A-4 . ST2151 . Spindle assembly checkpoint . Mitotic catastrophe . Caspase-3Combretastatin A-4 (CA-4), a natural stilbenoid isolated from Combretum caffrum, is a new vascular targeting agent (VTA) known for its antitumor activity due to its anti-tubulin properties. We investigated the molecular mechanisms leading to cell death in non-small cell lung cancer H460 cells induced by natural (CA-4) and synthetic stilbenoids (ST2151) structurally related to CA-4. We found that both compounds induced depolymerization and rearrangement of spindle microtubules, as well as an increasingly aberrant organization of metaphase chromosomes in a dose- and time-dependent manner. Prolonged exposition to ST2151 led cells to organize multiple sites of tubulin repolymerization, whereas tubulin repolymerization was observed only after CA-4 washout. H460 cells were arrested at a pro-metaphase stage, with condensed chromosomes and a triggered spindle assembly checkpoint, as evaluated by kinetochore localization of Bub1 and Mad1 antibodies. Persistent checkpoint activation led to mitochondrial membrane permeabilization (MMP) alterations, cytochrome c release, activation of caspase-9 and -3, PARP cleavage and DNA fragmentation. On the other hand, caspase-2, and -8 were not activated by the drug treatment. The ability of cells to reassemble tubulin in the presence of an activated checkpoint may be responsible for ST2151-induced multinucleation, a recognized sign of mitotic catastrophe. In conclusion, we believe that discovery of new agents able to trigger mitotic catastrophe cell death as a result of mitotic block and prolonged spindle checkpoint activation is particularly worthwhile, considering that tumor cells have a high proliferative rate and mitotic failure occurs irrespective of p53 status

Retinoid-related molecules (RRM) are novel agents with tumor-selective cytotoxic/antiproliferative activity, a different mechanism of action from classic retinoids and no cross-resistance with other chemotherapeutics. ST1926 and CD437 are prototypic RRMs, with the former currently undergoing phase I clinical trials. We show here that ST1926, CD437, and active congeners cause DNA damage. Cellular and subcellular COMET assays, H2AX phosphorylation (;-H2AX), and scoring of chromosome aberrations indicate that active RRMs produce DNA double-strand breaks (DSB) and chromosomal lesions in NB4, an acute myeloid leukemia (AML) cell line characterized by high sensitivity to RRMs. There is a direct quantitative correlation between the levels of DSBs and the cytotoxic/antiproliferative effects induced by RRMs. NB4.437r blasts, which are selectively resistant to RRMs, do not show any sign of DNA damage after treatment with ST1926, CD437, and analogues. DNA damage is the major mechanism underlying the antileukemic activity of RRMs in NB4 and other AML cell lines. In accordance with the S-phase specificity of the cytotoxic and antiproliferative responses of AML cells to RRMs, increases in DSBs are maximal during the S phase of the cell cycle. Induction of DSBs precedes inhibition of DNA replication and is associated with rapid activation of ataxia telangectasia mutated, ataxia telangectasia RAD3-related, and DNAdependent protein kinases with subsequent stimulation of the p38 mitogen-activated protein kinase. Inhibition of ataxia telangectasia mutated and DNA-dependent protein kinases reduces phosphorylation of H2AX. Cells defective for homologous recombination are particularly sensitive to ST1926, indicating that this process is important for the protection of cells from the RRM-dependent DNA damage and cytotoxicity. [Mol Cancer Ther 2008;7(9):2941 -54]

