Caspase-independent mitotic death (CIMD)

[Cell Cycle 7:8, 1001-1005; 15 April 2008]; ©2008 Landes Bioscience

Perspective

Caspase-independent mitotic death (CIMD) Katsumi Kitagawa* and Yohei Niikura Department of Molecular Pharmacology; St. Jude Children’s Research Hospital; Memphis, Tennessee USA

Key words: aneuploidy, Bub1, caspase-independent cell death (CIMD), mitosis, chromosomal instability, p73, spindle checkpoint

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occur.4 The spindle checkpoint (also called the mitotic spindle checkpoint, the mitotic checkpoint, or the spindle assembly checkpoint) is a surveillance system that can delay mitotic progression by transiently inhibiting the anaphase-promoting complex (APC), a ubiquitin-E3 ligase, in response to defective kinetochore-microtubule attachment (Fig. 1).5 Genes involved in the spindle checkpoint were first isolated from the budding yeast Saccharomyces cerevisiae and include MAD1, MAD2 and MAD3 (mitotic arrest-deficient);6 BUB1 and BUB3 (budding uninhibited by benzimidazole, a microtubule-depolymerizing drug);7 and MPS1 (monopolar spindle).8 Spindle checkpoint proteins and their functions are highly conserved between yeast and humans (the human homolog of Mad3 is BUBR1 and the designations of the other human homologs are the same as those of the yeast homologs), and defects in the spindle checkpoint result in aneuploidy.9 Consistent with a link between aneuploidy and cancer, several evidences indicate a role for the spindle checkpoint in tumorigenesis.3 Mutations in human homologs of Bub1 (BUB1 and BUBR1) were first found in subtypes of colorectal cancer cells that exhibited chromosome instability (CIN tumor cells).10 The CIN phenotype is associated with mutations in spindle checkpoint genes,11-13 decreased levels of spindle checkpoint proteins,14,15 and loss of spindle checkpoint activity.16,17 Mad2+/- mice develop lung tumors at high rates after a long latency.18 Bubr1+/- mice and Bub3/Rae1 heterozygotes are prone to tumor development.19,20 There are conflicting reports on the role of the spindle checkpoint in regulating apoptosis or cell death: in mice, when the function of Bub1 is compromised, cells escape apoptosis and continue to progress through the cell cycle, despite leaving mitosis with an altered spindle;21 however, mutations in bub1 cause chromosome missegregation and fail to block apoptosis in Drosophila,22 and Mad2-null mouse embryos undergo apoptosis at E6.5–E7.5.23 In these cases, cell death seems to occur in the subsequent G1 phase, but the role of the spindle checkpoint in cell death remains unclear. When cells cannot satisfy the spindle checkpoint after a long mitotic delay (known as adaptation to D-mitosis [delayed mitosis]24), cells undergo one of the following cell fates: (a) some die in mitosis, (b) some exit mitosis as viable cells but die by apoptosis in the G1 phase, or (c) some exit mitosis as viable cells but are tetraploid and reproductively dead.24 Microtubule inhibitors induce mitotic arrest by activating the spindle checkpoint and eventually causing cytotoxicity.25 Many reports have described the cytotoxicity of the inhibitors and resultant death as either apoptosis in G1 or reproductive death.25 However, questions about cell death during mitosis

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The spindle checkpoint, which monitors kinetochore-microtubule attachment, is required for high fidelity of chromosome transmission. A failure in this mechanism causes aneuploidy, thereby promoting progression to tumorigenesis. However, the cell death mechanism that prevents the aneuploidy caused by failure of the spindle checkpoint is yet unknown. We have recently identified a novel type of mitotic cell death, which we term caspaseindependent mitotic death (CIMD). In BUB1-deficient (but not MAD2-deficient) cells, CIMD is induced by conditions that activate the spindle checkpoint (i.e., cold shock or treatment with nocodazole, paclitaxel or 17-AAG [17-allylaminogeldanamycin]). CIMD depends on p73, a homolog of p53, but not on p53. It also depends on the apoptosis-inducing factor (AIF) and endonuclease G (Endo G), which are effectors of caspase-independent cell death. When BUB1 is completely depleted, aneuploidy occurs instead of CIMD. We propose that CIMD can be the cell death mechanism that protects cells from aneuploidy by inducing the death of cells prone to substantial chromosome missegregation. Our study also shows that previous evaluations of the spindle checkpoint activity in mutant or cancer cells by monitoring mitotic index could be misleading.

