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Toru Hirota

Publications and source records attributed to Toru Hirota.

At least 19 recordsLinked to original sources
PubMed2026

Plasticity of extrachromosomal DNA segregation during drug adaptation.

Uneven segregation during mitosis is a striking feature of extrachromosomal DNA (ecDNA). Because ecDNA lacks a centromere, it is thought to segregate stochastically, generating intratumoral heterogeneity in genomic copy number. Drug treatment can readily change ecDNA copy number, enabling cells to acquire drug resistance, yet whether these changes reflect static selection of pre-existing clones or active reconfiguration under stress remains unresolved. To address this, we develop a high-throughput framework combining single-cell DNA sequencing with cellular barcoding for clonal tracking. Single-cell cloning reveals that not all clones exhibit identical segregation modes even under drug-free conditions. Under treatment, resistant populations do not simply arise from pre-existing clones with favorable ecDNA states; instead, some clones actively reconfigure their segregation behavior to generate resistant cells. Thus, although ecDNA generally segregates stochastically, it can undergo nonrandom, actively regulated segregation under drug stress, raising the possibility of therapeutically targeting ecDNA segregation mechanisms to counteract adaptive resistance.

Extrachromosomal DNA↗
PubMed2006

Condensin I stabilizes chromosomes mechanically through a dynamic interaction in live cells.

BACKGROUND: Restructuring chromatin into morphologically distinct chromosomes is essential for cell division, but the molecular mechanisms underlying this process are poorly understood. Condensin complexes have been proposed as key factors, although controversial conclusions about their contribution to chromosome structure were reached by different experimental approaches in fixed cells or cell extracts. Their function under physiological conditions still needs to be defined. RESULTS: Here, we investigated the specific functions of condensin I and II in live cells by fluorescence microscopy and RNAi depletion. Photobleaching and quantitative time-lapse imaging showed that GFP-tagged condensin II bound stably to chromosomes throughout mitosis. By contrast, the canonical condensin I interacted dynamically with chromatin after completion of prophase compaction, reaching steady-state levels on chromosomes before congression. In condensin I-depleted cells, compaction was normal, but chromosomes were mechanically labile and unable to withstand spindle forces during alignment. However, normal levels of condensin II were not required for chromosome stability. CONCLUSIONS: We conclude that while condensin I seems dispensable for normal chromosome compaction, its dynamic binding after nuclear envelope breakdown locks already condensed chromatin in a rigid state required for mechanically stable spindle attachment.

Adenosine Triphosphatases↗
PubMed2005

Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin.

Histones are subject to numerous post-translational modifications. Some of these 'epigenetic' marks recruit proteins that modulate chromatin structure. For example, heterochromatin protein 1 (HP1) binds to histone H3 when its lysine 9 residue has been tri-methylated by the methyltransferase Suv39h (refs 2-6). During mitosis, H3 is also phosphorylated by the kinase Aurora B. Although H3 phosphorylation is a hallmark of mitosis, its function remains mysterious. It has been proposed that histone phosphorylation controls the binding of proteins to chromatin, but any such mechanisms are unknown. Here we show that antibodies against mitotic chromosomal antigens that are associated with human autoimmune diseases specifically recognize H3 molecules that are modified by both tri-methylation of lysine 9 and phosphorylation of serine 10 (H3K9me3S10ph). The generation of H3K9me3S10ph depends on Suv39h and Aurora B, and occurs at pericentric heterochromatin during mitosis in different eukaryotes. Most HP1 typically dissociates from chromosomes during mitosis, but if phosphorylation of H3 serine 10 is inhibited, HP1 remains chromosome-bound throughout mitosis. H3 phosphorylation by Aurora B is therefore part of a 'methyl/phos switch' mechanism that displaces HP1 and perhaps other proteins from mitotic heterochromatin.

Amino Acid Sequence↗
PubMed2005

The tumor suppressor WARTS activates the Omi / HtrA2-dependent pathway of cell death.

Drosophila tumor suppressor WARTS (Wts) is an evolutionally conserved serine / threonine kinase and participates in a signaling complex that regulates both proliferation and apoptosis to ensure the proper size and shape of the fly. Human counterparts of this complex have been found to be frequently downregulated or mutated in cancers. WARTS, a human homolog of Wts, is also known as tumor suppressor and mitotic regulator, but its molecular implications in tumorigenesis are still obscure. Here, we show that WARTS binds via its C-terminus to the PDZ domain of a proapoptotic serine protease Omi / HtrA2. Depletion of WARTS inhibited Omi / HtrA2-mediated cell death, whereas overexpression of WARTS promoted this process. Furthermore, WARTS can enhance the protease activity of Omi / HtrA2 both in vivo and in vitro. Activation of Omi / HtrA2-mediated cell death is thus a potential mechanism for the tumor suppressive activity of WARTS.

