Biomedical subjects

S Sakiyama

Publications and source records attributed to S Sakiyama.

At least 37 records · Page 2Linked to original sources

PubMed2001

Tissue-specific expression of a suicide gene for selective killing of neuroblastoma cells using a promoter region of the NCX gene.

The human NCX gene, a homologue of the murine neural crest homeobox (Ncx/Hox11L.1) gene whose expression is restricted to a subset of neural crest-derived tissues, was expressed in human neuroblastoma cells but not in other tumors or fibroblasts. A 4.5-kb genomic fragment in the 5'-flanking region of the NCX gene efficiently transcribed the fused luciferase reporter gene in human neuroblastoma cells but not in non-neuroblastoma cells. Sequential deletion of this regulatory region from the 5' side demonstrated that a 1.7-kb fragment upstream from the start codon retained the preferential promoter activity in neuroblastoma cells. The transcriptional activation by the NCX promoter was stronger than that by the SV40 T antigen promoter in human neuroblastoma cells. Transfection of neuroblastoma cells with the NCX promoter-linked herpes simplex virus-thymidine kinase (HSV-TK) gene increased their sensitivity to ganciclovir. The regulatory region of the NCX gene is thus useful for neuroblastoma-specific suicide gene therapy.

Cell Death↗

PubMed2001

Bystander effect in uracil phosphoribosyltransferase/5-fluorouracil-mediated suicide gene therapy is correlated with the level of intercellular communication.

We examined whether a suicide gene/prodrug system using the uracil phosphoribosyltransferase (UPRT) of E. coli origin and 5-fluorouracil (5-FU) could achieve a bystander effect in two rodent tumor cell lines, murine colon carcinoma (Colon 26) and rat gliosarcoma (9L) cells. Cytotoxicity tests of mixed populations consisting of parent and transduced cells showed that the bystander effect was not produced in Colon 26 cells in either the UPRT/5-FU system or the herpes simplex virus-thymidine kinase/ganciclovir system but a strong bystander effect was evidenced by both suicide gene systems in 9L cells. The expression level of connexin 43, a protein that constitutes gap junctions, was high in 9L but low in Colon 26 cells. A gap junction-permeable fluorescein dye could be transferred among 9L cells but hardly at all among Colon 26 cells. Taken together, the efficacy of the bystander effect in the UPRT/5-FU system can be affected by gap junction-mediated intercellular communication.

Animals↗

PubMed2001

The error-prone DNA polymerase zeta catalytic subunit (Rev3) gene is ubiquitously expressed in normal and malignant human tissues.

Mutagenesis induced by UV light and chemical agents in yeast is largely dependent on the function of Rev3, the catalytic subunit of DNA polymerase zeta that carries out translesion DNA synthesis. Human and mouse homologues of the yeast Rev3 gene have recently been identified, and inhibition of Rev3 expression in cultured human fibroblasts by Rev3 anti-sense was shown to reduce UV-induced mutagenesis, indicating that Rev3 also plays a crucial role in mutagenesis in mammalian cells. A common variant transcript with an insertion of 128-bp between nucleotides +139 and +140 is found in both human and mouse Rev3 cDNAs, but its biological significance has not been defined. We show here that the insertion variant is not translatable either under in vitro or in vivo conditions. We also found that the translational efficiency of Rev3 gene is enhanced by the 5' untranslated region that contains a putative stem-loop structure postulated to inhibit the translation. Since the human Rev3 gene is localized to chromosome 6q21, a region previously shown to contain genes involved in tumor suppression and cellular senescence, we examined its expression in various normal and malignant tissues. Rev3 and its insertion variant transcripts were ubiquitously detected in all 27 normal human tissues studied, with an additional variant species found in tissues with relatively high levels of Rev3 expression. Levels of Rev3 transcripts were similar in lung, gastric, colon and renal tumors compared to normal tissue counterparts. The data indicate that Rev3 expression is ubiquitous and is not dysregulated in malignancies.

