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Telling Cells How to Die Docetaxel Therapy in Cancer Cell Lines Héctor Hernández-Vargas1 José Palacios2 Gema Moreno-Bueno3,*

Abstract

1Spanish National Cancer Centre (CNIO); Madrid, Spain 2Servicio de Anatomía Patológica, HHUU Virgen del Rocío; Sevilla, Spain 3Instituto de Investigaciones Biomedicas (IIB)-Alberto Sols; Unidad de Biología Molecular y Celular del Cáncer; Madrid, Spain

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Taxanes (paclitaxel and docetaxel) are antimicrotubule agents used in the clinical practice for the treatment of solid tumors. The molecular mechanisms of taxane‑ dependent cytotoxicity are only partially known. In vitro, different modes of action have been established for paclitaxel according to its concentration. A similar phenomenon has been shown for other microtubule‑stabilizing drugs, including docetaxel. Recently, we described two different forms of mitotic exit in breast cancer cell lines exposed to docetaxel. The causes and consequences of the dual mechanism of cytotoxicity of this agent are discussed here.

*Correspondence to: Dra. Gema Moreno-Bueno; Instituto de Investigaciones Biomedicas (IIB)-Alberto Sols.; Unidad de Biología Molecular y Celular del Cáncer; C/ Arturo Duperier 4; 28029 Madrid, Spain; Email: [email protected]

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Antimicrotubule Agents and Known Mechanisms of Cell Death

Key words

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docetaxel, aberrant mitosis, mitotic slippage, apoptosis

Dynamic instability describes the process of slow growth and rapid shortening of microtubules by continuous addition and loss of tubulin at their ends.1 The precise regulation of this process is fundamental to the multiple functions of microtubules, especially those related with cell division.2 Several agents affecting microtubule dynamics have been shown to be effective against tumor cells. They are classified into those inducing either microtubule depolymerization or their polymerization. The last group is represented by taxanes, which include paclitaxel and docetaxel, both currently approved for clinical use.3,4 By binding to the b subunit of tubulin, these agents impair the dynamic of microtubules, promote their polymerization, and arrest the cells in mitosis. As a consequence, cells die by apoptosis through pathways still not fully characterized. Most studies, especially with paclitaxel, have shown cell cycle arrest in mitosis as a necessary step for cell death. However, it is also necessary that cells overcome this arrest, advancing to interphase without cytokinesis (a process called mitotic slippage), and entering a tetraploid or “pseudo‑G1” phase followed by apoptotic cell death.5,6 Mitotic arrest induced by taxanes is dependent on the activation of the spindle‑assembly checkpoint, which monitors any defects in microtubule‑kinetochore attachment or in the tension of the mitotic spindles. This checkpoint is usually weakened—but not absent—in human tumor cells, with cells able to respond with an initial cell cycle arrest but less able to maintain it.7,8 Therefore, tumor cells usually arrest in response to taxanes, but this arrest is weak or transitory permitting the mitotic slippage required for cells to die. Several questions still remain to be answered regarding the mechanisms of taxane‑ dependent cytotoxicity in tumor cells, including the induction of cell death without mitotic arrest in some experimental conditions, the dependence of these processes on transcriptional mechanisms, the importance of a functional p53 protein, and the exact mechanism(s) by which tumor cells die.

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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=4050

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Original manuscript submitted: 02/14/07 Manuscript accepted: 02/22/07

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Acknowledgements

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The authors thank P. Wachowicz, H. Peinado and M. Hergueta for their critical feedback and thoughtful suggestions. This work was supported by Ministerio de Educación y Ciencia, Grant Reference PTR1995-0753OP and Ministerio de Ciencia y Tecnologı’ a SAF2001-0065 and SAF2004-08258-C0201. G.M.B. is a junior investigator of the Ramón y Cajal Program (2004).

