Mechanisms of Drug-Induced Mitotic Catastrophe in Cancer Cells

Current Pharmaceutical Design, 2010, 16, 69-78 69 Mechanisms of Drug-Induced Mitotic Catastrophe in Cancer Cells

José Portugal*, Sylvia Mansilla and Marc Bataller

Instituto de Biologia Molecular de Barcelona, CSIC, Parc Cientific de Barcelona. Baldiri Reixach, 10, E-08028 Barcelona, Spain

Abstract: Mitotic catastrophe is a mechanism of cell death characterized by the occurrence of aberrant mitosis with the formation of large cells that contain multiple nuclei, which are morphologically distinguishable from apoptotic cells. Sometimes, mitotic catastrophe is used restrictively to indicate a type of cell death that occurs during or after a faulty mitosis leading to cell death, which takes place via necrosis or apoptosis, rather than a cell death itself. Several antitumor drugs and ionizing radiation are known to induce mitotic catastrophe, but precisely how the ensuring lethality is regulated or what signals are involved is barely characterized. The type of cell death resulting from antitumor therapy can be determined by the mechanism of action of the antitumor agent, dosing regimen of the therapy, and the genetic background in the cells being treated. Wild-type p53 promotes apoptosis or senescence, while mitotic catastrophe is independent of p53. Mitotic catastrophe can be regarded as a delayed response of p53-mutant tumors that are resistant to some damage. In this context, the elucidation of the mechanisms of treatment-induced mitotic catastrophe should contribute to an improvement of the antitumor therapy, because most of the solid tumors bear an inactive p53 protein. Keywords: Mitotic catastrophe, apoptosis, DNA-binding drugs, cell cycle, chemotherapy.

INTRODUCTION occurs as a result of the uncoupling of the onset of mitosis after Cell death is usually considered to occur by p53-mediated DNA replication. Therefore, it has been considered an abnormal apoptosis [1, 2]. However, recent advances have led to the appre- mitosis leading to cell death, rather than a form of cell death [19]. ciation of additional, non-apoptotic, cell death pathways, which can DNA damage activates p53, and there are grounds for considering be either p53-dependent or p53-independent [1, 3-5]. DNA damage that wild-type p53 can promote apoptosis or senescence, while it is appears to induce several cell death pathways, only part of which not required for mitotic catastrophe [3]. Mitotic catastrophe is fall within the classical definition of apoptosis. In response to DNA fundamentally regarded as different from apoptosis [3, 20], yet damage wild-type p53 becomes stabilized promoting cells to several in vivo and in vitro models have shown that the different undergo either apoptosis or cell-cycle arrest [1-3, 6]. Cell cycle pathways of cell death are somewhat associated, and mitotic catas- arrest can be used to repair any damage, while apoptotic response is trophe is sometimes accompanied by the release of pro-apoptotic a genetically controlled response for cells to commit suicide when proteins and caspase activation, which would entail that mitotic repair cannot be accomplished [7]. The absence of wild-type p53 catastrophe shares some events used to define apoptosis [12, 21, function is the most common molecular abnormality in cancer cells 22]. and it is thought to play a key role in carcinogenesis [1, 5], and p53- It is noteworthy that cells need to evade the apoptotic pathway null mice are highly prone to developing cancer [1]. to become malignant, while a weakened mitotic checkpoint — Chemotherapy and radiation are known to inhibit tumor cell which is frequently observed in cancer cells—, together with the proliferation. So far, the best-studied antiproliferative response to absence of functional p53, may prompt cells to undergo mitotic chemotherapeutic agents is apoptosis, and thus a prevalent model to catastrophe after chemotherapy [4, 5, 18]. In general, the suscep- explain how cancer therapy can reach its goals has been that tibility of tumor cells to apoptosis (defined as “a caspase-mediated antitumor drugs trigger the cells to undergo apoptotic death, often cell death with associated apoptotic morphology” [23], which is mediated by wild-type p53 [1, 7], and that, therefore, cells resistant commonly accompanied by functional wild-type p53 activity [7]) is to apoptosis may become resistant to chemotherapy [1, 6, 7]. How- severely distorted. This renders other forms of cell death, such as ever, other mechanisms of cells death such as mitotic catastrophe mitotic catastrophe and necrosis, more important. Moreover, after may be also important to explain the success in anticancer therapy treatment with drugs or radiation, some cells containing functional [1, 3, 4, 8] (Table 1). p53 may undergo growth arrest with phenotypic features of cell senescence, reviewed in refs. [24-26]. MITOTIC CATASTROPHE IN TUMOR CELLS We should query about what determines the mode of cell death Here, we review the recent advances in the understanding of induced by a particular antitumor drug. The answer is rather com- some molecular pathways operative in non-apoptotic and apoptotic plex, and we have to consider several factors such as the cell type, cell death and of any cross-talk among them. To this end, we pay the genetic background of the cell, the type of DNA damage and the attention to phenotypic changes and