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G2-arrested B lymphoma is prone to Wee1 inhibition
probably would not promote M phase entry (see below). To generalize our findings, we treated several human B- cell lymphoma lines, including Ramos, OCI-LY1, OCI- LY3, OCI-LY7 and DHL-16, and found that Ara-C treat- ment upregulated cyclinB1, cyclinA2, and pCDK1 expres- sion in all of them (Figure 1C, Online Supplementary Figure S3A). Additionally, we examined other players involved in DNA damage responses and found that Ara-C treatment activated Chk1 and Chk2 in most of the lymphoma lines examined. However, we did not detect obvious differ- ences in the levels of CDC25A or pCDC25C (Online Supplementary Figure S3B). Next, we examined the kinetics of cyclinB1/A2 upregulation. CyclinB1 and cyclinA2 were upregulated after 8 h of Ara-C treatment (data not shown), and significant induction occurred after 20 h or 24 h (Online Supplementary Figure S3C). Overall, our results showed a time-dependent effect of Ara-C treatment on
upregulating cyclinB1/A2.
Lastly, we showed that another DNA-damaging agent,
doxorubicin, can also upregulate cyclinB1/A2 in several B-
A
cell lymphoma lines (Figure 1D). We conclude that cyclinB1 and cyclinA2 upregulation appears to be an intrinsically programmed DNA damage response, which occurs in both activated primary B cells and various B-cell lymphomas.
Ara-C induces differential cell cycle arrest in different types of B cells
Since cyclins control cell cycle progression, we exam- ined how Ara-C affected cell cycling in mouse primary B cells and B-cell lymphomas in a time- and dose-dependent manner. Based on the Ara-C doses employed in previous studies,30 we chose to test the effects of treatment with 1 μM and 10 μM Ara-C. Six hours of Ara-C treatment did not affect cell cycle progression significantly in wt primary B cells regardless of Ara-C dosage (Figure 2A, top panel). In contrast, after 24 h of treatment, wt primary B cells pre- dominantly arrested in the S phase in the presence of 10 μM Ara-C, whereas, 1 μM Ara-C treatment caused a mod- est increase in the percentage of S and G2 phase-arrested
B
C
Figure 2. Different types of B cells are arrested in distinct phases of the cell cycle upon Ara-C treatment. (A) Primary B cells were arrested in the S phase of the cell cycle upon Ara-C treatment. Anti-CD40/IL-4 activated wt primary B cells were treated with 1 μM or 10 μM Ara-C for 6 or 24 h. (B) Mouse B-cell lymphomas were arrested in the G2 phase of the cell cycle upon Ara-C treat- ment. CH12 or G1XP lymphoma cells were treated with 1 μM Ara-C for 6 or 24 h. (C) Ramos lymphoma cells were treated with 1 μM Ara-C for 24 or 36 h. Cells were collected at indicated time points and fixed by 70% ethanol. After propidium iodide (PI) staining, cell cycles were determined by FACS (FL2- A). Data are representative results of three to five independent experiments.
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