Page 111 - Haematologica3

G2-arrested B lymphoma is prone to Wee1 inhibition
cemid, a microtubule-depolymerizing drug that blocks M phase progression (Figure 3B, Untreated versus Untreated plus colcemid). However, we did not detect a significant difference in cell cycle arrest between wt and p53 condi- tional knock-out primary B cells (Figure 3B). Ara-C treat- ment blocked M phase entry regardless of p53 genotype (Figure 3B). We, therefore, conclude that p53 deficiency does not affect cell cycle progression of primary B cells upon Ara-C treatment. This observation is consistent with our findings with G1XP lymphomas that are deficient in both Xrcc4 and Trp53, yet, we found that G1XP lym- phomas were arrested in the G2 phase, similar to p53-pro- ficient CH12 cells. These data suggest that Ara-C-induced G2 phase arrest is also independent of p53.
Increased cyclinA2 and cyclinB1 were not required for Ara-C-induced G2 phase arrest
To address whether increased cyclinA2 and cyclinB1 were required for Ara-C-induced G2 phase arrest,
cyclinA2, cyclinB1 or both were knocked down in CH12 cells by transient transfection of corresponding short hair- pin RNA. Western blot confirmed the reduced expression of cyclinA2, cyclinB1, or both in knocked down CH12 cells compared with scrambled controls (Online Supplementary Figure S6A). However, the reduction of cyclinA2, cyclinB1, or both had no obvious effects on Ara- C-induced G2 phase arrest (Online Supplementary Figure S6B). Additionally, the cleaved caspase3 was not affected by reduced expression of cyclinA2, cyclinB1 or both (Online Supplementary Figure S6A).
Wee1 inhibitor sensitized G2 phase-arrested lymphomas to Ara-C treatment
Ara-C treatment increased pCDK1 (Figure 1B), indicat- ing that pCDK1 may contribute to Ara-C-induced inhibitory effects on mitotic entry (Online Supplementary Figure S5). Since Wee1 kinase can phosphorylate CDK1, we employed Wee1 inhibitor to reduce pCDK1 in G1XP
Figure 4. Wee1 inhibitor (MK1775) enhances premature mitotic entry, mitotic catastrophe and apoptosis upon Ara-C treatment. (A, B) Wee1 inhibitor enhances premature mitotic entry of G2 phase-arrested B-cell lym- phomas upon Ara-C treatment. G1XP lymphoma cells were either untreated or treated with 1 μM Ara-C, 100 nM MK1775, or both for 24 h. Cells were collected and fixed by 70% ethanol. After propidium iodide (PI) and pH3 staining, cell cycles were determined by FACS (FL1-H/FL2-A). Statistical signifi- cance was calculated with one-way ANOVA, Tukey multiple comparison test, **P≤0.01 in (B). (C) Increased apopto- sis of B-cell lymphomas upon combined treatment with Ara-C and MK1775. G1XP lymphoma cells were treated as described in (A). pCDK1 and caspase3 were detected by western blot and β- actin was the loading control. (D) Increased mitotic catastrophe of B-cell lymphoma upon combined treatment with Ara-C and MK1775. G1XP lym- phoma cells were treated as described in (A). Cells undergoing mitotic catastro- phe (white arrow) were detected by flu- orescent microscopy with DAPI. (E) Increased cell death of Ara-C-induced G2 phase-arrested lymphomas upon MK1775 treatment. CH12 and G1XP lymphoma cells were treated as described in (A). Statistical significance was calculated with one-way ANOVA, Tukey multiple comparison test, *P≤0.05. (F) G1/S phase-arrested lym- phomas upon Ara-C treatment are not sensitive to MK1775 treatment. Ramos, Ly1, Ly7 and DHL-16 lym- phomas cells were either untreated or treated with 1 μM Ara-C, 100 nM MK1775, or both for 24 h. Cell numbers were counted and are presented as the percentage of the untreated group. Data are representative results of three independent experiments.
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