Characterization of NTera2/D1 cells as a model system for the investigation of cannabinoid function in human neurons and astrocytes.

The limited availability and potential to culture primary human brain cells means that there is still a need for cell lines that reliably model human neurons and glial cells. The human-derived NTera2/D1 (NT2) cell line is a promising tool from which both neuronal (NT2N) and astrocytic (NT2A) cells can be derived in vitro. Here we have investigated the potential to use this cell model to investigate the endocannabinoid system in the CNS. Through immunocytochemical characterization with a range of neuronal and glial markers, we found that these cell lines differentiate into cells with immature neuronal and astrocytic phenotypes, respectively. By real-time PCR, immunocytochemistry, and functional inhibition of cAMP accumulation, the cannabinoid 1 receptors were identified only on NT2N cells, consistent with high levels of expression of this receptor in neuronal cells of the CNS. No evidence of cannabinoid 2 receptor expression was found on any of the NT2 cell types. Both the precursors and the differentiated NT2N and NT2A cells demonstrated mRNA expression for the key enzymes involved in endocannabinoid synthesis and degradation. This work establishes a cannabinergic phenotype in NT2N and NT2A cells, providing an alternative human derived renewable cell model for investigation of cannabinoid receptor function and endocannabinoid synthesis and metabolism in the CNS.

Global mRNA analysis to determine a transcriptome profile of cancer stemness in a mouse model.

BACKGROUND: Striking similarities between stem cells and cancer cells have led to the concept of the existence of a cancer stem cell, a concept that has since been documented in many tumours including breast, brain and prostate tumours. Teratocarcinomas are malignant tumours occurring predominantly in the testes composed of undifferentiated stem cells and mature tissues. Cancer stemness was studied using the teratocarcinoma model of tumourigenesis. MATERIALS AND METHODS: The gene expression profile of murine embryonic stem cell lines was compared to its malignant counterpart, murine teratocarcinoma cell lines. Validation was performed using real-time quantitative PCR. RESULTS: A list of 1170 differentially expressed genes was obtained. Significant pathways involved in cancer stemness included oxidative stress and angiogenesis. Transcription factors and extracellular matrix molecules appeared prominently. CONCLUSION: Novel molecules have been highlighted including decorin, an extracellular matrix protein, which may provide opportunities for the investigation of innovative strategies in the future treatment of cancer.

Pharyngeal teratocarcinosarcoma: review of the literature and report of two cases.

Teratocarcinosarcomas are rare malignant neoplasms histologically characterized by the presence of benign and malignant epithelial and mesenchymal elements. They are seen almost exclusively in the sinonasal tract of men. We report two cases of teratocarcinosarcomas involving the posterior pharyngeal wall in a 55-year-old male and 60-year-old men. The tumors consisted of epithelial components including squamous, neuroendocrine, and glandular structures; neuroepithelium, and mesenchymal components with prominent rhabdomyoblastic, osteoblastic and chondroid differentiation. Immunohistochemical studies demonstrated markers characteristic of each component. The tumors were resected, and the patients received postoperative radiation therapy. One patient is alive with recurrent tumor 33 months after treatment and the other died 26 months after radiation therapy with distant metastasis.

Identification and partial characterization of soluble and membrane-bound KDN(deaminoneuraminic acid)-glycoproteins in human ovarian teratocarcinoma PA-1, and enhanced expression of free and bound KDN in cells cultured in mannose-rich media.

