ATM is activated by mitotic stress and suppresses centrosome amplification in primary but not in tumor cells

Centrosome amplification has been proposed to contribute to the development of aneuploidy and genome instability. Here, we show that Ataxia-Telangiectasia Mutated (ATM) is localized to the centrosome and co-purified with gamma-tubulin. The importance of ATM in centrosome duplication is demonstrated in Atm-deficient primary mouse embryonic fibroblasts that display centrosome amplification. Interestingly, centrosome amplification was not observed in tumor cell lines derived from Atm and p21 double deficient mouse. Our results also indicate that both p53 and p21 operate in the same pathway as ATM in regulating centrosome biogenesis. Finally, a potential role of ATM in spindle checkpoint regulation is demonstrated by which ATM protein is activated by mitotic stress. These results suggest a role of ATM in spindle checkpoint regulation and indicate that ATM suppresses genome instability and cellular transformation by regulating centrosome biogenesis.     

Targeted inhibition of Polo‐like kinase 1 by a novel small‐molecule inhibitor induces mitotic catastrophe and apoptosis in human bladder cancer cells

Bladder cancer is a common cancer with particularly high recurrence after transurethral resection. Despite improvements in neoadjuvant chemotherapy, the outcome of patients with advanced bladder cancer has changed very little. In this study, the anti‐tumour activities of a novel Polo‐like kinase 1 (PLK1) inhibitor (RO3280) was evaluated in vitro and in vivo in the bladder carcinoma cell lines 5637 and T24. MTT assays, colony‐formation assays, flow cytometry, cell morphological analysis and trypan blue exclusion assays were used to examine the proliferation, cell cycle distribution and apoptosis of bladder carcinoma cells with or without RO3280 treatment. Moreover, real‐time RT‐PCR and Western blotting were used to detect the expressions of genes that are related to these cellular processes. Our results showed that RO3280 inhibited cell growth and cell cycle progression, increased Wee1 expression and cell division cycle protein 2 phosphorylation. In addition, RO3280 induced mitotic catastrophe and apoptosis, increased cleaved PARP (poly ADP‐ribose polymerase) and caspase‐3, and decreased BubR1 expression. The in vivo assay revealed that RO3280 retarded bladder cancer xenograft growth in a nude mouse model. Although further laboratory and pre‐clinical investigations are needed to corroborate these data, our demonstration of bladder cancer growth inhibition and dissemination using a pharmacological inhibitor of PLK1 provides new opportunities for future therapeutic intervention.     

Molecular characterization of apoptosis induced by CARF silencing in human cancer cells

Collaborator of ARF (CARF) was cloned as an ARF-interacting protein and shown to regulate the p53-p21(WAF1)-HDM2 pathway, which is central to tumor suppression via senescence and apoptosis. We had previously reported that CARF inhibition in cancer cells led to polyploidy and caspase-dependent apoptosis, however, the mechanisms governing this phenomenon remained unknown. Thus, we examined various cell death and survival pathways including the mitochondrial stress, ataxia telangiectasia mutated (ATM)-ATR, Ras-MAP kinase and retinoblastoma cascades. We found that CARF is a pleiotropic regulator with widespread effects; its suppression affected all investigated pathways. Most remarkably, it protected the cells against genotoxicity; CARF knockdown elicited DNA damage response as evidenced by increased levels of phosphorylated ATM and γH2AX, leading to induction of mitotic arrest and eventual apoptosis. We also show that the CARF-silencing-induced apoptosis in vitro translates to in vivo. In a human tumor xenograft mouse model, treatment of developing tumors with short hairpin RNA (shRNA) against CARF via an adenovirus carrier induced complete suppression of tumor growth, suggesting that CARF shRNA is a strong candidate for an anticancer reagent. We demonstrate that CARF has a vital role in genome preservation and tumor suppression and CARF siRNA is an effective novel cancer therapeutic agent.     