molecular mechanisms dose of the drug used [5, 14, 24, 27, 28]. In general, tumors arising involved in the development of mitotic catastrophe. Mitotic from thymocytes, including T-cell lymphomas, spermatogonia, catastrophe is used to describe cell death that occurs during or after some hematological cancers, or bone marrow are often sensitive to a faulty mitosis, usually ending in polyploidy together with the the induction of apoptosis and display a clear overall response after formation of cells that contain multiple nuclei [9-14]. It has been treatment with DNA-damaging agents, which is generally p53- described as the principal form of cell death induced by ionizing dependent [1]. In contrast, for most solid tumors, which usually radiation [3, 9, 15, 16] and has been identified as the main response contain non-functional p53, the primary cell death pathway after to several antitumor drugs [3, 13, 14, 17, 18]. Mitotic catastrophe treatment with DNA-damaging agents may involve mitotic catastrophe [1, 4, 8]. *Address correspondence to this author at the Instituto de Biologia CELL CYCLE CHECKPOINTS AND THE RISE OF Molecular de Barcelona, CSIC, Parc Cientific de Barcelona. Baldiri MITOTIC CATASTROPHE Reixach, 10, E-08028 Barcelona, Spain; Tel: +34-93- 403 4959; Fax: +34- 93- 403 4979; E-mail: [email protected] Once DNA damage occurs, cells can stop progression through the cell cycle to allow for the damage to be repaired [29]. The phase

Table 1. Summary of Several Pathways of Cell Death Observed in Drug-Treated Cellsa

Apoptosis Mitotic catastrophe Necrosis

Definition and * Programmed cell death. * Cell death occurring during or after a *Identifies, in a negative fashion, cell characteristics * Cells shrink with blebbing of cell faulty mitosis. death lacking the features of apoptosis membranes. * Giant cells with two or more nuclei or autophagy. * Condensed chromatin and DNA and partially condensed chromatin. * Cells visible swell with breakdown of fragmentation. * Can lead to necrosis or apoptosis-like cell membrane. death (p53-independent). * Typical nuclei with vacuolization, and disintegrated cell organelles.

Associated genetic * Stimulated by cyclin D1 activation and * Stimulated by deficiencies in proteins * In general, it is not considered changes by Myc. involved in G1 and G2 checkpoints and genetically determined (this is open to * Can be inhibited by loss of wild-type in mitotic spindle assembly: p53, p21, debate). p53. Cdk1, Chk1, Chk2, etc. * Caspase activation. * Can follow caspase-dependent or – independent routes.

Detection methods *Sub-G1 peak in flow cytometry. * Cells with two or more nuclei detected *Early permeability to vital dyes. *Annexin-V-staining. by Microscopy or Laser Scanning * Staining with propidium iodide. Cytometry. *Accumulation in G2/M and *Internucleosomal laddering, etc. *Electron microscopy. polyploidy. aOther antiproliferative responses, such as autophagy and induced senescence, have been reported [3, 4, 55, 61]. of the cell cycle in which the cells arrest depends, in part, on their chromosome replication has not been completed, problems linked p53 status. Cells with wild-type p53 halt predominantly in G1, to DNA strand breaks might commit cells to die because they bear while cells with defective p53 (which occurs in more than 50% of chromosome deficiencies. Appropriate sensors detect incomplete human tumors) fail to arrest in G1 but rather arrest in S or G2 phase DNA replication, and the signal is then transmitted to kinases such [30, 31]. as ATR, which phosphorylate and activate other kinases such as Chk1 [32] and Fig. (1). The initiation of mitosis is turned on by the Cyclin-dependent kinases (Cdks) play a central role in checkpoint activation [32]. In the G1 phase of the cell cycle, DNA timely activation of the cyclin B-cdc2 complex [37, 38]. The checkpoint Chk1 and Chk2 kinases are essential for cell-cycle arrest damage activates p53, which in turns activates the expression of p21WAF1, a Cdk inhibitor. The induction of p21WAF1 is the major before mitosis in response to DNA damage and DNA replication blockage respectively [37-39]. Chk1 is phosphorylated at Ser317/345 mechanism underlying G1 arrest caused by DNA damage. Enhanced p21WAF1 expression can also lead to cell growth arrest in in response to DNA damage, and it seems to play a role at every G2 after DNA damage, by inhibiting the activity of cyclin- point in the cell cycle [40, 41]. Chk1 is subjected to multiple regulations, including transcriptional suppression by p53. A study dependent kinases, with p53 playing a critical role in halting cells at the G2 checkpoint [30]. It has been demonstrated that the inhibition using conditional Chk1 knock-out mice reveals that Chk1 deficiency causes severe cell death in proliferating somatic cells or knockout of genes in the p53 pathway, including the p53- inducible regulator p21WAF1, facilitates the G2 checkpoint abro- with large morphological abnormalities of their cell nuclei, in keeping with the occurrence of mitotic catastrophe [42]. Activation gation, promoting premature entry into mitosis [30]. It is evident that in tumor cells lacking active forms of p53, or in those where of cdc2 (also known as Cdk1) is regulated through a multifaceted process that includes the binding to cyclin B (Fig. (1)), whose level drugs have altered p53 gene expression [33], p21 overexpression WAF1 rises at late S phase, and peaks in mitosis [37, 43]. Just before has to follow p53-independent routes [24]. Besides, p21 - induced growth arrest is associated with the depletion of mitosis- mitosis, most cyclin B-cdc2 complexes exist in an inactive state because of the inhibitory phosphorylation of cdc2 on Thr14 and control proteins, leading to anomalous mitosis [10]. 