研究者詳細 - 佐谷　秀行

論文 - 佐谷　秀行

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The CD44 adhesion molecule and metastasis

Tanabe K. K., Saya H.

Critical Reviews in Oncogenesis 5 （ 2-3 ） 201 - 212 1994年

ISSN 0893-9675

Polyclonal uses of T-cell receptor (TCR)α and β genes for cytotoxic T lymphocytes in human metastatic melanoma

Seito D., Morita T., Masuoka K., Maeda T., Saya H., Itoh K.

International Journal of Cancer 58 （ 4 ） 497 - 502 1994年

ISSN 0020-7136

Identification of genetic structure and diversity of T-cell receptor (TCR)α and β genes for cytotoxic T lymphocytes (CTLs) infiltrating human cancers is important for the better understanding of molecular mechanisms of host defense at tumor sites. cDNAs of TCRα and β genes of 22 different melanoma-specific CTL clones established from the tumor-infiltrating lymphocytes of 2 patients were sequenced for analysis of their genetic structure and diversity. Vα7.2-Jα10-Cα was found in 4 of 22 clones, 2 of which also used the same β-chain. The other 20 clones showed different combinations of α and β use. At deduced amino-acid levels, 7 of 9 clones from one patient used a threonine residue at the 26th position in the complementarity-determining region (CDR) 1 of TCRα. Eight of 13 clones used a threonine at the 99th or a serine residue at the 100th position in CDR3 of TCRα. CTL clones with the same or different TCRαα showed the same or different patterns of cytotoxicity, respectively. These results suggest that CTLs usually do not demonstrate clonal expansion at tumor sites of metastatic melanoma's but rather that polyclonal T cells capable of binding to multiple melanoma determinants through CDR3 of TCRα accumulate in the tumor.

Increase in the capability of interleukin 2 gene-transduced renal cell carcinoma cells to induce cytotoxic lymphocytes.

Itoh K., Umezu Y., Morita T., Saya H., Seito D., Augustus L. B., Nakao M., Sakata M., Miyajima J., Masuoka K.

Kurume Medical Journal 41 （ 2 ） 53 - 63 1994年

ISSN 0023-5679

The immunogenicity of human cancer cells transfected with interleukin 2 (IL-2) gene, a potent vaccine candidate, has not yet been fully investigated. Human renal cell carcinoma (RCC) cells transduced with human IL-2 gene (RCC-IL-2) were investigated in vitro for the capability to induce lymphokine-activated killer (LAK) or cytotoxic T lymphocyte (CTL) activity in peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocyte (TIL). The RCC-IL-2 cells stimulated PBMC to demonstrate LAK activity, and also stimulated autologous TILs to proliferate and exhibit cytotoxicity relatively restricted to autologous tumor cells. In contrast, both parental RCC and RCC transduced with neomycin gene alone failed to induce these activities. These results indicate that RCC-IL-2 cells are more potent than the other RCC cells with regard to inducing cytotoxic lymphocytes against autologous tumor cells.

Bacterial expression of an active tyrosine kinase from a protein A/truncated c-src fusion protein

Saya Hideyuki, Lee Polly S Y, Nishi Toru, Izawa Ichiro, Nakajima Motowo, Gallick Gary E., Levin Victor A.

FEBS Letters 327 （ 2 ） 224 - 230 1993年07月

ISSN 0014-5793

The carboxy-terminal half of the c-src protein fused to the protein A moiety was expressed in bacteria. The protein A/truncated c-src fusion protein, which does not have SH2 and SH3 domains, is found in the periplasmic space allowing for a simple one-step purification and demonstrated high efficiency in autophosphorylation and exogeneous substrate phosphorylation. The missense mutation at codon 294 (Ile → Thr), which is located in the ATP-binding domain of the c-src, resulted in dramatic reduction of tyrosine kinase activity of the fusion protein. Using the fusion protein. we also revealed that staurosporin, a well-known kinase inhibitor, directly affects autophosphorylation of the C-terminal half of the c-src protein. This truncated c-src expression system provides a good source of enzyme for diverse experiments and is an ideal model for understanding the implication of structural alterations in the catalytic activity of the c-src kinase by site-directed mutagenesis experiments.

