Tera-2 Cells Research Articles

Synthesis and in vitro assessment of gold nanoparticles conjugated with extracts, sterols and pure compounds derived from marine sponges from the Indian and Pacific Oceans.

Gold nanoparticles (AuNPs) exhibit different physical properties compared to small molecules, bulk materials and other nanoparticles. Their synthesis using plant extracts, particularly polyflavonoids as phytoreductants, for the conversion of Au(iii) into Au(0) has been reported. In this study, AuNPs were synthesized with extracts, sterols and pure compounds derived from marine sponges using gold(iii) chloride trihydrate. Extracts, hexane (JDH) and ethyl acetate (JDE), sterols (JC-2) and jaspamide were obtained from Jaspis diastra. Pure compounds, namely, contignasterol, ansellone A, motuporamines A and MN100 (a synthetic analog of pelorol), were also used. JC-2 was characterized using NMR and GC-MS, and the major constituent was determined to be β-sitosterol. β-Sitosterol has shown great promise as an anti-cancer molecule, but its poor aqueous solubility and bioavailability coupled with low targeting efficacy limit its therapeutic efficacy. Transmission electron microscopy (TEM) images revealed the formation of spherical AuNPs conjugated with JDH, JDE, JC-2, ansellone and contignasterol with average diameters of 21.1 ± 3.0 nm, 20.7 ± 2.1 nm, 26.2 ± 1.2 nm, 33.3 ± 5.1 nm and 30.8 ± 5.5 nm, respectively. No particle formation was seen with motuporamines A and MN100. Zeta potential values indicated that AuNPs-JC-2 was more stable than AuNPs-JDE, AuNPs-JDH and AuNPs-ansellone. Based on IC50 values, the cytotoxicity of AuNPs-JDH increased in A172, TERA, HeLa and HepG2 cells but showed similar activity in HaCaT cells compared to JDH. The cytotoxicity of AuNPs-JDE decreased in A172 and HaCaT cells but increased in TERA1, HeLa and HepG2 cells compared to JDE. AuNPs-JC-2 showed enhanced cytotoxicity with a decrease in IC50 values from 3.37 ± 0.19 μg mL-1 to 0.52 ± 0.09 μg mL-1 in A172 and from 2.28 ± 0.20 μg mL-1 to 0.78 ± 0.28 μg mL-1 in TERA1 compared to JC-2. The synergistic action of sterols in AuNPs-JC-2 seemed to favour enhanced anti-cancer activity. The presence of sterols increased the ability of transforming Au(iii) into Au(0) to form AuNPs and further enhancing cellular uptake and, thus, anti-cancer activity. AuNPs-contignasterol displayed lower activity than contignasterol in the A172 cell line. No significant difference in activity was observed with AuNPs-ansellone A in the A172 and HaCaT cell lines compared to ansellone A.

A Comprehensive Analysis of Human Endogenous Retroviruses HERV-K (HML.2) from Teratocarcinoma Cell Lines and Detection of Viral Cargo in Microvesicles.

About 8% of our genome is composed of sequences from Human Endogenous Retroviruses (HERVs). The HERV-K (HML.2) family, here abbreviated HML.2, is able to produce virus particles that were detected in cell lines, malignant tumors and in autoimmune diseases. Parameters and properties of HML.2 released from teratocarcinoma cell lines GH and Tera-1 were investigated in detail. In most experiments, analyzed viruses were purified by density gradient centrifugation. HML.2 structural proteins, reverse transcriptase (RT) activity, viral RNA (vRNA) and particle morphology were analyzed. The HML.2 markers were predominantly detected in fractions with a buoyant density of 1.16 g/cm3. Deglycosylation of TM revealed truncated forms of transmembrane (TM) protein. Free virions and extracellular vesicles (presumably microvesicles—MVs) with HML.2 elements, including budding intermediates, were detected by electron microscopy. Viral elements and assembled virions captured and exported by MVs can boost specific immune responses and trigger immunomodulation in recipient cells. Sequencing of cDNA clones demonstrated exclusive presence of HERV-K108 env in HML.2 from Tera-1 cells. Not counting two recombinant variants, four known env sequences were found in HML.2 from GH cells. Obtained results shed light on parameters and morphology of HML.2. A possible mechanism of HML.2-induced diseases is discussed.

