Prognostic significance of sirtuin 2 protein nuclear localization in glioma: an immunohistochemical study.

The sirtuin 2 (SIRT2) protein is a member of the sirtuin family and homologous to Sir2 (silent information regulator 2) of Saccharomyces cerevisiae. To assess the pathobiological significance of SIRT2 protein expression and/or subcellular localization in human glioma, we examined SIRT2 protein expression in human gliomas using a polyclonal anti-SIRT2 antibody and immunohistochemistry. In this study, samples from 23 patients with glioblastoma (GB, grade IV), 8 patients with diffuse astrocytoma (DA, grade II) and 5 healthy individuals were examined. We established a SIRT2 labeling index (SIRT2-LI) that represents the percentage of cells with SIRT2 localized to the nucleus. The mean SIRT2-LI was 65.8±18.6 in GB samples, 41.2±22.8 in DA samples, and 28.6±12.3 in normal control samples. The SIRT2-LI of GB samples was significantly higher than that of normal control samples (P<0.01, Mann-Whitney's U-test) and that of DA samples (P<0.05). Moreover, the SIRT2-LI was positively correlated with malignant progression. Specifically, samples from patients with GB were divided into two groups, low SIRT2-LI (<60%) and high SIRT2-LI (≥60%), and the patients with low SIRT2-LI samples survived significantly longer than patients with high SIRT2-LI samples (P<0.05, Kaplan-Meier method and log-rank test). In conclusion, SIRT2-LI was indicative of glioma malignancy, and it may be predictive of GB patient survival.

DNA methylation-mediated silencing of nonsteroidal anti-inflammatory drug-activated gene (NAG-1/GDF15) in glioma cell lines.

Nonsteroidal anti-inflammatory drug-activated gene, NAG-1, a transforming growth factor-ß member, is involved in tumor progression and development. The association between NAG-1 expression and development and progression of glioma has not been well defined. Glioblastoma cell lines have lower basal expression of NAG-1 than other gliomas and normal astrocytes. Most primary human gliomas have very low levels of NAG-1 expression. NAG-1 basal expression appeared to inversely correlate with tumor grade in glioma. Aberrant promoter hypermethylation is a common mechanism for silencing of tumor suppressor genes in cancer cells. In glioblastoma cell lines, NAG-1 expression was increased by the demethylating agent, 5-aza-2'-deoxycytidine. To investigate whether the NAG-1 gene was silenced by hypermethylation in glioblastoma, we examined DNA methylation status using genomic bisulfite sequencing. The NAG-1 promoter was densely methylated in several glioblastoma cell lines as well as in primary oligodendroglioma tumor samples, which have low basal expression of NAG-1. DNA methylation at two specific sites (-53 and +55 CpG sites) in the NAG-1 promoter was strongly associated with low NAG-1 expression. The methylation of the NAG-1 promoter at the -53 site blocks Egr-1 binding and thereby suppresses Nag-1 induction. Treatment of cells with low basal NAG-1 expression with NAG-1 inducer also did not increase NAG-1. Incubation with a demethylation chemical increased Nag-1 basal expression and subsequent incubation with a NAG-1 inducer increased NAG-1 expression. We concluded from these data that methylation of specific promoter sequences causes transcriptional silencing of the NAG-1 locus in glioma and may ultimately contribute to tumor progression.

Primary intramedullary spinal cord germinoma.

A 21-year-old woman presented with an intramedullary spinal cord germinoma and a history of gait disturbance and elimination disorder. Magnetic resonance (MR) imaging demonstrated two isolated lesions, one located within the medulla between T9 and T11, and another at the cauda equina (L2 to L3 levels). After partial reduction of the intramedullary mass, histological findings revealed that the tumor was typical germinoma. Further MR imaging revealed no evidence of intracranial germinoma. Combined chemotherapy (carboplatin and etoposide) and whole spine radiation were performed. Follow-up MR imaging showed that the enhanced mass at the L2-L3 levels had disappeared. No recurrence of the tumor has been detected 3 years after the operation, and no dissemination into the cranial area was detected. Cisplatin and etoposide chemotherapy combined with radiotherapy is recommended for primary spinal germinoma, and is effective for inhibition of both tumor dissemination and recurrence.

