Characterization of magnesium requirement of human 5'-tyrosyl DNA phosphodiesterase mediated reaction

<p>Abstract</p> <p>Background</p> <p>Topo-poisons can produce an enzyme-DNA complex linked by a 3'- or 5'-phosphotyrosyl covalent bond. 3'-phosphotyrosyl bonds can be repaired by tyrosyl DNA phosphodiesterase-1 (TDP1), an enzyme known for years, but a complementary human enzyme 5'-tyrosyl DNA phosphodiesterase (hTDP2) that cleaves 5'-phosphotyrosyl bonds has been reported only recently. Although hTDP2 possesses both 3'- and 5'- tyrosyl DNA phosphodiesterase activity, the role of Mg²⁺in its activity was not studied in sufficient details.</p> <p>Results</p> <p>In this study we showed that purified hTDP2 does not exhibit any 5'-phosphotyrosyl phosphodiesterase activity in the absence of Mg²⁺/Mn²⁺, and that neither Zn²⁺or nor Ca²⁺can activate hTDP2. Mg²⁺also controls 3'-phosphotyrosyl activity of TDP2. In MCF-7 cell extracts and de-yolked zebrafish embryo extracts, Mg²⁺controlled 5'-phosphotyrosyl activity. This study also showed that there is an optimal Mg²⁺concentration above which it is inhibitory for hTDP2 activity.</p> <p>Conclusion</p> <p>These results altogether reveal the optimal Mg²⁺requirement in hTDP2 mediated reaction.</p

Generation of small molecules targeting hypoxia inducible factors

28th International Congress of the European-Respiratory-Society (ERS) -- SEP 15-19, 2018 -- Paris, FRANCEWOS: 000455567103418…European Respiratory So

Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

<div><p>The Hepatocyte Growth Factor (HGF)/c-Met signaling pathway regulates hepatocyte proliferation, and pathway aberrations are implicated in the invasive and metastatic behaviors of hepatocellular carcinoma (HCC). In addition to c-Met, heparin acts as a co-receptor to modulate pathway activity. Recently, anti-metastatic and anti-cancer effects of heparin have been reported. However, the role of heparin in the regulation of HGF signaling remains controversial and the effects of heparin on HGF-induced biological responses during hepatocarcinogenesis is not yet defined. In this study we determined the effects of heparin on HGF-induced activities of HCC cells and the underlying molecular mechanisms. Here, we report for the first time that heparin inhibits HGF-induced adhesion, motility and invasion of HCC cells. In addition, heparin reduced HGF-induced activation of c-Met and MAPK in a dose-dependent manner, as well as decreased transcriptional activation and expression of Early growth response factor 1 (Egr1). HGF-induced MMP-2 and MMP-9 activation, and MT1-MMP expression, also were inhibited by heparin. Stable knockdown of Egr1 caused a significant decrease in HGF-induced invasion, as well as the activation and expression of MMPs. Parallel to these findings, the overexpression of Egr1 increased the invasiveness of HCC cells. Our results suggest that Egr1 activates HGF-induced cell invasion through the regulation of MMPs in HCC cells and heparin inhibits HGF-induced cellular invasion via the downregulation of Egr1. Therefore, heparin treatment might be a therapeutic approach to inhibit invasion and metastasis of HCC, especially for patients with active HGF/c-Met signaling.</p></div

Overexpression of Egr1 induces MMP-9 activation, cell invasion and cell migration of HCC cells.

<p>The effect of Egr1 on the cell invasion of HCC cell lines were examined using SNU-449 transiently transfected with pCMV-6-AC-GFP Egr1 overexpression vector (<b>8A</b>). Equal amounts of wild type and Egr1 overexpressing SNU-449 cells were used for migration (<b>8B</b>) and invasion (<b>8C</b>) assay in the presence and absence of HGF. Results are expressed as fold differences of matrigel invaded cells. Zymographic gel showing the active MMP-9 bands in 24 h conditioned medium of cultured Egr1-overexpressing SNU-449 cells (<b>8D</b>). The graph compares the signal intensities obtained from zymography gels.</p

Heparin inhibits HGF-induced p-Met, p-MAPK, MMP-2 and MMP-9 activation in SK-HEP-1.

<p>In order to examine the time dependent effects of HGF, overnight starved SK-HEP-1 cells were grown in presence or absence of HGF for the time periods indicated. Total cell lysates were then analyzed by immunoblotting. Blots were probed with anti-p-Met, anti-c-Met, anti-pERK1/2, anti-ERK1/2 and anti-calnexin antibodies. From top to bottom: HGF stimulated c-Met phosphorylation; the amounts of c-Met protein present whole cell lysates; HGF induced ERK1/2 phosphorylation and ERK1/2 and c-Met total protein levels. Membranes were re-probed with ERK1/2 and c-Met antibody after stripping. The bottom panel verifies equal protein loading among the lanes (<b>3A</b>). SK-HEP1 cells left untreated or treated with HGF for 2 hours in the presence and absence of heparin. Total protein lysates were analyzed by immunoblotting. Membranes were blotted with anti-p-Met, anti-c-Met, anti-pERK1/2, anti-ERK1/2 antibodies, and anti-calnexin antibodies. The first panel shows the level of tyrosine phosphorylated c-Met; the amount of c-Met protein present in the each lane is shown in the second panel. The third and fourth panels show the amount of phosphorylated ERK1/2 level and the level of ERK1/2 protein in the whole cell lysates, respectively. Membranes were re-probed with ERK1/2 and c-Met antibody after stripping. The lower panels show the amount of calnexin as a loading control (<b>3B</b>). Zymographic gel showing active MMP-9 and MMP-2 bands in 24 h conditioned medium from cultured SK-HEP-1 cells left untreated or stimulated with HGF and heparin (<b>3C, 3D</b>).</p

Stable vector-mediated, inducible knockdown of Egr1.

<p>To achieve an inducible knockdown of Egr1, we firstly transfected SK-HEP-1 cells with pCDNA6/TR which express tet repressor. Stable cells were selected using Blasticidin before co-transfecting SK-HEP-1 T-REx cells with tetracycline–regulated pSUPER.retro.neo+GFP tet RNAi construct, which was directed against a sequence containing Egr1. Stable cells were selected using G418. After selection, clones were analyzed for downregulation of Egr1 after 24 h of tetracycline treatment. Ten-fold down-regulation of Egr1 was detected by immunoblotting in pSUPER.retro.neo+GFP tet/Egr1 clones (<b>6A</b>) but not in mock transfected clones (<b>6B</b>). The graph compares the signal intensities obtained from Egr1 bands.</p

Heparin inhibits HGF-induced Egr1 expression and the Egr1 promoter activity in a dose-dependent manner.

<p>HGF-induced activation of Egr1 mRNA and protein level (<b>2A, 2C</b>) and the effect of heparin on the HGF induced Egr1 expression in SK-HEP-1 cells were examined by RT-PCR and western blotting (<b>2B, 2D</b>). Egr1 transcriptional activity was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with pGL2-Luc-B-Egr1 plasmid construct and the pRL-TK <i>Renilia</i> luciferase. Relative luciferase activity was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042717#s4" target="_blank">Materials and Methods</a>. The firefly luciferase activity was normalized to <i>Renilia</i> luciferase activity (<b>2E, 2F</b>). Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.</p