5' Untranslated Regions↗

PubMed2001

Combinatory anti-tumor effects of electroporation-mediated chemotherapy and wild-type p53 gene transfer to human esophageal cancer cells.

Delivery of electric pulses to an established solid tumor augments the permeability of cell membrane and increases the susceptibility of tumors to an anti-cancer agent that is administered in the vicinity of tumors. Forced expression of the wild-type p53 gene in tumor cells that have non-functional p53 gene(s) can also enhance their sensitivity to a DNA-damaging agent. To investigate the feasibility of electroporation-mediated therapy for cancer, electric pulses were delivered to human esophageal tumors developed in nude mice after they received an anti-cancer agent and/or plasmid DNA containing the wild-type p53 gene. The growth of esophageal tumors was suppressed with electroporation-mediated chemotherapy compared with the treatment with an anti-cancer agent or electroporation alone. Intratumoral injection of the wild-type p53 gene into p53-mutated esophageal tumors followed by electroporation also inhibited tumor growth. When mice were administered with the wild-type p53 gene and an anti-cancer agent, subsequent electroporation produced a synergistic therapeutic effect. Combinatory transfer of plasmid DNA and a pharmacological agent by electroporation is thereby a possible therapeutic strategy for the treatment of solid tumors.

Animals↗

PubMed2001

[A novel neurotransmitter, DAN, mediates pain sensation in the spinal dorsal horn].

Differential screening-selected gene aberrative in neuroblastoma(DAN) belongs to a novel gene family(DAN family) that includes the head-inducing factor, Cerberus, and dorsaling factor, Gremlin. It has been suggested that DAN family members control diverse processes in growth, development and the cell cycle. Here, we demonstrate that DAN is produced in the small neurons of the dorsal root ganglion(DRG) and transported to the nerve terminals in the spinal dorsal horn in adult rats. Furthermore, intrathecal injection of an antibody to DAN suppressed pain sensations induced by the application of complete Freund's adjuvant and carageenan into the rat hindpaw, and the amount of DAN mRNA in the DRG neurons and of DAN in the spinal dorsal horn were increased in the inflammatory models. These data suggest that DAN in a novel neurotransmitter and/or modulator in the primary sensory nerve fibers for pain sensation.

Animals↗

PubMed2000

Overexpression of DAN causes a growth suppression in p53-deficient SAOS-2 cells.

It has been shown that the expression of DAN as well as Drm/Gremlin, a member of DAN/Cerberus family, is significantly down-regulated in rodent fibroblasts transformed with various oncogenes and overexpression of DAN results in the phenotypic reversion of the transformed phenotypes. In the present study, we examined the expression levels of DAN, BMP-2, BMP-4, and BMPRs (BMP receptors) in five human cell lines derived from bone and soft tissue tumors. Northern blot analysis revealed that DAN mRNA was detected in OS-KH and RMS-NK cells, but was not detectable in SAOS-2, NOS-1, and ASPS-KY cells. Transient overexpression of DAN in SAOS-2 cells, which lack functional p53 and pRB, resulted in a remarkable growth suppression without the induction of p21(Waf1). Interestingly, overexpression of DAN was associated with a reduction of alkaline phosphatase activity in SAOS-2 cells. Stable transfection of DAN in SAOS-2 cells caused a significant reduction of numbers of drug-resistant colonies, whereas the truncated form of DAN which lacked a possible signal peptide, completely lost this capability. Our results suggest that the secreted form of DAN exerts its growth-suppressive function in SAOS-2 cells in a p53-independent manner.

Alkaline Phosphatase↗

PubMed2000

Identification and characterization of a 500-kb homozygously deleted region at 1p36.2-p36.3 in a neuroblastoma cell line.

