Naoki Yokota

A Novel Zinc Finger Protein, Zic, Is Involved in Neurogenesis, Especially in the Cell Lineage of Cerebellar Granule Cells

Abstract: To clarify the mechanism of cerebellar development, we have cloned a gene, named zic, encoding a zinc finger protein that is expressed abundantly in granule cells throughout development of the cerebellum. zic has a significant homology to the zinc finger domain of the Caenorhabditis elegans tra1 gene, the Drosophila cubitus interruptus Dominant gene, and the human GLI oncogene. An in situ hybridization study revealed that zic showed a restricted expression pattern in the granule cells and their putative precursor cells. It is also expressed at an early embryonic stage in the dorsal half of the neural tube. The expression pattern and nuclear localization were confirmed by immunohistochemical study. Furthermore, the bacterially expressed zic protein containing the zinc finger domains bound to the GLI‐binding sequence. These findings suggest that zic is one of a number of nuclear factors involved in both differentiation in early development and maintenance of properties of the cerebellar granule cells.

Role of Wnt pathway in medulloblastoma oncogenesis

To clarify the roles of Wnt pathway in medulloblastoma oncogenesis, immunohistochemical staining of β‐catenin and Wnt‐1 and genomic analyses of CTNNB1 (β‐catenin) and AXIN1 (axin 1) were examined in 23 sporadic cases. Accumulation of β‐catenin in tumor cells was immunohistochemically proven in 5 cases; 2 cases showed positive immunoreactivity for Wnt‐1 and another 2 showed mutation of either CTNNB1 or AXIN1. AXIN1 mutation was in exon 3, corresponding to GSK‐3β binding site and CTNNB1 mutation was in exon 3, corresponding to its phosphorylation site. Disruption of these proteins could result in upregulation of the Wnt signaling and accumulation of β‐catenin, followed by cell proliferation and medulloblastoma oncogenesis.

Bystander effect-mediated gene therapy of gliomas using genetically engineered neural stem cells

Since neural stem cells (NSCs) have the ability to migrate toward a tumor mass, genetically engineered NSCs were used for the treatment of gliomas. We first evaluated the “bystander effect” between NSCs transduced with the herpes simplex virus-thymidine kinase (HSVtk) gene (NSCtk) and C6 rat glioma cells under both in vitro and in vivo conditions. A potent bystander effect was observed in co-culture experiments of NSCtk and C6 cells. In the intracranial co-implantation experiments in athymic nude mice and Sprague–Dawley rats, the animals co-implanted with NSCtk and C6 cells and treated with ganciclovir (GCV) showed no intracranial tumors and survived more than 100 days, while those treated with physiological saline (PS) died of tumor progression. We next injected NSCtk cells into the pre-existing C6 tumor in rats and treated them with GCV or PS. The tumor volume was serially measured by magnetic resonance imaging. The tumor disappeared in six out of nine rats in the NSCtk/GCV group, while all the rats treated with PS died of tumor progression by day 21. The results indicate the feasibility of a novel gene therapy strategy for gliomas through a bystander effect generated by intratumoral injection of NSCtk cells and systemic GCV administration.

Human SLITRK family genes: genomic organization and expression profiling in normal brain and brain tumor tissue

Slitrk family proteins are characterized as integral membrane proteins that have two leucine-rich repeat (LRR) domains and a carboxy-terminal domain that is partially similar to trk neurotrophin receptor proteins. The LRR domains are similar to those of slit proteins. In a previous study, we showed that mouse Slitrk genes are expressed predominantly in neural tissue and have neurite-modulating activity in cultured neuronal cells. Their expression profiles as well as their functions vary among the family members. In this paper, we characterized the human SLITRK1, SLITRK2, SLITRK3, SLITRK4, SLITRK5, and SLITRK6 genes. The six genes are located in three clusters, on 3q, 13q, and Xq, respectively. Their expression was detected mainly in the brain, but the expression profile of each SLITRK was unique. SLITRK expression was also investigated in various types of brain tumor tissue. The results showed that all SLITRK genes are differentially expressed in brain tumors, including astrocytoma, oligodendroglioma, glioblastoma, medulloblastoma, and supratentorial primitive neuroectodermal tumor (PNET). Particularly interesting findings were that SLITRK3 expression was enhanced in tissue from several different types of tumors and SLITRK6 expression was highly selective. These results suggest that the human SLITRK genes are useful molecular indicators of brain tumor properties.

