Cytokinesis arrest and multiple centrosomes in B cell chronic lymphocytic leukaemia

Abstract Cytokinesis failure leads to the emergence of tetraploid cells and multiple centrosomes. Chronic lymphocytic leukaemia (CLL) is the most common haematological malignancy in adults and is characterized by clonal B cell expansion. Here, we show that a significant number of peripheral blood CLL cells are arrested in cytokinesis and that this event occurred after nuclear envelope reformation and before cytoplasmic abscission. mRNA expression data showed that several genes known to be crucial for cell cycle regulation, checkpoint and centromere function, such as ING4, ING5, CDKN1A and CDK4, were significantly dysregulated in CLL samples. Our results demonstrate that CLL cells exhibit difficulties in completing mitosis, which is different from but may, at least in part, explain the previously reported accumulation of CLL cells in G0/1.

phenomenon remains incompletely understood. It is thought that numerical aberrations (aneuploidy) that are frequently found in tetraploid cells are connected to the presence of multiple centrosomes that block spindle geometry and, as a result, interfere with accurate chromosome segregation. 7 In this study, we report that a significant number of CLL cells are arrested in cytokinesis. Immunohistochemistry staining for known mitosis exit markers such as Tubulin, Actin and Polo-like kinase 1 (Plk1) was used to demonstrate that CLL cell doublets are retained at the stage of mitotic exit with multiple centrosomes in interphase.
Moreover, we found that expression levels of several genes known to regulate mitotic exit and centrosome function were significantly reduced in CLL samples. Importantly, the described cytokinesis defect is distinct from the previously reported accumulation of cells in G0/1.

| Isolation of CD19+ cells
Mononuclear cells (MNC) from 40 mL of peripheral blood (CLL patients) or 50 mL buffy coat (healthy donors) were separated by Ficoll-Hypaque density gradient centrifugation as described. 8 CD19+ B lymphocytes were positively selected using magnetic beads according to the manufacturer's instructions (Miltenyi Biotec, Germany), and beads were removed from cells using multi-sort release agent (Miltenyi Biotec). Samples were obtained following informed consent using protocols approved by the Regional Medical and Health Research Ethics Committee of South-East Norway.

| Statistical analysis
For all statistical analyses in the manuscript, t test was used. Quantitative data are presented as average AE SEM, and differences with P ≤ .05 using t test were considered significant.

| A significant number of CLL cells were arrested in cytokinesis
Cell cycle arrest is manifested as one of the key features of CLL 10 although the underlying molecular events remain incompletely understood. Here, we performed immunofluorescence staining for Actin and Tubulin on peripheral blood CD19+ B cells isolated from healthy donors and CLL patients (Supporting Information) and showed that approximately 30% of CLL cells were blocked at the cell cycle mitotic stage, more precisely at the step of cytokinesis ( Figure 1A-D). In contrast, we could not observe such a phenomenon in healthy donor samples ( Figure 1A). Using Lamin B1 and DAPI staining, we demonstrated that the nuclear envelope was closed and chromosomes were decondensed ( Figure 1B); however, in CLL doublet cells the cytoplasm was interconnected ( Figure 1A).
When we performed immunofluorescence staining for Plk1 and Aurora B, proteins known to regulate cytokinesis, 11,12 we found that Plk1 localized at the cytokinesis contractile ring in CLL doublets ( Figure 1C), which is a hallmark of cytokinesis. 11 Taken together, our results indicate that CLL cell doublets were in the cytokinesis stage of the cell cycle, specifically, after reformation of the nucleus, but before abscission and physical separation of the cytoplasm. 11,[13][14][15][16] To investigate whether CLL doublets can complete cytokinesis, we co-cultured them on CD40L feeder cells and performed live cell imaging. We observed that CLL doublets fluctuate back and forth and remained interconnected, a phenomenon we could not find in healthy donor cells (Figure 2A

| Basal TP53 expression levels were significantly altered in CLL samples
The heatmaps shown in Figure 3A contain several genes that encode proteins known to play a role in cell cycle control and to interact with TP53. As reduced TP53 levels have also been associated with accumulation of cells in cytokinesis, 25,26 we examined a potential link between TP53 dysregulation and the observed cytokinesis defect. In all analysed CLL samples, we found significantly reduced TP53 protein ( Figure 4A,C) and reduced mRNA expression levels compared to healthy donor samples (Supporting Information). In one CLL sample, we could not detect any TP53, and in 2 of 9 analysed CLL samples, TP53 migrated higher, which can possibly be explained by post-translational modifications ( Figure 4A). While it has previously been shown that TP53 is dysregulated in CLL cells, 27 to the best of our knowledge, we are the first to compare basal protein levels of TP53 between CLL and healthy donor CD19+ cells.