15 Tyr , whose phosphorylation is catalyzed by Wee1 and Myt1, Cells may proliferate once their repair is complete. Inhibition of while the dephosphorylation is catalyzed by Cdc25 phosphatases cell cycle checkpoints limits the time available for repair, thereby (Fig. (1)) [38]. forcing cells to prematurely progress through S and G2, and to undergo an aberrant mitosis [5, 34]. During mitotic catastrophe, a The mitotic spindle assembly checkpoint is responsible for defective mitosis results in a missegregation of chromosomes and ensuring the precise chromosome segregation between daughter aneuploidy, followed by cell division. Nuclear envelops form cells [8, 29, 38]. At the molecular level, this checkpoint prevents around individual chromosomes or groups of some chromosomes, advance to anaphase by producing inhibitors of APC (anaphase- resulting in the presence of large cells with multiple micronuclei promoting complex). Activation of this complex is induced at the (Table 1), which are markedly different from apoptotic cells [3, 10, beginning of mitosis [44]. APC inhibition is accomplished by the 13, 14, 35]. recruitment of checkpoint proteins, including Bub1, BubR1, Bub3, Mad1 and Mad2, to unattached kinetochores [45]. After all Impaired DNA replication checkpoint function in mammals kinetochores have properly attached (metaphase), signal generation may produce bulky chromosomal rearrangements. Recombination is silenced and the APC inhibitors decay through a barely known repair mechanisms appear to operate more efficiently during late S mechanism that may include the action of the Mad2 binding factor and G2 phase because of the presence of sister chromatids and Cmt2 [46]. APC remains inhibited, however, until the cell passes chromosomes [36]. DNA lesions that impede the equal segregation the metaphase checkpoint, after which activation of the ubiquitin and distribution of chromosomes are expected to elicit an intense degradation system brings about the transition from the metaphase response. When chromosome segregation starts at a stage at which Mechanisms of Drug-Induced Mitotic Catastrophe in Cancer Cells Current Pharmaceutical Design, 2010, Vol. 16, No. 1 71

Fig. (1). The G2/M transition is driven by the complex between cdc2 (Cdk1) and cyclin B. During interphase, the cdc2/CycB complex is inactivated through phosphorylation on Thr14, Tyr15 and Thr161. Activation of cdc2/CycB requires dephosphorylation at the Thr14 and Tyr15 residues before cells enter mitosis. Following drug-induced damage the G2/M arrest is initiated by phosphorylation of the cdc2/cyclin B-activating phosphatase Cdc25C, which is afterwards sequestrated in the cytoplasm by 14-3-3 proteins. Several p53 target genes ensure the maintenance of this arrest: p21WAF1 inhibits the cdc2/CycB complex, as well as its activating kinase CAK. GADD45 promotes dissociation of the cdc2/CycB complex; 14-3-3 sequesters cdc2/CycB in the cytoplasm; Cdc25C expression is repressed by p53. Chk1 and Chk2 can inactivate Cdc25C, preventing the cell from progressing into mitosis. to anaphase and subsequent progression through mitosis [44]. APC- checkpoint function by RNA interference specific for Mad2 or mediated ubiquitination of securin leads to activation of its binding BubR1 propitiates, in DNA-damaged cells, the escape from mitotic partner separase, which cleaves the cohesins that maintain the catastrophe [19]. Endoreplication may result from the abrogation of linkage between sister chromatids, leading to sister chromatid mitotic checkpoint control [48], which can be due to p21-mediated separation and anaphase onset. Furthermore, ubiquitination and depletion of checkpoint control proteins. Furthermore, the p21WAF1- degradation of cyclin B inactivates cdc2, thereby permitting exit induced decay of Cdk1 could interfere with control mechanisms from mitosis [47]. In this fashion, the mitotic checkpoint prevents that prevent illegitimate DNA replication by acting on the repli- aneuploidy by permitting unattached kinetochores on chromosomes cation-licensing factor (RLF). Polyploid cells can arise from the that would be missegregated to delay the irreversible transition failure of cytokinesis, which can be triggered by a lack of cyto- from metaphase to anaphase until they become appropriately kinesis-associated proteins [3]. Polyploidization occurring after attached. Prolonged inhibition of APC (which results in a prolonged release from p21WAF1 can also be attributed to direct inhibition of cdc2 activation) can end in mitotic catastrophe associated with p21 gene expression [10], which can result in micronucleation. centrosome overduplication [12]. WHEN CELLS ARE COMMITTED TO DIE: MITOTIC While a minimum strength of the mitotic checkpoint is essential CATASTROPHE VERSUS APOPTOSIS AND NECROSIS for cell viability to prevent chromosome loss, a weakened checkpoint contributes to tumor development by enhancing the frequency There are grounds for considering that mitotic catastrophe is of aneuploidies, without losing viability [19, 