24 research outputs found
A possible mechanism that underlies the generation of cell-specific patterns of histamine-induced Ca<sup>2+</sup> signals.
<p>The diagrams show the typical IP<sub>3</sub> and Ca<sup>2+</sup> dynamics observed in HeLa cells stimulated with histamine. Low-frequency sustained Ca<sup>2+</sup> oscillations tend to be observed in cells in which PLC-β1 is dominant (left), while high-frequency damped oscillations tend to be observed in cells in which PLC-β4 is dominant (right).</p
Identification of PLC isozymes involved in IP<sub>3</sub> generation in HeLa cells stimulated with histamine.
<p>(A) Representative traces of IRIS-1 signal changes (ΔR/R<sub>0</sub>; top) and Indo-5F signal changes (F/F<sub>0</sub>; bottom) observed in thapsigargin-treated HeLa cells. The horizontal broken lines indicate the baseline levels. (B) Traces of the mean ± SD IRIS-1 signal changes (ΔR/R<sub>0</sub>) observed in thapsigargin-treated HeLa cells after addition of 3 µM histamine (blue circles) or 3 µM histamine plus 2 mM Ca<sup>2+</sup> (green circles). (C–F) Effects of PLC isozyme knockdown on the IP<sub>3</sub> increase evoked by 3 µM histamine alone (C), the IP<sub>3</sub> increase evoked by 2 mM Ca<sup>2+</sup> alone (D), the first component of the IP<sub>3</sub> increase evoked by 3 µM histamine plus 2 mM Ca<sup>2+</sup>, and the second component of the IP<sub>3</sub> increase evoked by 3 µM histamine plus 2 mM Ca<sup>2+</sup>. Data are shown as means ± SD. The numbers of cells measured are shown in parentheses. Statistical analyses were performed by one-way ANOVA followed by Scheffe’s multiple comparison test. *P<0.05, **P<0.01, vs. the values in control siRNA-transfected cells.</p
Classification of cells depending on the time constants of peak amplitude decay of Ca<sup>2+</sup> spikes.
<p>Representative traces of Indo-5F signal changes (F/F<sub>0</sub>; top) and IRIS-1 signal changes (ΔR/R<sub>0</sub>; bottom) in cells showing sustained Ca<sup>2+</sup> oscillations (S-cell) (A) and damped oscillations (D-cell) (B). Three different concentrations of histamine (1, 3, and 10 µM) were sequentially applied to the same cells with an interval of 20 min. (C) Relationships between the histamine concentrations and the inverse time constants of Ca<sup>2+</sup> amplitude decay in S-cells (red) and D-cells (blue). (D) Relationships between the histamine concentrations and the Ca<sup>2+</sup> oscillation frequencies observed in S-cells (red) and D-cells (blue). (E–G) Comparisons of the Ca<sup>2+</sup> oscillation frequencies between S-cells (red) and D-cells (blue). The histamine concentrations were 1 µM (E), 3 µM (F), and 10 µM (G). (H–J) Comparisons of integrated IRIS-1 signals between S-cells (red) and D-cells (blue). The histamine concentrations were 1 µM (H), 3 µM (I), and 10 µM (J).</p
Specific primer sequences for RT-PCR analyses.
a<p>F and R denote foreword and reverse primers, respectively.</p
Reproducible cell-specific patterns in Ca<sup>2+</sup> and IP<sub>3</sub> responses in HeLa cells stimulated with histamine.
<p>(A) Venus fluorescence image of IRIS-1-expressing HeLa cells before histamine stimulation. Bar, 20 µm. (B) Changes in 460–510 nm emission of Indo-5F signals (F/F<sub>0,</sub> where F<sub>0</sub> is the basal level of F; top) and ECFP/Venus emission ratio of IRIS-1 signals (ΔR/R<sub>0</sub>; ΔR was defined as R – R<sub>0</sub>, where R<sub>0</sub> is the basal level of R; bottom) after repeated additions of 3 µM histamine with a 20-min interval in two different cells within the same field of view shown in (A). The horizontal broken lines indicate the baseline levels of the IRIS-1 and Indo-5F signals. The vertical broken lines indicate the onsets of stimulation.</p
siRNA sequences.
a<p>The GC% column indicates the GC content of each siRNA sequence.</p>b<p>The numbers in the position column denote where the sequence is located in the mRNA of the target gene (counted from the first nucleotide of the start codon) of human origin.</p
Effects of PLC-β1 or PLC-β4 knockdown on Ca<sup>2+</sup> and IP<sub>3</sub> dynamics evoked by histamine stimulation.
