tretinoin and Teratocarcinoma

To be maximally effective, therapy of cancer must be directed against both the resting stem cells and the proliferating cells of the cancer. The cell populations of both normal and cancer tissues consist of resting stem cells, proliferating transit-amplifying cells, terminally differentiating cells and dying (apoptotic) cells. The difference between normal tissue renewal and growth of cancers is that some of the transit-amplifying cells in the cancer population do not mature into terminally differentiating cells, but instead continue to proliferate and do not die (maturation arrest). Because of this the number of cancer cells increase, whereas the cell population of normal tissues remains a relatively constant. Conventional radiation treatment and chemotherapy kill the actively proliferating transit- amplifying cells of the cancer. Differentiation therapy, using specific targeted inhibitors of activation, effectively induces differentiation of the proliferating transit-amplifying cancer cells. However, even if the proliferating cancer cells are completely inhibited or eliminated, the cancer stem cells may restore the transit-amplifying population, so that clinical remission is usually temporary. The hypothesis presented in this paper is that successful cancer therapy must be directed against both the resting stem cells and the proliferating cells of the cancer. This may be possible if specific stem cell signals are inhibited using gene therapy, while at the same time attacking proliferating cells by conventional radiation treatment or chemotherapy. With advances in approaches using specific inhibitory RNA, such combination therapy may now be possible, but critical problems in delivering the inhibitory effect specifically to the cancer stem cells have yet to be worked out.

Topics: Animals; Breast Neoplasms; Cell Differentiation; Cell Lineage; Cell Proliferation; Embryonal Carcinoma Stem Cells; Female; Genetic Therapy; Humans; Leukemia; Models, Biological; Neoplasms; Neoplastic Stem Cells; RNA Interference; Stem Cells; Teratocarcinoma; Tretinoin

Cell therapy is a rapidly moving field with new cells, cell lines, and tissue-engineered constructs being developed globally. As these novel cells are further developed for transplantation studies, it is important to understand their safety profiles both prior to and posttransplantation in animals and humans. Embryonic carcinoma-derived cells are considered an important alternative to stem cells. The NTera2/D1 teratocarcinoma cell-line (or NT2-N cells) gives rise to neuron-like cells called hNT neurons after exposure to retinoic acid. NT2 cells form tumors upon transplantation into the rodent. However, when the NT2 cells are treated with retinoic acid to produce hNT cells, they terminally differentiate into post-mitotic neurons with no sign of tumorigenicity. Preliminary human transplantation studies in the brain of stroke patients also demonstrated a lack of tumorigenicity of these cells. This review focuses on the use of hNT neurons in cell transplantation for the treatment in central nervous system (CNS) diseases, disorders, or injuries and on the mechanism involved in retinoic acid exposure, final differentiation state, and subsequent tumorigenicity issues that must be considered prior to widespread clinical use.

Topics: Cell Culture Techniques; Cell Line, Tumor; Cell Transplantation; Clinical Trials as Topic; Embryonal Carcinoma Stem Cells; Humans; Neoplastic Stem Cells; Neurons; Surgical Procedures, Operative; Teratocarcinoma; Tretinoin

The human cytomegalovirus (HCMV) major immediate-early (MIE) gene is not transcribed in undifferentiated NTera-2 embryonal carcinoma cells, but is transcribed in their differentiated derivatives, offering a model with which to study the developmental regulation of the activity of a viral gene during the differentiation of these cells. The molecular mechanisms involved in the blockade of the MIE gene expression in undifferentiated NTera2 cells include covalent closure of the circular conformation of the viral genome, silencing of the viral MIE promoter by histone deacetylation, and increases in the expression of negatively regulating transcription factors responsible for the recruitment of the histone deacytylases around the viral MIE promoter (MIEP), resulting in repression of the MIEP in undifferentiated cells. The treatment of NTera2 cells with retinoic acid induces the differentiation of these cells. In HCMV-infected differentiated NTera2 cells, the MIEP becomes associated with hyperacetylated histones, which results in an open structure of chromatin, enhancing the access of DNA-binding factors which positively regulate MIE gene expression and viral replication. This model system contributes to an understanding of HCMV latency and reactivation in vivo in the cells of the myeloid lineage.

Topics: Antigens, Viral; Cell Differentiation; Cytomegalovirus; Gene Expression Regulation, Viral; Genes, Immediate-Early; Humans; Immediate-Early Proteins; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured; Virus Activation; Virus Latency

The NT2N cell system offers an attractive way to overcome some of the technical limitations inherent in working with primary neuronal cultures. In particular, it is possible to obtain large quantities of neurons with which to perform biochemical experiments, and the growth of neurites can be synchronized and controlled by varying the substrate on which the cells grow. In addition, because the differentiated NT2N neurons are derived from a mitotically active precursor cell line in vitro, it is possible to employ a variety of techniques, that are not otherwise available when working directly with postmitotic neurons, to obtain expression of foreign proteins. Because they are fully polarized, NT2N cells offer a way to study protein sorting to axons and dendrites at both the biochemical and the morphological level. Further characterization of NT2N cells is underway, and more efficient ways to obtain expression of foreign proteins will no doubt be found.

Topics: Base Sequence; Cell Compartmentation; Cell Differentiation; Cell Polarity; Culture Techniques; Gene Expression; Gene Transfer Techniques; Genetic Vectors; Humans; Molecular Sequence Data; Neurons; Recombinant Fusion Proteins; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured; Vaccinia virus

Hox genes have been shown to be important regulators of pattern formation in vertebrates. Retinoic acid has been shown to affect the expression of Hox genes in vitro and in vivo, and some of its effects on development correspond to changes in Hox gene expression. The idea that retinoic acid is not simply a powerful pharmocological agent, but rather that it plays an important role in creating the normal expression patterns of Hox genes, is provided by the recent identification of retinoic acid responsive enhancers near Hox genes.

Topics: Animals; Embryonic Induction; Enhancer Elements, Genetic; Gene Expression Regulation, Developmental; Genes, Homeobox; Humans; Nervous System; Receptors, Retinoic Acid; Teratocarcinoma; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

The lack of a convenient, easily maintained, and inexpensive in vitro human neuronal model to study neurodegenerative diseases prompted us to develop a rapid, 1-h differentiated neuronal cell model based on human NT2 cells and C3 transferase. Here, we describe the rapid differentiation of human neuronal NT2 cells, and the differentiation, transduction, and transfection of human SK-N-MC cells and rat PC12 cells to obtain cells with the morphology of differentiated neurons that can express exogenous genes of interest at high level.

Topics: ADP Ribose Transferases; Adrenal Gland Neoplasms; Animals; Botulinum Toxins; Cell Culture Techniques; Humans; Neuroblastoma; Neurogenesis; Neuronal Outgrowth; Neurons; PC12 Cells; Phenotype; Pheochromocytoma; Rats; Teratocarcinoma; Transfection; Tretinoin

This chapter describes the culture and propagation of murine embryonic stem cells, F9 and P19, and strategies for differentiation of these stem cells into neurons. Additional techniques are described for obtaining enriched populations of mature neurons from P19 cells and differentiation of F9 cells into serotonergic or catecholaminergic neurons. The protocols described herein can be used for dissection of the pathways such as gliogenesis and neurogenesis that are involved in differentiation of pluripotent stem cells such as F9 and P19 into glial cells or terminally differentiated neurons.

Topics: Animals; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Mice; Mouse Embryonic Stem Cells; Neural Stem Cells; Neurogenesis; Neurons; Phenotype; Teratocarcinoma; Tretinoin

Activation of peroxisome proliferator-activated receptor alpha (PPARα) has been reported to modulate cell proliferation, migration, and differentiation in astrocytes. In this study, we used a retinoic acid (RA)-induced differentiation model of NTERA-2/clone D1 (NT2) cells to explore the functional significance of PPARα in neuronal differentiation. We found that activating PPARα by Wy14643 accelerated neuronal differentiation via regulating the expression of neuronal markers. RT-PCR assays showed a significant increase in NeuroD expression and a decrease in nestin expression in cells treated concomitantly with RA and Wy14643 for 2 days compared to the levels in cells treated with RA alone. Expression of MAP2 protein, a mature neuronal marker, was markedly upregulated at day 10 of Wy14643 treatment, which was maintained after 21 days of neuronal formation. Corresponding to the changes in MAP2 expression, the expression of Cdk5 was upregulated with Wy14643 exposure from day 10 to day 21. Moreover, cells treated with Wy14643 displayed higher expression levels of phospho-ERK and phospho-p38 in the differentiation process than cell treated with RA alone. These results indicated that activation of PPARα accelerated neuronal differentiation through upregulating the expression of NeuroD, MAP2, and Cdk5 and downregulating the expression of nestin. MAPK signals, ERK and p38, might contribute to the accelerated differentiation process. These findings suggest that PPARα plays a role in regulating neuronal differentiation and may be beneficial for functional recovery from neurological disorders.