MP40-04 NOVEL SELECTIVE LYSINE-SPECIFIC DEMETHYLASE 1 INHIBITORS, NCL1 AND NCD38, SUPPRESS TESTICULAR TUMOR CELL PROLIFERATION

You have accessJournal of UrologyPenile & Testicular Cancer I (MP40)1 Sep 2021MP40-04 NOVEL SELECTIVE LYSINE-SPECIFIC DEMETHYLASE 1 INHIBITORS, NCL1 AND NCD38, SUPPRESS TESTICULAR TUMOR CELL PROLIFERATION Toshiki Etani, Taku Naiki, Nobuhiko Shimizu, Yusuke Noda, Takashi Nagai, Satoshi Nozaki, Keitaro Iida, Ryosuke Ando, Noriyasu Kawai, Satoru Takahashi, Takayoshi Suzuki, and Takahiro Yasui Toshiki EtaniToshiki Etani More articles by this author , Taku NaikiTaku Naiki More articles by this author , Nobuhiko ShimizuNobuhiko Shimizu More articles by this author , Yusuke NodaYusuke Noda More articles by this author , Takashi NagaiTakashi Nagai More articles by this author , Satoshi NozakiSatoshi Nozaki More articles by this author , Keitaro IidaKeitaro Iida More articles by this author , Ryosuke AndoRyosuke Ando More articles by this author , Noriyasu KawaiNoriyasu Kawai More articles by this author , Satoru TakahashiSatoru Takahashi More articles by this author , Takayoshi SuzukiTakayoshi Suzuki More articles by this author , and Takahiro YasuiTakahiro Yasui More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002055.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Lysine-specific demethylase 1 (LSD1), the first histone demethylase to be discovered, is a novel target in prostate cancer therapy. NCL1 and NCD38, novel selective cell-active inhibitors of LSD1, were discovered at our university. We analyzed the efficacy of LSD1 inhibitors in germ cell tumors in vitro and in vivo. METHODS: Various tests were used to uncover mechanism of germ cell tumor growth and anticancer effects of NCL1 and NCD38. Cell viability was assessed using a WST-8 assay with standard vehicle, NCL1 or NCD38 in NTERA2 and Tera-1 cells (human testicular embryonal carcinoma cell lines). Flow cytometry and western blotting were performed to assess apoptosis and the cell cycle after treatment of NTERA2 cells with NCL1 or NCD38. Subcutaneous and orthotopic tumors were induced by injecting nude mice with NTERA2 cells. Thereafter, mice were injected intraperitoneally with vehicle, NCL1 or NCD38, twice a week, and their subsequent growth recorded. TUNEL staining was used to detect apoptosis in tumors. Finally, tissue arrays were prepared from tissue specimens from 35 patients with seminoma who underwent orchiectomy. RESULTS: The WST assay revealed a reduction in the number of viable cells in a dose-dependent manner after NCL1 or NCD38 treatment. In western blotting, NCL1 or NCD38 treatment induced caspase-dependent apoptosis, while Oct 3/4 and SOX2 expression was decreased. In ChIP-qPCR analysis, LSD1 inhibition increased the methylation of promoter regions of SOX2 and Oct 3/4. In flow cytometry analysis, NCL1 or NCD38 significantly induced apoptosis in a dose-dependent manner. Subcutaneous tumor volumes and serum AFP levels were significantly lower in mice treated with NCL1 or NCD38 than in controls. TUNEL analysis showed that NCL1 and NCD38 treatment induced apoptosis in NTERA2 subcutaneous tumors. Tissue array analysis showed that LSD1 expression in human seminoma specimens was significantly higher than that in noncancerous specimens. CONCLUSIONS: Germ cell tumor growth was effectively suppressed with NCL1 or NCD38 treatment in vitro and in vivo without adverse events by regulating apoptosis and suppressing SOX2 and Oct4. These results indicate the potential of NCL1 and NCD38 as therapeutic agents for germ cell tumors. Source of Funding: Grants in Aid for Scientific Research (KAKENHI), Japan Society for the Promotion of Science (JSPS), Number 17K11152 © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e715-e716 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Toshiki Etani More articles by this author Taku Naiki More articles by this author Nobuhiko Shimizu More articles by this author Yusuke Noda More articles by this author Takashi Nagai More articles by this author Satoshi Nozaki More articles by this author Keitaro Iida More articles by this author Ryosuke Ando More articles by this author Noriyasu Kawai More articles by this author Satoru Takahashi More articles by this author Takayoshi Suzuki More articles by this author Takahiro Yasui More articles by this author Expand All Advertisement Loading ...

