Mitotic catastrophe occurs in the absence of apoptosis in p53-null cells with a defective G1 checkpoint

Cell death occurring during mitosis, or mitotic catastrophe, often takes place in conjunction with apoptosis, but the conditions in which mitotic catastrophe may exhibit features of programmed cell death are still unclear. In the work presented here, we studied mitotic cell death by making use of a UV-inactivated parvovirus (adeno-associated virus; AAV) that has been shown to induce a DNA damage response and subsequent death of p53-defective cells in mitosis, without affecting the integrity of the host genome. Osteosarcoma cells (U2OSp53DD) that are deficient in p53 and lack the G1 cell cycle checkpoint respond to AAV infection through a transient G2 arrest. We found that the infected U2OSp53DD cells died through mitotic catastrophe with no signs of chromosome condensation or DNA fragmentation. Moreover, cell death was independent of caspases, apoptosis-inducing factor (AIF), autophagy and necroptosis. These findings were confirmed by time-lapse microscopy of cellular morphology following AAV infection. The assays used readily revealed apoptosis in other cell types when it was indeed occurring. Taken together the results indicate that in the absence of the G1 checkpoint, mitotic catastrophe occurs in these p53-null cells predominantly as a result of mechanical disruption induced by centrosome overduplication, and not as a consequence of a suicide signal.     

Characterization of the p53-Dependent Postmitotic Checkpoint following Spindle Disruption

The p53 tumor suppressor gene product is known to act as part of a cell cycle checkpoint in G₁ following DNA damage. In order to investigate a proposed novel role for p53 as a checkpoint at mitosis following disruption of the mitotic spindle, we have used time-lapse videomicroscopy to show that both p53^+/+ and p53^−/− murine fibroblasts treated with the spindle drug nocodazole undergo transient arrest at mitosis for the same length of time. Thus, p53 does not participate in checkpoint function at mitosis. However, p53 does play a critical role in nocodazole-treated cells which have exited mitotic arrest without undergoing cytokinesis and have thereby adapted. We have determined that in nocodazole-treated, adapted cells, p53 is required during a specific time window to prevent cells from reentering the cell cycle and initiating another round of DNA synthesis. Despite having 4N DNA content, adapted cells are similar to G₁ cells in that they have upregulated cyclin E expression and hypophosphorylated Rb protein. The mechanism of the p53-dependent arrest in nocodazole-treated adapted cells requires the cyclin-dependent kinase inhibitor p21, as p21^−/− fibroblasts fail to arrest in response to nocodazole treatment and become polyploid. Moreover, p21 is required to a similar extent to maintain cell cycle arrest after either nocodazole treatment or irradiation. Thus, the p53-dependent checkpoint following spindle disruption functionally overlaps with the p53-dependent checkpoint following DNA damage.     

The involvement of MCT-1 oncoprotein in inducing mitotic catastrophe and nuclear abnormalities

Centrosome amplification and chromosome abnormality are frequently identified in neoplasia and tumorigenesis. However, the mechanisms underlying these defects remain unclear. We here identify that MCT-1 is a centrosomal oncoprotein involved in mitosis. Knockdown of MCT-1 protein results in intercellular bridging, chromosome mis-congregation, cytokinesis delay, and mitotic death. Introduction of MCT-1 oncogene into the p53 deficient cells (MCT-1-p53), the mitotic checkpoint kinases and proteins are deregulated synergistically. These biochemical alterations are accompanied with increased frequencies of cytokinesis failure, multi-nucleation, and centrosome amplification in subsequent cell cycle. As a result, the incidences of polyploidy and aneuploidy are progressively induced by prolonged cell cultivation or further promoted by sustained spindle damage on MCT-1-p53 background. These data show that the oncoprotein perturbs centrosome structure and mitotic progression, which provide the molecular aspect of chromsomal abnormality in vitro and the information for understanding the stepwise progression of tumors under oncogenic stress.     

Mechanism of cell death resulting from DNA interstrand cross-linking in mammalian cells

DNA interstrand cross-links (ICLs) are critical cytotoxic lesions produced by cancer chemotherapeutic agents such as the nitrogen mustards and platinum drugs; however, the exact mechanism of ICL-induced cell death is unclear. Here, we show a novel mechanism of p53-independent apoptotic cell death involving prolonged cell-cycle (G₂) arrest, ICL repair involving HR, transient mitosis, incomplete cytokinesis, and gross chromosomal abnormalities resulting from ICLs in mammalian cells. This characteristic ‘giant'' cell death, observed by using time-lapse video microscopy, was reduced in ICL repair ERCC1- and XRCC3-deficient cells. Collectively, the results illustrate the coordination of ICL-induced cellular responses, including cell-cycle arrest, DNA damage repair, and cell death.     

