Biomedical subjects

M Tagawa

Publications and source records attributed to M Tagawa.

At least 289 records · Page 16Linked to original sources

PubMedDate not supplied

Infection of human hepatocyte cell lines with hepatitis C virus in vitro.

Hepatocyte related cell lines, namely human embryonic hepatocyte cell line (WRL68) and hepatoblastoma cell line (Hep G2), were tested for their ability to support hepatitis C virus (HCV) replication in vitro. The replicative intermediate of the minus strand HCV RNA was newly transcribed from the inoculated virus, and was stable in WRL68 cells over a period of 62 days. HCV RNA detected in the culture medium at 62 days after infection suggested a long period of virus secretion from this cell line. In Hep G2, transcription of the minus strand HCV RNA was also detected until 39 days after infection, but transcription and secretion of viral progeny could not be demonstrated. Although HCV infection of WRL68 was not high, this cell line might prove to be a useful tool for studying HCV replication in vitro.

Base Sequence↗

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Syngeneic monoclonal antimelanoma antibodies and their application for analysis of tumor antigens, gene cloning, and in vitro/in vivo diagnosis.

In this article, we summarized syngeneic monoclonal antimelanoma antibodies and their application for chemical characterization of mouse melanoma antigens, cloning of genomic DNA controlling antigen expression, and in vivo/in vitro tumor diagnosis. The melanoma antigen is composed of a protein complex in association with GM3(NeuAc)-like sugar moiety. The GM3 structure expresses the cross-species epitopes shared in various mammalian species, whereas the mouse specific melanoma epitope is present on protein molecules. By using the monoclonal antimelanoma reactive with GM3 epitope, we developed a very sensitive sandwich radioimmunoassay system detecting soluble melanoma antigens equivalent to 10(2)-10(3) cells/ml. The antibody was also useful in imaging tumor in vivo. These results indicate that the antibody with cross-species reactivity has a potential for tumor targeting. The monoclonal antibody M562 recognizing protein molecule with species specific epitope but not other antimelanoma antibodies, however, effectively inhibited experimental lung metastasis of melanoma cells, indicating that the M562 epitope seems to possess important biological functions. Recently, the genomic DNA controlling the antigen expression was successfully isolated by DNA transfection and expression technique with monoclonal anti-melanoma M562 and the fluorescence-activated cell sorter. We also found that genomic DNA possesses transformation-related activity in NIH3T3 cells.

Amino Acids↗

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Antitumor effect of murine colon carcinoma cells retrovirally transduced with interleukin-4 and granulocyte macrophage-colony stimulating factor genes.

We have investigated the antitumor effect of murine colon carcinoma cells developed to produce cytokines. Retrovirally transduced cells were inoculated into syngeneic mice, and their survivals were examined. Inoculation of interleukin-4 (IL-4)- or granulocyte macrophage-colony stimulating factor (GM-CSF)-producing cells alone did not affect the survival of the mice. In the tumors derived from an admixture of IL-4 and GM-CSF producers, however, regression developed spontaneously, and prolonged survival of the challenged mice was consequently observed. Histological examination revealed predominant infiltration of neutrophils around the tumor of a mixed population of IL-4 and GM-CSF producers.

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Induction of acquired immunity in rats that have eliminated intracranial gliosarcoma cells by the expression of herpes simplex virus-thymidine kinase gene and ganciclovir administration.

We examined a possible antitumor response against 9L rat gliosarcoma cells induced by the expression of the herpes simplex virus-thymidine kinase (HSV-TK) gene and the ganciclovir (GCV) system. Based on the amount of the major histocompatibility complex (MHC) class I antigens expressed on 9L cells transduced by the HSV-TK gene (9L/HSV-TK) we selected two clones (clones H and L), which represent high and low expressors of class I antigens, respectively. By means of serial magnetic resonance imaging we followed the change of tumor volumes of each clone in syngeneic rats, and found that the intracranial tumor growth was inversely correlated with the expression of MHC class I antigens, although in vitro growths of the clones remained unchanged. Moreover, histological examination revealed significant lymphocyte infiltration in the 9L/HSV-TK tumor of high MHC expression but not in the wild-type tumor. The therapeutic effect of GCV on them was not different, but we observed a prolonged survival of the rats which had eliminated 9L/HSV-TK clone L tumors by the treatment of GCV and were rechallenged with the same cells compared with the survival of naive rats inoculated with clone L cells. These data collectively suggest that the immune response operates even in the brain previously described as an immunologically privileged site.

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Differential efficacy of suicide gene therapy by herpes simplex virus-thymidine kinase gene reflects the status of p53 gene in human esophageal cancer cells.

