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.

A potent Chk1 inhibitor is selectively cytotoxic in melanomas with high levels of replicative stress.

There are few effective treatments for metastatic melanoma. Checkpoint kinase 1 (Chk1) inhibitors are being trialled for their efficacy in enhancing conventional chemotherapeutic agents, but their effectiveness as single agents is not known. We have examined the effectiveness of two novel Chk1 selective inhibitors, AR323 and AR678, in a panel of melanoma cell lines and normal cell types.

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.

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.

Convergent antifouling activities of structurally distinct bioactive compounds synthesized within two sympatric Haliclona demosponges.

A wide range of sessile and sedentary marine invertebrates synthesize secondary metabolites that have potential as industrial antifoulants. These antifoulants tend to differ in structure, even between closely related species. Here, we determine if structurally divergent secondary metabolites produced within two sympatric haliclonid demosponges have similar effects on the larvae of a wide range of benthic competitors and potential fouling metazoans (ascidians, molluscs, bryozoans, polychaetes, and sponges).

Cyclin A/cdk2 coordinates centrosomal and nuclear mitotic events.

Cyclin A/cdk2 has a role in progression through S phase, and a large pool is also activated in G2 phase. Here we report that this G2 phase pool regulates the timing of progression into mitosis. Knock down of cyclin A by siRNA or addition of a specific cdk2 small molecule inhibitor delayed entry into mitosis by delaying cells in G2 phase.

Histone deacetylase inhibitors induce mitotic slippage.

Chromosomal passenger proteins have emerged as key players in the regulation of mitosis and cytokinesis. Histone deacetylase inhibitors (HDACi) are a class of anticancer drugs that induce aberrant mitosis and can overcome the spindle assembly checkpoint. Here, we investigate the mechanism by which HDACi disrupt normal mitotic progression and checkpoint function.

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.

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.

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.

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.

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.

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.

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.

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.

The C-terminal conserved domain of DNA-PKcs, missing in the SCID mouse, is required for kinase activity.

DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), has a phosphoinositol 3-kinase (PI 3-K) domain close to its C-terminus. Cell lines derived from the SCID mouse have been utilised as a model DNA-PKcs-defective system. The SCID mutation results in truncation of DNA-Pkcs at the extreme C-terminus leaving the PI 3-K domain intact.

Identification of a defect in DNA ligase IV in a radiosensitive leukaemia patient.

The major mechanism for the repair of DNA double-strand breaks (DSBs) in mammalian cells is non-homologous end-joining (NHEJ), a process that involves the DNA-dependent protein kinase [1] [2], XRCC4 and DNA ligase IV [3] [4] [5] [6]. Rodent cells and mice defective in these components are radiation-sensitive and defective in V(D)J-recombination, showing that NHEJ also functions to rejoin DSBs introduced during lymphocyte development [7] [8]. 180BR is a radiosensitive cell line defective in DSB repair, which was derived from a leukaemia patient who was highly sensitive to radiotherapy [9] [10] [11].

Double strand break rejoining by the Ku-dependent mechanism of non-homologous end-joining.

The DNA-dependent protein kinase functions in the repair of DNA double strand breaks (DSBs) and in V(D)J recombination. To gain insight into the function of DNA-PK in this process we have carried out a mutation analysis of Ku80 and DNA-PKcs. Mutations at multiple sites within the N-terminal two thirds of Ku80 result in loss of Ku70/80 interaction, loss of DNA end-binding activity and inability to complement Ku80 defective cell lines.

Targeted disruption of the catalytic subunit of the DNA-PK gene in mice confers severe combined immunodeficiency and radiosensitivity.

The DNA-dependent protein kinase is a mammalian protein complex composed of Ku70, Ku80, and DNA-PKcs subunits that has been implicated in DNA double-strand break repair and V(D)J recombination. Here, by gene targeting, we have constructed a mouse with a disruption in the kinase domain of DNA-PKcs, generating an animal model completely devoid of DNA-PK activity. Our results demonstrate that DNA-PK activity is required for coding but not for signal join formation in mice.

Molecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20.

The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is a member of a sub-family of phosphatidylinositol (PI) 3-kinases termed PIK-related kinases. A distinguishing feature of this sub-family is the presence of a conserved C-terminal region downstream of a PI 3-kinase domain. Mutants defective in DNA-PKcs are sensitive to ionising radiation and are unable to carry out V(D)J recombination.

Cellular localisation of the ataxia-telangiectasia (ATM) gene product and discrimination between mutated and normal forms.

The recently cloned gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is involved in DNA damage response at different cell cycle checkpoints and also appears to have a wider role in signal transduction. Antibodies prepared against peptides from the predicted protein sequence detected a approximately 350 kDa protein corresponding to the open reading frame, which was absent in 13/23 A-T homozygotes. Subcellular fractionation, immunoelectronmicroscopy and immunofluorescence showed that the ATM protein is present in the nucleus and cytoplasmic vesicles.

Defective signaling through the B cell antigen receptor in Epstein-Barr virus-transformed ataxia-telangiectasia cells.

A characteristic series of immunological abnormalities are observed in the human genetic disorder ataxia-telangiectasia (A-T). The recent cloning of a gene mutated in this syndrome provides additional evidence for a defect in intracellular signaling in A-T. We have investigated the possibility that signaling through the B cell antigen receptor is one manifestation of the A-T defect.

Rapamycin resistance in ataxia-telangiectasia.

The gene mutated in the human genetic disorder ataxia-telangiectasia (A-T) has been described recently (Savitsky et al., 1995a) and the complete coding sequence of this gene, ATM, has been reported (Savitsky et al., 1995b).

Defect in multiple cell cycle checkpoints in ataxia-telangiectasia postirradiation.

The recent description of a novel gene (ATM) mutated in ataxia-telangiectasia (A-T), with homologies to genes encoding proteins involved in both G1/S and G2/M checkpoint control, points to a common defect in cell cycle control in A-T operating through the cyclin-dependent kinases. In this report we demonstrate that cyclin-dependent kinases are resistant to inhibition by ionizing radiation exposure in A-T cells, and this appears to be due to insufficient induction of WAF1. Exposure of control lymphoblastoid cells to radiation during S phase and in G2 phase causes a rapid inhibition of cyclin A-Cdk2 and cyclin B-Cdc2 activities, respectively.

Genetic complementation of radiation response by 3' untranslated regions (UTR) of RNA.

The molecular basis of radiosensitivity was studied using a cDNA complementation approach to correct radiosensitivity in cells. Four cDNAs of sizes 1.6, 2.