Potential Regulatory Role of Human-Carboxylesterase-1 Glycosylation in Liver Cancer Cell Growth

We previously reported that human carboxylesterase 1 (CES1), a serine esterase containing a unique N-linked glycosyl group at Asn79 (N79 CES1), is a candidate serological marker of hepatocellular carcinoma (HCC). CES1 is normally present at low-to-undetectable levels in normal human plasma, HCC tumors, and major liver cancer cell lines. To investigate the potential mechanism underlying the suppression of CES1 expression in liver cancer cells, we took advantage of the low detectability of this marker in tumors by overexpressing CES1 in multiple HCC cell lines, including stable Hep3B cells. We found that the population of CES1-overexpressing (OE) cells decreased and that their doubling time was longer compared with mock control liver cancer cells. Using interactive transcriptome, proteome, and subsequent Gene Ontology enrichment analysis of CES1-OE cells, we found substantial decreases in the expression levels of genes involved in cell cycle regulation and proliferation. This antiproliferative function of the N79 glycan of CES1 was further supported by quantitative real-time polymerase chain reaction, flow cytometry, and an apoptosis protein array assay. An analysis of the levels of key signaling target proteins via Western blotting suggested that CES1 overexpression exerted an antiproliferative effect via the PKD1/PKCμ signaling pathway. Similar results were also seen in another HCC cell line (PLC/RFP/5) after transient transfection with CES1 but not in similarly treated non-HCC cell lines (e.g., HeLa and Tera-1 cells), suggesting that CES1 likely exerts a liver cell-type-specific suppressive effect. Given that the N-linked glycosyl group at Asn79 (N79 glycan) of CES1 is known to influence CES1 enzyme activity, we hypothesized that the post-translational modification of CES1 at N79 may be linked to its antiproliferative activity. To investigate the regulatory effect of the N79 glycan on cellular growth, we mutated the single N-glycosylation site in CES1 from Asn to Gln (CES1-N79Q) via site-directed mutagenesis. Fluorescence 2-D difference gel electrophoresis protein expression analysis of cell lysates revealed an increase in cell growth and a decrease in doubling time in cells carrying the N79Q mutation. Thus our results suggest that CES1 exerts an antiproliferative effect in liver cancer cells and that the single N-linked glycosylation at Asn79 plays a potential regulatory role. These functions may underlie the undetectability of CES1 in human HCC tumors and liver cancer cell lines. Mass spectrometry data are available via ProteomeXchange under the identifier PXD021573.

MP11-03 NOVEL SELECTIVE LYSINE-SPECIFIC DEMETHYLASE 1 INHIBITORS SUPPRESS TESTICULAR TUMOR CELL PROLIFERATION

INTRODUCTION AND OBJECTIVE: Lysine-specific demethylase 1 (LSD1), the first histone demethylase to be discovered, is a novel target in prostate cancer therapy. NCL1 and NCD38, novel selective cell-active inhibitors of LSD1, were discovered at our university. We analyzed the efficacy of LSD1 inhibitors in germ cell tumors in vitro and in vivo. METHODS: Various tests were used to uncover mechanism of germ cell tumor growth and anticancer effects of NCL1 and NCD38. Cell viability was assessed using a WST-8 assay with standard vehicle, NCL1 or NCD38 in NTERA2 and Tera-1 cells (human testicular embryonal carcinoma cell lines). Flow cytometry and western blotting were performed to assess apoptosis and the cell cycle after treatment of NTERA2 cells with NCL1 or NCD38. Subcutaneous tumors were induced by subcutaneously injecting nude mice with NTERA2 cells. Thereafter, mice were injected intraperitoneally with vehicle, NCL1 or NCD38, twice a week, and their subsequent growth recorded. TUNEL staining was used to detect apoptosis in tumors. Finally, tissue arrays were prepared from tissue specimens from 35 patients with seminoma who underwent orchiectomy. RESULTS: The WST assay revealed a reduction in the number of viable cells in a dose-dependent manner after NCL1 or NCD38 treatment. In western blotting, NCL1 or NCD38 treatment induced caspase-dependent apoptosis, while Oct4 and SOX2 expression was decreased. In flow cytometry analysis, NCL1 or NCD38 significantly induced apoptosis in a dose-dependent manner. Subcutaneous tumor volumes and serum AFP levels were significantly lower in mice treated with NCL1 or NCD38 than in controls. TUNEL analysis showed that NCL1 and NCD38 treatment induced apoptosis in NTERA2 subcutaneous tumors. No adverse effect was observed in body weights, blood tests, and hematoxylin-eosin staining of various organs in mice. Tissue array analysis showed that LSD1 expression in human seminoma specimens was significantly higher than that in noncancerous specimens. CONCLUSIONS: Germ cell tumor growth was effectively suppressed with NCL1 or NCD38 treatment in vitro and in vivo without adverse events by regulating apoptosis and suppressing SOX2 and Oct4. These results indicate the potential of NCL1 and NCD38 as therapeutic agents for germ cell tumors.Source of Funding: Grant-in-Aid for Scientific Research 17K11152

Ectopic activation of WNT signaling in human embryonal carcinoma cells and its effects in short- and long-term in vitro culture

