Masaki Hiramoto

KIF5B-RET fusions in lung adenocarcinoma

We identified in-frame fusion transcripts of KIF5B (the kinesin family 5B gene) and the RET oncogene, which are present in 1–2% of lung adenocarcinomas (LADCs) from people from Japan and the United States, using whole-transcriptome sequencing. The KIF5B-RET fusion leads to aberrant activation of RET kinase and is considered to be a new driver mutation of LADC because it segregates from mutations or fusions in EGFR, KRAS, HER2 and ALK, and a RET tyrosine kinase inhibitor, vandetanib, suppresses the fusion-induced anchorage-independent growth activity of NIH3T3 cells.

High-performance affinity beads for identifying drug receptors

We have developed a method using novel latex beads for rapid identification of drug receptors using affinity purification. Composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, the beads minimize nonspecific protein binding and maximize purification efficiency. We demonstrated their performance by efficiently purifying FK506-binding protein using FK506-conjugated beads, and found that the amount of material needed was significantly reduced compared with previous methods. Using the latex beads, we identified a redox-related factor, Ref-1, as a target protein of an anti-NF-κB drug, E3330, demonstrating the existence of a new class of receptors of anti-NF-κB drugs. Our results suggest that the latex beads could provide a tool for the identification and analysis of drug receptors and should therefore be useful in drug development.

Functional Interference of Sp1 and NF-κB through the Same DNA Binding Site

ABSTRACT Gene activation by NF-κB/Rel transcription factors is modulated by synergistic or antagonistic interactions with other promoter-bound transcription factors. For example, Sp1 sites are often found in NF-κB-regulated genes, and Sp1 can activate certain promoters in synergism with NF-κB through nonoverlapping binding sites. Here we report that Sp1 acts directly through a subset of NF-κB binding sites. The DNA binding affinity of Sp1 to these NF-κB sites, as determined by their relative dissociation constants and their relative efficiencies as competitor DNAs or as binding site probes, is in the order of that for a consensus GC box Sp1 site. In contrast, NF-κB does not bind to a GC box Sp1 site. Sp1 can activate transcription through immunoglobulin kappa-chain enhancer or P-selectin promoter NF-κB sites. p50 homodimers replace Sp1 from the P-selectin promoter by binding site competition and thereby either inhibit basal Sp1-driven expression or, in concert with Bcl-3, stimulate expression. The interaction of Sp1 with NF-κB sites thus provides a means to keep an elevated basal expression of NF-κB-dependent genes in the absence of activated nuclear NF-κB/Rel.

Bone marrow stroma from refractory anemia of myelodysplastic syndrome is defective in its ability to support normal CD34-positive cell proliferation and differentiation in vitro

We examined the supportive function of stromal cells from patients with refractory anemia (RA) of myelodysplastic syndrome (MDS) on CD34-positive hematopoietic cell proliferation and differentiation using a long-term bone marrow culture (LTMC) system. Primary marrow stromal cells were obtained from 11 MDS RA patients and 12 healthy volunteers, and freshly prepared CD34-positive bone marrow cells from a normal subject were inoculated onto the stroma. There seems to be three broad patterns of hematopoietic cell growth in the LTMCs. In one group, hematopoietic cells were maintained at near normal levels (type A). In the second group, the number of hematopoietic cells increased within the first 5-10 days of culture, but declined to low levels at 15-20 days of culture as compared with normal control (type B). In the third group, the incidence of hematopoietic cells steadily declined from the beginning of the culture (type C). Furthermore, apoptotic change of hematopoietic cells was very frequently observed in cultures with the type C stroma, which were especially defective for supporting CD34 + cell proliferation and differentiation. The expression of CD95 on hematopoietic cells was induced by the type C stroma, however, production of fas ligand by the stromal cells was not observed. These findings suggest a lack of hematopoietic supportive function in some cases of MDS RA and also indicate that there is heterogeneity of stromal function among MDS RA patients.

