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N Shindo-Okada

Publications and source records attributed to N Shindo-Okada.

At least 19 recordsLinked to original sources
PubMed2001

Isolation of a novel actin-related gene expressed in low-metastatic PC-14 human lung adenocarcinoma.

In this article, we report the identification of differentially expressed genes associated with the metastatic potential of PC-14 human lung adenocarcinoma. The mRNA differential display method was applied to compare mRNAs from six cloned PC-14 cell lines representing different metastatic potentials. A novel gene was identified as being expressed in low-metastatic cells but not in high-metastatic cells. Sequence analysis revealed that this novel gene has an open reading frame of 210 amino acid residues showing 89% amino acid identity with human Arp3, which is one of the actin-related proteins. This gene was designated the Arp11 gene (the 11th Actin-related gene). The Arp11 gene was mapped to human chromosome 7q32-36. Southern blot and PCR analyses show that all of the high-metastatic variant cells have not rearrangements and deletions of Arp11 allele. Expression of the Arp11 gene may regulate the metastatic potential of PC-14 human lung adenocarcinoma.

Actin-Related Protein 3↗
PubMed2001

Establishment of cell lines with high- and low-metastatic potential from PC-14 human lung adenocarcinoma.

This article reports the establishment of variant cell lines with high and low metastatic potential by repeated selection and the dilution plating technique. Five clones with high metastatic potential, Lu-2, Lu-7, Lu-4, Lu-1 and Lu-5, and four clones with low metastatic potential, 3S, 7S, 8S and 13S, were established from PC-14 human lung adenocarcinoma. The high-metastatic cell lines produced enhanced lung metastases, but the low-metastatic cell lines did not produce lung metastasis by injection into the tail vein of 5-week-old BALB / c nude mice. The high-metastatic cell lines produced enhanced tumors on both visceral and parietal pleurae, and enhanced metastases to the mediastinum and contralateral pleural cavity. The low-metastatic cell lines produced reduced tumors on both visceral and parietal pleurae and reduced metastases to the mediastinum and contralateral pleural cavity after injection into the left preceral cavity of the nude mice. When the nine variant cell lines and original PC-14 cells were embedded in collagen gels, the PC-14 cells and the low-metastatic cell lines gave rise to colonies with a dendritic morphology, and cells were tightly associated. The high-metastatic cell lines were more loosely associated and scattered into three-dimensional colonies. These nine cloned cell lines originated from heterogeneous populations of the parental PC-14 cells should be useful tools for studying the process of metastasis of lung cancer.

Adenocarcinoma↗
PubMed1998

Expression of the Elm1 gene, a novel gene of the CCN (connective tissue growth factor, Cyr61/Cef10, and neuroblastoma overexpressed gene) family, suppresses In vivo tumor growth and metastasis of K-1735 murine melanoma cells.

We previously isolated a partial cDNA fragment of a novel gene, Elm1 (expressed in low-metastatic cells), that is expressed in low-metastatic but not in high-metastatic K-1735 mouse melanoma cells. Here we determined the full-length cDNA structure of Elm1 and investigated the effect of Elm1 expression on growth and metastatic potential of K-1735 cells. The Elm1 gene encodes a predicted protein of 367 amino acids showing approximately 40% amino acid identity with the CCN (connective tissue growth factor [CTGF], Cyr61/Cef10, neuroblastoma overexpressed gene [Nov]) family proteins, which consist of secreted cysteine-rich proteins with growth regulatory functions. Elm1 is also a cysteine-rich protein and contains a signal peptide and four domains conserved in the CCN family proteins. Elm1 was highly conserved, expressed ubiquitously in diverse organs, and mapped to mouse chromosome 15. High-metastatic K-1735 M-2 cells, which did not express Elm1, were transfected with an Elm1 expression vector, and several stable clones with Elm1 expression were established. The in vivo growth rates of cells expressing a high level of Elm1 were remarkably slower than those of cells expressing a low level of Elm1. Metastatic potential of transfectants was reduced in proportion to the level of Elm1 expression. Thus, Elm1 is a novel gene of CCN family that can suppress the in vivo growth and metastatic potential of K-1735 mouse melanoma cells.

Animals↗
PubMed1997

Isolation of a novel Sry-related gene that is expressed in high-metastatic K-1735 murine melanoma cells.