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Detection of p53 alterations in human astrocytomas using frozen tissue sections for the polymerase chain reaction

Aka Koji, Bruner Janet M., Bondy Melissa L., Ligon Keith, Nishi Toru, del Giglio Auro, Moser Richard P., Levin Victor A., Saya Hideyuki

Journal of Neuro-Oncology 16 （ 2 ） 125 - 133 1993年06月

ISSN 0167-594X

The polymorphism of amino acid residue 72 on the human p53 tumor-suppressor gene is a useful marker for detecting intragenic loss of heterozygosity (LOH). We examined the LOH of the p53 gene in human malignant astrocytomas by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using DNA extracted from frozen tissue sections under histologic examination. Eleven of 16 informative cases (69%) of the malignant astrocytomas were found to have LOH in the p53 gene. Sequential frozen sections were analyzed by immunohistochemistry using anti-p53 antibody PAb1801 to detect overexpression of the p53 protein, which is presumably altered if it is detectable. Ten of the 11 cases that had LOH of the p53 gene overexpressed the p53 protein. Moreover, 4 of the 11 patients with LOH of the p53 gene developed a second neoplasm in addition to an astrocytoma, possibly indicating genetic instability in these patients. These data suggest that alterations of the p53 gene may play an important role in the genesis of malignant astrocytoma. The combination of the PCR-RFLP method and immunohistochemical analysis using frozen tissue sections is a practical diagnostic tool for examination of human malignancies, including astrocytomas.

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Expression of CD44R1 adhesion molecule in colon carcinomas and metastases

Tanabe K. K., Ellis L. M., Saya H.

The Lancet 341 （ 8847 ） 725 - 726 1993年03月

ISSN 0140-6736

CD44 is a cell surface adhesion molecule postulated to control lymphocyte recirculation by facilitating entry into lymphoid tissue. Tumour cells transfected to overexpress the epithelial variant CD44R1 readily gain access to lymph nodes and distant metastatic sites in animal models, possibly by mimicking circulating lymphocytes.. To investigate if human tumours display altered CD44 expression we performed reverse transcriptase a polymerase chain reaction (PCR) analysis of CD44 in 49 specimens from normal colonic mucosa, primary colon and rectal tumours, normal liver, and metastases of 20 patients. The haematopoietic variant CD44H was the principal isoform amplified in all of the specimens, However, 12/14 primary tumours and 16/16 metastatic tumours expressed CD44R1, compared with only 2 of 13 normal mucosa specimens. This increase in CD44R1 relative to CD44H expressed by human colon carcinoma cells may increase their metastatic potential. Moreover, the relative increase in PCR amplification of CD44R1 compared with that of CD44H may provide a sensitive method for detecting primary and metastatic colon carcinoma cells in small biopsy specimens.

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p53 protein immunostaining in routinely processed paraffin-embedded sections.

Bruner J. M., Connelly J. H., Saya H.

Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 6 （ 2 ） 189 - 194 1993年03月

ISSN 0893-3952

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human tumors. The altered protein product of the mutant gene is stabilized in tumor cells and can be detected using monoclonal antibody immunohistochemistry. We report a technique for immunostaining of the altered p53 protein in routinely processed paraffin-embedded tissue sections, comparing this method with a frozen-section immunostain method for concordance. Similarly processed tumor cell lines with known mutations or deletions of the p53 gene served as positive or negative controls. The results in 25 human gliomas and 20 colorectal carcinomas showed a 100% correspondence of positive reactivity in the colorectal carcinomas and an 83% correspondence in the gliomas. In no case did the paraffin-embedded sections react in the absence of frozen section reactivity (that is, there were no false positives). Three of 18 gliomas showed reactivity in frozen sections without reactivity in the companion paraffin-embedded sections. This discrepancy could have been caused by technical factors such as length of fixation time. We propose that this new paraffin-embedded section immunostaining method will be of value as a screening technique for the investigation of p53 mutations in archived human tumors. The data thus obtained may then be correlated with clinical information and perhaps be of value in diagnosis or predicting outcome for various human cancers.