KDN (Deaminoneuraminic acid, or deaminated neuraminic acid) is a minor but biosynthetically independent member of the sialic acid. Human occurrence of KDN has already been established, although its level is so little that it is often undetectable by conventional sialic acid analysis. Elevated expression of KDN in fetal cord blood cells and some malignant tumor cells have been reported. However, in mammalian cells and tissues KDN mostly occurs as the free sugar and little occurred conjugated to glycolipids and/or glycoproteins. A positive correlation between the ratio of free KDN/free Neu5Ac in ovarian adenocarcinomas and the stage of malignancy has been noted for diagnostic use. We hypothesized that elevated expression of KDN in mammalian systems may be closely related to elevated activities of enzymes involved in the formation of sialoglycoconjugates and/or aberrant supply of the precursor sugar, mannose, used in the biosynthesis of KDN. In this study we used human ovarian teratocarcinoma cells PA-1 to further analyze KDN expression in human cells. Major findings reported in this paper are, (i) a 30 kDa KDN-glycoprotein immunostainable with monoclonal antibody, mAb.kdn3G, (specific for the KDNalpha2 --> 3Galbeta1--> epitope) and sensitive to KDNase was identified in the membrane fraction of the cell: (ii) a 49 kDa KDN-glycoprotein that is not reactive with mAb.kdn3G but is sensitive to KDNase was identified in the soluble fraction: and (iii) PA-1 cells showed unique response to mannose added to the growth medium in that the levels of both free and bound forms of KDN are elevated. This is the first report on the identification of mammalian KDN-glycoproteins by chemical and biochemical methods.

Fate of embryonal carcinoma cells injected into postimplantation mouse embryos.

Embryonal carcinoma (EC) cells, stem cells of teratocarcinoma, represent an excellent model to study the developmental mechanisms that, inappropriately reactivated, can drive tumorigenesis. EC cells are very aggressive, and grow rapidly when injected into adult syngeneic mice. However, when injected into blastocysts, they revert to normality, giving rise to chimeric animals. In order to study the ability of postimplantation embryonic environment to "normalize" tumorigenic cells, and to study their homing, we transplanted F9, Nulli-SCC1, and P19 EC cells into 8 to 15-day allogenic CD1 mouse embryos, into allogenic CD1 newborns, and into syngeneic adult mice, and evaluated tumor formation, spreading, and homing. We found that, although at all embryonic stages successful transplantation occurred, the chances of developing tumors after birth increased with the time of injection of EC cells into the embryo. In addition, using enhanced green fluorescent protein-expressing F9 cells, we demonstrated that the cells not giving rise to tumors remained latent and could be tracked down in tissues during adulthood. Our data indicate that the embryonic environment retains a certain ability to "normalize" tumor cells also during post-implantation development. This could occur through yet unknown epigenetic signals triggering EC cells' differentiation.

Case report: Sinonasal teratocarcinosarcoma.

Sinonasal teratocarcinosarcoma (SNTCS) is a rare, teratoma-like lesion of the nasal cavity and paranasal sinus. To the best of our knowledge, SNTCS is highly malignant. We report a case of SNTCS arising in the nasal cavity of a 71-year-old male who complained of nasal obstruction and epistaxis. In spite of intensive treatment, the tumor recurred three times and the patient died from a local extension into the anterior cranial fossa 7 years after initial onset. The resected tumors consisted of variegated components, such as epithelial elements, including cystic, ductal and glandular structures occasionally associated with squamous differentiation, neuroectodermal ones exhibiting neural rosette formation, and mesenchymal ones with prominent rhabdomyoblastic differentiation. Immunohistochemical and ultrastructural studies clearly demonstrated characteristic cellular differentiation of each component. These three principal elements were often topographically related and showed morphological transition with each other. These findings suggest the derivation of divergent components from common progenitor cells. Although the cellular atypia of the primary lesion was inconspicuous, the recurrent tumors became anaplastic and mitotically active. Histologic anaplasia may be somewhat related with aggressiveness of recurrent lesions. Appropriate sampling of specimens, and awareness of this rare teratoid tumor are important to make the correct diagnosis.

IL-2 plasmid therapy of murine ovarian carcinoma inhibits the growth of tumor ascites and alters its cytokine profile.

