SecinH3 reduces proliferation of lung cancer cells expressing wild-type EGFR.

<p>A, structures of the inhibitors used in this study. B – C, proliferation of A549 (B) and H460 (C) cells was determined by MTT assay in the presence of the indicated inhibitors. ***, p<0.001 relative to DMSO-treated cells.</p

Effect of gefitinib and SecinH3 on EGFR signaling.

<p>A – D, serum-starved cells were stimulated with EGF for 5 min and probed by western blot for the activation of the indicated proteins. A, representative Western blot. B – D, Statistical evaluation of the phosphorylation of EGFR (B), IRS1 (C), and Akt (D) in EGF-stimulated H460 cells in presence of gefitinib, SecinH3, or a combination of both inhbitors. p indicates the phosphorylated, i.e. activated, form of the protein. The signals were normalized for the loading control Hsc70. The error bars give the standard error. *, p<0.05, **, p<0.01, ***, p<0.001 relative to EGF-treated cells except for those comparisons which are specified by brackets.</p

SecinH3 retards growth of H460 xenografts in vivo.

<p>Mice bearing subcutaneous H460 xenografts were treated with SecinH3 by daily intraperitoneal injections. A, tumor volume was determined each day. On day 9 the experiment had to be stopped because the tumor volume in the control animals exceeded 1 cm³. B, distribution of tumor volumes on day 9. C – D, the degree of apoptosis was analyzed in sections of tumors on day 9. C, the number of fragmented nuclei was determined by TUNEL staining. D, cleaved, i.e. activated, caspase-3 was detected by imunohistochemistry. Representative tumor sections are shown. 5× and 40× indicate the magnification of the objective. For all graphs, means and standard errors are shown (n = 14). *, p<0.05, **, p<0.01, ***, p<0.001.</p

Survivin and p27/Kip expression.

<p>A – C, H460 cells were treated overnight with 1 µM gefitinib, 15 µM SecinH3 or their combination. A, the expression of p27Kip and survivin was analyzed by western blot. B, C, Statistical evaluation of the western blot data shown in A. ** in B indicates p<0.01 relative to DMSO-treated controls as well as to gefitinib-treated cells. *** in C indicates p<0.001 relative to DMSO-treated controls. n = 3.</p

SecinH3 reduces IGF1R-dependent signaling and proliferation.

<p>A, H460 cells were serum-starved and treated overnight with 0.5 µM AG1024, 15 µM SecinH3 or their combination. After stimulation with IGF1 for 5 min the phosphorylation state of the indicated proteins was analyzed by western blot. The graphs summarize 3 experiments, the error bars give the standard error. *, p<0.05, **, p<0.01, ***, p<0.001 relative to IGF1-treated cells except for those comparisons which are specified by brackets. B, proliferation of H460 cells in the presence of the indicated inhibitors was determined by MTT assay. The numbers give the relative value of proliferation with untreated cells set as 1. The proliferation if an additive effect of SecinH3 and gefitinib is assumed (expected) is compared to the value found in the experiments (observed). Observed values below the expected values indicate synergism. ***, p<0.001.</p

Analysis of TET Expression/Activity and 5mC Oxidation during Normal and Malignant Germ Cell Development

<div><p>During mammalian development the fertilized zygote and primordial germ cells lose their DNA methylation within one cell cycle leading to the concept of active DNA demethylation. Recent studies identified the TET hydroxylases as key enzymes responsible for active DNA demethylation, catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. Further oxidation and activation of the base excision repair mechanism leads to replacement of a modified cytosine by an unmodified one. In this study, we analyzed the expression/activity of TET1-3 and screened for the presence of 5mC oxidation products in adult human testis and in germ cell cancers. By analyzing human testis sections, we show that levels of 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine are decreasing as spermatogenesis proceeds, while 5-methylcytosine levels remain constant. These data indicate that during spermatogenesis active DNA demethylation becomes downregulated leading to a conservation of the methylation marks in mature sperm. We demonstrate that all carcinoma in situ and the majority of seminomas are hypomethylated and hypohydroxymethylated compared to non-seminomas. Interestingly, 5-formylcytosine and 5-carboxylcytosine were detectable in all germ cell cancer entities analyzed, but levels did not correlate to the 5-methylcytosine or 5-hydroxymethylcytosine status. A meta-analysis of gene expression data of germ cell cancer tissues and corresponding cell lines demonstrates high expression of <i>TET1</i> and the DNA glycosylase <i>TDG,</i> suggesting that germ cell cancers utilize the oxidation pathway for active DNA demethylation. During xenograft experiments, where seminoma-like TCam-2 cells transit to an embryonal carcinoma-like state <i>DNMT3B</i> and <i>DNMT3L</i> where strongly upregulated, which correlated to increasing 5-methylcytosine levels. Additionally, 5-hydroxymethylcytosine levels were elevated, demonstrating that de novo methylation and active demethylation accompanies this transition process. Finally, mutations of <i>IDH1</i> (<i>IDH1</i>^R132) and <i>IDH2</i> (<i>IDH2</i>^R172) leading to production of the TET inhibiting oncometabolite 2-hydroxyglutarate in germ cell cancer cell lines were not detected.</p></div

Analyses of cytosine modifications in GCC cell lines.

<p>A) Absolute quantification of 5mC and 5hmC levels in GCC cell lines (TCam-2 =  seminoma; 2102EP, 833K3, NCCIT, H12.1, NT2/D1, GCT27, 1777N, 1411HP  =  EC; JEG-3 =  choriocarcinoma). Levels were quantified in triplicates. Standard deviation is given at the top of each bar. B) DNA dot blot analysis of 5fC (5fC) and 5caC (5caC) in indicated GCC cell lines. 250–62.5 ng of total DNA was spotted on a positive-charged nylon membrane. A methylene blue staining (MB) was used as loading control.</p

Analysis of TET expression/activity in three GCC cell lines.

<p>A) qRT-PCR analysis of TET1 -3 expression during 2–8 days of in vitro cultivation of TCam-2, 2102EP and JAR cells. Starting cell number: 1×10?4 cells. GAPDH was used as housekeeping gene and for data normalization. B) Measurement of total TET activity in whole protein lysate (WPL) of TCam-2, 2102EP and JAR cells at day 2 and 8 of in vitro cultivation. C) Relative quantification of 5hmC levels of TCam-2, 2102EP and JAR cells at day 2 and 8 of in vitro cultivation. Standard deviations are given at the top of each bar. Asterisks indicate a significant difference (p-value <0.05), while not significant changes are labelled as ‘n.s.’ (calculated by two-paired t-test).</p

Antibodies used in this study for IHC, western blotting and dot blotting.

<p>Antibodies used in this study for IHC, western blotting and dot blotting.</p

Analysis of ADD keyplayer expression and 5mC/5hmC levels after xenografting of TCam-2 and 2102EP cells.

<p>A) qRT-PCR analysis of indicated genes 1, 2, 4 and 6 weeks after xenografting of TCam-2 and 2102EP cells. GAPDH was used as housekeeping gene and for data normalization. Standard deviations are given at the top of each bar. B) IHC of DNMT3B 1, 2 and 6 weeks after xenografting of TCam-2 cells and 4 weeks after injection of 2102EP cells. Red arrows point at strongly DNMT3B positive cells. Scale bars: 100 µm. C) Relative quantification of 5mC and 5hmC levels of xenografted TCam-2 and 2102EP cells. Standard deviation is indicated above the bars. Significant changes are indicated as asterisks (p-value <0.05), while not significant changes are labelled as ‘n.s.’ (calculated by two-paired t-test).</p