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N Shinoura

Publications and source records attributed to N Shinoura.

At least 37 records · Page 2Linked to original sources
PubMed2000

Adenovirus-mediated transfer of p53 and Fas ligand drastically enhances apoptosis in gliomas.

Various therapeutic approaches toward killing glioma cells by inducing apoptosis have been developed, but these approaches are often hampered by anti-apoptotic mechanisms. In this study, we attempted to develop a technique that overrides the resistance toward apoptosis in glioma cells. To date, p53- and Fas-mediated apoptotic pathways have been shown to be different. Therefore, we carried out a gene therapy that combines the pro-apoptotic effect of these two different pathways. The recombinant adenoviruses (Advs) for p53 and Fas ligand (FL) (Adv-p53 and Adv-FL, respectively) were constructed. Transfecting the p53 gene into glioma cell lines (A-172 and U251 glioma cells) led to overexpression of Bax, a protein that induces permeability transition; at the same time, this transfection brought about an overexpression of Fas. To intensify Fas-mediated apoptosis, we transferred the FL gene together with the p53 gene by Adv-mediated gene transduction into A-172 and U251 cells. Coinfecting Adv-p53 and Adv-FL into A-172 cells, which are relatively resistant to apoptosis by infection with Adv-p53 or Adv-FL alone (Adv-p53, multiplicity of infection (MOI) of 100: 8.5 +/- 0.7%; Adv-FL, MOI of 100: 3.0 +/- 0.1%) resulted in a drastic enhancement of the percentage of apoptotic cells (Adv-p53 and Adv-FL, each at an MOI of 30: 24.2 +/- 0.9%). Coinfection with Adv-p53 and Adv-FL in U251 cells resulted in a similar enhancement of the percentage of apoptotic cells (Adv-p53 and Adv-FL, each at an MOI of 30: 59.0 +/- 2.3%) compared with that seen in U251 cells transfected with Adv-p53 or Adv-FL alone (Adv-p53, MOI of 30: 3.1 +/- 0.3%; Adv-FL, MOI of 30: 18.1 +/- 0.3%). Regardless of whether a cell line is resistant or sensitive to FL- and p53-mediated apoptosis, coinfection with Adv-p53 and Adv-FL dramatically enhanced the degree of apoptosis of glioma cells. Our results indicate that the coinfection approach can be used as a modality for the gene therapy of gliomas, overriding the apoptosis-resistant mechanisms in glioma cells.

Adenoviridae↗
PubMed2000

Adenovirus-mediated gene transduction of IkappaB or IkappaB plus Bax gene drastically enhances tumor necrosis factor (TNF)-induced apoptosis in human gliomas.

Tumor necrosis factor-alpha (TNF), which was initially supposed to be a promising cancer therapeutic reagent, does not kill most types of cancer cells partly due to the activation of an anti-apoptotic gene, NF-kappaB. NF-kappaB forms an inactive complex with the inhibitor kappa B alpha (IkappaBalpha), which is rapidly phosphorylated and degraded in response to various extracellular signals. To disrupt this protective mechanism, we introduced an inhibitor kappa B alpha (IkappaBdN) gene, a deletion mutant gene lacking the nucleotides for the N-terminal 36 amino acids of IkappaBalpha, into human glioma cells (U251, T-98G, and U-373MG) via an adenoviral (Adv) vector in addition to treatment of the glioma cells with recombinant TNF. Immunohistochemical analysis revealed that NF-kappaB was translocated to nuclei by TNF treatment in U251 and T-98G cells, but not in U-373MG cells. Neither transduction of IkappaBdN nor treatment with TNF protein alone induced apoptosis in U251 and T-98G cells, whereas both cell lines underwent drastic TNF-induced apoptosis after transduction of IkappaBdN. On the other hand, U-373MG cells were refractory to TNF-induced apoptosis even when they were transduced with the IkappaBdN gene. U-373MG cells underwent drastically increased apoptosis when co-transduced with the IkappaBdN and Bax gene in the presence of TNF. Adv-mediated transfer of IkappaBdN or IkappaBdN plus Bax may be a promising therapeutic approach to treat gliomas through TNF-mediated apoptosis.

Adenoviridae↗
PubMed2000

Transduction of a fiber-mutant adenovirus for the HSVtk gene highly augments the cytopathic effect towards gliomas.

