Kazuki Sasaki

STRUCTURE, MAPPING AND EXPRESSION OF A HUMAN NOR-1 GENE, THE THIRD MEMBER OF THE NUR77/NGFI-B FAMILY

We identified a human homologue of NOR-1 (neuron-derived orphan receptor) from the fetal brain. There are two transcripts for human NOR-1, encoding 626 amino acid residues with a calculated molecular mass of 68 kDa. The high homology between hNOR-1, mNur77/rNGFI-B/hTR3, and mNurr1/rRNR-1/hNOT indicated that these three orphan receptors form a distinct subfamily within the steroid/thyroid receptor superfamily. Human NOR-1 mRNA was detected in the adult heart and skeletal muscle as well as in the fetal brain, indicating that its expression is not restricted to events that occur during neural development. The hNOR-1 gene is more than 35 kilobases long and interrupted by seven introns. The exon-intron structure of the gene is generally conserved when compared with the steroid/thyroid receptor superfamily and is remarkably similar to that of the Nur77/NGFI-B genes. This suggests that the Nur77/NGFI-B family has evolved from a common ancestral gene. Fluorescence in situ hybridization (FISH) revealed that the gene is located on chromosome 9q.

Lipid Raft–Specific Knockdown of Src Family Kinase Activity Inhibits Cell Adhesion and Cell Cycle Progression of Breast Cancer Cells

Src family kinase (SFK) is known to control various cell functions, but the significance of the location of its activation was largely unknown. We herein revealed that SFK activation occurs in lipid rafts. Based on this finding, we have developed a lipid raft-targeted SFK inhibitory fusion protein (LRT-SIFP) that inhibits the SFK activity in lipid rafts. LRT-SIFP has a peptide inhibitor of SFK and a lipid raft-targeting sequence in which two cysteine residues are palmitoylated for clustering in lipid rafts. LRT-SIFP was found to inhibit cell adhesion and cell cycle progression of human breast cancer cell lines MCF-7 and MDA-MB231. On the other hand, the cell functions of MCF-7 cells were found to be not affected with a previously developed peptide inhibitor of SFK that lacks the lipid raft-targeting sequence. In addition, when we replaced the targeting sequence of LRT-SIFP with the consensus sequence for geranylgeranylation to make LRT-SIFP unable to cluster in lipid rafts, this mutated LRT-SIFP did not show any effect on the above cell functions of MCF-7 cells. Furthermore, in contrast to the breast cancer cell lines, LRT-SIFP did not show any inhibitory effect on cell adhesion and cell cycle progression of human normal cell line HEK293. The present lipid raft-specific knockdown of SFK activity would potentially be useful for selective cancer therapy to prevent tumorigenesis and metastasis of breast cancer cells.

Expression of NOR-1 and its closely related members of the steroid/thyroid hormone receptor superfamily in human neuroblastoma cell lines

Previously, we isolated a cDNA of neuron derived orphan receptor (NOR-1), a putative transcription factor with strong homologies to the orphan nuclear receptors NGFI-B and NURR1. In the present study, we examined the gene expression of NOR-1 as well as NGFI-B and NURR1 in human neuroblastoma cell lines by reverse transcription-polymerase chain reaction and nucleotide sequencing. Although the mRNAs of these orphan receptors were detected in all six neuroblastoma cell lines examined, basal expression levels of these genes varied among cell lines. Treatment with forskolin and 12-O-tetradecanoylphorbol-13-acetate rapidly increased the expression of all these genes in neuroblastoma NB-OK-1 cells. This induction did not require de novo protein synthesis, indicating that the NOR-1 gene as well as NGFI-B and NURR1 genes is an immediate-early gene. This is the first demonstration of NOR-1 gene expression in tumor cell lines.

Expression of the Putative Transcription Factor NOR-1 in the Nervous, the Endocrine and the Immune Systems and the Developing Brain of the Rat

NOR-1 is a novel member of the NGFI-B/RNR-1 subfamily within the nuclear receptor superfamily, and has been implicated in signal transduction mediated by various second messengers. To investigate the physiological role of NOR-1, we examined its gene expression in various adult rat tissues and developing rat brain by the quantitative reverse transcription-polymerase chain reaction using in vitro synthesized RNA as an internal standard. The NOR-1 gene was expressed in all tissues examined, but predominantly in the cerebral cortex and pituitary glands. Thymus, adrenal glands, spleen, epididymis, submandibular glands and deferent ducts showed moderate expression. In the brain, NOR-1 gene expression was developmentally regulated, with the peak levels on gestational day 18. These findings suggest a ubiquitous role of NOR-1 in signal transduction in diverse tissues. These findings also suggest that the nervous, endocrine and immune systems may be highly exposed to NOR-1-inducing stimuli under normal conditions in adult rats. Developing rat brain cells may most frequently receive the relevant signal on day 18 of gestation.

Decrease in neuron-restrictive silencer factor (NRSF) mRNA levels during differentiation of cultured neuroblastoma cells

Non-neuronal cells and undifferentiated neuronal progenitors express the neuron-restrictive silencer factor (NRSF), a silencer protein which represses neuronal gene transcription in these cell types. Neuroblastoma, a childhood tumor of neuroectodermal origin, shares some biological properties with neuronal progenitor cells and can acquire neuronal phenotypes in response to a variety of agents, including cyclic AMP and staurosporine. We report here that NRSF mRNA content was markedly decreased in a human neuroblastoma cell line following differentiation induced by staurosporine plus cyclic AMP, with a concomitant increase in mRNA levels of synapsin I, whose expression is restricted to neuronal cell types. Our novel finding suggests that NRSF expression is related to an undifferentiated state and regarded as a biochemical marker of neuronal differentiation in neuroblastoma cells.

