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X. Wang et al.
lymphomas and tested whether mitotic entry was affect- ed. Cell cycle analysis showed that Wee1 inhibitor enhanced the mitotic entry of Ara-C-induced G2 phase- arrested lymphomas (Figure 4A,B). Consistently, Wee1 inhibitor reduced the level of pCDK1 (Figure 4C). Furthermore, combined treatment of Ara-C and Wee1 inhibitor induced a higher level of cleaved caspase3 (Figure 4C), promoted more cells to undergo mitotic catastrophe (Figure 4D), and caused more cell death (Figure 4E).
Ara-C treatment resulted in G1 or S phase arrest in other lymphoma lines including DHL-16, Ly1, Ramos, and Ly7 (Online Supplementary Figure S7). Intriguingly, Wee1 inhibitor exhibited no effects on the level of mitotic entry (Online Supplementary Figure S7), cleaved caspase3 (Online Supplementary Figure S8) or cell death (Figure 4F) of the G1 or S phase-arrested lymphomas. Overall, our data suggest- ed that Wee1 inhibitor appears to preferentially promote
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mitotic entry, mitotic catastrophe and cell death in G2 phase-arrested lymphomas.
Newly acquired sensitivity to Wee1 inhibitor in doxorubicin-induced G2-phase-arrested lymphomas
To generalize our findings that Wee1 inhibitor may preferentially affect G2-phase-arrested lymphomas, we treated various lymphoma lines with doxorubicin and found that doxorubicin induced G2 phase-arrest in CH12, Ramos and Ly7 cells but G1 phase-arrest in Ly1 and DHL- 16 cells (Figure 5A). Consistently, we showed that com- bined treatment with doxorubicin and Wee1 inhibitor resulted in more cell death in G2 phase-arrested lym- phoma lines compared with doxorubicin alone (Figure 5B). In contrast, the combined treatment had no effects on the survival of non-G2 phase-arrested lymphoma lines (Figure 5C). In line with our findings in Ara-C-treated lym- phomas (Figure 4), we found that Wee1 inhibitor also
Figure 5. Newly acquired sensitivity to Wee1 inhibitor in doxorubicin-induced G2 phase-arrested lymphoma cells. (A) Cell cycle arrest of various lym- phoma lines upon doxorubicin treatment. CH12, Ramos, Ly7, Ly1 and DHL-16 were either untreated or treated with 100 nM doxorubicin, 100 nM MK1775, or both for 24 h. Cells were collected and fixed by 70% ethanol. After propidium iodide (PI) staining, cell cycles were determined by flow cytom- etry (FL2-A). (B) Increased cell death of doxoru- bicin-induced G2 phase-arrested lymphomas upon MK1775 treatment. CH12, Ramos and Ly7 lym- phomas cells were treated as described in (A). Statistical significance was calculated with one-way analysis of variance, Tukey multiple comparison test, *P≤0.05. (C) Non-G2 phase arrested lym- phomas, upon doxorubicin treatment, are not sen- sitive to MK1775 treatment. Ly1 and DHL-16 lym- phoma cells were treated as described in (A). Cell numbers were counted and presented as the per- centage of the untreated group. Data are represen- tative results of three independent experiments.
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haematologica | 2018; 103(3)