Introduction

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The molecular mechanisms that ensure accurate chromosome segregation during mitosis and meiosis are important to conserve euploidy in eukaryotes. Errors in these mechanisms (e.g., chromosome nondisjunction and chromosome loss) result in aneuploidy, and the phenotypic consequences of imbalances in chromosome number are usually profound. In humans, errors in chromosome segregation may play a role in the onset of neoplasia by causing the expression of recessive oncogenic phenotypes1 or by contributing to the development of specific types of aneuploidy.2 Mutations that cause genomic instability are currently considered to contribute to the development of cancer.3 Cell cycle checkpoints are cellular control systems that detect errors in the completion of ordered events in the cell cycle and prevent premature progression through the cell cycle when errors *Correspondence to: Katsumi Kitagawa; Department of Molecular Pharmacology; St. Jude Children’s Research Hospital; 332 N. Lauderdale Street; Memphis, Tennessee 38105-2794; Email: [email protected] Submitted: 01/31/08; Accepted: 02/11/08 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/5720 www.landesbioscience.com

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Bub1-mediated mitotic death

have remained. Although some evidence suggests that apoptosis occurs during mitosis,26-30 the relationship between apoptosis or programmed cell death during mitosis and the spindle checkpoint is unknown. We have recently identified a novel type of mitotic cell death that is independent of caspase activation and therefore termed it caspase-independent mitotic death (CIMD). CIMD occurs in BUB1-deficient cells that have defects in the kinetochore-microtubule attachment.31

When the Spindle Checkpoint Fails

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When cells have defects in kinetochore-microtubule attachment (e.g., when treated with microtubule inhibitors), the spindle checkpoint is activated and stops the cell cycle in early mitosis (Fig. 1). If Figure 1. The spindle checkpoint protects cells from chromosome missegregation caused by mitotic errors. When the checkpoint is activated by defects the spindle checkpoint is defective (Fig. 1), it cannot arrest the cells in kinetochore-microtubule attachment, it arrests the cell cycle by inhibiting in mitosis; therefore, even though all chromosomes are not aligned the anaphase-promoting complex (APC). APC inhibition causes securin to properly, premature mitotic exit occurs, followed by substantial accumulate, which in turn inhibits separase activity that targets cohesin. As a chromosome loss or gain (Fig. 2A). Consistent with this predic- result, cohesion is maintained and sister chromatids remain bound together. tion, we saw abnormal nuclei in Mad2-depleted HeLa cells treated Therefore, APC inhibition by the spindle checkpoint arrests cells in mitosis, thereby preventing aneuploidy. Defects in the mitotic spindle checkpoint with nocodazole (microtubule-deplolymerizing drug), paclitaxel result in cell death or aneuploidy, which may lead to tumorigenesis. (taxol, microtubule-stabilizing drug) and 17-allylaminogeldanamycin (17-AAG, an Hsp90 inhibitor that induces kinetochore defects), drugs that activate the spindle checkpoint.31,32 Surprisingly, substantial Bub1 depletion did not reduce the mitotic delay induced by these drugs,31 suggesting that the spindle checkpoint activity was not altered. However, a colony growth assay revealed that the treatments caused substantial lethality in Bub1depelted cells.31 The chromosomes in Bub1-depleted mitotic cells showed terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL)-positive signals,31 suggesting that these cells are dead or dying in mitosis before chromosomes are lost or gained. Thus, mitotic delay was not affected because cells were dead at that moment in mitosis and not because the spindle checkpoint activity was not altered (Fig. 2B). We found TUNEL-positive chromosomes in early mitosis (prophase, prometaphase or metaphase) only and not in late mitosis (anaphase or telophase).31 Because chromosomes are normally aligned (in metaphase) or before aligned, which means that they have not been lost or gained, there is no apparent reason for cells to die at this stage. We therefore thought that this mitotic cell death might be programmed cell death. Figure 2. (A) A model that describes how chromosome loss or nondisjunction occurs in Because DNA fragmentation occurs independent of spindle checkpoint-defective cells (MAD2-depleted cells or complete BUB1-depleted cells). caspase activity,31 we termed this cell death caspase- In spindle checkpoint-mutant cells, the spindle checkpoint is not activated even if there are defects in kinetochore-microtubule attachment. No mitotic delay occurs, which results in independent mitotic death (CIMD). premature exit from mitosis. Thus, substantial chromosome loss or nondisjunction occurs, Nocodazole or cold treatment depolymerizes micro- and presumably cell death follows. (B) A model that describes the same scenario in partial tubules, paclitaxel stabilizes microtubules, and 17-AAG BUB1-depleted cells. Here, defects in kinetochore-microtubule attachment induce lethal DNA is an Hsp90 inhibitor that induces kinetochore fragmentation. Because cells are still arrested in mitosis, the mitotic index is unchanged. defects.32 All these treatments (but not ICRF187, a top Therefore, the spindle checkpoint appears to be active (ON). II inhibitor) induce CIMD in BUB1-depleted cells.31 A common feature of CIMD induction by these treatments in BUB1CIMD is Programmed Cell Death depleted cells is defects in kinetochore-microtubule attachment. What is other evidence to show whether a type of cell death is Therefore, we conclude that defects in kinetochore-microtubule attachment, which activate the spindle checkpoint in normal cells, programmed/active or not? One is that death should occur rapidly. We found that DNA fragmentation starts 20 min after microtubule can induce CIMD in BUB1-deficient cells. 1002

