論文 - 戸田　正博

Suppression of glial tumor growth by expression of glial fibrillary acidic protein

Toda Masahiro, Miura Masayuki, Asou Hiroaki, Sugiyama Ichiro, Kawase Takeshi, Uyemura Keiichi

Neurochemical Research 24 （ 2 ） 339 - 343 1999年

ISSN  0364-3190

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<p>We examined the effect of expression of glial fibrillary acidic protein (GFAP) on the tumor growth of astrocytoma in vivo. When rat astrocytoma C6 cells were injected subcutaneously in athymic mice, the cells produced tumors that grew rapidly. The tumor growth of C6 cells transfected with GFAP cDNA was significantly reduced compared to that of control NeoC6 cells transfected only with the neomycin resistant gene. After implantation of C6 cells transfected with mutated GFAP cDNA at the phosphorylation sites, the tumor growth was suppressed similar to that of the wild GFAP transfectants. To determine whether the cell growth suppression by GFAP is specific for astroglial cells, we assessed the effect of GFAP on the cell growth of an L cell of fibroblast origin in vitro. By GFAP cDNA transfection, L cells showed morphological changes, but the cell growth was not reduced. These results suggest that GFAP is a critical regulator of the tumor growth of astrocytoma.</p>

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Attenuated, replication-competent herpes simplex virus type 1 mutant G207

Hunter William D., Martuza Robert L., Feigenbaum Frank, Todo Tomoki, Mineta Toshihiro, Yazaki Takahito, Toda Masahiro, Newsome Joseph T., Platenberg R. Craig, Manz Herbert J., Rabkin Samuel D.

Journal of Virology 73 （ 8 ） 6319 - 6326 1999年

ISSN  0022-538X

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<p>This study examined the safety of intracerebral inoculation of G207, an attenuated, replication-competent herpes simplex virus type 1 (HSV-1) recombinant, in nonhuman primates. Sixteen New World owl monkeys (Aotus nancyme [karyotype 1, formerly believed to be A. trivirgatus]), known for their exquisite susceptibility to HSV-1 infection, were evaluated. Thirteen underwent intracerebral inoculation with G207 at doses of 10⁷ or 10⁹ PFU, two were vehicle inoculated, and one served as an infected wild-type control and received 10³ PFU of HSV-1 strain F. HSV-1 strain F caused rapid mortality and symptoms consistent with HSV encephalitis, including fever, hemiparesis, meningitis, and hemorrhage in the basal ganglia. One year after G207 inoculation, seven of the animals were alive and exhibited no evidence of clinical complications. Three deaths resulted from nonneurologic causes unrelated to HSV infection, and three animals were sacrificed for histopathologic examination. Two animals were reinoculated with G207 (10⁷ PFU) at the same stereotactic coordinates 1 year after the initial G207 inoculation. These animals were alive and healthy 2 years after the second inoculation. Cerebral magnetic resonance imaging studies performed both before and after G207 inoculation failed to reveal radiographic evidence of HSV-related sequelae. Despite the lack of outwardly observable HSV pathology, measurable increases in serum anti-HSV titers were detected. Histopathological examination of multiple organ tissues found no evidence of HSV-induced histopathology or dissemination. We conclude that intracerebral inoculation of up to 10⁹ PFU of G207, well above the efficacious dose in mouse tumor studies, is safe and therefore appropriate for human clinical trials.</p>

Treatment of human breast cancer in a brain metastatic model by G207, a replication-competent multimutated herpes simplex virus 1

Toda Masahiro, Rabkin Samuel D., Martuza Robert L.

