Hideo Takeshima

Expression of tissue factor correlates with grade of malignancy in human glioma

Tissue factor (TF), a cell surface receptor of factor VII/VIIa, was initially recognized as an initiator of the extrinsic coagulation pathway. TF has recently been found to be expressed highly in certain types of malignant tumors. In addition, TF belongs to the interferon receptor family and is one of the immediate early genes, suggesting that TF may participate in the regulation of cell growth. However, the correlation between the expression of TF and cell growth is still unclear.

Cloning and functional characterization of the 5'-flanking region of the human monocyte chemoattractant protein-1 receptor (CCR2) gene. Essential role of 5'-untranslated region in tissue-specific expression

The human monocyte chemoattractant protein-1 receptor designated hCCR2 is an essential co-receptor in cell entry by the human immunodeficiency virus as well as a receptor for monocyte chemoattractant protein-1, a member of the family of C-C chemokines that mediate monocyte chemotaxis. To elucidate the molecular mechanisms underlying the transcriptional regulation of hCCR2, we cloned and sequenced the hCCR2 gene; it was approximately 8 kilobase pairs in length and consisted of three exons divided by two introns. In the 5′-flanking region, there were the typical mammalian promoter consensus elements, a CAAT box and a TATA box, resulting in a single transcription initiation site. In addition, we found clustered tissue-specific cis-regulatory elements such as GATA consensus sequences, Oct-1 binding sequences, and CAAT/enhancer-binding protein binding sequences. Luciferase assays with various promoter deletions and gel mobility shift assays indicated that threecis-regulatory elements located within the region from −89 to +118 are required for basal activity in THP-1 cells. One element is an octamer sequence 36-base pair upstream from the TATA box; it binds mainly to Oct-1 and is capable of increasing transcriptional activity. The other two elements, which are tandem recognition sites of the CAAT/enhancer-binding protein family, are located in the 5′-untranslated region and account for the transcriptional activation as well as the tissue specificity of hCCR2.

Impairment of cell adhesion by expression of the mutant neurofibromatosis type 2 (NF2) genes which lack exons in the ERM-homology domain

Neurofibromatosis 2 (NF2) is an inherited disorder characterized by a predisposition to multiple intracranial tumors. The protein encoded by the NF2 gene has striking similarities to ezrin, radixin and moesin (ERM) proteins which link membrane proteins to the cytoskeleton. Therefore, it can be speculated that the disruption of cytoskeletal organization by alterations in the NF2 gene is involved in the development of tumors. It has been reported that the majority of NF2 mutations were nonsense or frameshift mutations that result in premature termination of translation. To facilitate the detection of these mutations, we performed protein truncation test and found that 11 of 14 NF2 patients had truncational mutations (79%). Seven of the 11 patients (64%) had a splicing abnormality which lead to absence of exons in the ERM homology domain. To examine the biological significance of the exon-missing mutations in the ERM homology domain, we expressed the wild-type (wt-NF2) and the various mutant NF2s (mu-NF2s) in a fibroblast cell line by using both liposome-mediated transfection and nuclear microinjection of the expression plasmids. The wt-NF2 showed intense punctate staining in the perinuclear cytoplasm in addition to overall staining of the submembranous area, whereas the mu-NF2s lacking exons in the ERM homology domain showed granular staining at the perinuclear region without any accumulation at the submembrane region. Microinjection of wt-NF2 cDNA into the nucleus of VA13 cells revealed that wt-NF2 protein induced a progressive elongation of cell processes. Furthermore, cells that expressed mu-NF2 had decreased adhesion, which resulted in detachment from the substratum. These findings suggested that the exon-missing mutations in the ERM-homology domain may affect cell membrane–cytoskeleton signaling and consequently disrupt cell–to–cell or cell–to–matrix interaction.

Expression of citrulline–nitric oxide cycle in lipopolysaccharide and cytokine-stimulated rat astroglioma C6 cells

Nitric oxide (NO) is involved in many physiological and pathological processes in the brain. NO is synthesized from arginine by nitric oxide synthase (NOS), with citrulline generated as a by-product of the reaction. Thus, citrulline can by recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) via the citrulline-NO cycle. Rat astroglioma C6 cells were treated with bacterial lipopolysaccharide (LPS), interferon-gamma (IFNgamma) and tumor necrosis factor-alpha, and the expression of the enzymes of the citrulline-NO cycle was investigated by RNA blot and immunoblot analyses. NO production from arginine and citrulline was also assessed. iNOS mRNA and protein were induced 6-12 h after stimulation with LPS and cytokines and decreased at 24 h. AS mRNA increased up to 12 h and decreased at 24 h. AS protein increased gradually up to 48 h. On the other hand, AL mRNA remained unchanged by stimulation. NO production from arginine was enhanced by the treatment with LPS and cytokines. NO production was also observed when arginine was replaced by citrulline. These results indicate that NO production is enhanced in LPS- and cytokine-stimulated C6 cells due to induction of iNOS and that the citrulline-arginine recycling is important for NO production.

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.