29]. Therefore, tumors fundamentally different from apoptosis [3, 4, 8, 20], regardless of can display either a proficient or a compromised checkpoint. In this some opinions deeming it as a special case of p53-independent context, it is conceivable that after treatment with DNA damaging apoptosis [21]. In any case, a large variety of in vitro and a few in agents, cells with defective checkpoints may enter mitosis with vivo models have shown that the form of cell death can shift from abnormal DNA structures, which leads to activation of the mitotic mitotic catastrophe to apoptosis or necrosis, which appears to checkpoint and produces mitotic arrest [18, 19]. depend on the cell model examined, the antitumor agent used, and its dose [13, 14, 49-53]. In ovarian carcinomas, DNA damage The dynamics of mitotic catastrophe induced by DNA- triggers two distinct modes of cell death, apoptotic cell death or damaging agents in p53-deficient cancer cells has already been mitotic catastrophe, which are determined by the profile of proteins characterized [19]. Cells that enter mitosis with DNA damage arrest involved in the regulation of the cell cycle [54]. In this context, it is transiently at the metaphase for several hours without segregation worth mentioning mitotic catastrophe is sometimes accompanied by of chromosomes, subsequently dying from metaphase [12, 19]. In the release of pro-apoptotic proteins, such as cytochrome c, and by metaphase-arrested pre-catastrophic cells, the anaphase-promoting caspase activation [12, 22]. A key point regarding the requirements complex appears to be inactivated, while the spindle checkpoint is for apoptosis following DNA damage is to consider the activated after DNA damage. Moreover, suppression of the spindle 72 Current Pharmaceutical Design, 2010, Vol. 16, No. 1 Portugal et al. concentration of drug used. Doxorubicin-induced cell death appears skeleton-associated proapoptotic proteins, such as Bim and Bif to depend on both time and dose, with mitotic catastrophe being [18]. observed after 6 days, while apoptosis can occur within 12 h [13]. Necrosis has been observed as the final step in radiation- Apoptosis at rather high drug concentrations circumvents the need induced mitotic catastrophe [15] and after treatment with some for cell cycle progression, but the relevance of this swift apoptosis doses of antitumor drugs [14, 53, 61], but it is not necessarily the to the concentration that a tumor might be exposed to in vivo is no final step of mitotic catastrophe, particularly when caspase 3 less than doubtful [5]. Cell death at relatively small drug concen- remains functional [21, 22]. There is no clear molecular definition trations appears to be mediated by non-apoptotic mechanisms, for necrosis (Table 1), yet there are grounds for reconsidering while apoptosis requires higher concentrations. Therefore, apoptosis whether it is genetically regulated. A further biochemical under- may have little relevance to the survival of cells following some standing of this process may have important clinical implications kinds of damage [1]. [62]. Caspase activation is a main predictor of death and a clear feature of apoptosis, but the idea that ‘apoptosis equals caspase CELLULAR AND MOLECULAR MARKERS OF MITOTIC activation’ has been questioned [55]. Apoptosis can be defined as ‘a CATASTROPHE caspase-mediated cell death with associated apoptotic morphology’ While accumulating evidence suggests that tumor cell response [23], and applied, when appropriate, to the final fate of cells to radiotherapy and chemotherapy is not confined to apoptosis but suffering mitotic catastrophe [21, 22]. It has been considered that also includes other modes of cell death such as mitotic catastrophe mitotic catastrophe is fundamentally different from apoptosis [1, 3, 8, 20, 35], it is still necessary to establish unequivocal criteria because the overexpression of several anti-apoptotic genes [3] to differentiate among the various mechanisms of cell death [4, 55]. enhances the frequency of defective mitosis. In contrast, sometimes In the following paragraphs, we shall focus on the markers that mitotic catastrophe can be induced by DNA damage in the presence seem to be useful to establish the occurrence of mitotic catastrophe, of concomitant caspase activation, suggesting that it may constitute as well as on any connection between mitotic catastrophe and a special case of apoptosis [12, 21]. In some human cell lines, we apoptosis or necrosis. Moreover, some cross-talk seems to exist have shown that mitotic catastrophe results in cell death by caspase- between mitotic catastrophe, cell arrest and senescence [3, 10, 63]. dependent and caspase-independent mechanisms [14]. The presence In an attempt to better understand the differences between mitotic of caspase activities would mean that mitotic catastrophe could be catastrophe and other mechanisms of cell death, we review some of accompanied by key molecular events defining apoptosis [12, 21]. the parameters that can be used to uncover the presence of mitotic On the other hand, caspase-independent cell death pathways may catastrophe and whether mitotic catastrophe should be considered a protect the cells against potentially harmful cells if caspase- form of death or an irreversible trigger for death. mediated routes fail [56]. While the inhibition of caspases may A transient halt of DNA-damaged