Usefulness of non-contrast MRI three dimensional brain surface imaging using a T(2) weighted image.

MR brain surface imaging using a 3D T(1) fast spoiled gradient recalled acquisition in the steady state (FSPGR) sequence with contrast medium is useful for surgical planning. However, this method has several problems such as use of the contrast medium. In this paper we evaluated the usefulness of MR 3D brain surface imaging using a non-contrast enhanced 3D T(2) fast recovery fast spin echo (FRFSE) sequence as a new alternative method. This study includes 5 volunteers who underwent 3D volume rendering (VR) imaging using both a non-contrast enhanced 3D T(1)-SPGR sequence and a non-contrast enhanced 3D T(2)-FRFSE sequence, and 10 patients with brain tumors, who underwent preoperative 3D VR imaging using both a contrast enhanced 3D T(1)-SPGR sequence and a non-contrast enhanced 3D T(2)-FRFSE sequence. Four neurosurgeons assessed the visibility of sulci, gyri, and brain surface veins using a 4-point confidence scale on each VR image. 3D VR imaging using a 3D T(2)-FRFSE sequence was significantly superior to that using a 3D T(1)-SPGR sequence in the visualization of the sulci and gyri (p<0.05). In contrast, there was no significant difference between both sequences in the visualization of the brain surface veins. MR brain surface imaging using a non-contrast 3D T(2)-FRFSE sequence is at least equal or superior to 3D imaging using a contrast enhanced 3D T(1)-SPGR sequence in the preoperative planning.

A gene delivery system with a human artificial chromosome vector based on migration of mesenchymal stem cells towards human glioblastoma HTB14 cells.

Mesenchymal stem cells (MSCs) have been expected to become useful gene delivery vehicles against human malignant gliomas when coupled with an appropriate vector system, because they migrate towards the lesion. Human artificial chromosomes (HACs) are non-integrating vectors with several advantages for gene therapy, namely, no limitations on the size and number of genes that can be inserted. We investigated the migration of human immortalized MSCs bearing a HAC vector containing the herpes simplex virus thymidine kinase gene (HAC-tk-hiMSCs) towards malignant gliomas in vivo. Red fluorescence protein-labeled human glioblastoma HTB14 cells were implanted into a subcortical region in nude mice. Four days later, green fluorescence protein-labeled HAC-tk-hiMSCs were injected into a contralateral subcortical region (the HTB14/HAC-tk-hiMSC injection model). Tropism to the glioma mass and the route of migration were visualized by fluorescence microscopy and immunohistochemical staining. HAC-tk-hiMSCs began to migrate toward the HTB14 glioma area via the corpus callosum on day 4, and gathered around the HTB14 glioma mass on day 7. To test whether the delivered gene could effectively treat glioblastoma in vivo, HTB14/HAC-tk-hiMSC injected mice were treated with ganciclovir (GCV) or PBS. The HTB14 glioma mass was significantly reduced by GCV treatment in mice injected with HAC-tk-hiMSCs. It was confirmed that gene delivery by our HAC-hiMSC system was effective after migration of MSCs to the glioma mass in vivo. Therefore, MSCs containing HACs carrying an anticancer gene or genes may provide a new tool for the treatment of malignant gliomas and possibly of other tumor types.

Application of phase sensitive imaging (PSI) for hemorrhage diagnosis in pituitary adenomas.