Subacute sclerosing panencephalitis and chorioretinitis

This is a case report of a 10-year-old boy with subacute sclerosing panencephalitis (SSPE). He initially developed visual disturbance and macular degenerative changes of the right eye at the age of 8 years, followed by chorioretinitis of the left eye, and his neurological symptoms deteriorated rapidly from the age of 10 years. He was diagnosed as having SSPE, as judged on cerebrospinal fluid examination for measles virus RNA by reverse transcription-polymerase chain reaction (RT-PCR), at the second stage of Jabbours classification on admission. Although high intensity lesions were observed in the right occipital and temporal lobes, especially around the optic radiation, on T2-weighted brain MRI before the start of intrathecal interferon-alpha (IFN-alpha) therapy, they had disappeared at about two months after the treatment. Chorioretinitis (and/or macular degeneration) should be considered in the differential diagnosis of SSPE, permitting early IFN therapy.

Evaluation of residual tissues after adjuvant therapy in germ cell tumors.

Objective: Germ cell tumors are the tumors sensitive for adjuvant therapy such as radiotherapy and chemotherapy. We evaluated the pathological findings of these heterogeneous tumors to determine the persistence of residual viable tumor cells after adjuvant therapy. Patients and Methods: Between 1988 and 2005, we treated 31 patients with germinoma or germinoma with syncytiotrophoblastic giant cells (STGC) and 15 patients with non-germinomatous malignant germ cell tumors (NGMGCTs). All 46 patients received a combination of chemo- and radiotherapy. A second-look operation was performed in 3 of 31 patients with germinomas or germinomas with STGC and 12 of 15 patients with NGMGCTs. The follow-up period was 2–139 months (median 95) in patients with germinomas or germinomas with STGC (group 1) and 10–202 months (median 65) in NGMGCT patients (group 2). Results: Post-treatment, 3 group 1 and 12 group 2 patients manifested residual tumors. The pathological diagnosis in group 1 patients was mature teratoma, pineal cyst, and fibrous tissue with calcification; in group 2 it was yolk sac tumor (n = 1), immature teratoma (n = 3), mature teratoma (n = 4), and necrosis or fibrous tissue (n = 4). While no group 1 patients manifested tumor cells, MIB-1-positive viable tumor cells were present in resected tissues from one-third of the group 2 patients (3 immature teratomas and 1 yolk sac tumor). Conclusion: The absence of viable tumor cells in residual tissue indicates that the combination of cisplatin-based chemo- and radiotherapy was effective in our germinoma patients. On the other hand, in patients with NGMGCTs, these cells persisted despite this combination therapy.

The mechanism of growth-regulation of glioma cells by trapidil

SummaryTrapidil is a PDGF antagonist that can inhibit the proliferation of the PDGF-producing glioma cells, U251MG. As the mechanism of growth-regulation by trapidil remains unclear, we studied its effect on the growth of U251MG cells. We performed a cell cycle analysis and examined the intracellular transduction pathway and oncogene expression in serum-stimulated glioma cells with or without trapidil.After the serum starvation for 3 days, glioma cell proliferation was stimulated by the addition of serum. Cell cycle analysis showed that cell cycle perturbations induced by trapidil included a decreased transition rate from G0-G1 to S phase, suggesting that some metabolic event is required for progress through the G0-G1 phase and that this event is sensitive to trapidil. Internal signal transduction mechanisms are central in the molecular control of cell growth. One such regulator is the protein kinase C(PKC) system and the c-fos gene is likely to be a direct target of intracellular signal transduction pathways. Therefore, we hypothesize that the intracellular PKC activity and c-fos expression of the trapidil-treated cells are suppressed. We posit that trapidil affects the intracellular signal transduction pathway PKC activity and c-fos expression in cells stimulated with serum containing growth factors.

Secondary cervical dystonia following stereotactic radiosurgery in a patient with thalamic glioma

BACKGROUND Cervical dystonia associated with structural lesion is uncommon. We report the first patient with secondary CD after stereotactic radiosurgery for thalamic glioma. Possible network abnormalities relevant to manifestation of CD were discussed. CASE DESCRIPTION A 27-year-old woman complaining of headache and left motor weakness was found to have a thalamic tumor on the right side. The lesion was totally removed using transventricular approach. Histopathologically, tumor samples manifested features of anaplastic astrocytoma. She underwent stereotactic radiosurgery in addition to the conventional radiation and chemotherapy. Afterward, she returned to her usual life without any neurological deficits. Sixteen months postoperatively, the patient developed forced head tilting to the left side combined with chin lift. On the TWSTRS, she registered 15 for torticollis severity. The abnormal head posturing was alleviated by the sensory trick of touching her face with her right hand. Irregular-shaped lesion involving the thalamus, lenticular nuclei, midbrain, pons, and cerebellum was presented on magnetic resonance images. Steroid therapy effectively diminished the lesion size, and her abnormal head posturing was gradually ameliorated (TWSTRS severity scale = 3). CONCLUSION The clinical-neuroradiological course of the present case strongly suggested that the lesion detected long after the surgery was due to radiation necrosis. The present study may provide a critical information in understanding pathophysiological mechanisms of CD that may involve substantial interactions between olivocerebellar and basal ganglia-thalamocortical circuits.

Reporter/Functional gene transfer in rat brain.

Of the many methods and techniques for in vivo gene transfer, some have already been used in clinical trials. In most cases, genes are transferred into tissues using the infectivity of viral particles. However, viral systems have some known drawbacks (1,2). If an efficient and specific transfer method could be developed, naked plasmid DNA would be an ideal system for gene transfer. Plasmid-mediated methods would be economical and easy. Also, the transfer procedure could be easily repeated, as naked plasmid DNA has little antigenicity for the host (3,4).