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X. Wang et al.
aptamer-based multiplexed proteomic technology,27 SOMAscan, to identify the differentially expressed pro- teins in untreated versus Ara-C-treated B cells. Due to the breakthrough of SOMAmer,27 the SOMAscanTM proteom- ic assay enables us to quantify 1310 proteins across approximately eight logs of concentration as shown by previous studies.28,29 Among 1310 proteins tested, we found that there were only three proteins differentially expressed between untreated and Ara-C-treated primary B cells from wt or Xrcc4/p53 double conditional knockout mice.18 These were cyclinB1/cdk1, cyclinA2/cdk2 and importin B1 (IMB1), all of which were significantly upregulated (fold change ≥2, P<0.05) in Ara-C-treated primary activated B cells (Figure 1A and Online Supplementary Tables S1 and S2). We present the top ten upregulated/downregulated proteins in Ara-C-treated wt or double conditional knock-out primary B cells (Online Supplementary Figure S2A,B).
We recently established a unique mouse model by specifically deleting a NHEJ gene, Xrcc4, and Trp53, in ger-
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minal center B cells, which results in the spontaneous development of G1XP lymphomas.18 Cell lines were established from G1XP lymphomas. In line with our data from primary B cells, we found that cyclinB1 and cyclinA2 were also upregulated by Ara-C in our newly developed G1XP lymphomas and in CH12 lymphomas (Figure 1A).
We performed western blotting to validate the results of the SOMAscan assay. Consistently, we found that cyclinB1 and cyclinA2 were indeed upregulated in Ara-C- treated wt or p53 conditional knock-out primary B cells, and in CH12 or G1XP lymphoma cells (Figure 1B). Notably, we found that CDK1 phosphorylation (pCDK1) at Tyr15 was also enhanced by Ara-C treatment in pri- mary B cells or B lymphoma cells; in contrast, total pro- tein expression of CDK1 and CDK2 was not increased (Figure 1B). When CDK1 is phosphorylated at Tyr15 by Wee1 kinase, it is inactivated and blocks M phase entry. Thus, these data suggest that although cyclinB1 is upreg- ulated, the cyclinB1/CDK1 complex stays inactive and
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Figure 1. Upregulation of cyclinB1 and cyclinA2 in different types of B cells upon Ara- C treatment. (A) SOMAscan analysis of untreated or Ara-C-treated B cells. CH12 lym- phoma (n=3), G1XP lymphoma (n=3), wt (n=3) or Xrcc4/p53 conditional knockout (n=3) pri- mary B cells were treated with 10 μM Ara-C for 16 h. Cell lysates were prepared and subject to SOMAscan assay. Data are presented as mean±s.e.m. (B) Increased expression of cyclinB1, cyclinA2 and pCDK1 in different types of B cells upon Ara-C treatment. CH12 or G1XP lymphomas, wt or p53-/- primary B cells were treated with 1 μM Ara-C for 24 h. CyclinB1, CDK1, pCDK1, cyclinA2 and CDK2 were detected by western blot and β-actin as the loading control. (C) Ramos lymphomas were treated and analyzed as described in (B). (D) Upregulation of cyclinB1, cyclinA2 and pCDK1 in different types of B-cell lymphomas upon doxorubicin (DOX) treatment. CH12, Ramos and Ly1 lymphomas were treated with 100 nM doxorubicin for 24 h and analyzed as described in (B). Data are representative results of three independent experiments.
haematologica | 2018; 103(3)