Keishi Makino

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.

Interaction of NE-dlg/SAP102, a Neuronal and Endocrine Tissue-specific Membrane-associated Guanylate Kinase Protein, with Calmodulin and PSD-95/SAP90 A POSSIBLE REGULATORY ROLE IN MOLECULAR CLUSTERING AT SYNAPTIC SITES

NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase family protein, is known to bind to C-terminal ends ofN-methyl-d-aspartate receptor 2B (NR2B) through its PDZ (PSD-95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at synaptic sites in cultured rat hippocampal neurons, and their expressions increase in parallel with the onset of synaptogenesis. We have identified that NE-dlg/SAP102 interacts with calmodulin in a Ca2+-dependent manner. The binding site for calmodulin has been determined to lie at the putative basic α-helix region located around the src homology 3 (SH3) domain of NE-dlg/SAP102. Using a surface plasmon resonance measurement system, we detected specific binding of recombinant NE-dlg/SAP102 to the immobilized calmodulin with a K d value of 44 nm. However, the binding of Ca2+/calmodulin to NE-dlg/SAP102 did not modulate the interaction between PDZ domains of NE-dlg/SAP102 and the C-terminal end of rat NR2B. We have also identified that the region near the calmodulin binding site of NE-dlg/SAP102 interacts with the GUK-like domain of PSD-95/SAP90 by two-hybrid screening. Pull down assay revealed that NE-dlg/SAP102 can interact with PSD-95/SAP90 in the presence of both Ca2+ and calmodulin. These findings suggest that the Ca2+/calmodulin modulates interaction of neuronal membrane-associated guanylate kinase proteins and regulates clustering of neurotransmitter receptors at central synapses.

Influence of p53 mutations on prognosis of patients with glioblastoma.

The influence of p53 mutations on the biology of astrocytic tumors is controversial. p53 is thought to be inactivated in the early stage of gliomagenesis; however, what role its inactivation plays in the malignancy of gliomas remains unknown. To understand the significance of p53 inactivation, the authors identified the locus of p53 gene mutation in glioma samples at different stages of progression and studied the correlation between the mutation and clinical behavior.

Cloning and characterization of NE-dlg: A novel human homolog of the Drosophila discs large (dlg) tumor suppressor protein interacts with the APC protein

We have cloned a cDNA for a novel human homolog of the Drosophila discs large (dlg) tumor suppressor protein, termed NE-dlg (neuronal and endocrine dlg). Northern blot analysis revealed that the gene is highly expressed in neuronal and endocrine tissues. Fluorescence in situ hybridization (FISH) and radiation hybrid mapping studies localized the NE-dlg gene to chromosome Xq13. We also found that the NE-dlg gene encoded a 100 kDa protein. Immunolocalization studies using an NE-dlg antibody showed that the protein tended to be expressed in non-proliferating cells, such as neurons, cells in Langerhans islets of the pancreas, myocytes of the heart muscles, and the prickle and functional layer cells of the esophageal epithelium. Proliferative cells, including various cultured cancer cell lines and basal cells in the esophageal epithelium, showed little expression of the NE-dlg protein. In addition, yeast two-hybrid screening and in vitro binding assays revealed that the NE-dlg interacted with the carboxyl-terminal region of the APC tumor suppressor protein. These data suggest that NE-dlg negatively regulates cell proliferation through its interaction with the APC protein.

Epidemiological study of primary intracranial tumors: a regional survey in Kumamoto prefecture in southern Japan—20-year study

BackgroundThe increased use of neuro-imaging techniques, as well as various environmental factors, has been changing the incidence and the proportions of types of intracranial tumors. However, no accurate population-based epidemiological study of intracranial tumors in Japan has been reported. We evaluated recent trends in the occurrence of primary intracranial tumors among residents of Kumamoto prefecture, Japan.Methods and resultsWe surveyed 5,448 new cases of primary intracranial tumors that were diagnosed in Kumamoto prefecture between 1989 and 2008. The overall age-adjusted incidence rate was 14.09 (11.59 for males, 16.38 for females) per 100,000 population per year. The most common tumors were meningiomas (36.8%), followed by gliomas (19.5%), adenomas (17.8%), schwannomas (9.9%), and malignant lymphomas (3.6%). The number of cases of primary brain tumors, especially meningiomas and malignant gliomas, among the elderly has steadily increased and the incidence of asymptomatic intracranial tumors also increased. The number of asymptomatic meningiomas diagnosed per year was higher than that of symptomatic meningiomas in the years between 1997 and 2008. Furthermore, the incidence rate of brain lymphoma in Kumamoto prefecture is approaching that recorded in Western countries. On the other hand, the incidence rate of germ cell tumors is on the decline, approaching that recorded for children in Western countries.ConclusionEven though we adjusted the population in Kumamoto prefecture based on the Japanese population, increasing rates of several types of intracranial tumors were observed. These incidence rates are approaching those in Western countries.