We examined the effect of herpes simplex virus-thymidine kinase gene (HSV-TK)-mediated suicide gene therapy on human esophageal cancer. Two human lines, T.Tn cells which bears truncated p53 and TE2 cells with wild-type p53, were transduced with the HSV-TK gene and tested for their sensitivities to a prodrug, ganciclovir (GCV). The transduced cells, T.Tn/TK and TE2/TK, increased in vitro sensitivity to GCV compared with that of respective wild-type cells. However, the growth suppression of T.Tn/TK tumors induced by GCV was marginal in nude mice and the tumors regrew thereafter. In contrast, the growth of TE2/TK tumors was significantly inhibited by GCV and all the tumors disappeared. The status of the p53 gene of tumor cells thereby may influence the efficacy of the HSV-TK/GCV system.

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Polyadenylation signal facilitates the expression of foreign gene that is driven by an internal promoter located in the reverse orientation to long terminal repeat of retrovirus.

We examined whether the presence of a polyadenylation [poly(A)] signal in a retrovirus vector could affect the expression level of exogenous gene(s) that was controlled by an internal promoter. Three suicide genes were placed under a promoter of the human midkine gene, whose expression is elevated in lung cancer cells. Orientation of the internal transcriptional unit was designed to be opposite to the viral long terminal repeat. Expression of each suicide gene was greater in ecotropic packaging cells transfected with a retrovirus vector without a poly(A) signal than in those with a poly(A)-containing vector. Sensitivity to ganciclovir, a prodrug that becomes an active drug by herpes simplex virus-thymidine kinase, was significantly improved in retrovirally transduced lung cancer cells compared with wild-type cells. However, the sensitivity was much greater in the cells transduced with poly(A)-containing vector than in those with poly(A)-deleted construct. The presence of a poly(A) signal downstream of exogenous gene(s) therefore favors the expression of foreign gene(s) driven by an internal promoter.

Biotransformation↗

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Radiosensitivity of human breast cancer cells transduced with wild-type p53 gene is influenced by the p53 status of parental cells.

Induction of apoptosis with chemotherapeutic agents or radiation in tumours is frequently related to the status of those p53 gene of the tumours. To examine whether forced expression of the wild-type p53 gene in tumour cells can modulate their susceptibility to radiation and anti-cancer agents, we retrovirally transduced two types of human breast cancer cell lines, which respectively harboured a mutated p53 gene (OCUB-M) or wild-type p53 gene (YMB-1), with the wild-type p53 gene. Transduced cells which consistently expressed the wild-type p53 gene (OCUB-M/p53, YMB-1/p53) proliferated at the same rate as control cells which were transduced with the beta-galactosidase gene (OCUB-M/lacz, YMB-1/lacz). However, sensitivity to radiation was increased in OCUB-M/p53 cells but not in YMB-1/p53 cells. In vitro chemosensitivity to DNA-damaging anticancer agents such as cyclophosphamide and 5-fluorouracil was not influenced by the transduction of the wild-type p53 gene in either cells. Expression of the wild-type p53 gene in p53-mutated human breast cancer cells can therefore increase their sensitivity to radiation but not their chemosensitivity. Therapeutic effects following by the transduction of the wild-type p53 gene were not observed in breast cancer cells already bearing the wild-type p53 gene.

Antineoplastic Agents↗

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The p73 gene is not mutated in oligodendrogliomas which frequently have a deleted region at chromosome 1p36.3.

An allelic loss of the chromosome 1p36 region is frequently found in oligodendrogliomas, which suggests the presence of putative tumor suppressor gene(s) in the region. Since the p73 gene, which encodes a protein with significant homology with p53, is mapped to the 1p36.33 region, we examined genetic alterations of the p73 gene in oligodendrogliomas. We screened 10 specimens for mutation throughout the p73 coding regions by polymerase chain reaction (PCR)-single strand conformation polymorphism analysis and by sequencing aberrantly migrated PCR products. We found several polymorphic nucleotide changes, but no somatic mutations that caused an amino acid change. The p73 gene is thus unlikely to be a tumor suppressor gene for oligodendrogliomas.

Chromosome Deletion↗

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Electroporation-mediated transfer of cytokine genes into human esophageal tumors produces anti-tumor effects in mice.

