Takuyu Taki

Cell cycle-dependent expression of the mouseRad51 gene in proliferating cells

The mouseRad51 gene is a mammalian homologue of theEscherichia coli recA and yeastRAD51 genes, both of which are involved in homologous recombination and DNA repair in mitosis and meiosis. The expression of mouseRad51 mRNA was examined in synchronized mouse m5S cells. TheRad51 transcript was observed from late G1 phase through to M phase. During the period of late G1-S-G2, the RAD51 proteins were observed exclusively in nuclei. Activation by mitogens of T cell and B cell proliferation in spleen induced the expression ofRad51 mRNA. By immunohistochemical analyses, the mouse RAD51 protein was detected in proliferating cells: spermatogonia in testis, immature T cells in thymus, germinal center cells of the secondary lymphatic nodules of spleen and intestine, follicle cells in ovary and epithelial cells in uterus and intestine. It was also expressed in spermatocytes during early and mid-prophase of meiosis and in resting oocytes before maturation. Thus, mouseRad51 expression is closely related to the state of cell proliferation and is presumably involved in DNA repair coupled with DNA replication, as well as in meiotic DNA recombination in spermatocytes.

Microsatellite instability and mutated type II transforming growth factor-β receptor gene in gliomas

Microsatellite instability has been reported in familial cancer syndrome and in various kinds of human sporadic tumors. We investigated the replication error (RER) and mutation rate of the transforming growth factor-beta type II receptor (TGF-beta RII) gene to determine the frequency of the RER+ phenotype and elucidate the relation between the mutation of the TGF-beta RII gene and RER in the tumorigenesis of glioma. We screened genomic DNA from 40 gliomas, comprised from 24 glioblastomas (GB), 11 anaplastic astrocytomas (AA) and five astrocytomas (AS) and compared the results with DNA from corresponding leukocytes. Seven of the 40 (18%) gliomas had the RER+ phenotype: five (21%) of 24 GB and two (18%) of 11 AA. In six gliomas we detected mutation of the TGF-beta RII gene. Five (71%) of seven RER+ and one (3%) of 33 RER-tumors had one A deletion in the (A)10 repeat of the TGF-beta RII gene. No mutations were detected in the (GT)3 repeat area of the TGF-beta RII gene. As the normal cells of these glioma patients had no mutations, we concluded that the mutations were somatic. We posit that the observed mutations inactivate the receptor through a frameshift mutation resulting in protein truncation. Our data suggest that the TGF-beta RII (A)10 repeat may be one area of genomic instability in the early stages of malignant glioma tumorigenesis.

Fibronectin-mediated cell migration promotes glioma cell invasion through chemokinetic activity

In order to investigate the biological role of fibronectin in glioma cell invasion, we studied the relation between migratory responses or adhesiveness of glioma cells to fibronectin and the in vitro invasion in three human malignant glioma cell lines, A172, T98G and U373MG. All these cell lines chemotactically migrated in a dose-dependent manner to fibronectin in concentrations ranging from 0.5 to 10 µg/ml, with A172 cells showing the strongest migration and U373 cells the weakest. Checkerboard analyses demonstrated that A172 and T98G cells showed much stronger chemokinetic responses to fibronectin than U373MG cells. In contrast to the migratory responses, A172 and U373MG cells showed an almost equally high adhesion to fibronectin and T98G cells a low adhesion. The degree of expression of the integrin α5 subunit correlated well with the strength of glioma cell adhesion to fibronectin rather than that of migration to the molecule. Furthermore, the cell adhesion to fibronectin was almost completely inhibited by arginine-glycine-aspartic acid (RGD)-containing peptides, but the fibronectin-stimulated cell migration was only partially inhibited. An in vitro invasion assay disclosed that U373MG cells invaded the artificial basement membrane barrier the most and A172 cells the least. However, addition of fibronectin to the glioma cells markedly enhanced the invasive activity of A172 and T98G cells but had little effect on that of U373MG cells. These results indicate that fibronectin-stimulated migration can be one of the factors promoting invasiveness of glioma cells and that the chemokinetic activity of fibronectin may play a crucial role in glioma invasion through conferring motor-driving force on the glioma cells.