Molecular pathogenesis and management of gliomas

Kyritsis A. P., Saya H., Levin V. A.

Neuroimaging Clinics of North America 3 （ 4 ） 735 - 744 1993年

ISSN 1052-5149

Reduced tumorigenicity and cell motility of a gastric carcinoma cell line by introduction of wild-type p53 gene

Kyo E., Yokozaki H., Yanagihara K., Yasui W., Ito M., Sano T., Lee P. S., Saya H., Tahara E.

International Journal of Oncology 3 （ 2 ） 265 - 271 1993年

ISSN 1019-6439

Wild-type or mutant human p53 gene was transfected into a human gastric carcinoma cell line MKN-1 which shares a mutant p53 allele. Transfected wild- type p53 reduced the colony forming efficiency and tumorigenicity of MKN-1 cells. However, no difference in expression of cell adhesion molecule, oncogenes and growth factors was observed among parent, wild-type p53 and mutant p53 transfectants. In motility assay, the wild-type p53 transfectants relative to the parental or mutant p53 transfectants exhibited a decreased motility, and HGF had a greater effect on the motility of the mutant p53 transfectants, but very little effect on the motility of either the parental or wild-type transfectants. In invasion assay, mutant p53 transfectants revealed the increased invasion ability into collagen gel. These results suggest that allele loss and point mutation of p53 gene may play a critical role not only in growth but also in invasion of gastric carcinoma cells.

Proliferating cell nuclear antigen (PCNA) expression in chronic lymphocytic leukemia (CLL)

Giglio Auro Del, O'brien Susan, Ford Richard J., Manning John, Saya Hideyuki, Keating Michael, Johnston Dennis, Chamone Dalton Fisher, Deisseroth Albert B.

Leukemia and Lymphoma 10 （ 4-5 ） 265 - 271 1993年

ISSN 1042-8194

Chronic Lymphocytic Leukemia (CLL) is usually an indolent disorder which in some patients assumes an aggressive clinical course. In order to assess at presentation the prognosis of a given patient, several staging systems and prognostic variables have been proposed including the expression of the Proliferating Cell Nuclear Antigen (PCNA). PCNA is a 36 kd nuclear protein, the regulation of which is cell cycle-dependent. In CLL, PCNA levels correlate with cell proliferation, clinical stage and the lymphocyte doubling time (LDT). Furthermore, preliminary data suggests that PCNA expression may also predict response to Fludarabine-based chemotherapy. Since PCNA is a cofactor for Delta DNA polymerase, PCNA overexpression in CLL may also reflect the intrinsic DNA repair activity of the leukemic cells and thus their resistance to chemotherapy. Further studies aiming at modulation of PCNA expression in CLL cells may clarify this issue and may offer a future new therapeutic strategy with which to treat this disorder.

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Role and regulation of expression of 92-kDa type-IV collagenase (MMP-9) in 2 invasive squamous-cell-carcinoma cell lines of the oral cavity

Juarez J., Clayman G., Nakajima M., Tanabe K. K., Saya H., Nicolson G. L., Boyd D.

