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PubMed1984

Fibroblast feeder layers inhibit differentiation of retinoic acid-treated embryonal carcinoma cells by increasing the probability of stem cell renewal.

The appearance of differentiated cells in embryonal carcinoma (EC) cultures can be inhibited by culturing the cells on fibroblast feeder layers. To determine whether or not feeder layers act by increasing the probability of stem cell renewal, growth and differentiation were monitored in cultures of F9 (subclone OTF9 -63) EC cells exposed to retinoic acid (RA) in either the presence or absence of feeder layers. By measuring the fraction of laminin-positive TROMA 1-positive or alkaline phosphatase-negative cells, it was determined that the frequency of differentiated cells in RA-treated F9 cultures was reduced by 70-80% when cells were cultured on fibroblast feeder layers instead of gelatin-coated dishes. Experiments in which EC cells were cultured in close proximity to a feeder layer demonstrated that cell-cell contact was required for maximal inhibition of differentiation. The probability of stem cell renewal was determined by measuring the number of colony-forming cells in RA-treated cultures as a function of time. Analysis of the data demonstrated that the probabilities of stem cell renewal were 0.5 and 0.25 during the first and second 48 h periods, respectively, following addition of RA for cells cultured without feeder layers. Cultures maintained on feeder layers exhibited a stem cell renewal probability of 0.72. Thus, feeder layers reduce the frequency of differentiated cells in RA-treated cultures by increasing the probability of stem cell renewal. Determining the mechanism by which feeder layers counteract the effect of a chemically defined differentiation inducer should help to uncover the processes that regulate the probability of stem cell renewal.

Animals↗
PubMed1986

An anti-carbohydrate monoclonal antibody inhibits cell-substratum adhesion of F9 embryonal carcinoma cells.

A monoclonal rat IgM antibody (4C9) raised against F9 embryonal carcinoma cells reacted with fucosyl residues in poly-N-acetyllactosamine-type large carbohydrates of these cells (embryoglycan). The chemical properties and distribution of the antigen resembled those of SSEA-1. The monoclonal antibody was found to inhibit cell-substratum adhesion of F9 cells: in the presence of the antibody, cells grew as spherical cell aggregates on plastic dishes. When the antibody was added to the already spread cells, they displayed the initial sign of rounding up within 3 h; the rounding process was largely completed within 6 h. After removal of the antibody, cells resumed their normal morphology. The antibody could act in the presence of 2,4-dinitrophenol. In serum-free medium, F9 cells spread on plastic dishes coated with fibronectin or with laminin, and the process was also inhibited by the antibody. Immuno-electronmicroscopy revealed that 4C9 antigen was diffusely distributed over the cell surface of F9 cells. The distribution of the antigen was not altered generally after culturing with the antibody for 6 h. Another monoclonal rat IgM antibody, which did not react with embryoglycan and resembled anti-Forssman, did not inhibit cell-substratum adhesion of F9 cells, in spite of its reactivity to the cells. Thus, a glycoprotein with fucosyl (poly)-N-acetyllactosamine structure appears to be involved in cell-substratum adhesion of F9 cells.

2,4-Dinitrophenol↗
PubMed1989

The protein composition of the nuclear matrix of murine P19 embryonal carcinoma cells is differentiation-stage dependent.

The protein composition of the nuclear matrix of murine P19 embryonal carcinoma (EC) cells was compared with that of clonal derivatives of P19 EC differentiated in vitro, and with that of P19 EC cells induced to differentiate with retinoic acid (RA). Several major differences in nuclear matrix protein composition were found between the cell lines tested. Some polypeptides were found to occur only in EC cells, whereas others proved to be restricted to one or more of the differentiated derivatives. During RA treatment of EC cells a transient expression of some matrix proteins was observed. Several new proteins appeared, and others disappeared. Our data indicate that the protein composition of the nuclear matrix is a sensitive gauge for the differentiation state of cells.

Animals↗
PubMed1989

Presumptive neurons derived by differentiation of a human embryonal carcinoma cell line exhibit tetrodotoxin-sensitive sodium currents and the capacity for regenerative responses.

Cloned human embryonal carcinoma cells (NTERA-2 cl.D1) differentiate into neuron-like cells upon exposure to retinoic acid. Using whole-cell patch-clamp techniques, these putative neurons exhibited rapidly activating and inactivating inward currents upon depolarization as well as outward currents. The electrical characteristics and tetrodotoxin (TTX) sensitivity of the inward currents suggest that they were sodium currents. By contrast, only outward potassium currents were seen in the undifferentiated stem cells. Under current clamp conditions, the neuron-like cells showed regenerative responses. The peaks of these responses never exceeded the O-mV level, perhaps due to the low mean inward current density of 93.8 +/- 17.8 (SEM) microA/cm2:n = 9. The electrophysiological characteristics of these human teratocarcinoma-derived neuron-like cells were consistent with our previous identification of these cells as neurons, but suggest that they may resemble immature embryonic, rather than adult, neurons.

Action Potentials↗
PubMed1987

Effects of tunicamycin on the differentiation of F9 cells induced by either retinoic acid or retinoic acid and dibutyryl cyclic AMP.