Cell therapy with encapsulated xenogeneic tumor cells secreting β-endorphin for treatment of peripheral pain

The purpose of this study was to assess whether xenogeneic tumor cells secreting β-endorphin and immunologically isolated in polymer capsules could survive and continue to reduce pain when transplanted into the spinal cerebro-spinal fluid (CSF) space of rats. Also, a silicone container for polymer capsules was designed for the clinical application of this method of cell therapy. The mouse tumor cell lines, proopiomelanocortin gene transfected Neuro2A which secrete β-endorphin, were enclosed in polymer capsules at a density of 5 × 106 mL, and transplanted into the spinal CSF space from the occipito-atlantal junction of male Sprague-Dawley rats. Three analgesiometric tests - the tail pinch test, the hot plate test, and electrical stimulation test - showed that the rats with encapsulated Neuro2A (n = 6) were significantly less sensitive to pain after transplantation than control animals (n = 8). The analgesia induced by the encapsulated cells secreting β-endorphin was attenuated by the opiate antagonist naloxone. Morphological study revealed that the encapsulated cells survived for 1 mo after transplantation into the CSF space. An in vitro experiment on cultured capsules (3 cm long) with a silicone container (Kaneka Medics Co) showed that the encapsulated Neuro2A (5 × 106 mL) could secrete peptides for 1 mo. The results of this study indicate that immunologically isolated xenogeneic tumor cells can secrete opiate in the CSF space, and that a silicone container may help the application of this method to the treatment of cancer pain.

Anti-proliferative effects of TNP-470 on human malignant gliomain vivo: potent inhibition of tumor angiogenesis

SummaryA novel angiogenesis inhibitor TNP-470 was investigated for its anti-tumor activity against malignant gliomas bothin vitro andin vivo. TNP-470 cytostatically inhibited the growth in all of the seven glioma cell lines in culture including anticancer drug resistant cells. The 50% inhibitory concentrations (IC50) of these glioma cell lines were 10 to 30 Μg/ml and they were 10 to 20 times higher than IC50 of normal endothelial cells. TNP-470 (30 mg/kg, i.p., every other day) also significantly inhibited the tumor growth of T98G-transplanted nude mice. Microscopically, tumor vessels after the treatment of the tumor-bearing mice with TNP-470 became fewer in number and smaller in diameter than those without treatment. Furthermore, there appeared extensive necrotic areas in the tumor with TNP-470. These results indicate that TNP-470 is a potent angiogenesis inhibitor for malignant gliomas. In addition, the studies of labeling index of BrdU and Ki67 suggest that TNP-470 may act mainly on tumor endothelial cells, thus resulting in reduction of the tumor growth.

Homozygous deletions of p16INK4A/MTS1 and p15INK4B/MTS2 genes in glioma cells and primary glioma tissues

The p16INK4A/MTS1 (p16) and p15INK4B/MTS2 (p15) genes map to 9p21 where genetic alterations have been frequently reported in various human tumors. Using the polymerase chain reaction (PCR), we investigated the loss of these genes on primary glioma samples and cultured glioma cells. All or any of three exons of the p16 gene were homozygously delted in 11 (35.5%) of 31 glioblastomas, none of 9 anaplastic astrocytomas and 5 astrocytomas, and in all 6 human glioma cell lines. Exon 2 of the p15 gene was homozygously deleted in 4 (12.9%) of 31 glioblastomas, but not in lower grade gliomas. It was homozygously deleted in 5 (83.3%) of 6 glioma cell lines. In 12 short-term cultures of cells derived from primary glioma samples, 5 (41.7%) and 2 (16.7%) glioblastoma-derived cells had homozygous deletion of all or any of the three exons of the p16 gene and exon 2 of the p15 gene, respectively. The deletion pattern of these genes in cultured cells was completely consistent with that seen in the primary tumors. Furthermore, two long-term cultures retained both genes that were identical to those in the original tumor tissues. Our results indicate that loss of the p16 and p15 genes may be involved in tumor progression in human gliomas, especially in the development of glioblastoma, that this loss may give growth advantage to the cells in culture, and that it is not the result of culture artifacts.

In vivo etoposide-resistant C6 glioma cell line: Significance of altered DNA topoisomerase II activity in multi-drug resistance