Suicide gene therapy utilizing the herpes simplex thymidine kinase (HSVtk) / ganciclovir (GCV) system has been performed to kill cancer cells. However, the low transduction efficiency of HSVtk gene into cancer cells critically limits its efficacy in cancer treatment in clinical situations. To improve delivery of the HSVtk gene into cancer cells, we transduced U-87MG and U-373MG glioma cells with adenovirus (Adv) vectors with a fiber mutant, F / K20, which has a stretch of 20 lysine residues added at the C-terminus of the fiber, for the HSVtk gene (Adv-TK-F / K20), and compared the cytopathic effect of Adv-TK-F / K20 with that of the Adv for HSVtk with wild-type fiber (Adv-TK). The cytopathic effect of Adv-TK-F / K20 in U-87MG and U-373MG cells was approximately 140 and 40 times, respectively, stronger than that of Adv-TK. At the same multiplicity of infection (MOI) in each cell line, Adv-TK-F / K20 induced a higher degree of apoptosis (U-87MG, 35%; U-373MG, 77%) than Adv-TK (U-87MG, 0.11%; U-373MG, 27%) in U-87MG (MOI 0.03) and U-373MG cells (MOI 0.1). Cleavage of poly(ADP-ribose)polymerase (PARP) was more marked in the cells that were infected with Adv-TK-F / K20 than in cells that were infected with Adv-TK. These results indicate that gene therapy utilizing Adv-TK-F / K20 may be a promising therapeutic modality for the treatment of gliomas.

Adenoviridae↗
PubMed2000

Adenovirus-mediated transfer of Fas ligand gene augments radiation-induced apoptosis in U-373MG glioma cells.

Most malignant astrocytomas (gliomas) express a high level of Fas, whereas the surrounding normal tissues such as neurons and astrocytes express a very low level of Fas. Thus, transduction of Fas ligand would selectively kill malignant astrocytoma cells. On the other hand, glioma cells harboring p53 mutation have been reported to be resistant to conventional therapies including radiation. To override the resistance mechanism of glioma cells with p53 mutation to radiation, we transduced U-373MG malignant astrocytoma (glioma) cells harboring mutant p53 with Fas ligand via an adenovirus (Adv) vector in combination with X-ray irradiation, and evaluated the degree of apoptosis. The degree of apoptosis in U-373MG cells infected with the Adv for Fas ligand (Adv-FL) and treated with irradiation (81%) was much higher than that in U-373MG cells infected with Adv-FL and not treated with irradiation (0.8%) or that in U-373MG cells infected with the control Adv for lacZ and treated with irradiation (5.0%). In U-373MG cells infected with Adv-FL, irradiation increased the expression of Fas ligand. Coincident with the increase in Fas ligand, there was a marked reduction in the caspase-3 level and a marked increase in the cleaved form of poly(ADP-ribose) polymerase (PARP), which are downstream components of Fas ligand-mediated apoptosis. This suggests that the enhanced activation of caspase-3 by the transduction of Fas ligand combined with irradiation, induced extensive apoptosis in U-373MG cells. In summary, transduction of Fas ligand may override the resistance mechanism to radiotherapy in glioma cells harboring p53 mutation.

Adenoviridae↗
PubMed2000

[Astroblastoma].

Explore the source record for details and available documents.

Adult↗
PubMed1999

High level calcineurin activity predisposes neuronal cells to apoptosis.

Calcineurin is a Ca(2+)/calmodulin-dependent protein phosphatase that is abundantly expressed in several specific areas of the brain, which are exceptionally vulnerable to stroke, epilepsy, and neurodegenerative diseases. In this study, we assessed the effects of high level activity of calcineurin on neuronal cells. Virus-mediated high level constitutive activity of calcineurin rendered neuronal cells susceptible to apoptosis induced by serum reduction or by a brief exposure to calcium ionophore. Adenovirus-mediated, high level forced activity of calcineurin induced cytochrome c/caspase-3-dependent apoptosis in neurons. Preincubation with the calcineurin inhibitors cyclosporin A and FK506 reduced susceptibility to apoptosis. High level constitutive expression of Bcl-2 or CrmA or incubation with a specific caspase-3 inhibitor inhibited the calcineurin-induced apoptosis. These data indicate that high level constitutive activity of calcineurin predisposes neuronal cells to cytochrome c/caspase-3 dependent apoptosis even under sublethal conditions.

Adenoviruses, Human↗
PubMed1999

Adenovirus-mediated transfer of p33ING1 with p53 drastically augments apoptosis in gliomas.

