Tomokazu Aoki

Isolation of a novel mouse gene MA-3 that is induced upon programmed cell death.

Typical programmed cell death requires de novo macromolecular synthesis and shares common morphological changes referred to as apoptosis. To elucidate the molecular mechanism of apoptosis, we isolated cDNA clones that are induced in various types of apoptosis by the differential display method. Among such clones, the MA-3 mRNA was induced in all apoptosis-inducible cell lines tested so far, including thymocytes, T cells, B cells and pheochromocytoma. The nucleotide sequence of the MA-3 cDNA predicted an amino acid (aa) sequence of 469 aa, which did not reveal significant similarity to any known proteins and functional aa motifs in databases. The MA-3 mRNA was strongly expressed in the thymus although small amounts of the MA-3 mRNA were ubiquitously expressed in mouse adult tissues. The MA-3 gene was highly conserved during evolution and cross-hybridization bands were found not only in vertebrates but also in Drosophila melanogaster.

Expression of Interleukin-1β Converting Enzyme Gene Family and bcl-2 Gene Family in the Rat Brain following Permanent Occlusion of the Middle Cerebral Artery

Recent investigations have been suggesting that some neuronal subpopulations may die via programmed cell death after focal ischemic injury. To clarify the possible roles of the genes involved in the cell-death program, this study examined the expression of three members of the interleukin-1β converting enzyme (Ice) gene family (Ice, Nedd2, and Yama/CPP32) and two members of the bcl-2 gene family (bcl-2 and bcl-x) in the rat brain after permanent occlusion of the middle cerebral artery. Northern blot analysis revealed a transient induction of Nedd2 mRNA 8 h after the ischemic insult (3.8-fold) and an increase in Yama/CPP32 mRNA 16 to 24 h after the insult (5.8-fold at 24 h), whereas the expression of Ice remained constant. The expression of bcl-2 and bcl-x remained constant after the ischemic insult. Taking into account the key role of the Ice gene family in the execution of programmed cell death, the induction of Ice gene family might play a causative role in apoptotic cell death.

Conservation of the biochemical mechanisms of signal transduction among mammalian Notch family members

Mouse Notch1, which plays an important role in cell fate determination in development, is proteolytically processed within its transmembrane domain by unidentified γ-secretase-like activity that depends on presenilin. To study this proteolytic event, we established a cell-free Notch cleavage assay system using the membrane fraction of fibroblast transfectants of various Notch constructs with deletion of the extracellular portion (Notch ΔE). The cytoplasmic portion of Notch1 ΔE was released from the membrane upon incubation at 37°C, which was inhibited by the specific γ-secretase inhibitor, MW167, or by overexpression of dominant negative presenilin1. Likewise, other members of mouse Notch family were proteolytically cleaved in a presenilin-dependent, MW167-sensitive manner in vivo as well as in the cell-free Notch ΔE cleavage assay system. All four members of the mouse Notch family migrated to the nucleus and activated the transcription from the promoter carrying the RBP-J consensus sequences after they were released from the membrane. These results demonstrate the conserved biochemical mechanism of signal transduction among mammalian Notch family members.

Phase II Study of Single-agent Bevacizumab in Japanese Patients with Recurrent Malignant Glioma

