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Increase of oligodendrocyte progenitor cells after spinal cord injury

Ishii Ken, Toda Masahiro, Nakai Yoko, Asou Hiroaki, Watanabe Masahiko, Nakamura Masaya, Yato Yoshiyuki, Fujimura Yoshikazu, Kawakami Yutaka, Toyama Yoshiaki, Uyemura Keiichi

Journal of Neuroscience Research 65 （ 6 ） 500 - 507 2001年09月

ISSN 0360-4012

The reaction of oligodendrocyte progenitor cells (OPCs) after spinal cord injury (SCI) is poorly understood. In this study, we examined oligodendroglial reactions after contusion SCI in adult rats by immunohistochemistry. OPCs were identified by staining with monoclonal antibodies (mAbs) A2B5 and O4. Each of the A2B5-, O4-positive OPCs and galactocerebroside-positive oligodendrocytes dramatically increased in the lesion of the dorsal posterior funiculus. Bromodeoxyuridine (BrdU) incorporation studies showed that most O4-positive cells in the lesion were labeled with BrdU, suggesting that these OPCs were proliferative. In contrast, the expression of myelin basic protein was decreased in the lesion compared with controls that received laminectomy only. From the injured cord, OPCs were isolated by immunopanning with mAb A2B5. We observed an increased number of OPCs from the injured spinal cords compared with those isolated from controls and unoperated animals. After several days in culture, the OPCs from the lesion expressed galactocerebroside. These results suggest that OPCs are induced and can differentiate following SCI in the adult rat. © 2001 Wiley-Liss, Inc.

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Combination suicide/cytokine gene therapy as adjuvants to a defective herpes simplex virus-based cancer vaccine

Toda M., Martuza R. L., Rabkin S. D.

Gene Therapy 8 （ 4 ） 332 - 339 2001年

ISSN 0969-7128

We have used syngeneic, established bilateral subcutaneous tumor models to examine the antitumor activity of herpes simplex virus (HSV) vectors, including the induction of an immune response against non-inoculated distant tumors. In such a model with CT26 murine colon adenocarcinoma, unilateral intratumoral inoculation of replication-deficient HSV-1 tsK inhibited the growth of both the inoculated and noninoculated established tumors. To enhance this limited antitumor immune response, we generated a defective HSV vector, dvIL12-tk encoding both interleukin-12 (IL-12) and HSV thymidine kinase (TK), with tsK as the helper virus. In a 'suicide gene' strategy, ganciclovir (GCV) treatment after intratumoral inoculation of dvlacZ-tk/tsK, encoding E. coli lacZ instead of IL-12, resulted in enhanced antitumor activity. Antitumor activity was also enhanced by local expression of IL-12 from dvIL-12-tk/tsK. The combination of IL-12 cytokine therapy with GCV treatment was the most efficacious approach, with significantly greater inhibition of tumor growth than IL-12 or TK + GCV alone. These results illustrate the power of combining different cancer therapy approaches; 'suicide gene' therapy, cytokine therapy, and HSV vector infection. HSV vectors are particularly well suited to this because they can accommodate the insertion of large and multiple gene sequences.

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Expression of the neural RNA- binding protein Musashi1 in human gliomas

Toda Masahiro, Iizuka Yukihiko, Yu Wangjie, Imai Takao, Ikeda Eiji, Yoshida Kazunari, Kawase Takeshi, Kawakami Yutaka, Okano Hideyuki, Uyemura Keiichi

GLIA 34 （ 1 ） 1 - 7 2001年

ISSN 0894-1491

Tumor cells arising from a particular tissue may exhibit the same gene expression patterns as their precursor cells. To test this proposition, we have analyzed the expression of a neural RNA-binding protein, Musashi1, in primary human central nervous system (CNS) tumors. In rodents, Musashi1 is expressed predominantly in proliferating multipotent neural precursor cells, but not in newly generated postmitotic neurons. The expression of Musashi1 is downregulated with the successive progression of neurogenesis. In normal adult human tissues, we detected low levels of Musashi1 expression in brain and testis by RT-PCR analysis. In an RNA panel of 32 cancer tissues and cell lines, elevated expression of Musashi1 was seen in all five malignant gliomas studied, in contrast to the slight expression seen in other tumor cells, including those in several melanomas and a prostate cancer. Western blot analysis showed strong Musashi1 expression in malignant gliomas compared with nonneoplastic brain tissue. Glioblastomas, the most malignant form of glioma, showed higher Musashi1 expression than less malignant gliomas by immunohistochemical analysis. Tumors with strong Musashi1 expression tended to have high proliferative activity. Thus, the expression of Musashi1 correlated with the grade of the malignancy and proliferative activity in gliomas. These results suggest that primary CNS tumors may share gene expression patterns with primitive, undifferentiated CNS cells and that Musashi1 may be a useful marker for the diagnosis of CNS tumors. © 2001 Wiley-Liss, Inc.

