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Although it has been reported that Bcl-2 phosphorylation is associated with certain types of apoptosis, there is much controversy over the functional significance of and the kinases responsible for the phosphorylation. In this study, we examined whether Bcl-2 is phosphorylated by CDC2 kinase, a master regulator of G 2 /M transition in the eukaryotic cell cycle. When CDC2 was activated by okadaic acid in HL-60 cells, Bcl-2 phosphorylation was readily induced. The phosphorylation was correlated with the accumulation of cells in G 2 /M phases, but was not proportional to the level of apoptosis. Furthermore, we found that Bcl-2 was phosphorylated during G 2 /M phases of normal cell cycle. The ability of CDC2 to phosphorylate Bcl-2 was confirmed by in vitro kinase assay with a highly purified CDC2-cyclin B complex. Using synthetic peptides and mutant cell lines, we identified threonine 56, one of two consensus sites for CDC2 within the Bcl-2 sequence, as a residue phosphorylated by CDC2. Mutation at threonine 56 abrogated the cell cycle inhibitory effect of Bcl-2 without affecting anti-apoptotic function. These results suggest that two distinct functions of Bcl-2 (anti-apoptosis and cell cycle inhibition) are differentially regulated by post-translational mechanisms such as phosphorylation. CDC2-mediated phosphorylation of Bcl-2 may play some physiological roles in the negative regulatory events during mitosis.

Anti-CD20 antibody rituximab is now essential for the treatment of CD20-positive B-cell lymphomas. Decreased expression of CD20 is one of the major mechanisms underlying both innate and acquired resistance to rituximab. In this study, we show that histone deacetylase (HDAC) inhibitors augment the cytotoxic activity of rituximab by enhancing the surface expression of CD20 antigen on lymphoma cells. HDAC inhibitors, valproic acid (VPA) and romidepsin, increased CD20 expression at protein and mRNA levels in B-cell lymphoma cell lines with relatively low CD20 expression levels. The VPA-mediated increase in CD20 expression occurred at 1 mM, which is clinically achievable and safe, but insufficient for inducing cell death. Chromatin immunoprecipitation assays revealed that HDAC inhibitors transactivated the CD20 gene through promoter hyperacetylation and Sp1 recruitment. HDAC inhibitors potentiated the activity of rituximab in complementdependent cytotoxic assays. In mouse lymphoma models, HDAC inhibitors enhanced CD20 expression along with histone hyperacetylation in transplanted cells, and acted synergistically with rituximab to retard their growth. The combination with HDAC inhibitors may serve as an effective strategy to overcome rituximab resistance in B-cell lymphomas.

Summary. To maintain the fidelity and integrity of blood formation, the cell cycle is under strict regulation during haematopoietic cell differentiation. To elucidate the molecular mechanisms of cell cycle regulation during haematopoiesis, we examined cell cycle control gene expression during lineage-specific differentiation from CD34 1 progenitor cells. Expression of cyclin-dependent kinases (cdks) and cyclins, except cdk4, was generally suppressed in CD34 1 cells freshly isolated from the bone marrow of healthy volunteers. Among four major cdk inhibitors, p16 was expressed more highly in CD341 cells than in CD34-negative bone marrow mononuclear cells, whereas the amounts of p21 and p27 transcripts increased in the CD34 2 population. The behaviour of cell cycle control genes during haematopoietic differentiation was classified into four patterns: (i) universal upregulation (cdc2, cdk2, cyclin A, cyclin B and p21); (ii) upregulation in specific lineages (cyclin D1, cyclin D3 and p15); (iii) no induction or stable expression (cdk4, cyclin D2, cyclin E and p27); and (iv) universal downregulation (p16). Lineage-specific changes included the sustained elevation of cdc2 and cyclin A during erythroid differentiation, cyclin D1 and p15 induction in myeloid lineage and selective upregulation of cyclin D3 in megakaryocytes. Blocking induction of cyclin D3 resulted in the inhibition of megakaryocytic differentiation. These results suggest that the expression of cell cycle control genes is distinctively regulated in a lineage-dependent manner, reflecting the cell cycle characteristics of each lineage. Some of these genes play an essential role in the process of differentiation itself.

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