Akira Tamase

Identification of tumor-initiating cells in a highly aggressive brain tumor using promoter activity of nucleostemin

Controversy remains over whether the cancer stem cell (CSC) theory applies to all tumors. To determine whether cells within a highly aggressive solid tumor are stochastically or hierarchically organized, we combined a reporter system where the nucleostemin (NS) promoter drives GFP expression (termed NS-GFP) with a mouse brain tumor model induced by retroviral Ras expression on a p16Ink4a/p19Arf-deficient background. The NS-GFP system allowed us to monitor the differentiation process of normal neural stem/precursor cells by analyzing GFP fluorescence intensity. In tumor-bearing mice, despite the very high frequency of tumorigenic cells, we successfully identified the NS-GFP+ cells as tumor-initiating cells (T-ICs). The clonal studies conclusively established that phenotypical heterogeneity can exist among the cells comprising a genetically homogeneous tumor, suggesting that this aggressive brain tumor follows the CSC model. Detailed analyses of the NS-GFP+ brain tumor cells revealed that T-ICs showed activation of the receptor tyrosine kinase c-Met, which functions in tumor invasiveness. Thus, the NS-GFP system provides a powerful tool to elucidate stem cell biology in normal and malignant tissues.

Identification of Stem Cells During Prepubertal Spermatogenesis via Monitoring of Nucleostemin Promoter Activity

The nucleostemin (NS) gene encodes a nucleolar protein found at high levels in several types of stem cells and tumor cell lines. The function of NS is unclear but it may play a critical role in S‐phase entry by stem/progenitor cells. Here we characterize NS expression in murine male germ cells. Although NS protein was highly expressed in the nucleoli of all primordial germ cells, only a limited number of gonocytes showed NS expression in neonatal testes. In adult testes, NS protein was expressed at high levels in the nucleoli of spermatogonia and primary spermatocytes but at only low levels in round spermatids. To evaluate the properties of cells expressing high levels of NS, we generated transgenic reporter mice expressing green fluorescent protein (GFP) under the control of the NS promoter (NS‐GFP Tg mice). In adult NS‐GFP Tg testes, GFP and endogenous NS protein expression were correlated in spermatogonia and spermatocytes but GFP was also ectopically expressed in elongated spermatids and sperm. In testes of NS‐GFP Tg embryos, neonates, and 10‐day‐old pups, however, GFP expression closely coincided with endogenous NS expression in developing germ cells. In contrast to a previous report, our results support the existence in neonatal testes of spermatogonial stem cells with long‐term repopulating capacity. Furthermore, our data show that NS expression does not correlate with cell‐cycle status during prepuberty, and that strong NS expression is essential for the maintenance of germline stem cell proliferation capacity. We conclude that NS is a marker of undifferentiated status in the germ cell lineage during prepubertal spermatogenesis.

NKX2.2 Suppresses Self-Renewal of Glioma-Initiating Cells

Glioblastoma (GBM) is the most aggressive and destructive form of brain cancer. Animal models that can unravel the mechanisms underlying its progression are needed to develop rational and effective molecular therapeutic approaches. In this study, we report the development of mouse models for spontaneous gliomas representing distinct progressive stages of disease that are governed by defined genetic alterations. Neural stem/progenitor cell (NPC)-specific constitutive Ras activation in vivo plus p53 deficiency led to development of primarily anaplastic astrocytoma (grade III), whereas combined loss of p53 plus p16(Ink4a)/p19(Arf) led to development of GBM (grade IV) at 100% penetrance within 6 weeks. These glioma models showed enhanced stem cell properties (stemness) accompanied by malignant progression. Notably, we determined that, in our models and in human specimens, downregulation of the homeodomain transcription factor NKX2.2, which is essential for oligodendroglial differentiation, was correlated with increased tumor malignancy. NKX2.2 overexpression by GBM-derived glioma-initiating cells (GIC) induced oligodendroglial differentiation and suppressed self-renewal capacity. By contrast, Nkx2.2 downregulation in mouse NPCs accelerated GBM formation. Importantly, the inhibitory effects of NXK2.2 on GIC self-renewal were conserved in human cells. Thus, our mouse models offer pathobiologically significant advantages to investigate the nature of brain tumors, with improved opportunities to develop novel mechanism-based therapeutic approaches.