Gamma-actin is involved in regulating centrosome function and mitotic progression in cancer cells

Reorganization of the actin cytoskeleton during mitosis is crucial for regulating cell division. A functional role for γ-actin in mitotic arrest induced by the microtubule-targeted agent, paclitaxel, has recently been demonstrated. We hypothesized that γ-actin plays a role in mitosis. Herein, we investigated the effect of γ-actin in mitosis and demonstrated that γ-actin is important in the distribution of β-actin and formation of actin-rich retraction fibers during mitosis. The reduced ability of paclitaxel to induce mitotic arrest as a result of γ-actin depletion was replicated with a range of mitotic inhibitors, suggesting that γ-actin loss reduces the ability of broad classes of anti-mitotic agents to induce mitotic arrest. In addition, partial depletion of γ-actin enhanced centrosome amplification in cancer cells and caused a significant delay in prometaphase/metaphase. This prolonged prometaphase/metaphase arrest was due to mitotic defects such as uncongressed and missegregated chromosomes, and correlated with an increased presence of mitotic spindle abnormalities in the γ-actin depleted cells. Collectively, these results demonstrate a previously unknown role for γ-actin in regulating centrosome function, chromosome alignment and maintenance of mitotic spindle integrity.     

Radiation-induced centrosome overduplication and multiple mitotic spindles in human tumor cells

The centrosome is a highly regulated organelle and its proper duplication is indispensable for the formation of bipolar mitotic spindles and balanced chromosome segregation. To elucidate a possible linkage between centrosome duplication and radiation-induced nuclear damage, we examined centrosome dynamics in U2-OS osteosarcoma cells following gamma-irradiation. Nearly all control cells contained one or two centrosomes, and at mitosis more than 97% of the cells displayed typical bipolar spindles. In contrast, over 20% of cells at 48 h after 10 Gy gamma-irradiation contained more than two centrosomes, and 60% of the mitotic cells showed aberrant spindles organized by multiple poles. Remarkably, the cells with multiple centrosomes frequently exhibited changes in size and/or morphology of the nucleus, including micronuclei formation. We conclude that abnormal centrosome duplication could be one of the key events involved in nuclear fragmentation and perhaps even cell death following irradiation.     

Caspase-3 is not activated in seizure-induced neuronal necrosis with internucleosomal DNA cleavage

A caspase-3-activated DNase produces internucleosomal DNA cleavage (DNA laddering). We determined whether caspase-3 is activated by lithium-pilocarpine-induced status epilepticus in six brain regions with necrosis-induced DNA laddering. The thymuses of adult rats given methamphetamine or normal saline were used as controls for apoptosis. Some 6-8 h after methamphetamine treatment, thymocytes showed apoptosis by electron-microscopic examination, positive terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), DNA laddering, cleavage of caspase-3 into its active p17 subunit, active caspase-3 immunoreactivity, and a 25-fold increase in caspase-3-like activity. Six hours after SE, necrotic neurons by electron-microscopic examination in hippocampus, amygdala and piriform, entorhinal and frontal cortices showed no TUNEL and no DNA laddering. Twenty-four hours after seizures, most necrotic neurons were negative for TUNEL, some were positive, but all regions showed DNA laddering. However, 6 and 24 h after seizures, active caspase-3 immunoreactivity was negative, caspase-3-like activity did not increase, and western blot analysis failed to show the p17 subunit. In addition, 24 h after seizures,microdialytic perfusion of carbobenzoxy-valyl-alanyl-aspartyl (O-methylester) fluoromethylketone was not neuroprotective. Thus, caspase-3 is not activated in brain regions with seizure-induced neuronal necrosis with DNA laddering. Either caspase-activated DNase is activated by another enzyme, or a caspase-independent DNase is responsible for the DNA cleavage.     

Overcoming resistance to mitochondrial apoptosis by BZML‐induced mitotic catastrophe is enhanced by inhibition of autophagy in A549/Taxol cells

Objectives

Our previous in vitro study showed that 5‐(3, 4, 5‐trimethoxybenzoyl)‐4‐methyl‐2‐(p‐tolyl) imidazol (BZML) is a novel colchicine binding site inhibitor with potent anti‐cancer activity against apoptosis resistance in A549/Taxol cells through mitotic catastrophe (MC). However, the mechanisms underlying apoptosis resistance in A549/Taxol cells remain unknown. To clarify these mechanisms, in the present study, we investigated the molecular mechanisms of apoptosis and autophagy, which are closely associated with MC in BZML‐treated A549 and A549/Taxol cells.

Methods

Xenograft NSCLC models induced by A549 and A549/Taxol cells were used to evaluate the efficacy of BZML in vivo. The activation of the mitochondrial apoptotic pathway was assessed using JC‐1 staining, Annexin V‐FITC/PI double‐staining, a caspase‐9 fluorescence metric assay kit and western blot. The different functional forms of autophagy were distinguished by determining the impact of autophagy inhibition on drug sensitivity.

