Faculty Profiles - OUCHIDA Mamoru

Cytologic changes in two cervical carcinoma cell lenes after transfection of the wild-type p53 genes. Reviewed

Iwasaka T, Zheng PS, Ouchida M, Yamasaki H, Yokoyama M, Sugimori H

Acta Obset. Gynecol. Scand. 75 ( 9 ) 797 - 803 1996

Cytologic changes in two cervical carcinoma cell lines after transfection of the wild-type p53 gene

Tsuyoshi Iwasaka, Peng-Sheng Zheng, Mamoru Ouchida, Humio Yamasaki, Masatoshi Yokoyama, Hajime Sugimori

Acta Obstetricia et Gynecologica Scandinavica 75 ( 9 ) 797 - 803 1996

Language：English Publishing type：Research paper (scientific journal) Publisher：Wiley-Blackwell Publishing Ltd

Background. To examine morphological changes in cervical carcinoma cells after induction of overexpression of wild-type (wt) p53. Method. A dexamethazone-inducible wt-p53 cDNA was introduced into two cervical carcinoma cell lines (TMCC-1 and ME180) and morphological changes were examined under a phase contrast microscope and following Papanicolaou staining. Results. TMCC-1 clones obtained by transfection with wt-p53 gene had an altered morphology especially after induction of p53 expression by treatment with dexamethazone. These changes were characterized by cell flattening, multinucleation, micronucleation, and huge giant cells, yet the parental TMCC-1 had none of these morphological changes even after treatment with dexamethazone. Similar cellular changes were observed in ME180 clones obtained by transfection of wt-p53 gene. Conclusion. On the basis of our observations, we propose that the growth inhibition induced by expression of wt-p53 in these cervical carcinoma cell lines carrying HPV DNA sequences is not the result of G1 arrest but rather relates to a blockade in the M phase of the cell cycle.

DOI： 10.3109/00016349609054706

Loss of tumorigenicity of Ewing's sarcoma cells expressing antisense RNA to EWS-fusion transcripts. International journal

M Ouchida, T Ohno, Y Fujimura, V N Rao, E S Reddy

Oncogene 11 ( 6 ) 1049 - 54 1995.9

Language：English Publishing type：Research paper (scientific journal)

Cytogenetic analysis of Ewing's sarcoma, primitive neuroectodermal tumors and Askin tumors revealed characteristic translocations t(11;22) or t(21;22). Molecular analysis of these translocations revealed 5'-region of EWS gene (from band 22q12) is fused to the 3'-region of either Fli-1 gene (from band 11q24) or erg gene (from band 21q22). Functional characterization of the EWS-Fli-1 and EWS-erg chimeric proteins suggested that they function as transcriptional activators. In order to develop therapeutic agents, it is essential to know whether expression of the EWS-fusion gene products is coupled to tumorigenicity of Ewing's sarcoma cells and if targeting the EWS-fusion products results in loss of tumorigenicity of Ewing's sarcoma cells. For this reason, we have made stable Ewing's sarcomas expressing antisense EWS-Fli-1 or EWS-erg expression plasmids. Expression of antisense EWS fusion transcripts resulted in a significant loss of endogenous EWS-Fli-1 and EWS-erg proteins in Ewing's sarcoma cells. These cells expressing antisense EWS fusion transcripts showed loss of anchorage independent growth and tumorigenicity in nude mice unlike the parental Ewing's sarcoma cells. These results demonstrate the necessity of a certain threshold level of expression of EWS-fusion products in the clonogenicity and tumorigenicity of Ewing's sarcoma cells and therefore emphasizes the importance of targeting the EWS-fusion products as a therapy for Ewing family of tumors.

TLS/FUS fusion domain of TLS/FUS-erg chimeric protein resulting from the t(16;21) chromosomal translocation in human myeloid leukemia functions as a transcriptional activation domain. International journal

D D Prasad, M Ouchida, L Lee, V N Rao, E S Reddy

Oncogene 9 ( 12 ) 3717 - 29 1994.12

Language：English Publishing type：Research paper (scientific journal)

EWS and TLS/FUS genes, which code for RNA binding proteins are involved in a wide variety of human solid tumors. The TLS/FUS gene is involved both in human myxoid liposarcomas which carry a characteristic chromosomal translocation, t(12;16)(q13;p11) and in human myeloid leukemias with recurrent chromosomal translocation, t(16;21)(p11:q22). The TLS/FUS gene is fused to a transcriptional repressor, CHOP (in human myxoid liposarcomas) or transcriptional activator, erg (in human myeloid leukemias). To understand better the functional role of TLS/FUS-erg in human myeloid leukemias, we have cloned the TLS/FUS and TLS/FUS-erg cDNAs and studied the functional properties of their gene products. TLS/FUS protein binds to RNA in vitro and shows preferential binding to poly G. Both the amino- and the carboxy- terminal regions of TLS/FUS containing the conserved RNA binding motifs are needed for poly G specific RNA binding activity. The TLS/FUS fusion domain (TFD) appears to regulate the DNA binding activity of TLS/FUS-erg chimeric protein which shows weaker transcriptional activation properties compared to normal erg proteins. Mutational analysis of the TLS/FUS-erg chimeric protein reveals TFD to function as a transcriptional activation domain thus replacing the amino terminal transcriptional activation domain of the erg protein. Therefore alterations in both DNA binding and transcriptional activation properties of aberrant erg proteins may be responsible for the genesis of t(16;21) chromosomal translocation-bearing human myeloid leukemias.