OBJECTIVE: Intratumoral hemorrhaging is a common occurrence in pituitary adenomas. Asymptomatic pituitary apoplexies have become more frequently diagnosed due to recent advances in magnetic resonance (MR) imaging. The purpose of this study was to investigate the usefulness of phase sensitive imaging (PSI) in the diagnosis of hemorrhages within pituitary adenomas. PATIENTS AND METHODS: PSI methodology was applied to 28 patients with surgically diagnosed pituitary macroadenomas, and compared with conventional methods. No patients presented with sudden onset of apoplectic symptoms. A 3.0 T MR unit was used to examine all patients before surgery. RESULTS: Seventeen of 28 cases exhibited a hemorrhage component on PSI. However, hemorrhaging was demonstrated in 13 of 28 cases by T2-weighted imaging (T2WI). Hemorrhaging was detected in pituitary adenomas by PSI, but not by T2WI, in four of 17 cases. Poor visualization of the hemorrhage in the sellar turcica was a drawback, which was due to PSI artifacts. CONCLUSION: We suggest that PSI be used to provide diagnostic assistance for pituitary apoplexies.

The cyclooxygenase inhibitor sulindac sulfide inhibits EP4 expression and suppresses the growth of glioblastoma cells.

EP4 expression in human glioblastoma cells correlates with growth on soft agar. The cyclooxygenase inhibitor sulindac sulfide first altered specificity protein-1 (Sp-1) and early growth response gene-1 expression, then increased the expression of nonsteroidal anti-inflammatory drug-activated gene 1 and activating transcription factor 3, and then decreased EP4 expression. EP4 suppression was dependent on blocking the Sp-1 binding sites in the human EP4 promoter. Mutation in the Sp-1 sites in EP4 altered the promoter activity and abolished sulindac sulfide effects. The inhibitory effect of sulindac sulfide on EP4 expression was reversed by PD98059, a mitogen-activated protein/extracellular signal-regulated kinase kinase-1/extracellular signal-regulated kinase inhibitor. Sp-1 phosphorylation was dependent on sulindac sulfide-induced Erk activation. Chromatin immunoprecipitation assay confirmed that Sp-1 phosphorylation decreases Sp-1 binding to DNA and leads to the suppression of EP4. Inhibition of cell growth on soft agar assay was found to be a highly complex process and seems to require not only the inhibition of cyclooxygenase activity but also increased expression of nonsteroidal anti-inflammatory drug-activated gene 1 and activating transcription factor 3 and suppression of EP4 expression. Our data suggest that the suppression of EP4 expression by sulindac sulfide represents a new mechanism for understanding the tumor suppressor activity.

Cerebral ischemia promotes rich pseudopalisading necrosis in the rat c6 glioblastoma model.

The effect of hypoxia on glioma growth including pathological changes was investigated in an experimental model of brain ischemia in the rat C6 glioma model. C6 glioma cells were inoculated into the subcortex of adult Wistar rats. Focal cerebral ischemia near the implanted glioma area was induced by permanent middle cerebral artery occlusion (PMCAO). Ten days later, the rats were sacrificed to compare tumor volume of C6 glioma without PMCAO (control group) versus C6 glioma with PMCAO (hypoxia group). The histological features were also observed. The mean tumor volume in the hypoxia group was significantly larger than that in the control group. The most prominent histological finding in the hypoxia group was abundant formation of pseudopalisading around the necrotic areas. Immunohistological examinations showed intensive staining for vascular endothelial growth factor and hypoxia-inducible factor in these pseudopalisading cells. These findings suggest that cerebral ischemia positively modulates glioma mass growth by the formation of pseudopalisading necrosis, a characteristic histological finding of glioblastoma.

Aberrant promoter methylation and expression of the imprinted PEG3 gene in glioma.

Glioma includes astrocytoma, oligodendroglioma, ependymoma and glioblastoma. We previously reported the epigenetic silencing of paternally expressed gene 3 (PEG3) in glioma cell lines. In this study, we investigated methylation of an exonic CpG island in the promoter region and the expression of PEG3 gene in 20 glioma and 5 non-tumor tissue samples. We found wide variations in the methylation level. Hypomethylaiton and hypermethylation was found in 3 and 4 glioma tissue samples, respectively. Monoallelic expression, which is an evidence of an imprinted gene, was maintained in eight out of nine informative cases which have T/C polymorphisms in PEG3. The lower gene expression, which suggested epigenetic silencing of PEG3, was confirmed statistically in glioblastoma using quantitative reverse-transcription polymerase chain reaction. Interestingly, we found higher expression of PEG3 in two out of three oligodendrogliomas. A negative correlation between the methylation level and gene expression was shown by regression analysis. These results suggest that the abnormal regulation of PEG3 is associated with several glioma subtypes and that it plays an important role in tumorigenesis.

