Cyclin A/cdk2 regulates adenomatous polyposis coli-dependent mitotic spindle anchoring.

Mutations in adenomatous polyposis coli (APC) protein is a major contributor to tumor initiation and progression in several tumor types. These mutations affect APC function in the Wnt-beta-catenin signaling and influence mitotic spindle anchoring to the cell cortex and orientation. Here we report that the mitotic anchoring and orientation function of APC is regulated by cyclin A/cdk2. Knockdown of cyclin A and inhibition of cdk2 resulted in cells arrested in mitosis with activation of the spindle assembly checkpoint. The mitotic spindle was unable to form stable attachments to the cell cortex, and this resulted in the spindles failing to locate to the central position in the cells and undergo dramatic rotation. We have demonstrated that cyclin A/cdk2 specifically associates with APC in late G2 phase and phosphorylates it at Ser-1360, located in the mutation cluster region of APC. Mutation of APC Ser-1360 to Ala results in identical off-centered mitotic spindles. Thus, this cyclin A/cdk2-dependent phosphorylation of APC affects astral microtubule attachment to the cortical surface in mitosis.

In vitro studies of cells grown on the superconductor PrO(x)FeAs.

The recent discovery of arsenic-based high temperature superconductors has reignited interest in the study of superconductor: biological interfaces. However, the new superconductor materials involve the chemistry of arsenic and their toxicity remains unclear [Hand, E., 2008. Nature 452 (24), 922]. In this study the possible adverse effects of this new family of superconductors on cells have been examined. Cell culture studies in conjunction with microscopy and viability assays were employed to examine the influence of arsenic-based superconductor PrO(x)FeAs (x=0.75) material in vitro. Imaging data revealed that cells were well adhered and spread on the surface of the superconductor. Furthermore, cytotoxicity studies showed that cells were unaffected during the time-course of the experiments, providing support for the biocompatibility aspects of PrO(x)FeAs-based superconductor material.

Caffeine promotes apoptosis in mitotic spindle checkpoint-arrested cells.

The spindle assembly checkpoint arrests cells in mitosis when defects in mitotic spindle assembly or partitioning of the replicated genome are detected. This checkpoint blocks exit from mitosis until the defect is rectified or the cell initiates apoptosis. In this study we have used caffeine to identify components of the mechanism that signals apoptosis in mitotic checkpoint-arrested cells. Addition of caffeine to spindle checkpoint-arrested cells induced >40% apoptosis within 5 h. It also caused proteasome-mediated destruction of cyclin B1, a corresponding reduction in cyclin B1/cdk1 activity, and reduction in MPM-2 reactivity. However, cells retained MAD2 staining at the kinetochores, an indication of continued spindle checkpoint function. Blocking proteasome activity did not block apoptosis, but continued spindle checkpoint function was essential for apoptosis. After systematically eliminating all known targets, we have identified p21-activated kinase PAK1, which has an anti-apoptotic function in spindle checkpoint-arrested cells, as a target for caffeine inhibition. Knockdown of PAK1 also increased apoptosis in spindle checkpoint-arrested cells. This study demonstrates that the spindle checkpoint not only regulates mitotic exit but apoptosis in mitosis through the activity of PAK1.

Inhibition of S/G2 phase CDK4 reduces mitotic fidelity.

Cyclin-dependent kinase 4 (CDK4)/cyclin D has a key role in regulating progression through late G(1) into S phase of the cell cycle. CDK4-cyclin D complexes then persist through the latter phases of the cell cycle, although little is known about their potential roles. We have developed small molecule inhibitors that are highly selective for CDK4 and have used these to define a role for CDK4-cyclin D in G(2) phase. The addition of the CDK4 inhibitor or small interfering RNA knockdown of cyclin D3, the cyclin D partner, delayed progression through G(2) phase and mitosis. The G(2) phase delay was independent of ATM/ATR and p38 MAPK but associated with elevated Wee1. The mitotic delay was because of failure of chromosomes to migrate to the metaphase plate. However, cells eventually exited mitosis, with a resultant increase in cells with multiple or micronuclei. Inhibiting CDK4 delayed the expression of the chromosomal passenger proteins survivin and borealin, although this was unlikely to account for the mitotic phenotype. These data provide evidence for a novel function for CDK4-cyclin D3 activity in S and G(2) phase that is critical for G(2)/M progression and the fidelity of mitosis.