Human embryonal carcinoma (EC) cells comprise the pluripotent stem cells of malignant non-seminomatous germ cell tumors (GCTs) and represent the malignant counterpart of embryonic stem cells (ESCs). WNT/β-catenin signaling has been implicated in regulating adult and embryonic stem cells although its role in EC cells is less investigated. Here, we studied WNT signaling in a panel of representative pluripotent and nullipotent human EC cell lines. We found that EC cell lines show distinct levels of intrinsic WNT signaling and respond differently to ectopic WNT activation. Short-term activation of WNT signaling induced a differentiation-response in the pluripotent EC cells (NT2 and NCCIT) whereas the nullipotent EC cells (TERA1 and 2102Ep) were refractory and maintained high levels of OCT4 and SSEA4 expression. Long-term activation of WNT signaling in NCCIT and, to a lesser extent, TERA1 cells led to (re)gain of OCT4 expression and a switch from SSEA4 to SSEA1 surface antigens ultimately resulting in OCT4+/SSEA4−/SSEA1+ profile. Cisplatin treatment indicated that the OCT4+/SSEA4−/SSEA1+ NCCIT cells became more resistant to chemotherapy treatment. Our findings are of particular interest for the GCT and ES cell biology and shed light on the role of WNT signaling in human EC cells.

Cytotoxic and apoptotic effects of cyproterone acetate against cancer cells and normal cells

Back ground: Cyproterone acetate (CPA) is a steroidal anti-androgen inhibits the testosterone and DHT action it is used as a medicine for prostate cancer by association with the androgen receptors located on the surface of prostate cells, thus preventing the association of testosterone with receptors.
 Objective: Investigation of the anticancer activities of Cyproterone acetate against cancer cell lines testicular (Tera-1), macrophage (RAW 264.7) in comparison to non- tumorigenic fetal hepatic cell line (WRL-68).
 Material and method: The cytotoxic effect of CPA was investigated according to selected parameters including: MTT assay as assay of cell function to determined cell viability, high content screening (HCS) technique for the apoptosis of cell. Only the most cytotoxic concentration of CPA and the most sensitive cells as assayed by MTT was selected to complete the other test: (HCS). (MTT) assay was carried out at the Centre of Biotechnology Research’s, Al- NahrainUniversityBaghdad,Iraq . The HCS assay was performed at the Centre for Natural Product Research and Drug Discovery, Department of Pharmacology,Faculty of Medicine, University of Malaya, KualaLumpur, Malaysia
 Results: The most significant cytotoxic effect of Cyproterone acetate towards 2 cancer cell lines was obtained when its concentration was 1.25mg/mL. The Tera-1 Cells were more sensitive to Cyproterone acetate compared with RAW264.7 and WRL-68. cells. There was a significant decrease in valid cell count, nuclear intensity and mitochondrial membrane potential (MMP) when CPA (400 μg/mL)was used compared with doxorubicin (20 μg/mL) as a standard. Also, there was a significant increase in membrane permeability and cytochrome C releasing when CPA (400μg/ml) was used compared with positive control. 
 Conclusion: CPA showed cytotoxic effects against the Tera-1 and RAW264.7 cancer cell lines while the WRL-68. Cells was not affected as determined in-vitro by the MTT assay. The HCStechnique also showed toxic effect towards Tera-1.

CD30-Redirected Chimeric Antigen Receptor T Cells Target CD30+ and CD30- Embryonal Carcinoma via Antigen-Dependent and Fas/FasL Interactions.

Tumor antigen heterogeneity limits success of chimeric antigen receptor (CAR) T-cell therapies. Embryonal carcinomas (EC) and mixed testicular germ cell tumors (TGCT) containing EC, which are the most aggressive TGCT subtypes, are useful for dissecting this issue as ECs express the CD30 antigen but also contain CD30-/dim cells. We found that CD30-redirected CAR T cells (CD30.CAR T cells) exhibit antitumor activity in vitro against the human EC cell lines Tera-1, Tera-2, and NCCIT and putative EC stem cells identified by Hoechst dye staining. Cytolytic activity of CD30.CAR T cells was complemented by their sustained proliferation and proinflammatory cytokine production. CD30.CAR T cells also demonstrated antitumor activity in an in vivo xenograft NOD/SCID/γcnull (NSG) mouse model of metastatic EC. We observed that CD30.CAR T cells, while targeting CD30+ EC tumor cells through the CAR (i.e., antigen-dependent targeting), also eliminated surrounding CD30- EC cells in an antigen-independent manner, via a cell-cell contact-dependent Fas/FasL interaction. In addition, ectopic Fas (CD95) expression in CD30+ Fas- EC was sufficient to improve CD30.CAR T-cell antitumor activity. Overall, these data suggest that CD30.CAR T cells might be useful as an immunotherapy for ECs. Additionally, Fas/FasL interaction between tumor cells and CAR T cells can be exploited to reduce tumor escape due to heterogeneous antigen expression or to improve CAR T-cell antitumor activity. Cancer Immunol Res; 6(10); 1274-87. ©2018 AACR.