We have evaluated whether i.p. murine ovarian tumors could be treated with an IL-2 plasmid DNA complexed with the cationic lipid, (+/-)-N-(2-hydroxyethyl)-N,N-dimethyl-2, 3-bis(tetradecyloxy)-1-propanaminium bromide/dioleoylphosphatidylethanolamine (DMRIE/DOPE). Reporter gene studies were initially conducted in which mice bearing i.p. murine ovarian teratocarcinoma (MOT) were injected i.p. with reporter gene plasmid DNA (pDNA):DMRIE/DOPE. Histochemical analyses revealed that transfection occurred primarily in the tumor cells of the ascites, with only a minority of other ascitic cells or surrounding tissues transfected. IL-2 levels in the MOT ascites were determined after i. p. injection of either IL-2 pDNA:DMRIE/DOPE or recombinant IL-2 protein. IL-2 was detected in tumor ascites for up to 10 days after a single i.p. injection of IL-2 pDNA:DMRIE/DOPE, but was undetectable 24 h after a single i.p. injection of IL-2 protein. In an antitumor efficacy study, MOT tumor-bearing mice injected i.p. with IL-2 pDNA:DMRIE/DOPE on days 5, 8, and 11 after tumor cell implant had a significant inhibition of tumor ascites (p = 0.001) as well as a significant increase in survival (p = 0.008). A cytokine profile of the MOT tumor ascites revealed that mice treated with IL-2 pDNA:DMRIE/DOPE had an IL-2-specific increase in the levels of IFN-gamma and GM-CSF. Taken together, these findings indicate that i. p. treatment of ovarian tumors with IL-2 pDNA:DMRIE/DOPE can lead to an increase in local IL-2 levels, a change in the cytokine profile of the tumor ascites, and a significant antitumor effect.

GABA(A) receptors expressed in undifferentiated human teratocarcinoma NT2 cells differ from those expressed by differentiated NT2-N cells.

During CNS development, changes occur in expression of GABA(A) receptor subunit subtypes and GABA(A) receptor pharmacological and biophysical properties. We used reverse transcription PCR and whole-cell-recording techniques to determine whether GABA(A) receptor expression and function also changed during retinoic acid-induced differentiation of human Ntera 2 (NT2) teratocarcinoma cells into neuron-like cells (NT2-N cells). In undifferentiated NT2 cells only alpha5, beta3, gamma3, and pi subtype mRNAs were detected. NT2 GABA(A) receptor currents had a maximal amplitude of 52 pA and an EC(50) of 4.0 microM, were relatively insensitive to enhancement by zolpidem and diazepam, and were enhanced by loreclezole and inhibited by lanthanum, zinc, and furosemide. In contrast, in NT2-N cells after 13 weeks of retinoic acid treatment, all GABA(A) receptor subtype mRNAs were detected. Maximal peak whole-cell currents were approximately 50-fold larger than NT2 cell currents, and the GABA EC(50) was higher (39.7 microM). In 13 week NT2-N cells, diazepam, zolpidem, loreclezole, and lanthanum had only small effects on GABA(A) receptor currents, and the zinc IC(50) for current inhibition was significantly higher than that for NT2 cells. In a previous study, we showed that NT2-N cells after 5 weeks of retinoic acid treatment had moderate peak currents, GABA EC(50,) and zinc IC(50) but that currents were robustly enhanced by diazepam, zolpidem, and loreclezole. During differentiation of NT2 cells to NT2-N cells, GABA(A) receptors underwent changes in subunit expression and pharmacology that were similar to many of the developmental changes in GABA(A) receptors that occur in CNS neurons.

Cyanine fluorochrome-labeled antibodies in vivo: assessment of tumor imaging using Cy3, Cy5, Cy5.5, and Cy7.

Monoclonal antibodies to two different targetable antigens were conjugated to each of four commercially available cyanine fluorochromes. Equal amounts of all four antibodies were coinjected into tumor-bearing animals and imaged. Small, superficial tumors were adequately labeled using all four fluorochromes. Large tumors were labeled well only by Cy7, probably due to self-masking and dilution effects. Cy7 was superior to other cyanine fluorochromes for visualizing structures located deep within the animal.

Complement-regulatory proteins in ovarian malignancies.

