Masayuki Nitta

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.

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.

Dependence of Paclitaxel Sensitivity on a Functional Spindle Assembly Checkpoint

Paclitaxel stabilizes microtubules, causing mitotic arrest and activating the spindle assembly checkpoint. We determined whether suppression of the checkpoint genes Mad2 and BubR1 affects paclitaxel resistance and whether overexpression of Mad2 protein in checkpoint-defective cells enhances paclitaxel sensitivity. Suppression of Mad2 and BubR1 in paclitaxel-treated cancer cells abolished checkpoint function, resulting in paclitaxel resistance that correlated with suppression of cyclin-dependent kinase-1 activity. In contrast, overexpression of Mad2 in cells with a checkpoint defect attributable to low Mad2 expression restored checkpoint function, resulting in enhanced paclitaxel sensitivity that correlated with enhanced cyclin-dependent kinase-1 activity. However, overexpression of Mad2 failed to enhance paclitaxel sensitivity via checkpoint activation in Mad2-independent checkpoint-defective and -intact cells. Thus, checkpoint function is required for paclitaxel sensitivity. These findings show that any molecules that could interfere with the spindle assembly checkpoint could generate paclitaxel resistance in any patient.

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.

Transcriptional blockade induces p53-dependent apoptosis associated with translocation of p53 to mitochondria

The tumor suppressor p53 functions as a transcriptional activator to induce cell cycle arrest and apoptosis in response to DNA damage. Although p53 was also shown to mediate apoptosis in a manner independent of its transactivation activity, the mechanism and conditions that trigger such cell death have remained largely unknown. We have now shown that inhibition of RNA polymerase II-mediated transcription by α-amanitin or RNA interference induced p53-dependent apoptosis. Inhibition of pol II-mediated transcription resulted in down-regulation of p21Cip1, which was caused by both transcriptional suppression and protein degradation, despite eliciting p53 accumulation, allowing the cells to progress into S phase and then to undergo apoptosis. This cell death did not require the transcription of p53 target genes and was preceded by translocation of the accumulated p53 to mitochondria. Our data thus suggested that blockade of pol II-mediated transcription induced p53 accumulation in mitochondria and was the critical factor for eliciting p53-dependent but transcription-independent apoptosis.

A novel human nucleoside diphosphate (NDP) kinase, Nm23-H6, localizes in mitochondria and affects cytokinesis

Nucleoside diphosphate kinases (NDP kinases) are enzymes known to be conserved throughout evolution and have been shown to be involved in various biological events, in addition to the “housekeeping” phosphotransferase activity. We present the molecular cloning of a novel human NDP kinase gene, termed Nm23‐H6. Nm23‐H6 gene has been mapped at chromosome 3p21.3 and is highly expressed in heart, placenta, skeletal muscle, and some of the cancer cell lines. Recombinant Nm23‐H6 protein has been identified to exhibit functional NDP kinase activity. Immunolocalization studies showed that both endogenous and inducibly expressed Nm23‐H6 proteins were present as short, filament‐like, perinuclear radical arrays and that they colocalized with mitochondria. Cell fractionation study also demonstrated the presence of Nm23‐H6 protein in a mitochondria‐rich fraction. Moreover, induction of overexpression of Nm23‐H6 in SAOS2 cells, using the Cre‐loxP gene activation system, resulted in growth suppression and generation of multinucleated cells. Flow cytometric analysis also demonstrated that the proportion of cells with more than 4N DNA content increased to 28.1% after induction of Nm23‐H6, coinciding with the appearance of multinucleated cells. These observations suggest that Nm23‐H6, a new member of the NDP kinase family, resides in mitochondria and plays a role in regulation of cell growth and cell cycle progression. J. Cell. Biochem. 76:254–269, 1999.

K858, a novel inhibitor of mitotic kinesin Eg5 and antitumor agent, induces cell death in cancer cells.

The aim of this study was to investigate the mechanism of inhibition of Eg5 (kinesin spindle protein), a mitotic kinesin that plays an essential role in establishing mitotic spindle bipolarity, by the novel small molecule inhibitor K858. K858 was selected in a phenotype-based forward chemical genetics screen as an antimitotic agent, and subsequently characterized as an inhibitor of Eg5. K858 blocked centrosome separation, activated the spindle checkpoint, and induced mitotic arrest in cells accompanied by the formation of monopolar spindles. Long-term continuous treatment of cancer cells with K858 resulted in antiproliferative effects through the induction of mitotic cell death, and polyploidization followed by senescence. In contrast, treatment of nontransformed cells with K858 resulted in mitotic slippage without cell death, and cell cycle arrest in G(1) phase in a tetraploid state. In contrast to paclitaxel, K858 did not induce the formation of micronuclei in either cancer or nontransformed cells, suggesting that K858 has minimal effects on abnormalities in the number and structure of chromosomes. K858 exhibited potent antitumor activity in xenograft models of cancer, and induced the accumulation of mitotic cells with monopolar spindles in tumor tissues. Importantly, K858, unlike antimicrotubule agents, had no effect on microtubule polymerization in cell-free and cell-based assays, and was not neurotoxic in a motor coordination test in mice. Taken together, the Eg5 inhibitor K858 represents an important compound for further investigation as a novel anticancer therapeutic.