Human Gene Therapy 9 （ 15 ） 2177 - 2185 1998年10月

ISSN  1043-0342

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<p>We investigated the therapeutic efficacy of G207, a replication-competent multimutated herpes simplex virus type 1, for the treatment of human malignant mammary tumors metastatic to the brain. In vitro studies demonstrated that G207 efficiently destroyed three of four human malignant breast cancer cell lines. MDA-MB-435 was most susceptible and MDA-MB-231 was least susceptible to G207. In athymic mice harboring subcutaneous or intracerebral MDA-MB-435 cells, intraneoplastic inoculation of G207 caused growth inhibition and/or prolonged survival. In contrast, G207 had minimal effects on MDA-MB-231 subcutaneous tumor growth or survival in the intracerebral tumor model. The efficacy of G207 therapy in vivo correlated well with the susceptibility of the human cancer cells to G207 in vitro. Histological studies indicate that G207 replication is restricted to tumor cells in vivo and does not occur in the surrounding brain tissue. These results suggest that G207 shows particular promise for use as a novel antineoplastic agent for metastatic brain tumors and that in vitro testing may predict which tumors will be most responsive in vivo.</p>

In situ cancer vaccination

Toda Masahiro, Martuza Robert L., Kojima Hidefumi, Rabkin Samuel D.

Journal of Immunology 160 （ 9 ） 4457 - 4464 1998年05月

ISSN  0022-1767

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<p>Intratumoral inoculation of replication-competent, attenuated herpes simplex virus (HSV) mutants inhibits tumor growth by direct cytotoxic viral replication and induction of a tumor-specific immune response. To boost the antitumor response, we describe a defective HSV vector encoding IL-12 as an adjuvant to in situ vaccination by the replicaton-competent HSV helper virus. The defective HSV vector system consists of defective particles containing tandem repeats of the cytokine genes (p40 and p35) in combination with a HSV helper virus. Heterodimeric IL-12 was expressed and secreted after IL-12 defective vector infection of tumor cells. In a syngeneic, bilateral established tumor model with CT26 murine colon carcinoma, unilateral intratumoral inoculation with an IL-12 defective/replication-competent HSV vector combination significantly reduced tumor growth of the inoculated and noninoculated contralateral tumors. This antitumor effect was significantly greater than with a lacZ-defective/replication-competent HSV vector combination, which itself was significantly greater than the mock inoculation. Efficacy is associated with enhancement of tumor-specific CTL activity, including specificity against the CT26 immunodominant MHC class I restricted Ag AH1, and IFN-γ production. There was no significant tumor growth inhibition after lntratumoral inoculation of s.c. CT26 tumors in athymic mice. We conclude that this defective HSV vector system is an effective method for cytokine gene delivery to tumors in situ and IL-12 expression in tumors synergizes the antitumor activity mediated by the replication-competent HSV helper virus.</p>

Effect of a neuron-specific actin-binding protein, drebrin A, on cell-substratum adhesion

Ikeda Keiro, Shirao Tomoaki, Toda Masahiro, Asada Hideo, Toya Shigeo, Uyemura Keiichi

Neuroscience Letters 194 （ 3 ） 197 - 200 1995年07月

ISSN  0304-3940

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<p>Drebrin A expression was induced in non-neuronal L cells via transfection with a vector containing the cDNA of rat drebrin A. Following treatment with colcemid (5 μg/ml) and cytochalasin D (0.5 μg/ml), most L cells collapsed into round cells, while drebrin expressing cells were resistant to the treatment, keeping their cell shapes. Simultaneously, actin filaments and microtubules were disrupted in both cell lines. By quantitative analysis, in the presence of cytochalasin D, the extent of cell spreading and cell attachment in drebrin expressing cells was significantly higher than that in control cells. These results suggest that drebrin A modulates cell-substratum adhesion. © 1995.</p>

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Development of oligodendrocyte and myelination in the central nervous system.

Asou H., Murakami K., Toda M., Uyemura K.

