Lenalidomide upregulates p21 in CLL cells. (A) CLL cells from 4 patient samples were cocultured with Fibroblasts_CD154 and IL-4/IL-10, and exposed to 10 µM lenalidomide or the equivalent volume of DMSO. After 24 hours, the cells were collected and analyzed for the expression levels of p21 messenger RNA (mRNA) by QuantiGene. The expression of hypoxanthine guanine phosphoribosyltransferase was monitored as housekeeping gene and used to normalize p21 expression level for each sample. *P < .05 (Student t test, mean ± SEM; n = 4). (B-C) CLL cells were cocultured with HeLa_CD154 and IL-4/IL-10 and exposed to increasing amounts of lenalidomide or vehicle control. In parallel, HeLa_CD154 alone were cultured with increasing doses of lenalidomide as a control. After 24 hours, the cells were collected and protein extracted for the analysis of p21, p53, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression by immunoblot. In (B), the results from 3 CLL samples are shown, along with the HeLa_CD154 control, for which the same amount of total protein as the CLL cells was run. In (C), the densitometry analysis of p21 and p53 expression is presented for all 3 patients from (B). The intensity of the target proteins were normalized to GAPDH levels. One-way ANOVA and Tukey’s multiple comparison test were used to determine statistically significant differences from control. *P < .05; ***P < .001 (mean ± SEM; n = 3). (D) CLL cells from a sample deficient in functional p53 (p53 def) and from a sample with functional p53 (p53 WT) were exposed to either γ-irradiation (1 Gy) or 3 µM lenalidomide followed by 8 hours incubation, at which point the cells were collected for protein extraction and detection of p21, p53, and GAPDH by immunoblot. (E-F) CLL cells from 16 patients were cocultured with HeLa_CD154 and IL-4/IL-10 and exposed to 3 µM lenalidomide or DMSO. After 3 days, the cells were collected, lysed, and analyzed for p21 and GAPDH expression by immunoblot. (E) Shows the immunoblot results, while (F) presents the densitometry analysis quantifying the expression levels of p21 protein for all patients presented in (E). The expression levels of p21 have been normalized to GAPDH. ***P < .001 (Student t test). (G) CLL cells were cocultured with HeLa_CD154 and IL-4/IL-10 and exposed to 3 µM lenalidomide or an equivalent volume of DMSO. At day 3, a fraction of the cells were collected, lysed, and analyzed for the expression of p21 and GAPDH by immunoblot, as described above. At day 6, viable cell counts were performed by flow cytometry as described in “Methods.” The percent decrease in proliferation for each patient measured at day 6 (100 × [expansion fold_{CTRL-treated samples} − expansion fold_{lenalidomide-treated samples}] / expansion fold_{CTRL-treated samples}) is presented in function of the percent increase in p21 protein expression measured by densitometry analysis as above (100 × [p21_{CTRL-treated samples} – p21_{lenalidomide-treated samples}] / p21_{CTRL-treated samples}). Each dot represents data from 1 CLL patient (n = 22; Pearson r = 0.52).