Loss of heterozygosity of the distal region of chromosome 1p where tumor suppressor gene(s) might harbor is frequently observed in many human cancers including neuroblastoma (NBL) with MYCN amplification and poor prognosis. We have identified for the first time a homozygously deleted region at the marker D1S244 within the smallest region of overlap at 1p36.2-p36.3 in two NBL cell lines, NB-1 and NB-C201 (MASS-NB-SCH1), although our genotyping has suggested the possibility that both lines are derived from the same origin. The 800-kb PAC contig covering the entire region of homozygous deletion was made and partially sequenced (about 60%). The estimated length of the deleted region was 500 kb. We have, thus far, identified six genes within the region which include three known genes (DFF45, PGD, and CORT) as well as three other genes which have been reported during processing our present project for the last 3(1/2) years (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14). They include the genes related to apoptosis, glucose metabolism, ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule and biogenesis of peroxisome. At least three genes (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14) were differentially expressed at high levels in favorable and at low levels in unfavorable subsets of primary neuroblastoma. Since the 1p distal region is reported to be imprinted, those differentially expressed genes could be the new members of the candidate NBL suppressor, although RT-PCR-SSCP analysis has demonstrated infrequent mutation of the genes so far identified. Full-sequencing and gene prediction for the region of homozygous deletion would elucidate more detailed structure of this region and might lead to discovery of additional candidate genes. Oncogene (2000) 19, 4302 - 4307

Carrier Proteins↗

PubMed2000

Transduction of the human deoxycytidine kinase gene in rodent tumor cells induces in vivo growth retardation in syngeneic hosts.

Deoxycytidine kinase (dCK) mediates the phosphorylation of nucleoside analogues that can be used as anti-cancer agents. We examined whether susceptibility of mouse colon carcinoma (Colon 26) and rat gliosarcoma (9L) cells to 1-beta-D-arabiofuranosylcytonsine (AraC), a chemotherapeutic agent, can be increased after the tumor cells were transduced with the human dCK gene. Expression of the dCK gene in both cell lines conferred increased sensitivity in vitro to AraC. Although their proliferation rates in vitro remained the same as those of parental cells, tumor growth of the transduced cells in syngeneic host animals was unexpectedly retarded compared with that of respective parental cells. In contrast, the growth of the transduced cells was not different from that of parental cells, when they were inoculated in T cell-defective nude mice. A histological examination revealed infiltration of eosinophils into the dCK gene-transduced but not into parental Colon 26 tumor. These data suggest that a therapeutic gene, when expressed in xenogenic animals, can be a tumor antigen which is recognized by a host defense system.

Animals↗

PubMed2000

Therapeutic efficacy of the suicide gene driven by the promoter of vascular endothelial growth factor gene against hypoxic tumor cells.

We examined whether herpes simplex virus thymidine kinase (HSV-TK) gene expression driven by the promoter of the vascular endothelial growth factor (VEGF) gene that is activated by hypoxia is effective in killing highly metastatic Lewis lung carcinoma A11 cells under hypoxic conditions. We isolated the promoter region encompassing the hypoxia response element (HRE) of the mouse VEGF gene. To assess the hypoxia responsiveness of the VEGF promoter, A11 cells were transiently transfected with luciferase reporter plasmids. Exposure of the transfectants to hypoxia resulted in a 2-3-fold induction of luciferase activity. Deletion of the HRE site abolished VEGF promoter activity under both normoxic and hypoxic conditions. We constructed a retroviral vector harboring the HSV-TK or green fluorescence protein (GFP) gene under the control of the VEGF promoter. A11 cells transfected with vector harboring the VEGF promoter fused to the HSV-TK gene [A11(HRE/TK) cells] were more sensitive to ganciclovir than cells transfected with the control vector harboring the VEGF promoter alone, and the sensitivity of the A11(HRE/TK) cells was increased by exposure to hypoxia followed by reoxygenation. Culturing A11 cells transfected with vector harboring the VEGF promoter fused to the GFP gene under hypoxic conditions resulted in an increase in the expression of GFP. Monitoring GFP expression and vascularity in the A11 transfectant tumors revealed up-regulation of GFP expression in poorly vascularized regions. Administration of ganciclovir to mice bearing s.c. tumors formed by A11(HRE/TK) cells resulted in regression of the tumors. These results suggest a possible application of the suicide gene driven by the VEGF promoter to cancer gene therapy that efficiently targets hypoxic tumor cells.