Results

Our data showed that BZML also exhibited desirable anti‐cancer activity against drug‐resistant NSCLC in vivo. Moreover, BZML caused ROS generation and MMP loss followed by the release of cytochrome c from mitochondria to cytosol in both A549 and A549/Taxol cells. However, the ROS‐mediated apoptotic pathway involving the mitochondria that is induced by BZML was only fully activated in A549 cells but not in A549/Taxol cells. Importantly, we found that autophagy acted as a non‐protective type of autophagy during BZML‐induced apoptosis in A549 cells, whereas it acted as a type of cytoprotective autophagy against BZML‐induced MC in A549/Taxol cells.

Conclusions

Our data suggest that the anti‐apoptosis property of A549/Taxol cells originates from a defect in activation of the mitochondrial apoptotic pathway, and autophagy inhibitors can potentiate BZML‐induced MC to overcome resistance to mitochondrial apoptosis.

     

Increased apoptosis of parasympathetic but not enteric neurons in mice lacking GFRalpha2

Enteric neurons, unlike sympathetic and sensory neurons that require target-derived neurotrophins for survival, do not undergo classical caspase-3-mediated programmed cell death (PCD) during normal development. Whether parasympathetic neurons in the pancreas, which originate from a subpopulation of enteric nervous system (ENS) precursors, or other parasympathetic neurons undergo PCD during normal mammalian development is unknown. In GFRalpha2-deficient mice, many submandibular and intrapancreatic parasympathetic neurons are missing but whether this is due to increased neuronal death is unclear. Here we show that activated caspase-3 and PGP9.5 doubly positive neurons are present in wild-type mouse pancreas between embryonic day E15 and birth. Thus, in contrast to ENS neurons, intrapancreatic neurons undergo PCD via apoptosis during normal development. We also show that, in GFRalpha2-deficient mice, most intrapancreatic neurons are lost during this late fetal period, which coincides with a period of increased apoptosis of the neurons. Since the percentage of BrdU and Phox2b doubly positive cells in the fetal pancreas and the number of intrapancreatic neurons at E15 were similar between the genotypes, impaired precursor proliferation and migration are unlikely to contribute to the loss of intrapancreatic neurons in GFRalpha2-KO mice. Caspase-3-positive neurons were also found in GFRalpha2-deficient submandibular ganglia around birth, suggesting that parasympathetic neurons depend on limited supply of (presumably target-derived neurturin) signaling via GFRalpha2 for survival.     

PPARgamma1 synthesis and adipogenesis in C3H10T1/2 cells depends on S-phase progression, but does not require mitotic clonal expansion

Adipogenesis is typically stimulated in mouse embryo fibroblast (MEF) lines by a standard hormonal combination of insulin (I), dexamethasone (D), and methylisobutylxanthine (M), administered with a fresh serum renewal. In C3H10T1/2 (10T1/2) cells, peroxisome proliferator-activated receptor gamma1 (PPARgamma1) expression, an early phase key adipogenic regulator, is optimal after 36 h of IDM stimulation. Although previous studies provide evidence that mitotic clonal expansion of 3T3-L1 cells is essential for adipogenesis, we show, here, that 10T1/2 cells do not require mitotic clonal expansion, but depend on cell cycle progression through S-phase to commit to adipocyte differentiation. Exclusion of two major mitogenic stimuli (DM without insulin and fresh serum renewal) from standard IDM protocol removed mitotic clonal expansion, but sustained equivalent PPARgamma1 synthesis and lipogenesis. Different S-phase inhibitors (aphidicolin, hydroxyurea, l-mimosine, and roscovitin) each arrested cells in S-phase, under hormonal stimulation, and completely blocked PPARgamma1 synthesis and lipogenesis. However, G2/M inhibitors effected G2/M accumulation of IDM stimulated cells and prevented mitosis, but fully sustained PPARgamma1 synthesis and lipogenesis. DM stimulation with or without fresh serum renewal elevated DNA synthesis in a proportion of cells (measured by BrdU labeling) and accumulation of cell cycle progression in G2/M-phase without complete mitosis. By contrast, standard IDM treatments with fresh serum renewal caused elevated DNA synthesis and mitotic clonal expansion while achieved equivalent level of adipogenesis. At most, one-half of the 10T1/2 mixed cell population differentiated to mature adipocytes, even when clonally isolated. PPARgamma was exclusively expressed in the cells that contained lipid droplets. IDM stimulated comparable PPARgamma1 synthesis and lipogenesis in isolated cells at low cell density (LD) culture, but in about half of the cells and with sensitivity to G1/S, but not G2/M inhibitors. Importantly, growth arrest occurred in all differentiating cells, while continuous mitotic clonal expansion occurred in non-differentiating cells. Irrespective of confluence level, 10T1/2 cells differentiate after progression through S-phase, where adipogenic commitment induced by IDM stimulation is a prerequisite for PPARgamma synthesis and subsequent adipocyte differentiation.     