Topics: Analysis of Variance; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cyclin-Dependent Kinase 5; Gene Expression Regulation, Neoplastic; Humans; Microtubule-Associated Proteins; Nestin; Neurons; PPAR alpha; Prostaglandins E, Synthetic; Signal Transduction; Teratocarcinoma; Time Factors; Tretinoin

Mouse F9 cells differentiate into primitive endoderm (PrE) following the activation of the canonical WNT-β-catenin pathway. The upregulation of Wnt6 and activation of β-catenin-TCF-LEF-dependent transcription is known to accompany differentiation, but the Frizzled (FZD) receptor responsible for transducing the WNT6 signal is not known. Eight of the 10 Fzd genes were found to be expressed in F9 cells, with Fzd7 being the most highly expressed, and chosen for further analysis. To alter steady-state Fzd7 levels and test the effect this has on differentiation, siRNA and overexpression approaches were used to knock-down and ectopically express the Fzd7 message, respectively. siRNA knock-down of Fzd7 resulted in reduced DAB2 levels, and the overexpression activated a TCF-LEF reporter, but neither approach affected differentiation. Our focus turned to how canonical WNT6 signaling was attenuated to allow PrE cells to form parietal endoderm (PE). Dkk1, encoding a WNT antagonist, was examined and results showed that its expression increased in F9 cells treated with retinoic acid (RA) or overexpressing Wnt6. F9 cells overexpressing human DKK1 or treated with DKK1-conditioned medium and then treated with RA failed to differentiate, indicating that a negative feedback loop involving WNT6 and DKK1 attenuates canonical WNT-β-catenin signaling, thereby allowing PE cells to differentiate.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Apoptosis Regulatory Proteins; beta Catenin; Cell Differentiation; Cell Line, Tumor; Culture Media, Conditioned; Endoderm; Feedback, Physiological; Frizzled Receptors; Gene Expression Regulation, Neoplastic; Genes, Reporter; Hepatocyte Nuclear Factor 1-alpha; Intercellular Signaling Peptides and Proteins; Lymphoid Enhancer-Binding Factor 1; Mice; Proto-Oncogene Proteins; Receptors, G-Protein-Coupled; RNA, Small Interfering; Signal Transduction; Teratocarcinoma; Tretinoin; Wnt Proteins

Cell differentiation is a central process in development and in cancer growth and dissemination. OCT4 (POU5F1) and NANOG are essential for cell stemness and pluripotency; yet, the mechanisms that regulate their expression remain largely unknown. Repetitive elements account for almost half of the Human Genome; still, their role in gene regulation is poorly understood. Here, we show that the dioxin receptor (AHR) leads to differentiation of human carcinoma cells through the transcriptional upregulation of Alu retrotransposons, whose RNA transcripts can repress pluripotency genes. Despite the genome-wide presence of Alu elements, we provide evidences that those located at the NANOG and OCT4 promoters bind AHR, are transcribed by RNA polymerase-III and repress NANOG and OCT4 in differentiated cells. OCT4 and NANOG repression likely involves processing of Alu-derived transcripts through the miRNA machinery involving the Microprocessor and RISC. Consistently, stable AHR knockdown led to basal undifferentiation, impaired Alus transcription and blockade of OCT4 and NANOG repression. We suggest that transcripts produced from AHR-regulated Alu retrotransposons may control the expression of stemness genes OCT4 and NANOG during differentiation of carcinoma cells. The control of discrete Alu elements by specific transcription factors may have a dynamic role in genome regulation under physiological and diseased conditions.

Topics: Alu Elements; Animals; Basic Helix-Loop-Helix Transcription Factors; Carcinoma; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Gene Expression Regulation, Neoplastic; Humans; Mice; MicroRNAs; Nanog Homeobox Protein; Octamer Transcription Factor-3; Promoter Regions, Genetic; Receptors, Aryl Hydrocarbon; RNA Polymerase III; Teratocarcinoma; Teratoma; Transcription, Genetic; Tretinoin

The proteins in the syntaxin family are known to mediate fusion of cytoplasmic vesicles to the target membrane, yet subpopulations of certain syntaxins, including syntaxin4, translocate across the cell membrane in response to external stimuli. Here, we show that extracellularly presented syntaxin4 impacts cell behavior and differentiation in teratocarcinoma F9 cells. While undifferentiated F9 cells extruded a small subpopulation of extracellular syntaxin4 at the lateral cell membrane, the induction of differentiation with all-trans retinoic acid (RA) abolished this localized expression pattern. We found that the cells that were stimulated in a non-directional fashion by extracellular syntaxin4 displayed a flattened shape and retained a substrate-bound morphology even under a long-term, serum-starved cultivation. Such a cellular response was also elicited by a circular peptide composed of the potential functional core of syntaxin4 (AIEPQK; amino acid residues 103~108) (ST4n1). While the proliferation and metabolism were not affected in these cells, cell-cell interaction became weakened and the expression of vinculin, a regulator of both intercellular and cell-substrate adhesion molecules, was altered. We also found that the expressions of several differentiation markers were up-regulated in cells stimulated with extracellular syntaxin4 and that syntaxin3, another family member, was most prominent. Intriguingly, forced expression of syntaxin3 induced the spread morphology in F9 cells, indicating that syntaxin3 partly mediates the function of extracellular syntaxin4. These results demonstrate the involvement of a non-directional stimulation of extracellular syntaxin4 in the functional and morphological differentiation of F9 cells.

Topics: Animals; Cell Adhesion; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Mice; Qa-SNARE Proteins; Teratocarcinoma; Tretinoin

Aberrant activation of the Wnt/β-catenin signaling pathway is a common event in human tumor progression. Wnt signaling has also been implicated in maintaining a variety of adult and embryonic stem cells by imposing a restraint to differentiation. To understand the function and mechanism of Wnt/β-catenin signaling on the pathogenesis of teratocarcinoma, we used the mouse teratocarcinoma P19 cell line as a model in vitro. Gsk3β specific inhibitor (SB216763) was used to activate Wnt/β-catenin signaling. All trans-retinoic acid (RA) was used to induce P19 cell differentiation. At different culture times, gene expression was examined by immunofluorescence staining, quantitative real-time PCR, and Western-blotting; BrdU incorporation assays were performed to measure P19 cell proliferation. Small interference RNA technology was used to downregulate c-myc expression. The results showed that SB216763 induced the nuclear translocation of β-catenin, upregulated the expression of c-myc and pluripotency related genes, oct4, sox2 and nanog, and blocked cell differentiation induced by all trans-RA. The proliferation of P19 cells was significantly enhanced by SB216763, as well as c-myc overexpression. C-myc downregulation inhibited P19 cell proliferation caused by activation of Wnt/β-catenin signaling and induced P19 cell differentiation. In conclusion, activation of the Wnt/β-catenin pathway could promote the proliferation and inhibit the differentiation of mouse teratocarcinoma cells by upregulation of c-myc expression.

Topics: Animals; beta Catenin; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Embryonal Carcinoma Stem Cells; Fluorescent Antibody Technique; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Homeodomain Proteins; Indoles; Maleimides; Mice; Nanog Homeobox Protein; Octamer Transcription Factor-3; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-myc; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Messenger; SOXB1 Transcription Factors; Teratocarcinoma; Time Factors; Transfection; Tretinoin; Up-Regulation; Wnt Signaling Pathway

Nitric oxide (NO) plays a key role in neurogenesis as a regulator of cell proliferation and differentiation. NO is synthesized from the amino acid L-arginine by nitric oxide synthases (NOS1, NOS2, and NOS3), which are encoded by separate genes and display different tissue distributions. We used an in vitro model of RA-induced neural differentiation of NT2 cells to examine which of the three NO-synthesizing enzymes is involved in this process. The results revealed a transient induction of NOS3 (known as the constitutively expressed endothelial nitric oxide synthase; eNOS) during the time course of the RA treatment. The peak of gene expression and the nuclear presence of NOS3 protein coincided with cell cycle exit of NT2-derived neuronal precursors. The subsequent analysis of cytosine methylation and histone H3 acetylation of the human NOS3 5' regulatory sequences indicated that epigenetic modifications, especially upstream of the proximal promoter (-734 to -989, relative to exon 2 TSS at +1), were also taking place. NOS1 was expressed only in the differentiated neurons (NT2-N), whereas NOS2 was not expressed at all in this cellular model. Thus, a burst of NO production, possibly required to inhibit neural cell proliferation, was generated by the transient expression of NOS3. This pattern of gene expression, in turn, required epigenetic remodeling of its regulatory region.

Topics: 5' Untranslated Regions; Acetylation; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Chromatin Immunoprecipitation; CpG Islands; DNA Methylation; Enzyme Induction; Gene Expression Regulation, Developmental; Histones; Humans; Nerve Tissue Proteins; Neurogenesis; Neuroglia; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Ornithine; Protein Processing, Post-Translational; RNA, Messenger; Teratocarcinoma; Tretinoin; Triazenes

Activity-dependent neuroprotective protein (ADNP) and the homologous protein ADNP2 provide cell protection. ADNP is essential for brain formation, proper brain development and neuronal plasticity, all reported to be impaired in the schizophrenia patient brains. Furthermore, reduction in ADNP expression affects social interactions, a major hallmark of schizophrenia. To evaluate a possible involvement of ADNP and ADNP2 in the pathophysiology of schizophrenia in humans, we measured relative brain mRNA transcripts of both proteins compared with control subjects. Quantitative real time polymerase chain reaction in postmortem hippocampal specimens from normal control subjects exhibited a significant ADNP to ADNP2 transcript level correlation (r=0.931, p<0.001), also apparent in a neuroglial model system. In contrast, in the hippocampus of matched schizophrenia patients, this correlation (r=0.637, p=0.014) was drastically decreased in a statistically significant manner (p=0.03), mirroring disease-associated increased ADNP2 transcripts. In the prefrontal cortex of schizophrenia patients the correlation between ADNP and ADNP2 mRNA levels was apparently higher than in the hippocampus (r=0.854, p<0.001), but did not reach a significant difference (p=0.25). Thus, imbalance in ADNP/ADNP2 expression in the brain may impact disease progression in schizophrenia.

