The development of safe vectors for gene therapy requires fail-safe mechanisms to terminate therapy or remove genetically altered cells. The ideal ''suicide switch'' would be nonimmunogenic and nontoxic when uninduced and able to trigger cell death independent of tissue type or cell cycle stage. By using chemically induced dimerization, we have developed powerful death switches based on the cysteine proteases, caspase-1 ICE (interleukin-1␤ converting enzyme) and caspase-3 YAMA. In both cases, aggregation of the target protein is achieved by a nontoxic lipid-permeable dimeric FK506 analog that binds to the attached FK506-binding proteins, FKBPs. We find that intracellular cross-linking of caspase-1 or caspase-3 is sufficient to trigger rapid apoptosis in a Bcl-x L -independent manner, suggesting that these conditional proapoptotic molecules can bypass intracellular checkpoint genes, such as Bcl-x L , that limit apoptosis. Because these chimeric molecules are derived from autologous proteins, they should be nonimmunogenic and thus ideal for long-lived gene therapy vectors. These properties should also make chemically induced apoptosis useful for developmental studies, for treating hyperproliferative disorders, and for developing animal models to a wide variety of diseases.

The mitogen-activated protein kinase (MAPK) family of kinases connects extracellular stimuli with diverse cellular responses ranging from activation or suppression of gene expression to the regulation of cell mortality, growth, and differentiation. The MAPK family has been studied extensively; however, the role of these kinases in cell growth and cell-cycle control has become increasingly complex. Patterns have begun to emerge from these studies that show the functions of MAPK subfamilies at different stages of the cell cycle. Their patterns of subcellular localization and movement during the cell cycle are subfamily-specific and have raised many questions about possible cell-cycle functions that have yet to be demonstrated. This article will compare and contrast our current understanding of the functions and localization patterns of the MAPK subfamilies (ERK, BMK, p38, and JNK) in cell-cycle control.

The JNK family members JNK1 and JNK2 regulate tumor growth and are essential for transformation by oncogenes such as constitutively activated Ras. The mechanisms downstream of JNK that regulate cell cycle progression and transformation are unclear. Here we show that inhibition of JNK2, but not JNK1, with either a dominant-negative mutant, a pharmacological inhibitor, or RNA interference caused an accumulation of mammalian cells with 4N DNA content. When observed by immunofluorescence, these cells progressed to metaphase without apparent defects in spindle formation or chromosome alignment to the metaphase plate, suggesting that the 4N accumulation is a result of postmetaphase defects. Consistent with this prediction, when JNK activity was suppressed, we observed defects in central spindle formation and chromosome segregation during anaphase. In contrast, cyclin-dependent kinase 1 activity, cyclin B1 protein, and Polo-like kinase 1 protein turnover remained intact when JNK was inhibited. In addition, continued inhibition of JNK activity did not block reentry into subsequent cell cycles but instead resulted in polyploidy. This evidence suggests that JNK2 functions in maintaining the genomic stability of mammalian cells by signaling that is independent of cyclin-dependent kinase 1/cyclin B1 down-regulation. JNK11 and JNK2 are members of the mitogen-activated protein kinase family, which also includes the prototypical family members extracellular signal-regulated kinase and p38. Mitogen-activated protein kinases are components of signal transduction pathways that connect extracellular stimuli to intracellular responses such as modulation of cell viability, cell cycle regulation, and gene expression (1-3). JNK1 and JNK2 are ubiquitously expressed and are generally considered to share redundant functions in apoptosis and transformation and differential functions in T cell differentiation (3-6). However, recent studies have shown that the stress-induced proapoptotic function previously attributed to both isoforms is actually a function specific to JNK1 (7,8). The function of JNK2, therefore, has become less clear. This suggests that JNK functions in transformation may also be isoform-specific and should be addressed in a way that differentiates between the contributions of JNK1 and JNK2.Ras-activating mutations have been identified in close to 30% of human cancers (9). Transformation by Ras requires JNK activity, which has made JNK an attractive target for cancer therapy (10 -12). Targeting JNK for cancer therapy is also supported by studies showing that JNK activity is elevated in human tumors and that loss of JNK function inhibits tumor growth in mice (13-16). The transforming mechanism downstream of JNK is not entirely clear, but it may work through the phosphorylation of c-Jun, which in turn regulates transcription of cell cycle regulators. In addition to this pathway, JNK may have a more direct function in cell cycle regulation. We have shown that JNK localizes to centrosomes and is active in this compartment from...

Exposure to the natural mineral fiber asbestos causes severe lung-damaging fibrosis and cancer, yet it continues to be used as an industrial insulating material throughout the world. When cultured human lung cells are exposed to asbestos, individual fibers are engulfed into the cytoplasm where they induce significant mitotic aberrations leading to chromosomal instability and aneuploidy. The mechanisms of how asbestosis ultimately leads to lung cancer remain unclear. However, our experiments indicate that intracellular asbestos fibers induce aneuploidy and chromosome instability by binding to a subset of proteins that include regulators of the cell cycle, cytoskeleton, and mitotic process. Moreover, precoating of fibers with protein complexes efficiently blocked asbestos-induced aneuploidy in human lung cells without affecting their uptake by cells. These results provide new evidence that asbestos fibers can contribute to significant spindle damage and chromosomal instability by binding to proteins needed for the assembly and regulation of the cytoskeleton or the cell cycle.

Aurora‐B and –C kinases are members of the Aurora serine/threonine kinase family of mitotic regulators. Aurora‐B kinase is evolutionarily conserved from yeast to humans and has multiple functions in chromosome condensation, cohesion, biorientation, and in cytokinesis. In contrast, Aurora‐C kinase has only been found in mammals, is upregulated in some tumor cell lines and tissues, and has a unique physiological role in spermiogenesis. Despite these known functions, little is known about the function of Aurora‐C in mitosis. We have explored Aurora‐C function in mitosis, and have found that it interacts with several known substrates of Aurora‐B: the chromosomal passenger complex (CPC) proteins Inner Centromere Protein (INCENP), Survivin, and Borealin. We have also found that Aurora‐C, like Aurora‐B, phosphorylates the centromeric histone Centromere Protein‐A (CENP‐A), and Borealin in vitro. These molecular mechanisms are consistent with our observation that in the absence of Aurora‐B, Aurora‐C is sufficient for proper centromeric localization of the CPC proteins and timely prometaphase progression. Thus, Aurora‐C shares many mitotic substrates and is capable of performing mitotic functions previously attributed to only Aurora‐B. This work was supported by NIH Grant CA 41424 to BRB and The Huffington Foundation Grant to BRB and RAM.