International Journal of Cancer 55 （ 1 ） 10 - 18 1993年

ISSN 0020-7136

The present study was undertaken to determine the role of the metalloproteinase MMP-9 in the invasive phenotype of squamous-cell carcinoma of the oral cavity and the regulation of its expression. Zymographic analysis of conditioned medium from 2 highly invasive squamous-cell-carcinoma cell lines indicated large amounts of an enzyme which was indistinguishable, in size (92 kDa) from the MMP-9 pro-enzyme. Conversion of the 92-kDa gelatinase into a lower-molecular-weight species (84 kDa), identical in size to the activated gelatinase, was evident when both cell lines, which are avid secretors of urokinase, were cultured in the presence of plasminogen. Penetration of an extracellular-matrix-coated filter was dramatically reduced in the presence of the collagenase inhibitor, tissue inhibitor of metalloproteinase-2, suggesting a critical role for MMP-9 in the invasive process. Immunohistochemical studies demonstrating the presence of MMP-9 in tumor cells of resected squamous-cell cancers suggested that secretion of this collagenase by cells in vitro was reflective of the in vivo setting. Since several phorbol-ester response elements are present in the MMP-9 promoter, we determined the role of protein-kinase-C pathways in the regulation of MMP-9 expression in cultured SCC. Treatment of cells with PMA resulted in a more-than-20-fold increase in the level of protein and mRNA. Conversely, culturing of cells in the presence of the protein-kinase-C inhibitor, calphostin-C, led to a dose-dependent decrease in the amount of MMP-9 mRNA and protein, suggesting that the constitutive expression of this collagenase reflects activation of this signal transduction pathway. In summary, our data suggest that, for a sub-population of squamous-cell carcinomas, secreted MMP-9 is an important determinant of the invasive phenotype, and that the expression of this metalloproteinase is regulated by protein-kinase-C pathways.

Novel variants of CD44 arising from alternative splicing

Tanabe K. K., Nishi T., Saya H.

Molecular Carcinogenesis 7 （ 4 ） 212 - 220 1993年

ISSN 0899-1987

CD44 is a cell-surface glycoprotein postulated to play a role in a variety of biological processes, including lymphocyte homing and tumor-cell metastasis. Several isoforms of CD44 have been identified in human cells, and the genesis of some of these isoforms has been attributed to alternative splicing. In the study presented here we amplified three novel transcript variants of CD44 from human cell lines using a reverse transcriptase-polymerase chain reaction strategy. Two of the novel isoforms differed from previously described CD44 isoforms as a result of alternative splicing that occurred at previously reported splice junctions. The third novel CD44 isoform was generated from a previously unreported alternative splice junction near the 5' end of the open reading frame. Southern blot analysis of genomic DNA revealed that these novel isoforms and all of the previously described CD44 isoforms arose from alternative splicing. The capability of cells to modify their CD44 alternative splicing pattern was demonstrated in MCF-7 cells, which altered their CD44-isoform expression pattern in response to treatment with hyaluronidase. A better understanding of mechanisms regulating CD44 alternative splicing may provide insights into diverse processes, including tumor-cell metastasis annd lymphocyte homing.

Molecular, genetic, and biologic aspects of primary brain tumors

Steck P. A., Bruner J. M., Pershouse M. A., Hadi A., Conrad C. A., Yung W. K A, Saya H.

Cancer Bulletin 45 （ 4 ） 296 - 303 1993年

ISSN 0740-820X

Epidemiology, cytogenetics, and molecular biology of brain tumors

Kyritsis A. P., Saya H.

Current Opinion in Oncology 5 （ 3 ） 474 - 480 1993年

ISSN 1040-8746

Prognostic value of proliferating cell nuclear antigen expression in chronic lymphoid leukemia

Del Giglio Auro, O'Brien Susan, Ford Richard, Saya Hideyuki, Manning John, Keating Michael, Johnston Dennis, Khetan Rainer, El-Naggar Adel, Deisseroth Albert

Blood 79 （ 10 ） 2717 - 2720 1992年05月

ISSN 0006-4971

Chronic lymphocytic leukemia (CLL) is a usually indolent disease that can assume an aggressive clinical course in some patients. To develop assays that would be predictive of how a particular patient's disease would evolve, we studied the expression of proliferating cell nuclear antigen (PCNA) by Western blotting in 40 patients with CLL. The concentration of PCNA, a cofactor for δDNA-dependent DNA polymerase, is indicative of the proliferative state of the cell. Significantly lower PCNA levels were observed in earlier stage CLL when compared with more advanced disease. The leukemic cell proliferative rate, assessed by lymphocyte doubling time and flow cytometry, also correlated significantly with the level of PCNA expression. These results suggest that a high level of PCNA in the cells of CLL patients at presentation identifies a subgroup of patients whose CLL cells have a higher proliferative activity and who may, therefore, have a potentially shorter survival.

