The Seminoma cell line TCam-2 is Sensitive to HDAC Inhibitor Depsipeptide but Tolerates Various Other Chemotherapeutic Drugs and Loss of NANOG Expression

Genes, Chromosomes and Cance

TCam-2 but not JKT-1 cells resemble Seminoma in Cell Culture

Cell and Tissue Researc

NANOG promoter methylation and expression correlation during normal and malignant human germ cell development

Epigenetic

Gene expression within the amplified region.

<p>A) Expression histogram indicating the relative expression of genes mapped within the amplified region in a series of SE and EC. The stars indicate genes significantly differentially expressed between SE and EC (according to Mann Whitney U test). Most of the genes are represented by multiple specific probes; B) DNA-FISH result for SOX2 in NCCIT cells. A centromere 12 (C12) specific probe is used as control. Red dye (Cye3) shows SOX2 probe. For C12 probe green dye (FITC) is used. The blue background color is DAPi. Multiple red spots for SOX2 are detectable in each cell containing two green spots for C12, indicating SOX2 amplification. Magnification used was 630x. DNA-FISH was performed on cut tissue section with a thickness of 4 micron. This results in possible heterogeneity of the probe sizes detected. This issue was not a limitation as the purpose of this experiment was to determine the copy numbers and the size or intensity of the region. The FISH (BAC) probes were ordered at BACPAC Resources Center (BPRC) online:bacpac.chori.org. They were verified and confirmed at the department of genetics.</p

OCT3/4 and SOX2 knockdown in NCCIT.

<p>A) Percentage of positive cells for OCT3/4 and SOX2 in cells with reduced OCT3/4 and SOX2 levels compared to cells transfected with control siRNA in the NCCIT cells in three different time-points post transfection based on immunohistochemistry; The controls are set to 100 in all cases. B) Relative expression pattern of 32 genes representing targets for pluripotency and differentiation (ectoderm, mesoderm and endoderm). The expression levels are normalized based on the housekeeping gene <i>HPRT</i>; C) Percentage of living NCCIT cells with reduced level of OCT3/4 and SOX2 compared to cells transfected with control siRNA at each time point based on Trypan blue measurement; D) Percentage of positive NCCIT cells for Ki67 in untreated cells, cells transfected with control siRNA and cells with reduced levels of OCT3/4 and SOX2 at 48+12 h post transfection; E) Percentage of positive cells for Caspase 3 in untreated NCCIT cells, Cells transfected with control siRNA show a reduced level of OCT3/4 and SOX2 at all three time points after the transfection; F) FACS analysis with Propidium Iodide staining in cells transfected with control siRNA, cells transfected with SOX2 and OCT3/4 siRNA at 60 h post transfection. NCCIT cells transfected with control siRNA show the presence of 78% living cells, while cells transfected with SOX2 siRNA show 51% living cells, NCCIT cells with OCT3/4kd show 62% live cells. G) Annexin V assay results in cells with siRNA control and cells with reduced SOX2 and OCT3/4 at 60 h after transfection. In the control, percentages of living-annexin negative cells are 89.6%, while dead-annexin positive cells are 12.2%. In SOX2kd cells, the amount of living-annexin negative cells is 57.5% while the amount of dead-annexin positive cells is 17.2%. In cells transfected with OCT3/4 siRNA, living-annexin negative cells are 68.3% while dead-annexin positive cells are 12.8%. The differences between the percentages of live and dead cells in all experiments are due to using independent and various methods in order to prove apoptosis and different sensitivity of the materials and methods used. As it is demonstrated, OCT3/4 knock-down also induces apoptosis, however, this effect is not as dramatic as SOX2 knock-down. The FACS analysis were performed in triplicate.</p

Schematic representation of the hypothetical indirect regulation of SOX2 by p53 status.

<p>Wild type p53 results in induction of expression of miR-145 and miR-34a. Subsequently, miR-34a and mir-145 down-regulates pluripotency genes, including SOX2, the latter via Wnt signaling.</p

Expression of BLIMP1/PRMT5 and concurrent histone H2A/H4 arginine 3 dimethylation in fetal germ cells, CIS/IGCNU and germ cell tumors

<p>Background: Most testicular germ cell tumors arise from intratubular germ cell neoplasia unclassified (IGCNU, also referred to as carcinoma in situ), which is thought to originate from a transformed primordial germ cell (PGC)/gonocyte, the fetal germ cell. Analyses of the molecular profile of IGCNU and seminoma show similarities to the expression profile of fetal germ cells/gonocytes. In murine PGCs, expression and interaction of Blimp1 and Prmt5 results in arginine 3 dimethylation of histone H2A and H4. This imposes epigenetic modifications leading to transcriptional repression in mouse PGCs enabling them to escape the somatic differentiation program during migration, while expressing markers of pluripotency.</p><p>Results: In the present study, we show that BLIMP1 and PRMT5 were expressed and arginine dimethylation of histones H2A and H4 was detected in human male gonocytes at weeks 12–19 of gestation, indicating a role of this mechanism in human fetal germ cell development as well. Moreover, BLIMP1/PRMT5 and histone H2A and H4 arginine 3 dimethylation was present in IGCNU and most seminomas, while downregulated in embryonal carcinoma (EC) and other nonseminomatous tumors. </p><p>Conclusion: These data reveal similarities in marker expression and histone modification between murine and human PGCs. Moreover, we speculate that the histone H2A and H4 arginine 3 dimethylation might be the mechanism by which IGCNU and seminoma maintain the undifferentiated state while loss of these histone modifications leads to somatic differentiation observed in nonseminomatous tumors.</p