The relationship between microtubular dynamics, dismantling of pericentriolar components and induction of apoptosis was analysed after exposure of H460 non-small lung cancer cells to anti-mitotic drugs. The microtubule destabilising agent, combretastatin-A4 (CA-4) led to microtubular array disorganization, arrest in mitosis and abnormal metaphases, accompanied by the presence of numerous centrosome-independent "star-like" structures containing tubulin and aggregates of pericentrosomal matrix components like gamma-tubulin, pericentrin and ninein, whereas the structural integrity of centrioles was not affected by treatment. On the contrary, in condition of prolonged exposure or high concentrations of CA-4 such aggregates never formed. Treatment with 7.5 nM CA-4, which produced a high frequency "star-like" aggregates, was accompanied by mitotic catastrophe commitment characterized by translocation of the proapoptotic Bim protein to mitochondria activation of caspases-3/9 and DNA fragmentation as a result of either prolonged metaphase arrest or attempt of cells to divide. Drug concentrations which fail to block cells at mitosis were also unable to activate apotosis. A detailed time-course analysis of cell cycle arrest and apoptosis indicated that after CA-4 washout the number of metaphases with "star-like" structures decreased as a function of time and arrested cells proceeded in anaphase. After 4 h, the multiple alpha- and gamma-tubulin aggregates coalesced into two well-defined spindles in a bipolar mitotic spindle organization. Overall, our findings suggest that the maintenance of microtubular integrity plays a relevant role in stabilising the pericentriolar matrix, whose dismantling can be associated with apoptosis after exposure to microtubule depolymerising agents.

The BH3-only Bcl-2 subfamily member Bim is a well known apoptosis promoting protein. However, the mechanisms upstream of mitochondrion membrane permeability by which Bim is involved in apoptosis have been poorly investigated, particularly in response to agents capable of interfering with the cytoskeleton architecture and arresting cells in mitosis. Based on the observation that Bim is sequestered on the microtubule-array by interaction with the light chain of dynein, we have investigated upon depolymerisation, whether Bim could be involved in the commitment of apoptosis. With this purpose H460 Non Small Lung Cancer Cells (NSLC) were treated with the microtubule damaging agent combretastatin-A4 (CA-4) (7.5 nM; 8-48 h), and various parameters were investigated. Upon treatment, cells arrested in mitosis and died through a caspase-3-dependent mitotic catastrophe. Transient knock down of Bim drastically reduced apoptosis, indicating that this protein was involved in cell death as induced by microtubules disorganisation. In response to increasing conditions of microtubules depolymerisation, we found that the protein level of Bim was strongly upregulated in a time-dependent manner at transcriptional level. Furthermore, Bim was released from microtubule-associated components. Bim was translocated to mitochondria, even in a condition of protein synthesis inhibition, where it showed a markedly increased interaction with Bcl-2. In turn, the fraction of Bax bound to Bcl-2 decreases in response to treatment, thereby indicating that Bim possibly promotes Bax release from the pro-survival protein Bcl-2. Overall, we demonstrated that Bim is required for the CA-4-induced cell death in the H460 lung cancer cell line via activation of the mitochondrial signalling pathway. Defining the contribution of Bim to the mechanism of apoptosis may offer some different clues in view of developing new strategies for chemotherapy with CA-4, underlining the relevance of the cytoskeleton integrity in the apoptotic response.

Chromosome lagging at anaphase and migration of both sister chromatids to the same pole, i.e. nondisjunction, are two chromosome-segregation errors producing aneuploid cell progeny. Here, we developed an assay for the simultaneous detection of both chromosome-segregation errors in the marsupial PtK1 cell line by using multiplex fluorescence in situ hybridization with specific painting probes obtained by chromosome flow sorting. No differential susceptibility of the six PtK1 chromosomes to undergo nondisjunction and/or chromosome loss was observed in ana-telophase cells recovering from a nocodazole- or a monastrol-induced mitotic arrest, suggesting that the recurrent presence of specific chromosomes in several cancer types reflects selection effects rather than differential propensities of specific chromosomes to undergo missegregation. Experiments prolonging metaphase duration during drug recovery and inhibiting Aurora-B kinase activity on metaphase-aligned chromosomes provided evidence that some type of merotelic orientations was involved in the origin of both chromosome-segregation errors. Visualization of mero-syntelic kinetochore-microtubule attachments (a merotelic kinetochore in which the thicker microtubule bundle is attached to the same pole to which the sister kinetochore is connected) identified a peculiar malorientation that might participate in the generation of nondisjunction. Our findings imply random missegregation of chromosomes as the initial event in the generation of aneuploidy in mammalian somatic cells.