The p53 tumor suppressor gene is an important target for the gene therapy of cancers, and clinical trials targeting this gene have been conducted. Some cancers, however, are refractory to p53 gene therapy. Therefore, it has been combined with other therapies, including chemotherapy and radiotherapy, to enhance the cytopathic effect of p53 induction. The p33ING1 gene cooperates with p53 to block cell proliferation. In this study, we investigated whether adenovirus (Adv)-mediated coinduction of p33ING1 and p53 enhances apoptosis in glioma cells (U251 and U-373 MG), which showed no genetic alterations but low expression levels of p33ING1. Although the single infection of Adv for p33ING1 (Adv-p33) at a multiplicity of infection (MOI) of 100, or Adv for p53 controlled by myelin basic protein (MBP) promoter (Adv-MBP-p53), a glioma-specific promoter, at a MOI of 50, did not induce apoptosis in U251 and U-373 MG glioma cells; coinfection of Adv-p33 and Adv-MBP-p53 at the same MOIs induced drastically enhanced apoptosis in both cell lines. Apoptosis was not induced in NGF-treated PC-12 cells infected with a high MOI (300) of Adv-p33 nor in those coinfected with Adv-p33 (100) and Adv-MBP-p53 (50). Coinfection of Adv-p33 and Adv-MBP-p53 demonstrated morphological mitochondrial damage during the initial stage of apoptosis, which likely led to apoptotic cell death. Our results indicate that this coinfection approach can be used as a modality for the gene therapy of gliomas, sparing damage to normal tissues.

Adenoviridae↗
PubMed1999

Relative level of expression of Bax and Bcl-XL determines the cellular fate of apoptosis/necrosis induced by the overexpression of Bax.

The Bax protein plays a critical role in the apoptosis of cancers induced by radiotherapy or chemotherapy, which induce both apoptosis and necrosis. We transduced various glioblastoma cells with the Bax gene via an adenoviral vector and found that A-172 cells led to necrotic cell death, while U251 cells apoptotic cell death, even though a similar level of Bax protein was introduced. A-172 cells displayed a much higher constitutive expression of the Bcl-XL protein compared with that of U251 cells. Upon simultaneous overexpression of the Bcl-XL and Bax proteins in the U251 cells, Bax-induced apoptosis of U251 cells was suppressed and an increase in the number of necrotic cells was seen. Moreover, induction of a higher amount of Bax protein in A-172 cells increased the percentage of apoptotic cells. In conclusion, if a cancerous cell expresses a high enough amount of Bax to undergo death, apoptosis will be induced. If a cancerous cell expresses a level of Bcl-XL which prevents Bax-induced apoptosis, the overexpression of Bax leads to necrotic cell death.

Apoptosis↗
PubMed1999

Expression level of Bcl-2 determines anti- or proapoptotic function.

Bcl-2 is an oncogene with antiapoptotic function. However, Bcl-2 is converted to a Bax-like death effector by caspases, suggesting that the expression of Bcl-2 may not favor the growth of cancers. We introduced the Bcl-2 gene to gliomas via adenovirus (Adv; Adv-Bcl-2) with the Adv for Fas (Adv-Fas) and the Adv for Fas ligand (Adv-FL) to evaluate the antiapoptotic function of Bcl-2. In U251 glioblastoma cells, Bcl-2 at a low level of expression repressed apoptosis induced by Adv-Fas and Adv-FL, whereas Bcl-2 at a high level of expression did not. On the other hand, Bcl-X(L) showed antiapoptotic function against Fas-mediated apoptosis, irrespective of its expression level. In glioblastoma cells, induction of Bcl-2 alone at a high level induced apoptosis, whereas induction of Bcl-X(L) alone did not. As the multiplicity of infection of Adv-Bcl-2 was increased, the quantity of a cleaved product of Bcl-2 increased. Induction of caspase-inhibitory genes (CrmA and p35) inhibited apoptosis induced by Adv-Bcl-2. Induction of Bcl-2 led to alteration of the membrane potential and structure of the mitochondria. In summary, although Bcl-2 at a low level of expression was antiapoptotic, Bcl-2 at a high level of expression was proapoptotic to Fas-mediated apoptosis. Overexpression of Bcl-X(L) was consistently antiapoptotic to Fas-mediated apoptosis.

Apoptosis↗
PubMed1999

Highly augmented cytopathic effect of a fiber-mutant E1B-defective adenovirus for gene therapy of gliomas.