Mitotic catastrophe: a special case of apoptosis

Mitotic catastrophe is a poorly defined type of cell death linked to the abnormal activation of cyclin B/Cdk1. Here we propose that a conflict in cell cycle progression or DNA damage can lead to mitotic catastrophe, provided that cell cycle checkpoints are inhibited, in particular the DNA structure checkpoints and the spindle assembly checkpoint. Two subtypes of mitotic catastrophe can be distinguished. First, mitotic catastrophe can kill the cell during or close to the metaphase, in a p53-independent fashion, as this occurs in Chk2-inhibited heterokarya generated by fusion. Second, mitotic catastrophe can occur after failed mitosis, during the activation of the polyploidy checkpoint, in a partially p53-dependent fashion. In these conditions, cells die as a result of caspase activation and mitochondrial membrane permeabilization that constitute hallmarks of apoptosis. Prevention of caspase activation and/or mitochondrial damage avoids mitotic catastrophe, indicating that this form of cell death indeed constitutes a special case of apoptosis. Importantly, the suppression of mitotic catastrophe can favor asymmetric division and the generation of aneuploid cells. This delineates a molecular pathway through which failure to arrest the cell cycle and inhibition of apoptosis can favor the occurrence of cytogenetic abnormalities which are likely to participate in oncogenesis.     

Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins

Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-x_L by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.     

Effects of conditional depletion of topoisomerase II on cell cycle progression in mammalian cells

Topoisomerase II (Topo II) that decatenates newly synthesized DNA is targeted by many anticancer drugs. Some of these drugs stabilize intermediate complexes of DNA with Topo II and others act as catalytic inhibitors of Topo II. Simultaneous depletion of Topo IIα and Topo IIβ, the two isoforms of mammalian Topo II, prevents cell growth and normal mitosis, but the role of Topo II in other phases of mammalian cell cycle has not yet been elucidated. We have developed a derivative of p53-suppressed human cells with constitutive depletion of Topo IIβ and doxycycline-regulated conditional depletion of Topo IIα. The effects of Topo II depletion on cell cycle progression were analyzed by time-lapse video microscopy, pulse-chase flow cytometry and mitotic morphology. Topo II depletion increased the duration of the cell cycle and mitosis, interfered with chromosome condensation and sister chromatid segregation and led to frequent failure of cell division, ending in either cell death or restitution of polyploid cells. Topo II depletion did not change the rate of DNA replication but increased the duration of G₂. These results define the effects of decreased Topo II activity, rather than intermediate complex stabilization, on the mammalian cell cycle.     

Effects of conditional depletion of topoisomerase II on cell cycle progression in mammalian cells

Topoisomerase II (Topo II) that decatenates newly synthesized DNA is targeted by many anticancer drugs. Some of these drugs stabilize intermediate complexes of DNA with Topo II and others act as catalytic inhibitors of Topo II. Simultaneous depletion of Topo IIα and Topo IIβ, the two isoforms of mammalian Topo II, prevents cell growth and normal mitosis, but the role of Topo II in other phases of mammalian cell cycle has not yet been elucidated. We have developed a derivative of p53-suppressed human cells with constitutive depletion of Topo IIβ and doxycycline-regulated conditional depletion of Topo IIα. The effects of Topo II depletion on cell cycle progression were analyzed by time-lapse video microscopy, pulse-chase flow cytometry and mitotic morphology. Topo II depletion increased the duration of the cell cycle and mitosis, interfered with chromosome condensation and sister chromatid segregation and led to frequent failure of cell division, ending in either cell death or restitution of polyploid cells. Topo II depletion did not change the rate of DNA replication but increased the duration of G₂. These results define the effects of decreased Topo II activity, rather than intermediate complex stabilization, on the mammalian cell cycle.Key words: topoisomerase II, mitosis, G₂, conditional knockdown, S phase, mitotic catastrophe     

Down-regulation of Wild-type p53-induced Phosphatase 1 (Wip1) Plays a Critical Role in Regulating Several p53-dependent Functions in Premature Senescent Tumor Cells

