An important question related to cells undergoing endomitosis is whether the mitotic cyclins and kinases are altered to promote this cell cycle. Studies in megakaryocytic cell lines suggested that a reduction in activity of the cyclin B-dependent Cdc2 kinase is an important part of megakaryocyte polyploidization (Datta et al., 1996; Zhang et al., 1996). The work of Datta et al., carried out with a clone of HEL cells, had shown a reduction in Cdc2 kinase activity with polyploidy, even though levels of cyclin B1 in this system were apparently unaltered (Datta et al., 1996). Other investigations with synchronized megakaryocytic cell lines found cyclin B1, but not Cdc2, to be reduced in polyploidizing cells (Zhang et al., 1996). In all of these studies, cell cycle protein levels were determined by Western blot analyses of equally loaded proteins derived from diploid, actively proliferating cells, as compared to polyploidizing cells. In contrast to cyclin B1, the levels of cyclin A per micrograms protein in these latter cell lines were similar between diploid and polyploid cells, suggesting that the frequency of cells entering S phase in these populations was similar. Zhang et al. have also shown an increased destruction of cyclin B1, but not cyclin A, by the ubiquitin-proteosome pathway, both in a polyploidizing megakaryocytic cell line and in high ploidy primary murine megakaryocytes. These authors suggested that the anaphase promoting complex is functional in these cells and that the potentially accelerated or premature destruction of cyclin B may allow re-entry into the S phase of the cell cycle, thus promoting polyploidization (Zhang et al., 1998). In accordance with the above-described results in megakaryocytic cell lines, it is important to note that in yeast, activation of the degradation system, which lowers the levels of cyclin B, was found to be essential for re-entry into S phase in cells that do not enter anaphase due to an experimentally-induced mitotic blockage (Zachariae and Nasmyth, 1996). In mitotic cells, one of the licensing factors that permits cells to exit anaphase and to enter cytokinesis involves the degradation of cyclin B by the Anaphase Promoting Complex (APC) and inactivation of Cdc2 kinase (Adachi and Laemmli, 1994). This feature would be important in cells that undergo polyploidization by the endomitotic cell cycle. Cyclin B1 and Cdc2 were also examined in primary megakaryocytes. Cyclin B1 was detected in TPO-treated polyploidizing human megakaryocytes by immunohistochemistry, flow cytometry, immunoprecipitation, and Western blotting (Vitrat et al., 1998). The immunofluorescence microscopy studies showed association of cyclin B1 with the mitotic spindle in human as well as murine polyploid megakaryocytes during early mitosis (Nagata et al., 1997; Vitrat et al., 1998). Analysis by flow cytometry, involving DNA staining and reaction of the human cells with an antibody to cyclin B1, indicated that cyclin B1 is expressed in megakaryocytes of all ploidy classes. Careful examination of these flow cytometry data, however, revealed that cyclin B1 was not detectable during S phase, while the ratio of cyclin B1 to ploidy level (cyclin B/DNA content) was significantly greater in the low ploidy cells as compared to high ploidy ones (Vitrat et al., 1998). This may be due to the existence of a higher fraction of resting cells (in which cyclin B1 is never elevated) in the highly polyploid population. Alternatively, or in addition, this could reflect an accelerated degradation of cyclin B in the high ploidy cells in accordance with an earlier report on a megakaryocytic cell line (Zhang et al., 1998). In a very recent study with TPO-treated murine megakaryocytes, cyclin B1 levels were also followed by flow cytometry analysis of cells of different ploidy class (Crow et al., 2001), using a different antibody to cyclin B1 than the one used in the study of human cells (Vitrat et al., 1998). This investigation represents a unique effort to obtain large quantities of megakaryocytes selected from mouse bone marrow. The authors determined that on average, 36% of 8n-32n megakaryocytes expressed abundant cyclin B1 during G2/M and that the level of cyclin B1 per G2/M increased linearly with ploidy (after correction for cell size). This later conclusion contradicts somewhat the above-described reports on human samples. Crow et al. also reported that cyclin B expression oscillated normally in murine megakaryocytes undergoing endomitosis, although cyclin B1 never really disappeared. It is worth pointing out that the flow cytometry method of detection must rely on a careful choice of cyclin B antibody which should display specific staining by Western blotting, as all proteins (specific and others) will react with the antibody and display a positive signal in a ploidy class examined by flow cytometry. Western blot assays were also used to explore whether the levels of mitotic regulators are comparable in diploid and polyploid megakaryocytes of primary origin. Analyses of murine or human megakaryocytes indicated that high ploidy cells and diploid/tetraploid megakaryocytes or diploid, nonmegakaryocytic bone marrow cells, all contained similar levels of cyclin B1. Cdc2 levels in these populations were equivalent or somewhat reduced in the diploid cells (Vitrat et al., 1998; Bassini et al., 1999; Crow et al., 2001). In these investigations, however, the cells examined were not synchronized and the percentage of cycling cells in each population (diploid vs. polyploid) was not determined. This parameter might influence the profile of cyclins detected. Nevertheless, it has become clear that polyploidizing megakaryocytes contain significant levels of cyclin B1. Immunoprecipitation of cyclin B1-associated proteins showed a comparable level of H1 histone kinase activity in nocodazole-blocked human polyploid megakaryocytes as compared to the 2n/4n population, but Cdc2-associated ones were not examined in this study (Vitrat et al., 1998). A recent report on human primary, TPO-treated megakaryocytes that were subjected to immunohistochemistry, further supported the conclusion that cyclin B1 was associated with the mitotic spindle during polyploidization, but also indicated that it was undetectable at anaphase (Roy et al., 2001). This too sustained earlier findings (indicated as data not shown) that cyclin B1 is significantly reduced at anaphase of polyploidizing TPO-treated mouse megakaryocytes (Nagata et al., 1997). Roy et al., hence, deduced that the APC component responsible for cyclin B degradation is active in polyploidizing human megakaryocytes (Roy et al., 2001), as previously concluded with a megakaryocytic cell line and TPO-treated murine bone marrow cells (Zhang et al., 1998). Whether cyclin B1 degradation is accelerated in primary megakaryocytes undergoing endomitosis, as compared to proliferating ones, remains to be explored. Gu et al. examined non-TPO treated human bone marrow by immunohistochemistry and electron microscopy, and reported significant staining with an antibody to Cdc2 in all megakaryocytes and other bone marrow cells. Anti cyclin B1, on the other hand, showed negligible staining in megakaryocytes but strong staining in granulocytes, monocytes, and macrophages (Gu et al., 1993; Wang et al., 1995). Since only 15% of bone marrow megakaryocytes are actively involved in polyploidization (Wang et al., 1995), it is possible that the majority of cells examined were in a quiescent state.