Objective This single-arm, open-label, Phase II study evaluated the efficacy and safety of single-agent bevacizumab, a monoclonal antibody against vascular endothelial growth factor, in Japanese patients with recurrent malignant glioma. Methods Patients with histologically confirmed, measurable glioblastoma or World Health Organization Grade III glioma, previously treated with temozolomide plus radiotherapy, received 10 mg/kg bevacizumab intravenous infusion every 2 weeks. The primary endpoint was 6-month progression-free survival in the patients with recurrent glioblastoma. Results Of the 31 patients enrolled, 29 (93.5%) had glioblastoma and 2 (6.5%) had Grade III glioma. Eleven (35.5%) patients were receiving corticosteroids at baseline; 17 (54.8%) and 14 (45.2%) patients had experienced one or two relapses, respectively. The 6-month progression-free survival rate in the 29 patients with recurrent glioblastoma was 33.9% (90% confidence interval, 19.2–48.5) and the median progression-free survival was 3.3 months. The 1-year survival rate was 34.5% with a median overall survival of 10.5 months. There were eight responders (all partial responses) giving an objective response rate of 27.6%. The disease control rate was 79.3%. Eight of the 11 patients taking corticosteroids at baseline reduced their dose or discontinued corticosteroids during the study. Bevacizumab was well-tolerated and Grade ≥3 adverse events of special interest to bevacizumab were as follows: hypertension [3 (9.7%) patients], congestive heart failure [1 (3.2%) patient] and venous thromboembolism [1 (3.2%) patient]. One asymptomatic Grade 1 cerebral hemorrhage was observed, which resolved without treatment. Conclusion Single-agent bevacizumab provides clinical benefit for Japanese patients with recurrent glioblastoma.

Apoptosis induced by selenium in human glioma cell lines

Several studies have shown that selenium can inhibit tumorigenesis in tissues. However, little is known about the mechanism and the effect of selenium on DNA, especially in brain tumor cells. In this study we examined the biological effect of selenium on human glioma cell lines (A172 and T98G). Selenium exhibited an antiproliferative effect on these cell lines (and induced the typical ladder pattern of DNA fragmentation commonly found in apoptosis), which were prevented by catalase. Few effects of selenium on NTI4 fibroblasts were found. These findings demonstrate that selenium may induce, by apoptosis, cell death of human glioma cell lines, which are resulting from free radical oxygen forming.

Management of glioblastoma.

Glioblastoma multiforme (GBM) are among the most devastating neoplasms claiming the lives of patients within a median of 1 year after diagnosis. Treatment of GBM requires a multidisciplinary approach. Treatments include surgery, radiotherapy, chemotherapy and so on. Temozolomide (TMZ) has emerged as an active agent against malignant gliomas. On the basis of the work by the European Organisation for Research and Treatment of Cancer/National Cancer Institute of Canada, concurrent radiotherapy and the oral alkylating agent TMZ followed by adjuvant TMZ has become the standard of care for patients with newly diagnosed GBM, although the methylation status of the O6-mehylguanine-DNA methyltransferase promoter is predictive for survival of GBM patients. Gliadel is a biodegradable polymer wafer impregnated with carmustine. Gliadel has been one of the few treatment modalities to demonstrate a statistical benefit in patients with malignant glioma. These new FDA approved drugs advanced the treatment of malignant glioma, but more progress is needed. Patients require improvements in chemotherapy, surgery, radiotherapy, molecular targeted therapy, immunotoxin using the convection-enhanced delivery and more.

A Multicenter Phase I/II Study of the BCNU Implant (Gliadel ® Wafer) for Japanese Patients with Malignant Gliomas

Carmustine (BCNU) implants (Gliadel® Wafer, Eisai Inc., New Jersey, USA) for the treatment of malignant gliomas (MGs) were shown to enhance overall survival in comparison to placebo in controlled clinical trials in the United States and Europe. A prospective, multicenter phase I/II study involving Japanese patients with MGs was performed to evaluate the efficacy, safety, and pharmacokinetics of BCNU implants. The study enrolled 16 patients with newly diagnosed MGs and 8 patients with recurrent MGs. After the insertion of BCNU implants (8 sheets maximum, 61.6 mg BCNU) into the removal cavity, various chemotherapies (including temozolomide) and radiotherapies were applied. After placement, overall and progression-free survival rates and whole blood BCNU levels were evaluated. In patients with newly diagnosed MGs, the overall survival rates at 12 months and 24 months were 100.0% and 68.8%, and the progression-free survival rate at 12 months was 62.5%. In patients with recurrent MGs, the progression-free survival rate at 6 months was 37.5%. There were no grade 4 or higher adverse events noted due to BCNU implants, and grade 3 events were observed in 5 of 24 patients (20.8%). Whole blood BCNU levels reached a peak of 19.4 ng/mL approximately 3 hours after insertion, which was lower than 1/600 of the peak BCNU level recorded after intravenous injections. These levels decreased to less than the detection limit (2.00 ng/mL) after 24 hours. The results of this study involving Japanese patients are comparable to those of previous studies in the United States and Europe.