We have established an in vivo etoposide-resistant glioma cell line (C6/VP) from C6 rat glioma cells by stepwise exposure to increasing doses of etoposide. The C6/VP cells were 10 times more resistant to etoposide than the parental C6 cells. In addition C6/VP cells demonstrated cross-resistance to vincristine and vinblastine, but not to ADM or m-AMSA. Interestingly, the cells had collateral sensitivity to ACNU, cisDDP and Ara-C. The C6/VP cells did not express the MDR gene or p-glycoprotein, while they showed 16 times less topoisomerase II catalytic activity compared to the C6 cells. Although there was no significant difference between C6 and C6/VP cells in amounts of topoisomerase II in nuclear extracts, the C6/VP cells had 2.9 times higher amounts of the enzyme than C6 cells in nuclear scaffold prepared from a relatively low-salt buffer (0.5 M NaCl). Northern blot analysis demonstrated that mRNAs of topoisomerase IIα isoforms were expressed both in C6 and C6/VP cells, and that the amounts of topoisomerase IIα in C6/VP cells were 14 times greater than in C6 cells. The total uptake of etoposide in tumor tissues derived from C6/VP cells was 3 times less than those derived from parental C6 cells. These results indicate that the C6/VP acquired a multi-drug resistance phenotype by a reduction of the catalytic activity of topoisomerase II and/or diminished accumulation of drugs. This phenotype did not involve the p-glycoprotein. Alterations of topoisomerase II in the C6/VP cells also were accompanied by an increased amount of the topoisomerase IIα isoform, most of which was localized in the nuclear scaffold (matrix). This suggests that altered binding of topoisomerase II to topologically organized DNAs in the nuclear scaffold may be the molecular basis of this multi-drug resistance phenotype.

A “Sling Swing Transposition” Technique With Pedicled Dural Flap for Microvascular Decompression in Hemifacial Spasm

BACKGROUND: The key to successful microvascular decompression of the neurovascular compression syndrome is maintaining the separation between the nerve and the offending vessel. OBJECTIVE: We describe a transposition technique in which a local pedicled dural flap, fashioned from the petrous posterior surface, is used to retract the offending vessel away from the root exit zone of the facial nerve in hemifacial spasm cases. METHODS: We conducted a retrospective review of microvascular decompression operations in which the offending vessel was transposed and then retained by a local pedicled dural flap made from the dura of the petrous posterior surface. RESULTS: This technique was used in 7 consecutive cases of the most recently operated series. Postoperatively, complete symptom relief was achieved in 100% of the patients without any significant surgical complications. CONCLUSION: To our knowledge, this is the first report in which an autologous anatomic structure in the cerebellopontine angle, such as petrous dura mater, is used in the microvascular decompression of the facial nerve. This is a simple yet robust method and can be considered an option for the treatment of hemifacial spasm caused by arterial compression. ABBREVIATIONS: AICA, anterior inferior cerebellar artery HFS, hemifacial spasm IAC, internal auditory canal MVD, microvascular decompression PICA, posterior inferior cerebellar artery RExZ, root exit zone VA, vertebral artery

Microsurgical Maneuvers under Side-Viewing Endoscope in the Treatment of Skull Base Lesions.

The objective of the present study is to elucidate the feasibility of surgical maneuvers under the side-viewing endoscope during skull base tumor removal. The study focused on 51 patients who underwent tumor removal with the assistance of a side-viewing endoscope. The side-viewing endoscope enabled visualization and removal of residual tumors obscured by the skull base bone, cranial nerves, and other vital structures after a microscopic procedure. If the surgical field is surrounded by the dura or skull base tissue, not only curettage of a tumor but also semisharp dissection and bipolar coagulation are shown to be feasible. In the subarachnoid space, however, the primary feasible surgical maneuver was suctioning of the tumor. The extent of skull base resection could be reduced in 25 cases and additional tumor removal became possible in 47 cases. Application of the side-viewing endoscope enabled removal of the tumor compartment, the exposure of which has conventionally required an extensive skull base resection. This technique is a promising option for the treatment of skull base tumors.

Unruptured Internal Carotid-Posterior Communicating Artery Aneurysm Splitting the Oculomotor Nerve: A Case Report and Literature Review

Objective To report a rare case of unruptured internal carotid-posterior communicating artery (IC-PC) aneurysm splitting the oculomotor nerve treated by clipping and to review the previously published cases. Case Presentation A 42-year-old man suddenly presented with left oculomotor paresis. Three-dimensional digital subtraction angiography (3D DSA) demonstrated a left IC-PC aneurysm with a bulging part. During surgery, it was confirmed that the bulging part split the oculomotor nerve. After the fenestrated oculomotor nerve was dissected from the bulging part with a careful microsurgical technique, neck clipping was performed. After the operation, the symptoms of oculomotor nerve paresis disappeared within 2 weeks. Conclusions We must keep in mind the possibility of an anomaly of the oculomotor nerve, including fenestration, and careful observation and manipulation should be performed to preserve the nerve function during surgery, even though it is very rare.