Cyclin A/Cdk2 regulates Cdh1 and claspin during late S/G2 phase of the cell cycle.

Whereas many components regulating the progression from S phase through G2 phase into mitosis have been identified, the mechanism by which these components control this critical cell cycle progression is still not fully elucidated. Cyclin A/Cdk2 has been shown to regulate the timing of Cyclin B/Cdk1 activation and progression into mitosis although the mechanism by which this occurs is only poorly understood. Here we show that depletion of Cyclin A or inhibition of Cdk2 during late S/early G2 phase maintains the G2 phase arrest by reducing Cdh1 transcript and protein levels, thereby stabilizing Claspin and maintaining elevated levels of activated Chk1 which contributes to the G2 phase observed. Interestingly, the Cyclin A/Cdk2 regulated APC/C(Cdh1) activity is selective for only a subset of Cdh1 targets including Claspin. Thus, a normal role for Cyclin A/Cdk2 during early G2 phase is to increase the level of Cdh1 which destabilises Claspin which in turn down regulates Chk1 activation to allow progression into mitosis. This mechanism links S phase exit with G2 phase transit into mitosis, provides a novel insight into the roles of Cyclin A/Cdk2 in G2 phase progression, and identifies a novel role for APC/C(Cdh1) in late S/G2 phase cell cycle progression.

The histone deacetylase inhibitor MGCD0103 has both deacetylase and microtubule inhibitory activity.

Histone deacetylase inhibitors (HDACis) are currently in trial or are in clinical use for the treatment of a number of tumor types. The clinical efficacy of HDACis can be partly attributed to the modulation of the cell cycle by the HDACis. Here, we have examined the effects of N-(2-aminophenyl)-4-((4-pyridin-3-ylpyrimidin-2-ylamino)methyl)benzamide (MGCD0103), a class I-selective histone deacetylase inhibitor, on the cell cycle and cell killing. Surprisingly, MGCD0103 treatment failed to initiate a G(1)-phase arrest but caused marked accumulation of cells in G(2)/M at 6 and 12 h after treatment and was cytotoxic 24 h after treatment. These cell cycle effects were considerably distinct from the effects of suberic bishydroxamic acid, a representative of the pan-isoform HDACi used in this study. MGCD0103 shared the ability of the pan-isoform HDACi to trigger defective mitosis and promote mitotic slippage. Likewise, it also specifically targeted tumor cells and was nontoxic to normal nontransformed cells. However, MGDC0103 also seemed to disrupt normal microtubule spindle formation, whereas HDACis generally have only a minor effect on spindle formation. The effect of MGCD0103 on spindle formation was shown to be a consequence of microtubule destabilization. This is the first example of an HDACi with microtubule destabilizing activity, and the combined effects of this drug have advantages for its therapeutic use.

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.

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.

Deficiency in the catalytic subunit of DNA-dependent protein kinase causes down-regulation of ATM.

Previous reports have suggested a connection between reduced levels of the catalytic subunit of DNA-dependent protein kinases (DNA-PKcs), a component of the nonhomologous DNA double-strand breaks end-joining system, and a reduction in ATM. We studied this possible connection in other DNA-PKcs-deficient cell types, and following knockdown of DNA-PKcs with small interfering RNA, Chinese hamster ovary V3 cells, lacking DNA-PKcs, had reduced levels of ATM and hSMG-1, but both were restored after transfection with PRKDC. Atm levels were also reduced in murine scid cells. Reduction of ATM in a human glioma cell line lacking DNA-PKcs was accompanied by defective signaling through downstream substrates, post-irradiation. A large reduction of DNA-PKcs was achieved in normal human fibroblasts after transfection with two DNA-PKcs small interfering RNA sequences. This was accompanied by a reduction in ATM. These data were confirmed using immunocytochemical detection of the proteins. Within hours after transfection, a decline in PRKDC mRNA was seen, followed by a more gradual decline in DNA-PKcs protein beginning 1 day after transfection. No change in ATM mRNA was observed for 2 days post-transfection. Only after the DNA-PKcs reduction occurred was a reduction in ATM mRNA observed, beginning 2 days post-transfection. The amount of ATM began to decline, starting about 3 days post-treatment, then it declined to levels comparable to DNA-PKcs. Both proteins returned to normal levels at later times. These data illustrate a potentially important cross-regulation between the nonhomologous end-joining system for rejoining of DNA double-strand breaks and the ATM-dependent damage response network of pathways, both of which operate to maintain the integrity of the genome.