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low enough to release phosphorylated p73 from BUB1 to bind DNA. However, we have not yet been able to visualize the interaction between BUB1 and p73 by immunoprecipitation (unpublished data). Therefore, we propose a model that assumes the existence of a mediator protein X (Fig. 3), according to which BUB1 phosphorylates protein X instead of p73. The phosphorylated protein X can bind to p73 to activate it. According to this model, overexpression of protein X should induce CIMD. Unknown and known proteins that physically interact with BUB1, such as BUBR1,35 BUB3,36,37 CENPE,35 RAE138 and Blinkin,39 are candidates for protein X and should be tested to validate the model.

Potential Role of CIMD in Tumorigenesis

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Figure 3. A model that describes how partial depletion of BUB1 induces CIMD. Defects in kinetochore-microtubule attachment (KT-MT defects) activate BUB1. The activated BUB1 binds to protein X and phosphorylates it. The phosphorylated protein X cannot bind to p73 while it is bound to BUB1. When BUB1 is depleted, protein X can bind to p73 to activate its transcriptional activity.

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What is the physiological function of CIMD? Cold treatment (at 23°C), which depolymerizes microtubules, instead of treatment with microtubule inhibitors induces CIMD in Bub1-depelted cells.31 Cells in a human body can occasionally be exposed to this stress. We have not yet determined the pathway that leads to DNA fragmentation from Bub1 depletion, but defects or changes in any other factors in this pathway could induce CIMD when the cells are exposed to room temperature or a lower temperature. We hypothesize that CIMD protects cells from aneuploidy, which leads to tumorigenesis; conversely, when CIMD fails, tumorigenesis increases in BUB1-deficient animals. Although Bub1+/- mice do not exhibit spontaneous tumorigenesis,40 Bub1 hypomorphic mice (Bub1H/H and Bub1-/H) that express the Bub1 protein at 20%–30% of the levels of Bub1+/+ mice are more prone to spontaneous tumorigenesis than are Bub1+/+ mice.40 It is therefore possible that CIMD may occur in Bub1+/- but not in the Bub1 hypomorphic mice. We are currently testing mouse embryonic fibroblast (MEF) cells derived from these Bub1 mutant mice38 for CIMD. If spontaneous tumorigenesis in Bub1+/- mice is suppressed by CIMD, then the reduction or deletion of p73 in Bub1+/- mice should result in increased spontaneous tumorigenesis. These genetic analyses will determine the in vivo function of CIMD.