Nek2 localizes to multiple sites in mitotic cells, suggesting its involvement in multiple cellular functions during the cell cycle.

Nek2 is a mammalian protein kinase that is structurally homologous to NIMA, a mitotic regulator in Aspergillus nidulans. To understand the possible cellular processes in which Nek2 participates during the cell cycle, we investigated the expression and subcellular localization of Nek2 in mitotic cells. The Nek2 protein levels were observed to be regulated in a cell cycle stage-specific manner in cultured cells. The cell cycle stage specificity of Nek2 expression was also confirmed in cells undergoing mitosis in vivo. Nek2 proteins were localized in both the nucleus and cytoplasm throughout the cell cycle, but exhibited dynamic changes in distribution, depending on the cell cycle stage. Nek2 was associated with chromosomes from prophase to metaphase and then was dissociated upon entering into anaphase. Nek2 then appeared at the midbody of the cytoplasmic bridge at telophase. Nek2 was also associated with the centrosome throughout the cell cycle as observed previously by others. Additionally, the nuclear localization of Nek2 was increased during S phase. Such dynamic behavior of Nek2 suggests that Nek2 may be a mitotic regulator that is involved in diverse cell cycle events.     

Apoptosis signal-regulating kinase 1 is involved not only in apoptosis but also in non-apoptotic cardiomyocyte death

The molecular basis of myocardial cell death in the ischemia-reperfused heart still remains to be clarified. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays an important role in stress-induced apoptosis. We studied ASK1(-/-) mice to examine the role of ASK1 in ischemia-reperfusion injury. In the wild-type heart, ischemia-reperfusion resulted in necrotic injury, whereas infarct size was drastically reduced in the ASK1(-/-) heart. The necrotic injury was not accompanied with any evidence of apoptosis such as an increase in TUNEL-positive cells, DNA fragmentation or the activation of caspase-3. ASK1(-/-) cardiomyocytes were more resistant to H(2)O(2)- or Ca(2+)-induced apoptotic and non-apoptotic cell death compared with wild-type cells. These data suggest that ASK1 is involved in necrosis as well as apoptosis and that ASK1-dependent necrosis is likely to contribute to myocardial cell death in the ischemia-reperfused heart.     

Induction of apoptosis by Centaurea nerimaniae extract in HeLa and MDA-MB-231 cells by a caspase-3 pathway

We investigated the cytotoxic and apoptotic effects of a methanol extract of Centaurea nerimaniae, a plant endemic in Turkey, on HeLa and MDA-MB-231 cells. Eight concentrations of C. nerimaniae extract were applied to cells, and cytotoxic effects were measured using the xCELLigence system. The TUNEL assay was used to assess apoptotic cell death and immunohistochemistry was used to determine active caspase-3 using the effective cytotoxic doses of the extract. Doses of 1.42 mg/ml C. nerimaniae inhibited the growth of HeLa cells and 3.67 mg/ml C. nerimaniae inhibited the growth of MDA-MB-231 cells in a dose- and time-dependent manner. The apoptotic indexes for HeLa and MDA-MB-231 cells were increased significantly compared to control groups. Immunohistochemistry showed that the number of caspase-3 immunostained cells increased in the extract treatment groups for both HeLa and MDA-MB-231 cells. In the MDA-MB-231 cell line, caspase-3 immunostaining was observed in nuclei and/or cytoplasm in the extract treated group. Caspase-3 activation was greater in HeLa cells than in MDA-MB-231 cells. We found that the extract of C. nerimaniae had a strong antiproliferative effect and induced apoptosis via caspase-3; MDA-MB-231 cancer cells were more resistant than HeLa cells.     

