Takashi Tamiya

Proton magnetic resonance spectroscopy reflects cellular proliferative activity in astrocytomas.

Abstract We examined whether proton magnetic resonance spectroscopy (MRS) could provide accurate information on histological grade and cell proliferation in astrocytomas. We studied 23 patients with astrocytomas: five grade II, 10 grade III and eight with grade IV (glioblastoma multiforme). We performed proton MRS and determined the Ki-67 labeling index (LI), a tumour proliferation marker, in the same areas of the astrocytomas, and examined the statistical relationship between proton MRS and Ki-67 LI. The N-acetylaspartate (NAA)/creatine-phosphocreatine (Cr) and NAA/choline (Cho)-containing compound ratios were always significantly lower and the Cho/Cr ratios significantly higher than those for normal brain. The Cho/Cr ratio correlated positively and the NAA/Cho ratio inversely with Ki-67 LI. These findings suggest that the Cho signal in proton MRS reflects cellular proliferation. In Kaplan-Meier survival analysis, there was no significant difference between high (> 2.0, 14 cases) and low (< 2.0, 9 cases) Cho/cr ratio groups.

Peritumoral brain edema in intracranial meningiomas: effects of radiological and histological factors.

OBJECTIVEWe examined the radiological and histological features influencing the development of peritumoral brain edema (PTBE) among patients with meningiomas. METHODSFactors causing PTBE were retrospectively analyzed for 125 patients with primary intracranial meningiomas. These factors included tumor size, tumor location, brain-tumor interface, signal intensity on T2-weighted scans, contrast enhancement, and cyst formation (as observed on magnetic resonance imaging scans), as well as tumor vascularity and blood supply (as observed in digital subtraction angiography studies). We defined the edema/tumor volume ratio as the edema index, and we used this index to evaluate PTBE. RESULTSA relationship between the tumor size and the volume of PTBE was observed. Convexity and middle fossa meningiomas demonstrated the greatest increases in mean edema indices. Meningothelial, anaplastic, microcystic, and angiomatous subtypes exhibited higher edema indices than did other types. Multivariate analysis demonstrated two significant radiological factors: cortical penetration (as defined by the disappearance of the arachnoid layer on magnetic resonance imaging scans) (relative risk, 2.067;P = 0.0148) and vascular supply from the pial-cortical arteries (as observed on angiograms) (relative risk, 2.087;P = 0.0082). CONCLUSIONTumor infiltration into adjacent brain parenchyma and a pial-cortical blood supply are critical factors for the development of PTBE among patients with meningiomas.

Malignant astrocytomas with homozygous CDKN2/p16 gene deletions have higher Ki-67 proliferation indices

p16 is involved in a cell-cycle regulatory cascade that includes cyclin-dependent kinase 4 (cdk4), cyclin D1 and pRb. Alterations of each of these components have been described in primary human glioblastoma multiforme (GBM) or GBM cell lines, and alterations of the individual components of this pathway appear inversely correlated with one another. While this suggests that disruption of any individual component has similar oncogenic effects, homozygous deletions of the CDKN2/p16 gene are the most common genetic alteration. We investigated the relationship between homozygous CDKN2/ p16 deletions and cellular proliferation in 50 primary astrocytomas (2 WHO grade I pilocytic astrocytoma, 15 grade II astrocytomas, 20 grade III anaplastic astrocytomas and 13 grade IV GBMs). Using a comparative multiplex PCR assay, homozygous deletions of the CDKN2/p16 gene were detected in 5 anaplastic astrocytomas (25%) and 6 GBMs (46%), but in none of the lower-grade tumors. Ki-67 immunohistochemistry was used to assess the number of proliferating cells in the same samples used for molecular genetic analysis. In both anaplastic astrocytomas and GBMs, Ki-67 proliferation indices were significantly higher in tumors with CDKN2/p16 deletions (20%) than in those without deletions (10%; p = 0.0001). These results suggest that homozygous CDKN2/p16 deletions in high-grade astrocytomas may have a more deleterious effect on cell cycle control than the other aberrations in the p16-cdk4-cyclin D1-pRb pathway, and may provide one explanation for why homozygous CDKN2/p16 deletions are more common genetic events in high-grade astrocytomas than RB mutations or CDK4 amplification.

