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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.

DZ, a benzodiazepine known to affect centrosome separation at prophase, leads to a higher degree of mitotic arrest in HeLa cells than in primary human fibroblasts. In fact, differently from fibroblasts, which undergo a transient block in prophase-to-prometaphase transition, a high proportion of tumor cells attempt to escape from the DZ-imposed mitotic block, fail to undergo complete mitosis and die by mitotic failure. DZ-treated samples showed certain biochemical hallmarks of apoptosis, such as induction of the proapototic Bax protein, mitochondrial alterations assessed by JC-1 staining and TEM analysis, PARP cleavage, and DNA fragmentation. However, in DZ-treated cells, we observed a very low or absent caspase activation as shown by immunofluorescence and immunoblot experiments with antibodies directed to activated caspases and by staining with the pancaspase inhibitor FITC-VAD-FMK. Experiments on mitochondrial depolymerization and apoptosis induction carried out in the presence of specific inhibitors of caspase-2 and caspase-3/7 indicated a caspase-independent apoptotic process induced by DZ. Accordingly, TEM analysis of treated cells revealed ultrastructural features resembling those reported for caspase-independent apoptosis. In conclusion, we hypothesize that HeLa cells override the prophase block imposed by DZ, producing a high rate of aberrant pro-metaphases, which, in turn, activates caspase-independent, apoptosis-like mitotic catastrophe.