T cells signaled by NF-kappa B- dendritic cells are sensitized not anergic to subsequent activation.

Paradoxically, while peripheral self-tolerance exists for constitutively presented somatic self Ag, self-peptide recognized in the context of MHC class II has been shown to sensitize T cells for subsequent activation. We have shown that MHC class II(+)CD86(+)CD40(-) DC, which can be generated from bone marrow in the presence of an NF-kappa B inhibitor, and which constitutively populate peripheral tissues and lymphoid organs in naive animals, can induce Ag-specific tolerance. In this study, we show that CD40(-) human monocyte-derived dendritic cells (DC), generated in the presence of an NF-kappa B inhibitor, signal phosphorylation of TCR zeta, but little proliferation or IFN-gamma in vitro. Proliferation is arrested in the G(1)/G(0) phase of the cell cycle. Surprisingly, responding T cells are neither anergic nor regulatory, but are sensitized for subsequent IFN-gamma production. The data indicate that signaling through NF-kappa B determines the capacity of DC to stimulate T cell proliferation. Functionally, NF-kappa B(-)CD40(-)class II(+) DC may either tolerize or sensitize T cells. Thus, while CD40(-) DC appear to "prime" or prepare T cells, the data imply that signals derived from other cells drive the generation either of Ag-specific regulatory or effector cells in vivo.

Histone deacetylase inhibitors specifically kill nonproliferating tumour cells.

Conventional chemotherapeutic drugs target proliferating cells, relying on often small differences in drug sensitivity of tumour cells compared to normal tissue to deliver a therapeutic benefit. Consequently, they have significant limiting toxicities and greatly reduced efficacy against nonproliferating compared to rapidly proliferating tumour cells. This lack of selectivity and inability to kill nonproliferating cells that exist in tumours with a low mitotic index are major failings of these drugs. A relatively new class of anticancer drugs, the histone deacetylase inhibitors (HDI), are selectively cytotoxic, killing tumour and immortalized cells but normal tissue appears resistant. Treatment of tumour cells with these drugs causes both G1 phase cell cycle arrest correlated with increase p21 expression, and cell death, but even the G1 arrested cells died although the onset of death was delayed. We have extended these observations using cells that were stably arrested by either serum starvation or expression of the cyclin-dependent kinase inhibitor p16(ink4a). We report that histone deacetylase inhibitors have similar cytotoxicity towards both proliferating and arrested tumour and immortalized cells, although the onset of apoptosis is delayed by 24 h in the arrested cells. Both proliferating and arrested normal cells are unaffected by HDI treatment. Thus, the histone deacetylase inhibitors are a class of anticancer drugs that have the desirable features of being tumour-selective cytotoxic drugs that are equally effective in killing proliferating and nonproliferating tumour cells and immortalized cells. These drugs have enormous potential for the treatment of not only rapidly proliferating tumours, but tumours with a low mitotic index.

Analysis of checkpoint responses to histone deacetylase inhibitors.

Cell cycle checkpoints respond to a wide range of stresses to prevent compromise to the integrity of the cell. The best studied checkpoints are those induced by genotoxic agents that cause DNA damage. Histone deacetylase inhibitors not only increase the acetylation state of chromatin histones, but they also perturb the cell cycle, causing both G1 and G2 phase arrests, the latter by initiating a checkpoint response. In this chapter we will describe the analysis of the histone deacetylase inhibitor-sensitive G2 checkpoint using synchronized cell populations.