A rat glioblastoma model with diffuse leptomeningeal gliomatosis induced by intracarotid injection of C6 glioma cells.

OBJECTIVE: A reproducible brain tumor model using experimental animals is required to study biological behavior and develop more potent antineoplastic drugs and effective therapeutic modalities. In this work, we attempted to establish diffuse leptomeningeal gliomatosis in the rat by intracarotid injection of C6 glioma cells. METHODS: Intracarotid injection of 1 x 10(7) C6 glioma cells in Wistar rats was performed to establish a primary diffuse leptomeningeal gliomatosis model. Ki-67 and matrix metalloproteinases (MMPs) immunohistochemistry staining were used to study the biological behavior of the developed tumor. Methodology, physical findings and histopathological features were also discussed. RESULTS: Leptomeningeal gliomas grew in all Wistar rats after the administration of 1 x 10(7) C6 glioma cells. Intracranial hypertension, weight loss and cachexia developed, and the median survival time was 18.0 +/- 2.9 days. The glioma mass distributed throughout the ventricles, the leptomeningeal regions in the brain and the brainstem, with typical pathological features of glioblastoma. The immunohistochemistry stainings showed high Ki-67 labeling index (42.1 +/- 10.3%), and concomitant overexpression of MMP-2 and MMP-9 suggested proliferation, invasion and angiogenesis potential. DISCUSSION: The advantage of the intracarotid injection route is the absence of an operative scar in the cranium. This established animal model is a novel model of primary diffuse leptomeningeal gliomatosis. This model probably can be used for pre-clinical testing in the progression of glioblastoma.

Induction of cyclooxygenase-2 expression by interleukin-1beta in human glioma cell line, U87MG.

Cyclooxygenase-2 (COX-2) is up-regulated in most high-grade gliomas, and high COX-2 expression is associated with aggressive character and poor prognosis. However, the effect of COX-2 in human glioma cell lines is not well known. This study examined the effect of several stimuli, including interleukin-1beta (IL-1beta) and carcinogens, on COX-2 induction in normal astrocyte cells and human glioma cell lines U87MG, A172, and T98G. IL-1beta-induced COX-2 expression strongly at both protein and messenger ribonucleic acid levels in only the U87MG cells of the glioma cell lines. Furthermore, carcinogen induced COX-2 expression. Similar findings were also observed in normal human astrocyte cells. The U87MG glioma cell line is a good model for COX-2 induction in glioma cell lines.

Nonsteroidal anti-inflammatory drug-activated gene (NAG-1/GDF15) expression is increased by the histone deacetylase inhibitor trichostatin A.

Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is a putative tumor suppressor whose expression can be increased by drug treatment. Glioblastoma is the most common central nervous system tumor, is associated with high morbidity and mortality, and responds poorly to surgical, chemical, and radiation therapy. The histone deacetylase inhibitors are under current consideration as therapeutic agents in treating glioblastoma. We investigated whether trichostatin A (TSA) would alter the expression of NAG-1 in glioblastoma cells. The DNA demethylating agent 5-aza-dC did not increase NAG-1 expression, but TSA up-regulated NAG-1 expression and acted synergistically with 5-aza-dC to induce NAG-1 expression. TSA indirectly increases NAG-1 promoter activity and increases NAG-1 mRNA and protein expression in the T98G human glioblastoma cell line. TSA also increases the expression of transcription factors Sp-1 and Egr-1. Small interfering RNA experiments link NAG-1 expression to apoptosis induced by TSA. Reporter gene assays, specific inhibition by small interfering RNA transfections, and chromatin immunoprecipitation assays indicate that Egr-1 and Sp-1 mediate TSA-induced NAG-1 expression. TSA also increases the stability of NAG-1 mRNA. TSA-induced NAG-1 expression involves multiple mechanisms at the transcriptional and post-transcriptional levels.