Topics: Adult; Analysis of Variance; Animals; Brain; Cell Line, Tumor; Disease Progression; Female; Gene Expression Regulation; Homeodomain Proteins; Humans; Male; Mice; Middle Aged; Nerve Tissue Proteins; Postmortem Changes; RNA, Messenger; Schizophrenia; Statistics as Topic; Teratocarcinoma; Tretinoin

The NTERA2 cl D1 (NT2) cell line, derived from human teratocarcinoma, exhibits similar properties as embryonic stem (ES) cells or very early neuroepithelial progenitors. NT2 cells can be induced to become postmitotic central nervous system neurons (NT2N) with retinoic acid. Although neurons derived from pluripotent cells, such as NT2N, have been characterized for their neurotransmitter phenotypes, their potential suitability as a donor source for neural transplantation also depends on their ability to respond to localized environmental cues from a specific region of the CNS. Therefore, our study aimed to characterize the regional transcription factors that define the rostocaudal and dorsoventral identity of NT2N derived from a monolayer differentiation paradigm using quantitative PCR (qPCR). Purified NT2N mainly expressed both GABAergic and glutamatergic phenotypes and were electrically active but did not form functional synapses. The presence of immature astrocytes and possible radial glial cells was noted. The NT2N expressed a regional transcription factor code consistent with forebrain, hindbrain and spinal cord neural progenitors but showed minimal expression of midbrain phenotypes. In the dorsoventral plane NT2N expressed both dorsal and ventral neural progenitors. Of major interest was that even under the influence of retinoic acid, a known caudalization factor, the NT2N population maintained a rostral phenotype subpopulation which expressed cortical regional transcription factors. It is proposed that understanding the regional differentiation bias of neurons derived from pluripotent stem cells will facilitate their successful integration into existing neuronal networks within the CNS.

Topics: Cell Differentiation; Cell Line, Tumor; Humans; Induced Pluripotent Stem Cells; Neoplastic Stem Cells; Neural Stem Cells; Phenotype; Teratocarcinoma; Transcription Factors; Tretinoin; Tumor Microenvironment

Retinoids play key roles in differentiation, growth arrest, and apoptosis and are increasingly being used in the clinic for the treatment of a variety of cancers, including neuroblastoma. Here, using a large-scale RNA interference-based genetic screen, we identify ZNF423 (also known as Ebfaz, OAZ, or Zfp423) as a component critically required for retinoic acid (RA)-induced differentiation. ZNF423 associates with the RARalpha/RXRalpha nuclear receptor complex and is essential for transactivation in response to retinoids. Downregulation of ZNF423 expression by RNA interference in neuroblastoma cells results in a growth advantage and resistance to RA-induced differentiation, whereas overexpression of ZNF423 leads to growth inhibition and enhanced differentiation. Finally, we show that low ZNF423 expression is associated with poor disease outcome in neuroblastoma patients.

Topics: Adolescent; Antineoplastic Agents; Cell Differentiation; Child; Child, Preschool; Chromatin Immunoprecipitation; DNA-Binding Proteins; Female; Humans; Infant; Infant, Newborn; Male; Neuroblastoma; Prognosis; Promoter Regions, Genetic; Proteins; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Survival Rate; Teratocarcinoma; Tretinoin; Zinc Fingers

The mouse F9 teratocarcinoma cell line is a model that can be manipulated to imitate one of the earliest epithelial-mesenchymal transitions in mouse development. When cells are treated with Retinoic Acid they differentiate into primitive endoderm and into parietal endoderm with the addition of dibutyryl cAMP. Parietal endoderm also develops when undifferentiated cells express a constitutively active (CA) form of Galpha13(Q226L). Differentiation is accompanied by a translocation of beta-catenin to the nucleus and considerable changes to the cytoskeleton and cell morphology. ERM proteins facilitate rearrangements to the F-actin cytoskeleton, and at least one, moesin, is essential for cell survival. In this study we found that moesin translocated to the nucleus during RA-induced differentiation, and sequence analysis identified putative nuclear localization signals in the protein. In the absence of RA, transient over-expression of rat moesin or the distantly related zebrafish homologue in F9 cells induced primitive endoderm. Furthermore, no apparent beta-catenin was seen in the nucleus of cells over-expressing zebrafish moesin. Our previous results have shown that depleting F9 cells of moesin using an antisense morpholino strategy caused them to detach from the substrate unless they expressed CA-Galpha13(Q226L). This CA-Galpha13 signalling maintained cell survival, but at the expense of differentiation. We now report that over-expressing zebrafish moesin in mouse moesin-depleted F9 cells not only ensured cell survival, but also induced differentiation to primitive endoderm. Together, the results suggest a new role for moesin, acting in a signalling pathway facilitating the differentiation of extraembryonic endoderm.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; beta Catenin; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Endoderm; Male; Mice; Microfilament Proteins; Molecular Sequence Data; Signal Transduction; Teratocarcinoma; Testicular Neoplasms; Transfection; Tretinoin; Zebrafish

Activity-dependent neuroprotective protein (ADNP) 2 (KIAA0863; ZNF508) gene, a homeobox-profile containing gene, was identified in a screen for homologous proteins to ADNP. The human ADNP2 contains 1131 amino acid residues with a molecular weight of 122.8 KDa. In silico analysis indicated that ortholgs to ADNP2 exist in different phyla, suggesting that ADNP2 might be evolutionary conserved. Here, we began to explore the molecular and functional characterization of ADNP2. Results showed that the mouse ADNP2 mRNA is ubiquitously expressed in distinct normal tissues with increased expression in the brain, particularly in the cerebral cortex. During development, a relatively high level of ADNP2 gene expression was found in the embryonic mouse brain and was sustained throughout embryogenesis and adulthood. An increase in the mRNA was detected in differentiated P19 neuronal/glial-like cells as compared with the non-differentiated cells. To gain insight into ADNP2 function, ADNP2-deficient cell lines were established by the RNA silencing (small interfering RNA) technology. ADNP2 deficiency significantly changed the toxicity induced by hydrogen peroxide in P19 embryonic carcinoma cells, similar to what would be predicted for ADNP deficiency. These findings represent an initial characterization of ADNP2 and suggest that this gene product may have an important function in brain by playing a role in cellular survival pathways.

Topics: Amino Acid Sequence; Analysis of Variance; Animals; Animals, Newborn; Cell Differentiation; Cell Line, Tumor; Cell Survival; Embryo, Mammalian; Female; Gene Expression Regulation, Developmental; Homeodomain Proteins; Hydrogen Peroxide; Male; Mice; Nerve Tissue Proteins; Oxidative Stress; RNA, Small Interfering; Teratocarcinoma; Time Factors; Tretinoin

Rex1 (Zfp42), first identified as a gene that is transcriptionally repressed by retinoic acid (RA), encodes a zinc finger transcription factor expressed at high levels in F9 teratocarcinoma stem cells, embryonic stem cells, and other stem cells. Loss of both alleles of Rex1 by homologous recombination alters the RA-induced differentiation of F9 cells, a model of pluripotent embryonic stem cells. We identified Suppressor of Cytokine Signaling-3 (SOCS-3) as a gene that exhibits greatly increased transcriptional activation in RA, cAMP, and theophylline (RACT)-treated F9 Rex1(-/-) cells (approximately 25-fold) as compared to wild-type (WT) cells ( approximately 2.5-fold). By promoter deletion, mutation, and transient transfection analyses, we have shown that this transcriptional increase is mediated by the STAT3 DNA-binding elements located between -99 to -60 in the SOCS-3 promoter. Overexpression of STAT3 dominant-negative mutants greatly diminishes this SOCS-3 transcriptional increase in F9 Rex1(-/-) cells. This increase in SOCS-3 transcription is associated with a four- to fivefold higher level of tyrosine-phosphorylated STAT3 in the RACT-treated F9 Rex1(-/-) cells as compared to WT. Dominant-negative Src tyrosine kinase, Jak2, and protein kinase A partially reduce the transcriptional activation of the SOCS 3 gene in RACT-treated F9 Rex1 null cells. In contrast, parathyroid hormone peptide enhances the effect of RA in F9 Rex1(-/-) cells, but not in F9 WT. Thus, Rex1, which is highly expressed in stem cells, inhibits signaling via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, thereby modulating the differentiation of F9 cells.

Topics: Animals; Binding Sites; Bucladesine; Cyclic AMP-Dependent Protein Kinases; Embryonal Carcinoma Stem Cells; Gene Expression Regulation, Neoplastic; Genes, Reporter; Janus Kinase 2; Mercaptoethanol; Mice; Microarray Analysis; Neoplastic Stem Cells; Parathyroid Hormone; Peptides; Promoter Regions, Genetic; Proto-Oncogene Proteins pp60(c-src); Receptors, Retinoic Acid; RNA, Messenger; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Teratocarcinoma; Theophylline; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Tretinoin

We have shown previously that neonatal testicular gonocytes express platelet-derived growth factor receptors (PDGFR) alpha and beta. We report the expression of a novel PDGFRbeta (V1-PDGFRbeta) transcript in gonocytes of 3-d-old rat testes. V1-PDGFRbeta nucleotide sequence spans from intron 6 to exon 23 of the PDGFRbeta gene, and is predicted to encode a protein lacking part of the extracellular domain. V1-PDGFRbeta transcripts are expressed preferentially in developing gonads. The embryonic teratocarcinoma F9 cells, in which differentiation is driven by retinoic acid (RA), express V1-PDGFRbeta, but not wild-type PDGFRbeta. Green fluorescent protein-tagged V1-PDGFRbeta localized mainly in cytosol of F9, MA-10, and COS-1 cells. FLAG and green fluorescent protein-tagged V1-PDGFRbeta displayed tyrosine kinase activities and contain phosphotyrosine residues, suggesting that V1-PDGFRbeta is a cytosolic tyrosine kinase. Treatment of F9 cells with RA induced V1-PDGFRbeta gene expression, concomitant with changes in morphology and increased mRNA expression of collagen IV and laminin B1, suggesting that V1-PFGRbeta is involved in cell differentiation. Similarly, treatment of postnatal d 3 rat gonocytes with RA induced a dose-dependent increase in V1-PDGFRbeta expression together with an increase in c-kit and Stra8, markers of more differentiated germ cells and a concomitant decrease in GFRalpha1, a marker of spermatogonial stem cells. However, an excess of V1-PDGFRbeta inhibited RA-mediated collagen IV and laminin B1 expression and altered both RA-dependent and RA-independent morphological changes in F9 cells, while increasing cell survival. These results suggest that the expression of V1-PDGFRbeta is tightly regulated during differentiation and that it may play an active role in germ cell differentiation.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Survival; Chlorocebus aethiops; COS Cells; Female; Genetic Variation; Germ Cells; Glioma; Green Fluorescent Proteins; Humans; Male; Mice; Mice, Inbred BALB C; Pregnancy; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Receptor, Platelet-Derived Growth Factor beta; Teratocarcinoma; Testis; Tretinoin