Animals↗

PubMed2000

Identification of the homozygously deleted region at chromosome 1p36.2 in human neuroblastoma.

BACKGROUND: We have identified for the first time a homozygously deleted region within the smallest region of overlap at 1p36.2-3 in two neuroblastoma cell lines. PROCEDURE: The 800-kb PAC contig covering the entire homozygously deleted region was made and sequenced. To date, approximately 70% of sequencing has been accomplished, and the estimated length of the deleted region was 500 kb. RESULTS: Currently, we have found six genes within the region, which include three known genes as well as three other genes that have been reported during processing of our present project for the last 3(1/2) years. We report here the results of expression and mutation analyses of those genes. CONCLUSIONS: Full sequencing for the region of homozygous deletion as well as further analyses of the genes mapped within the region may reveal whether or not there is a neuroblastoma suppressor gene as proposed by the Knudson's two-hit hypothesis.

Child, Preschool↗

PubMed2000

Hunting the subset-specific genes of neuroblastoma: expression profiling and differential screening of the full-length-enriched oligo-capping cDNA libraries.

BACKGROUND: Neuroblastoma (NBL) has a distinct nature in different prognostic subgroups. PROCEDURE: To understand the molecular mechanism of NBL's genesis and biology as well as that of the neural crest development, we constructed full-length-enriched cDNA libraries by an oligo-capping method from two different subsets of primary NBL, one with favorable biology and the other with MYCN amplification. RESULTS: Sequencing analysis of these libraries revealed that the expression profile was markedly different between both subsets. To identify the genes differentially expressed between the subsets, semi-quantitative RT-PCR analyses are proceeding. CONCLUSION: So far, 54 transcripts have been found to be expressed at high levels in favorable NBLs, and significantly at low levels in unfavorable NBLs.

Gene Expression Regulation, Neoplastic↗

PubMed2000

Molecular cloning and expression analysis of the human DA41 gene and its mapping to chromosome 9q21.2-q21.3.

DA41 was previously identified as one of the DAN-binding proteins, via a yeast-based two-hybrid screening strategy. In the present study, we cloned a human homolog of DA41 cDNA. Structural analysis revealed that human DA41 cDNA consisted of 2,861 nucleotides in length and encoded a protein of 589 amino acids, with a predicted molecular mass of 62.4kDa. Human DA41 exhibited an 86% amino acid sequence identity to rat DA41, indicating the evolutionarily conserved structure and function of DA41. A database search for DA41-related protein(s) identified mouse PLIC-1, PLIC-2, frog XDRP1, and yeast DSK2. DA41 and each DA41-related protein contain a ubiquitin-like domain in their amino-terminal regions. DA41 was expressed ubiquitously in adult human tissues, with relatively higher levels in pituitary gland, adrenal gland, kidney, thymus, and placenta. Fluorescence in situ hybridization (FISH) revealed that DA41 was mapped to human chromosome 9q21.2-q21.3, a position overlapping the candidate tumor suppressor locus for bladder cancer.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Brain imaging in childhood- and adolescence-onset schizophrenia associated with obsessive-compulsive symptoms.

OBJECTIVE: Childhood- and adolescence-onset schizophrenic patients with obsessive-compulsive symptoms (OCS) constitute a specific subgroup of schizophrenia. We performed magnetic resonance imaging in this group seeking evidence of neurodevelopmental insults. METHOD: Thirty-two schizophrenic patients were compared with 19 controls. Schizophrenic subjects were divided into 15 patients with OCS (SOCS+ group; onset at 15.5 +/- 1.6 years) and 17 without OCS (SOCS- group; onset at 15.3 +/- 1.3 years). Areas of the hippocampus, frontal lobe, corpus callosum and putamen were analysed morphometrically. RESULTS: The left hippocampus was significantly smaller in the SOCS+ group than in the SOCS- and control groups. CONCLUSION: Reduced size of the left hippocampus in the SOCS+ group supports a neurodevelopmental etiology in this subgroup.

