Tetsuro Morisaki

Roles of aurora-A kinase in mitotic entry and G2 checkpoint in mammalian cells

Background: Various mitotic events are controlled by Cdc2‐cyclin B and other mitotic kinases. Aurora/Ipl1‐related mitotic kinases were proved to play key roles in mitotic progression in diverse lower organisms. Aurora‐A is a mammalian counterpart of aurora/Ipl1‐related kinases and is thought to be a potential oncogene. However, the regulation of aurora‐A activation and the commitment of aurora‐A in the progression of G2‐M phase are largely unknown in mammalian cells.

A human homolog of Drosophila warts tumor suppressor, h-warts, localized to mitotic apparatus and specifically phosphorylated during mitosis

We identified a human homolog of Drosophila warts tumor suppressor gene, termed h‐warts, which was mapped at chromosome 6q24‐25.1. The h‐warts protein has a serine/threonine kinase domain and is localized to centrosomes in interphase cells. However, it becomes localized to the mitotic apparatus, including spindle pole bodies, mitotic spindle, and midbody, in a highly dynamic manner during mitosis. Furthermore, h‐warts is specifically phosphorylated in cells at mitotic phase, most likely by Cdc2 kinase. These findings suggest that h‐warts functions as a component of the mitotic apparatus and is involved in proper progression of mitosis.

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 G1 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 G1 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 G1 tetraploidy checkpoint and entered S phase to achieve a DNA content of 8N. This impairment of G1 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 G1 tetraploidy checkpoint.

WARTS tumor suppressor is phosphorylated by Cdc2/cyclin B at spindle poles during mitosis.

Identification of physiological substrates for Cdc2/cyclin B is crucial for understanding the functional link between mitotic events and Cdc2/cyclin B activation. A human homologue of the Drosophila warts tumor suppressor, termed WARTS, is a serine/threonine kinase and a dynamic component of the mitotic apparatus. We have found that Cdc2/cyclin B forms a complex with a fraction of WARTS in the centrosome and phosphorylates the Ser613 site of WARTS during mitosis. Immunocytochemical analysis has shown that the S613‐phosphorylated WARTS appears in the spindle poles at prometaphase and disappears at telophase. Our findings suggest that Cdc/cyclin B regulates functions of WARTS on the mitotic apparatus.

Serine protease Omi/HtrA2 targets WARTS kinase to control cell proliferation

The serine protease Omi/HtrA2 was initially regarded as a proapoptotic molecule that proteolyses several proteins to induce cell death. Recent studies, however, indicate that loss of Omi protease activity increases susceptibility to stress-induced cell death. These complicated findings suggest that the protease activity of Omi is involved not only in apoptosis but also in cellular homeostasis. However, the targets which Omi uses to mediate this novel process are unknown. Previously, we showed that WARTS (WTS)/large tumor-suppressor 1 mitotic kinase interacts with the protein/discs-large protein/zonula (PDZ) domain of Omi and promotes its protease activity. We now report that WTS is a substrate for Omi protease activity, thus it is not only a regulator but also a downstream target of this protease. Interaction with Omi PDZ domain is required for WTS to be proteolysed. When caspase-9-deficient mouse embryonic fibroblasts (MEFs) were treated with staurosporine, WTS was proteolysed by activated endogenous Omi without induction of cell death. Therefore, protease activity of Omi and proteolysis of WTS are not necessarily required for cell death. We found that depletion of Omi from HeLa cells results in accelerated cell proliferation despite no significant change in the duration of mitosis. The depletion of WTS showed the same effect on S phase progression. Therefore, WTS proteolytic fragment(s) generated by Omi may act as an inhibitor of G1/S progression. Our data reveal a role for Omi-mediated processing of WTS in negative regulation of cell cycle progression at interphase, suggesting a novel function of Omi other than apoptosis.

NE-dlg, a mammalian homolog of Drosophila dlg tumor suppressor, induces growth suppression and impairment of cell adhesion: Possible involvement of down-regulation of β-catenin by NE-dlg expression

Membrane‐associated guanylate kinases (MAGUKs) are known to function as scaffolds for forming multiprotein complexes at the synaptic junctions of neuronal cells and at sites of epithelial cell‐cell contact. In Drosophila, mutations of the lethal (1)‐discs large (dlg) gene, which encodes a MAGUK protein, leads to post‐synaptic structure defects in neuronal cells and neoplastic overgrowth of epithelial cells. We previously showed that NE‐dlg (neuronal and endocrine dlg), a human homolog of the dlg, plays a crucial role in formation of synaptic structure in human neuronal cells. Here we demonstrate that NE‐dlg, similar to Drosophila dlg, is involved in regulation of cell cycle progression and adhesive ability of non‐neuronal cells. Overexpression of NE‐dlg in proliferating cells including various cancer cell lines induced growth suppression and impairment of cell adhesive ability. Furthermore, NE‐dlg overexpression caused the down‐regulation of β‐catenin in cancer cells regardless of mutations in the APC (adenomatous polyposis coli) gene. The PDZ domains of NE‐dlg were found to be essential for the growth suppression, loss of adhesive property and down‐regulation of β‐catenin. We propose that NE‐dlg regulates the cell growth and adhesive ability by controlling the level of β‐catenin through an APC‐independent pathway. Inactivation of NE‐dlg may therefore contribute to development and/or progression of human neoplasms. Int. J. Cancer 86:480–488, 2000.

Extended Resection Combined with Intraoperative Radiotherapy

Since 1984, intraoperative radiotherapy (IORT) combined with extended resection for pancreatic cancer has been performed in our clinic to prevent local recurrence. Following extended resection, a dose of 30 Gy of 9–12 MeV electrons is administered to the operative field, including the paraaortic area from the diaphragm above to the inferior mesenteric artery below. The 5-year survival rate was 19.4% in all 33 cases, 26.6% in patients who had macroscopic tumor clearance, and 20.3% in patients with stage IVa tumor according to the Japanese classification. In autopsies of 7 patients who underwent the combined therapy, only 2 had local recurrence enclosed by thick, firm connective tissue. There was no local recurrence in 2 patients who underwent noncurative resection. Enhanced local control induced by the combined therapy, however, has only a limited impact on overall survival because of system disease progression, especially hepatic metastases. These results suggest that a combination of IORT and extended resection should be performed on selected patients to control local recurrence and that anticancer treatment for metastases of the liver must be established as soon as possible for the cure of pancreatic cancer.