Alternative splicing of neurofibromatosis type 1 gene transcript in malignant brain tumors

Mochizuki H., Nishi T., Bruner J. M., Lee P. S Y, Levin V. A., Saya H.

Molecular Carcinogenesis 6 （ 2 ） 83 - 87 1992年

ISSN 0899-1987

The neurofibromatosis type 1 (NF1) gene encodes a 360-residue region showing significant homology to the catalytic domains of both mammalian GTPase-activating protein (GAP) and yeast IRA protein. The product of the GAP-related domain of the NF1 gene (NF1-GRD) has been shown to stimulate ras GTPase and consequently to inactivate ras protein. We previously reported that the NF1-GRD has two types of transcripts, type I and type II, which are generated by an alternative splicing mechanism, and that the differential splicing of the NF1-GRD may be related to differentiation of neuroectodermal cells. Here we examined the differential expression of type I and type II transcripts of NF1-GRD in clinical samples of supratentorial malignant brain tumors by the RNA-polymerase chain reaction (PCR) method using frozen tissue sections. Our observations revealed that normal cerebrum predominantly expressed the type II NF1-GRD transcript, whereas primitive neuroectodermal tumors predominantly expressed the type I transcript. Additionally, although the type I/type II ratio in astrocytomas varied widely among tissue samples, all glioblastomas showed higher type I/type II ratios that adjacent brain samples. The RNA-PCR analysis using frozen tissue sections is a useful and sensitive method for detecting genetic markers in clinical tissue samples.

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Structural alterations of the p53 gene in human leukemias

Miwa Hiroshi, Kita Kenkichi, Saya Hideyuki, Otsuji Akira, Masuya Masahiro, Nishii Kazuhiro, Morita Norihisa, Takakura Nobuyuki, Ohishi Koushi, Nasu Kaori, Nakase Kazunori, Shirakawa Shigeru

Leukemia Research 16 （ 11 ） 1105 - 1112 1992年

ISSN 0145-2126

We have investigated alterations of the p53 gene in human leukemias by polymerase chain reaction-mediated restriction fragment length polymorphism analysis. This method detects the codon 72 polymorphism of the p53 gene, allowing identification of loss of heterozygosity (LOH) of the p53 gene. In this study, at least two specimens were obtained from each patient to compare the allele status at different points of clinical course. Of 21 cases examined 7 were heterozygous at this polymorphic site and were evaluable for LOH study. Only one patient of Philadelphia chromosomepositive acute lymphoblastic leukemia (ALL) lost the heterozygosity in the specimen of her last relapse. Leukemic cells from her repeated relapses including the last one revealed to have the clonally rearranged immunoglobulin H chain gene of the same size, indicating that alteration of the p53 gene in this patient might account for the lethal relapse as a clonal evolution event. Northern-blot analysis of the p53 gene showed that one Gase of CD7(+) ALL had altered p53 mRNA. Overall, it is demonstrated that alterations of the p53 gene might be involved in the pathogenesis or progression of at least some human leukemias, although the alterations in leukemias seemed to be not as frequent as in solid tumors.