The retinoid-related molecules (RRMs) ST1926 [(E)-3-(4'-hydroxy-3'-adamantylbiphenyl-4-yl)acrylic acid] and CD437 (6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid) are promising anticancer agents. We compared the retinoic acid receptor (RAR) trans-activating properties of the two RRMs and all-trans-retinoic acid (ATRA). ST1926 and CD437 are better RARgamma agonists than ATRA. We used three teratocarcinoma cell lines to evaluate the significance of RARgamma in the activity of RRMs: F9-wild type (WT); F9gamma-/-, lacking the RARgamma gene; F9gamma51, aF9gamma-/-derivative, complemented for the RARgamma deficit. Similar to ATRA, ST1926 and CD437 activate cytodifferentiation only in F9-WT cells. Unlike ATRA, ST1926 and CD437 arrest cells in the G2/M phase of the cell cycle and induce apoptosis in all F9 cell lines. Our data indicate that RARgamma and the classic retinoid pathway are not relevant for the antiproliferative and apoptotic activities of RRMs in vitro. Increases in cytosolic calcium are fundamental for apoptosis, in that intracellular calcium chelators abrogate the process. Comparison of the gene expression profiles associated with ST1926 and ATRA in F9-WT and F9gamma-/-indicates that the RRM activates a conspicuous nonretinoid response in addition to the classic and RAR-dependent pathway. The pattern of genes regulated by ST1926 selectively, in a RARgamma-independent manner, provides novel insights into the possible molecular determinants underlying the activity of RRMs in vitro. Furthermore, it suggests that RARgamma-dependent responses are relevant to the activity of RRMs in vivo. Indeed, the receptor hinders the antitumor activity in vivo, in that both syngeneic and immunosuppressed SCID mice bearing F9gamma-/- tumors have increased life spans after treatment with ST1926 and CD437 relative to their F9-WT counterparts.

Topics: Adamantane; Animals; Antineoplastic Agents; Calcium; Cell Death; Cell Differentiation; Cells, Cultured; Chlorocebus aethiops; Cinnamates; COS Cells; Cytosol; Disease Models, Animal; G2 Phase; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, SCID; Mitosis; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Retinoids; RNA, Messenger; Teratocarcinoma; Tretinoin

Human teratocarcinoma NTera2/cloneD1 (NT2) cells are able to generate postmitotic neurons in response to retinoic acid (RA) and for this reason these cells provide an important tool to study human neurogenesis in vitro. We have obtained neurons by treating NT2 aggregated cells with RA for solely 14 days. RT-PCR assays showed that NT2 cells express mRNAs of several neural bHLH genes such as Hes1, Ngn1, Mash1, NeuroD, Math1 and Pax6, just in the early days of RA exposure. In particular, we reported for the first time that RA treatment was followed by a modulation of endogenous Ngn1 and Math1 transcripts. RT-PCR and Western blotting experiments also demonstrated expression of typical neuronal markers such as GluR, MAP2, Tau and NeuN. Knowledge of the expression pattern of the different neuronal genes during NT2 commitment could be used to investigate alterations in the molecular pathways involved in the human neuronal differentiation.

Topics: Antineoplastic Agents; Biomarkers; Blotting, Western; Cell Aggregation; Cell Culture Techniques; Cell Differentiation; Cell Line, Tumor; Clone Cells; DNA Primers; Helix-Loop-Helix Motifs; Humans; Nerve Tissue Proteins; Neurons; Reverse Transcriptase Polymerase Chain Reaction; Teratocarcinoma; Transcription Factors; Tretinoin

Aberrant activation of the Wnt signaling pathway is a common event in human tumor progression. Wnt signaling has also been implicated in maintaining a variety of adult and embryonic stem cells by imposing a restraint to differentiation. To understand the effect of Wnt signaling on the differentiation of epithelial cells, we used mouse teratocarcinoma F9 cells as a model. The F9 cells can be differentiated into visceral endoderm (VE) resembling absorptive columnar epithelial cells. We performed comparative gene expression analysis on retinoic acid-differentiated and undifferentiated F9 cells and confirmed that markers of VE and intestinal epithelium were induced upon differentiation. The induction of these markers by retinoic acid was reduced in the presence of Wnt, although Wnt alone did not change their expression. This suggests that Wnt signaling inhibited the differentiation of F9 cells by altering gene expression. This inhibition was also reflected in the morphology of the F9 cells as their apical-basal polarity was disrupted by inclusion of Wnt during differentiation. These results support a model in which Wnt modulates the expression of genes required for normal terminal differentiation of the stem cells. However, it follows that progenitor cells must escape from Wnt signaling to attain the differentiated state. Accordingly, we found that differentiated F9 cells no longer responded to Wnt and that a blockade in Wnt signaling occurred upstream of Axin. Consistent with this, Wnt negative regulators, such as Dickkopf-1 and Disabled-2, were induced upon the differentiation of F9 cells. We propose that a similar system to produce Wnt inhibitors regulates homeostasis of certain stem cell compartments in vivo.

Topics: Animals; Biomarkers; Cell Differentiation; Cell Line, Tumor; Cell Polarity; Endoderm; Gene Expression Regulation, Neoplastic; Mice; Proto-Oncogene Proteins; Signal Transduction; Teratocarcinoma; Tretinoin; Viscera; Wnt Proteins; Zebrafish Proteins

The effect of RNA interference (RNAi) induced by synthetic small interfering RNAs (siRNAs) on proliferating mammalian cells appears to last for approximately 3-7 days after its induction. Here we show that the RNAi activity induced by a synthetic 21-nucleotide siRNA duplex in postmitotic neurons, mouse primary hippocampal neurons and neurons that differentiated from mouse embryonal carcinoma P19 cells persists for at least 3 weeks, suggesting long-lasting RNAi activity in mammalian neurons. In addition, we also show that an apoptotic (or antiviral) pathway triggered by long dsRNAs is generated during neuronal differentiation of P19 cells, by which the sequence-specific RNAi activity involving long dsRNA appears to be masked.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Division; Cell Line, Tumor; Cells, Cultured; Cytarabine; Gene Expression Profiling; Hippocampus; Mice; Mice, Inbred ICR; Neurons; RNA, Double-Stranded; RNA, Small Interfering; Teratocarcinoma; Time Factors; Tretinoin

We have previously shown that an F9 teratocarcinoma retinoic acid receptor beta(2) (RARbeta(2)) knockout cell line exhibits no growth arrest in response to all-trans-retinoic acid (RA), whereas F9 wild type (Wt), F9 RARalpha(-/-), and F9 RARgamma(-/-) cell lines do growth arrest in response to RA. To examine the role of RARbeta(2) in growth inhibition, we analyzed the cell cycle regulatory proteins affected by RA in F9 Wt and F9 RARbeta(2)(-/-) cells. Flow microfluorimetry analyses revealed that RA treatment of F9 Wt cells greatly increased the percentage of cells in the G1/G0 phase of the cell cycle. In contrast, RA did not alter the cell cycle distribution profile of RARbeta(2)(-/-) cells. In F9 Wt cells, cyclin D1, D3, and cyclin E protein levels decreased, while cyclin D2 and p27 levels increased after RA treatment. Compared to the F9 Wt cells, the F9 RARbeta(2)(-/-) cells exhibited lower levels of cyclins D1, D2, D3, and E in the absence of RA, but did not exhibit further changes in the levels of these cell cycle regulators after RA addition. Since RA significantly increased the level of p27 protein (approximately 24-fold) in F9 Wt as compared to the F9 RARbeta(2)(-/-) cells, we chose to study p27 in greater detail. The p27 mRNA level and the rate of p27 protein synthesis were increased in RA-treated F9 Wt cells, but not in F9 RARbeta(2)(-/-) cells. Moreover, RA increased the half-life of p27 protein in F9 Wt cells. Reduced expression of RARbeta(2) is associated with the process of carcinogenesis and RARbeta(2) can mediate the growth arrest induced by RA in a variety of cancer cells. Using both genetic and molecular approaches, we have identified some of the molecular mechanisms, such as the large elevation of p27, through which RARbeta(2) mediates these growth inhibitory effects of RA in F9 cells.