Phase II clinical study on intraoperative photodynamic therapy with talaporfin sodium and semiconductor laser in patients with malignant brain tumors

OBJECT The objective of the present study was to perform a prospective evaluation of the potential efficacy and safety of intraoperative photodynamic therapy (PDT) using talaporfin sodium and irradiation using a 664-nm semiconductor laser in patients with primary malignant parenchymal brain tumors. METHODS In 27 patients with suspected newly diagnosed or recurrent primary malignant parenchymal brain tumors, a single intravenous injection of talaporfin sodium (40 mg/m(2)) was administered 1 day before resection of the neoplasm. The next day after completion of the tumor removal, the residual lesion and/or resection cavity were irradiated using a 664-nm semiconductor laser with a radiation power density of 150 mW/cm(2) and a radiation energy density of 27 J/cm(2). The procedure was performed 22-27 hours after drug administration. The study cohort included 22 patients with a histopathologically confirmed diagnosis of primary malignant parenchymal brain tumor. Thirteen of these neoplasms (59.1%) were newly diagnosed glioblastomas multiforme (GBM). RESULTS Among all 22 patients included in the study cohort, the 12-month overall survival (OS), 6-month progression-free survival (PFS), and 6-month local PFS rates after surgery and PDT were 95.5%, 91%, and 91%, respectively. Among patients with newly diagnosed GBMs, all these parameters were 100%. Side effects on the skin, which could be attributable to the administration of talaporfin sodium, were noted in 7.4% of patients and included rash (2 cases), blister (1 case), and erythema (1 case). Skin photosensitivity test results were relatively mild and fully disappeared within 15 days after administration of photosensitizer in all patients. CONCLUSIONS Intraoperative PDT using talaporfin sodium and a semiconductor laser may be considered as a potentially effective and sufficiently safe option for adjuvant management of primary malignant parenchymal brain tumors. The inclusion of intraoperative PDT in a combined treatment strategy may have a positive impact on OS and local tumor control, particularly in patients with newly diagnosed GBMs. Clinical trial registration no.: JMA-IIA00026 (https://dbcentre3.jmacct.med.or.jp/jmactr/App/JMACTRS06/JMACTRS06.aspx?seqno=862).

Targeting EGFR Induced Oxidative Stress by PARP1 Inhibition in Glioblastoma Therapy

Despite the critical role of Epidermal Growth Factor Receptor (EGFR) in glioblastoma pathogenesis [1], [2], EGFR targeted therapies have achieved limited clinical efficacy [3]. Here we propose an alternate therapeutic strategy based on the conceptual framework of non-oncogene addiction [4], [5]. A directed RNAi screen revealed that glioblastoma cells over-expressing EGFRvIII [6], an oncogenic variant of EGFR, become hyper-dependent on a variety of DNA repair genes. Among these, there was an enrichment of Base Excision Repair (BER) genes required for the repair of Reactive Oxygen Species (ROS)-induced DNA damage, including poly-ADP ribose polymerase 1 (PARP1). Subsequent studies revealed that EGFRvIII over-expression in glioblastoma cells caused increased levels of ROS, DNA strand break accumulation, and genome instability. In a panel of primary glioblastoma lines, sensitivity to PARP1 inhibition correlated with the levels of EGFR activation and oxidative stress. Gene expression analysis indicated that reduced expression of BER genes in glioblastomas with high EGFR expression correlated with improved patient survival. These observations suggest that oxidative stress secondary to EGFR hyper-activation necessitates increased cellular reliance on PARP1 mediated BER, and offer critical insights into clinical trial design.

Mechanism of hyperploid cell formation induced by microtubule inhibiting drug in glioma cell lines

Checkpoint mechanism plays a crucial role in ensuring genomic integrity during cell cycle. Loss of checkpoint function is known to induce genomic instability and to alter ploidy of dividing cells. In this study, we examined mechanisms of hyperploid formation in glioma cells by treatment with nocodazole, which activates spindle assembly checkpoint by inhibiting microtubule polymerization. By prolonged nocodazole treatment, U251MG human glioma cell, which has a p53 mutation, underwent transient arrest at mitosis, and subsequently exited from mitotic arrest (termed ‘mitotic slippage’) followed by DNA replication without cytokinesis, resulting in hyperploid formation. Additionally, the heterogeneity in the number of centrosomes per cell increased during the hyperploid formation, suggesting that these hyperploid cells have genomic instability. By employing LN382 glioma cell that has a temperature-sensitive p53 mutation, we found that the activation of p53 prevents hyperploid formation after the prolonged nocodazole treatment. Furthermore, staurosporine, an inhibitor for a broad range of serine/threonine kinases including cdc2, was found to enhance hyperploid formation in U251MG cells by accelerating the induction of mitotic slippage. Interestingly, inhibitors specific for cdc2 kinase prevented the G2 to M transition but did not accelerate mitotic slippage, suggesting that staurosporine-sensitive kinases other than cdc2 are required for maintenance of spindle assembly checkpoint. Moreover, the enhancement of hyperploid formation by staurosporine was also blocked by p53-dependent G1 checkpoint. These results suggest that abrogation of G1 checkpoint is a critical factor for formation of hyperploid cells after the mitotic slippage.