Druggable Oncogene Fusions in Invasive Mucinous Lung Adenocarcinoma

Purpose: To identify druggable oncogenic fusions in invasive mucinous adenocarcinoma (IMA) of the lung, a malignant type of lung adenocarcinoma in which KRAS mutations frequently occur. Experimental Design: From an IMA cohort of 90 cases, consisting of 56 cases (62%) with KRAS mutations and 34 cases without (38%), we conducted whole-transcriptome sequencing of 32 IMAs, including 27 cases without KRAS mutations. We used the sequencing data to identify gene fusions, and then performed functional analyses of the fusion gene products. Results: We identified oncogenic fusions that occurred mutually exclusively with KRAS mutations: CD74-NRG1, SLC3A2-NRG1, EZR-ERBB4, TRIM24-BRAF, and KIAA1468-RET. NRG1 fusions were present in 17.6% (6/34) of KRAS-negative IMAs. The CD74-NRG1 fusion activated HER2:HER3 signaling, whereas the EZR-ERBB4 and TRIM24-BRAF fusions constitutively activated the ERBB4 and BRAF kinases, respectively. Signaling pathway activation and fusion-induced anchorage-independent growth/tumorigenicity of NIH3T3 cells expressing these fusions were suppressed by tyrosine kinase inhibitors approved for clinical use. Conclusions: Oncogenic fusions act as driver mutations in IMAs without KRAS mutations, and thus represent promising therapeutic targets for the treatment of such IMAs. Clin Cancer Res; 20(12); 3087–93. ©2014 AACR.

Substance P-induced cadherin expression and its signal transduction in a cloned human corneal epithelial cell line

Although the absence of Substance P (SP), a neurotransmitter in the trigeminal nerve, has been speculated as a cause for developing neurotrophic keratitis, its exact pathogenesis is still not clarified. In a previous report, we showed with electron microscopic examination that epithelial cell attachment was weakened in denervated corneas. In this study, SV40‐transformed human corneal epithelial cells (HCE‐Ts) were used to explore the molecular mechanisms responsible for mediating regulation of E‐cadherin expression in response to Substance P receptor stimulation. Expression of the mRNAs for specific SP receptors, neurokinin (NK)‐1R, NK‐2R, and NK‐3R, was demonstrated with RT‐PCR. The cells were treated with various concentrations of SP in vitro, and the expression of an adhesion molecule E‐cadherin was analyzed by immunofluorescence, immunoblotting, and enzyme‐linked immunosorbent assay (ELISA) using an anti‐E‐cadherin antibody. E‐cadherin expression was increased by SP in a dose‐dependent manner both in the cytosolic fraction and in the cell membrane fraction. This increase in E‐cadherin expression was completely inhibited by Calphostin C (PKC inhibitor) and KN‐62 (CaMK inhibitor), but not by H‐89 (PKA inhibitor), indicating that SP‐induced E‐cadherin expression involves the activation of protein kinase C (PKC) and calmodulin kinase (CaMK). SP did not affect cell proliferation at all. All these findings indicate that SP induced E‐cadherin expression through PKC and CaMK activation and suggest that a lack of SP may account in part for the pathogenesis of neurotrophic keratitis. J. Cell. Physiol. 182:189–195, 2000.

Direct purification of multiple ATF/E4TF3 polypeptides from HeLa cell crude nuclear extracts using DNA affinity latex particles

We developed a method using affinity latex particles to rapidly and efficiently purify DNA-binding proteins directly from crude cell extracts. The particles are composed of a styrene core and a polyglycidyl methacrylate surface, to which DNA oligomers were immobilized by means of epoxy groups. Multiple polypeptides were copurified, which bound to the ATF/E4TF3-binding site from crude nuclear extracts of HeLa cells, within a few hours. Affinity-purified polypeptides stimulated transcription in vitro from a promoter in which ATF/E4TF3-binding sites were present. At least eight polypeptides with molecular masses of 116, 80, 65, 60, 55, 47, 45, and 43 kDa were copurified. About 2 micrograms of the 43-kDa protein was purified directly from 8 mg of crude nuclear extracts. All the polypeptides directly bound to the same DNA sequence and were thought to form a family. The results indicated that the particles are useful for quickly purifying various DNA-binding proteins directly from crude cell extracts.

