Overexpression of Endogenous Retroviruses and Malignancy Markers in Neuroblastoma Cell Lines by Medium-Induced Microenvironmental Changes

Neuroblastoma (NB) is the commonest solid tumor outside the central nervous system in infancy and childhood with a unique biological heterogeneity. In patients with advanced, metastasizing neuroblastoma, treatment failure and poor prognosis is often marked by resistance to chemo- or immunotherapy. Thus, identification of robust biomarkers seems essential for understanding tumor progression and developing effective therapy. Here, we have studied the expression of human endogenous retroviruses (HERV) as potential targets in NB cell lines during stem-cell medium-induced microenvironmental change. Quantitative PCR revealed that relative expression of the HERV-K family and HERV-W1 ENV were increased in all three NB cell lines after incubation in stem-cell medium. Virus transcriptome analyses revealed the transcriptional activation of three endogenous retrovirus elements: HERV-R ENV (ERV3-1), HERV-E1 and HERV-Fc2 ENV (ERVFC1-1). Known malignancy markers in NB, e.g. proto-oncogenic MYC or MYCN were expressed highly heterogeneously in the three investigated NB cell lines with up-regulation of MYC and MYCN upon medium-induced microenvironmental change. In addition, SiMa cells exclusively showed a phenotype switching from loosely-adherent monolayers to low proliferating grape-like cellular aggregates, which was accompanied by an enhanced CD133 expression. Interestingly, the overexpression of HERV was associated with a significant elevation of immune checkpoint molecule CD200 in both quantitative PCR and RNA-seq analysis suggesting tumor escape mechanism in NB cell lines after incubation in serum-free stem cell medium.

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Expansive growth of two glioblastoma stem-like cell lines is mediated by bFGF and not by EGF

Radiology and Oncology ◽

10.2478/raon-2013-0063 ◽

2013 ◽

Vol 47 (4) ◽

pp. 330-337 ◽

Cited By ~ 17

Author(s):

Neza Podergajs ◽

Narve Brekka ◽

Bernhard Radlwimmer ◽

Christel Herold-Mende ◽

Krishna M. Talasila ◽

...

Keyword(s):

Stem Cell ◽

Cell Lines ◽

Basal Medium ◽

Growth Stimulation ◽

Growth Conditions ◽

Stem Cell Markers ◽

Egfr Amplification ◽

Egfr Expression ◽

Stem Cell Medium ◽

Cell Medium

Abstract Background. Patient-derived glioblastoma (GBM) stem-like cells (GSCs) represent a valuable model for basic and therapeutic research. GSCs are usually propagated in serum-free Neural Basal medium supplemented with bFGF and EGF. Yet, the exact influence of these growth factors on GSCs is still unclear. Recently it was suggested that GBM stemlike cells with amplified EGFR should be cultured in stem cell medium without EGF, as the presence of EGF induced rapid loss of EGFR amplification. However, patient biopsies are usually taken into culture before their genomic profiles are defined. Thus, an important question remains whether GBM cells without EGFR amplification also can be cultured in stem cell medium without EGF. Meterials and methods. To address this question, we used two heterogeneous glioblastoma GSC lines (NCH421k and NCH644) that lack EGFR amplification. Results. Although both cell lines showed very low EGFR expression under standard growth conditions, bFGF stimulation induced higher expression of EGFR in NCH644. In both cell lines, expression of the stem cell markers nestin and CD133 was higher upon stimulation with bFGF compared to EGF. Importantly, bFGF stimulated the growth of both cell lines, whereas EGF had no effect. We verified that the growth stimulation by bFGF was either mediated by proliferation (NCH421k) or resistance to apoptosis (NCH644). Conclusions. We demonstrate that GSC cultures without EGFR amplification can be maintained and expanded with bFGF, while the addition of EGF has no significant effect and therefore can be omitted.