Electroporation facilitates transfer of chemicals or plasmid DNA from extracellular milieu into cells by increasing the permeability of the cell membrane. Delivery of electric pulses to established tumors thereby can improve the susceptibility of tumors to an anti-cancer agent administered. We examined whether electroporation-mediated transfer of cytokine genes into solid tumors could produce anti-tumor effects in the tumor-bearing mice. Plasmid DNA containing cytokine genes were injected into human esophageal T.Tn tumors developed in nude mice and electric pulses were then delivered. Administration of murine GM-CSF or human IL-2 gene followed by electroporation significantly suppressed the subsequent growth of T.Tn tumors and prolonged the survival of the inoculated mice. In contrast, electroporation-mediated introduction of a control gene, human GM-CSF gene, whose products do not bind to murine GM-CSF receptors, did not achieve any anti-tumor effects. In vivo transfection of cytokine genes with electroporation could be a possible therapeutic strategy for established solid tumors.

Animals↗

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Study of irradiation effects on cytokine secretion from retrovirally-transduced tumor cells: a model for tumor vaccination.

We have examined the effects of irradiation on the cytokine secretion from genetically modified human esophageal and gastric carcinomas. Both cell lines were transduced retrovirally to secrete interleukin-2, interleukin-6 and granulocyte-macrophage colony-stimulating factor, respectively. The metabolism of all the transduced cells was partially inhibited by 6 Gy, and greatly inhibited by 20 Gy irradiation. Cytokine productions, however, was not affected by 6 Gy in many cases, and continued to be detected even after 60 Gy irradiation. The analysis of the dose and time of irradiation in each cytokine producer is useful for the designing of tumor vaccine using cytokine gene transfer.

Cell Division↗

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Therapeutic potency of transduction with herpes simplex virus thymidine kinase gene against multidrug resistant mouse leukemia cells.

In order to examine the therapeutic potency of ganciclovir (GCV) against multidrug resistant tumour cells by transduction with the herpes simplex virus thymidine kinase (HSV-TK) gene, vincristine (VCR)-resistant mouse leukemia P388 cells (P388/VCR) were transduced retrovirally with the HSV-TK gene. In vitro, P388/VCR cells expressing HSV-TK (P388/VCR/TK) were approximately 200-fold more sensitive to GCV than P388/VCR cells. Age-matched CDF1 mice were intraperitoneally inoculated with P388/VCR or P388/VCR/TK cells and were treated with either GCV or VCR. The results showed that VCR had little therapeutic effect against P388/VCR/TK-bearing mice, whereas GCV significantly increased the life span of the mice. These results imply the potential value of HSV-TK gene transduction followed by GCV treatment in killing multidrug-resistant tumor cells.

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Inability to induce the alteration of tumorigenicity and chemosensitivity of p53-null human pancreatic carcinoma cells after the transduction of wild-type p53 gene.

We investigated the therapeutic benefits of p53 expression by the transduction of wild-type p53 gene into p53-null human pancreatic carcinoma cells (AsPC-1). Induction of p21WAF1/CIP1 protein was observed in p53 gene-transduced AsPC-1 cells, showing the proper function of integrated p53 gene. However, the cell growth in vitro of transduced cells was not different from that of parent cells, and the tumor growth of transduced cells inoculated into nude mice was unchanged compared with that of wild-type cells. Moreover, the in vitro sensitivity to 4 different kinds of anticancer agents including cisplatin, etoposide, 5-fluorouracil and paclitaxel, was not modulated by the expression of wild-type p53 gene. Thus, the data presented here suggest that the expression of wild-type p53 gene in p53-null tumor cells does not consistently produce the therapeutic effects previously reported.

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Discordant production of released exogenous protein and infectious virions from retrovirus-packaging cells used for gene transduction.

We examined the correlation between the amount of exogenous protein secreted and that of its viral transcript from PA317 packaging cells transduced with a cytokine gene. Amphotropic packaging cells for the retrovirus expressing murine interleukin-15 (IL-15) gene were cloned, and we examined 22 clones for both the amount of IL-15 secreted with enzyme-linked immunosorbent assay and the IL-15 transcript released as infectious virions by slot blot-hybridization analysis. The study revealed that there was no statistical correlation between them, and suggests that the integrated transcript is used discordantly. Thus, the examination of secreted protein from packaging cells cannot be substituted for laborious assays to determine the amount of virion transcripts when isolating high titer packaging cells.

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Impaired in vivo tumor growth of human pancreatic carcinoma cells retrovirally transduced with GM-CSF gene.

We have examined the antitumor effect of human pancreatic carcinoma cells (AsPC-1) retrovirally transduced with mouse granulocyte macrophage-colony stimulating factor (GM-CSF) gene in nude mice. Growth retardation of the subcutaneous tumors of GM-CSF-producing AsPC-1 cells was observed, although their in vitro proliferation was not different from that of wild-type cells. Histological examination revealed infiltration of monocytic cells into the tumor of GM-CSF-producing cells, and they were shown to be mainly CD11b positive cells by immunohistochemical staining. The survival of the mice inoculated intraperitoneally with GM-CSF- producing AsPC-1 cells was significantly prolonged compared with that of the mice inoculated with wild-type AsPC-1 cells. Thus, the expression of GM-CSF gene in human pancreatic cells induced an antitumor effect in vivo even in the mature T cell-deficient condition.