Apoptosis↗
PubMed2005

Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells.

Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's alpha kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its alpha kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila MEI-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its alpha kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. Our results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin.

Amino Acid Sequence↗
PubMed2005

Over-expression of Aurora-A targets cytoplasmic polyadenylation element binding protein and promotes mRNA polyadenylation of Cdk1 and cyclin B1.

Aurora-A is a centrosomal serine-threonine kinase that regulates mitosis. Over-expression of Aurora-A has been found in a wide range of tumors and has been implicated in oncogenic transformation. However, how Aurora-A over-expression contributes to promotion of carcinogenesis remains elusive. Immunohistochemical analysis of breast tumors revealed that over-expressed Aurora-A is not restricted to the centrosomes but is also found in the cytoplasm. This over-expressed Aurora-A appeared to be phosphorylated on Thr288, which is known to be required for its enzymatic activation. In analogy to Aurora-A's role in oocyte maturation and the early embryonic cell cycle, here we investigated whether ectopically over-expressed Aurora-A can similarly stimulate polyadenylation of mRNA in human somatic cultured cells by interacting with a human ortholog of cytoplasmic polyadenylation element binding protein, h-CPEB. In vitro experiments revealed that Aurora-A binds directly to, and phosphorylates, h-CPEB. We found that polyadenylation of mRNA tails of cyclin B1 and Cdk1 was synergistically stimulated when Aurora-A and h-CPEB were over-expressed, and they were further promoted in the presence of an Aurora-A activator Ajuba. Our results suggest a function of ectopically over-expressed Aurora-A that might be relevant for carcinogenesis.

Amino Acid Sequence↗
PubMed2004

Distinct functions of condensin I and II in mitotic chromosome assembly.

Condensin is a protein complex associated with mitotic chromosomes that has been implicated in chromosome condensation. In vertebrates, two types of condensin complexes have recently been identified, called condensin I and II. Here, we show that in mammalian cells condensin II associates with chromatin in prophase, in contrast to condensin I which is cytoplasmic and can thus interact with chromosomes only after nuclear envelope breakdown. RNA interference experiments in conjunction with imaging of live and fixed cells revealed that condensin II is required for chromosome condensation in early prophase, whereas condensin I appears to be dispensable at this stage. By contrast, condensin I is required for the complete dissociation of cohesin from chromosome arms, for chromosome shortening and for normal timing of progression through prometaphase and metaphase, whereas normal condensin II levels are dispensable for these processes. After depletion of both condensin complexes, the onset of chromosome condensation is delayed until the end of prophase, but is then initiated rapidly before nuclear envelope breakdown. These results reveal that condensin II and I associate with chromosomes sequentially and have distinct functions in mitotic chromosome assembly.

Cell Cycle Proteins↗
PubMed2004

Cre-loxP-controlled periodic Aurora-A overexpression induces mitotic abnormalities and hyperplasia in mammary glands of mouse models.

Aurora-A, a serine/threonine mitotic kinase, was reported to be overexpressed in various human cancers, and its overexpression induces aneuploidy, centrosome amplification and tumorigenic transformation in cultured human and rodent cells. However, the underlying mechanisms and pathological settings by which Aurora-A promotes tumorigenesis are largely unknown. Here, we created a transgenic mouse model to investigate the involvement of Aurora-A overexpression in the development of mammary glands and tumorigenesis using a Cre-loxP system. The conditional expression of Aurora-A resulted in significantly increased binucleated cell formation and apoptosis in the mammary epithelium. The surviving mammary epithelial cells composed hyperplastic areas after a short latency. Induction of Aurora-A overexpression in mouse embryonic fibroblasts prepared from the transgenic mice also led to aberrant mitosis and binucleated cell formation followed by apoptosis. The levels of p53 protein were remarkably increased in these Aurora-A-overexpressing cells, and the apoptosis was significantly suppressed by deletion of p53. Given that no malignant tumor formation was found in the Aurora-A-overexpressing mouse model after a long latency, additional factors, such as p53 inactivation, are required for the tumorigenesis of Aurora-A-overexpressing mammary epithelium. Our findings indicated that this mouse model is a useful system to study the physiological roles of Aurora-A and the genetic pathways of Aurora-A-induced carcinogenesis.