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341 FELINE EMBRYONIC STEM-LIKE CELLS DERIVED FROM IN VITRO-PRODUCED BLASTOCYSTS RETAIN IN VITRO DIFFERENTIATION POTENTIAL

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab341 ◽

2015 ◽

Vol 27 (1) ◽

pp. 259

Author(s):

T. Tharasanit ◽

N. Tiptanavattana ◽

P. Phakdeedindan ◽

M. Techakumphu

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Cell Lines ◽

Es Cells ◽

Embryonic Stem ◽

In Vitro Differentiation ◽

Stem Cell Medium ◽

Cell Medium ◽

Embryonic Stem Cell Medium

Embryonic stem (ES) cells are pluripotent cells that can differentiate into all 3 germ layers, including endoderm, mesoderm, and ectoderm. Embryonic stem cells are generally divided into 2 types, naïve and primed-state, depending on their signaling pathways. Domestic cat is a useful animal model for the study of human diseases because many genetic and infectious diseases in the cat are analogous with similar aetiology to human diseases. The cat can also be used as a research model for reproductive physiology and conservation of wild felids. Until recently, information on establishment of feline ES cells is limited. The objectives of this study were to isolate cat ES cells from in vitro-produced blastocysts and to examine the effect of different concentrations of basic fibroblast growth factor (bFGF) on the expression of pluripotent genes. Inner cell masses (ICM) from cat blastocysts (n = 40, Day 7 after in vitro fertilization) that were matured, fertilized, and cultured entirely in vitro, were isolated by immunosurgery and plated on mitmycin-treated mouse embryonic fibroblasts. The ICM (n = 20) were then cultured in embryonic stem cell medium containing 1000 IU mL–1 of leukemia inhibitory factor (LIF) and different bFGF concentrations (0, 4, 10, and 20 ng mL–1). The ICM outgrowths at 7 days postplating were collected and analysed for expression of pluripotent genes (SOX-2, OCT-4, and NANOG). Results showed that transcription levels of all 3 pluripotent genes were higher in ICM outgrowths cultured in 20 ng mL–1 of bFGF compared with the lower concentrations. For isolation of ES cells, ICM (n = 20) were cultured in embryonic stem cell medium supplemented with 1000 IU mL–1 of LIF and 20 ng mL–1 of bFGF due to the results obtained from the above experiment. Established ES cells were characterised by detecting alkaline phosphatase (AP) activity and expression of ES markers (SOX-2, OCT-4, SSEA-4) at protein level, and karyotyped at passage 20 and 40. In vitro differentiation into embryoid bodies (EB) was induced by the hanging drop technique, and EB samples (n = 5 for each time point) were tested for the expression of TTR, AFP, T (Bracyury), NKX2.5, MAP-2, and NESTIN genes at 0, 7, and 14 days of culture. A total of 3 ES-like cell lines were established with a typical ES morphology, such as a well-defined colony, a large nucleus to cytoplasm ratio with 1 to 2 prominent nucleoli. The 3 ES-like cell lines were passaged up to 40 times with a normal diploid karyotype (n = 38). They were strongly positive for AP, SOX-2, OCT-4, and SSEA-4. Following EB culture, cell aggregation and cystic-like structure were observed. The EB samples also expressed all differentiation markers. This study reports that feline ES-like cell lines can be generated from in vitro-produced feline blastocysts. The ES cell lines can be repeatedly passaged indicating self-renewal ability, and gene expression of the EB demonstrates cellular differentiation into all 3 germ layers.

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Infection of Nonhuman Primate Cells by Pig Endogenous Retrovirus

Journal of Virology ◽

10.1128/jvi.74.16.7687-7690.2000 ◽

2000 ◽

Vol 74 (16) ◽

pp. 7687-7690 ◽

Cited By ~ 56

Author(s):

Juergen H. Blusch ◽

Clive Patience ◽

Yasuhiro Takeuchi ◽

Christian Templin ◽

Christian Roos ◽

...

Keyword(s):

Risk Assessment ◽

Cell Lines ◽

Nonhuman Primate ◽

Endogenous Retrovirus ◽

Papio Hamadryas ◽

Endogenous Retroviruses ◽

Human Donor ◽

Baboon Model ◽

Primate Cell ◽

Perv Transmission

ABSTRACT The ongoing shortage of human donor organs for transplantation has catalyzed new interest in the application of pig organs (xenotransplantation). One of the biggest concerns about the transplantation of porcine grafts into humans is the transmission of pig endogenous retroviruses (PERV) to the recipients or even to other members of the community. Although nonhuman primate models are excellently suited to mimic clinical xenotransplantation settings, their value for risk assessment of PERV transmission at xenotransplantation is questionable since all of the primate cell lines tested so far have been found to be nonpermissive for PERV infection. Here we demonstrate that human, gorilla, and Papio hamadryas primary skin fibroblasts and also baboon B-cell lines are permissive for PERV infection. This suggests that a reevaluation of the suitability of the baboon model for risk assessment in xenotransplantation is critical at this point.