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Tumor growth inhibition by intratumoral inoculation of defective herpes simplex virus vectors expressing granulocyte - Macrophage colony-stimulating factor

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

Molecular Therapy 2 （ 4 ） 324 - 329 2000年10月

ISSN 1525-0016

To evaluate the potential of defective herpes simplex virus (HSV) amplicon vectors as in vivo cytokine gene transfer vehicles for active immunotherapy, we generated a defective HSV vector that encodes the murine granulocyte - macrophage colony-stimulating factor (GM-CSF) gene, using a replication-defective HSV as helper virus. A variety of murine tumor cell lines were efficiently infected in vitro with the defective GM-CSF vector (dvGM), and this led to the synthesis and secretion of murine GM-CSF. In an established bilateral subcutaneous tumor model with Harding-Passey murine melanoma, unilateral intratumoral inoculation of dvGM significantly inhibited tumor growth of both the inoculated and noninoculated contralateral tumors. This tumor inhibition was dose-dependent and resulted in increased survival of the dvGM-treated mice. Inoculation of a lacZ-expressing defective vector had no effect on tumor growth. We conclude that this defective HSV vector system offers an effective method for cytokine gene delivery in vivo and that GM-CSF expression in tumors has antitumor activity.

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Comparison of Fas- versus perforin-mediated pathways of cytotoxicity in TCR- and Thy-1-activated murine T cells

Kojima Hidefumi, Toda Masahiro, Sitkovsky Michail V.

International Immunology 12 （ 3 ） 365 - 374 2000年

ISSN 0953-8178

T cell-mediated cytotoxicity can be triggered by cross-linking of TCR or Thy-1 surface proteins. While the TCR-triggered signaling initiates both perforin- and Fas ligand (FasL)-Fas-mediated mechanisms of cytotoxicity, it was not clear which mechanism was utilized by Thy-1-triggered signals and which pathway of cytotoxicity was triggered at low levels of antigen expression. It is shown that glycophosphatidylinositol-linked surface glycoprotein Thy-1 preferentially activates FasL-Fas- but not perforin-mediated cytotoxicity. This is explained by the lesser intensity of Thy-1-mediated signaling in T cells. The data suggest that Thy-1-triggered Fas-mediated cytotoxicity is completely dependent on cross-talk between Thy-1 and TCR signals since mutations in TCR-CD3 complex molecules or inhibition of tyrosine kinases or calcineurin abolished or strongly inhibited Thy-1-triggered FasL-Fas-mediated cytotoxicity. Lower concentrations of antigenic peptide or levels of cross-linking with anti-TCR-CD3 mAb are required to trigger Fas-mediated than perforin-mediated cytotoxicity by different cytotoxic T lymphocyte (CTL) lines and clones, and it is shown that cross-linking of Thy-1 is much less efficient in triggering accumulation of second messengers (intracellular Ca²⁺) than cross-linking of TCR on CTL. Taken together, these data reflect the possibility of differential activation of FasL and/or perforin pathways of cytotoxicity depending on the nature of activating stimuli and surface receptor.

Suppression of an actin-binding protein, drebrin, by antisense transfection attenuates neurite outgrowth in neuroblastoma B104 cells

Toda Masahiro, Shirao Tomoaki, Uyemura Keiichi

Developmental Brain Research 114 （ 2 ） 193 - 200 1999年05月

ISSN 0165-3806

Drebrins, actin-binding proteins, are dominantly expressed during embryogenesis and accumulated in neurite processes of postmigratory neurons. While the cytoskeletal proteins are the important factors for regulating neurite outgrowth, the cellular mechanism in neurons is still unclear. To address the role of drebrins in the neurite outgrowth, we have studied the effect of suppression of drebrin on a rat neuroblastoma B104 cell line, which constitutively expresses drebrin. Deprivation of serum or addition of gangliosides in the culture medium induced remarkable neurite outgrowth of B104 cells. We transfected B104 cells with an antisense construct of human drebrin E cDNA and found that the drebrin expression was significantly reduced in the stable antisense cell lines. In response to serum deprivation and gangliosides treatment, their ability to extend neurite processes was significantly attenuated. In contrast, the cell proliferation of the antisense transfectants was arrested by serum deprivation similar to control B104 cells. These data suggest that the drebrins are required for neurite outgrowth in neuronal cells.

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Herpes simplex virus as an in situ cancer vaccine for the induction of specific anti-tumor immunity

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

Human Gene Therapy 10 （ 3 ） 385 - 393 1999年02月

ISSN 1043-0342

The success of cancer gene therapy is likely to require the targeting of multiple antitumor mechanisms. One strategy involves the use of attenuated, replication-competent virus vectors, such as herpes simplex virus type 1 (HSV-1) mutant G207, which is able to replicate in human tumor cells with resultant cell death and tumor growth inhibition, yet is nonpathogenic in normal tissue. In this study, we demonstrate that infection of established tumors with G207 also induces a highly specific systemic anti-tumor immune response. In a syngeneic, bilateral established subcutaneous tumor model, with mouse CT26 colorectal carcinoma cells in BALB/c mice or M3 melanoma cells in DBA/2 mice, unilateral intratumoral inoculation with G207 caused a significant reduction in the growth of both the inoculated and contralateral noninoculated tumors. This elicited anti-tumor response is dependent on viral infection of the tumor, as intradermal inoculation of G207 in BALB/c mice had no effect on CT26 tumor growth. Treatment of subcutaneous CT26 tumors by intratumoral inoculation of G207 induced a tumor-specific T cell response, CD8⁺ cytotoxic T lymphocyte (CTL) activity was generated that recognized a dominant 'tumor-specific' major histocompatibility complex (MHC) class I-restricted epitope (AH1) from CT26 cells. In immune-competent animals, G207 is acting as an in situ tumor vaccine. Therefore, intratumoral G207 inoculation is able to inhibit tumor growth both by local cytotoxic viral replication in tumor cells and induction of a systemic anti-tumor immune response.

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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

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.

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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

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.

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

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.

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

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.

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

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.

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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

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.

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

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.

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

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.

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

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.

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