THE EWS GENE, INVOLVED IN EWING FAMILY OF TUMORS, MALIGNANT-MELANOMA OF SOFT PARTS AND DESMOPLASTIC SMALL ROUND-CELL TUMORS, CODES FOR AN RNA-BINDING PROTEIN WITH NOVEL REGULATORY DOMAINS

T OHNO, M OUCHIDA, L LEE, Z GATALICA, VN RAO, ESP REDDY

ONCOGENE 9 ( 10 ) 3087 - 3097 1994.10

Language：English Publishing type：Research paper (scientific journal) Publisher：STOCKTON PRESS

The EWS gene, which maps to band q12 of human chromosome 22, is involved in a wide variety of human solid tumors including Ewing sarcoma, related primitive neuroectodermal tumors, malignant melanoma of soft parts and desmoplastic small round cell tumors. In these tumors, the EWS is fused to genes encoding transcriptional activators/repressors, like Fli-1 or erg or ATF 1 or wt1. To better understand the function of the EWS protein, we cloned the EWS cDNA. Sequence analysis of this cDNA revealed differential splicing invoving two exons encoding 72 amino acids. Both alternatively spliced transcripts, EWS and EWS-b, are expressed in a variety of cells. Because EWS proteins contain putative conserved RNA binding motifs, we studied the RNA binding properties of the EWS protein. The EWS-b protein binds to RNA in vitro and, specifically, to poly G and poly U. The RNA binding activity was localized to the carboxy terminal 86 amino acids, which constitute RGG box. Thus the amino terminal domain of EWS (NTD-EWS), which is involved in chromosome translocation may regulate the specificity of RNA binding activity of EWS. An EWS-erg chimeric protein, which is found in Ewing's sarcoma cells, functions as a transcriptional activator. Mutational analysis of EWS-erg chimeric protein revealed that NTD-EWS functions as a regulatory domain for the transcriptional activation properties of EWS-erg chimeric protein.

Web of Science

PCR法によるＡＢＯ式血液型（糖転移酵素の遺伝子型判定).

袖崎賢一郎, 的場梁次, 大内田守

法医学の実際と研究 36 33 - 37 1993

Correlation between HPV Positivity and state of the p53 gene in cervical Carcinoma cell lines

Iwasaka T, Oh-uchida M, Matsuo N, Yokoyama M, Fukuda K, Hara K, Fukuyama K, Hori K, Sugimori H

48, 104-109 1993

Detection of human papillomavirus genome and analysis of expression of c-myc and Ha-ras oncogenes in invasive cervical carcinomas

Tsuyoshi Iwasaka, Masatoshi Yokoyama, Mamoru Oh-uchida, Norito Matsuo, Kouichi Hara, Kouzou Fukuyama, Toru Hachisuga, Kouichi Fukuda, Hajime Sugimori

Gynecologic Oncology 46 ( 3 ) 298 - 303 1992

Language：English Publishing type：Research paper (scientific journal)

Invasive carcinomas of the uterine cervix of 38 patients were examined for the presence of human papillomavirus (HPV) genomes and for the state of the c-myc and Ha-ras oncogenes. A combination of Southern blot hybridization and polymerase chain reaction revealed the presence of the genome of HPV type 16 in 17 tumors (45%), that of HPV type 18 in 3 tumors (8%), and that of unknown types in 16 others (42%), while no viral DNA sequences were detected in 2 tumors. Of the 38 tumors, c-myc amplification was found in only 1 tumor, while there was no Ha-ras amplification. Overexpression of the c-myc gene was observed in 15 (44%) of the 34 tumors analyzed, while there was no overexpression of Ha-ras. Of the 23 squamous cell carcinomas analyzed, relapse-free rates at 24 months were 55% in tumors with c-myc overexpression and 100% in case of tumors with no c-myc overexpression, respectively. The results suggest the possibility that activation of the c-myc oncogene is involved in tumor progression. © 1992.