Premature or drug-induced senescence is a major cellular response to chemotherapy in solid tumors. The senescent phenotype develops slowly and is associated with chronic DNA damage response. We found that expression of wild-type p53-induced phosphatase 1 (Wip1) is markedly down-regulated during persistent DNA damage and after drug release during the acquisition of the senescent phenotype in carcinoma cells. We demonstrate that down-regulation of Wip1 is required for maintenance of permanent G₂ arrest. In fact, we show that forced expression of Wip1 in premature senescent tumor cells induces inappropriate re-initiation of mitosis, uncontrolled polyploid progression, and cell death by mitotic failure. Most of the effects of Wip1 may be attributed to its ability to dephosphorylate p53 at Ser¹⁵ and to inhibit DNA damage response. However, we also uncover a regulatory pathway whereby suppression of p53 Ser¹⁵ phosphorylation is associated with enhanced phosphorylation at Ser⁴⁶, increased p53 protein levels, and induction of Noxa expression. On the whole, our data indicate that down-regulation of Wip1 expression during premature senescence plays a pivotal role in regulating several p53-dependent aspects of the senescent phenotype.     

Abnormal mitosis triggers p53-dependent cell cycle arrest in human tetraploid cells

Erroneously arising tetraploid mammalian cells are chromosomally instable and may facilitate cell transformation. An increasing body of evidence shows that the propagation of mammalian tetraploid cells is limited by a p53-dependent arrest. The trigger of this arrest has not been identified so far. Here we show by live cell imaging of tetraploid cells generated by an induced cytokinesis failure that most tetraploids arrest and die in a p53-dependent manner after the first tetraploid mitosis. Furthermore, we found that the main trigger is a mitotic defect, in particular, chromosome missegregation during bipolar mitosis or spindle multipolarity. Both a transient multipolar spindle followed by efficient clustering in anaphase as well as a multipolar spindle followed by multipolar mitosis inhibited subsequent proliferation to a similar degree. We found that the tetraploid cells did not accumulate double-strand breaks that could cause the cell cycle arrest after tetraploid mitosis. In contrast, tetraploid cells showed increased levels of oxidative DNA damage coinciding with the p53 activation. To further elucidate the pathways involved in the proliferation control of tetraploid cells, we knocked down specific kinases that had been previously linked to the cell cycle arrest and p53 phosphorylation. Our results suggest that the checkpoint kinase ATM phosphorylates p53 in tetraploid cells after abnormal mitosis and thus contributes to proliferation control of human aberrantly arising tetraploids.     

Paclitaxel Induces Primary and Postmitotic G1 Arrest in Human Arterial Smooth Muscle Cells

Paclitaxel (PTX), a microtubule-active drug, causes mitotic arrest leading to apoptosis in certain tumor cell lines. Here we investigated the effects of PTX on human arterial smooth muscle cell (SMC) cells. In SMC, PTX caused both (a) primary arrest in G1 and (b) post-mitotic arrest in G1. Post-mitotic cells were multinucleated (MN) with either 2C (near-diploid) or 4C (tetraploid) DNA content. At PTX concentrations above12 ng/ml, MN cells had 4C DNA content consistent with the lack of cytokinesis during abortive mitosis. Treatment with 6-12 ng/ml PTX yielded MN cells with 2C DNA content. Finally, 1-6 ng/ml of PTX, the lowest concentrations that affected cell proliferation, caused G1 arrest without multinucleation. It is important that PTX did not cause apoptosis in SMC. The absence of apoptosis could be explained by mitotic exit and G1 arrest as well as by low constitutive levels of caspase expression and by p53 and p21 induction. Thus, following transient mitotic arrest, SMC exit mitosis to form MN cells. These post-mitotic cells were subsequently arrested in G1 but maintained normal elongated morphology and were viable for at least 21 days. We conclude that in SMC PTX causes post-mitotic cell cycle arrest rather than cell death.     

p53-Independent Apoptosis and p53-Dependent Block of DNA Rereplication Following Mitotic Spindle Inhibition in Human Cells

We have studied the response of human transformed cells to mitotic spindle inhibition. Two paired cell lines, K562 and its parvovirus-resistant KS derivative clone, respectively nonexpressing and expressing p53, were continuously exposed to nocodazole. Apoptotic cells were observed in both lines, indicating that mitotic spindle impairment induced p53-independent apoptosis. After a transient mitotic delay, both cell lines exited mitosis, as revealed by flow-cytometric determination of MPM2 antigen and cyclin B1 expression, coupled to cytogenetic analysis of sister centromere separation. Both cell lines exited mitosis without chromatid segregation. K562 p53-deficient cells further resumed DNA synthesis, giving rise to cells with a DNA content above 4C, and reentered a polyploid cycle. In contrast, KS cells underwent a subsequent G1 arrest in the tetraploid state. Thus, G1 arrest in tetraploid cells requires p53 function in the rereplication checkpoint which prevents the G1/S transition following aberrant mitosis; in contrast, p53 expression is dispensable for triggering the apoptotic response in the absence of mitotic spindle.     