To identify genes differentially expressed in association with metastatic potential of K-1735 mouse melanoma cells, the mRNA differential display method was applied to compare mRNAs from high- and low-metastatic K-1735 cells. A novel gene was identified as being expressed in high-metastatic cells but not in low-metastatic cells. Sequence analysis revealed that this gene had an open reading frame of 538 amino acid residues containing a Sry high-mobility group (HMG) box domain, known as a DNA binding motif, particularly in the Sox family genes. This gene showed the highest homology to the human SOX9 gene, which is the responsible gene for an inherited disease, campomelic dysplasia. Thus, this gene was designated the Sox21 gene (the 21st Sox family gene). The Sox21 gene was mapped to mouse Chromosome 15, to which neither genes containing the HMG box region nor the loci of hereditary diseases similar to campomelic dysplasia have been previously mapped. This gene was highly conserved and specifically expressed in the brain. These results suggest that expression of the Sox21 gene is involved in the development of nerve systems and may function to enhance the metastatic potential of K-1735 mouse melanoma cells of nerve cell origin.

Amino Acid Sequence↗
PubMed1997

[Mechanism of action of azatyrosine: recent process].

Azatyrosine is known to convert ras, raf or c-erbB-2-transformed NIH3T3 cells to a normal phenotype. We attempted to identify the signal-transduction process triggered by oncogenic c-ErbB-2 that was inhibited by azatyrosine. Azatyrosine did not suppress activation of Ras induced by introduction of c-ErbB-2. However, it inhibited increases in phosphorylation of c-Raf-1 induced by oncogenic c-ErbB-2. Furthermore, azatyrosine inhibited activation of the 12-O-tetradecanoylphorbol-13-acetate (TPA) response element in response to stimulation by oncogenic c-ErbB-2. These results suggest that this agent acts downstream of Ras in signal transduction from oncogenic c-ErbB-2 to nuclear factors. Moreover, we found that azatyrosine was incorporated into proteins instead of tyrosine. The simultaneous presence of a high concentration of tyrosine inhibited the conversion to a normal phenotype of transformed cells by azatyrosine. These results strongly suggest that incorporation of azatyrosine into proteins might convert the transformed cells in to cells with a normal phenotype.

3T3 Cells↗
PubMed1996

Identification of genes differentially expressed in association with metastatic potential of K-1735 murine melanoma by messenger RNA differential display.

To identify genes differentially expressed in association with the metastatic potential of K-1735 mouse melanoma cells, the mRNA differential display method was applied to compare mRNAs from high- and low-metastatic K-1735-derived cells. Three of the high- and three of the low-metastatic clones were used to reduce the false positives in the initial screening, and Southern blot screening against reverse transcription-PCR products was used to confirm that cDNA fragments detect differential expression between high- and low-metastatic cells. By using 256 different combinations of modified long arbitrary primers which provide broad screening of expressed genes, approximately 12,000 cDNA fragments were amplified from mRNA of each cell line. Among them, eight genes were identified as being expressed in either high- or low-metastatic cells using Northern blot analysis. Integrin alpha6 and two unknown genes were expressed in high-metastatic cells, whereas beta-tropomyosin, macrophage colony-stimulating factor, inhibin/activin betaB subunit, and two unknown genes were expressed in low-metastatic cells. These results indicate that the acquisition of metastatic potential in tumor cells was regulated by activation and/or inactivation of several specific genes, such as those for cell adhesion molecule, cytoskeletal protein, and growth factors.

Animals↗
PubMed1996

Cloning of the RhoB gene from the mouse genome and characterization of its promoter region.

Rho proteins have been implicated in a variety of cytoskeletal functions, but it is unclear how Rho proteins regulate these cellular functions and how Rho proteins are regulated. In this study, we cloned the rhoB gene from the mouse genome and characterized its promoter region. The predicted amino acid sequence was identical to that encoded by the human rhoB gene. A site for initiation of transcription was found at position -376 relative to the site for initiation of translation. Deletion analysis of the 5'-flanking region of the rhoB gene revealed that the minimum region of the promoter was located between positions -507 and -376. Northern blotting analysis showed that the expression of the mouse rhoB gene was induced by serum, suggesting that expression of the rhoB gene might be controlled by some signal-responsive element(s).

3T3 Cells↗
PubMed1992

Isolation of genes specifically expressed in flat revertant cells derived from activated ras-transformed NIH 3T3 cells by treatment with azatyrosine.

We previously reported that mouse NIH 3T3 cells transformed by transfection of activated human c-Ha-ras become apparently normal upon treatment with the antibiotic azatyrosine. The revertant cells maintain their normal phenotype during prolonged culture in the absence of azatyrosine, although activated p21ras is still expressed. The normal phenotype induced by azatyrosine could be due to activation of expression of some cellular gene(s) in the cells that results in suppression of ras function. To identify the genes with increased expression in the revertant cells, we adopted differential screening of recombinants from a phage cDNA library made from mRNA of the revertant cells, hybridized with 32P-labeled cDNAs made from mRNAs of the ras-transformed NIH 3T3 cells and the revertant cells. Two clones thus isolated were found to be almost identical to the ras recision gene (rrg), which was identified as a tumor-suppressor gene by Contente et al. [Contente, S., Kenyon, K., Rimoldi, D. & Friedman, R. M. (1990) Science 249, 796-798]. Other genes identified were the collagen type III and rhoB genes. Approximately half the clones were found to contain a sequence corresponding to that of the murine retrovirus-like intracisternal A particle. We speculate that azatyrosine activates several cellular genes in the ras-transformed cells and that some of these genes, including rrg, act cooperatively to counteract ras function, resulting in reversion of the ras-transformed cells to the normal phenotype.