DOI： 10.1016/0090-8258(92)90220-D

Altered Structure and Expression of the p 53 Gene in Human Neuroepithelial Tumors

Tabuchi Kazuo, Fukuyama Kouzou, Mineta Toshihiro, Mamoru Oh-Uchida, Hori Katsuji

neurologia medico-chirurgica 32 ( 10 ) 725 - 732 1992

Language：English Publishing type：Research paper (scientific journal)

The p53 gene, located on chromosome 17p13.1, may be important in the pathogenesis of human neuroepithelial tumors, because it is a tumor suppressor gene and genetic alteration is essential for cer tain human cells to acquire the neoplastic phenotype. The structure and expression of the p53 gene were investigated in cultured human glioma cells and biopsied specimens of neuroepithelial tumors. Immunocytochemical examination of p53 gene expression revealed positive nuclear staining in six of seven glioma cell lines tested. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis demonstrated unequivocal heterogeneity of migration rate in p53 bands. Pulse-chase analysis clearly showed an increased half-life of p53 in cultured human glioma cells. These abnormalities are presumably due to genetic alterations in the p53 gene. Nucleotide substitutions in exon 5, 7, or 8 of the p53 gene could be detected by polymerase chain reaction-single strand conformational polymorphic analysis in four of seven (57%) human glioma cell lines, and nine of 29 (31%) biopsied specimens of neuroepithelial tumors examined. The present results indicate that genetic alterations in the p53 gene are responsible for the tumorigenesis of at least some human neuroepithelial tumors. © 1992, The Japan Neurosurgical Society. All rights reserved.

DOI： 10.2176/nmc.32.725

Suppression of anchorage-independent growth of human glioblastoma cell by major histocompativility complex class I gene-Transfection.

Momozaki N, Oh-uchida M, Tabuchi K, Ikezaki K, Hori K

J. Neurosurg. 76 845 - 849 1992

ヒト神経膠腫における癌抑制遺伝子の単離と遺伝子および遺伝子産物の分子生物学的解析

大内田守

佐川がん研究助成振興財団研究報告集 2 13 1991

Transformation‐specific Decrease of Phosphorylation of 80K Protein, a Substrate of Protein Kinase C, in NIH3T3 Cells

Mamoru Oh‐uchida, Kazumi Yano, Shoko Kawamoto, Kenji Shimizu

Japanese Journal of Cancer Research 81 ( 8 ) 799 - 806 1990

Language：English Publishing type：Research paper (scientific journal)

Phosphorylation in normal and transformed NIH3T3 cells of the 80K protein, a specific substrate for protein kinase C, was compared by means of two‐dimensional gel analysis. We obtained evidence that NIH3T3 cells transformed by the c‐raf or H‐ras oncogene maintained a decreased level of phosphorylation of the 80K protein, with or without phorbol ester (TPA)‐stimulation, at all concentrations of serum tested while normal NIH3T3 cells maintained an elevated level of phosphorylation of the 80K protein. Furthermore, NIH3T3 cells transformed by N‐ras, K‐ras, src, mos or polyoma middle T antigen exhibited a decreased level of phosphorylation of the 80K protein. These events were confirmed by an analysis of a hormone‐inducible H‐ras transformant. Thus, phosphorylation of the 80K protein is inversely correlated with cellular transformation. Copyright © 1990, Wiley Blackwell. All rights reserved

DOI： 10.1111/j.1349-7006.1990.tb02648.x

Activated c-raf-1 gene from human stomach cancer

グリオーマにおける癌抑制遺伝子の単離及びその解析

大内田守

日本脳神経財団報告書 6 73 1990

Changes in the patterns of RNA synthesis in early embryogenesis of Xenopus laevis. Reviewed

Shiokawa K, Misumi Y, Tashiro K, Nakakura N, Yamana K, Oh-uchida M

Cell Differ Dev. 28 ( 1 ) 17 - 25 1989

K. Shimizu, Y. Nakatsu, M. Oh-uchida, S. Nomoto, M. Sekiguchi

Japanese Journal of Cancer and Chemotherapy 14 ( 6 ) 2140 - 2146 1987.6

We previously isolated a novel human transforming sequence from a primary stomach cancer and identified the gene as an activated version of the c-raf-1 gene which is the human homologue of v-raf, a viral oncogene encoding a serine/threonine-specific protein kinase. Of 57 kbp of the human sequence isolated, a region of 40 kbp was found to be the minimum functional unit for the transforming activity, because a cosmid clone harboring this region was capable of inducing foci upon transfection. The size of the transcript of the transforming c-raf-1 gene was estimated to be about 2.8kb. Analyses of cDNA clones of this gene revealed that the gene was generated by substitution of the 5'-sequence (exons 1-5) of the normal c-raf-1 gene with an unrelated human sequence. We identified a region in the genomic clone where the rearrangement had occurred. The rearranged EcoRI fragment was detected in all the primary transformants obtained from two independent transfections, suggesting that the recombination had occurred in the primary cancer. The substituted cDNA sequence is composed of an open reading frame, which joins to exon 6 of the c-raf-1 gene in an in-phase manner. The substituted open reading frame encodes an extremely hydrophobic polypeptide. Thus, the putative product of the transforming gene seems to have a hydrophobic stretch ahead of the ser/thr-protein kinase domain of the c-raf-1 gene product. These results suggest that the truncation or replacement of the amino-terminal domain of the c-raf-1 protein leads to constitutive activation of the protein kinase residing in the downstream domain.