cells in G2/M, which is easy retard cell death associated with metaphase, death may occur to detect by conventional flow cytometry, and the subsequent entry through other routes [14, 57]. A significant activation of caspase 2 of cells into mitosis is required for mitotic catastrophe to occur and caspase 3 is observed in MDA-MB-231 breast carcinoma cells (Fig. (2A)). The absence of functional p53 (or a direct inhibition of —which bear non-functional p53— that undergo mitotic catas- its tumor suppressor function), as well as changes in p21WAF1, is trophe following treatment with doxorubicin, but not with bis- required to pass through the G2/M checkpoint. A simple time- anthracycline WP631 [14]. In MCF-7/VP cells, which do not dependent analysis of the expression of these genes (e.g., by express functional caspase 3, mitotic catastrophe is also induced quantitative real-time PCR) or the protein levels by immunoblotting after treatment [14]. This would indicate that caspases are not (Western blot) is useful in checking the behavior of these proteins mandatory for the occurrence of cell death after a defective mitosis. (Fig. (2B)). However, the changes in these protein levels are not Activated caspases lead to cell death through apoptotic pathways, sufficient to evaluate the final fate of cells after entering mitosis, while caspase-independent mitotic catastrophe may induce several yet these changes can be followed in turn by changes in cell rounds of abnormal mitosis, with polyploidy plus multinucleation, distribution, including a halt in G2 and polyploidization — ending in cell death via necrosis. Given that caspases also have occurring upon release from p21 [10]—, which can easily be other functions (for example, cytokine production, proliferation of studied by conventional cytometry (Fig. (2A)). An indirect way to T-lymphocytes, and differentiation of progenitor cells during evaluate that the cells are located in mitosis is to measure the extent erythropoiesis), the activation of caspases should be deemed as a of G2/M checkpoint abrogation using a mitosis-specific antibody frequent, but not sufficient, indicator of apoptosis [4]. against phosphorylated (Ser10) Histone H3, which can be assayed Proteins linking the mitotic checkpoint and the apoptotic by immunoblotting. program are scarcely documented [3, 21, 48]. We do not know, for Abnormal mitosis in irradiated or drug-treated cells may follow example, to what extent activation of the mitotic checkpoint can be several pathways, yet the final step is commonly the formation of considered per se pro-apoptotic, or it may generate death-inducing nuclear envelopes around individual clusters of missegregated signals that can be suppressed by apoptotic inhibitors acting during chromosomes (micronuclei) [16, 34, 49]. Multinuclei cells that mitosis. Therefore, mitotic slippage without silencing the pro-death originate from mitotic catastrophe can be distinguished from signal may lead to the inactivation or elimination of any apoptotic apoptotic cells by their morphology [34](Fig. (2C)), since the latter inhibitor, thus starting apoptosis. Specific spindle checkpoint contain fragmented nuclei accompanied by shrunken cytoplasm and proteins might mediate the correct balance between cell death and condensed chromatin, while cells that have undergone mitotic survival signals during mitosis, and this could explain why mitotic catastrophe are large and contain uncondensed chromosomes in cells are highly susceptible to chemotherapeutic agents that perturb absence of nuclear margination (Table 1) [3, 48]. Various this balance [8, 58, 59]. Another possibility is that a proapoptotic microscopy techniques have been employed to morphologically signal is not generated until slippage into a pseudo-G1 phase [18]. analyze polyploid cells arising after treatment, but also to determine An entry into the pseudo-G1 phase with an incompletely silenced whether they are multinucleate cells. These include fluorescence mitotic checkpoint may induce the expression of proapoptotic pro- microscopy, confocal laser microscopy and electron microscopy [8, teins, since gene transcription is inhibited during mitosis [60]. 13, 49, 50, 52]. LSC (Laser Scanning Cytometry) permits a direct Instead, mitotic slippage, after sustained mitotic arrest, may pro- analysis of multinucleated cells. Cells can be re-located, measured duce changes in the organization of microtubules and the cyto- and photographed (Fig. (2A)). LSC measures multicolor fluore- skeleton, which result in the release of microtubule- or cyto- scence and light scatter on a single cell basis. By using a strategy in which specific populations are identified, including bi-nucleate and Mechanisms of Drug-Induced Mitotic Catastrophe in Cancer Cells Current Pharmaceutical Design, 2010, Vol. 16, No. 1 73