Critical Function of AP-2gamma/TCFAP2C in Mouse Embryonic Germ Cell Maintenance

<p>Formation of the germ cell lineage involves multiple processes, including repression of somatic differentiation and reacquisition of pluripotency as well as a unique epigenetic constitution. The transcriptional regulator Prdm1 has been identified as a main coordinator of this process, controlling epigenetic modification and gene expression. Here we report on the expression pattern of the transcription factor Tcfap2c, a putative downstream target of Prdm1, during normal mouse embryogenesis and the consequences of its specific loss in primordial germ cells (PGCs) and their derivatives. Tcfap2c is expressed in PGCs from Embryonic Day 7.25 (E 7.25) up to E 12.5, and targeted disruption resulted in sterile animals, both male and female. In the mutant animals, PGCs were specified but were lost around E8.0. PGCs generated in vitro from embryonic stem cells lacking TCFAP2C displayed induction of Prdm1 and Dppa3. Upregulation of Hoxa1, Hoxb1, and T together with lack of expression of germ cell markers such Nanos3, Dazl, and Mutyh suggested that the somatic gene program is induced in TCFAP2C-deficient PGCs. Repression of TCFAP2C in TCam-2, a human PGCresembling seminoma cell line, resulted in specific upregulation of HOXA1, HOXB1, MYOD1, and HAND1, indicative of mesodermal differentiation. Expression of genes indicative of ectodermal, endodermal, or extraembryonic differentiation, as well as the finding of no change to epigenetic modifications, suggested control by other factors. Our results implicate Tcfap2c as an important effector of Prdm1 activity that is required for PGC maintenance, most likely mediating Prdm1-induced suppression of mesodermal differentiation.</p

Overview mutations found and immunohistochemistry.

<p>Ex, exon; ND, not done; NA, not available; mutations indicated in bold, other samples wild type unless indicated otherwise; DG, dysgerminoma; GB, gonadoblastoma; CIS, carcinoma in situ; TE, teratoma (im: immature); YST yolk sac tumor; itSE, intratubular seminoma;</p><p>seq, sequence; LC, LightCycler; c-KIT LC, LightCycler melting curve results; case numbers in bold are bilateral cases.</p>§<p>tumor/precursor percentage below 50%.</p>‡<p>as determined by FISH on gonadal tissue.</p><p>I789I: heterozygous synonymous SNP, rs.5578615.</p><p>P567P: homozygous synonymous SNP, rs.1873778.</p

High mRNA expression of splice variant <i>SYK</i> short correlates with hepatic disease progression in chemonaive lymph node negative colon cancer patients

<div><p>Objective</p><p>Overall and splice specific expression of Spleen Tyrosine Kinase (<i>SYK</i>) has been posed as a marker predicting both poor and favorable outcome in various epithelial malignancies. However, its role in colorectal cancer is largely unknown. The aim of this study was to explore the prognostic role of <i>SYK</i> in three cohorts of colon cancer patients.</p><p>Methods</p><p>Total messenger RNA (mRNA) expression of <i>SYK</i>, <i>SYK(T)</i>, and mRNA expression of its two splice variants <i>SYK</i> short <i>(S)</i> and <i>SYK</i> long <i>(L)</i> were measured using quantitative reverse transcriptase (RT-qPCR) in 240 primary colon cancer patients (n = 160 patients with chemonaive lymph node negative [LNN] and n = 80 patients with adjuvant treated lymph node positive [LNP] colon cancer) and related to microsatellite instability (MSI), known colorectal cancer mutations, and disease-free (DFS), hepatic metastasis-free (HFS) and overall survival (OS). Two independent cohorts of patients with respectively 48 and 118 chemonaive LNN colon cancer were used for validation.</p><p>Results</p><p>Expression of <i>SYK</i> and its splice variants was significantly lower in tumors with MSI, and in <i>KRAS</i> wild type, <i>BRAF</i> mutant and <i>PTEN</i> mutant tumors. In a multivariate Cox regression analysis, as a continuous variable, increasing <i>SYK(S)</i> mRNA expression was associated with worse HFS (Hazard Ratio[HR] = 1.83; 95% Confidence Interval[CI] = 1.08–3.12; p = 0.026) in the LNN group, indicating a prognostic role for <i>SYK(S)</i> mRNA in patients with chemonaive LNN colon cancer. However, only a non-significant trend between <i>SYK(S)</i> and HFS in one of the two validation cohorts was observed (HR = 4.68; 95%CI = 0.75–29.15; p = 0.098).</p><p>Conclusion</p><p>In our cohort, we discovered <i>SYK(S)</i> as a significant prognostic marker for HFS for patients with untreated LNN colon cancer. This association could however not be confirmed in two independent smaller cohorts, suggesting that further extensive validation is needed to confirm the prognostic value of <i>SYK(S)</i> expression in chemonaive LNN colon cancer.</p></div