Adolescent↗

PubMed2000

Protective immunity is induced in murine colon carcinoma cells by the expression of interleukin-12 or interleukin-18, which activate type 1 helper T cells.

We investigated the antitumor effects induced by the production of interleukin-12 (IL-12) or IL-18, which influence the function of T helper type 1 cells, in murine colon carcinoma cells (Colon 26). Retrovirally transduced cells with IL-12 genes that encoded both p35 and p40 (Colon 26/IL-12) lost their tumorigenicity when inoculated subcutaneously or intraperitoneally into syngeneic immunocompetent mice. Moreover, the mice that had rejected the Colon 26/IL-12 cells generated protective immunity to wild-type (wt) cells when subsequently challenged. Colon 26 cells transduced with the IL-18 gene (Colon 26/IL-18) could not form subcutaneous tumors in immunocompetent mice, and the mice became resistant to inoculated wt cells. Immunohistochemical analysis revealed that the numbers of blood vessels in Colon 26/IL-12 or Colon 26/IL-18 tumors were markedly reduced, and that the expression of adhesion molecules such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 increased on the endothelium in the stroma of Colon 26/IL-12 tumors. The loss of tumorigenicity of Colon 26/IL-12 or Colon 26/IL-18 cells was not observed in immunocompromised mice. However, the survival days of the immunocompromised mice inoculated with Colon 26/IL-12 but not Colon 26/IL-18 cells were significantly longer than those inoculated with wt cells. The secretion of cytokines that stimulate T helper type 1 cells from tumor cells can thereby induce an antitumor response. However, the effector cells involved in these antitumor effects could differentially migrate to the tumors, and the inhibition of angiogenesis may partially contribute to the antitumor responses observed.

Animals↗

PubMed2000

Impaired tumorigenicity of human pancreatic cancer cells retrovirally transduced with interleukin-12 or interleukin-15 gene.

We examined the antitumor effect of locally secreted interleukin (IL)-12 or IL-15 on human pancreatic cancer cells (AsPC-1). We subcutaneously inoculated AsPC-1 cells retrovirally transduced with IL-12 or IL-15 cDNA into nude mice. Tumors derived from these cells showed retarded growth compared with those from wild-type (wt) cells. Nude mice inoculated intraperitoneally with the cytokine producers survived longer than those injected with wt cells. These cytokine producers were also tested for their tumor growth in severe combined immunodeficient mice. The tumor growth of IL-12 producers was similarly suppressed as found in nude mice, but the average tumor volumes of IL-15 producers were not statistically different from those of wt tumors. In nude mice that were administered anti-asialo GM1 antibody before the inoculation of the tumor cells, growth retardation of tumors of IL-12 producers remained the same as in untreated animals, but that of IL-15 producers was markedly reduced. Immunohistochemical analysis revealed that CD11b+ cells migrated into the tumors of cytokine producers and that the number of CD31+ endothelial cells within the tumors was not different between IL-12 producers and wt cells. Taken together with other data, it is possible that granulocytes are candidate cells for the IL-12-mediated antitumor effect, and that natural killer cells and gammadelta T cells are involved in the IL-15-induced antitumor effect. We did not observe synergistic effects of these cytokines to suppress subcutaneous tumors.

Animals↗

PubMed2000

Expression of Escherichia coli uracil phosphoribosyltransferase gene in murine colon carcinoma cells augments the antitumoral effect of 5-fluorouracil and induces protective immunity.