KRIBB53 binds to OCT4 and enhances its degradation through the proteasome, causing apoptotic cell death of OCT4-positive testicular germ cell tumors.

We hypothesized that octamer-binding transcription factor 4 (OCT4) inhibition would have therapeutic benefits in testicular germ cell tumors (TGCT). To identify inhibitors of OCT4, a chemical library was screened using a luciferase reporter system under the control of an OCT4 response element. A compound named KRIBB53 was identified based on its blocking of OCT4-dependent luciferase activation. When NCCIT cells were exposed to KRIBB53, the expression levels of OCT4 target genes, such as NANOG and USP44, were inhibited with an IC50 of 13 and 15 μM, respectively. In addition, the levels of OCT4 were decreased by exposing NCCIT cells to KRIBB53, and pretreating the cells with the proteasomal inhibitor MG132 reversed the KRIBB53-induced OCT4 degradation. Biotinyl-KRIBB53 was synthesized and showed comparable activity to KRIBB53 in OCT4 downregulation. Using affinity chromatography assay, KRIBB53 was shown to associate with OCT4 in vitro. Furthermore, the drug affinity responsive target stability (DARTS) assay confirmed unmodified KRIBB53 binding to OCT4. KRIBB53 selectively inhibited proliferation of TGCT cells such as NCCIT and Tera-1 cells but not that of immortalized normal cells. Finally, the administration of KRIBB53 at 30 mg/kg reduced tumor volumes by 77% in the mice xenografted with NCCIT cells relative to their vehicle-treated counterparts. Immunoblotting assays showed that expression of OCT4 was lower in KRIBB53-treated tumor tissues than in control tissues. We provide the first report, to our knowledge, of an OCT4 inhibitor that binds to OCT4 and induces its degradation.

Abstract 923: Human embryonic stem cells exhibit altruistic cell death that release death signals having potent anti-cancer activity

Abstract Background: We recently described the altruistic stem cell (ASC) phenotype in human embryonic stem cells (hESCs) as well as in mesenchymal stem cells (1, 2). The ASC phenotype was characterized by altered state of p53/MDM2 feedback system that permitted the cells to transiently acquire an unstable state of low p53 allowing the cells to survive in extreme microenvironment (1). Interestingly, we noted that ASCs spontaneously returned to its basal state of high p53 after a period of two weeks (1, 2). In this study, we speculate that this return of p53 levels to basal state could be a safety mechanism to prevent malignant transformation of ASCs. We also speculate that ASCs may release soluble factors like high mobility group protein B1 (HMGB-1) that could selectively target the cells exhibiting abnormal p53 e.g. cancer cells. Here, we further characterized ASC phenotype and its fate. Methods: hESC, BG01 cells were exposed to extreme hypoxia followed by reoxygenation and then ABCG2+/SSEA3+ cells were flow cytometry sorted. This specific subpopulation is enriched in ASCs and could be cultured in vitro for two weeks (1). The post- hypoxia treated ABCG2+/SSEA3+ cells were maintained in serum free media, and subjected to apoptosis, and senescence assays. The conditioned media (CM) of these cells and subjected to HMGB-1 measurement by ELISA. To measure bystander apoptosis, teratocarcinoma cells (Tera-2) were treated with the CM followed by measurement of apoptosis. Results: Here we confirmed that p53 and MDM2 in ABCG2+/SSEA3+ cells exhibited return of oscillation between days 19-24. This was associated increase in apoptosis, and senescence of these cells. Apoptosis/senescence was decreased when treated with either Pifithrin α, an inhibitor of p53, or by siRNA silencing of p53, suggesting p53-dependent apoptosis. Additionally, treatment with Nutlin-3, an inhibitor of MDM2 (1) led to a 10-fold increase in apoptosis indicating that high MDM2 was required to prevent the apoptosis of ASCs. Thus, ASCs underwent spontaneous apoptosis (altruistic cell death) due to the return of p53/MDMD2 oscillation. We suggest that this return of p53 and associated apoptosis could be a safety mechanism to prevent malignant transformation of ASCs. Next, we found that the CM of ABCG2+/SSEA3+ cells contained high levels of HMGB-1 compared to the parental BG01 cells. Importantly, CM treatment led to p53 dependent apoptosis of Tera-2 cells. Furthermore, CM treatment also inhibited the growth of Tera-2 xenografts in NOD/SCID mice. Conclusion: Our results indicate that ASCs undergoes altruistic cell death, and releases death signal mediated by HMGB1 that can target neighboring cells exhibiting abnormal p53 including cancer cells. We suggest that this unique property of ASCs could be exploited as a novel cancer therapeutic. 1. Das B et al, Stem Cells, 2012; 30(8):1685-95. 2. Pal B et al, Cancer Research, 2016; Volume 76, Issue 14 Supplement, pp. 251 Note: This abstract was not presented at the meeting. Citation Format: Bidisha Pal, Seema Bhuyan, Jaishree Garhyan, Herman Yeger, Bikul Das. Human embryonic stem cells exhibit altruistic cell death that release death signals having potent anti-cancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 923. doi:10.1158/1538-7445.AM2017-923