ER stress-induced caspase-12 activation is inhibited by PKC in neuronal cells

Caspase-12 is activated when the cells are exposed to excess levels of various stimuli, which cause endoplasmic reticulum (ER) stress. Protein kinase C (PKC) plays an important role in many signaling pathways in cells, and the activation of PKC has multiple actions in the signaling function of the ER. This study examined whether or not phorbol 12, 13-dibutyrate (PDBu)-induced PKC activation modulates caspase-12 cleavage and its processing, using a wild type caspase-12 overexpressing neuronal cell line, known as Cas-12 cells. The thapsigargin treatment induced caspase-12 fragmentation in the Cas-12 cells. This was inhibited by PKC, which had previously been stimulated by PDBu. The PDBu treatment attenuated the ER stress-induced translocation of caspase-12 from the ER to the cytoplasm. The caspase-3 specific inhibitor blocked caspase-12 fragmentation, and purified caspase-12 was cleaved by the active caspase-3 in vitro, suggesting thatcaspase-12 might be a substrate for caspase-3. In addition, the PDBu treatment influenced the decrease of active caspase-3 fragment. These results suggest that an ER stress induces the activation of caspase-12 via caspase-3, and that PKC regulates both caspase-12 and caspase-3 activations in Cas-12 cells     

Interaction between caspase-3 and caspase-5 in the stretch-induced programmed cell death in the human periodontal ligament cells

In our previous studies, programmed cell death (PCD) was induced in human periodontal ligament (PDL) cells, through activation of caspase-3 and upregulation of CASP5 gene (encoding caspase-5 protein), in response to mechanical stretch loading. The aim of this study is to explore the relationship between the inflammatory caspase, caspase-5, and the apoptotic executioner protein, caspase-3, in human PDL cells. Here, we found that cyclic stretching upregulated the activity and the protein expression level of caspase-3 and -5 and the addition of the caspase-3 inhibitor or caspase-5 inhibitor significantly inhibited the stretch-induced PCD. Meanwhile, the inhibition of caspase-5 inhibited the activation of caspase-3 and vice versa. The result of coimmunoprecipitation also demonstrated that the expression of caspase-3 was immunoprecipitated with caspase-5. Thus, our study revealed that the in vitro application of cyclic stretching induced PCD by activation of caspase-3 and -5 in human PDL cells, and these two caspases could interact with each other after mechanical stretch loading. The study may facilitate further studies on the mechanism of stretch-induced PCD and help us understand the force-related periodontal homeostasis and remodeling better.     

Alpha-lactalbumin unfolding is not sufficient to cause apoptosis, but is required for the conversion to HAMLET (human alpha-lactalbumin made lethal to tumor cells)

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex of human alpha-lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis-like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from alpha-lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of alpha-lactalbumin is sufficient to induce cell death. We used the bovine alpha-lactalbumin Ca(2+) site mutant D87A, which is unable to bind Ca(2+), and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET-like complex in the presence of oleic acid. BAMLET (bovine alpha-lactalbumin made lethal to tumor cells) and D87A-BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca(2+)site, as HAMLET maintained a high affinity for Ca(2+) but D87A-BAMLET was active with no Ca(2+) bound. We conclude that partial unfolding of alpha-lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca(2+)-binding site is not required for conversion of alpha-lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca(2+)site.     

A decrease in intracellular zinc level precedes the detection of early indicators of apoptosis in HL-60 cells

Low extracellular zinc concentrations have been associated with the induction of apoptosis. To assess the relationship between intracellular zinc concentration and the rate of apoptosis, cells were grown in media containing 0.5, 25, or 50 M zinc and analyzed by flow cytometry or fluorescence microscopy. Cells grown in 0.5 M zinc medium over 48 h showed a successive decrease in intracellular zinc concentration measured by the zinc-specific fluorophore, zinquin. After 18 h in 0.5 M zinc medium, rhodamine 123 retention decreased. However, the addition of 10 M zinc to the 0.5 M medium before 16 h in culture restored rhodamine retention in the cells. After 30 h there was an increase in the number of cells cultured in 0.5 M zinc medium that bound annexin V-FITC. These data indicated that decreased intracellular zinc concentration preceded early markers of apoptosis, with alterations in mitochondrial transmembrane potential preceding the loss of polarity in the cell membrane.     