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inhibitors or 17-AAG are added, and several hours later the cells collapse.31 Programmed cell death should also depend on the factors that induce cell death actively; in other words, by removing these factors, lethality should be suppressed. We found that CIMD depends on the apoptosis-inducing factor (AIF) and endonuclease G (Endo G), both DNases, and their depletion suppresses the lethality of CIMD.31 Furthermore, CIMD depends not on p53 but on its homolog p73, a putative tumor suppressor and checkpoint protein.31 These findings strongly suggest that CIMD is programmed cell death. As a transcriptional factor, p73 is similar to p53.33 However, p73 has specific targets, and thereby specific roles,33 and CIMD appears to be one of them. It would be of interest to find which genes p73 regulates during CIMD. DNA microarray can help identify the genes that work downstream of p73 in the CIMD pathway.

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Partial (but not Complete) BUB1 Depletion and Defects in Kinetochore-Microtubule Attachment Induce CIMD

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Meraldi and Sorger (2005) reported that a small amount of BUB1 remaining in the cell is sufficient to induce mitotic delay, but when BUB1 is completely depleted, premature mitotic exit occurs.34 We confirmed their finding with the observation that CIMD does not occur when BUB1 is almost completely depleted; the remaining BUB1 appears to be required to induce CIMD.31 Many cells with abnormal nuclei did not result from CIMD,31 which raises the possibility that CIMD protects cells from aneuploidy by killing those that are destined to have abnormal nuclei. How does partial but not complete depletion of BUB1 activate p73? A simple model is that BUB1 kinase might be activated because of defects in kinetochore-microtubule attachment, and BUB1 might phosphorylate p73 to activate it but binding of BUB1 to p73 might silence the transcriptional activity of p73. Therefore, a certain level of BUB1 is needed to phosphorylate p73, but this level should be www.landesbioscience.com

Applications for Cancer Therapy Microtubule inhibitors and 17-AAG are used as anticancer drugs.25,41,42 Because CIMD occurs in the presence of these inhibitors and 17-AAG, it appears to be a mechanism to kill cancer cells. We could use the CIMD assay (i.e., BUB1 depletion and TUNEL assay) to screen by using BUB1-depleted cells (BUB1 shRNA stable transformants) for drugs or inhibitors that induce defects in kinetochore-microtubule attachment, as microtubule inhibitors or 17-AAG do (Fig. 4, novel inhibitors #1). Also, BUB1 kinase inhibitors (not yet published or available) should work synergistically with microtubule inhibitors and 17-AAG (Fig. 4, novel inhibitors #2). Thus, it would be useful to screen chemical compound or small-molecule libraries for BUB1 kinase inhibitors. Also, small interfering RNA (siRNA) screens to identify the factors that affect CIMD will identify the genes that function downstream of BUB1 (Fig. 4, novel inhibitors #3). Elucidation of the CIMD pathway will help identify more compounds that can be used as anticancer agents.

The Spindle Checkpoint Activity Assesed by Mitotic Index Our finding of CIMD revealed that there is a caveat for assessing mitotic index when microtubule inhibitors or 17-AAG are used. For

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example, when the spindle checkpoint activity of a mutant cell or a cancer cell line is tested, even if the reduction of mitotic index is not seen, it is too early to conclude that the spindle checkpoint activity is intact. The TUNEL-assay has to be performed because CIMD might occur. Bub1 levels are often reduced in several human cancers, including colorectal esophageal and gastric cancers.14,43,44 Thus, the spindle checkpoint activity assessed previously in cancer cells has to be reevaluated. Also, proteins assumed to be not required for spindle checkpoint activity may induce CIMD instead. Although the role of BUB1 in cell cycle progression, chromosome stability and cancer biology is vital, little is known about the substrates of the BUB1 kinase. Identification of substrates for BUB1 kinase will be helpful for studies on several areas of biology.

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Figure 4. Pathway showing how and where the treatments used in our studies act on our proposed model. The anticancer drug 17-AAG, microtubule inhibitors, and cold shock induce defects in the kinetochore-microtubule attachment, which lead to CIMD in Bub1-deficient cells. (#1) This assay identifies inhibitors that are similar to these anticancer agents. (#2) Bub1 inhibitors (that can induce CIMD in mitotically dividing caner cells in combination with 17-AAG and microtubule inhibitors) are likely to synergistically work as anticancer drugs with 17-AAG and microtubule inhibitors. (#3) An siRNA against the factor that functions downstream of BUB1 inhibits CIMD. siRNA screens will be able to fill up the pathway.

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