Phase II study of ifosfamide, carboplatin, and etoposide in patients with a first recurrence of glioblastoma multiforme

OBJECT The prognosis of recurrent glioblastoma multiforme (GBM) remains unsatisfactory. The authors conducted a Phase II study of ifosfamide, carboplatin, and etoposide (ICE) for a first recurrence of GBM to determine whether it prolonged a patients good-quality life. METHODS This trial was an open-label, single-center Phase II study. Forty-two patients with a first GBM relapse after surgery followed by standard radiotherapy (60 Gy) and first-line temozolomide- or nimustine-based chemotherapy were eligible to participate. The primary end point was progression-free survival at 6 months after the ICE treatment (PFS-6), and secondary end points were response rate, toxicity, and overall survival. Chemotherapy consisted of ifosfamide (1000 mg/m(2) on Days 1, 2, and 3), carboplatin (110 mg/m(2) on Day 1), etoposide (100 mg/m(2) on Days 1, 2, and 3), every 6 weeks. RESULTS Progression-free survival at 6 months after ICE treatment was 35% (95% CI 22-50%). The median duration of PFS was 17 weeks (95% CI 10-24 weeks). The response rate was 25% (95% CI 9-34%). Adverse events were generally mild and consisted mainly of alopecia. CONCLUSIONS This regimen was well tolerated and has some activity and could be one of the options for patients with recurrent GBM.

Expression of heat shock proteins in the developing rat retina

Expression of three heat shock proteins (HSPs), HSP70, HSP90, and immunoglobulin heavy chain binding protein (Bip) was examined in the developing rat retina using Northern blot analysis. The expression of the inducible form of HSP70 remained uniformly low throughout the perinatal period until P5 and increased rapidly at P7. On the other hand, the constitutive form of HSP70, HSP90, and Bip were expressed constitutively in the rat retina throughout the developmental stage except P3-P5, at which a transient decrease of the expression was observed. The increase of inducible HSP70 mRNA at P7 may correspond to the functional maturation of photoreception in the visual nervous system and may be one of the stress responses to photostimulation. The potential roles of each HSP during development of the rat visual system are discussed.

Advantages of high b-value diffusion-weighted imaging to diagnose pseudo-responses in patients with recurrent glioma after bevacizumab treatment

BACKGROUND The diagnosis of pseudo-responses after bevacizumab treatment is difficult. Because diffusion-weighted imaging (DWI) is associated with cell density, it may facilitate the differentiation between true- and pseudo-responses. Furthermore, as high b-value DWI is even more sensitive to diffusion, it has been reported to be diagnostically useful in various clinical settings. MATERIALS AND METHODS Between September 2008 and May 2011, 10 patients (5 males, 5 females; age range 6-65 years) with recurrent glioma were treated with bevacizumab. All underwent pre- and post-treatment MRI including T2- or FLAIR imaging, post-gadolinium contrast T1-weighted imaging, and DWI with b-1000 and b-4000. Response rates were evaluated by MacDonald- and by response assessment in neuro-oncology working group (RANO) criteria. We also assessed the response rate by calculating the size of high intensity areas using high b-value diffusion-weighted criteria. Prognostic factors were evaluated using Kaplan-Meier survival curves (log-rank test). RESULTS It was easier to identify pseudo-responses with RANO- than MacDonald criteria, however the reduction of edema by bevacizumab rendered the early diagnosis of tumor progression difficult by RANO criteria. In some patients with recurrent glioma treated with bevacizumab, high b-value diffusion-weighted criteria did, while MacDonald- and RANO criteria did not identify pseudo-responses at an early point after the start of therapy. DISCUSSION AND CONCLUSION High b-value DWI reflects cell density more accurately than regular b-value DWI. Our findings suggest that in patients with recurrent glioma, high b-value diffusion-weighted criteria are useful for the differentiation between pseudo- and true responses to treatment with bevacizumab.