Sequential induction of mitotic catastrophe followed by apoptosis in human leukemia MOLT4 cells by imidazoacridinone C-1311

Imidazoacridinone C-1311 is a DNA-targeting antitumor intercalator/alkylator currently undergoing Phase II clinical trials. Here, we elucidated the sequence of death responses to C-1311 in human leukemia MOLT4 cells using drug concentration (30 nM) that causes near complete cell growth inhibition at 48 h. Early (6-12 h) responses included transient accumulation of cells at the G2/M border followed by also transient rise in several mitotic markers. Mitotic attempts were largely abnormal, resulting in numerous multinucleated cells (peaking at 24-39 h and declining markedly at later times). These events, indicative of mitotic catastrophe, were not associated with immediate cell death. The fraction of necrotic cells did not exceed 3%. Also, the classical manifestations of apoptosis were marginal at 24 h and their progression clearly followed the decline in the fraction of mitotic and multinucleated cells. Quantification of several apoptotic markers (including phosphatidylserine externalization, apoptotic DNA breaks, mitochondrial dysfunction, caspase activation, and cell membrane integrity) showed a considerable progression and the shift from early to late apoptosis at later times. At 72 h, >80% of cells were apoptotic. Collectively, these findings show that C-1311-induced mitotic catastrophe is not the ultimate death event but rather a step precipitating delayed, albeit massive, apoptotic responses.     

The novel actin/focal adhesion-associated protein MISP is involved in mitotic spindle positioning in human cells

Accurate mitotic spindle positioning is essential for the regulation of cell fate choices, cell size and cell position within tissues. The most prominent model of spindle positioning involves a cortical pulling mechanism, where the minus end-directed microtubule motor protein dynein is attached to the cell cortex and exerts pulling forces on the plus ends of astral microtubules that reach the cortex. In nonpolarized cultured cells integrin-dependent, retraction fiber-mediated cell adhesion is involved in spindle orientation. Proteins serving as intermediaries between cortical actin or retraction fibers and astral microtubules remain largely unknown. In a recent genome-wide RNAi screen we identified a previously uncharacterized protein, MISP (C19ORF21) as being involved in centrosome clustering, a process leading to the clustering of supernumerary centrosomes in cancer cells into a bipolar mitotic spindle array by microtubule tension. Here, we show that MISP is associated with the actin cytoskeleton and focal adhesions and is expressed only in adherent cell types. During mitosis MISP is phosphorylated by Cdk1 and localizes to retraction fibers. MISP interacts with the +TIP EB1 and p150^glued, a subunit of the dynein/dynactin complex. Depletion of MISP causes mitotic arrest with reduced tension across sister kinetochores, chromosome misalignment and spindle multipolarity in cancer cells with supernumerary centrosomes. Analysis of spindle orientation revealed that MISP depletion causes randomization of mitotic spindle positioning relative to cell axes and cell center. Together, we propose that MISP links microtubules to the actin cytoskeleton and focal adhesions in order to properly position the mitotic spindle.     

Functional analysis of phosphorylation of the mitotic centromere-associated kinesin by Aurora B kinase in human tumor cells

Mitotic centromere-associated kinesin (MCAK) is the best characterized member of the kinesin-13 family and plays important roles in microtubule dynamics during mitosis. Its activity and subcellular localization is tightly regulated by an orchestra of mitotic kinases, such as Aurora B. It is well known that serine 196 of MCAK is the major phosphorylation site of Aurora B in Xenopus leavis extracts and that this phosphorylation regulates its catalytic activity and subcellular localization. In the current study, we have addressed the conserved phosphorylation site serine 192 in human MCAK to characterize its function in more depth in human cancer cells. Our data confirm that S192 is the major phosphorylation site of Aurora B in human MCAK and that this phosphorylation has crucial roles in regulating its catalytic activity and localization at the kinetochore/centromere region in mitosis. Interfering with this phosphorylation leads to a delayed progression through prometa- and metaphase associated with mitotic defects in chromosome alignment and segregation. We show further that MCAK is involved in directional migration and invasion of tumor cells, and interestingly, interference with the S192 phosphorylation affects this capability of MCAK. These data provide the first molecular explanation for clinical observation, where an overexpression of MCAK was associated with lymphatic invasion and lymph node metastasis in gastric and colorectal cancer patients.