Differential expression of HERV-K (HML-2) proviruses in cells and virions of the teratocarcinoma cell line Tera-1.

Human endogenous retrovirus (HERV-K (HML-2)) proviruses are among the few endogenous retroviral elements in the human genome that retain coding sequence. HML-2 expression has been widely associated with human disease states, including different types of cancers as well as with HIV-1 infection. Understanding of the potential impact of this expression requires that it be annotated at the proviral level. Here, we utilized the high throughput capabilities of next-generation sequencing to profile HML-2 expression at the level of individual proviruses and secreted virions in the teratocarcinoma cell line Tera-1. We identified well-defined expression patterns, with transcripts emanating primarily from two proviruses located on chromosome 22, only one of which was efficiently packaged. Interestingly, there was a preference for transcripts of recently integrated proviruses, over those from other highly expressed but older elements, to be packaged into virions. We also assessed the promoter competence of the 5’ long terminal repeats (LTRs) of expressed proviruses via a luciferase assay following transfection of Tera-1 cells. Consistent with the RNASeq results, we found that the activity of most LTRs corresponded to their transcript levels.

Aza-deoxycytidine induces apoptosis or differentiation via DNMT3B and targets embryonal carcinoma cells but not their differentiated derivatives

Background:Teratocarcinoma is a malignant male germ cell tumour, which contains stem cells and differentiated cancer tissues. DNMT3B has been shown to be highly expressed in human teratocarcinoma stem cells, and to mediate cytotoxicity of Aza-deoxycytidine (Aza-dC) in a pluripotent stem cell line NTERA2.Methods:We have established DNMT3B or POU5F1 (hereafter referred to as OCT4) knockdown in teratocarcinoma stem cells N2102Ep and TERA1 and in the pluripotent NTERA2 by a doxycycline-inducible system, and tested the cytotoxicity induced by Aza-dC.Results:Silencing of DNMT3B led to apoptosis of human teratocarcinoma stem cells N2102Ep and TERA1. Further, we found that induction of apoptosis or differentiation in NTERA2 and human embryonic stem cells by Aza-dC requires DNMT3B. To test whether Aza-dC inhibits proliferation of differentiated teratocarcinoma cells, we depleted OCT4 expression in N2102Ep and TERA1 cells treated with Aza-dC. Treatment with Aza-dC reduced cell number of differentiated cells to a lesser extent than their undifferentiated parental stem cells. Moreover, in contrast to the stem cells, Aza-dC failed to induce apoptosis of differentiated cells.Conclusions:Our finding suggests that DNMT3B acts as an antiapoptotic gene in teratocarcinoma stem cells, and mediates apoptosis and differentiation of human pluripotent stem cells induced by Aza-dC, and that Aza-dC specifically induces apoptosis of teratocarcinoma stem cells.

Cobalt and Nickel Stabilize Stem Cell Transcription Factor OCT4 through Modulating Its Sumoylation and Ubiquitination

Stem cell research can lead to the development of treatments for a wide range of ailments including diabetes, heart disease, aging, neurodegenerative diseases, spinal cord injury, and cancer. OCT4 is a master regulator of self-renewal of undifferentiated embryonic stem cells. OCT4 also plays a crucial role in reprogramming of somatic cells into induced pluripotent stem (iPS) cells. Given known vivo reproductive toxicity of cobalt and nickel metals, we examined the effect of these metals on expression of several stem cell factors in embryonic Tera-1 cells, as well as stem cells. Cobalt and nickel induced a concentration-dependent increase of OCT4 and HIF-1α, but not NANOG or KLF4. OCT4 induced by cobalt and nickel was due primarily to protein stabilization because MG132 stabilized OCT4 in cells treated with either metals and because neither nickel nor cobalt significantly modulated its steady-state mRNA level. OCT4 stabilization by cobalt and nickel was mediated largely through reactive oxygen species (ROS) as co-treatment with ascorbic acid abolished OCT4 increase. Moreover, nickel and cobalt treatment increased sumoylation and mono-ubiquitination of OCT4 and K123 was crucial for mediating these modifications. Combined, our observations suggest that nickel and cobalt may exert their reproductive toxicity through perturbing OCT4 activity in the stem cell compartment.