A novel human protein of the maternal centriole is required for the final stages of cytokinesis and entry into S phase

Centrosomes nucleate microtubules and contribute to mitotic spindle organization and function. They also participate in cytokinesis and cell cycle progression in ways that are poorly understood. Here we describe a novel human protein called centriolin that localizes to the maternal centriole and functions in both cytokinesis and cell cycle progression. Centriolin silencing induces cytokinesis failure by a novel mechanism whereby cells remain interconnected by long intercellular bridges. Most cells continue to cycle, reenter mitosis, and form multicellular syncytia. Some ultimately divide or undergo apoptosis specifically during the protracted period of cytokinesis. At later times, viable cells arrest in G1/G0. The cytokinesis activity is localized to a centriolin domain that shares homology with Nud1p and Cdc11p, budding and fission yeast proteins that anchor regulatory pathways involved in progression through the late stages of mitosis. The Nud1p-like domain of centriolin binds Bub2p, another component of the budding yeast pathway. We conclude that centriolin is required for a late stage of vertebrate cytokinesis, perhaps the final cell cleavage event, and plays a role in progression into S phase.     

Persistent expression of Nqo1 by p62-mediated Nrf2 activation facilitates p53-dependent mitotic catastrophe

Prolonged mitosis due to aberrant chromosome segregation permits cells to enter the G1 phase without cytokinesis and subsequently triggers the p53-dependent cell death program, known as mitotic catastrophe. Cells which fail to go through mitotic catastrophe create aneuploidy, posing a risk of oncogenesis. In the present report, we show that p62-mediated non-canonical activation of Nrf2 leads to the persistent expression of Nqo1, which plays a critical role for p53 stabilization during mitotic catastrophe. With prolonged exposure to nocodazole, a microtubule-depolymerizing agent, p62-deficient HCT116 cells exhibited an accumulation of a polyploid population with a limited appearance of apoptotic cells, which was attributable to the attenuated stabilization of p53. Combinatorial gene manipulation analysis verified that the regulatory cascade with a hierarchy of p62–Keap1–Nrf2–Nqo1 is required for p53 stabilization for mitotic catastrophe. This is consistent with the role of Nqo1 as a gatekeeper for proteasomal degradation of p53. Thus, we demonstrate for the first time the functional connection between the non-canonical Nrf2 pathway and p53-dependent cell death program upon prolonged mitosis.     

Dephosphorylation of Plk1 occurs through PP2A-B55/ENSA/Greatwall pathway during mitotic DNA damage recovery

Recovery from DNA damage is critical for cell survival. However, serious damage cannot be repaired, leading to cell death for prevention of abnormal cell growth. Previously, we demonstrated that 4N-DNA accumulates via the initiation of an abnormal interphase without cytokinesis and that re-replication occurs during a prolonged recovery period in the presence of severe DNA damage in mitotic cells. Mitotic phosphorylated Plk1 is typically degraded during mitotic exit. However, Plk1 has unusually found to be dephosphorylated in mitotic slippage without cytokinesis during recovery from mitotic DNA damage. Here, we investigated how Plk1 dephosphorylation is established during recovery from mitotic DNA damage. Mitotic DNA damage activated ATM and Chk1/2 and repressed Cdk1 and Greatwall protein kinase, followed by PP2A activation through the dissociation of ENSA and PP2A-B55. Interaction between Plk1 and PP2A-B55α or PP2A-B55δ was strongly induced during recovery from mitotic DNA damage. Moreover, the depletion of PP2A-B55α and/or PP2A-B55δ by siRNA transfection led to the recovery of Plk1 phosphorylation and progression of the cell cycle into the G₁ phase. Therefore, to adapt to severe DNA damage, the activated Greatwall/ENSA signaling pathway was repressed by ATM/Chk1/2, even in mitotic cells. Activation of the PP2A-B55 holoenzyme complex induced the dephosphorylation of Plk1 and Cdk1, and finally, mitotic slippage occurred without normal chromosome segregation and cytokinesis.     