Ovarian cancer has features that makes it well-suited for MAb adjuvant immunotherapy. Several of the MAbs used in clinical trials mediate cancer cell destruction by activation of complement (C). In this study, therefore, we examined the ability of ovarian-tumor cells to resist C attack. We found that the C regulators membrane cofactor protein (MCP, CD46) and protectin (CD59) were strongly expressed in the tumor cells in all 28 benign and malignant tumors examined. Decay-accelerating factor (DAF; CD55) was more heterogeneously expressed, and only 75% of the tumors exhibited a moderate amount of DAF in the tumor cells. In adenoma cells, CD59 and DAF were preferentially located apically, while in adenocarcinoma cells they were expressed also at the basolateral cell surface. The ovarian-carcinoma cell lines SK-OV-3, Caov-3, SW626 and PA-1 expressed both the 58- and the 68-kDa isoforms of MCP. DAF was present as a glycosyl-phosphatidylinositol(GPI)-anchored 70-kDa glycoprotein. The surface-expression level of DAF varied, and correlated with the vulnerability of the cells to C-mediated lysis. CD59 was expressed as a GPI-linked 19- to 25-kDa protein exhibiting multiple glycosylation variants. The surface expression of CD59 correlated with the amount of the main 1.9 + 2.1-kb CD59 mRNA transcripts. Neutralization of CD59 with an anti-CD59 MAb significantly enhanced C-mediated killing of the cell lines. Low expression of C regulators on the PA-1 teratocarcinoma cell line was associated with high sensitivity to C lysis. Thus, the expression of C regulators on malignant ovarian cells may constitute a tumor escape mechanism, and is a critical parameter to be examined when MAb therapy is being considered.

Cytoplasmic ribonucleoprotein complexes containing human LINE-1 protein and RNA.

P40 is the protein encoded by the first open reading frame (ORF1) of the human LINE-1 (L1Hs) retrotransposon; it is 338 amino acids long, has a leucine zipper motif and has been found in human teratocarcinoma cell lines and some tumor cells. In this report, we describe properties of p40 in the human teratocarcinoma cell lines NTera2D1 and 2102Ep. The results indicate that: (i) most of p40 occurs in large multimeric cytoplasmic complexes, (ii) L1Hs RNA is associated with the p40 complexes, (iii) the complexes are dissociated by ribonuclease and (iv) p40 is a novel RNA-binding protein. Cross-linking experiments with full-length and truncated p40 produced in Escherichia coli also showed that: (i) p40 itself can form a multimeric complex larger than 250 kDa, (ii) the leucine zipper motif and the region conserved among the predicted ORF1 polypeptides of mammalian LINE-1s participate in complex formation and (iii) the amino terminal region is important for the stability of complex formation. Analysis of the amino acid sequence of p40 suggests that long segments of the molecule can assume an alpha-helical configuration including the leucine zipper and the conserved region. The evidence presented here suggests that the p40 complex is a ribonucleoprotein complex containing L1Hs RNA(s) and that protein-protein interactions in which alpha-helix structures participate, for example coiled-coils, may occur in the complex.

Cloning, chromosomal localization, and tissue expression of autotaxin from human teratocarcinoma cells.

Autotaxin, a potent human tumor cell motility-stimulating exophosphodiesterase, was isolated and cloned from the human teratocarcinoma cell line NTera2D1. The deduced amino acid sequence for the teratocarcinoma autotaxin has 94% identity to the melanoma-derived protein, 90% identity to rat brain phosphodiesterase I/nucleotide pyrophosphatase (PD-I alpha), and 44% identity to the plasma cell membrane marker PC-I. Utilizing polymerase chain reaction screening of the CEPH YAC library, we localized the autotaxin gene to human chromosome 8q23-24. Northern blot analysis of relative mRNA from multiple human tissues revealed that autotaxin mRNA steady state expression is most abundant in brain, placenta, ovary, and small intestine.

Expression of Cdx-2 in the mouse embryo and placenta: possible role in patterning of the extra-embryonic membranes.

