Figure 3. CD44 activates the PI3K/AKT and MAPK/ERK pathways and increased MCL-1 expression.
CLL-cells were incubated with anti-CD44 antibody (BU75, 10μg/ml) or with isotype control antibody (anti-mouse IgG2, 10μg/ml) for 30 minutes, washed and then cross-linked with secondary goat anti-mouse antibody (1μg/ml). (A) Cell lysates (>90% CLL cells) were obtained at baseline and after 5 and 30 minutes of stimulation. The blots were probed with anti-phospho-Akt (ser473) and anti-phospho-ERK1/2 (Thr202/Tyr204) antibody as indicated. Equal protein loading was confirmed by probing for total AKT and ERK1/2. (B) An extended cohort of both M-CLL and U-CLL samples (n=20), showed increased AKT phosphorylation on average of 2.37±1.3 fold compared to control (p=0.0002). shown are Box and Whisker plots for densitomtery quantification of fold change increase in phospho-Akt (ser473) levels, after 30 minutes of CD44 engagement, both in M-CLL and U-CLL cells compared to control and normalized to total AKT (M-CLL samples n= 9, mean=2.45±1.57 Vs. U-CLL samples n= 11, mean=2.31±1.11 One-tailed T-test p=0.4).(C) Western blots of cell lysates obtained 24 hours after indicated stimulation were probed with antibodies against MCL-1, BCL-XL, BCL-2, and tubulin (loading control). (D) An extended cohort of both M-CLL and U-CLL samples (n=22), showed an increased MCL-1 expression on average of 1.45±0.66 fold compared to control (p=0.004). Shown are Box and Whisker plots for densitomtery quantification of fold change increase in MCL-1 levels, 30 minutes after CD44 engagement, both in M-CLL and U-CLL cells compared to control and normalized to tubulin (M-CLL n=10, mean=1.2±0.6, Vs. U-CLL=12, mean=1.65±0.66. One-tailed T-test p=0.06). (E) mRNA levels of MCL-1 by quantitative PCR after 6 hours of stimulation. The mean and standard deviation from 5 independent experiments is shown.