Topics: Animals; Antineoplastic Agents; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Cysteine Endopeptidases; Gene Expression Regulation; Mice; Mice, Knockout; Multienzyme Complexes; Proteasome Endopeptidase Complex; Receptors, Retinoic Acid; RNA, Messenger; S-Phase Kinase-Associated Proteins; Teratocarcinoma; Tretinoin; Tumor Suppressor Proteins; Ubiquitins; Up-Regulation

In our previous studies, the TR4 orphan nuclear receptor (TR4) has been demonstrated to suppress retinoic acid (RA)-induced transactivation via a negative feedback control mechanism and in situ analysis showed that TR4 is extensively expressed in mouse brain, especially in regions where the cells are proliferating. To further study the potential roles of TR4 during cell differentiation, a tetracycline-inducible system with anti-sense TR4 in teratocarcinoma P19 cell lines was generated to analyze the retinoic acid-induced differentiation of these cells. The results indicated that the expression of TR4 reduced by doxycycline anti-sense TR4 would alter the retinoic acid-induced differentiation pathway that results in the changes of cell morphology and cell cycle profile. Unexpectedly, our data further indicated that the RA-induced apoptosis, judging by DNA fragmentation, could also be altered by the induction of anti-sense TR4. Together, these findings provide the first in vivo evidence that an orphan nuclear receptor, such as TR4, may play major roles in the RA-mediated apoptosis or differentiation in P19 cells.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Line, Tumor; Doxycycline; Humans; Mice; Receptors, Steroid; Receptors, Thyroid Hormone; Recombinant Proteins; Teratocarcinoma; Tretinoin

Retinoic acid (RA) causes differentiation of mouse F9 embryonic carcinoma cell line into primitive and parietal (with dibutiril-cAMP) endoderm. The role of AP-1 transcription factor during RA-induced differentiation was studied in F9 cell line. It was shown that differentiated cells acquired protein complexes, which are specifically bound to well characterized AP-1 32P-labeled binding sites from collagenase (Col-AP-1) and c-jun (Jun2-AP-1) promoters. These complexes contain c-Fos/c-Jun with Col-AP-1 site and c-Jun/ATF-2 with Jun2-AP-1 site as revealed by supershift analysis. DNA-binding activity of these complexes is high in parietal endoderm but low-detectable in undifferentiated cells. DNA-binding activity of AP-1 transcription factor correlates with increased expression of c-fos and c-jun genes. RT-PCR analysis showed an increase in steady-state level of c-fos and c-jun gene transcription at the stage of parietal endoderm (terminally differentiated F9 cells). Transcription of immediate early c-fos and c-jun genes and DNA-binding activity of c-Fos/c-Jun complex are serum dependent. The rate of c-fos and c-jun gene transcription and DNA-binding activity of c-Fos/c-Jun complex decreased in serum-starved cells, but was rapidly induced upon stimulation with serum. Undifferentiated F9 cells contain a very low level of c-fos mRNA, with may be a consequence of repressive chromatin structure in promoter region. Histone deacetylase (HDAC) activity is necessary to restrict expression of specific number of genes, also HDAC inhibitors are well known inductors of differentiation and anticancer agents. Frow cytometry analysis showed a decreased rate of proliferation of F9 cells after their incubation with HDAC inhibitors, sodium butirate and trichostatin A. Also, these ihibitors induced the transcription of c-fos gene. So, we conclude that HDAC activity may be necessary to sustain a high proliferative rate of undifferentiated F9 cells.

Topics: Animals; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Enzyme Inhibitors; Flow Cytometry; Gene Expression Regulation, Neoplastic; Histone Deacetylases; Hydroxamic Acids; Mice; Protein Binding; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Teratocarcinoma; Transcription Factor AP-1; Transcription, Genetic; Tretinoin

Synaptophysin, one of the major proteins on synaptic vesicles, is ubiquitously expressed throughout the brain. Synaptophysin and synapsin I, another synaptic vesicle protein, are also expressed by retinoic acid-induced neuronally differentiated P19 teratocarcinoma cells. Here, we show that inhibition of histone deacetylase activity in P19 cells is sufficient to activate transcription of the synaptophysin and synapsin I genes, indicating that neuronal differentiation and impairment of histone deacetylases results in a similar gene expression pattern. The transcription factor REST, a repressor of neuronal genes in non-neuronal tissues, has been shown to function via recruitment of histone deacetylases to the transcription unit, indicating that modulation of the chromatin structure via histone deacetylation is of major importance for REST function and neuron-specific gene transcription. Furthermore, REST has been shown to be the major regulator of neuronal expression of synapsin I. Here, we have identified a functional binding site for REST in the first intron of the human synaptophysin gene indicating that REST blocks human synaptophysin gene transcription through an intronic neuron-specific silencer element. The synaptophysin promoter is, however, devoid of neuron-specific genetic elements and directs transcription in both neuronal and non-neuronal cells. Using a dominant-negative approach we have identified the transcription factor Sp1 as one of the regulators responsible for constitutive transcription of the human synaptophysin gene.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cells, Cultured; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression Regulation; Humans; Hydroxamic Acids; Introns; Male; Mice; Molecular Sequence Data; Neurons; Promoter Regions, Genetic; Repressor Proteins; Sp1 Transcription Factor; Synapsins; Synaptophysin; Teratocarcinoma; Testicular Neoplasms; Transcription Factors; Transcription, Genetic; Tretinoin

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (ER) membrane through a protein-conducting channel called translocon. Sec61alpha, a multispanning membrane translocon protein, has been implicated as being essential for translocation of polypeptide chains into the cisterns of the ER. Sec61alpha forms a protein complex with collagen and Hsp47, an ER-resident heat shock protein that binds specifically to collagen. However, it is not known whether Sec61alpha is ubiquitously produced in collagen-producing F9 teratocarcinoma cells or under heat shock treatment. Furthermore, the production and utilization of Sec61alpha may depend on the stage of cell differentiation. Cultured F9 teratocarcinoma cells are capable of differentiation in response to low concentrations of retinoic acid. This differentiation results in loss of tumorigenicity. Mouse F9 cells were grown in culture medium at 37 degrees C and 43 degrees C (heat shock treatment) treated or not with retinoic acid, and labeled in certain instances with 35S-methionine. Membrane-bound polysomes of procollagen IV were then isolated. Immunoprecipitation and Western blot analysis were performed using polyclonal antibodies against collagen IV, Hsp47 and Sec61alpha. Under retinoic acid-untreated conditions, F9 cells produced undetectable amounts of Sec61alpha. Sec61alpha, Hsp47 and type IV collagen levels were increased after retinoic acid treatment. Heat shock treatment did not alter Sec61alpha levels, suggesting that Sec61alpha production is probably not affected by heat shock. These data indicate that the enhanced production of Sec61alpha in retinoic acid-induced F9 teratocarcinoma cells parallels the increased synthesis of Hsp47 and collagen type IV.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Cell Differentiation; Collagen Type IV; Electrophoresis, Polyacrylamide Gel; Heat-Shock Proteins; HSP47 Heat-Shock Proteins; Luminescent Measurements; Membrane Proteins; Mice; SEC Translocation Channels; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Connexin-mediated gap junctions and open hemichannels in nonjunctional membranes represent two biologically relevant mechanisms by which neural progenitors can coordinate their response to changes in the extracellular environment. NT2/D1 cells are a teratocarcinoma progenitor line that can be induced to differentiate terminally into functional hNT neurons and NT-G nonneuronal cells. Clinical transplants of hNT neurons and experimental grafts of NT2/D1 progenitors or hNT neurons have been used in cell-replacement therapy in vivo. Previous studies have shown that NT2/D1 cells express connexin 43 (Cx43) and that NT2/D1 progenitors are capable of dye transfer. To determine whether NT2/D1 progenitors and differentiated hNT cultures express other connexins, Cx26, Cx30, Cx32, Cx36, Cx37, Cx43, and Cx46.6 mRNA and protein were analyzed. NT2/D1 progenitors express Cx30, Cx36, Cx37, and Cx43. hNT/NT-G cultures express Cx36, Cx37, and de novo Cx46.6. Cx26 and Cx32 were not expressed in NT2/D1 or hNT/NT-G cells. NT2/D1 progenitors formed functional gap junctions as assessed by dye coupling as well as open hemichannels in nonjunctional membranes as assessed by dye-uptake studies. Dye coupling was inhibited by the gap junction blocker 18alpha-glycyrrhetinic acid. Hemichannel activity was inhibited by the dual-specificity chloride channel/connexin hemichannel inhibitor flufenamic acid but not by the chloride channel inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Both dye coupling and dye uptake were substantially reduced following differentiation of NT2/D1 progenitors. We conclude that the pattern of connexin expression in NT2/D1 cells changes over the course of differentiation corresponding with a reduction in biochemical coupling and hemichannel activity in differentiated cells.

Topics: Antineoplastic Agents; Base Sequence; Blotting, Western; Cell Communication; Cell Differentiation; Connexin 26; Connexins; Gap Junctions; Gene Expression; Humans; Immunohistochemistry; Molecular Sequence Data; Neurons; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Homology; Stem Cells; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Retinoids, a group of natural and synthetic analogues of vitamin A (retinol), modulate the differentiation of many cell types. Retinoids are also used for the prevention and treatment of cancer. The actions of retinoids are generally mediated by the retinoic acid receptors (RARs alpha, beta, and gamma) and the retinoid X receptors (RXRs alpha, beta, and gamma). One of the RARs, RARbeta, is expressed at reduced levels in many human carcinomas, and F9 RARbeta(2)(-/-) cells do not growth arrest in response to RA. To determine if RARbeta(2) regulates the expression of a unique set of genes, through the use of subtractive hybridization and DNA array analysis, we have identified and characterized genes that are differentially expressed in F9 RARbeta(2)(-/-) teratocarcinoma cells. These genes, which encode transcription factors, cell surface signal transduction molecules, and metabolic enzymes, include c-myc, FOG1, GATA6, glutamate dehydrogenase, glutathione S-transferase homologue (p28), Foxq1, Hic5, Meis1a, Dab2, midkine, and the PDGF-alpha receptor. These genes are regulated specifically by RARbeta(2) in F9 wild-type (Wt) cells as indicated by their expression profiles in F9 RARbeta(2)(-/-) cells as compared to F9 Wt, RARalpha(-/-), or RARgamma(-/-) cells, and their responsiveness to specific retinoid receptor agonists. The basal expression levels of some of these genes, such as c-myc, are higher in the F9 RARbeta(2)(-/-) cells than in F9 Wt in the absence of exogenous retinoids, suggesting that RARbeta(2) can inhibit gene expression in the absence of a ligand. The RARbeta(2) target genes are transcriptionally activated by retinol, as well as RA, in F9 Wt cells. Because the lack of RARbeta(2) alters both the control of proliferation and differentiation in F9 cells, the genes that we have characterized may mediate key effects of RA, via RARbeta(2), on these processes.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Antineoplastic Agents; Apoptosis Regulatory Proteins; Bucladesine; Carrier Proteins; Cell Differentiation; Cell Line, Tumor; Cytokines; Cytoskeletal Proteins; DNA-Binding Proteins; Forkhead Transcription Factors; GATA6 Transcription Factor; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Glutamate Dehydrogenase; Glutathione Transferase; Homeodomain Proteins; Humans; Intracellular Signaling Peptides and Proteins; LIM Domain Proteins; Midkine; Myeloid Ecotropic Viral Integration Site 1 Protein; Neoplasm Proteins; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Phosphodiesterase Inhibitors; Proto-Oncogene Proteins c-myc; Receptor, Platelet-Derived Growth Factor alpha; Receptors, Retinoic Acid; Teratocarcinoma; Theophylline; Trans-Activators; Transcription Factors; Transcriptional Activation; Tretinoin; Tumor Suppressor Proteins

