Spindle damaging agents are widely used in cancer chemotherapy, but their mechanism of action in cell killing by apoptosis induction is still incompletely understood. The impact of spindle checkpoint functionality and of several of its components and of functional p53 on the efficacy of spindle damaging agents to induce apoptosis in cancer cells was investigated. A main determinant of apoptosis induction is the duration of mitotic arrest upon spindle damage. Mitotic arrest depends on normal levels of each of the spindle checkpoint proteins Mad1, Mad2, BubR1 and Bub1 and several more not tested here.
Surprisingly, however, the induction of apoptosis is governed by specific spindle checkpoint proteins upon different types of spindle damage. Mad2 is required for cell death upon treatment with nocodazole, taxol and monastrol, which alter spindle dynamics or polarity, respectively. In contrast, Mad1 is needed for nocodazole-induced apoptosis, but is dispensable for apoptosis upon taxol or monastrol treatment (Figure 49). p53 is absolutely required for apoptosis induction by spindle damaging agents in HCT116 colon carcinoma cells, whereas HeLa cervix carcinoma cells induce apoptosis effectively despite lacking functional p53. Therefore the role of p53 in spindle damage-induced apoptosis is likely to be cell- or cancer type-specific and dependent on other factors like components of apoptotic or cell cycle arrest signaling pathways as well.
Spindle damage-induced apoptosis is dependent on prolonged mitotic arrest followed by mitotic slippage
My experiments with HeLa cells show that apoptosis is induced upon mitotic slippage after at least 24 h of nocodazole or taxol treatment. However, in mitotically arrested cells with normal or reduced levels of Mad2, BubR1 or Bub1, only basal levels of caspase 3 activity can be detected. So far this effect has not been resolved, but mounting evidence points to the existence of a cytoprotective mechanism acting in mitosis in form of a survival pathway comprising survivin and bcl-2. While survivin’s main function in untransformed cells lies in the regulation of mitosis, it is probably promoting survival in many cancer cell lines (Altieri 2006) and its levels peak in mitosis (Li et al. 1998). Bcl-2 is phosphorylated at several residues upon mitotic arrest and some of these phosphorylations have been
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proteolytic degradation (Breitschopf et al. 2000, Deng et al. 2004), but it was also reported that the level of bcl-2 expression in mitochondria rather than its phosphorylation state could regulate the sensitivity of cancer cells to taxol in vitro (Brichese et al. 2002). Upon mitotic slippage, survivin levels drop dramatically due to loss of a stabilizing Cdk1-mediated phosphorylation (Li et al. 1998, O’Connor et al. 2000, Shin et al. 2003) and mitotic bcl-2 phosphorylation is also lost, leading to increased apoptosis. Furthermore, transcription of proapoptotic proteins can be carried out in pseudo-G1 phase following mitotic slippage and accelerate apoptosis in these cells (Blagosklonny 2006). Interestingly, several recent reports link caspases, mitosis and the spindle checkpoint. An inhibitory phosphorylation at threonine 125 conferred by Cdk1/Cyclin B keeps caspase 9 activity in check during mitosis and upon spindle damage (Allan and Clarke 2007), while caspase 3 expression and activation at the G2/M transition in unstressed cells could enable immediate action upon extensive damage in mitosis (Hsu et al. 2006). Furthermore, caspase 3-dependent cleavage of Bub1 and BubR1 during an undisturbed mitosis or upon treatment with spindle damaging agents promotes mitotic slippage and caspase-insensitive mutants of both proteins increase apoptotic cell death in tetraploid cells by prolonging mitotic arrest (Baek et al. 2005, Kim et al. 2005). Since these hints have yet to give rise to a model linking spindle checkpoint proteins and apoptotic proteins, survivin is to date the only well documented spindle checkpoint protein with ties to the apoptotic machinery. Our results indicate a proapoptotic role of Mad2 upon spindle damage, which could be tested in various assays to find a connection between Mad2 and its activation of proapoptotic or inhibition of antiapoptotic proteins. Direct interactions could be detectable by immunoprecipitation and purification of complexes from cancer cells or an approach via a yeast two-hybrid system. An interaction of Mad2 and survivin is conceivable, since these proteins come into close proximity at the kinetochore. However, Mad2’s links to apoptosis could be indirect and mediated via a complex pathway involving several proteins and comprising many signaling steps.
Differential roles of spindle checkpoint proteins in spindle damage-induced apoptosis
Our new finding is that differential sensitivity to taxol- or monastrol-induced apoptosis is dependent on Mad1 or Mad2. Future experiments might help to clarify the role of all known spindle checkpoint proteins in response to spindle damaging agents with different
Discussion
modes of action like Vinca alkaloids or Eg5 inhibitors. Nocodazole is not used as a medication, but Vinca alkaloids like vincristines and vinblastines employ a similar mechanism of action and are commonly used anticancer agents. Eg5 inhibitors induce a monopolar spindle and a resulting lack of tension due to a lack of amphitelic attachment of the mitotic spindle and are therefore mechanistically similar to taxol in terms of spindle checkpoint activation and apoptosis induction. The drug-induced monopolar spindle itself appears to be intact at superficial inspection, but is probably less stable than its bipolar counterpart (our unpublished observations).