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Neurofibromatosis type 1 (NF1) gene

Nishi Toru, Saya Hideyuki

Cancer and Metastasis Reviews 10 （ 4 ） 301 - 310 1991年12月

ISSN 0167-7659

The gene responsible for neurofibromatosis type 1 (NF1), a common autosomal dominantly inherited disease, has been isolated. A region of NF1 gene product has been demonstrated to share structural and functional similarities with the mammalian GTPase activating protein (GAP) and the yeast IRA proteins. Thus, the NF1 protein is thought to play a role in signal transduction by stimulating the conversion of the Ras protein from a GTP-bound active form to a GDP-bound inactive form. The increased risk of malignant tumors in neuroectodermal tissues of NF1 patients may be caused by disruption of growth and differentiation regulatory functions of the NF1 gene. A second type of the NF1-GAP related domain (NF1-GRD) transcript, which has an extra 21-amino-acid insert in the center of the previously reported first type transcript, has been described. This insert significantly changes the hydrophilicity and secondary structure of the central region of NF1-GRD, therefore, suggesting it also changes its function. Alternative splicing is the most likely mechanism by which these two types of transcripts arise. The NF1-GRD alternative splicing has been shown to be intimately involved in differentiation of neuroectodermal tissues. Aberrant regulation of the alternative splicing may contribute to tumor formation in neuroectodermal tissue.

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Effect of herbimycin a on growth and pp60^c-src activity in human colon tumor cell lines

Garcia R., Parikh N. U., Saya H., Gallick G. E.

Oncogene 6 （ 11 ） 1983 - 1989 1991年11月

ISSN 0950-9232

The effect of herbimycin A, an ansamycin antibiotic which inhibits cellular transformation by retroviral tyrosine kinases, on the monolayer growth of seven colon tumor cell lines and one cell line established from normal colonic mucosa, CCL239, was examined. Each colon tumor cell line tested showed dose-dependent growth inhibition in response to herbimycin A. A 125 ng ml^-1 dose of the antibiotic caused > 40% growth inhibition in all colon tumor cell lines after two cell doublings. In contrast, at similar herbimycin A concentrations only 12% inhibition was observed in 'normal' CCL239 cells. No major morphologic changes were observed at the light microscopic level in any of the tumor cell lines or CCL239 cells in response to treatment with herbimycin A. Studies using the HT29 colon adenocarcinoma cell line showed dosedependent inactivation of pp60^c-src by herbimycin A, resulting in decreased autophosphorylation, enolase phosphorylation and steady-state levels, which correlated with cellular growth inhibition. Herbimycin A-induced reductions in pp60^c-src kinase activity preceded changes in pp60^c-src steady-state levels. Growth and pp60^c-src inhibition were reversible following removal of herbimycin A from cell culture media. Our results suggest that regulation of pp60^c-src tyrosine kinase activity may be important in growth control of colon tumor cells.

Differential expression of two types of the neurofibromatosis type 1 (NF1) gene transcripts related to neuronal differentiation

Nishi Toru, Lee Polly S Y, Oka Koji, Levin Victor A., Tanase Sumio, Morino Yoshimasa, Saya Hideyuki

Oncogene 6 （ 9 ） 1555 - 1559 1991年09月

ISSN 0950-9232

A 360 residue region encoded by the neurofibromatosis type 1 (NF1) gene shows significant homology to the catalytic domains of both mammalian GTPase-activating proteins (GAP) and yeast IRA proteins. This GAP-related domain of the NF1 gene (NF1-GRD), like the GAP and IRA protein, has been reported to mediate hydrolysis of Ras-bound GTP to GDP, resulting in inactivation of Ras protein. In the present study, we identified two different types of NF1-GRD cDNA. One (type I) is identical to the previously reported sequence, and the other (type II) contained an additional 63 bp insertion that encodes for a region of 21 amino acids in the center of the NF1-GRO molecule. Alternative splicing is the most likely mechanism by which these two types of transcripts arise. Our observations reveal that the type I transcript is predominantly expressed in undifferentiated cells, whereas the type II transcript predominates in differentiated cells. Furthermore, the expression pattern of type I and type II NF1-GRD mRNA immediately changed in SH-SY5Y neuroblastoma cells when neuronal differentiation programs were induced by retinoic acid treatment. We propose that the differential expression of type I and type II NF1-GRD transcripts might be an 'on/off' switch that regulates the catalytic activity of the NF1 gene product, which plays an important role in the regulation of neuronal differentiation.

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論文 - 佐谷　秀行