Adefective response to DNA damage is observed in several human autosomal recessive ataxias with oculomotor apraxia, including ataxia-telangiectasia. We report that senataxin, defective in ataxia oculomotor apraxia (AOA) type 2, is a nuclear protein involved in the DNA damage response. AOA2 cells are sensitive to H2O2, camptothecin, and mitomycin C, but not to ionizing radiation, and sensitivity was rescued with full-length SETX cDNA. AOA2 cells exhibited constitutive oxidative DNA damage and enhanced chromosomal instability in response to H2O2. Rejoining of H2O2-induced DNA double-strand breaks (DSBs) was significantly reduced in AOA2 cells compared to controls, and there was no evidence for a defect in DNA single-strand break repair. This defect in DSB repair was corrected by full-length SETX cDNA. These results provide evidence that an additional member of the autosomal recessive AOA is also characterized by a defective response to DNA damage, which may contribute to the neurodegeneration seen in this syndrome.

Cells can respond to DNA damage by activating checkpoints that delay cell cycle progression and allow time for DNA repair. Chemical inhibitors of the G 2 phase DNA damage checkpoint may be used as tools to understand better how the checkpoint is regulated and may be used to sensitize cancer cells to DNA-damaging therapies. However, few inhibitors are known. We used a cell-based assay to screen natural extracts for G 2 checkpoint inhibitors and identified debromohymenialdisine (DBH) from a marine sponge. DBH is distinct structurally from previously known G 2 checkpoint inhibitors. It inhibited the G 2 checkpoint with an IC 50 of 8 M and showed moderate cytotoxicity (IC 50 ‫؍‬ 25 M) toward MCF-7 cells. DBH inhibited the checkpoint kinases Chk1 (IC 50 ‫؍‬ 3 M) and Chk2 (IC 50 ‫؍‬ 3.5 M) but not ataxiatelangiectasia mutated (ATM), ATM-Rad3-related protein, or DNA-dependent protein kinase in vitro, indicating that it blocks two major branches of the checkpoint pathway downstream of ATM. It did not cause the activation or inhibition of different signal transduction proteins, as determined by mobility shift analysis in Western blots, suggesting that it inhibits a narrow range of protein kinases in vivo.

A phenotypic cell-based assay for inhibitors of the G (2) DNA damage checkpoint was used to screen plant extracts from the US National Cancer Institute Natural Products Repository. It revealed activity in a methanol extract from the common ragweed Ambrosia artemisiifolia. Assay-guided fractionation led to the identification of the sesquiterpene lactones psilostachyins A and C as novel checkpoint inhibitors. Elimination of their alpha,beta-unsaturated carbonyl group caused a loss of activity, suggesting that the compounds can bind covalently to target proteins through Michael addition. Psilostachyins A and C also blocked cells in mitosis and caused the formation of aberrant microtubule spindles. However, the compounds did not interfere with microtubule polymerization in vitro. The related sesquiterpene lactones psilostachyin B, paulitin and isopaulitin were also isolated from the same extract but showed no checkpoint inhibition. The identification of the target(s) of psilostachyins A and C may provide further insight into the signalling pathways involved in cell cycle arrest and mitotic progression.

Checkpoints activated in response to DNA damage cause arrest in the G 1 and G 2 phases of the cell cycle. Inhibitors of the G 2 checkpoint may be used as tools to study this response and also to increase the effectiveness of DNA-damaging therapies against cancers lacking p53 function. Using a cell-based assay for G 2 checkpoint inhibitors, we have screened extracts from the NCI National Institutes of Health Natural Products Repository and have identified 13-hydroxy-15-oxozoapatlin (OZ) from the African tree Parinari curatellifolia. Flow cytometry with a mitosis-specific antibody showed that checkpoint inhibition by OZ was maximal at 10 M, which released 20% of irradiated MCF-7 cells expressing defective p53 and 30% of irradiated HCT116p53 ؊/؊ cells from G 2 arrest. OZ additively increased the response to the checkpoint inhibitors isogranulatimide and debromohymenialdisine, but it did not augment the effects of UCN-01 or caffeine. Unlike other checkpoint inhibitors, OZ did not inhibit ataxia-telangiectasia mutated (ATM), ATM and Rad3-related (ATR), Chk1, Chk2, Plk1, or Ser/ Thr protein phosphatases in vitro. Treatment with OZ also caused G 2 -arrested and cycling cells to arrest in mitosis in a state resembling prometaphase. In these cells, the chromosomes were condensed and scattered over disordered mitotic spindles. The results demonstrate that OZ is both a G 2 checkpoint inhibitor and an antimitotic agent.In response to DNA damage, cell cycle progression pauses to allow time for DNA repair. This checkpoint response helps to maintain the integrity of the genome, and loss of checkpoints may enable the accumulation of mutations during carcinogenesis (1, 2). Cell cycle arrest is achieved by inhibiting the activities of cyclin-dependent kinases that govern entry into S phase or mitosis. In G 1 phase, DNA damage leads to the stabilization and nuclear localization of the p53 transcription factor, resulting in up-regulation of p21Waf1/Cip1 , an inhibitor of G 1 cyclindependent kinase activities (3-6). The G 2 checkpoint operates through a different mechanism, and the details of this pathway are beginning to emerge (7). In the current model, regulation is exerted through a phosphorylation cascade and by control of the subcellular localization of enzymes and their substrates. ATM 1 and ATR kinases play a role in the early signaling of DNA damage and cause phosphorylation of Chk1 and Chk2 kinases (8 -12). Phosphorylation of the phosphatase Cdc25C by Chk1 and Chk2 creates a 14 -3-3 binding site and inhibits its phosphatase activity in vitro, preventing its activation of Cdc2 kinase, the master regulator of mitosis (13-15). Cdc2 activity is also regulated by inhibitory phosphorylation by Wee1 and Myt1 kinases (16,17).Inhibitors of the G 2 checkpoint, such as caffeine, 2-aminopurine, pentoxifylline, staurosporine, and UCN-01, have been studied extensively in tissue culture model systems (18 -22). More recently, we have identified isogranulatimide (IGR) and debromohymenialdisine (DBH) as checkpoint inhibitors from marine inv...

Using a chemical genetics screen, we have identified ent-15-oxokaurenoic acid (EKA) as a chemical that causes prolonged mitotic arrest at a stage resembling prometaphase. EKA inhibits the association of the mitotic motor protein centromeric protein E with kinetochores and inhibits chromosome movement. Unlike most antimitotic agents, EKA does not inhibit the polymerization or depolymerization of tubulin. To identify EKA-interacting proteins, we used a cell-permeable biotinylated form that retains biological activity to isolate binding proteins from living cells. Mass spectrometric analysis identified six EKA-binding proteins, including Ran-binding protein 2, a kinetochore protein whose depletion by small interfering RNA causes a similar mitotic arrest phenotype.

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A major interest of mine is the use of cell-based screening of natural products to identify new chemicals that affect cell-cycle progression.I find it attractive that these chemicals can serve as tools to study biological mechanisms and that they may have therapeutic potential. My work has identified a structural class of compounds that arrest cells at an early stage of mitosis. It was an exciting step forward for us to be able to chemically modify the compound described in this paper in a way that allowed the identification of its binding partners and the visualization of its binding sites within the cell. This paper illustrates a chemical genetics approach to studying the complex pathways in mitosis specifically, but the strategy may be applied to protein target identification in other systems as well.(Read Rundle's article on p 443.