Defining the chemotherapeutic targets of histone deacetylase inhibitors.

The use of many conventional chemotherapeutic drugs is often severely restricted due to dose-limiting toxicities, as these drugs target the destruction of the proliferating fraction of cells, often with little specificity for tumor cells over proliferating normal body tissue. Many newer drugs attempt to overcome this shortcoming by targeting defective gene products or cellular mechanisms that are specific to the tumor, thereby minimizing the toxicity to normal tissue. Histone deacetylase inhibitors are an example of this type of tumor-directed drug, having significant toxicity for tumors but minimal effects on normal tissue. These drugs can affect the transcriptional program by modifying chromatin structure, but it is not yet clear whether specific transcriptional changes are directly responsible for their tumor-selective toxicity. Recent evidence suggests that transcriptional changes underlie their cytostatic activity, although this is not tumor-selective and affects all proliferating cells. Here we present evidence that supports an alternative mechanism for the tumor-selective cytotoxicity of histone deacetylase inhibitors. The target is still likely to be the chromatin histones, but rather than transcriptional changes due to modification of the transcriptionally active euchromatin, we propose that hyperacetylation and disruption of the transcriptionally inactive heterochromatin, particularly the centromeric heterochromatin, and the inability of tumor cells to cell cycle arrest in response to a specific checkpoint, underlies the tumor-selective cytotoxicity of these drugs.

Tumor cell-selective cytotoxicity by targeting cell cycle checkpoints.

Cell cycle checkpoints act to protect cells from external stresses and internal errors that would compromise the integrity of the cell. Checkpoints are often defective in cancer cells. Drugs that target checkpoint mechanisms should therefore be selective for tumor cells that are defective for the drug-sensitive checkpoint. Histone deacetylase inhibitors typify this class of agents. They trigger a G2-phase checkpoint response in normal cells but are cytotoxic in tumor cells in which this checkpoint is defective. In this study, we investigated the molecular basis of the tumor-selective cytotoxicity of these drugs and demonstrated that it is due to the disruption of two cell cycle checkpoints. The first is the histone deacetylase inhibitor-sensitive G2-phase checkpoint, which is defective in drug-sensitive cells and permits cells to enter an aberrant mitosis. The second is the drug-dependent bypass of the mitotic spindle checkpoint that normally detects aberrant mitosis and blocks mitotic exit until the defect is rectified. The disruption of both checkpoints results in the premature exit of cells from an abortive mitosis followed by apoptosis. This study of histone deacetylase inhibitors demonstrates that drugs targeting cell cycle checkpoints can provide the selectivity and cytotoxicity desired in effective chemotherapeutic agents.

Functional link between BLM defective in Bloom's syndrome and the ataxia-telangiectasia-mutated protein, ATM.

Chromosome aberrations, genomic instability, and cancer predisposition are hallmarks of a number of syndromes in which the defective genes recognize and/or repair DNA damage or are involved in some aspect of DNA processing. We report here direct interaction between BLM, mutated in Bloom's Syndrome (BS), and ATM, mutated is ataxia-telangiectasia, and we have mapped the sites of interaction. Full-length BLM cDNA corrected sister chromatid exchange (SCE) and radiosensitivity in BS cells. Mitotic phosphorylation of BLM was partially dependent on ATM, and phosphorylation sites on BLM were identified. A phosphospecific antibody against one of these sites (Thr-99) revealed radiation-induced phosphorylation, which was defective in ataxia-telangiectasia cells. Stable cell lines expressing phosphorylation site mutants failed to correct radiosensitivity in BS cells but corrected SCE. These mutants also sensitized normal control cells to radiation and increased radiation-induced chromosome aberrations but did not cause SCE numbers to increase. These data suggest that ATM and BLM function together in recognizing abnormal DNA structures by direct interaction and that these phosphorylation sites in BLM are important for radiosensitivity status but not for SCE frequency.