3T3 Cells↗
PubMed1992

Inhibition of chemical carcinogenesis in vivo by azatyrosine.

A single painting of 7,12-dimethylbenz[a]anthracene on the skin of transgenic mice harboring the human protooncogene c-Ha-ras induced papillomas at 100% incidence after 20 weeks (M. Izawa et al., unpublished data). Application of L-beta-(5-hydroxy-2-pyridyl)alanine (azatyrosine) to the skin at a dose of 2 mg/mouse once every 3 days after initiation with 7,12-dimethylbenz[a]anthracene greatly reduced the percentage incidence, number per mouse, and size of papillomas. Injection of methylnitrosourea i.p. into transgenic mice induced papillomas in the forestomach after 12 weeks (2 to 12 papillomas/mouse) at 100% incidence (K. Ando et al., Cancer Res., 52: 978-982, 1992). Administration of azatyrosine i.p. at a dose of 2 mg/mouse once every 2 days for 12 weeks after initiation with methylnitrosourea completely prevented the formation of forestomach papillomas. These results clearly indicated that azatyrosine inhibits chemical carcinogenesis in vivo.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1992

Azatyrosine inhibits neurite outgrowth of PC12 cells induced by oncogenic Ras.

An antibiotic, azatyrosine [L-beta-(5-hydroxy-2-pyridyl) alanine], specifically converts ras-, raf- or c-erbB2 (neu)-transformed NIH3T3 cells to apparently normal phenotype. The reversion induced by azatyrosine is permanent, and the phenotype of the revertant cells does not change even after prolonged culture in the absence of azatyrosine [N. Shindo-Okada, O. Manabe, H. Nagahara & S. Nishimura (1989). Mol. Carcinogen., 2, 159-167]. In the present study, we found that neurite outgrowth of PC12 cells induced by expression of either the ras or raf oncogenes was inhibited by addition of azatyrosine to the medium. Azatyrosine also inhibited neurite outgrowth induced by microinjection of oncogenic Ras protein into PC12 cells. The dose dependency was much the same for the two systems, inhibition of neurite outgrowth of PC12 cells and reversion of the transformed NIH3T3 cells. Microinjection of azatyrosine into the cells was as effective as addition to the medium, indicating that the target of azatyrosine is intracellular. In contrast, neurite outgrowth induced by nerve growth factor, which has been shown to be mediated by normal Ras [N. Hagag, S. Halegouna & M. Viola (1986). Nature, 319, 680-682], was found to be resistant to azatyrosine. Azatyrosine also showed no effect on neurite outgrowth induced by a membrane-permeant cyclic AMP analog through another pathway. These findings suggest that azatyrosine sensitivity is the result of abnormal signal transduction by oncogenic Ras. It was shown that azatyrosine also inhibited differentiation-associated growth arrest of PC12 cells induced by oncogenic Ras. In Ras-induced neurite outgrowth, the azatyrosine-sensitive process was found to be completed in the first 6-9 h, and is probably essential for the commitment of PC12 cells to differentiation rather than to growth.

Alanine↗
PubMed1989

Permanent conversion of mouse and human cells transformed by activated ras or raf genes to apparently normal cells by treatment with the antibiotic azatyrosine.

Azatyrosine [L-beta-(5-hydroxy-2-pyridyl)-alanine], an antibiotic isolated from Streptomyces chibanensis, inhibited the growth of NIH 3T3 cells transformed by the activated human c-Ha-ras gene but did not significantly inhibit the growth of normal NIH 3T3 cells. Surprisingly, upon treatment with azatyrosine most of the transformed cells apparently became normal. These apparently normal cells, named revertant cells, grew in the presence of azatyrosine and stopped growing when they reached confluency, and their normal phenotype persisted during prolonged culture in the absence of azatyrosine. The revertant cells did not grow in soft agar and scarcely proliferated in nude mice. The human c-Ha-ras gene present in transformed NIH 3T3 cells was still present in the revertant cells and was expressed to the same extent as in the original transformed cells, producing the same amount of activated p21. Treatment with azatyrosine caused similar conversion of NIH 3T3 cells transformed by activated c-Ki-ras, N-ras, or c-raf to apparently normal cells, but NIH 3T3 cells transformed by hst or ret were not exclusively converted by azatyrosine. Human pancreatic adenocarcinoma cells, which are known to contain an amplified activated c-Ki-ras gene and an amplified c-myc gene, were also converted to flat and giant revertant cells by treatment with azatyrosine.