Tunicamycin (0.5 micrograms/ml) inhibited differentiation of F9 cells treated either with retinoic acid or with retinoic acid and dibutyryl cyclic AMP, as monitored by the activity of alkaline phosphatase and expression of cytokeratins. On the other hand, the pattern of the polysaccharide chain synthesis changed drastically with the treatment irrespective of the presence of tunicamycin. Therefore, phenotypes induced with retinoic acid are dissociated into two categories, one that is directly induced by the drug and the other that is induced indirectly by a mechanism in which glycoproteins play a role.

Animals↗
PubMed1990

Induction of human embryonal carcinoma cell differentiation using N,N'-hexamethylene bisacetamide in vitro.

Morphological differentiation of a pluripotent cloned human embryonal carcinoma cell line, NEC 14, was induced with the addition of N,N'-hexamethylene bisacetamide (10(-2) M) for 3 days in vitro. Once differentiated, the NEC 14 cells temporarily lost both their proliferative capacity in vitro and their tumorigenic potential in the nude mouse. When culture was continued, the differentiated derivatives occasionally began to proliferate 4 weeks after the removal of the inducer, but the loss of tumorigenic potential was not recovered. Besides morphology and loss of proliferative and tumorigenic potential, the differentiated derivatives expressed both differentiation markers, HLA antigens, and intermediate filaments. This system is useful in the selection of differentiation modifiers in cancer therapy.

Acetamides↗
PubMedDate not supplied

Molecular analysis of an ataxia telangiectasia T-cell clone with a chromosomal translocation t(14;18)--evidence for a breakpoint in the T-cell receptor delta-chain gene.

We established a clonal T-cell line with a reciprocal chromosomal translocation t(14;18)(q11;q23) from a patient with ataxia telangiectasia (AT) and T-cell chronic lymphocytic leukemia (T-CLL). The tumor cells and the derived T-cell line were compared with respect to phenotype, karyotype, and rearrangement pattern. Restriction fragment analyses of the T-cell receptor (TCR)-delta gene, which is located within the TCR-alpha gene on chromosome 14q11, indicated that the breakpoint is located within the TCR-delta locus, splitting the TCR-delta gene between the variable and joining segments. This specific chromosomal translocation was only detected in the derived T-cell line and may be involved in the genesis of T-cell malignancies in AT.

Ataxia Telangiectasia↗
PubMed2005

Expression profiles of mouse dendritic cell sarcoma are similar to those of hematopoietic stem cells or progenitors by clustering and principal component analyses.

We isolated and screened two tumor cell clones DD1 and DG6 with different capacity of metastasis from the same parent cell line, a mouse dendritic cell (DC) sarcoma, using limited dilution method. The genome-wide expressions of DD1 and DG6 cells were detected by Affymetrix's MOE-430A microarray. The expression profiles related with mouse DC development were downloaded from GEO at NCBI and ArrayExpress at EBI database. In order to compare the expression of DC sarcoma and DC developmental arrays which was performed by MG-U74av2, we had screened the best matched probesets between MOE-430A and MG-U74av2 according to the probe identities from Affymetrix technical annotation. After the normalization of 11 housekeeping genes across the 34 arrays (2 DC sarcoma and 32 DC developmental arrays), all these expression profiles were analyzed by the methods of hierarchical clustering, principal component analysis, nearest-neighborhood, and self-organizing maps. The results indicate that expression profiles of DC sarcoma are closer to those of the DC progenitors and hematopoietic stem cells from bone marrow compared with the sorted DCs from spleen. The results support the hypothesis that cancers (tumors or sarcomas) arise from stem cells. It is suggested that the DC sarcomas are more similar to the DC progenitors and hematopoietic stem cells than the relative mature DCs in gene expressions on the large-scale.

Animals↗
PubMed2006

Deconstructing the molecular portrait of basal-like breast cancer.

Gene-expression profiling has revealed several molecular subtypes of breast cancer, which differ in their pathobiology and clinical outcomes. Basal-like tumors are a newly recognized subtype of breast cancer, which express genes that are characteristic of basal epithelial cells, such as the basal cytokeratins, and are associated with poor relapse-free and overall survival. However, the genetic and epigenetic alterations that are responsible for the biologically aggressive phenotype of these estrogen receptor-negative and HER2/ErbB2-negative tumors are not well understood, thereby hindering efforts to develop targeted therapies. Here, we focus on new insights into the molecular pathogenesis of basal-like breast cancer and explore how these discoveries might impact the treatment of these poor-prognosis tumors.

BRCA1 Protein↗
PubMed2003

Formation of neurospheres from human embryonal carcinoma stem cells.

Embryonal carcinoma (EC) stem cells derived from germ cell tumours are valuable tools for the study of embryogenesis and closely resemble embryonic stem cells. When human TERA2.cl.SP12 EC cells are exposed to retinoic acid and grown as adherent monolayers, approximately 10-15% of cells commit toward becoming neurons whilst the remainder of cells produce non-neuronal cell types. Using established protocols it is possible to isolate and purify neurons from these cultures but such a process takes several weeks and the numbers of neurons produced are relatively low. In this study, we describe the development of novel procedures to enhance neuronal productivity with dramatically increased efficiency, which will be of value for research purposes and drug discovery programmes.