<p>(A) Representative traces of Indo-5F signal changes (F/F<sub>0</sub>; top) and IRIS-1 signal changes (ΔR/R<sub>0</sub>; bottom) in siRNA-treated cells stimulated with 3 µM histamine. The siRNAs used are shown on the left. The horizontal broken lines indicate the baseline levels of the IRIS-1 and Indo-5F signals. The vertical broken lines indicate the onsets of stimulation. Stacked histograms of the inverse time constants for exponential decay of the Ca<sup>2+</sup> oscillation amplitude (B), Ca<sup>2+</sup> oscillation frequencies (C), and integrated IP<sub>3</sub> signals (D) of cells treated with control siRNA (top row), PLCβ1KD siRNAs (second row), and PLCβ4KD siRNAs (third row). The results for the two siRNAs are shown in different colors. The means ± SD are shown at the bottom. The numbers of cells measured are shown in parentheses. Statistical analyses were performed by one-way ANOVA followed by Scheffe’s multiple comparison test. **P<0.01, vs. the values in control siRNA mGC-transfected cells.</p
Effects of PLC-β1 or PLC-β4 overexpression on Ca<sup>2+</sup> and IP<sub>3</sub> dynamics evoked by histamine stimulation.
<p>(A) Western blotting analyses of cell lysates prepared from HeLa cells transfected with PLC-β1-IRES-mRFP (lane 2) or PLC-β4-IRES-mRFP (lane 4). Non-transfected cells were used as controls (lanes 1 and 3). (B) Representative traces of Indo-5F signal changes (F/F<sub>0</sub>; top) and IRIS-1 signal changes (ΔR/R<sub>0</sub>; bottom) in transfected cells stimulated with 3 µM histamine. The plasmid DNAs used to transfect the cells are shown on the left. The horizontal broken lines indicate the baseline levels of IRIS-1 and Indo-5F signals. The vertical broken lines indicate the onsets of stimulation. (B–D) Histograms for the inverse time constants for exponential decay of the Ca<sup>2+</sup> oscillation amplitude (B), Ca<sup>2+</sup> oscillation frequencies (C), and integrated IP<sub>3</sub> signals (D) in cells expressing mRFP (top row), PLC-β1 and mRFP (second row), and PLC-β4 and mRFP (third row). The means ± SD are shown at the bottom. The numbers of cells measured are shown in parentheses. Statistical analyses were performed by one-way ANOVA followed by Scheffe’s multiple comparison test. **P<0.01, vs. the values in IRES-mRFP transfected cells.</p
Dasatinib suppresses peritoneal dissemination of SGC cells and their association with stromal fibroblasts in vivo.
<p>A, 44As3 cells were intraperitoneally injected into nude mice and DMSO or dasatinib was administered via intraperitoneal injection. The number of mesentery nodules was calculated as described in the Materials and Methods. Bars show mean ± SEM (<i>n</i> = 10). *, <i>p</i><0.005 by Mann-Whitney test. B, Representative macroscopic views of metastatic tumor nodules (arrowheads) formed in the mesentery. C, Immunofluorescence analysis of the mouse mesenteries bearing tdTomato-labeled 44As3 tumor nodules. Arrowheads denote the regions where FSP1 positive stromal fibroblasts were accumulated around tumor nodules. D, Mesentery nodules were stained with hematoxylin and eosin and anti-αSMA antibody for histological examination.</p
Stromal Fibroblasts Mediate Extracellular Matrix Remodeling and Invasion of Scirrhous Gastric Carcinoma Cells
<div><p>Scirrhous gastric carcinoma (SGC) has the worst prognosis of all gastric cancers, owing to its rapid expansion by invasion and frequent peritoneal dissemination. Due to the increased proliferation of stromal fibroblasts (SFs) that occurs within SGC lesions and the peritoneal metastatic sites, SFs have been proposed to support the progression of this disease. However, the biological and molecular basis and the pathological role of the intercellular interaction between SGC cells and SFs remain largely unknown. In this study, we investigated the role of SFs in the invasion of the extracellular matrix (ECM) by SGC cells. When SGC cells were cocultured with SFs derived from SGC tissue on three-dimensional (3D) Matrigel, they were attracted together to form large cellular aggregates that invaded within the Matrigel. Time-lapse imaging revealed that this process was associated with extensive contraction and remodeling of the ECM. Immunofluorescence and biochemical analysis showed that SGC cells stimulate phosphorylation of myosin light chain and actomyosin-mediated mechanical remodeling of the ECM by SFs. By utilizing this assay system for inhibitor library screening, we have identified several inhibitors that potently suppress the cooperation between SGC cells and SFs to form the invasive structures. Among them, a Src inhibitor dasatinib impaired the interaction between SGC cells and SFs both in vitro and in vivo and effectively blocked peritoneal dissemination of SGC cells. These results indicate that SFs mediate mechanical remodeling of the ECM by SGC cells, thereby promoting invasion and peritoneal dissemination of SGC.</p></div