Hypofractionated stereotactic radiotherapy with CyberKnife for nonfunctioning pituitary adenoma: high local control with low toxicity

The aim was to evaluate the clinical outcome of hypofractionated stereotactic radiotherapy (SRT) with CyberKnife for nonfunctioning pituitary adenoma. From October 2000 to March 2009, 100 patients with nonfunctioning pituitary adenoma were treated with hypofractionated SRT. Forty-three patients were male, and 57 were female. The patients ages ranged from 16 to 82 years (median, 59 years). Five patients were medically inoperable, and 1 refused surgery; the remaining 94 were recurrent cases or those receiving postoperative adjuvant SRT. No patients had a history of previous cranial radiotherapy. Tumor volume ranged from 0.7 to 64.3 mL (median, 5.1 mL). The marginal doses were 17.0 to 21.0 Gy for the 3-fraction schedule and 22.0 to 25.0 Gy for the 5-fraction schedule. Toxicities were evaluated with the Common Terminology Criteria for Adverse Events version 4.0. The median follow-up period for living patients was 33 months (range, 18-118.5 months). The 3-year overall survival and local control rates were 98% and 98%, respectively. In-field and out-field tumor regrowth were observed in 3 and 2 patients, respectively. Transient cyst enlargement occurred in 3 cases. A post-SRT grade 2 visual disorder occurred in 1 patient. Symptomatic post-SRT hypopituitarism was observed in 3 of 74 patients who had not received hormone replacement therapy after surgery. CyberKnife SRT involving 21 Gy in 3 fractions or 25 Gy in 5 fractions is safe and effective for surgical treatment of nonfunctioning pituitary adenoma. Hypofractionated SRT appears useful for protecting the visual nerve and neuroendocrine function, especially for tumors located near the optic pathways and large tumors.

Monitoring of singlet oxygen is useful for predicting the photodynamic effects in the treatment for experimental glioma.

Purpose: Singlet oxygen (1O2) generated in photodynamic therapy (PDT) plays a very important role in killing tumor cells. Using a new near-IR photomultiplier tube system, we monitored the real-time production of 1O2 during PDT and thus investigated the relationship between the 1O2 production and photodynamic effects. Experimental Design: We did PDT in 9L gliosarcoma cells in vitro and in an experimental tumor model in vivo using 5-aminolevulinic acid and nanosecond-pulsed dye laser. During this time, we monitored 1O2 using this system. Moreover, based on the 1O2 monitoring, we set the different conditions of laser exposure and investigated whether they could affect the tumor cell death. Results: We could observe the temporal changes of 1O2 production during PDT in detail. At a low fluence rate the 1O2 signal gradually decreased with a low peak, whereas at a high fluence rate it decreased immediately with a high peak. Consequently, the cumulative 1O2 at a low fluence rate was higher, which thus induced a strong photodynamic effect. The proportion of apoptosis to necrosis might therefore be dependent on the peak and duration of the 1O2 signal. A low fluence rate tended to induce apoptotic change, whereas a high fluence rate tended to induce necrotic change. Conclusions: The results of this study suggested that the monitoring of 1O2 enables us to predict the photodynamic effect, allowing us to select the optimal laser conditions for each patient.

Incidentally identified syringomyelia associated with Chiari I malformations: is early interventional surgery necessary?

OBJECTIVEThe purpose of this study was to analyze clinical data and magnetic resonance imaging (MRI) findings for patients with asymptomatic, incidentally identified syringomyelia associated with Chiari I malformations who were monitored for more than 10 years, and to clarify the natural history of these lesions. METHODSThe clinical records of nine patients who had not been surgically treated and were regularly subjected to neurological and MRI examinations were analyzed. In MRI studies, the axial diameter of the syrinx at the widest level, the longitudinal extent of the syrinx, and the extent of tonsillar herniation into the spinal canal were analyzed. As a control, MRI findings for 11 patients with symptomatic syringomyelia associated with Chiari I malformations who had been surgically treated were also analyzed, and these MRI parameters were statistically compared between the asymptomatic and symptomatic groups. RESULTSOne patient underwent surgery, because of neurological changes, 7 years after the first visit. None of the remaining patients demonstrated any neurological change during the follow-up period (11.2 ± 0.7 yr), and all of them have been faring well without surgery. No statistically significant differences in MRI findings between the asymptomatic and symptomatic groups were observed. CONCLUSIONThe long-term clinical courses of patients with asymptomatic, incidentally identified syringomyelia associated with Chiari I malformations were observed to be benign. MRI parameters did not provide predictable values to recommend interventional surgery. Unless changes in neurological or MRI findings are detected, early interventional surgery is not necessary.