Antigens, Surface↗
PubMed2001

Progenitor cells from patients with advanced phase chronic myeloid leukaemia respond to STI571 in vitro and in vivo.

STI571 targets p210(BCR-ABL) in chronic myeloid leukaemia (CML). In vitro, STI571 reduces self-replication (replating ability) by chronic-phase CML CFU-GM. Here, we studied CFU-GM in advanced-phase (accelerated and blast crisis) CML. The numbers and self-replication of CFU-GM in advanced phase were greater than in the chronic phase. Self-replication by CFU-GM from advanced phase patients was reduced by STI571 or IFN alfa to the same extent as in the chronic phase. The reduced replating ability induced by STI571 correlated with that induced by IFN alpha (r=0.73). STI571 treatment in vivo also reduced replating ability and the numbers of CFU-GM/ml of blood.

Antineoplastic Agents↗
PubMed2001

Human pluripotent stem cells: a progress report.

The derivation of diploid human pluripotent stem cell lines from either human blastocysts or embryonic gonads in 1998 attracted a great deal of interest because of the widespread potential applications of these cells in research and in regenerative medicine. Since the initial reports, there has been some progress in the characterisation of blastocyst-derived stem cells, and some technical advances in their manipulation. Conditions for differentiation in vitro of pluripotent stem cells from either blastocysts or gonads have been defined. In some studies, committed progenitor cell populations have been isolated from mixed cultures of differentiating ES cells.

Bioethics↗
PubMed2005

Using the chorions of fertilized zebrafish eggs as a biomaterial for the attachment and differentiation of mouse stem cells.

The development of proper biomaterials is critical for the success of cell therapy and modern tissue engineering. Here, we extruded the yolk and remaining inner mass from fertilized zebrafish eggs and used the resulting chorions as a biomaterial for the differentiation and attachment of mouse P19 embryonic carcinoma (EC) cells. Cells inserted into the chorion showed the spontaneous formation of embryoid body due to the repulsive cell adhesion of the chorion and differentiated specifically into neural cells and cardiomyocytes. In contrast, dissolved chorion extracellular matrix (ECM) conferred enhanced cell attachment on it, suggesting that a unique property of the zebrafish chorion with nanoporous structure appears to be responsible for the simple and controllable embryoid formation for stem cell differentiation. These results indicate that chorions from fertilized zebrafish eggs may be used as an extracellular matrix alternative and applied for stem cell differentiation to specific cell lineages.

Animals↗
PubMed2006

SUMOylation of Tr2 orphan receptor involves Pml and fine-tunes Oct4 expression in stem cells.

The Tr2 orphan nuclear receptor can be SUMOylated, resulting in the replacement of coregulators recruited to the regulatory region of its endogenous target gene, Oct4. UnSUMOylated Tr2 activates Oct4, enhancing embryonal carcinoma-cell proliferation, and is localized to the promyelocytic leukemia (Pml) nuclear bodies. When its abundance is elevated, Tr2 is SUMOylated at Lys238 and seems to be released from the nuclear bodies to act as a repressor. SUMOylation of Tr2 induces an exchange of its coregulators: corepressor Rip140 replaces coactivator Pcaf, which switches Tr2 from an activator to a repressor. This involves dynamic partitioning of Tr2 into Pml-containing and Pml-free pools. These results support a model where SUMOylation-dependent partitioning and differential coregulator recruitment contribute to the maintenance of a homeostatic supply of activating, as opposed to repressive, Tr2, thus fine-tuning Oct4 expression and regulating stem-cell proliferation.

Animals↗
PubMed2006

Overexpression of Aurora-A kinase promotes tumor cell proliferation and inhibits apoptosis in esophageal squamous cell carcinoma cell line.

Aurora-A kinase, a serine/threonine protein kinase, is a potential oncogene. Amplification and overexpression of Aurora-A have been found in several types of human tumors, including esophageal squamous cell carcinoma (ESCC). It has been demonstrated that cells overexpressing Aurora-A are more resistant to cisplatin-induced apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. In this report, we showed that overexpression of Aurora-A through stable transfection of pEGFP-Aurora-A in human ESCC KYSE150 cells significantly promoted cell proliferation and inhibited cisplatin- or UV irradiation-induced apoptosis. Cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP) in Aurora-A overexpressing cells were substantially reduced after cisplatin or UV treatment. Furthermore, we found that silencing of endogenous Aurora-A kinase with siRNA substantially enhanced sensitivity to cisplatin- or UV-induced apoptosis in human ESCC EC9706 cells. In parallel, overexpression of Aurora-A potently upregulated the expression of Bcl-2. Moreover, the knockdown of Bcl-2 by siRNA abrogated the Aurora-A's effect on inhibiting apoptosis. Taken together, these data provide evidence that Aurora-A overexpression promoting cell proliferation and inhibiting apoptosis, suggesting a novel mechanism that is closely related to malignant phenotype and anti-cancer drugs resistance of ESCC cells.

Apoptosis↗