Uracil phosphoribosyltransferase (UPRT) of Escherichia coli origin can convert 5-fluorouracil (5-FU), a chemotherapeutic agent widely used for solid tumors, to an active intermediate, 5-fluorouridine-5'-monophosphate, as mammalian orotate phosphoribosyltransferase does. To examine whether the E. coli UPRT gene expressed in tumor cells can confer increased sensitivity to 5-FU, we retrovirally transduced Colon 26 cells, a murine colon carcinoma cell line, with the UPRT gene (Colon 26/UPRT cells) and tested the in vivo antitumoral effect of 5-FU in syngeneic immunocompetent mice. After 5-FU administration, tumors of Colon 26/UPRT cells regressed, whereas those of wild-type cells were unaffected. The mice that once eliminated Colon 26/UPRT tumors after 5-FU treatment rejected wild-type cells that were subsequently inoculated but not irrelevant syngeneic tumor cells. This suicide gene/prodrug system was less efficient in nude mice, suggesting that mature alphabeta T cells play a role in the antitumoral effect. The cytotoxicity mediated by the bystander effect was marginal in this system, contrary to the herpes simplex virus-thymidine kinase gene/ganciclovir system. Therefore, expression of the UPRT gene in tumor cells followed by 5-FU administration is a possible strategy for cancer gene therapy, but potentiation of the bystander effect is required for its therapeutic application.

Adenocarcinoma↗

PubMed2000

Treatment of rat experimental brain tumors by herpes simplex virus thymidine kinase gene-transduced allogeneic tumor cells and ganciclovir.

Transfer of the herpes simplex virus thymidine kinase (HSVtk) gene, followed by administration of ganciclovir (GCV), generates the "bystander effect," in which HSVtk-negative wild-type cells are killed by GCV, as are HSVtk-expressing cells. Our previous study demonstrated that intracranial 9L gliomas could be efficiently treated due to this bystander effect by injecting the 9L glioma cells transduced with the HSVtk gene in the vicinity of the preimplanted wild-type 9L glioma and then administering GCV. For a possible clinical application of the bystander effect-mediated cell killing, we tested HSVtkgene-transduced allogeneic C6 glioma cells (C6tk) instead of syngeneic 9L glioma cells transduced with the HSVtk gene. Fisher rats were implanted intracranially with wild-type 9L glioma cells, subsequently injected with C6tk cells at the same brain coordinate, and thereafter treated with GCV or saline. When the rats were treated with GCV, a significant retardation of tumor growth was observed by serial magnetic resonance imaging, although this growth retardation was less prominent than that observed with 9L glioma cells transduced with the HSVtk gene; consequently, survival was prolonged (P < .01). Tumors that received C6tk cells contained almost no HSVtk-positive cells after treatment with GCV. Rejection of allogeneic tumor cells, although possibly incomplete in the brain, can also contribute to the safety of this therapeutic strategy.

Animals↗

PubMed2000

Immunological responsiveness to interleukin-2-producing brain tumors can be restored by concurrent subcutaneous transplantation of the same tumors.

The central nervous system shows tolerance for activated host immune reactions, and this relative unresponsiveness may lessen the efficacy of an immunotherapy for brain tumors. Using interleukin-2 (IL-2)-producing 9L rat gliosarcoma cells (9L/IL-2), we examined whether secretion of IL-2 from subcutaneous (s.c.) and/or intracerebral (i.c.) tumors can elicit augmented immunological responses to brain tumors. Syngeneic rats could reject 9L/IL-2 cells inoculated s.c., but developed 9L/IL-2 brain tumors by i.c. inoculation. The growth of i.c. 9L/IL-2 tumors was, however, significantly retarded compared with that of i.c. wild-type tumors. The growth of i.c. wild-type tumors was significantly suppressed when the rats concurrently received 9L/IL-2 cells s.c. Moreover, most of the rats that were inoculated i.c. with 9L/IL-2 cells did not develop brain tumors when concurrently injected s.c. with 9L/IL-2 cells. Immunohistochemical analysis on i.c. 9L/IL-2 tumors, when the rats were concurrently inoculated s.c. with 9L/IL-2 cells, revealed that migration of CD4+ or CD8+ T cells, monocytes/microglias, and macrophages was markedly augmented to a similar level as found in the s.c. 9L/IL-2 tumors. These results showed that systemic immune responses to brain tumor were induced in an immunologically privileged site by concurrent s.c. inoculation of the same tumors that produce IL-2. The present study may also raise the possibility of a therapeutic strategy for brain tumors by the combinatory expression of IL-2 gene using s.c. immunization followed by direct gene transfer into brain tumors.

Animals↗