Reed-Sternberg cells form by abscission failure in the presence of functional Aurora B kinase

Abstract

Large multinucleated Reed-Sternberg cells (RS) and large mononucleated Hodgkin cells (H) are traditionally considered to be the neoplastic population in classical Hodgkin lymphoma, (cHL) and postulated to promote the disease. However, the contribution of these larger cells to the progression of cHL remains debatable. We used established cHL cell lines and cHL cellular fractions composed of small mononucleated cells only or enriched in large RS/H cells to investigate RS/H cell origin and to characterize the cells which they derive from. We confirm that the small mononucleated cells give rise to RS/H cells, and we show that the latter proliferate significantly more slowly than the small cells. By using live-cell imaging, we demonstrate that binucleated RS cells are generated by failure of abscission when a few small cells attempt to divide. Finally, our results reveal that the small mononucleated cells are chromosomally unstable, but this is unlikely to be related to a malfunctioning chromosomal passenger protein complex. We propose that the small mononucleated cells, rather than the RS/H cells, are the main drivers of cHL.

Fig 1. Binucleated RS cells form by failure of abscission.

Time lapse video imaging of KMH2 cells undergoing cell division. Shown are selected DIC still images. Time is in hours:minutes:seconds. Time zero is anaphase in A,B,D,E and timepoint after nuclear envelope breakdown in C. A. 88% of the cells divide successfully giving rise to two daughter cells. B. 3.2% of the cells seem to complete cell division but maintain an intercellular bridge connecting the two cells (white arrow) that later broadens up leading to the formation of binucleated cells. C. 8% of cells undergo apoptotic death during mitosis. D. < 1% of sister cells appear to fuse but actually remain separate, as the cells round up separately for the following mitosis. E. Small mononucleated cell undergoing cell division and failing cytokinesis after completing furrow ingression. The two daughter cells are still connected by the midbody (white arrows), when the furrow regresses. Black arrow points at chromosomal bridges during anaphase. Size bar is 10 μm.

Fig 2. Aurora B activity is normal in cHL small and large cells.

A-F. Immunofluorescence images of small mononucleated cells (A-E) and large cell undergoing mitosis (F). A. Cell in prometaphase with Aurora B kinase properly localized at centromeres and phospho-Ser10-histone H3 on chromatin. B. One cell in telophase (left) and one cell in the last stages of cytokinesis (right). Aurora B localizes properly in the central spindle or in the thin intercellular bridge that connects the two sister cells, respectively. As expected, signal for phospho-Ser10-histone H3 is only observed in the cell on the left where nuclear envelope has not reformed yet. C. Cell in prometaphase with most of the chromosomes aligned at the metaphase plate and one mono-oriented chromosome on the spindle pole with the unattached kinetochore showing prominent accumulation of auto-phosphorylated Aurora B (white arrow). Asterisk indicates a spindle pole that is unspecifically labeled by the P-T232-Aurora B antibody. D. Cell in the last stages of cytokinesis with chromatin present in the intercellular bridge (white arrowhead). Auto-phosphorylated Aurora B accumulates on the intercellular bridge revealing that Aurora B has been properly activated at a time when abscission is taking place. E. Cell in telophase with chromatin bridges staining positive for phospho-Ser10-histone H3. F. Large cell with a double metaphase plate showing auto-phosphorylated Aurora B and therefore active kinase on centromeres. Asterisks are the spindle poles. Size bar is 5 μm.