Animals↗

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Antitumor effect of human pancreatic cancer cells transduced with cytokine genes which activate Th1 helper T cells.

We have examined antitumor effect of human pancreatic carcinoma cells (AsPC-1) retrovirally transduced with interleukin-12 (IL-12), IL-15 or IL-18 gene in nude mice. The tumor growth of IL-12-expressing AsPC-1 cells was significantly retarded and that of IL-15-expressing cells was also impeded compared with that of wild-type cells, although their in vitro cell growth remained unchanged. However, the expression of IL-18 in AsPC-1 cells did not generate any antitumor effect since the tumor growth of the transduced cells was the same as that of wild-type cells. Thus, the differential actions of these cytokines on non-T cells can generate a variety of antitumor effect in nude mice, although their actions on T cells lineage favor the stimulation of Th1-type helper T cells.

Animals↗

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Impaired tumorigenicity and decreased liver metastasis of murine neuroblastoma cells engineered to secrete interleukin-2 or granulocyte macrophage colony-stimulating factor.

We have examined the antitumor effect of murine neuroblastoma cells (C1300) engineered to produce cytokines. Retrovirally transduced cells with human interleukin-2 (IL-2) or murine GM-CSF gene, but not murine IL-4 gene, abolished their tumorigenicity in syngeneic mice, although their in vitro growth rate and expression of class I antigens of the major histocompatibility complex were unchanged. Inoculation of wild-type cells into the mice, which had rejected IL-2 or GM-CSF producers, did not develop tumors, indicating that protective immunity was induced. In an experimental hematogenous metastasis model, we found that the numbers of metastatic foci in the liver caused by intravenous administration of IL-2 or GM-CSF producers were significantly reduced compared with those by the injection of wild-type or vector virus-transduced cells. No significant differences in their adhesiveness to extracellular matrices and ability to differentiate were observed among parent and transduced cells. Thus, these results indicate that IL-2 or GM-CSF secretion, in the vicinity of neuroblastoma cells, produced antitumor effect and reduced metastatic ability.

Animals↗

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Inhibition of experimental lung metastasis of murine colon carcinoma cells depends on the amount of interleukin-2 secreted from the transduced cells.

We examined the antitumor effect of low and high interleukin-2 (IL-2) producers of murine colon carcinoma cells (Colon 26) which were generated by transduction with IL-2 gene in an experimental lung metastasis model using syngeneic mice. Intravenous injection of the low IL-2 producer cells formed multiple lung metastatic foci and the survival of the mice was not different from that of the mice injected with wild-type cells. However, the mice administrated with the high producer cells survived significantly longer. Subcutaneous inoculation of the low producers, although it caused the development of local tumors at the inoculation sites in some of the mice tested, inhibited lung metastasis of wild-type cells subsequently inoculated and prolonged the survival of the mice rechallenged with Meth A cells, syngeneic fibrosarcoma cells. In contrast, inoculation of the high producers did not cause the development of subcutaneous tumors and inhibited the experimental metastasis of parental but not Meth A cells inoculated thereafter. Thus, the amount of secreted IL-2 from tumor cells differentially influences antitumor effects by inducing tumor specific and nonspecific immunity.

Animals↗

PubMedDate not supplied

Induction of T cell dependent acquired immunity in syngeneic mice by the combined expression of interleukin-4 and granulocyte macrophage-colony stimulating factor gene in murine colon carcinoma cells.

The inoculation of cytokine-producing tumor cells into syngeneic animals can produce antitumor effects. We previously reported that injection of a mixed population of murine colon carcinoma (Colon 26) cells transduced with interleukin-4 (IL-4) gene with granulocyte macrophage-colony stimulating factor (GM-CSF) gene developed subcutaneous tumors in syngeneic mice, but these tumors thereafter regressed spontaneously (Oncology 1997;54:69-73). In this study, we found the generation of tumor-specific acquired immunity in the mice which had rejected the tumors of a mixed population of IL-4 and GM-CSF producer cells, and that the immunity was mediated by mature T cells. Moreover, local secretion of both IL-4 and GM-CSF in the vicinity of tumors was found to be crucial for this rejection. These data suggest that enhanced host defense brought about by the combined expression of IL-4 and GM-CSF genes in tumor cells is a potential therapeutic strategy.

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