Progression of the cell cycle and control of apoptosis (programmed cell death) are thought to be intimately linked processes, acting to preserve homeostasis and developmental morphogenesis. Although proteins that regulate apoptosis have been implicated in restraining cell-cycle entry and controlling ploidy (chromosome number), the effector molecules at the interface between cell proliferation and cell survival have remained elusive. Here we show that a new inhibitor of apoptosis (IAP) protein, survivin, is expressed in the G2/M phase of the cell cycle in a cycle-regulated manner. At the beginning of mitosis, survivin associates with microtubules of the mitotic spindle in a specific and saturable reaction that is regulated by microtubule dynamics. Disruption of survivin-microtubule interactions results in loss of survivin's anti-apoptosis function and increased caspase-3 activity, a mechanism involved in cell death, during mitosis. These results indicate that survivin may counteract a default induction of apoptosis in G2/M phase. The overexpression of survivin in cancer may overcome this apoptotic checkpoint and favour aberrant progression of transformed cells through mitosis.

Molecular chaperones, especially members of the heat shock protein 90 (Hsp90) family, are thought to promote tumor cell survival, but this function is not well understood. Here, we show that mitochondria of tumor cells, but not most normal tissues, contain Hsp90 and its related molecule, TRAP-1. These chaperones interact with Cyclophilin D, an immunophilin that induces mitochondrial cell death, and antagonize its function via protein folding/refolding mechanisms. Disabling this pathway using novel Hsp90 ATPase antagonists directed to mitochondria causes sudden collapse of mitochondrial function and selective tumor cell death. Therefore, Hsp90-directed chaperones are regulators of mitochondrial integrity, and their organelle-specific antagonists may provide a previously undescribed class of potent anticancer agents.

The interface between apoptosis (programmed cell death) and the cell cycle is essential to preserve homeostasis and genomic integrity. Here, we show that survivin, an inhibitor of apoptosis over-expressed in cancer, physically associates with the cyclindependent kinase p34 cdc2 on the mitotic apparatus, and is phosphorylated on Thr 34 by p34 cdc2 -cyclin B1, in vitro and in vivo. Loss of phosphorylation on Thr 34 resulted in dissociation of a survivincaspase-9 complex on the mitotic apparatus, and caspase-9-dependent apoptosis of cells traversing mitosis. These data identify survivin as a mitotic substrate of p34 cdc2 -cyclin B1 and suggest that survivin phosphorylation on Thr 34 may be required to preserve cell viability at cell division. Manipulation of this pathway may facilitate the elimination of cancer cells at mitosis.

Here we investigate the role of the control of apoptosis in normal cell division. We show that interference with the expression or function of the apoptosis inhibitor survivin causes caspase-dependent cell death in the G2/M phase of the cell cycle, and a cell-division defect characterized by centrosome dysregulation, multipolar mitotic spindles and multinucleated, polyploid cells. Use of a dominant-negative survivin mutant or antisense survivin complementary DNA disrupts a supramolecular assembly of survivin, caspase-3 and the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 within centrosomes, and results in caspase-dependent cleavage of p21. Polyploidy induced by survivin antagonists is accentuated in p21-deficient cells, and corrected by exogenous expression of p21. These findings show that control of apoptosis and preservation of p21 integrity within centrosomes by survivin are required for normal mitotic progression.

Plasminogen binding to cell surfaces results in enhanced plasminogen activation, localization of the proteolytic activity of plasmin on cell surfaces, and protection of plasmin from alpha 2-antiplasmin. We sought to characterize candidate plasminogen binding sites on nucleated cells, using the U937 monocytoid cell as a model, specifically focusing on the role of cell-surface proteins with appropriately placed lysine residues as candidate plasminogen receptors. Lysine derivatives with free alpha-carboxyl groups and peptides with carboxy-terminal lysyl residues were effective inhibitors of plasminogen binding to the cells. One of the peptides, representing the carboxy-terminal 19 amino acids of alpha 2-antiplasmin, was approximately 5-fold more effective than others with carboxy-terminal lysines. Thus, in addition to a carboxy-terminal lysyl residue, other structural features of the cell-surface proteins may influence their affinity for plasminogen. Affinity chromatography has been used to isolate candidate plasminogen receptors from U937 cells. A major protein of Mr 54,000 was recovered and identified as alpha-enolase by immunochemical and functional criteria. alpha-Enolase was present on the cell surface and was capable of binding plasminogen in ligand blotting analyses. Plasminogen binding activity of a molecular weight similar to alpha-enolase also was present in a variety of other cell types. Carboxypeptidase B treatment of alpha-enolase abolished its ability to bind plasminogen, consistent with the presence of a C-terminal lysyl residue. Thus, cell-surface proteins with carboxy-terminal lysyl residues appear to function as plasminogen binding sites, and alpha-enolase has been identified as a prominent representative of this class of receptors.