Metal ion selectivity for formation of the calmodulin-metal-target peptide ternary complex studied by surface plasmon resonance spectroscopy.

Ion selectivities for Ca(2+) signaling pathways of 33 metal ions were examined based on the Ca(2+)-dependent on/off switching mechanism of calmodulin (CaM): Ca(2+) ion-induced selective binding of CaM-Ca(2+) ion complex to the target peptide was observed as an increase in surface plasmon resonance (SPR) signals. As the target peptide, M13 of 26-amino-acid residues derived from skeletal muscle myosin light-chain kinase was immobilized in the dextran matrix, over which sample solutions containing CaM and each metal ion were injected in a flow system. Large changes in SPR signals were also observed for Sr(2+), Ba(2+), Cd(2+), Pb(2+), Y(3+) and trivalent lanthanide ions, thereby indicating that not only Ca(2+) but also these metal ions induce the formation of CaM-M13-metal ion ternary complex. No SPR signal was, however, induced by Mg(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+) and all monovalent metal ions examined. The latter silent SPR signal indicates that these ions, even if they bind to CaM, are incapable of forming the CaM-M13-metal ion ternary complex. Comparing the obtained SPR results with ionic radii of those metal ions, it was found that all cations examined with ionic radii close to or greater than that of Ca(2+) induced the formation of the CaM-metal-M13 ternary complex, whereas those with smaller ionic radii were not effective, or much less so. Since these results are so consistent with earlier systematic data for the effects of various metal ions on the conformational changes of CaM, it is concluded that the present SPR analysis may be used for a simple screening and evaluating method for physiologically relevant metal ion selectivity for the Ca(2+) signaling via CaM based on CaM/peptide interactions.

Toxohormones responsible for cancer cachexia syndrome in nude mice bearing human cancer cell lines

Abstractu2003Toxohormones are tumor-derived factors that induce cancer cachexia syndrome in tumor-bearing animals. Nude mice bearing tumors induced by eight human cancer cell lines with this activity were studied for cytokine production and expression of a newly identified gene, ob, which has the ability to control body weight. A melanoma cell line, SEKI, and a neuroepithelioma cell line, NAGAI, produced a large amount of the cytokine, leukemia-inhibitory factor (LIF). A uterine carcinoma cell line, Yumoto, produced a large amount of interleukin 6 (IL-6), and an oral cavity carcinoma cell line, OCC-1C, concomitantly produced LIF, IL-6, and IL-11. Reverse transcription polymerase chain reaction studies revealed that ob gene mRNA was not expressed in any of these cell lines, suggesting that the gene does not have a role as a tumor product responsible for cancer cachexia in this model. These findings suggest that in four of eight animal models in which cancer cachexia syndrome developed, LIF, IL-6, or possibly IL-11 produced by cancer cells may be toxohormones, but in the remaining four cancer cell lines the mechanism responsible for cachexia syndrome remains unknown.

Screening method for antagonists that inhibit the binding of calmodulin to a target peptide using surface plasmon resonance

Abstract A new screening method was developed for evaluating effects of calmodulin (CaM) antagonists on the interaction between CaM and its target protein in the Ca 2+ -signaling pathway. A binding of Ca 2+ -CaM to the target peptide, M13, derived from myosin light-chain kinase (MLCK) is monitored by surface plasmon resonance (SPR) technique. When a sample solution containing Ca 2+ -CaM was injected into a flow cell with M13 immobilized on the SPR sensor surface, the SPR signal largely increased and leveled-off within 3xa0min. By adding W-7, a CaM antagonist, into the sample solution, the SPR signal at the equilibrium state decreased. This decrease in the SPR signal is due to the binding of W-7 to Ca 2+ -CaM, thereby inhibiting the specific interaction between Ca 2+ -CaM and M13. In the case of other antagonists such as trifluoperazine, prenylamine and bepridil, upon increasing the concentration of these antagonists, the initial rate of the increase in the SPR signals decreased, and the signals reached the same value under the equilibrium state. These IC 50 values obtained by the present method were consistent with ones obtained earlier by MLCK activity itself. The present method was thus capable of finding antagonists inhibiting the interaction between Ca 2+ -CaM and MLCK. The applicability of the present method for evaluating the effect of endocrine disrupting chemicals toward the Ca 2+ -signaling pathway was also examined and discussed.

Differentiation of cultured neuroblastoma induced by staurosporine and cyclic AMP: methods for assessing a neuronal phenotype

In cultured neuronal cells, neurite outgrowth provides a prominent morphological marker for assessing neurotrophic activity derived from different sources. Permanent cell lines are often used for such studies; however, the process of differentiation should be evaluated biochemically or electrophysiologically to determine whether the affected cells acquire a neuronal phenotype. We have reported that low-dose staurosporine acts synergistically with cyclic AMP to induce neuronal differentiation in a human neuroblastoma cell line. This protocol describes a method for examining phenotypic changes in cultured cells.

Long-term regulation of synapsin I gene expression and neuronal morphology by cyclic AMP and low-dose staurosporine

We examined the phenotypic changes of neuroblastoma cells chronically treated with cAMP and nanomolar concentrations of staurosporine. These agents, given together, produced cells with a neuronal morphology and a delayed increase (approximately 10 days) in synapsin I mRNA levels. Dopamine-beta hydroxylase mRNA was upregulated within 24 h. We provide evidence that low-dose staurosporine acts cooperatively with cyclic AMP in the acquisition of mature neuronal phenotypes.