Fig. (2). Characterization of mitotic catastrophe in drug-treated cells. Various representative examples are shown. A: Changes in the cell cycle distribution in drug-treated cells analyzed by flow cytometry. The presence of G2/M accumulation and polyploidy (p) are indicated. The insert displays a multinucleated cell located from the polyploidy cell population and photographed using Laser Scanning Cytometry. B: Changes in protein levels in Jurkat T lymphocytes and MDA-MB231 breast carcinoma cells treated with bis-anthracycline WP631. Representative Western blots showing time-dependent changes in the levels of proteins, indicated in the figure, in the presence of WP631 —MDA-MB-231 cells lack of functional p53—. -actin or tubulin are shown as loading controls. C: HCT-116 cells bearing two nuclei after treatment (b) or multiple nuclei (m) observed by optical microscopy. D: Examples of mitotic figures observed by optical microscopy in Jurkat T cells treated with WP631 (Leichman’s staining; photographed at 1000x magnification). Mitotic figures are identified as follows: P, aberrant prophase (c-mitosis); M, multipolar metaphase; A, multipolar anaphase (adapted from [63]). E: Flow cytometry analysis of breast carcinoma cells treated with nanomolar concentrations of bis-antracycline WP631. Cells were stained with Annexin-V-fluorescein and propidium iodide (PI). The presence of necrosis, either primary, detected by loss of the membrane ability to exclude propidium iodide (Annexin-V -/PI +), or secondary (Annexin-V +/PI + cells) is shown together with apoptotic-like cells (Annexin-V +/PI -) (adapted from ref. [14]). multinucleate cells [64], these populations can then be easily scored proteins, such as the mitosis initiator Chk2, the centromere protein as cells undergoing mitotic catastrophe [14, 50]. CENP-A, the spindle checkpoint control proteins Mad2 and BubR1, A variety of anomalous mitotic figures can be found in drug- and the DNA damage-induced checkpoint kinase Chk1 have been treated cells (Fig. (2D)). For example, lagging anaphases or telo- reported to change their levels in drug-treated cells entering mitotic phases are observed after treatment of some cell lines with anthra- catastrophe [10, 12, 13, 66, 67]. These results suggest that antitu- cyclines [10, 63], but also with other drugs [8, 65, 66], indicating mor drugs may induce depletion of some proteins controlling problems during chromosome segregation. Usually, c-mitosis (a mitosis, thus contributing to abnormal nuclear division and term for randomly distributed chromosomes throughout the cells) is subsequent cell death in, at least, certain cell types [4, 13, 37]. observed (Fig. (2D)) in drug-treated cells entering mitosis without Nevertheless, although the characterization of proteins partici- having duplicated their centrosomes [10, 63]. pating in the different steps of mitosis helps us to understand the At the molecular level, one way to determine what is occurring fate of cells entering mitosis with damaged or incompletely in cells suffering mitotic catastrophe is to analyze, in drug-treated replicated DNA, expression changes in the genes mentioned above cells, the expression of genes involved in mitosis and compare this cannot be considered specific markers of mitotic catastrophe. This with untreated cells (normal mitosis). A time-course analysis of the is because similar changes can be produced by, or during, other changes in the expression of mitosis-associated proteins permits to events in the cell cycle progression. At present, we do not know of determine the presence of mitotic catastrophe [13, 66, 67]. Several any direct, or even surrogate, hallmark of mitotic catastrophe. In this context, it is of outstanding interest to inquire about whether 74 Current Pharmaceutical Design, 2010, Vol. 16, No. 1 Portugal et al. mitotic catastrophe may present molecular markers, which would UCN-01 arrests selectively p53-defective cells at the G2 checkpoint make it easier to differentiate from other cell death mechanisms [5], leading to mitotic catastrophe [35]. Validation of target such as apoptosis, necrosis or senescence. It has been hypothesized inhibition in clinical studies involving Chk1 inhibitors presents a that alternative non-mitotic pathways may be in operation during unique challenge, as it requires demonstration of successful the development of mitotic catastrophe [48]. Interestingly, several suppression of an activated Chk1-mediated signaling event by the meiosis-specific genes, are up-regulated in some tumor cells inhibitor. Although it is frequently stated that Chk1 phosphorylates undergoing mitotic catastrophe, suggesting that some regulators of Ser216 of cdc25C following DNA damage, this site is constitutively meiosis can be involved in mitotic catastrophe [48, 68]. For phosphorylated by a yet unidentified kinase in undamaged cells, example, the principal meiotic regulator MOS is post-transcrip- making it an unreliable marker for Chk1 inhibition [73]. tionally up-regulated in p53-mutant lymphoma cells after irradia- The outcome of mitotic catastrophe as a consequence of cell tion, and its highest expression is directly proportional to both the treatments with drugs that bind to DNA, either covalently or rever- extent of the arrest and the number of endopolyploid cells [68]. It sibly with high affinity, has been characterized, which includes a remains to be fully demonstrated whether meiotic genes can be remarkable response to relatively low doses of cisplatin [5], ‘specific markers’ during mitotic failure. mentioned in a previous paragraph, and to several anthracyclines [13, 14, 51, 67]. In a study of the changes induced by treatment MITOTIC CATASTROPHE CAN BE INDUCED BY with moderate doses of doxorubicin in several human solid tumor CHEMOTHERAPY cell lines, it was established that fourteen cell lines, derived from 10 Drug-treated cells can undergo ‘slippage’ through the mitotic different types of cancer and treated for 2 