E2F-1 is capable of promoting both cell cycle progression and apoptosis. The latter is important for suppressing untoward expansion of proliferating cells. In this study, we investigated its underlying mechanisms. E2F-1-induced apoptosis was accompanied by caspase-9 activation and inhibited by a specific inhibitor of caspase-9 in K562 sublines overexpressing E2F-1. E2F-1 enhanced the expression of Apaf-1 without the cytosolic accumulation of cytochrome c. Apaf-1-deficient melanoma cell lines were resistant to E2F-1, indicating that Apaf-1 is an essential element of E2F-1-mediated apoptosis. Finally, we isolated the promoter region of the Apaf-1 gene and found a putative binding site for E2F. A chromatin immunoprecipitation assay revealed that E2F-1 bound to Apaf-1 promoter upon E2F-1 overexpression, suggesting that Apaf-1 is under transcriptional regulation of E2F-1. These data demonstrate a novel mechanism of apoptosis in which an increase in Apaf-1 levels results in direct activation of caspase-9 without mitochondrial damage, leading to the initiation of a caspase cascade.

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.

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.

A B S T R A C T Human plasma a2-plasmin inhibitor in fibrinolytic states was studied using immunochemical methods and radioiodinated plasminogen. The concentration and activity of plasma a2-plasmin inhibitor decreased when urokinase was added to plasma in vitro or infused intravenously in man. The decrease was associated with the appearance of plasmin-a2-plasmin inhibitor complex which subsequently disappeared from the circulation in a short time. A decrease of other major inhibitors, such as a2-macroglobulin and a1-antitrypsin, was not observed when the amount of urokinase added or infused was relatively small, and conversion of plasminogen to plasmin was not extensive. The formation of plasmina2-macroglobulin complex was observed only when plasma plasminogen was activated with a larger amount of urokinase, and after most of the a2-plasmin inhibitor was consumed by forming complexes with plasmin. The formation of plasmin-al-antitrypsin complex was not observed even in the highly activated plasma unless exogenous plasmin was added to the plasma. a2-Plasmin inhibitor was the only inhibitor of which the concentration in plasma was significantly decreased in patients with disseminated intravascular coagulation and fibrinolysis among the major plasmin inhibitors in plasma. The most reactive inhibitor for regulating plasma fibrinolysis very likely is a2-plasmin inhibitor.

IntroductionIntermolecular end-to-middle domain pairing between a thrombin-exposed 'A' polymerization site in Invest. 1995Invest. . 96:1053Invest. -1058