Antitumor effect of humanized anti-interleukin-6 receptor antibody (tocilizumab) on glioma cell proliferation: Laboratory investigation

OBJECT Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates diverse physiological functions, including cell proliferation and survival. Recent studies have shown that IL-6 expression is often elevated in response to several types of glioma. Although IL-6 is said to play an important role in glioma, the involvement of IL-6 signaling has been quite controversial. The aim of this study was to evaluate the involvement of IL-6 signaling in glioma and the inhibitory effect of IL-6 signaling on glioma tumor proliferation. METHODS The expression of IL-6 receptors (IL-6Rs) was evaluated in glioma tissues by means of immunohistochemical analysis, and the involvement of IL-6 signaling in glioblastoma multiforme (GBM) U87MG cell proliferation was also determined. In addition, to examine the inhibitory effect of IL-6 signaling on glioma cell proliferation, the authors investigated the effects of tocilizumab, the humanized anti-human IL-6R antibody in U87MG cells. RESULTS Increased immunoreactivity for IL-6R was predominantly found in the cytoplasm of endothelial cells in all GBM samples. Inhibition of IL-6 signaling by both IL-6- and IL-6R-specific small interfering RNA and AG490, a specific inhibitor of JAK2 phosphorylation, suppressed glioma cell proliferation. Furthermore, tocilizumab, a clinically developed humanized anti-human IL-6R antibody, exerted an antiproliferative effect on cells from the GBM cell line U87MG via the IL-6R-dependent JAK-STAT3 pathway. CONCLUSIONS The IL-6 signaling pathway plays an important role in glioma cell proliferation, and tocilizumab exerts an antitumor effect in U87MG glioma cells. These results may bring new insight into the molecular pathogenesis of glioma and may lead to a new therapeutic intervention.

C-kit expression in germinoma: an immunohistochemistry-based study.

SummaryIn our immunohistochemical study of 25 human primary intracranial germinomas and germinomas with syncytiotrophoblastic giant cells (STGC), we stained the same sections for c-kit and placental alkaline phosphatase (PLAP). Immunohistochemical expression was graded using a semi-quantitative scoring system where 3+ = 51–75%, and 4+ = 76–100%. Of the 25 cases, 7 (28%) were graded 3+ and 18 (72%) 4+ for c-kit; 8 (32%) were 3+ or 4+ for PLAP. All 3 cases negative for PLAP-staining were strongly positive and all embryonal carcinomas, immature teratomas, and yolk sac tumors were negative for c-kit staining. The soluble isoform of c-kit (s-kit) is reportedly detectable in cerebral spinal fluid of patients with germinomas and germinomas with STGC. C-kit and s-kit may be powerful tumor markers for germinomas with or without STGC.

Primary intracranial plasma-cell granuloma: a case report and review of the literature.

BACKGROUND Plasma-cell granulomas, which are characterized by the non-neoplastic proliferation of plasma cells, are primarily found in the lungs and upper respiratory tracts, and are extremely rare in the central nervous system. METHODS An intracranial tumor of an 11-year-old boy was evaluated by histologic and radiologic examination. RESULTS An 11-year-old boy had a 2-month history of mild headache and nausea. A computed tomography scan showed a round, slightly high-density mass surrounded by marked edema in the left frontal lobe. On magnetic resonance imaging (MRI), the mass had a slightly high signal intensity on the T1-weighted image and marked low signal intensity on the T2-weighted image. It was heterogenously enhanced with gadolinium-DTPA. Microscopic examination demonstrated a non-neoplastic mixed cell population with a predominance of plasma cells. Immunohistochemical staining revealed that it was characterized by a polyclonal plasma cell population. CONCLUSION Our extensive search of the literature indicated this to be the ninth reported case of intracranial plasma cell granulation. The MRI was very useful for evaluating the extent of the intracranial lesions. The tumor was removed surgically and did not recur during a 2-year follow-up with no radiation therapy.

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.

Recurrent intracranial germinoma outside the initial radiation field: A single-institution study

Between 1975 and 2005, we treated 52 newly diagnosed germinoma patients. Until 1991, patients with pure germinomas or germinomas with syncytiotrophoblastic giant cells (STGCs) received whole-brain radiotherapy only. Of the 52 patients, 30 were treated with a reduced radiation volume and combined chemotherapy; seven of these received local irradiation with 24 Gy, two received whole-brain (30 Gy) plus local irradiation (20 Gy), 16 received extended local irradiation delivered to the whole ventricles (30 Gy) plus local (20 Gy) irradiation, and five received extended local irradiation (24 Gy). Of the 30 patients treated with a reduced radiation volume and combined chemotherapy, four experienced tumor recurrence; three patients had been treated with 24 Gy of local radiotherapy and one had received extended local (30 Gy) plus local (20 Gy) irradiation in addition to chemotherapy. In these patients, the delivered radiotherapy was inadequate and the origin of the recurrent tumors was outside the radiation field. None of the patients who had received at least 24 Gy of whole ventricle radiotherapy combined with chemotherapy experienced tumor recurrence. In combination with chemotherapy, the delivery of irradiation covering the ventricles effectively reduced the incidence of tumor recurrence in patients with germinomas or germinomas with STGCs.