Keio Journal of Medicine 44 （ 2 ） 47 - 52 1995年06月

ISSN  0022-9717

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<p>We demonstrated the cell lineage of oligodendrocytes from the glial precursor cells to mature oligodendrocytes forming myelin sheath around the axon. There are several different stages of oligodendrocyte development in vito. So far there are no precise data about their morphological changes during oligodendrocyte development, but by the analysis using SEM and immunostaining, the characteristic morphological changes with serial expression of cell markers were observed in each developmental steps of oligodendrocyte. We have also clearly demonstrated how oligodendrocytes wrap around the axon by using video time-lapse movies. These results will be useful for understanding the exact cellular mechanism of myelination in the CNS.</p>

Cell growth suppression of astrocytoma C6 cells by glial fibrillary acidic protein cDNA transfection

Toda Masahiro, Miura Masayuki, Asou Hiroaki, Toya Shigeo, Uyemura Keiichi

Journal of Neurochemistry 63 （ 5 ） 1975 - 1978 1994年11月

ISSN  0022-3042

　概要を見る

<p>The cellular functions of the intermediate filament family including glial fibrillary acidic protein (GFAP) are not well known yet beyond their roles as structural elements of cells. Expression of GFAP, which is specific in astrocytes and regulated developmentally, suggests its involvement in cell growth and differentiation of astrocytes. We transfected murine GFAP cDNA into a rat astrocytoma C6 cell line to assess the specific effect of GFAP on cells. Two stable GFAP-transfected cell lines, GFC6-5 and GFC6-6, exhibited a series of morphological and growth characteristics that distinguish them from their counterparts, i.e., NeoC6 cells transfected only with the neomycin- resistant gene, and native C6 cells. Both GFC6-5 and GFC6-6 cells showed elongated cell shapes with extended processes rich in GFAP, markedly suppressed cell growth, and decreased bromodeoxyuridine uptake. Western blot analysis revealed a remarkable increase of GFAP expression in GFC6-5 and GFC6-6 compared with that in NeoC6 and C6, in contrast to similar vimentin expression in all cell lines. The results indicate that the expression of GFAP has dramatic effects on cell morphology and cell growth suppression in C6 cells, suggesting that GFAP may function as a tumor suppressor in astrocytoma.</p>

GFAP transfected cells produce laminin, leading to neurite outgrowth promotion

Toda M., Asou H., Miura M., Toya S., Uyemura K.

NeuroReport 5 （ 15 ） 1969 - 1972 1994年

ISSN  0959-4965

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<p>Accumulating evidence indicates the importance of astrocytes in neuronal development and regeneration. While glial fibrillary acidic protein (GFAP) is believed to mediate the morphology of developing astrocytes, its precise function remains unknown. To analyse the function of GFAP in astrocytes, we established GFAP-expressing cell lines by transfection of mouse GFAP cDNA into mouse fibroblast L cells. Stable transfectants expressed GFAP uniformly in the cytoplasm with no phenotypic changes and exhibited extended processes rich in GFAP. GFAP-expressing cells significantly promoted the neurite outgrowth of rat cerebral cortex neurones in the co-culture system. Analysis of the products of GFAP-expressing cells revealed an increase in production of laminin, but not fibronectin. These results suggest that L cells expressing GFAP increase laminin production, leading to promotion of neurite outgrowth.</p>

Molecular-Cloning of cDNA Encoding Human Drebrin E and Chromosomal Mapping of Its Gene

Toda M., Shirao T., Minoshima S., Shimizu N., Toya S., Uyemura K.

Biochemical and Biophysical Research Communications 196 （ 1 ） 468 - 472 1993年10月

ISSN  0006-291X

　概要を見る

<p>Drebrins are novel actin-binding proteins in the brain which are developmentally regulated. Three isoforms: two embryonic types (El and E2) and an adult type (A) are generated by alternative RNA splicing from a single drebrin gene in the chicken brain. A full length cDNA clone of human drebrin E has been isolated from a cDNA library of human fetus brain. The clone is 2596 base pairs in length and contains an open reading frame of 1947 nucleotides encoding a protein of 649 amino acids. The deduced amino acid sequence, except for the internal 138-nucleotide sequence (ins2), exhibits 88% homology with rat drebrin A. Spot blot hybridization using flow-sorted human chromosomes provides evidence that the gene encoding human drebrin protein locates on human chromosome 5. © 1993 Academic Press. All rights reserved.</p>

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