Emergent endovascular treatment of ruptured vertebral artery dissecting aneurysms

The goal of this study was to evaluate the results of endovascular and surgical treatments for ruptured vertebral artery dissecting aneurysms (VADAs) to determine which treatment is preferable. We evaluated the cases of 25 consecutive patients with ruptured VADAs treated in our institution. From 1992 to 1997, five patients were treated surgically. Since 1998, 20 patients with VADAs have been treated with endovascular therapy. The goal of the treatment was to exclude the aneurysm from the circulation. Among the five patients undergoing surgery, three aneurysms were treated with proximal clipping, one with trapping, and one with dome clipping. None of the patients were treated during the acute stage of rupture. Transient complications occurred in two patients. Of the 20 patients treated through the endovascular approach, 15 were treated within 24xa0h of rupture, but 12 had rebleeding before treatment. Eighteen aneurysms were occluded, along with the affected vertebral artery (VA), by using detachable coils (internal trapping), and one was occluded with the VA preserved. A stent-assisted occlusion of one aneurysm was done in a patient who had a contralateral hypoplastic VA. In both groups, the outcome of each patient depended greatly on the patient’s condition before treatment and whether there was rebleeding. No posttreatment bleeding occurred. All procedures were effective, but endovascular treatment was less invasive and easier to use during the acute stage of subarachnoid hemorrhage. Although this report does not describe a controlled study, we found that endovascular treatment is preferable for treating ruptured VADAs in the acute stage.

Experimental gene therapy for brain tumors using adenovirus-mediated transfer of cytosine deaminase gene and uracil phosphoribosyltransferase gene with 5-fluorocytosine.

Transduction of the cytosine deaminase (CD) gene into tumor cells followed by administration of 5-fluorocytosine (5-FC), called 5-FC/CD gene therapy, was created as suicide gene therapy for various cancers. The uracil phosphoribosyltransferase (UPRT) gene, which is absent from mammalian cells, directly converts 5-fluorouracil (5-FU) to 5-fluorouridine 5-monophosphate. We evaluated whether the coexpression of CD and UPRT genes could generate a synergistic antitumor effect on experimental brain tumors. In vitro study showed that 9L cells, transduced with the UPRT gene by an adenovirus, were 16 times more sensitive to 5-FU, and CD + UPRT-transduced cells were 6,000 times more sensitive to 5-FC than parent cells, indicating that the acquisition of CD and UPRT further increased the 5-FC sensitivity of 9L cells compared with cells transduced with CD alone. In a rat brain tumor model, decreased amounts of CD and UPRT vectors were inoculated into the tumors to detect any additional effect of UPRT. CD and UPRT coexpression followed by 5-FC administration showed an antitumor effect as detected by sequential magnetic resonance imaging. This therapy significantly prolonged animal survival. These results suggest that 5-FC/CD + UPRT gene therapy can enhance the antitumor effect of 5-FC/CD gene therapy. Consequently, this approach might be a more feasible modality for the treatment of malignant brain tumors.

In vivo efficacy and toxicity of 5-fluorocytosine/cytosine deaminase gene therapy for malignant gliomas mediated by adenovirus.

We evaluated the therapeutic efficacy and neurotoxicity of adenovirus-mediated transduction of the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) for experimental malignant brain tumors. The 5-FC sensitivity in 9 L cells infected by an adenovirus vector expressing CD (AdexCACD) was increased 1700-fold compared with control cells. Rats bearing 9 L brain tumors were treated with an intratumoral injection of AdexCACD followed by intraperitoneal administration of 5-FC. The rats demonstrated remarkable inhibition of tumor growth by magnetic resonance imaging, and 7 of 10 rats survived for >90 days. To evaluate the potential side-effects of the 5-FC/CD gene therapy, rats were treated with an intracerebral injection of AdexCACD into the right basal ganglia and with 5-FC. The magnetic resonance imaging showed a highly enhanced area on the gadollinium-enhanced T1-weighted image at 18 days postinjection. Pathologically, this corresponded to an area of necrosis with surrounding apoptotic cells. In addition, there was demyelination and gliosis with enlargement of the lateral ventricles. These results suggest that the 5-FC/CD gene therapy may provide an anticancer effect for malignant brain tumors in humans, but also show that there are neurotoxic effects on normal brain tissue.

The relationship between peritumoral brain edema and the expression of vascular endothelial growth factor and its receptors in intracranial meningiomas

We examined the radiological and histological features of, and the influences of the expression of VEGF and its two major receptors, Flt-1 and Flk-1, on the development of peritumoral brain edema (PTBE) in patients with intracranial meningiomas. The expressions of VEGF and VEGF receptors in the immunohistochemical study were analyzed in relation to several factors, including tumor size, location, vascularity, and blood supply, as seen on digital subtraction angiographic studies. The edema volume (P = 0.0003) and edema index (P < 0.0001) had a significantly positive relation to VEGF expression. The positivity of Flt-1 and Flk-1 was mainly observed in tumor vessels; 44 cases (37.2%) were positive for the Flt-1 antibody and 37 cases (31.4%) for the Flk-1 antibody. The mean value of the edema index of the positive-Flt-1 group (5.220 ± 11.586) was significantly higher than that of the negative-Flt-1 group (1.782 ± 2.559) (P < 0.0001). The mean value of the edema index of the positive-Flk-1 group (3.925 ± 5.870) was slightly higher than that of the negative-Flk-1 group (2.671 ± 8.136) (P < 0.0001). Our data suggest that the expressions of VEGF and VEGF receptors positively relate to each other and to the formation of PTBE in patients with meningiomas.