Differential responses to genotoxic agents between induced pluripotent stem cells and tumor cell lines

Given potential values of induced pluripotent stem (iPS) cells in basic biomedical research and regenerative medicine, it is important to understand how these cells regulate their genome stability in response to environmental toxins and carcinogens. The present study characterized the effect of Cr(VI), a well-known genotoxic agent and environmental carcinogen, on major molecular components of DNA damage response pathways in human iPS cells. We compared the effect of Cr(VI) on human iPS cells with two established cell lines, Tera-1 (teratoma origin) and BEAS-2B (lung epithelial origin). We also studied the effect of hydrogen peroxide and doxorubicin on modulating DNA damage responses in these cell types. We demonstrated that ATM and p53 phosphorylation is differentially regulated in human iPS cells compared with Tera-1 and BEAS-2B cells after exposure to various genotoxic agents. Moreover, we observed that inhibition of CK2, but not p38, promotes phosphorylation of p53S392 in iPS cells. Combined, our data reveal some unique features of DNA damage responses in human iPS cells.

Cytoplasmic NANOG-Positive Stromal Cells Promote Human Cervical Cancer Progression

Tumor development has long been known to resemble abnormal embryogenesis. The embryonic stem cell gene NANOG, a divergent homeodomain transcription factor that is independent of leukemia inhibitory factor, has been reported to be expressed in germ cells and in several tumor types. However, the short-term expression and role of NANOG in cervical cancer remain unclear. In the present study, we demonstrate that NANOG exhibits cellular shuttling behavior and increasing stromal distribution during the progression of cervical cancer. Our molecular data using RT-PCR and restriction enzyme digestion show that NANOG is mainly transcribed from the NANOG gene in cervical cancer. In addition, IHC using confocal microscopy suggests that mesenchymal stem cells (MSCs) are one type of cytoplasmic NANOG-positive cells in cervical cancer stroma. Co-culture of cervical cancer-derived MSCs with SiHa cells showed increased proliferation characteristics in vitro and enhanced tumor growth in vivo. Our results show, for the first time to our knowledge, that MSCs are a source of cytoplasmic NANOG expression in the cervical cancer stroma and that they participate in the progression of cervical cancer both in vitro and in vivo. Our study provides evidence that NANOG is a cervical cancer progression marker and also serves as a starting point for a more extensive exploration of the cellular translocation of NANOG and the multifunctionality of the stromal microenvironment.

Abstract 1465: Histone deacetylase inhibitor suberoylanilide hydroxamic acid blocks epithelial-mesenchymal transition exhibited by Tera-2 embryonal carcinoma cells with a stem cell phenotype

Abstract The process of epithelial-mesenchymal transition (EMT) is associated with tumor progression. Tumor cells that undergo EMT have altered morphology and acquire a stem cell phenotype. Although there are numerous studies that demonstrate that shRNA approaches targeting specific transcription factors can block EMT, there are few pharmacological agents yet identified that can alter the process of EMT. Recent evidence suggests that the silencing of E-cadherin during EMT is dependent upon the activity of specific histone deacetylyase enzymes, which are known to regulate gene transcription. We used Tera-2 (HTB 106) embryonal carcinoma cells grown as 3 dimensional spheroids as a model of a tumor type characterized by a cancer stem cell phenotype. We observed that the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat; Zolinza®, Merck), which is FDA approved for treatment of cutaneous T cell lymphoma, significantly inhibited expression of over 55 distinct genes associated with EMT including those regulating responses to growth factors, adhesion, motility, and genes associated with maintenance/survival of stem cells, including Alpha 5 Integrin/fibronectin receptor alpha (ITGA5), Caldesmon 1 (CALD1), Epidermal growth factor receptor (EGFR), Forkhead box C2 (MFH-1, mesenchyme forkhead 1) (FOXC2), Glycogen synthase kinase 3 beta (GSK3 beta), Jagged 1 (JAG1), Integrin-linked kinase (ILK), Matrix metallopeptidase 2 (MMP2), Notch 1 homolog (NOTCH1), Platelet-derived growth factor receptor beta (PDGFb), Snail homolog 1 (SNAI1), Snail homolog 2 (SNAI2), Transcription factor 4 (TCF4), transforming growth factorβ1 (TGFβ1), Twist homolog 1 (Twist1), Wingless-type MMTC integration site family, member 5b (WNT5b), and Zinc finger E-box binding homeobox 2 (ZEB2). SAHA also altered the morphology of Tera-2 cells when cultured as adherent cells, which was associated with inhibited of both invasion, and anchorage independent growth in soft agar. In addition, SAHA inhibited the self-renewal capacity of Tera-2 tumor spheroids, which was associated with changes in expression of stem cell surface markers. Studies are ongoing to identify the HDAC enzymes that regulate expression of genes associated with the process of EMT and maintenance/survival of Tera-2 tumor spheroids that are targeted by SAHA. These studies suggest that HDAC inhibitors may be promising agents to reverse the process of EMT and may be potentially effective as inhibitors of tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1465.