Animals↗
PubMed2004

Roles of polo-like kinase 1 in the assembly of functional mitotic spindles.

BACKGROUND: The stable association of chromosomes with both poles of the mitotic spindle (biorientation) depends on spindle pulling forces. These forces create tension across sister kinetochores and are thought to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint. Polo-like kinase 1 (Plk1) has been implicated in regulating centrosome maturation, mitotic entry, sister chromatid cohesion, the anaphase-promoting complex/cyclosome (APC/C), and cytokinesis, but it is unknown if Plk1 controls chromosome biorientation. RESULTS: We have analyzed Plk1 functions in synchronized mammalian cells by RNA interference (RNAi). Plk1-depleted cells enter mitosis after a short delay, accumulate in a preanaphase state, and subsequently often die by apoptosis. Spindles in Plk1-depleted cells lack focused poles and are not associated with centrosomes. Chromosomes attach to these spindles, but the checkpoint proteins Mad2, BubR1, and CENP-E are enriched at many kinetochores. When Plk1-depleted cells are treated with the Aurora B inhibitor Hesperadin, which silences the spindle checkpoint by stabilizing microtubule-kinetochore interactions, cells degrade APC/C substrates and exit mitosis without chromosome segregation and cytokinesis. Experiments with monopolar spindles that are induced by the kinesin inhibitor Monastrol indicate that Plk1 is required for the assembly of spindles that are able to generate poleward pulling forces. CONCLUSIONS: Our results imply that Plk1 is not essential for mitotic entry and APC/C activation but is required for proper spindle assembly and function. In Plk1-depleted cells spindles may not be able to create enough tension across sister kinetochores to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2004

Spindle checkpoint function is required for mitotic catastrophe induced by DNA-damaging agents.

Mitotic catastrophe is an important mechanism for the induction of cell death in cancer cells by antineoplastic agents that damage DNA. This process is facilitated by defects in the G1 and G2 checkpoints of the cell cycle that are apparent in most cancer cells and which allow the cells to enter mitosis with DNA damage. We have now characterized the dynamics of mitotic catastrophe induced by DNA-damaging agents in p53-deficient cancer cells. Cells that entered mitosis with DNA damage transiently arrested at metaphase for more than 10 h without segregation of chromosomes and subsequently died directly from metaphase. In those metaphase arrested precatastrophic cells, anaphase-promoting complex appeared to be inactivated and BubR1 was persistently localized at kinetochores, suggesting that spindle checkpoint is activated after the DNA damage. Furthermore, suppression of spindle checkpoint function by BubR1 or Mad2 RNA interference in the DNA damaged cells led to escape from catastrophic death and to subsequent abnormal mitosis. Dysfunction of the spindle checkpoint in p53-deficient cancer cells is thus likely a critical factor in resistance to DNA-damaging therapeutic agents.

Base Sequence↗
PubMed2004

Regulation of sister chromatid cohesion between chromosome arms.

Sister chromatid separation in anaphase depends on the removal of cohesin complexes from chromosomes. In vertebrates, the bulk of cohesin is already removed from chromosome arms during prophase and prometaphase, whereas cohesin remains at centromeres until metaphase, when cohesin is cleaved by the protease separase. In unperturbed mitoses, arm cohesion nevertheless persists throughout metaphase and is principally sufficient to maintain sister chromatid cohesion. How arm cohesion is maintained until metaphase is unknown. Here we show that small amounts of cohesin can be detected in the interchromatid region of metaphase chromosome arms. If prometaphase is prolonged by treatment of cells with microtubule poisons, these cohesin complexes dissociate from chromosome arms, and arm cohesion is dissolved. If cohesin dissociation in prometaphase-arrested cells is prevented by depletion of Plk1 or inhibition of Aurora B, arm cohesion is maintained. These observations imply that, in unperturbed mitoses, small amounts of cohesin maintain arm cohesion until metaphase. When cells lacking Plk1 and Aurora B activity enter anaphase, chromatids lose cohesin. This loss is prevented by proteasome inhibitors, implying that it depends on separase activation. Separase may therefore be able to cleave cohesin at centromeres and on chromosome arms.