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Mitotic Exit after Exposure to Taxanes Recently, we described a dual mechanism of action for docetaxel in breast cancer cell lines,9 that was suggested by a biphasic growth inhibition curve. The presence of at least two mechanisms of action was confirmed by the observation of different cell cycle and cell death profiles in both cell lines, and further studied by gene expression microarrays performed after two concentrations of docetaxel. The most striking differences were found at the level of cell cycle. Low concentrations (2–4 nM) induced aberrant mitosis followed by aneuploidy, whereas higher concentrations (100 nM) induced sustained mitotic arrest followed by mitotic slippage (Fig. 1). Although mitotic arrest and mitotic slippage are well characterized responses to this agent, there are also some previous reports Cell Cycle

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Figure 1. Exit from mitosis after exposure to docetaxel. The effect of docetaxel on cell cycle is represented here after a synchronization experiment. Cells normally replicating their DNA from G1 (2n) to G2 (4n) are then subjected to low and high concentrations of the drug. Cells not receiving treatment exit normal mitosis (M) to start a new cell cycle from G1 (1). In contrast, low and high concentrations of docetaxel induce transient (3) or prolonged (2) arrest in mitosis, respectively. The respective cell cycle profiles (obtained by DNA staining with propidium iodide) are shown.

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Docetaxel Therapy in Cancer Cell Lines

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indicating that docetaxel is able to induce aberrant mitosis at lower concentrations.10,11 However, most studies indicating a dual mechanism of action of taxanes had been reported with paclitaxel.12‑15 In accordance to our findings with docetaxel, Ikui et al reported that higher concentrations of paclitaxel induce a longer spindle‑assembly checkpoint in HeLa cells. In contrast, low concentrations of paclitaxel cause mitotic delay followed by premature dissociation of p55CDC/CDC20 from Mad2 and BubR1 and abrogation of the spindle‑assembly checkpoint, leading to aneuploidy.16 As represented in Figure 1, mitotic exit will be produced through at least two possible processes depending on drug concentration. Concentrations known to stabilize the microtubules, induce a prolonged activation of the spindle‑assembly checkpoint (the mitotic index was maximal after 24 hours). This is followed by mitotic slippage, and manifested by multinucleation. In contrast, low concentrations of docetaxel will affect the dynamics of microtubules, inducing a transient activation of the spindle‑assembly checkpoint. In the breast cancer cells that we evaluated, this checkpoint is not strong enough to stop the segregation of chromosomes, and this is manifested by aneuploidy.17 Aneuploidy is known to be the result of aberrant mitosis which can be caused by divisions in the presence of a multipolar spindle, as has been described for docetaxel. However, it can also be the result of previous defects in cytokinesis or centrosome amplification, by defects in chromosome cohesion, by spindle attachment defects, or by impairment of the mitotic checkpoint response.7 Interestingly, although docetaxel has been described to interfere with centrosome www.landesbioscience.com

Figure 2. Transcriptional responses to docetaxel. Gene expression profiles of selected transcripts after treatment of MCF7 cells with 2 concentrations of docetaxel (4 nM and 100 nM), and different time points (8, 24 and 48 hours). Gene expression is represented as units relative to nontreated cells.

duplication during S phase, cells synchronized in G2 and treated with 2 nM docetaxel during 6 hours, were still able to show aneuploidy after passing through mitosis. This suggests that mechanisms independent of centrosome duplication are related with the induction of aberrant mitosis in this context. Our gene expression data supports some of these previous findings. Favoring the stronger mitotic arrest with higher concentrations of docetaxel, an induction of mitosis‑related genes (AURKA, PLK1) was observed in this condition (Fig. 2). Checkpoint‑related genes (BUB1, MAD2L1) showed a similar profile in the first time points of treatment. However, as can be seen in Figure 2, a downregulation of these transcripts was observed after 48 hours. This can be related to a release from the spindle checkpoint and the induction of mitotic slippage. Although exit from a normal mitosis is controlled by ubiquitin‑dependent proteolysis and phosphorylation/dephosphorylation events, it is possible that transcriptional regulation participates in the prolonged activation of the spindle‑assembly checkpoint after

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Together, these data point to a mechanism regulating the different consequences of docetaxel exposure according to the duration of mitotic arrest. This mechanism can be partially regulated at the transcriptional level by changes both in p21 and checkpoint genes.