Telomerase is active in about 90% of cancers and contributes to the immortality of cancer cells by maintaining the lengths of the ends of chromosomes. Undifferentiated embryonic human teratocarcinoma (HT) cells were found to express high levels of hTERT, the catalytic subunit of telomerase, and the hTERT promoter was unmethylated in these cells. Retinoic acid (RA)-induced differentiation led to hTERT gene silencing and increased methylation of the hTERT promoter. Treatment with trichostatin A, a histone deacetylase inhibitor, resulted in hTERT reactivation only in very early differentiating HT cells. After methylation patterns had been established within the hTERT promoter region in late differentiating cells, 5-azacytidine, a common demethylating agent, activated the hTERT gene but trichostatin A had no effect on hTERT transcription. These studies suggest that histone deacetylation is involved in early hTERT gene down-regulation and that DNA methylation may maintain silencing of the hTERT gene in these cells.

Topics: Antineoplastic Agents; Azacitidine; Catalytic Domain; Cell Differentiation; DNA (Cytosine-5-)-Methyltransferases; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Humans; Hydroxamic Acids; Methylation; Promoter Regions, Genetic; Telomerase; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

To bind DNA and to be retained in the nucleus, PBX proteins must form heterodimeric complexes with members of the MEINOX family. Therefore the balance between PBX and MEINOX must be an important regulatory feature. We show that overexpression of PREP-1 influences the level of PBX-2 protein maintaining the PREP-1-PBX balance. This effect has important functional consequences. F9 teratocarcinoma cells stably transfected with PREP-1 had an increased DNA binding activity to a PREP-PBX-responsive element. Because PREP-1 binds DNA efficiently only when dimerized to PBX, the increased DNA binding activity suggests that the level of PBX might also have increased. Indeed PREP-1-overexpressing cells had a higher level of PBX-2 and PBX-1b proteins. PBX-2 increase did not depend on increased mRNA level or a higher rate of translation but rather because of a protein stabilization process. Indeed, PBX-2 level drastically decreased after 3 h of cycloheximide treatment in control but not in PREP-1-overexpressing cells and the proteasome inhibitor MG132 prevented PBX-2 decay in control cells. Hence, dimerization with PREP-1 appears to decrease proteasomal degradation of PBX-2. Retinoic acid induces differentiation of F9 teratocarcinoma cells with a cascade synthesis of HOX proteins. In PREP-1-overexpressing cells, HOXb1 induction was more sustained (3 days versus 1 day) and the induced level of MEIS-1b, another TALE (three amino acid loop extension) protein involved in embryonal development, was higher. Thus an increase in PREP-1 leads to changes in the fate-determining HOXb1 and has therefore important functional consequences.

Topics: Amino Acid Sequence; Blotting, Northern; Cell Differentiation; Cell Nucleus; Cycloheximide; Cysteine Endopeptidases; Cytoplasm; Dimerization; DNA; Homeodomain Proteins; Immunoblotting; Kinetics; Leupeptins; Models, Biological; Molecular Sequence Data; Multienzyme Complexes; Myeloid Ecotropic Viral Integration Site 1 Protein; Neoplasm Proteins; Proteasome Endopeptidase Complex; Protein Binding; Protein Biosynthesis; Protein Synthesis Inhibitors; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Teratocarcinoma; Time Factors; Transfection; Tretinoin; Tumor Cells, Cultured

Expression levels of over 8,900 murine genes were examined, using cDNA microarrays, during the differentiation of F9 cells into parietal endoderm following exposure to retinoic acid/dibutyryl cAMP. Gene induction and repression over the time course exhibited a biphasic pattern consistent with a transition from undifferentiated F9 cells to primitive endoderm and finally parietal endoderm. A 6-h induction with retinoic acid/cAMP/cycloheximide resulted in 109 candidate immediate response genes. During a 9-day time course 516 genes were selected as being significantly induced/repressed. Several of these genes had been previously identified as having altered expression patterns in F9 cells undergoing differentiation by retinoic acid/cAMP. Functional characterization of these genes demonstrated that the majority were transcription factors while others included surface antigens and genes involved in intracellular transport. Cluster analysis, utilizing both a hierarchical algorithm and self-organizing map, resulted in very similar gene clusters. Our studies revealed an extremely complex set of interacting signals that decide between cell death, differentiation, cell cycle withdrawal, and ultimately the traits associated with the terminal differentiated parietal endoderm cell type. The sets of genes identified here can now be modulated in a rational way to try to understand their role in differentiation.

Topics: Animals; Bucladesine; Cell Differentiation; Embryonal Carcinoma Stem Cells; Endoderm; Gene Expression; Gene Expression Profiling; Humans; Mice; Multigene Family; Neoplastic Stem Cells; Oligonucleotide Array Sequence Analysis; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Cultured murine F9 teratocarcinoma stem cells resemble pluripotent stem cells of the inner cell mass of the mouse blastocyst and, depending upon their treatment, can be induced to differentiate along the primitive endoderm, the parietal endoderm (PE), or the visceral endoderm (VE) pathway. The Rex-1 gene encodes a zinc finger family transcription factor which is expressed at high levels in undifferentiated F9 stem cells, embryonic stem cells, and in other types of stem cells. To examine whether the Rex-1 protein plays a role in F9 cell differentiation, homologous recombination was employed to generate F9 cell lines which lack both alleles of Rex-1. F9 wild type cells in monolayer culture require both retinoic acid and cyclic AMP analogs to differentiate into PE, whereas the F9 Rex-1(-/-) cells differentiate into PE, as assessed by several molecular markers, including thrombomodulin and laminin B1, in the presence of RA alone. The F9 Rex-1(-/-) cells do not completely differentiate into VE after RA treatment in aggregate culture; they do not express alpha-fetoprotein, a definitive marker of VE differentiation. These results indicate that the Rex-1 transcription factor regulates the differentiation of F9 stem cells along several distinct cell lineages found in the early embryo.

Topics: Animals; Cell Differentiation; DNA-Binding Proteins; Embryonal Carcinoma Stem Cells; Endoderm; Mice; Mutagenesis, Site-Directed; Neoplastic Stem Cells; Parietal Cells, Gastric; Pluripotent Stem Cells; Sequence Deletion; Teratocarcinoma; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Viscera

In search of a cellular model suitable for studying molecular events contributing to brain disorders, we have characterised the expression and functionality of dopamine receptors in human teratocarcinoma NT2 cells. The cells were differentiated by a 4-week retinoic acid treatment, followed by a 3-week mitotic inhibitor treatment in the absence of retinoic acid. The messages of two D(2)-like family members, D(2L) and D(3), were expressed in undifferentiated NT2 cells. The retinoic acid treatment resulted in increased expression of both spliced variants of the D(2) receptor, D(2L) and D(2S) isoforms and a significant induction of D(1) and D(5) gene transcripts. The same treatment turned off expression of the D(3) gene. Further induction of the D(5) gene was observed in the post-mitotic NT2N neurons. The NT2N cells stained positively for D(2) and D(5) receptor proteins, and the intracellular cyclic AMP level increased in response to forskolin, dopamine and the D(1)-receptor agonist SKF-81297. Furthermore, dopamine was ineffective in the presence of the D(2) receptor agonist PPHT and the D(1) receptor antagonist cis-(z)-flupenthixol. These results indicated that upon ligand/agonist/antagonist binding, the receptors could be coupled to the adenylyl cyclase system, hence were functional. To our knowledge, NT2 is the only human immortalized cell line expressing functional dopamine receptors of both families.

Topics: Adenylyl Cyclases; Cell Differentiation; Cell Line, Transformed; Cyclic AMP; Dopamine Agonists; Dopamine Antagonists; Gene Expression Regulation, Developmental; Humans; Ligands; Models, Neurological; Protein Binding; Receptors, Dopamine; RNA, Messenger; Teratocarcinoma; Tretinoin

The synapsin family consists of three neuronal-specific phosphoproteins associated with dynamic reorganization of the neuronal cytoskeleton. Synapsin I and II are implicated in axonal and synaptic differentiation, formation and maintenance, whereas the function of synapsin III is not as well defined. We report a significant transcriptional upregulation of all three synapsins (synapsin I, 2.1-fold; synapsin II, 2.6-fold; and synapsin III, 5.5-fold) by retinoic acid-induced differentiation of NTera-2cl.D1 cells, a human paradigm for neuronal differentiation. The observed stronger regulation of synapsin III might be due to still active neurite elongation and a rather early state of presynaptic maturation at the time-point investigated, as synapsin III was previously found to be highly enriched in growth cones and during early synaptic development.