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Endogenous retroviruses are a source of enhancers with oncogenic potential in acute myeloid leukaemia

10.1101/772954 ◽

2019 ◽

Author(s):

Özgen Deniz ◽

Mamataz Ahmed ◽

Christopher D. Todd ◽

Ana Rio-Machin ◽

Mark A. Dawson ◽

...

Keyword(s):

Cell Lines ◽

Leukemia Cell ◽

Endogenous Retrovirus ◽

Epigenetic Silencing ◽

Endogenous Retroviruses ◽

Transcriptional Networks ◽

Hematopoietic Malignancy ◽

Additional Layer ◽

Leukemia Cell Lines ◽

Acute Myeloid

AbstractAcute myeloid leukemia (AML) is a highly aggressive hematopoietic malignancy, defined by a series of genetic and epigenetic alterations, which result in deregulation of transcriptional networks. One understudied but important source of transcriptional regulators are transposable elements (TEs), which are widespread throughout the human genome. Aberrant usage of these sequences could therefore contribute to oncogenic transcriptional circuits. However, the regulatory influence of TEs and their links to disease pathogenesis remain unexplored in AML. Using epigenomic data from AML primary samples and leukemia cell lines, we identified six endogenous retrovirus (ERV) families with AML-associated enhancer chromatin signatures that are enriched in binding of key regulators of hematopoiesis and AML pathogenesis. Using both CRISPR-mediated locus-specific genetic editing and simultaneous epigenetic silencing of multiple ERVs, we demonstrate that ERV deregulation directly alters the expression of adjacent genes in AML. Strikingly, deletion or epigenetic silencing of an ERV-derived enhancer suppressed cell growth by inducing apoptosis in leukemia cell lines. Our work reveals that ERVs are a previously unappreciated source of AML enhancers that have the potential to play key roles in leukemogenesis. We suggest that ERV activation provides an additional layer of gene regulation in AML that may be exploited by cancer cells to help drive tumour heterogeneity and evolution.

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Expression of stem cell factor and c-kit in human neuroblastoma. The Children's Cancer Group

Blood ◽

10.1182/blood.v84.10.3465.3465 ◽

1994 ◽

Vol 84 (10) ◽

pp. 3465-3472 ◽

Cited By ~ 94

Author(s):

PS Cohen ◽

JP Chan ◽

M Lipkunskaya ◽

JL Biedler ◽

RC Seeger

Keyword(s):

Stem Cell ◽

Neural Crest ◽

Cell Lines ◽

Stem Cell Factor ◽

Growth Regulation ◽

Neuroblastoma Cell ◽

Receptor Expression ◽

Human Neuroblastoma ◽

Neuroblastoma Cell Lines ◽

Simultaneous Expression

Abstract During development, mice with mutations of stem cell factor (SCF) or its receptor c-kit exhibit defects in melanogenesis, as well as hematopoiesis and gonadogenesis. Because melanocytes derive from neural crest cells, the role of SCF and c-kit was investigated in the neural crest-derived childhood tumor neuroblastoma. Using reverse transcription-polymerase chain reaction analysis, simultaneous expression of steady-state mRNA for the SCF ligand and its receptor c- kit was found in 14 of 14 (100%) human neuroblastoma cell lines and clones and in 8 of 18 (45%) human neuroblastoma tumor samples. Functional blockade of c-kit receptors in the cell lines SK-N-BE(2) and SH-SY5Y using the mouse monoclonal anti-c-kit antibody SR-1 resulted in a significant decrease in cellular growth rate when measured by either 3H-thymidine incorporation or clonogenicity. In addition, higher levels of c-kit mRNA expression were associated with parental neuroblastoma cell lines and subclones with a neuronal (N) differentiation phenotype, whereas lower levels of c-kit mRNA were associated with neuroblastoma cell line subclones having a schwannian/glial/melanocytic pattern of differentiation. However, the differentiation phenotype of neuroblastoma cell lines was not directly altered when c-kit expression was blocked using the SR-1 antibody. In summary, these data indicate that c-kit receptor expression may play a significant role in the growth regulation of the two neuroblastoma cell lines examined and suggest that c-kit may also play a similar role in neuroblastoma growth regulation in vivo. Simultaneous expression of SCF and c-kit mRNA in both neuroblastoma cell lines and tumors implies that c-kit may act as part of an autocrine growth loop in conjunction with endogenous production of SCF in this disease.