| NuMA protein levels were reduced in CLL patient samples
Proteomics analysis performed on a subset of CLL samples indicated that NuMA protein expression levels might be altered in CLL (data not shown). As NuMA is known to be required for proper assembly and maintenance of the mitotic spindle, and non-functional NuMA has been shown to cause defects in mitosis exit and inability to complete cytokinesis, 28,29 we analysed NuMA protein levels in our cohort of CLL samples. Indeed, we found a significant reduction (50%) of NuMA in 8 of 9 analysed CLL compared to 5 healthy donor samples ( Figure 4A,B).
In addition, we detected cytoplasmic leakage of NuMA in CLL samples, while this was not the case in healthy donor samples ( Figure 4D,E). These results further support that CLL cells exploit mitosis defects since under physiological conditions, NuMA localizes to the nucleus during interphase and is dispersed throughout the cytoplasm during mitosis. 30,31 F I G U R E 4 TP53 and NuMA protein levels were dysregulated in chronic lymphocytic leukaemia (CLL) samples. (A) Protein lysates of CD19+ cells (10 lg) isolated from CLL samples (n = 9) and healthy donor control were analysed for NuMA and TP53 protein levels by immunoblotting. For healthy donors, a total of 5 samples were analysed for NuMA and TP53 protein levels; however, only tone representative sample has been included in the present blot.

| DISCUSSION
In the present study, we report for the first time that a significant number of CLL cells are arrested in cytokinesis. CLL cells exhibit supernumerary centrosomes and decreased expression levels of genes encoding proteins involved in cell cycle regulation and mitotic progression such as CENPO, PSME3 and CDKN1A. In contrast, genes encoding CDC16, CDK4 and CENPT, which are known to be involved in centrosome assembly and duplication, were increased in CLL samples.
The evidence that CLL cells were arrested in cytokinesis is based on Actin, Tubulin and Plk-1 staining of a joint cytoplasmic bridge, which is distinct from the previously described overrepresentation of cells in the G0/1 cell cycle phase. The described Actin, Tubulin and Plk1 localization corresponds to late steps of cytokinesis, more precisely, to the step after nuclear envelope reformation and chromosome decondensation, but before cytoplasmic abscission by the contractile ring between the 2 daughter cells. 11,[13][14][15][16] Previous studies have linked cytokinesis defects, multiple centrosomes and accumulation of cells in G0/1 to be a consequence of dysregulation of important players in cell cycle regulation. 34 Centrosome amplification can be induced via deregulation of the centrosome duplication process in interphase [35][36][37][38][39][40][41][42][43] or through mitotic defects resulting in G0/1 cells with tetraploid cells. 44 The latter can be experimentally induced by overexpression of Plk1, Aurora A and Aurora B. 41,44 Our data also indicate that centrosome aberrations in malignant cells can arise via mitotic defects. The described cytokinesis failure leading to cells that harbour supernumerary centrosomes resembles the findings described for overexpression of Plk1, Evi1, Aurora A and Aurora B. 41,44 The TP53 pathway has been extensively studied in CLL, and chromosomal aberrations such as deletion of 17p13 are known to cause loss of TP53 function. 45 In addition, decreased TP53 expression and activity independent of 17p13 deletions have been associated with deletion of the ATM gene, which increases MDM2 activity and in turn leads to decreased TP53 pathway activity 45,46 .
In the present study, we detected significantly reduced basal TP53 protein levels in all analysed CLL samples, although only 2 of the 20 patients included in our study are known to harbour a TP53 mutation. Down-regulation of TP53 is known as one cause to bypass anaphase checkpoint and DNA damage response during mitosis exit, which under normal conditions is active and prevents F I G U R E 5 Chronic lymphocytic leukaemia (CLL) cells exhibited multiple Pericentrin-positive centrosomes. Representative images of primary CD19+ cells stained with Lamin B1 (red), Pericentrin (green) and DAPI (blue). All analysed patient samples (n = 11) displayed multiple centrosomes, while this was not the case for any of the analysed healthy donor samples (n = 4). Scale bar 10 lm cells from acquiring chromosomal aberrations. Moreover, reduced TP53 levels lead to multiple centrosomes and accumulation of cells in cytokinesis. 26,41 Reduced TP53 protein levels may contribute to the cytokinesis defect described in the present study, a notion supported by our finding that siRNA-mediated knockdown of TP53 in healthy donor CD19+ cells led to an accumulation of cells arrested in cytokinesis.
NuMA is important for maintenance of the mitotic spindle, and non-functional NuMA has been shown to cause defects in mitosis exit and completion of cytokinesis. 28,29 These studies support our finding that reduced NuMA levels may contribute to the cytokinesis arrest described in the present study. Previous studies performing RNAi-mediated knockdown linked reduced NuMA protein levels to reduced CDKN1A (p21) mRNA expression levels, while it does not affect expression of TP53 regulated pro-apoptotic genes. 28, 29 We also showed that CDKN1A expression levels were reduced in CLL compared to healthy donor samples, while pro-apoptotic genes such as TP53I3, ING4 and ING5 were increased.
One key characteristic of CLL, as assessed by flow cytometrybased analysis is the accumulation of clonal B cells arrested in the early G0/1 phase of the cell cycle. 47,48 A possible explanation why the cytokinesis defect described here has not previously been reported is that conventional flow cytometry does not easily distinguish between cells in the G1 or cytokinesis part of the cell cycle and that a majority of cells in cytokinesis are counted as G1 cells because of the break of the cytoplasmic bridge. 49 In conclusion, our results provide novel insights into the cell cycle dysregulation that occurs in CLL. Centrosome amplification, secondary to a cytokinesis defect, might be one important contributing factor to chromosomal instability in CLL. Additionally, it may be a critical

CONF LICT OF I NTEREST
The authors declare no conflict of interest.