days with doxorubicin, checkpoints to undergo mitotic catastrophe, although different indu- underwent 70–90% growth inhibition, and drug treatments cers of mitotic catastrophe appear to elicit different mechanisms increased the fraction of micronucleate cells in almost all of the cell and routes. A survey of the different routes that can be followed by lines, while only two cell lines displayed morphological charac- cells undergoing mitotic catastrophe is shown in Fig. (3). teristics of apoptosis [51]. These results indicate that mitotic death Numerous antitumor drugs, including spindle assembly inhibi- may be more common than apoptosis among solid tumor cell lines tors and DNA damaging agents, can activate the spindle checkpoint exposed to moderate doses of doxorubicin [13, 51]. [18]. Some alkylating agents induce centrosome amplification, A human cDNA array containing various oncogenes and tumor resulting in the formation of multi-polar spindles when damaged suppressor genes has been used, together with RT-PCR, to quantify cells enter mitosis [65]. Sensitivity of tumor cells to the induction gene expression profiles in Jurkat T lymphocytes treated with of mitotic catastrophe by anti-microtubular agents might be due to clinically relevant concentrations of daunorubicin [27] or bis- the concentration-dependent accumulation of deficiencies in the anthracycline WP631 [63]. Changes in gene expression after mitotic spindle checkpoint, [3, 28, 60]. The primary target of Vinca treatment suggest that cell-cycle arrest and cell death follow distinct alkaloids and the taxanes is the mitotic spindle thus these molecules pathways depending on the drug concentration. In this regard, disrupt mitosis directly [3, 28]. While the Vinca alkaloids are exposure of Jurkat T lymphocytes containing functional p53 to high microtubule-depolymerizing agents, taxanes hyperpolymerize the (IC75) daunorubicin concentrations results in cell-cycle arrest in G1 microtubules. Studies on the microtubule-stabilizing agent followed by almost immediate apoptosis [27]. Low concentration of paclitaxel (Taxol) have identified numerous cellular and molecular the anthracycline (IC50) cause G2 arrest and senescence-like growth effects, such as induction of cytokines and tumor-suppressor genes, arrest, while features of necrosis and apoptosis are observed only indirect cytotoxicity due to secretion of tumor necrosis factor, large after longer incubation periods [27]. Treatments with nanomolar activation of signal-transduction pathways, and selective activity concentrations of bis-anthracycline WP631 result in arrest at the against cells lacking functional p53 [8]. In human arterial smooth G2/M checkpoint and a transient senescence-like phenotype in the muscle cells, it causes the rise of post-mitotic multinucleated cells presence of DNA synthesis. Afterwards, cells enter mitosis, become with either near-diploid or tetra-ploid DNA content, in agreement polyploid, show aberrant mitotic figures, and they die through with the lack of cytokinesis during abortive mitosis [69]. mitotic catastrophe [63]. Cell death is accompanied by changes in Docetaxel (Taxotere) is a microtubule-stabilizing taxane used the expression profile of various oncogenes and tumor suppressor for the treatment of breast and prostate cancers. It has a higher genes, including the down-regulation of p53 and p21WAF1 [63]. antitumor activity compared with paclitaxel [28]. Treatment of HCT-116 colon cells deleted for p21WAF1 show a superior breast carcinoma cells with docetaxel causes the inhibition of major sensitivity to several DNA damaging agents [4, 74], in keeping with cellular events and induces cell death through mitotic catastrophe the ability of p21WAF1 to prevent replication of damaged DNA and [52, 53]. From experiments using breast carcinoma cells, two mitosis [3]. mechanisms of action have been suggested: induced aberrant In HT-29 human colon carcinoma cells, Imidazoacridone C- mitosis followed by necrosis (at low nanomolar concentrations of 1311, a drug that intercalates into DNA, delays the progression of docetaxel) and mitotic arrest plus apoptosis (at higher concen- cells through the S phase followed by G2 arrest, G2 to M transit trations) [28]. The molecular profiling of docetaxel cytotoxicity in and cell death through mitotic catastrophe [66], which is evidenced two breast carcinoma cells reveals, through an ontogeny analysis, by various biochemical and morphological criteria such as acti- changes in genes related to multiple cell functions, including cell vation of Cdk1 kinase, the presence of the mitotic epitope MPM-2, cycle control and apoptosis [53]. and the condensation of chromatin into mitotic chromosomes in Cyclin-dependent kinases (Cdks) and checkpoint kinases drug-treated cells. The presence of mitotic chromosomes and (Chks) have been considered very promising therapeutic targets in MPM-2 mitotic marker in drug-treated HT-29 cells suggests that human malignancies. An obvious concern, however, is whether cells progress from G2 at least until the metaphase–anaphase blocking these kinases would preferentially affect to cancer cells, transition. and not to normal cells [70-72]. Chk1 is primarily responsible for Breast carcinoma cells elicit diverse responses to drug arrest of cells in S and G2 phase [39]. Inhibition of Chk1 is clearly treatments, which depend on both the drug used and