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A stem cell medium containing neural stimulating factor induces a pancreatic cancer stem-like cell-enriched population

International Journal of Oncology ◽

10.3892/ijo.2014.2603 ◽

2014 ◽

Vol 45 (5) ◽

pp. 1857-1866 ◽

Cited By ~ 12

Author(s):

YUSAKU WATANABE ◽

KIYOSHI YOSHIMURA ◽

KOICHI YOSHIKAWA ◽

RYOICHI TSUNEDOMI ◽

YOSHITARO SHINDO ◽

...

Keyword(s):

Pancreatic Cancer ◽

Stem Cell ◽

Stem Cell Medium ◽

Cell Medium ◽

Stimulating Factor

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Generation of large pig and bovine blastocysts by culturing in human induced pluripotent stem cell medium

Zygote ◽

10.1017/s0967199415000088 ◽

2015 ◽

Vol 24 (2) ◽

pp. 236-244 ◽

Cited By ~ 1

Author(s):

Qing-Shan Gao ◽

Long Jin ◽

Suo Li ◽

Hai-Ying Zhu ◽

Qing Guo ◽

...

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

State University ◽

North Carolina State University ◽

Stem Cell Medium ◽

Induced Pluripotent ◽

Cell Medium

SummaryWe investigated the effect of human induced pluripotent stem cell (hiPS) medium on porcine somatic cell nuclear transfer and bovine in vitro fertilized early blastocysts, in comparison with North Carolina State University (NCSU)-37 medium and in vitro culture (IVC)-II medium. After 2 days of culture, the diameter of the portion of the blastocyst that was extruded from the zona pellucid dramatically differed between porcine blastocysts cultured in hiPS medium and those cultured in NCSU-37 medium (221.47 ± 38.94 μm versus 481.87 ± 40.61 μm, P < 0.01). Moreover, the diameter of the portion of the blastocyst significantly differed between bovine blastocysts cultured in hiPS medium and those cultured in IVC-II medium (150.30 ± 29.49 μm versus 195.58 ± 41.59 μm, P < 0.01). Furthermore, the total number of cells per porcine and bovine blastocyst was more than two-fold higher in blastocysts cultured in hiPS medium than in those cultured in NCSU-37 medium (44.33 ± 5.28 and 143.33 ± 16.05, P < 0.01) or IVC-II medium (172.12 ± 45.08 and 604.83 ± 242.64, P < 0.01), respectively. These results indicate that hiPS medium markedly improves the quality of porcine and bovine blastocysts.

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Human endogenous retrovirus-W envelope (syncytin) is expressed in both villous and extravillous trophoblast populations

Journal of General Virology ◽

10.1099/vir.0.81412-0 ◽

2006 ◽

Vol 87 (7) ◽

pp. 2067-2071 ◽

Cited By ~ 26

Author(s):

A. Muir ◽

A. M. L. Lever ◽

A. Moffett

Keyword(s):

Cell Lines ◽

First Trimester ◽

Endogenous Retrovirus ◽

Endogenous Retroviruses ◽

Extravillous Trophoblast ◽

Human Endogenous Retrovirus ◽

Human Trophoblast ◽

Human Endogenous Retroviruses ◽

Normal Tissues ◽

Villous Trophoblast

The placenta is unique amongst normal tissues in transcribing numerous different human endogenous retroviruses at high levels. In this study, RT-PCR and immunohistochemistry were used to investigate the expression of syncytin in human trophoblast. Syncytin transcripts were found in first-trimester trophoblast cells with both villous and extravillous phenotypes and also in the JAR and JEG-3 choriocarcinoma cell lines. Syncytin protein was detected in villous trophoblast and in all extravillous trophoblast subpopulations of first- and second-trimester placental tissues. It was also present in ectopic trophoblast from tubal implantations. This study confirms that syncytin is expressed widely by a variety of normal human trophoblast populations, as well as choriocarcinoma cell lines.

