Induction of polyploidy and apoptosis after exposure to high concentrations of the spindle poison nocodazole

Abstract

The proportions of aneuploid/polyploid versus euploid cells formed after treatment with spindle poisons like nocodazole are of course dependent on the relative survival of cells with numerical chromosome aberrations. This work aimed at studying the survival of polyploid cells formed after treatment with a nocodazole concentration sufficient to significantly decrease tubulin polymerization (0.1 microg/ml). First, normal primary lymphocytes were analysed and the following complementary chromosomal parameters were quantified: mitotic index, frequency of abnormal mitoses, polyploid metaphases and apoptotic cells. The results clearly indicate a positive correlation between abnormal mitotic figures, apoptosis and the induction of polyploidy. They therefore led to a single cell approach in which both apoptosis and polyploidy induction could be scored in the same cell. For this purpose, actively proliferating cells are required and two human leukaemic cell lines were used, KS (p53-positive) and K562 (p53-negative), which have a near-triploid karyotype. Cells were separated into an apoptotic and a viable fraction by means of annexin-V staining and flow cytometry. In KS, treatment with nocodazole induced a similar fraction of hexaploid cells in both the viable and apoptotic fraction, but no dodecaploid cells were ever observed. In contrast, a population of dodecaploid cells (essentially viable) was clearly observed in the K562 cell line. The results in KS, as compared with K562, confirm that wild-type p53 can prevent further cycling of polyploid cells by blocking rereplication. The most probable explanation for these data is that not only the mitotic spindle but also interphase microtubules are sensitive to nocodazole treatment. Our data thus strongly suggest that besides the G(1)/S checkpoint under the control of p53, the G(2)/M transition may be sensitive to depolymerization of microtubules, possibly under the control of Cdc2, Bcl-2, Raf-1 and/or Rho.

STI-571 (Imatinib Mesylate) Enhances the Apoptotic Efficacy of Pyrrolo-1,5-Benzoxazepine-6, a Novel Microtubule-Targeting Agent, in Both STI-571-Sensitive and -Resistant Bcr-Abl-Positive Human Chronic Myeloid Leukemia Cells

“…6B, the apoptotic potential of nocodazole was significantly enhanced by STI-571-mediated inhibition of polyploidy. The formation of polyploidy in K562 cells following exposure to nocodazole was described previously by Verdoodt et al (1999). A 10-fold increase in nocodazole (10 M) strengthened the mitotic block induced by nocodazole and minimized mitotic slippage and polyploid formation.…”

“…As demonstrated with other microtubule-targeting agents (MTAs), we showed that activation of c-Jun NH 2 -terminal kinase (McGee et al, 2002), together with the phosphorylation and inactivation of the antiapoptotic proteins Bcl-2 and Bcl-x L are a prerequisite for PBOX-6-mediated apoptosis (McGhee et al, 2004). In addition, we demonstrate herein that the PBOX compounds induce the formation of polyploid cells, a characteristic shared with other MTAs (Verdoodt et al, 1999). Generally, in response to MTAs, cells enter mitosis, transiently arrest, and exit without undergoing cytokinesis.…”

Heterozygous Disruption of the TATA-Binding Protein Gene in DT40 Cells Causes Reduced cdc25B Phosphatase Expression and Delayed Mitosis

“…Delayed mitosis can lead to DNA rereplication and apoptotic death. DNA rereplication caused by delayed mitosis is naturally blocked by p53, while apoptosis is p53 independent (6,74). As DT40 cells do not express p53 (66), TBP-Het cells can undergo DNA rereplication and also exhibit a high proportion of apoptotic cells.…”

Cell death by mitotic catastrophe: a molecular definition

“…The absence of functional p53 is permissive for the generation/survival of polyploid cells in a number of in vitro systems; HeLa cells proliferating in vitro without any treatment other than antisense p53 (Iotsova and Stehelin, 1995), human leukemia cell lines treated with nocodazole ( (Verdoodt et al, 1999), U87MG glioma cells exposed to 1,3-bis(2choroethyl)-1-nitrosourea (Wu et al, 2001a), Msh2-deficient fibroblasts treated with cisplatin (Strathdee et al, 2001), the 32D murine myeloid cell line manipulated to overexpress c-myc (Yin et al, 1999), murine T cells subjected to mitotic arrest (Baek et al, 2003), or mouse embryonic fibroblasts treated with IC261, an inhibitor of casein kinase-1d and -e (Behrend et al, 2000), or fused with polyethylene glycol (Castedo et al, 2001), just to give a few examples. The overexpression of Aurora-A, Aurora-B and Plk causes cell division defects with consequent multinucleation (mostly tetraploidization) and an increase in centrosome number.…”