Involvement of activin/BMP system in development of human pituitary gonadotropinomas and nonfunctioning adenomas

Roles of activin/bone morphogenetic protein (BMP) system in the pathogenesis of human pituitary adenoma remain unknown although these factors stimulate follicle-stimulating hormone (FSH) secretion in the normal pituitary. Here we demonstrated that type-I and -II subunit mRNAs of activin/BMP receptors are expressed in Pit-1-negative FSH-producing (FSH-oma) and nonfunctioning pituitary adenomas (NF-oma). Basal levels of serum FSH standardized by luteinizing hormone (LH) were markedly high in FSH-omas in contrast to NF-omas. However, gonadotropin-releasing hormone (GnRH)-induced increment of FSH standardized by that of LH was not changed in FSH-omas, suggesting that imbalanced FSH secretion by FSH-oma is not attributable to GnRH regardless of the expression of GnRH receptor. Although activin betaA subunit was detected in neither adenoma, the betaB subunit was expressed highly in FSH-omas and, to lesser extent, in NF-omas. As for BMPs, BMP-6 and -7 were detected in NF-omas while BMP-4 and -15 were not detected in either type of adenoma. In the presence of pituitary activin/BMP system, the levels of co-expressing follistatin mRNA in the tumors were reduced in FSH-oma compared with NF-oma, suggesting that endogenous follistatin is involved in FSH overproduction through inhibition of activin/BMP system independently of GnRH.

Inhibition of Poly(ADP-Ribose) Polymerase Attenuates Cerebral Vasospasm After Subarachnoid Hemorrhage in Rabbits

Background and Purpose— Poly(ADP-ribose) polymerase (PARP) is important in modulating inflammation, which has been implicated in cerebral vasospasm after subarachnoid hemorrhage (SAH). We investigated the role of PARP in vasospasm using 3-aminobenzamide (3-AB), a PARP inhibitor, in a rabbit model. Methods— Twenty-four New Zealand White rabbits were divided into 4 groups: (1) no treatment (control group, n=6); (2) blood injection without pretreatment (SAH-only group, n=6); (3) blood injection with pretreatment by vehicle (SAH+vehicle group, n=6); and (4) blood injection with pretreatment by 3-AB (SAH+3-AB group, n=6). We used the single-hemorrhage model of SAH, injecting autologous arterial blood into the cisterna magna. Angiography was performed before (baseline) and after (day 2) SAH, and the diameter of the basilar artery (BA) was measured. Animals were euthanatized after the second angiogram. After perfusion and fixation, the brains were cut into sections for hematoxylin and eosin and immunohistochemical staining for poly(ADP-ribosyl)ation. Results— In the control group, there were no differences in the BA lumen caliber between baseline and day 2 (96.8±10.4%). Cerebral vasospasm in the SAH+3-AB group (88.2±6.2%) was remarkably attenuated in comparison with that in the SAH-only group (64.9±8.0%) and the SAH+vehicle group (65.6±10.8%). The BA in the SAH+3-AB group showed less corrugation of the tunica elastica interna than that in the SAH-only and SAH+vehicle groups. Staining for poly(ADP-ribosyl)ation was markedly inhibited in smooth muscle and adventitial cells of the BA in the SAH+3-AB group compared with other groups. Conclusions— Inhibiting ADP-ribosylation attenuates cerebral vasospasm after SAH in rabbits, and PARP activation may play an important role in the development of cerebral vasospasm.

Apoptosis induction with 5-fluorocytosine/cytosine deaminase gene therapy for human malignant glioma cells mediated by adenovirus

Previously, we evaluated the therapeutic efficacy of the adenovirus-mediated transduction of the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) for malignant gliomas. However, the molecular pathways that mediate the 5-FC/CD gene therapy-induced cell death remains to be elucidated. In this study, we examined the induction of apoptosis and the role of caspases in 5-FC/CD gene therapy using human malignant glioma cells [Gli36Δ5 (mutated p53) and U87MG (wild p53)]. The treatment with 5-FC/CD gene-therapy-induced apoptosis both in Gli36Δ5 cells and in U87MG cells according to flow cytometric analysis. Immunoblot analysis revealed that caspases 3 and 9 were processed in response to 5-FC/CD in a concentration- and time-dependent manner, but caspase 8 was not. Each caspase 3 and 9 inhibitor significantly reduced apoptosis triggered by 5-FC/CD, but the caspase 8 inhibitor did not affect apoptosis induction. 5-FC/CD significantly promoted the release of cytochorme c from mitochondria in a concentration-dependent manner. These results indicate that 5-FC/CD gene therapy induces apoptosis in human malignant glioma cells and that the apoptotic cell death is mediated by the activation of mitochondrial caspase cascades involving caspases 3 and 9. This is the first report concerning the apoptotic mechanism of 5-FC/CD gene therapy, and these findings could be used to increase the efficacy of suicide gene therapy systems for the treatment of malignant glioma.