Synthesis of a new [6]-gingerol analogue and its protective effect with respect to the development of metabolic syndrome in mice fed a high-fat diet

To determine the effects of a [6]-gingerol analogue (6G), a major chemical component of the ginger rhizome, and its stable analogue after digestion in simulated gastric fluid, aza-[6]-gingerol (A6G), on diet-induced body fat accumulation, we synthesized 6G and A6G. Mice were fed either a control regular rodent chow, a high-fat diet (HFD), or a HFD supplemented with 6G and A6G. Magnetic resonance imaging adiposity parameters of the 6G- and A6G-treated mice were compared with those of control mice. Supplementation with 6G and A6G significantly reduced body weight gain, fat accumulation, and circulating levels of insulin and leptin. The mRNA levels of sterol regulatory element-binding protein 1c (SREBP-1c) and acetyl-CoA carboxylase 1 in the liver were significantly lower in mice fed A6G than in HFD control mice. Our findings indicate that A6G, rather than 6G, enhances energy metabolism and reduces the extent of lipogenesis by downregulating SREBP-1c and its related molecules, which leads to the suppression of body fat accumulation.

Patterns of age‐dependent changes in the numbers of lymph follicles and germinal centres in somatic and mesenteric lymph nodes in growing C57Bl/6 mice

The timing of the first appearance of lymph follicles and germinal centres in various lymph nodes, and the ways in which numbers of these and IgM‐synthesising cells increase within the nodes, were investigated in male and female C57Bl/6N mice aged from 4 d to 16 wk. The lymphoid organs examined were the Peyers patches, spleen, somatic (submandibular, deep cervical, brachial, axillary, inguinal and popliteal) and visceral (mesenteric and lumbar) lymph nodes. Primary follicles appeared in most somatic lymph nodes 6 d after birth. The number of follicles per node then increased rather sharply in larger lymph nodes and slowly in smaller nodes, up to 28 d of age, reaching a level which varied according to the location of the node. Thereafter, the number of follicles in the somatic lymph nodes increased only slightly to moderately, reaching a peak or plateau at 8–12 wk. In the mesenteric (ileocaecal) nodes, primary follicles first appeared at 12 d, then increased linearly during the suckling period and after weaning to reach a plateau at 8 wk of age. Germinal centres appeared in the submandibular and mesenteric nodes at 28 d and their numbers increased consistently in the latter, while remaining low in the former. The impact of possible ‘natural’ exogenous antigen stimulation of the various lymph nodes was estimated from the presence of IgM‐synthesising cells and germinal centres. Differences between the patterns of age‐dependent changes in the numbers of lymph follicles observed in the somatic and mesenteric lymph nodes during their ontogeny are discussed in relation to differences in the magnitude of the exogenous antigen stimulatory effect. We also found that the variations in the numbers of lymph follicles produced in somatic lymph nodes at different locations during the first 28 d after birth reflected differences in the dimensions of the body regions drained by a particular somatic lymph node at this stage of development.

Establishment of stromal cell line from an MDS RA patient which induced an apoptopic change in hematopoietic and leukemic cells in vitro

OBJECTIVE We previously reported on the heterogeneity of bone marrow stromal cell function in supporting hematopoietic cell proliferation and differentiation in vitro among refractory anemia (RA) of myelodysplastic syndrome (MDS) patients. Interestingly, stromal cells from some MDS RA patients induced an apoptotic change in CD34+ hematopoietic cells. However, the mechanism responsible for this action was unclear. MATERIALS AND METHODS In the present study, we established a cloned stromal cell line (LS801) from an MDS RA patient by introducing recombinant SV40-adenovirus vector containing the SV40 early gene. RESULTS LS801 induced an apoptotic change in CD34+ cells from normal subjects and cloned leukemic cells in a coculture system. When hematopoietic cells were cocultured but kept separate from LS801 by a 0.45-microm Millipore membrane to prevent their attachment, the action of LS801 in inducing apoptosis of hematopoietic cells was inhibited. Additionally, no production of fas ligand, tumor necrosis factor alpha or interferon gamma in LS801 was observed. CONCLUSION These findings suggest that the novel stromal cell line LS801 will shed light on research into the mechanism underlying the apoptotic changes induced by stromal cells in hematopoietic cells.