Polo-like kinase 1 inactivation following mitotic DNA damaging treatments is independent of ataxia telangiectasia mutated kinase

Polo-like kinase 1 (Plk1) is an important regulator of several events during mitosis. Recent reports show that Plk1 is involved in both G2 and mitotic DNA damage checkpoints. Ataxia telangiectasia mutated kinase (ATM) is an important enzyme involved in G2 phase cell cycle arrest following interphase DNA damage, and inhibition of Plk1 by DNA damage during G2 occurs in an ATM-/ATM-Rad3-related kinase (ATR)-dependent fashion. However, it is unclear how Plk1 is regulated in response to M phase DNA damage. We found that treatment of mitotic cells with DNA damaging agents inhibits Plk1 activity primarily through dephosphorylation of Plk1, which occurred in both p53 wild-type and mutant cells. Inhibition of Plk1 is not prevented by caffeine pretreatment that inhibits ATM activity and also occurs in ATM mutant cell lines. Furthermore, ATM mutant cell lines, unlike wild-type cells, fail to arrest after mitotic DNA damaging treatments. The phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, reduces Plk1 dephosphorylation following mitotic DNA damaging treatments, suggesting that the PI3K pathway may be involved in regulating Plk1 activity. Earlier studies showed that inhibition of Plk1 by G2 DNA damage occurs in an ATM-dependent fashion. Our results extend the previous studies by showing that ATM is not required for dephosphorylation and inhibition of Plk1 activity following mitotic DNA damage, and also suggest that Plk1 is not a principal regulator or mediator of the mitotic DNA damage response.     

Inhibition of p53-Dependent,but Not p53-Independent,Cell Death by U19 Protein from Human Herpesvirus 6B

Infection with human herpesvirus (HHV)-6B alters cell cycle progression and stabilizes tumor suppressor protein p53. In this study, we have analyzed the activity of p53 after stimulation with p53-dependent and -independent DNA damaging agents during HHV-6B infection. Microarray analysis, Western blotting and confocal microscopy demonstrated that HHV-6B-infected cells were resistant to p53-dependent arrest and cell death after γ irradiation in both permissive and non-permissive cell lines. In contrast, HHV-6B-infected cells died normally through p53-independet DNA damage induced by UV radiation. Moreover, we identified a viral protein involved in inhibition of p53 during HHV-6B-infection. The protein product from the U19 ORF was able to inhibit p53-dependent signaling following γ irradiation in a manner similar to that observed during infection. Similar to HHV-6B infection, overexpression of U19 failed to rescue the cells from p53-independent death induced by UV radiation. Hence, infection with HHV-6B specifically blocks DNA damage-induced cell death associated with p53 without inhibiting the p53-independent cell death response. This block in p53 function can in part be ascribed to the activities of the viral U19 protein.     

A role of the mitotic spindle checkpoint in the cellular response to DNA replication stress

Replication stress is a frequent and early event during tumorigenesis. Whereas the cellular responses to a persistent block of replication fork progression have been extensively studied, relatively little is known about how cells respond to low-intensity replication stress. However, transient replication fork perturbations are likely to occur even more frequently in tumor cells than a permanent replication arrest. We report here that transient, low intensity replication stress leads to a rapid activation of the DNA replication checkpoint but to a significantly delayed apoptotic response in a small but significant number of cells. This late apoptotic response was independent of p53 and we found evidence for cell death during mitosis in a proportion of cells. To further explore the role of p53 in the response to replication stress, we analyzed mouse embryonic fibroblasts (MEFs) deficient of p53 in comparison to wild-type or p63- or p73-deficient MEFs. We detected a significant increase of apoptosis and morphological signs of failed mitosis such as multinucleation in p53-deficient MEFs following replication stress, but not in wild-type or p63- or p73-deficient cells. Multinucleated p53-deficient MEFs frequently retained cyclin B1 expression indicating a persistently activated mitotic spindle checkpoint. Collectively, our results suggest that the cellular response to replication stress involves the mitotic spindle checkpoint in a proportion of cells. These findings imply that the mitotic spindle checkpoint may act in concert with DNA damage and cell-cycle checkpoints as an early anti-tumor barrier and provide a possible explanation for its frequent relaxation in human cancer.