Basal ganglia germinoma: diagnostic value of MR spectroscopy and (11)C-methionine positron emission tomography.

We herein report a 12-year-old girl with a basal ganglia germinoma who presented with right-sided hemiparesis after a minor head trauma. Magnetic resonance (MR) imaging revealed a minimally enhanced lesion involving the left putamen, thalamus, and corona radiata. The lesion showed low-signal intensity on T1-, and high intensity on T2- and diffusion-weighted imaging. The MR signal in the adjacent globus pallidum was also low on T2-weighted imaging. MR spectroscopy on the lesion showed a large lactate/lipid/macromolecule peak with a decreased NAA/Cr ratio, but no increase in the Cho/Cr ratio. However, posttraumatic infarction at the territory of lateral lenticulostriate artery was ruled out 1 month later. This was based on progression of the hemiparesis and neuroimaging results, including an increased Cho/Cr ratio and weak uptake on (11)C-methionine positron emission tomography of the basal ganglia lesion. Stereotaxic brain biopsy confirmed the diagnosis of germinoma.

Regulation of EP4 expression via the Sp-1 transcription factor: inhibition of expression by anti-cancer agents.

For glioblastomas, COX-2 expression is linked to poor survival. COX-2 effects are mediated by the receptors EP2 and EP4, whose regulation is poorly understood. The expression of EP4, and activation or inhibition of EP4 activity in human glioblastoma T98G cells, was found to correlate with growth on soft agar. Chemoprevention drugs, troglitazone (TGZ) and some COX inhibitors, significantly suppressed EP4 expression in T98G cells in a dose dependant manner. Specificity protein 1 (Sp-1) binding sites, located within region -197 to -160 of the human EP4 promoter, are important for the transcription initiation of the human EP4 gene and are responsible for the EP4 suppression by TGZ. Mutation in the Sp-1 sites altered the promoter activity of luciferase constructs and TGZ effects on the promoter. The inhibitory effect of TGZ on EP4 expression was reversed by PD98059, a MEK-1/Erk inhibitor. Immunoprecipitation-Western blot analysis detected Sp-1 phosphorylation that was dependent on TGZ-induced Erks activation. ChIP assay confirmed that Sp-1 phosphorylation decreases its binding to DNA and as a result, leads to the suppression of EP4 expression. Thus, we propose that the expression of EP4 is regulated by Sp-1, but phosphorylation of Sp-1 induced by TGZ suppresses this expression. This represents a new and unique mechanism for the regulation of the EP4 receptor expression.

Metastasis of carcinoid to the arch of the axis in a multiple endocrine neoplasia patient: a case report.

BACKGROUND CONTEXT: Carcinoid tumors eventually metastasize to the spine, and epidural spinal cord compression is a relatively frequent neurologic complication of carcinoid. However, a case of multiple endocrine neoplasia type 1 (MEN1) presenting with spinal cord compression as a result of a metastatic carcinoid tumor has not been reported previously. PURPOSE: To report an extremely rare case of MEN1 presenting with spinal cord compression by metastatic carcinoid tumor. STUDY DESIGN: Case report. METHODS: A 51-year-old man, with a past history of thymoma, insulinoma, and gastric carcinoid presented with neck pain. Neuroradiological examination revealed that a tumor around the arch of the axis compressed the spinal cord with osteoblastic changes. RESULTS: After hemilaminectomy of the axis and removal of the tumor followed by irradiation, the patient returned to his previous job. Histological examination confirmed metastatic carcinoid tumor. CONCLUSIONS: Spinal metastasis of carcinoid tumor occurred in a multiple endocrine neoplasia patient, and it is significant to note that carcinoid metastasis is one of differential diagnoses for osteoblastic lesions.

Establishment of experimental glioma models at the intrinsic brainstem region of the rats.