Topics: Cell Aggregation; Cell Differentiation; Cell Movement; Central Nervous System; Culture Techniques; Gene Expression Regulation, Developmental; Growth Cones; Humans; Models, Biological; Neural Pathways; Neurites; Neurofilament Proteins; Neurons; Neuropeptides; Phosphoproteins; Synapses; Synapsins; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured; Up-Regulation

The morphogen retinoic acid promotes the formation of primitive endoderm in mouse F9 teratocarcinoma cells as does the stimulation of the Frizzled-1 pathway. We investigated whether the beta-catenin/Lef-Tcf-sensitive transcriptional pathway activated by Frizzled-1 plays a role in the retinoic acid-induced pathway to primitive endoderm formation. An analysis of Lef-Tcf-sensitive transcription reveals increased transcription at 1 and 4 h post-treatment with retinoic acid. The stimulation of Lef-Tcf-sensitive transcription as well as the formation of primitive endoderm was accompanied by the stabilization of beta-catenin as observed in activation of the Frizzled-1 pathway. Transient transfection of F9 cells with an expression vector harboring a dominant-negative mutant of Tcf4 resulted in the attenuation of both the increase in Lef-Tcf-sensitive transcription and formation of primitive endoderm in response to the morphogen. Clones stably transfected to express the dominant-negative Tcf4 displayed a block in retinoic acid-induced activation of Lef-Tcf-sensitive transcription and primitive endoderm formation. These data reveal the obligate role of the beta-catenin/Lef-Tcf transcriptional pathway in the action of the morphogen retinoic acid.

Topics: Animals; beta Catenin; Cells, Cultured; Cytoskeletal Proteins; DNA-Binding Proteins; Endoderm; Lymphoid Enhancer-Binding Factor 1; Mice; TCF Transcription Factors; Teratocarcinoma; Trans-Activators; Transcription Factor 7-Like 2 Protein; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

An alternative source of cells for neural transplantation and brain repair that has many characteristics of immature neurons is the hNT neuron, derived from an embryonal human teratocarcinoma (NTera2) cell line that is terminally differentiated in vitro with retinoic acid. The majority of hNT neurons are GABAergic in cell culture. We have determined the calcium-binding protein (CBP) phenotypes of hNT neurons for three CBPs, calretinin (CR), calbindin D-28K (CB), and parvalbumin (PV), in cell culture and after transplantation into the rat striatum. In cell culture, 95% of all cell profiles were human nuclear matrix antigen (NuMA) positive. PV-positive hNT neurons constituted 50% of all neuron-like profiles, with CB+ and CR+ constituting 14 and 6% of cells, respectively. In contrast, when the striatal grafts were examined after 30 days survival using confocal microscopy, only 10% of hNT neurons immunopositive for NuMA were PV+; 19% were CB+/NuMA+, approximately the same percentage as was seen in vitro, and 82% of grafted hNT neurons were CR+. These results suggest that hNT neurons can be subdivided into at least three subpopulations based on the CBP phenotype that they express and that there is a CBP phenotypic shift following transplantation. Three related hypotheses are proposed to account for this phenotypic shift of hNT neurons after transplantation: (a) selective survival of the CR+ subpopulation of hNT neurons, (b) selective transitory quiescence of the transplanted PV+ cells due to transplantation stress, or (c) dedifferentiation of the hNT neurons following transplantation, which may allow them to respond to local environmental cues during the engraftment process.

Topics: Animals; Antigens, Nuclear; Brain Tissue Transplantation; Calcium-Binding Proteins; Cell Cycle Proteins; Cell Differentiation; Cells, Cultured; Corpus Striatum; Cryopreservation; Embryonal Carcinoma Stem Cells; Female; Fetal Tissue Transplantation; Graft Survival; Humans; Neoplastic Stem Cells; Neurons; Nuclear Matrix-Associated Proteins; Nuclear Proteins; Phenotype; Rats; Rats, Sprague-Dawley; Stroke; Teratocarcinoma; Transplantation, Heterologous; Tretinoin; Tyrosine 3-Monooxygenase

Herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein ICP0 stimulates the initiation of lytic infection and reactivation from quiescence in human fibroblast cells. These functions correlate with its ability to localize to and disrupt centromeres and specific subnuclear structures known as ND10, PML nuclear bodies, or promyelocytic oncogenic domains. Since the natural site of herpesvirus latency is in neurons, we investigated the status of ND10 and centromeres in uninfected and infected human cells with neuronal characteristics. We found that NT2 cells, a neuronally committed human teratocarcinoma cell line, have abnormal ND10 characterized by low expression of the major ND10 component PML and no detectable expression of another major ND10 antigen, Sp100. In addition, PML is less extensively modified by the ubiquitin-like protein SUMO-1 in NT2 cells compared to fibroblasts. After treatment with retinoic acid, NT2 cells differentiate into neuron-like hNT cells which express very high levels of both PML and Sp100. Infection of both NT2 and hNT cells by HSV-1 was poor compared to human fibroblasts, and after low-multiplicity infection yields of virus were reduced by 2 to 3 orders of magnitude. ICP0-deficient mutants were also disabled in the neuron-related cell lines, and cells quiescently infected with an ICP0-null virus could be established. These results correlated with less-efficient disruption of ND10 and centromeres induced by ICP0 in NT2 and hNT cells. Furthermore, the ability of ICP0 to activate gene expression in transfection assays in NT2 cells was poor compared to Vero cells. These results suggest that a contributory factor in the reduced HSV-1 replication in the neuron-related cells is inefficient ICP0 function; it is possible that this is pertinent to the establishment of latent infection in neurons in vivo.

Topics: Cell Differentiation; Cell Nucleus Structures; Centromere; Chromosomal Proteins, Non-Histone; Fibroblasts; Fluorescent Antibody Technique; Gene Deletion; Gene Expression Regulation, Viral; Herpesvirus 1, Human; Humans; Immediate-Early Proteins; Kinetics; Mutation; Neurons; Superinfection; Teratocarcinoma; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured; Ubiquitin-Protein Ligases; Virus Activation; Virus Replication

An expression sequence tag identified in a screen for genes upregulated by retinoic acid induced neuronal differentiation of the human teratocarcinoma cell line Ntera2/D1 was found in close genomic proximity to a region of high sequence homology to the septin subfamily of GTPase genes. We could show that the tag corresponds to the 3' untranslated region of this novel gene named septin 3 and cloned three isoforms A (2191 bp), B (4378 bp), and C (1896 bp) from human Ntera2/D1 cDNA. We present the genomic localization and organization on chromosome 22q13.2, a chromosomal hot spot for translocations implicated in leukemia. Interestingly, MSF the closest paralog of septin 3 is a fusion partner in a therapy-related acute myeloid leukemia. Quantitative PCR confirmed the upregulation of the putative septin by neuronal differentiation and northern blotting showed only one band corresponding to sep3B with a neurospecific expression pattern in adult human tissues.

Topics: Cell Differentiation; Chromosomes, Human, Pair 22; Cloning, Molecular; GTP Phosphohydrolases; Humans; Molecular Sequence Data; Nerve Tissue Proteins; Neurons; Organ Specificity; Phylogeny; Physical Chromosome Mapping; Protein Isoforms; Septins; Sequence Homology, Amino Acid; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Through the use of microarray analysis it was discovered that the nuclear receptor coregulator, receptor interacting protein 140 (RIP140), was induced early during all-trans retinoic acid (RA)-induced differentiation of human embryonal carcinoma cells. A rapid, fourfold induction of RIP140 mRNA was detected within 3 h of RA treatment in human embryonal carcinoma and MCF-7 human breast cancer cells. RIP140 protein levels were induced within 6 h of RA treatment. The RA induction of RIP140 mRNA did not require de novo protein synthesis, consistent with RIP140 being a direct transcriptional target of retinoid receptors. Promoter/enhancer elements directly upstream of the RIP140 coding region supported RA-induced transcription of a luciferase gene. In addition the ability of overexpressed RIP140 to repress ligand activated retinoid receptors was confirmed. The finding that RIP140 is a direct transcriptional target of RA is one of the first examples of acute transcriptional regulation of a nuclear receptor coactivator or corepressor. These data are consistent with a model by which RA induction of RIP140 supplies a negative feedback signal toward ligand-activated retinoid receptors.

Topics: Adaptor Proteins, Signal Transducing; Breast Neoplasms; Carcinoma, Embryonal; Feedback; Female; Gene Expression Regulation, Neoplastic; Humans; Models, Genetic; Nuclear Proteins; Nuclear Receptor Interacting Protein 1; Receptors, Cytoplasmic and Nuclear; Teratocarcinoma; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

The positions of nucleosomes in the proximal 5' regions of the coordinately regulated murine entactin/nidogen and laminin gamma1 genes have been identified in four different transcriptional states--constitutively off, basal, induced, and constitutively induced. In the entactin gene a 450 base pair (bp) region of open chromatin is present between three positioned nucleosomes and the transcriptional start site in the basal, induced, and constitutively induced states. Additionally there is a 200 bp open chromatin region at approximately -2.1 kbp that is only present in the induced and constitutively induced states. In the laminin gamma1 gene, a 650 bp region of nucleosome-free chromatin is present between nucleosomes positioned at approximately -750 and +120 in all transcriptionally active states. These results suggest that basal co-expression of these genes requires sites present in these near upstream regions. The induction to high levels appears to involve additional sites and possibly the production of new and/or the modification of existing trans-acting factors.