Recurrent intracranial esthesioneuroblastoma outside the initial field of radiation with progressive dural and intra-orbital invasion

SummaryA 55-year-old man presented with esthesioneuroblastoma in the right paranasal sinuses and orbita, extending into the right anterior and middle cranial fossa. He received a partial resection of tumour and post-operative radiotherapy, which was set with a central focus on the right orbit. Five years later, he came to our hospital with a complaint of left exophthalmos. Neuro-imaging revealed that the tumour recurred on the opposite side of the primary lesion, which was out side the irradiated field, with progressive invasion of the left temporal dura. The residual tumour in the irradiated field had reduced in size. He received gross total resection and post-operative radiotherapy. We would like to emphasize that radiotherapy is an important adjuvant therapy for esthesioneuroblastoma, and that the field setting for radiation therapy is extremely important.

Nucleostemin in Injury-Induced Liver Regeneration

The high regenerative capacity of liver contributes to the maintenance of its size and function when injury occurs. Partial hepatectomy induces division of mature hepatocytes to maintain liver function, whereas severe injury stimulates expansion of undifferentiated hepatic precursor cells, which supply mature cells. Although several factors reportedly function in liver regeneration, the precise mechanisms underlying regeneration remain unclear. In this study, we analyzed expression of nucleostemin (NS) during development and in injured liver by using transgenic green fluorescent protein reporter (NS-GFP Tg) mice. In neonatal liver, the hepatic precursor cells that give rise to mature hepatocytes were enriched in a cell population expressing high levels of NS. In adult liver, NS was abundantly expressed in mature hepatocytes and rapidly upregulated by partial hepatectomy. Severe liver injury promoted by a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine induced the emergence of NS-expressing ductal epithelial cells as hepatic precursor cells. NS knockdown inhibited both hepatic colony formation in vitro and proliferation of hepatocytes in vivo. These data strongly suggest that NS plays a critical role in regeneration of both hepatic precursor cells and hepatocytes in response to liver injury.

Giant Subependymoma Developed in a Patient with Aniridia: Analyses of PAX6 and Tumor‐relevant Genes

We observed an unusually large subependymoma in a female patient with congenital aniridia. To analyze the genetic mechanisms of tumorigenesis, we first examined the paired box 6 (PAX6) gene using both tumor tissue and peripheral lymphocytes. Tumor suppressor activity has been proposed for PAX6 in gliomas, in addition to its well‐known role in the eye development. Using genomic quantitative PCR and loss of heterozygosity analysis, we identified hemizygous deletions in the 5′‐region of PAX6. In lymphocytes, the deletion within PAX6 spanned from between exons 6 and 7 to the 5′‐upstream region of the gene, but did not reach the upstream gene, RNC1, which is reported to be associated with tumors. The subependymoma had an additional de novo deletion spanning from the intron 4 to intron 6 of PAX6, although we could not completely determine whether these two deletions are on the same chromosome or not. We also examined other potentially relevant tumor suppressor genes: PTEN, TP53 and SOX2. However, we detected no exonic mutations or deletions in these genes. Collectively, we speculate that the defect in PAX6 may have contributed to the extremely large size of the subependymoma, due to a loss of tumor suppressor activity in glial cell lineage.

Cavernous Sinus Dural Arteriovenous Fistula Patients Presenting With Headache as an Initial Symptom