Three mouse homologues of the Drosophila homeotic gene Caudal (Cad) have been described. They are currently designated Cdx-1, Cdx-2, and Cdx-4. Cdx-1 and 2 are both strongly expressed in the adult mid- and hindgut, while Cdx-1 and 4 have been shown to be activated in the embryonic primitive streak. Using a polyclonal antibody against a fusion protein containing the amino terminal 109 amino acids of murine Cdx-2, we here describe the topographical location of the gene product from early cleavage to 12.5 days of embryonic development. Cdx-2 expression begins at 3.5 days and is confined to the trophectoderm, being absent from the inner cell mass. Subsequently, staining is located in the extra-embryonic ectoderm adjacent to the epiblast, but sparing the more superficially placed polar, as well as the mural trophoblastic cells. Continuing expression in the fetal membranes involves the chorion, the allantoic bud, and, at even later stages, the spongiotrophoblast. From 8.5 days, Cdx-2 begins to be expressed in embryonic tissues, principally (unlike Cdx-1) in the posterior part of the gut from its earliest formation, as well as in the tail bud and in the caudal part of the neural tube. Cdx-2 is, therefore, transcribed well before any other membrane of the Cad homologue group and of the related Hox-C group; its expression in the extra-embryonic membranes and in the hindgut reflects the phylogenetic relationship between the cloaca and the chorio-allantois and suggests the possibility that homeobox genes may be involved in placental development and/or patterning.

Expression of syndecan-1 changes during the differentiation of visceral and parietal endoderm from murine F9 teratocarcinoma cells.

F9 teratocarcinoma stem cells treated with retinoic acid differentiate in suspension into embryoid bodies with an outer layer of visceral endoderm surrounding a core of largely undifferentiated cells. The visceral endoderm-containing embryoid bodies, when plated onto an extracellular matrix coating, give rise to parietal endoderm outgrowth. These in vitro cell cultures mimic both geometrically and biochemically the differentiation of visceral and parietal endoderm in the early mouse embryo and, thus, were used as a model system for the study of molecular and cellular mechanisms underlying the differentiation of the extraembryonic endoderm lineages. We have investigated the expression of syndecan-1, an integral membrane proteoglycan that binds to multiple components of the extracellular matrix and basic FGF, during visceral endoderm differentiation and parietal endoderm outgrowth. Syndecan-1 immunostaining is detected on all cell surfaces in the undifferentiated embryoid bodies and in the differentiating embryoid bodies prior to the formation of the visceral endoderm. Following the differentiation of visceral endoderm, syndecan-1 localizes predominantly to the basal surface of this epithelial layer, while syndecan-1 staining in the core of differentiated embryoid bodies is faint. Quantitation of cell associated syndecan-1 indicates that syndecan-1 is down-regulated during embryoid body differentiation. However, northern analysis shows that the amounts of steady-state syndecan-1 mRNA are the same in undifferentiated versus differentiated embryoid bodies, suggesting post-transcriptional regulation of syndecan-1 expression in the differentiating embryoid body. Analysis of syndecan-1 distribution in the outgrowth culture by immunofluorescence demonstrates that syndecan-1 is absent from the cell surface of parietal endoderm. However, a substantial amount of syndecan-1 is detected inside parietal endoderm cells. While all three cell types release syndecan-1 ectodomain into the culture medium, the parietal endoderm outgrowth releases more syndecan-1 ectodomain than the differentiated embryoid body. These data suggest that the post-transcriptional control and post-translational shedding of syndecan-1 from the cell surface are developmentally regulated during the differentiation of visceral to parietal endoderm and the migration of parietal endoderm.

Proliferation, cell death, and neuronal differentiation in transplanted human embryonal carcinoma (NTera2) cells depend on the graft site in nude and severe combined immunodeficient mice.