its concen- associated with the phenotypic occurrence of G2 checkpoint tration [14, 52, 53]. The presence of wild-type p53 has been abrogation in living cells, thereby it has been suggested as an correlated in MCF-7 cells with the occurrence of senescence after important target for drug discovery and development efforts [71]. In treatment with doxorubicin [67, 75]. In MDA-MB-231 and MCF- certain cell types, the percentage of cells with G2 DNA content 7/VP breast carcinoma cells, doxorubicin induces polyploidy, decreases soon after the addition of UCN-01 (7-hydroxystauro- formation of multinucleated cells, and cell death by mitotic sporine), which may inhibit Chk and ATM kinases [35, 73]. So far, catastrophe [14]. In both cell lines, the cytotoxic effect of bis- Mechanisms of Drug-Induced Mitotic Catastrophe in Cancer Cells Current Pharmaceutical Design, 2010, Vol. 16, No. 1 75

Fig. (3). Pathways leading to mitotic catastrophe and cell death after DNA damage. Several key events occurring after a faulty mitosis are represented. DNA damage in p53-deficient cells can result in an exit from mitosis without cytokinesis, or in cell death occurring during mitosis. The relationship of mitotic catastrophe with apoptosis and/or necrosis is indicated (reproduced, with permission, from ref. [4]). anthracycline WP631, which binds to DNA with a higher affinity induction of cell death, which can occur via apoptosis or mitotic than doxorubicin, is superior to that of doxorubicin [27], and a catastrophe (Table 1), and by senescence-like terminal proliferation transient halt in G2/M is observed. This is followed by mitotic arrest [24]. Since understanding the genetic and biological catastrophe [27]. Drug-induced changes in the expression of c-myc causation of cancer can generate new targets for therapy [1, 72], and p21WAF1, and in their respective protein levels, have been establishing the consequences of treatment with DNA-binding documented. They depend on the breast cell line tested, the drug drugs remains of the utmost importance in elucidating the used and its concentration [14]. Activation of caspase 2 and caspase molecular changes in tumor cells undergoing mitotic catastrophe. 3 was observed in doxorubicin-treated MDA-MB231 cells, but For several decades, apoptosis has been considered the key these protease activities were not observed in drug-treated MCF- mechanism of cell death in drug-treated cancer cells, as well as of 7/VP cells [14]. These observations imply that differences in the tumor remission in patients after treatment with various antitumor contribution of caspase-dependent and caspase-independent agents [1, 7, 20], but accumulating evidence, reviewed here, processes to cell death depend on both the cytotoxic agent and the indicates that tumor response to chemotherapy includes mitotic cell type, and that activation of caspases is not mandatory for the catastrophe, among other modes of cell death [1, 3, 4]. Mitotic occurrence of cell death via mitotic catastrophe [14]. catastrophe might be the main mechanism of tumor selectivity in The induction of the multidrug-resistance MDR1 protein as a the search for clinically useful antitumor drugs, because cell death consequence of treatment with ionizing radiation might impede through mitotic catastrophe can elicit a more intense, longer-lasting apoptosis [76], but this block in apoptosis is followed by a high effect in vivo [3]. increase in the fraction of cells undergoing senescence or mitotic Remarkably, apoptosis and senescence are regulated largely by catastrophe [3]. Besides, in cells expressing a different multidrug- wild-type p53, while mitotic catastrophe is not [24, 78, 79]. Given resistance protein (MRP-1), the treatment with some anthracyclines that apoptosis is frequently inactivated due to a non-functional p53 results in both the remission of the pump efflux and the rise of a protein [1, 5], the commitment of cells to mitotic catastrophe can be cell population that is committed to die through mitotic catastrophe considered advantageous in cancer treatment. DNA damage does [77]. Although the mechanisms that link drug-resistance, its not just delays mitotic exit, but blocks it leading to the formation of abolition, and cell death routes, are not fully understood, these binucleated cells and mitotic catastrophe (Figs. (2) and (3)) [4, 59]. results are in keeping with that mitotic catastrophe is sufficient to It is noteworthy that, in contrast to apoptosis and senescence, which account for cell death in the absence of apoptosis. are physiological antiproliferative responses, mitotic catastrophe is enhanced, rather than inhibited, by cellular changes that occur ON THE ROLE OF MITOTIC CATASTROPHE IN THE during tumor development (Table 1). Morphological evidences of CLINICAL TREATMENT OF CANCER mitotic catastrophe (Table 1), have been observed in some patients The overriding consequences of treatment with chemo- after chemotherapy [80]. Nevertheless, the presence of micronuclei therapeutic drugs are determined by factors responsible for the occurs not only in tumor cells after treatment but also in normal 76 Current Pharmaceutical Design, 2010, Vol. 16, No. 1 Portugal et al. lymphocytes, and micronuclei frequencies seem to be subject to REFERENCES inter-individual variability [81]. 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Received: June 18, 2009 Accepted: June 30, 2009