Animals↗
PubMed2004

Tumor suppressor WARTS ensures genomic integrity by regulating both mitotic progression and G1 tetraploidy checkpoint function.

Defects in chromosomes or mitotic spindles activate the spindle checkpoint, resulting in cell cycle arrest at prometaphase. The prolonged activation of spindle checkpoint generally leads to mitotic exit without segregation after a transient mitotic arrest and the consequent formation of tetraploid G(1) cells. These tetraploid cells are usually blocked to enter the subsequent S phase by the activation of p53/pRb pathway, which is referred to as the G(1) tetraploidy checkpoint. A human homologue of the Drosophila warts tumor suppressor, WARTS, is an evolutionarily conserved serine-threonine kinase and implicated in development of human tumors. We previously showed that WARTS plays a crucial role in controlling mitotic progression by forming a regulatory complex with zyxin, a regulator of actin filament assembly, on mitotic apparatus. However, when WARTS is activated during cell cycle and how the loss of WARTS function leads to tumorigenesis have not been elucidated. Here we show that WARTS is activated during mitosis in mammalian cells, and that overexpression of a kinase-inactive WARTS in Rat1 fibroblasts significantly induced mitotic delay. This delay resulted from prolonged activation of the spindle assembly checkpoint and was frequently followed by mitotic slippage and the development of tetraploidy. The resulting tetraploid cells then abrogated the G(1) tetraploidy checkpoint and entered S phase to achieve a DNA content of 8N. This impairment of G(1) tetraploidy checkpoint was caused as a consequence of failure to induce p53 expression by expressing a kinase-inactive WARTS. WARTS thus plays a critical role in maintenance of ploidy through its actions in both mitotic progression and the G(1) tetraploidy checkpoint.

Actins↗
PubMed2004

Down-regulation of nuclear protein ICBP90 by p53/p21Cip1/WAF1-dependent DNA-damage checkpoint signals contributes to cell cycle arrest at G1/S transition.

Checkpoints, which monitor DNA damage and regulate cell cycle progression, ensure genomic integrity and prevent the propagation of transformed cells. DNA damage activates the p53-dependent checkpoint pathway that induces expression of p21Cip1/WAF1, resulting in cell cycle arrest at G1/S transition by inhibition of cdk activity and DNA replication. ICBP90 was identified as a nuclear protein that binds to the TopoII alpha gene promoter and is speculated to be involved in DNA replication. ICBP90 expression is cell cycle regulated in normal cells but stably high throughout cell cycle in various cancer cell lines. We here demonstrate that ICBP90 expression is down-regulated by the p53/p21Cip1/WAF1-dependent DNA damage checkpoint signals. The reduction of ICBP90 appeared to be caused by both transcriptional suppression and protein degradation. Adenoviral expression of p21Cip1/WAF1 directly led to ICBP90 reduction in p53-/- HCT116 cells without DNA damage. Furthermore, ICPB90 depletion by RNA interference significantly blocked G1/S transition after DNA damage in HeLa cells. The down-regulation of ICBP90 is an important mechanism for cell cycle arrest at G1/S transition, which is induced by the activation of a p53/p21Cip1/WAF1-dependent DNA-damage checkpoint. Deregulation of ICBP90 may impair the control of G1/S transition during checkpoint activation and lead to genomic instability.

Amanitins↗
PubMed2003

Activation of m-calpain is required for chromosome alignment on the metaphase plate during mitosis.

Calpains form a superfamily of Ca(2+)-dependent intracellular cysteine proteases with various isoforms. Two isoforms, micro- and m-calpains, are ubiquitously expressed and known as conventional calpains. It has been previously shown that the mammalian calpains are activated during mitosis by transient increases in cytosolic Ca(2+) concentration. However, it is still unknown whether the activation of calpains contributes to particular events in mitosis. With the use of RNA interference (RNAi), we investigated the roles of calpains in mitosis. Cells reduced the levels of m-calpain, but not mu-calpain, arrested at prometaphase and failed to align their chromosomes at the spindle equator. Specific peptidyl calpain inhibitors also induced aberrant mitosis with chromosome misalignment. Although both m-calpain RNAi and calpain inhibitors affected neither the separation of centrosomes nor the assembly of bipolar spindles, Mad2 was detected on the kinetochores of the misaligned chromosomes, indicating that the prometaphase arrest induced by calpain inhibition is due to activation of the spindle assembly checkpoint. Furthermore, when calpain activity was inhibited in cells having monopolar spindles, chromosomes were clustered adjacent to the centrosome, suggesting that calpain activity is involved in a polar ejection force for metaphase alignment of chromosomes. Based on these findings, we propose that activation of m-calpain during mitosis is required for cells to establish the chromosome alignment by regulating some molecules that generate polar ejection force.