Downregulation of miRNA-200c Links Breast Cancer Stem Cells with Normal Stem Cells

Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters, miR-200c-141, miR-200b-200a-429, and miR-183-96-182 were downregulated in human BCSCs, normal human and murine mammary stem/progenitor cells, and embryonal carcinoma cells. Expression of BMI1, a known regulator of stem cell self-renewal, was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly, miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells.

Loopy Chromatin Brings Distant DNA to Bear on Silencing Promoter Genes

Loopy Chromatin Brings Distant DNA to Bear on Silencing Promoter Genes

PcG Proteins, DNA Methylation, and Gene Repression by Chromatin Looping

Many DNA hypermethylated and epigenetically silenced genes in adult cancers are Polycomb group (PcG) marked in embryonic stem (ES) cells. We show that a large region upstream (∼30 kb) of and extending ∼60 kb around one such gene, GATA-4, is organized—in Tera-2 undifferentiated embryonic carcinoma (EC) cells—in a topologically complex multi-loop conformation that is formed by multiple internal long-range contact regions near areas enriched for EZH2, other PcG proteins, and the signature PcG histone mark, H3K27me3. Small interfering RNA (siRNA)–mediated depletion of EZH2 in undifferentiated Tera-2 cells leads to a significant reduction in the frequency of long-range associations at the GATA-4 locus, seemingly dependent on affecting the H3K27me3 enrichments around those chromatin regions, accompanied by a modest increase in GATA-4 transcription. The chromatin loops completely dissolve, accompanied by loss of PcG proteins and H3K27me3 marks, when Tera-2 cells receive differentiation signals which induce a ∼60-fold increase in GATA-4 expression. In colon cancer cells, however, the frequency of the long-range interactions are increased in a setting where GATA-4 has no basal transcription and the loops encompass multiple, abnormally DNA hypermethylated CpG islands, and the methyl-cytosine binding protein MBD2 is localized to these CpG islands, including ones near the gene promoter. Removing DNA methylation through genetic disruption of DNA methyltransferases (DKO cells) leads to loss of MBD2 occupancy and to a decrease in the frequency of long-range contacts, such that these now more resemble those in undifferentiated Tera-2 cells. Our findings reveal unexpected similarities in higher order chromatin conformation between stem/precursor cells and adult cancers. We also provide novel insight that PcG-occupied and H3K27me3-enriched regions can form chromatin loops and physically interact in cis around a single gene in mammalian cells. The loops associate with a poised, low transcription state in EC cells and, with the addition of DNA methylation, completely repressed transcription in adult cancer cells.

Human Endogenous Retrovirus Family HERV-K(HML-2) RNA Transcripts Are Selectively Packaged into Retroviral Particles Produced by the Human Germ Cell Tumor Line Tera-1 and Originate Mainly from a Provirus on Chromosome 22q11.21

The human germ cell tumor line Tera-1 produces retroviral particles which are encoded by the human endogenous retrovirus family HERV-K(HML-2). We show here, by quantitative reverse transcriptase PCR, that HML-2 gag and env RNA transcripts are selectively packaged into Tera-1 retroviral particles, whereas RNAs from cellular housekeeping genes and from other HERV families (HERV-H and HERV-W) are nonselectively copackaged. Assignment of cloned HML-2 gag and env cDNAs from Tera-1 retroviral particles to individual HML-2 loci in the human genome demonstrated that HML-2 RNA transcripts packaged into Tera-1 retroviral particles originate almost exclusively from an HML-2 provirus on chromosome 22q11.21. Based on relative cloning frequencies, this provirus was the most active among a total of eight transcribed HML-2 loci identified in Tera-1 cells. These data suggest that at least one HML-2 element, that is, the HML-2 provirus on 22q11.21, has retained the capacity for packaging RNA into HML-2-encoded retroviral particles. Given its elevated transcriptional activity and the presence of a full-length Gag open reading frame, the 22q11.21 HML-2 provirus may also significantly contribute to Gag protein and thus particle production in Tera-1 cells. Our findings provide important clues to the generation and biological properties of HML-2-encoded particles. In addition, copackaging of non-HML-2 HERV transcripts in HML-2-encoded particles should inform the debate about endogenous retroviral particles putatively encoded by non-HML-2 HERV families that have previously been described for other human diseases, such as multiple sclerosis.