < .001). (B) After 48 hours with lenalidomide (0.5μM) or vehicle control, transcription was inhibited by the addition of ActD and 107 cells were collected for each time point over a 4.5-hour time course. The percentage of RNA remaining from each time point is plotted. Lenalidomide increases CD154 mRNA stability as demonstrated by significantly diminished decay after ActD treatment of CLL cells compared with vehicle control (n = 6, P = .002). (C) CLL B cells were transfected with CD154 luciferase reporter plasmid along with 1 μg of pCMV-renilla reporter vector or pGL3 empty reporter plasmid (pGL3-basic) as a negative control. After 48 hours with lenalidomide 0.5μM or vehicle control, luciferase activity was determined and corrected for transfection efficiency using renilla activity (n = 13, P = .001 for lenalidomide vs vehicle control). (D) Nuclear extracts from lenalidomide- or vehicle control-treated CLL cells were resolved on SDS–polyacrylamide gel electrophoresis and subjected to Western blotting with anti-NFATc1 or anti–c-Rel antibody. Nuclear protein Brg1 was used as an internal loading control. (E) Lenalidomide treatment of CLL B cells enhanced promoter binding of NFATc1 (n = 3, P < .001) and c-Rel (n = 3, P < .001) to the CD154 promoter by chromatin immunoprecipitation (ChIP). PCR to detect the CD154 promoter region (CD154-κB) was performed on the precipitated DNA (n = 3, P = .004). Error bars represent SD. *Statistical significance.