A Dual Mechanism Implies Dual Outcomes

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Induction of apoptosis has been described as a main mechanism of cell death after antimicrotubule agents, implicating different downstream events including the dysregulation of cell cycle‑related proteins (particularly p34cdc‑2),21 the activation of the jun N‑terminal kinase (JNK)22 and Raf‑1 kinase23,24 �� pathways, and the final induction of a misbalance of the Bcl‑2 family of proteins.22,24‑27 �� However, two different effects on mitosis that we described were also followed by different types of cell death (Fig. 3). Although 100 nM docetaxel induced a clear pattern of early apoptosis by Annexin‑V staining at short time points (24 hours or less), cell death was lower with 2–4 nM concentrations, and it was mainly represented by necrosis.9 This is consistent with reports showing that cells undergoing aberrant mitosis eventually result in reduced clonogenic survival,28,29 usually due to nonapoptotic mechanisms.30 In this sense, some groups have described alternative caspase‑independent mechanisms of cell death after taxane therapy.31 More recently, it was shown that autophagy can be an important form of programmed cell death after treatment of MCF7 breast cancer cells with paclitaxel analogs and docetaxel, by using MAP I LC3 antibody as an specific marker of autophagosomes.32 Therefore, the most likely scenery is a combination of apoptotic and nonapoptotic mechanisms. In fact, a known mediator of apoptosis after taxane therapy is the cell death receptor CD95/ FAS. Our own gene expression data shows that the induction of FAS is a common response to docetaxel. Moreover, this response was dose‑ and cell line‑independent (Fig. 2). However, it is possible that nonapoptotic pathways override the induction of FAS after aberrant mitosis induced by low concentrations of docetaxel. One possible mechanism triggering cell death in this context is the induction of DNA damage. It is likely that aberrant mitosis creates chromosomal breakage, interchromosomal concatenation and a lethal genetic imbalance that leads to cell death.5,33 In this sense, we have observed accumulation of phospho‑H2AX after docetaxel, especially in the p53‑non functional MDA‑MB‑231 cells, at low concentrations of docetaxel. In addition, it was recently shown that DNA damage can be also involved in the activation of the G1 checkpoint after mitotic slippage induced by antimitotic agents.34

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Figure 3. Model of cell outcome according to drug concentration. A model for the dual mechanism of action of docetaxel is represented in the diagram. The main effects and outcomes are shown in a color gradient, corresponding to the increasing concentrations of the drug. The hypothetic correlation between induction of the spindle‑assembly checkpoint (S‑A‑checkpoint) and accumulation of p53 protein is shown, as well as the inverse correlation between p53, and the induction of p53 targets.

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taxane exposure. In support of this view, the results of Chen et al. suggest that the mitotic slippage induced by microtubule‑stabilizing drugs may result from altered transcriptional regulation of G2/M and spindle checkpoint genes when higher concentrations of drug are used.15 Finally, we observed a different expression profile of known p53 targets when comparing low and high concentrations of docetaxel in the p53‑functional cell line MCF7. Specifically, p21 and TP53INP1 were induced at earlier time points with lower concentrations. In addition, there was an inverse correlation between p53 protein accumulation and p21 transcript and protein expression (Figs. 2 and 3). This observation supports a model in which p53 protein accumulation is a marker of the timing of mitotic arrest.18 In addition, the earlier induction of p21 after low concentrations of docetaxel may be related with the mechanism of aberrant mitosis, as has been reported.19 In fact, previous studies have suggested that p21 is not required for the mitotic arrest in response to paclitaxel, but instead it could have a role in facilitating the exit from abnormal mitosis.20

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Concluding Remarks The impairment of microtubule dynamics results in cell cycle alterations and cell death. Both effects can be the result of independent activities of docetaxel. However, our observations point to the possibility that the mechanism of cell death is the direct consequence of the previous type of mitotic exit induced by this agent. Therefore, the understanding of the mechanisms underlying docetaxel cytotoxicity will rest on a better knowledge of the link between checkpoint activation and cell cycle arrest, and the subsequent induction of cell death. The elucidation of these mechanisms should consider the signaling pathways, and possible crosstalk among those pathways, activated in a concentration‑dependent manner.

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32. Górka M, Daniewski WM, Gajkowska B, Lusakowska E, Godlewski MM, Motyl T. Autophagy is the dominant type of programmed cell death in breast cancer MCF‑7 cells exposed to AGS 115 and EFDAC, new sequiterpene analogs of paclitaxel. Anti‑Cancer Drugs 2005; 16:777‑88. 33. Fujikawa‑Yamamoto K, Ohodoi C, Yamagishi H, Zong ZP, Murakami M, Yamaguchi N. Lack of synchrony among multiple nuclei induces partial DNA fragmentation in V79 cells polyploidized by demecolcine. Cell Proliferation 1999; 32:337‑49. 34. Quignon F, Rozier L, Lachages AM, Bieth A, Simili M, Debatisse M. Sustained mitotic block elicits DNA breaks: One‑step alteration of ploidy and chromosome integrity in mammalian cells. Oncogene 2006; 26:165‑72.

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