BACKGROUND: Embryonal carcinoma cell lines have been used to study the induction and progression of tumors, the mechanisms governing lineage commitment in the central nervous system, and the developmental biology of neurons and glia. Here, we have used a human embryonal carcinoma cell line (NTera2/cl.D1 or NT2 cells) that resembles neural progenitor cells to study how an in vivo environment influences and regulates the fate of these cells. EXPERIMENTAL DESIGN: To understand the mechanisms that coordinately regulate the proliferation, death, and differentiation of NT2 cells, we examined these processes by transplanting human NT2 cells in the brains and peripheral tissues (liver, muscle) of immunodeficient mice. RESULTS: We demonstrate that the proliferation, differentiation, and death of NT2 cells were modulated by the anatomical site into which the NT2 grafts were implanted. The NT2 cells continued to proliferate and undergo cell death but showed a very limited capacity to differentiate into neurons after implantation into the subarachnoid space and superficial neocortex. At this site, the NT2 cell grafts rapidly formed bulky tumors that were lethal within 70 days postimplantation. Further, NT2 cell grafts in the lateral ventricles, liver, and muscle behaved in a similar manner. In contrast, NT2 cells implanted into the caudoputamen ceased proliferating and showed no evidence of necrosis or apoptosis after postimplantation survival intervals of more than 20 weeks. This occurred in parallel with the progressive differentiation of large numbers of NT2 cells into postmitotic, immature, neuron-like cells. CONCLUSIONS: These results suggest that signal molecules or other "cues" (e.g., cell-cell contacts) capable of regulating the proliferation, death, and differentiation of human NT2 cells are biologically active in the adult mouse caudoputamen. Thus, the transplantation of human NT2 cells into the central nervous system of immunodeficient mice may serve as an in vivo model system for studies of the formation and re-modeling of the developing central nervous system.

Cell surface glycoproteins: biochemical, immunological and molecular biological studies.

This article briefly summarizes the author's contribution to the study of cell surface glycoproteins. At first, much effort was devoted to developing analytical methods, especially exoglycosidases and endoglycosidases. An endo-beta-N-acetylglucosaminidase found in Streptococcus pneumoniae was the first example of an endoglycosidase acting on glycoproteins. A combination of radioactive labeling, glycosidase digestion and lectin affinity chromatography enabled the characterization of carbohydrate moieties of cell surface glycoproteins derived from cultured cells. Application of the method to teratocarcinoma stem cells and preimplantation mouse embryos led to the discovery of marked changes in the carbohydrate moieties of cell surface glycoproteins during embryogenesis. Combining biochemical and immunohistochemical methods, an overall picture was obtained for carbohydrate changes in early embryogenesis of the mouse, and molecular biological approaches have been adopted to determine their biological significance. Furthermore, some core proteins carrying developmentally regulated carbohydrate markers were found and characterized by molecular cloning. One example of such a protein is embigin, which enhances integrin-mediated cell-substratum adhesion and established the existence of a new group in the immunoglobulin superfamily. Carbohydrate immunochemical markers useful in the analysis of mouse embryogenesis were also found to be effective in the classification of human carcinomas with respect to metastatic potential.

Developmentally regulated expression of two novel platelet-derived growth factor alpha-receptor transcripts in human teratocarcinoma cells.

Two novel platelet-derived growth factor (PDGF) alpha-receptor transcripts of 1.5 kilobases and 5.0 kilobases are expressed in the human teratocarcinoma cell line Tera-2 only while the cells are in an undifferentiated state. After retinoic acid-induced differentiation, expression of these mRNAs is completely shut off and instead, the cells express a single 6.4-kilobase mRNA species which is also expressed in many other cell types. The 1.5-kilobase mRNA initiates within intron 12, contains the correctly spliced exons 13, 14, 15, and 16, and contains a cryptic exon, designated teratocarcinoma cryptic exon, at the 3' end. Teratocarcinoma cryptic exon contains a functional polyadenylation signal. Exons 13 to 16 correspond to the first tyrosine kinase domain and to part of the interkinase domain of the PDGF alpha-receptor. Recently, a splice variant lacking exon 14 was identified. These results show that a combination of alternative promoter usage and alternative splicing of the human PDGF alpha-receptor gene occur in a developmentally regulated fashion. In vitro translation of the 1.5-kilobase mRNA generates protein products which can be specifically immunoprecipitated with a PDGF alpha-receptor-specific antibody. The significance of the expression of this transcript for the growth factor-independent proliferation of undifferentiated Tera-2 cells is unclear. Expression of PDGF alpha-receptor transcripts containing the cryptic exon may be useful as a marker for undifferentiated stem cells in human teratocarcinomas.