Potent Bystander Effect in Suicide Gene Therapy Using Neural Stem Cells Transduced with Herpes Simplex Virus Thymidine Kinase Gene

Objective: The herpes simplex virus thymidine kinase (HSVtk)/ganciclovir suicide gene therapy system has been considered as one of the most promising therapeutic strategies for malignant gliomas. We have been using HSVtk gene-transduced neural stem cells (NSCtk) that possess an ability to migrate toward a tumor mass for the treatment of experimental brain tumors. In the present study, we evaluated the potency of anti-tumor effect mediated by the bystander effect between NSCtk and C6 glioma cells in the HSVtk/ganciclovir suicide gene therapy system. Methods: NSCtk and C6 glioma cells were mixed at various ratios (NSCtk:C6 cell ratios of 1:1 to 1:64) and the bystander effect was evaluated both under in vitro and in vivo conditions. Results: In vitro co-culture experiment showed a complete tumor growth inhibition at the NSCtk:C6 ratios as low as 1:16. In vivo co-implantation study in the rat brain showed no visible tumors at the NSCtk:C6 ratios as low as 1:16 and all those rats survived more than 100 days. Conclusion: The results clearly demonstrated an extremely potent bystander effect between NSCtk and C6 cells, and the minimum number of NSCtk cells needed for the treatment of tumors was roughly estimated.

Protein kinase Cδ and α are involved in the development of vasospasm after subarachnoid hemorrhage

Abstract We have previously shown the enhanced activity of protein kinase C in the membrane fraction of the canine vasospastic artery after subarachnoid hemorrhage, which increased with progression of angiographic vasospasm. This study examined identification of protein kinase C isoforms in the canine basilar artery, and the changes in expression and/or translocation of each isoform during the development of vasospasm. Vasospasm was produced by using the “two-hemorrhage” canine model in the basilar artery, and angiographic progression of vasospasm was assessed consecutively. Two isoforms, protein kinase Cα and δ were identified in basilar arteries by Western blotting. Densitometric analysis showed that the expression of protein kinase Cδ in the membrane fraction was significantly increased in the earlier stage, and protein kinase Cα was increased later as vasospasm progressed. These results indicate that protein kinase Cδ and α isoforms may play a significant role in the development and maintenance of vasospasm.

Identification of Differentially Expressed Genes in Rat Hippocampus After Transient Global Cerebral Ischemia Using Subtractive cDNA Cloning Based on Polymerase Chain Reaction

Background and Purpose— The purpose of this study is to identify new molecules that play important roles in the phenomena that occur in the hippocampus after transient global cerebral ischemia, as clues to better understanding of the mechanisms. Methods— A subtractive cDNA library was established by suppression subtractive hybridization of rat hippocampal tissues after transient global cerebral ischemia. With differential screening of the library, upregulated fragments were identified. The mRNA expression levels of selected genes were measured with semiquantitative reverse transcriptase polymerase chain reaction (PCR). Results— Among more than 100 isolated fragments, approximately half were determined to be identical to known sequences. The rest showed high homology to known sequences, and only 2 did not exhibit homology to any known sequences. The expression of 5 genes identified in this study increased in 24 hours after ischemia to a level twice as high as that in sham-operated controls. These included furin, prosaposin, synaptotagmin IV, heat shock protein 105, and the neutral and basic amino acid transporter (NBAT). The increases in the mRNA expression levels of the genes except NBAT, as revealed by semiquantitative reverse transcription PCR, were statistically significant at both 6 and 24 hours after ischemia. Conclusions— Genes isolated are thought to be associated with production of proteins necessary for degeneration, neuroprotection, and reconstruction of neurons. How the expression of these genes relates to functional changes after ischemia remains to be determined. PCR-based subtractive cDNA cloning is demonstrated to be a useful tool for analyzing in vivo gene expression in animal ischemia models.