It will be of great interest to reconcile the observed differences in the requirement of spindle checkpoint components for the induction of mitotic arrest on the one hand and the requirement of a subset of specific spindle checkpoint components for spindle damage-induced apoptosis on the other hand. For instance, both Mad1 and Mad2 are required for the induction of mitotic arrest upon nocodazole, taxol and monastrol treatment, but while either Mad1 or Mad2 downregulation confer resistance to nocodazole, only reduced levels of Mad2 protect cells also against taxol- or monastrol-induced apoptosis. Therefore, not the spindle checkpoint proficiency per se, but the status of individual spindle checkpoint components might be of great prognostic value in cancer chemotherapy (Figure 49). One explanation for the observed differences might be the existence of at least three different categories of spindle checkpoint proteins: 1. Proteins with a function in spindle checkpoint signaling inducing mitotic arrest (e.g. Bub1, Bub3). 2. Proteins with a function in spindle checkpoint signaling inducing mitotic arrest and a role in kinetochore independent mitotic timing (e.g. Mad2, BubR1). 3. Proteins with a function in spindle checkpoint signaling inducing mitotic arrest and positively or negatively regulating apoptosis upon treatment with spindle damaging agents (e.g. Mad2, survivin). For survivin it has been shown, that its mitotic and prosurvival functions are separable, because they depend on the subcellular localization of survivin governed by a nuclear export signal (Colnaghi et al. 2006). Some evidence points to a similar role of its subcellular localization for the Mad2 protein. It was reported that mislocalization of the Mad1/Mad2 complex by the viral Tax protein from the nucleus to the cytoplasm correlates with spindle checkpoint impairment and resistance to chemotherapeutic agents in adult T-cell leukemia cell lines (Kasai et al. 2003). The same mislocalization pattern for Mad2 was reported for tissue samples and spindle checkpoint impairment was found in 75% of the investigated cell lines derived from testicular germ cell tumors, which also showed reduced MAD2 expression levels (Fung et al. 2007).
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cytoplasm could negatively affect spindle checkpoint function, since the nucleus is dissolved in mitosis. Also, it has to be noted that the proapoptotic role of Mad2 in spindle damage-induced apoptosis might stem from Mad2’s terminal position in spindle checkpoint signaling, but might also indicate a putative spindle checkpoint independent function in apoptosis.
Figure 49: Spindle damage-induced apoptosis is differentially regulated by Mad1 and Mad2. In HCT116 cell lines different kinds of spindle damage, i.e. spindle depolymerization, spindle stabilization and a monopolar spindle, induce mitotic arrest followed by mitotic slippage. The mitotic arrest is shortened in MAD1 knockdown and in MAD2+/- cells, but not in wild typic and TP53-/- cells (short-term reaction, 16 h).
HCT116 wt cells are sensitive to all kinds of spindle damage, whereas HCT116 TP53-/- cells are resistant.
Lowered levels of Mad2 confer resistance to all kinds of spindle damage, whereas reduced levels of Mad1 confer resistance to nocodazole-induced spindle depolymerization, but not to taxol-induced spindle stabilization or monastrol-induced spindle monopolarity (long-term reaction, 48 h). This suggests that not the ability of a cancer to induce and maintain mitotic arrest by spindle checkpoint activation, but rather the distinct apoptosis-inducing properties of the spindle checkpoint components determine chemotherapy responsivity.
Apoptosis induction due to altered spindle dynamics was most intensely studied for taxol.
However, the mechanisms of apoptosis induction by various agents impinging on spindle dynamics are still only poorly understood. Apparently, at higher doses of these agents, cell killing depends on spindle checkpoint-mediated mounting of a prolonged mitotic arrest and
Discussion
subsequent mitotic slippage, while at lower doses cell death occurs independent of the spindle checkpoint or traversal through mitosis (Wang et al. 2000). Spindle disruption leads to intrinsic apoptosis, which mainly depends on members of the bcl-2 family. Bax and/or bak are activated by conformational changes and translocate to the mitochondria, bim is released from microtubules, bad is activated by phosphorylation and each of these alterations inhibits bcl-2 and bcl-XL, tipping the scales toward apoptosis (Bhalla 2003).
Apoptosis induction by Eg5/KSP inhibitors employs similar mechanisms (Tao et al. 2005, Tao et al. 2007, Vijapurkar et al. 2007). Spindle damage induces stress kinases, but their relative contribution to activation or inhibition of apoptosis remains controversial (Bhalla 2003, Mollinedo and Gajate 2003). The mechanism of apoptosis induction upon spindle damage remains largely unresolved and might differ depending on the kind of spindle damage and its extent. Future experiments have to uncover the links between the spindle checkpoint and apoptosis, characterize the signaling pathways involved and discriminate between different cases or different amounts of spindle damage. This could be achieved by employing isogenic cell systems, chemical compounds with different modes of action on the mitotic apparatus and monitoring of apoptotic signaling over time, preferably in single cells, not in cell populations.
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