Alanine↗
PubMed1986

Queuine analogues. Their synthesis and inhibition of growth of mouse L5178Y cells in vitro.

A variety of analogues of queuine (7-[(3S,4R,5S)-4,5-dihydroxycyclopent-1-en-3-ylaminomethyl]- 7 -deazaguanine), i.e., those with 7-N-substituted aminomethyl side chains and those in which the oxygen function at the 6 position of the 7-deazaguanine ring was replaced by sulfur, were synthesized and tested for ability to act as substrates for tRNA-guanine transglycosylase and for inhibitory effects on growth of mouse L5178Y leukemic cells in vitro. Of the compounds tested, analogues with sulfur at the 6 position of the 7-deazaguanine ring in place of oxygen or with an N-o-hydroxyphenyl, N-m-hydroxyphenyl, or iodoacetyl group in the 7-aminomethyl side chain in place of the naturally occurring cyclopentene diol moiety markedly inhibited the growth of cells at concentrations of 1-10 micrograms/mL, although queuine itself had practically no effect at a concentration of 100 micrograms/mL.

Animals↗
PubMed1983

Characterization and analysis of oncofetal tRNA and its possible application for cancer diagnosis and therapy.

We determined the primary structures of various tumor-specific tRNAs as well as of their normal counterparts by postlabeling RNA-sequencing procedures. The results clearly indicated that tumor-specific tRNAs are mostly formed by undermodification of hypermodified nucleosides located in the anticodon loop: no new tRNA transcripts have so far been found in tumor cells. Among the modified nucleosides affected by tumorigenesis, queuosine and Y base are the most interesting. In various tumor tRNAPhe species, hydroxy Y base located next to the anticodon is undermodified to form hypomodified hydroxy Y base lacking methyl and carboxymethyl groups. This Y base analog should be a good marker for analyzing the state of cancer cells. Queuosine, located in the first position of the anticodon, is partly or completely replaced by guanosine in all tumor cells tested so far. The amount of G-tRNA decreased markedly when the cells differentiated into mature erythroid cells, with concomitant increase of Q-tRNA. This indicates that the presence of G-tRNA is closely related to the state of the cells, not merely to the fast growth rate of tumor cells. The enzyme tRNA-guanine transglycosylase, which is a key enzyme in biosynthesis of queuosine in tRNA (inserting Q base into tRNA by a transglycosylase reaction), is active in both tumor cells and normal cells. Administration of chemically synthesized Q base to tumor-bearing mice resulted in complete conversion of G-tRNA to Q-tRNA in tumor cells, indicating that exogenously added Q base is effectively incorporated into G-tRNA. Various Q base analogs that can be used as substrates for tRNA-guanine transglycosylase were synthesized chemically. These compounds may be used for clinical cancer diagnosis, since they should be incorporated selectively into tRNA in tumor cells. In addition to use as cancer chemotherapeutic reagents, it should be possible to develop new Q base analogs that induce miscoding or blocking of protein synthesis after insertion into G-tRNA.

Animals↗
PubMed1981

Nucleotide sequences of two aspartic acid tRNAs from rat liver and rat ascites hepatoma.

The nucleotide sequences of the major tRNAAsp from rat liver and a minor tRNAAsp present specifically in rat ascites hepatoma were determined by combined use of several postlabeling procedures. The sequence of rat liver tRNAAsp was determined to be pU-C-C-U-C-G-U-U-A-G-U-A-phi-A-G-U-G-G-D-G-A-G-U-A-U-C-C-C-C-G-C-U-C-manQ-U-C-A -m5C-G-C-G-G-G-A-G-A-m5C-m5C-G-G-G-G-T-phi-C-G-A-U-U-C-C-C-C-G-A-C-G-G-G-G-A-G- C-C-AOH. The nucleotide sequence of rat ascites hepatoma TRNAAsp was found to be the same as that of rat liver tRNAAsp, except that mannose-attached queuosine (manQ) present in the wobbling position of rat liver tRNAAsp was replaced by normal guanosine in rat ascites hepatoma tRNAAsp. The other modified nucleosides in the two tRNAAsp species were the same. Thus it was concluded that the new isoacceptor species of tRNAAsp that appears in the tumor tissue is due to hypomodification with respect to Q in the posttranscriptional process. The most notable feature on the sequence of the mammalian tRNAAsp is that cytidine is present in the position next to the 5'-end of the anticodon, because the only known tRNAs having cytidine in this position are cytoplasmic initiator tRNAs from multicellular eukaryotic organisms.

Animals↗