An E1B 55-kDa gene-defective adenovirus (Adv), ONYX-015, has been reported to be a highly useful replication-competent Adv that shows cytopathic effect for cancers with an abnormal p53 gene, without damaging normal tissues. In this study, we combined this Adv (Adv-E1AdB) with a fiber mutation, F/K20, which has a stretch of 20 lysine residues added at the COOH-terminus of the fiber and shows high transduction efficiency to gliomas. In U-373 MG glioma cells, the transduction efficiency of Adv-F/ K20 for lacZ was nine times higher than that of the Adv with wild-type fiber (Adv-F/wt) for lacZ. At a multiplicity of infection of 30, the replication efficiency of Adv-E1AdB-F/K20 was 11 times higher than that of Adv-E1AdB with wt fiber (Adv-E1AdB-F/wt). The ED50 value of AdvE1AdB-F/K20 to U-373 MG cells, which is a measure of the in vitro cytopathic effect, was 32 times greater than that of Adv-E1AdB-F/wt. injection of Adv-E1AdB-F/K20 suppressed the in vivo growth of tumors. The antitumoral effect of Adv-E1AdB-F/K20 was remarkably stronger than that of Adv-E1AdB-F/wt. A greater quantity of replicated virus protein (hexon) by infection with Adv-E1AdB-F/K20 was demonstrated in vitro and in vivo, compared with that of Adv-E1AdB-F/wt. In conclusion, gene therapy using Adv-E1AdB-F/K20, which drastically augmented the antitumoral effect of Adv-E1AdB, will be a promising therapeutic approach for gliomas.

Adenovirus E1B Proteins↗
PubMed1998

Construction, propagation, and titer estimation of recombinant adenoviruses carrying proapoptotic genes.

Generation of a recombinant adenovirus (Adv) that induces the constitutive expression of an apoptotic gene has been extremely difficult owing to severe apoptotic damage to the host cell. In this study, 293 cells were transduced with the caspase-inhibiting CrmA gene (293-CrmA cells), and used as host cells to generate Adv carrying apoptosis-inducing genes (proapoptotic genes). The 293-CrmA cells proved to be highly efficient for the construction of recombinant Adv carrying genes encoding Fas and Fas ligand. Moreover, the 293-CrmA line produced an ample quantity of these recombinant viruses. Because the conventional 293 plaque formation assay did not reflect the actual number of cells infected with the Adv carrying the proapoptotic gene, a determination of the Adv DNA copy number introduced into target cells was necessary to evaluate the quantity of infective virus. The techniques described here should be widely applicable for the construction of a recombinant Adv, in ample quantity, and for the estimation of the quantity of recombinant Adv produced.

Adenoviridae↗
PubMed1998

Generation of fiber-mutant recombinant adenoviruses for gene therapy of malignant glioma.

Recombinant adenovirus (Adv)-mediated gene transduction is a powerful technology for cancer gene therapy. In this article, we report the generation of a fiber-mutant Adv vector, using the Adv genomic DNA-terminal protein complex (DNA-TPC) cotransfection method. First, a fiber-mutant construct in a plasmid carrying the right-side two-thirds of the human adenovirus type 5 (Ad5) genome (pTR) was cotransfected with Ad5 DNA-TPC, yielding the recombinant Adv with the desired fiber mutation. The DNA-TPC from the mutant Adv was then utilized to produce a second-step recombinant Adv with an expression cassette in the place of E1. By this procedure, we generated a fiber mutant, F/K20, that has a linker and a stretch of 20 lysine residues added at the C terminus of the fiber. By using Adv carrying a reporter lacZ gene (AxCAZ2) with either F/K20 or wild-type fiber (F/wt), we examined the transduction efficiency of F/K20-Adv. No significant difference in the transduction efficiency between F/K20 and F/wt-Adv was observed for a human fibroblast line, WI-38, or various tumor cell lines, including melanoma, prostate, esophageal, and pancreatic cancer lines. In clear contrast, F/K20-Adv showed a remarkably enhanced efficiency in genetic transduction of human glioma cells. In all four human glioma lines tested, the multiplicities of infection (MOIs) for transduction of 50% of the population (ED50) were decreased with F/K20-Adv compared with F/wt-Adv: 7-fold for T98G, 14-fold for U251, 9-fold for U373, and 42-fold for U87 cells. Therefore, we attempted to apply F/K20-Adv for gene therapy of malignant glioma. Glioma cells infected with F/K20-Adv carrying genes for interleukin 2 or interleukin 12 produced a high level of each cytokine at a much lower MOI than did cells infected with F/wt-Adv. Infection with F/K20-Adv carrying the wild-type p53 tumor suppressor gene resulted in an enhanced level of p53 protein expression and an increased incidence of F/K20-Adv in transduction efficiency for malignant glioma, providing promising tools for gene therapy.

Adenoviridae↗
PubMed1998

Apoptosis by retrovirus- and adenovirus-mediated gene transfer of Fas ligand to glioma cells: implications for gene therapy.