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Human endogenous retrovirus transcription profiles of the kidney and kidney-derived cell lines

Journal of General Virology ◽

10.1099/vir.0.031518-0 ◽

2011 ◽

Vol 92 (10) ◽

pp. 2356-2366 ◽

Cited By ~ 9

Author(s):

Sonja Haupt ◽

Michele Tisdale ◽

Michelle Vincendeau ◽

Mary Anne Clements ◽

David T. Gauthier ◽

...

Keyword(s):

Cell Culture ◽

Cell Line ◽

Cell Lines ◽

Kidney Tissue ◽

Human Kidney ◽

Endogenous Retrovirus ◽

Endogenous Retroviruses ◽

Human Endogenous Retrovirus ◽

Tissue Samples ◽

Transcription Pattern

The human genome comprises approximately 8–9 % of human endogenous retroviruses (HERVs) that are transcribed with tissue specificity. However, relatively few organs have been examined in detail for individual differences in HERV transcription pattern, nor have tissue-to-cell culture comparisons been frequently performed. Using an HERV-specific DNA microarray, a core HERV transcription profile was established for the human kidney comparing 10 tissue samples. This core represents HERV groups expressed uniformly or nearly so in non-tumour kidney tissue. The profiles obtained from non-tumour tissues were compared to 10 renal tumour tissues (renal cell carcinoma, RCC) derived from the same individuals and additionally, to 22 RCC cell lines. No RCC cell line or tumour-specific differences were observed, suggesting that HERV transcription is not altered in RCC. However, when comparing tissue transcription to cell line transcription, there were consistent differences. The differences were irrespective of cancer state and included cell lines derived from non-tumour kidney tissue, suggesting that a specific alteration of HERV transcription occurs when establishing cell lines. In contrast to previous publications, all known HERV-derived tumour antigens, including those identified in RCC, were expressed both in multiple RCC cell lines and several non-tumour tissue-derived cell lines, a result that contrasts with findings from patient samples. The results establish the core kidney transcription pattern of HERVs and reveal differences between cell culture lines and tissue samples.

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Identification of a native novel oncolytic immunoglobulin on exfoliated colon epithelial cells: A bispecific heterodimeric chimera of IgA/IgG

10.1101/140673 ◽

2017 ◽

Author(s):

George P. Albaugh ◽

Sudhir K. Dutta ◽

Vasantha Iyengar ◽

Samina Shami ◽

Althaf Lohani ◽

...

Keyword(s):

Stem Cell ◽

Direct Method ◽

Stem Cell Markers ◽

Distinct Cell ◽

Stem Cell Medium ◽

Stool Samples ◽

Low Levels ◽

Colonic Cells ◽

Cell Medium ◽

A Direct Method

ABSTRACTUnderstanding the nature of cell surface markers on exfoliated colonic cells is a crucial step in establishing criteria for a normally functioning mucosa. We have found that colonic cells isolated from stool samples (SCSR-010 Fecal Cell Isolation Kit, NonInvasive Technologies, Elkridge, MD), preserved at room temperature for up to one week, with viability of >85% and low levels of apoptosis (8% - 10%) exhibit two distinct cell size subpopulations, in the 2.5μM– 5.0 μM and 5.0μM-8.0μM range. In addition to IgA, about 60% of the cells expressed a novel heterodimeric IgA/IgG immunoglobulin that conferred a broad-spectrum cell mediated cytotoxicity against tumor cells. In a cohort of 58 subjects the exclusive absence of this immunoglobulin in two African-Americans was suggestive of a germline deletion. Serial cultures in stem cell medium retained the expression of this heterodimer. Since a majority of the cystic cells expressed the stem cell markers Lgr5 and Musashi-1 we termed these cells as gastrointestinal progenitor stem cells (GIP-C**). CXCR-4, the cytokine co-receptor for HIV was markedly expressed. These cells also expressed CD20, IgA, IgG, CD45, and COX-2. We assume that they originated from mature columnar epithelium by dedifferentiation. Our observations indicate that we have a robust noninvasive method to study mucosal pathophysiology and a direct method to create a database for applications in regenerative medicine.

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