Calcium↗
PubMed2003

Identification of a subunit of a novel Kleisin-beta/SMC complex as a potential substrate of protein phosphatase 2A.

Protein phosphatase 2A (PP2A) holoenzymes consist of a catalytic C subunit, a scaffolding A subunit, and one of several regulatory B subunits that recruit the AC dimer to substrates. PP2A is required for chromosome segregation, but PP2A's substrates in this process remain unknown. To identify PP2A substrates, we carried out a two-hybrid screen with the regulatory B/PR55 subunit. We isolated a human homolog of C. elegans HCP6, a protein distantly related to the condensin subunit hCAP-D2, and we named this homolog hHCP-6. Both C. elegans HCP-6 and condensin are required for chromosome organization and segregation. HCP-6 binding partners are unknown, whereas condensin is composed of the structural maintenance of chromosomes proteins SMC2 and SMC4 and of three non-SMC subunits. Here we show that hHCP-6 becomes phosphorylated during mitosis and that its dephosphorylation by PP2A in vitro depends on B/PR55, suggesting that hHCP-6 is a B/PR55-specific substrate of PP2A. Unlike condensin, hHCP-6 is localized in the nucleus in interphase, but similar to condensin, hHCP-6 associates with chromosomes during mitosis. hHCP-6 is part of a complex that contains SMC2, SMC4, kleisin-beta, and the previously uncharacterized HEAT repeat protein FLJ20311. hHCP-6 is therefore part of a condensin-related complex that associates with chromosomes in mitosis and may be regulated by PP2A.

Adenosine Triphosphatases↗
PubMed2003

Aurora-A kinase maintains the fidelity of early and late mitotic events in HeLa cells.

Aurora-A, a member of the Aurora/Ipl1-related kinase family, is overexpressed in various types of cancer and considered to play critical roles in tumorigenesis. To better understand the pathological effect of Aurora-A activation, it is first necessary to elucidate the physiological functions of Aurora-A. Here, we have investigated the roles of Aurora-A in mitotic progression with the small interfering RNA, antibody microinjection, and time lapse microscopy using human cells. We demonstrated that suppression of Aurora-A by small interfering RNA caused multiple events to fail in mitosis, such as incorrect separation of centriole pairs, misalignment of chromosomes on the metaphase plate, and incomplete cytokinesis. Antibody microinjection of Aurora-A into late G2 cells induced dose-dependent failure in separation of centriole pairs at prophase, indicating that Aurora-A is essential for proper separation of centriole pairs. When we injected anti-Aurora-A antibodies into prometaphase cells that had separated their centriole pairs, chromosomes were severely misaligned on the metaphase plate, indicating that Aurora-A is required for proper movement of chromosomes on the metaphase plate. Furthermore, inhibition of Aurora-A at metaphase by microinjected antibodies prevented cells from completing cytokinesis, suggesting that Aurora-A also has important functions in late mitosis. These results strongly suggest that Aurora-A is essential for many crucial events during mitosis and that the phosphorylation of a series of substrates by Aurora-A at different stages of mitosis may promote diverse critical events in mitosis to maintain chromosome integrity in human cells.

Antibodies↗
PubMed2003

Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells.

Aurora family kinases contribute to regulation of mitosis. Using RNA interference in synchronized HeLa cells, we now show that Aurora-A is required for mitotic entry. We found that initial activation of Aurora-A in late G2 phase of the cell cycle is essential for recruitment of the cyclin B1-Cdk1 complex to centrosomes, where it becomes activated and commits cells to mitosis. A two-hybrid screen identified the LIM protein Ajuba as an Aurora-A binding protein. Ajuba and Aurora-A interact in mitotic cells and become phosphorylated as they do so. In vitro analyses revealed that Ajuba induces the autophosphorylation and consequent activation of Aurora-A. Depletion of Ajuba prevented activation of Aurora-A at centrosomes in late G2 phase and inhibited mitotic entry. Overall, our data suggest that Ajuba is an essential activator of Aurora-A in mitotic commitment.

Animals↗