OBJECTIVES: As the treatment of human intrinsic brainstem gliomas remains challenging, experimental glioma models are needed. METHODS: We developed a rat model of intrinsic brain stem glioma that uses a stereotactic frame to fix the head for the delivery of C6 glioma cells to target sites via a permanently implanted cannula. We inoculated the rat midbrain, pons or cerebral cortex with 5 x 10(4) cells suspended in 1 microl culture medium over the course of 2 minutes. RESULTS: Three days post-implantation, tumor formation was visible in the periaqueductal gray matter in the midbrain and the tegmentum of the pons. On the tenth day, the tumor diameter exceeded over 2 mm; there was no tumor cell seeding into the cerebrospinal fluid space. The tumor manifested the histological features typical of glioblastoma; Ki-67 labeling index was 32%. DISCUSSION: Because in our model the cannula is permanently implanted, additional inocula can be delivered. Here we detail our rat brainstem glioma model and discuss its usefulness for the investigation of these tumor in humans.

Cerebral venous sinus thrombosis associated with iron deficiency: two case reports.

Two patients presented with cerebral venous sinus thrombosis (CVST) associated with iron deficiency. A 14-year-old man had thrombosis extending from the end of the superior sagittal sinus to the left transverse sinus. Severe dehydration after competitive sport had induced CVST. The laboratory findings showed severe iron deficiency anemia which persisted for 1 year. A 47-year-old man had thrombosis in the entire superior sagittal sinus. Dehydration caused by poor nutrition had induced CVST. The laboratory findings showed transient iron deficiency in the acute phase. Both patients received conservative treatment for dehydration and iron supplementation. These two cases indicate that iron deficiency is a risk factor for CVST.

Relationship of hypotension and cerebral vasospasm in patients with aneurysmal subarachnoid hemorrhage.

OBJECTIVES: Changes in systemic arterial blood pressure and the degree of cerebral vasospasm were investigated in 125 patients with aneurysmal subarachnoid hemorrhage. METHODS: Systemic arterial blood pressure was measured every 2 hours in each patient for a period of more than 2 weeks, and a fall in systemic blood pressure (FBP) was defined as a decrease of >40 mmHg of systolic blood pressure between two consecutive measurements. RESULTS: A total of 91 FBPs occurred in 52 (41.6%) of 125 patients despite specific post-operative management to prevent hypovolemia. Five (5.5%) of the 91 FBPs occurred just before the onset of symptomatic vasospasm. Symptomatic vasospasm was observed in 36 (69.2%) of 52 patients with FBP and in 32 (43.8%) of 73 patients without FBP (p<0.01, chi-squared test). A hypodense area on computed tomographic scans in association with cerebral vasospasm was observed in 25 (48.1%) of 52 patients with FBP and in 21 (28.8%) of 73 patients without FBP (p<0.05). DISCUSSION: We conclude that FBP might result from delayed cerebral vasospasm and/or brain dysfunction owing to subarachnoid hemorrhage itself.

A case of Tolosa-Hunt syndrome affecting both the cavernous sinuses and the hypophysis, and associated with C3 and C4 aneurysms.

BACKGROUND: We describe the first case of Tolosa-Hunt syndrome that is associated with C3 and C4 aneurysms. CASE DESCRIPTION: The patient, a female aged 58 years, had diplopia and right retroorbital pain. Magnetic resonance imaging revealed an enlargement of the hypophysis and bilateral cavernous sinuses, particularly on the right side. Cerebral angiography demonstrated ICA aneurysms of the left C3 and right C4 portions. These symptoms were immediately alleviated after initiation of prednisolone therapy, but recurred after the dose was tapered off. Radiological examination revealed an enlargement of the hypophysis whereas the right C4 aneurysm had decreased in size and no blood flow was apparent in the ipsilateral ophthalmic artery. A biopsy was performed and the results showed a focal inflammatory change. Steroid therapy was represcribed, and after a follow-up period of 10 months without therapy, the patient has been free of symptoms. CONCLUSION: We conclude that bilateral ICA aneurysms might be directly induced by inflammatory infiltration into intracavernous ICAs.