Cavernous sinus (CS) dural arteriovenous fistula (dAVF) patients presenting with only headache as an initial symptom are not common. Patients with CS-dAVF commonly present with symptoms related to their eyes. In all three patients, headache was the initial symptom. Other symptoms related to the eyes developed 1 - 7 months after headache. In one patient, headache was controlled by sumatriptan succinate, but not diclofenac sodium or loxoprofen sodium. In another patient, headache was controlled by loxoprofen sodium. In the third patient, headache was improved by stellate ganglion block. In all patients, magnetic resonance angiography (MRA) in the early stage of the clinical course showed abnormal blood flow in the CS. However, reflux to the superior ophthalmic vein (SOV) was not detected. As treatment, transarterial and transvenous embolizations were necessary for one patient, and transvenous embolization was performed for another patient with significant blood flow to the SOV and cortical veins. On the other hand, manual compression of the bilateral carotid arteries at the neck resulted in disappearance of the fistula in the third patient. In all patients, the symptoms improved after the disappearance of blood reflux to the CS. The refluxed blood to the CS might cause elevation of the CS pressure and stimulate the trigeminal nerve in the dural membrane, resulting in headache before developing reflux in an anterior direction. CS-dAVF could induce both migraine and common headache. In cases with blood reflux to the CS on magnetic resonance imaging and/or MRA even without eye symptoms, a differential diagnosis of CS-dAVF should be taken into consideration.

Pseudoaneurysm formation due to rupture of intracranial aneurysms: Case series and literature review.

Background Intracranial pseudoaneurysm formation due to a ruptured non-traumatic aneurysm is extremely rare. We describe the radiological findings and management of pseudoaneurysms due to ruptured cerebral aneurysms in our case series and previously reported cases. Patients and methods Four additional and 20 reported patients presenting with subarachnoid hemorrhage (SAH) are included. Radiological findings and clinical features of these patients were reviewed. Results In our series, three-dimensional computed tomographic angiography (3D-CTA) and/or angiography showed an irregular- or snowman-shaped cavity extending from the parent artery. The radiological examination additionally revealed delayed filling and retention of contrast medium. These findings were the same as previously reported cases. One patient underwent direct clipping of the true aneurysm. For the other three patients with aneurysms at the basilar and anterior communicating arteries, the true portion of the aneurysm was embolized with platinum coils. During the procedures, care was taken not to insert the coils into the distal pseudoaneurysm portion to prevent rupture. The review of 24 cases revealed that the location of the aneurysms was most frequent in the anterior communicating artery (41.7%), and 86.7% of patients were in a severe stage of SAH (>Grade 3 in WFNS or Hunt & Kosnik grading) implying abundant SAH. Conclusions Pseudoaneurysm formation in SAH after non-traumatic aneurysm rupture is rare. However, in cases with an irregular-shaped aneurysm cavity, pseudoaneurysm formation should be taken into consideration.

Agenesis of the Left Internal Carotid Artery Associated with Dolichoectatic Intracranial Arteries

A 28-year-old man without a significant medical history visited our hospital complaining of a headache. Computed tomography (CT) demonstrated thick, calcified vertebral artery (VA) and basilar artery (BA), despite the patient being young. Magnetic resonance angiography demonstrated the absence of the left internal carotid artery (ICA). The right ICA, the bilateral VA, and the BA were well developed and dolichoectatic. CT revealed the absence of the carotid canal on the left side. The condition was diagnosed as congenital agenesis of the left ICA with dolichoectatic changes in 3 other arteries. In a young patient with thick, calcified intracranial arteries, close examination is necessary, because vascular anomalies such as ICA agenesis may exist.

Late Seizures after Stroke in Clinical Practice: The Prevalence of Non-convulsive Seizures

Objective The prevalence of the non-convulsive type of late seizure after stroke is unknown. The aim of the present study was to clarify the characteristics of late seizure in clinical practice, mainly focusing on the prevalence of non-convulsive seizure. Methods A total of 178 consecutive patients who were admitted and diagnosed with late seizure after stroke were retrospectively enrolled, and the data of 127 patients for whom the complete seizure was observed by a bystander were analyzed. Clinical information was obtained from the medical records and nursing notes. Results A non-convulsive seizure was observed in 37 patients (29%). A focal seizure and its secondary generalization accounted for 79% of the seizure types. Status epilepticus was observed in 60 patients (47%), including 11 patients (9%) without convulsion. The patients with non-convulsive seizures were significantly younger than those with convulsive seizures, but there were no other significant differences between the two groups with respect to sex, classification or the lesion of stroke. Conclusion There was a high rate of non-convulsive seizures in patients with late seizure after stroke. A non-convulsive seizure may be caused by any type or location of preceding stroke. More attention is needed in the differential diagnosis of neurological deterioration after stroke.