Astrocytic tumors frequently express Fas/APO-1 (Fas), in sharp contrast to surrounding normal brain cells, providing a potential window through which selective killing of tumor cells could be pursued. To assess this possibility, we transduced Fas into U251, a glioma cell line resistant to anti-Fas antibody-mediated apoptosis, and obtained transfectants with high levels of Fas expression. Anti-Fas antibody showed significantly enhanced cytotoxicity for the transfectants, suggesting that U251 cells maintained an intercellular cascade of Fas-mediated apoptosis. When U251 transfectants with high-level Fas expression were transduced with Fas ligand-encoding gene via retrovirus, they were unaffected by exposure to anti-Fas antibody or Fas ligand adenovirus (Adeno-FL). Thus, retroviral induction of Fas ligand into the glioma cells with high levels of Fas led to the selection of cells that were resistant to Fas-dependent apoptosis. These resistant U251 transfectants were susceptible to FADD adenovirus (Adeno-FADD)-induced apoptosis, indicating that a cascade of death signals was blocked at the steps between Fas ligand and FADD. As for adenoviral transduction of Fas ligand into gliomas, gliomas with a relatively high level of expression of Fas were remarkably sensitive to Adeno-FL-induced apoptosis. Besides, Adeno-FADD induced pronounced apoptosis in all glioma cells. Our data suggest the possibility of using adenovirus-mediated transduction of Fas ligand and FADD genes as a therapeutic approach to target gliomas.

Adenoviridae↗
PubMed1997

Brain tumors: detection with C-11 choline PET.

PURPOSE: To evaluate the effectiveness of positron emission tomography (PET) with carbon-11 choline in brain tumor imaging. MATERIALS AND METHODS: A rat glioma cell line (C6) was incubated with C-14 choline; the time course of uptake and metabolism was determined in vitro. C-11 choline was injected intravenously in tumor-bearing rats; the time course of distribution in organs was determined. C-11 choline also was injected intravenously in 20 patients (aged 6-86 years) with brain tumors and two volunteers (aged 38 and 58 years); distribution of the tracer in the brain was determined. Regional cerebral blood flow was measured by using oxygen-15 water on the same day. RESULTS: C-14 choline was metabolized to phosphoryl choline in glioma cells. The uptake of C-11 choline by glioblastoma cells was three to four times higher than that in the rat brain. All brain tumors took up more C-11 choline than did normal brain; thus, brain tumors that were not treated, as well as those that were treated with surgery or radiation therapy, were depicted. The tumor-normal brain uptake ratio of C-11 choline in brain tumor did not correlate with the O-15 water regional blood flow in the corresponding area. CONCLUSION: C-11 choline PET can depict brain tumors effectively. This method was clinically useful in patients who had undergone surgery.

Adult↗
PubMed1997

PET imaging of brain tumor with [methyl-11C]choline.

UNLABELLED: This article describes a new method of [11C]choline synthesis for intravenous injection. We aimed at the utilization of this compound for brain tumor imaging with PET. METHODS: After [11C]carbon dioxide production in a cyclotron and the subsequent [11C]methyl iodide synthesis, [methyl-11C]choline was synthesized by the reaction of [11C]methyl iodide with "neat" dimethylaminoethanol at 120 degrees C for 5 min. Purification was achieved by evaporation of the reactants followed by passage of the aqueous solution of the product through a cation-exchange resin cartridge. The time required for overall chemical processing, excluding the cyclotron operation, was 15 min. Radiochemical yield was > 98%. Radiochemical purity was > 98%. Chemical purity was > 90% (dimethylaminoethanol was the only possible impurity). Specific radioactivity of the product was > 133 GBq/mumol. The whole body distribution was examined in rabbits with PET. Clinical studies were performed in patients with brain tumor using PET after intravenous injection of 370 MBq of [11C]choline. RESULTS: In rabbits,[11C]choline was taken up from blood by various tissues very rapidly, and the radioactivity remaining in blood became almost negligible 5 min after intravenous injection. Taking advantage of this characteristic, we obtained stable tissue distribution images of human brain using PET. In patients with brain tumor, PET produced clearly delineated positive images of the tumors. CONCLUSION: Carbon-11-choline can be used for obtaining clear images of brain tumor in PET.

Adenoma↗
PubMed1996

Malignant schwannoma metastasizing to the dura mater--case report.

A 58-year-old female, with a previous history of radical resection of malignant schwannoma arising in the maxillary sinus, presented with a metastasis to the dura mater following lung metastases 15 years after the initial diagnosis. A large mass at the left temporal region was totally removed immediately after an intratumoral hemorrhage. She subsequently died of massive hemoptysis. Although the prognosis is poor for patients with malignant schwannoma, aggressive treatment including radical surgery combined with radiotherapy and chemotherapy is recommended.

Brain Neoplasms↗