Topics: Animals; Bucladesine; Cell Differentiation; Chromatin; Chromosome Mapping; Gene Expression Regulation, Neoplastic; Genes; Laminin; Membrane Glycoproteins; Mice; Multiple Myeloma; Neoplasm Proteins; Nucleosomes; Teratocarcinoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Using a PCR-based cDNA subtractive hybridization method (L. Diatchenko et al., Proc. Natl. Acad. Sci. USA, 93: 6025-6030, 1996), we cloned a cDNA fragment of a novel gene that is highly expressed in F9-10; F9-10 is an F9 teratocarcinoma stem cell line that expresses high levels of exogenous Hoxa-1 mRNA and protein in comparison to F9 wild-type stem cells, which do not express endogenous Hoxa-1 mRNA in the absence of retinoic acid (RA). Rapid amplification of cDNA ends was used to clone the full-length cDNA of this gene, designated HA1R-62 (Hoxa1 regulated-62). We have shown that HA1R-62 is also a RA-responsive gene and that it is expressed (mRNA size, approximately 4.3 kb) in adult mouse thymus, lung, kidney, and ovary as well as in 12.5-day mouse embryos. DNA sequence analysis and in vitro translation experiments have shown that HA1R-62 encodes a protein with a molecular mass of approximately 26 kDa. Elucidation of the function of the HA1R-62 gene product will provide new insights into the functions of RA and homeobox genes.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Mice; Molecular Sequence Data; Neoplasm Proteins; Protein Biosynthesis; Proteins; Repressor Proteins; Reticulocytes; RNA, Messenger; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured; Ubiquitin-Protein Ligases; Up-Regulation

The metallothionein (MT)3 gene is expressed predominantly in the brain and the organs of the reproductive system, and fails to respond to metal ions in vivo. A CTG repeat was proposed to function as a potential repressor element in nonpermissive cells, and a sequence similar to the JC virus silencer element was found to function as a negative element in permissive primary astrocytes. The objective of this study was to characterize further the mechanisms governing cell-type specific MT-3 gene transcription. We searched for a suitable cell line expressing the MT-3 gene to be used for determination of MT-3 promoter tissue specificity, and showed that MT-3 expression is activated during neuroectodermal differentiation of P19 cells induced by retinoic acid to levels similar to those found in whole brain. Deletion of the CTG repeat or of the JC virus silencer did not promote MT-3 promoter activity in nonpermissive cells, or enhance expression in permissive cells. We identified MT-3 promoter sequences interacting with liver and brain nuclear proteins, as assayed by DNase I footprinting analyses and electrophoretic mobility shift assay, and assessed the role of these sequences in the regulation of MT-3 expression by cotransfection experiments. We generated stable transfectants in permissive C6 and nonpermissive NIH-3T3 cells, and analysed the methylation status of the MT-3 gene. These studies show that regulation of tissue-specific MT-3 gene expression does not appear to involve a repressor, and suggest that other mechanisms such as chromatin organization and epigenetic modifications could account for the absence of MT-3 gene transcription in nonpermissive cells.

Topics: 3T3 Cells; Animals; Base Sequence; Cell Differentiation; Cells, Cultured; Chlorocebus aethiops; COS Cells; DNA; Gene Expression Regulation, Neoplastic; Humans; Metallothionein; Metallothionein 3; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Neoplasms; Neoplasms, Experimental; Neuroglia; Neurons; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; RNA, Messenger; RNA, Neoplasm; Species Specificity; Teratocarcinoma; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) induces the differentiation of many murine teratocarcinoma cell lines such as F9 and P19. In F9 cells, the level of the cellular retinoic acid binding protein I (CRABP I) mRNA is greatly reduced after exposure of the cultured cells to exogenous RA. In P19 cells, the level of CRABP I mRNA is greatly increased after RA exposure. We have identified a 176-bp region in the murine CRABP I promoter, between -2.9 and -2.7 kb 5' of the start site of transcription, which acts as an enhancer in undifferentiated F9 stem cells and through which RA effects inhibition of CRABP I transcription. Within this region are two footprinted sites at -2763 and -2834. This 176-bp regulatory region does not function to enhance CRABP I transcription in P19 stem cells. Several DNA sequences within these two footprinted regions bind proteins from F9 nuclear extracts but not from P19 nuclear extracts (e.g., FP1B, FP1A, and FP2B), as assessed by gel shift assays. This 176-bp CRABP I genomic region has not been sequenced previously and functionally analyzed in cultured cells because it was not present in the murine CRABP I clones used for the promoter analyses reported earlier by another laboratory. The function of this enhancer may be to reduce the expression of the CRABP I gene in specific embryonic cell types in order to regulate the amount of RA to which the cells are exposed.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Nucleus; DNA Footprinting; Enhancer Elements, Genetic; Fibroblasts; Gene Expression Regulation, Neoplastic; Mice; Molecular Sequence Data; Neoplasm Proteins; Promoter Regions, Genetic; Receptors, Retinoic Acid; RNA, Messenger; RNA, Neoplasm; Teratocarcinoma; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured

p16(INK4a), p15(INK4b), p18(INK4c) and p19(INK4d) comprise a family of cyclin-dependent kinase inhibitors and tumor suppressors. We report that the INK4 proteins share the ability to arrest cells in G1, and interact with CDK4 or CDK6 with similar avidity. In contrast, only p18 and particularly p19 are phosphorylated in vivo, and each of the human INK4 proteins shows unique expression patterns dependent on cell and tissue type, and differentiation stage. Thus, the INK4 proteins harbor redundant as well as non-overlapping properties, suggesting distinct regulatory modes, and diverse roles for the individual INK4 family members in cell cycle control, cellular differentiation, and multistep oncogenesis.

Topics: Binding, Competitive; Carrier Proteins; Cell Cycle Proteins; Cell Differentiation; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p18; Cyclin-Dependent Kinase Inhibitor p19; Cyclin-Dependent Kinases; Enzyme Inhibitors; G1 Phase; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, p16; Humans; Multigene Family; Organ Specificity; Phosphorylation; Protein Binding; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Recombinant Fusion Proteins; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

After traumatic injury to the central nervous system (CNS), various cytokines orchestrate the physiological responses of injured neurons and glial cells. The control of these intercellular signals is of major interest from a medical point of view. Since the transcriptional activator retinoic acid (RA) is known to regulate gene expression of cytokines in various cell culture systems we investigated the role of RA signaling in glial cells. The transcriptional activity of RA-induced genes is largely determined by the distribution of RA, which in turn depends on the local oxidation of retinaldehyde (RAL). This is synthesized from retinol or internalized as a component of vitamin A. Using high-pressure liquid chromatography and an RA-sensitive reporter cell line, we showed that OLN-93 cells, which serve as a model system for CNS oligodendrocytes, convert all-trans-RAL to the biologically active form all-trans-RA, but neither oxidize 9-cis-RAL nor isomerize RA enzymatically. The oligodendrocyte cell line expresses a cytosolic aldehyde dehydrogenase with an apparent molecular weight of 54-57 kDa and pI of 5.3-5.7. As indicated by a zymography bioassay, this enzyme is responsible for RA synthesis. The reaction requires NAD+ as cosubstrate and can be inhibited by disulfiram and citral. No other RA-producing enzyme activities were detected. These findings are in accordance with a putative role for retinoid signaling in neuroglial interactions in the CNS.

Topics: Aldehyde Dehydrogenase; Alitretinoin; Animals; Biological Assay; Cell Line; Chromatography, High Pressure Liquid; Oligodendroglia; Rats; Receptors, Retinoic Acid; Teratocarcinoma; Transfection; Tretinoin; Tumor Cells, Cultured

Recently, we showed expression of apolipoprotein E (apoE) in human neuronal-type cells such as neuroblastoma SK N SH-SY 5Y cells. In this model, a negative effect of neuronal differentiation on apoE synthesis was suspected. To check this hypothesis, we studied the regulation of apoE in human postmitotic neurons. The presence of apoE was investigated in undifferentiated human teratocarcinoma NT2/D1 (NT2) cells and during their differentiation into postmitotic hNT neurons induced by retinoic acid (RA) treatment. Before differentiation, apoE protein and mRNA were detected in NT2 cells by Western blotting and RT-PCR experiments. Immunofluorescence study showed that apoE was present in all cells. For longer times of RA treatment (3 weeks), the apoE labeling became heterogeneous: only some cells were immunopositive and among them were some differentiating cells in which apoE was located in both cellular body and neuritic process. Interestingly, terminally differentiated hNT cells no longer expressed apoE. These results demonstrate that neuronal precursor and differentiating cells were able to synthesize apoE while the fully neuronal differentiation exerted a negative effect on apoE neuronal expression. Our results are compatible with a weak expression of apoE in neurons of adult brains.

Topics: Antineoplastic Agents; Apolipoproteins E; Cell Differentiation; Cellular Senescence; Gene Expression; Humans; Mitosis; Neurons; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stem Cells; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Human NTera-2/clone D1 teratocarcinoma cells are induced by retinoic acid (RA) to differentiate into postmitotic cells with morphological and biochemical characteristics of embryonic human neurones. Currently only limited information concerning peptide-contents and neurotransmitter pools of these cells is available. Zeller and Strauss [Int. J. Dev. Neurosci. 1995;13(5):437] described an increase in choline acetyltransferase (ChAT) activity in RA-treated, but not in untreated NTera-2 cells, suggesting the induction of a cholinergic phenotype during treatment with RA. In the present study we investigated the effect of RA-differentiation on the amount of the neurotransmitters acetylcholine (ACh), and dopamine in NTera-2 in order to specify the transmitter phenotype induced by RA-differentiation. We found that a 4-week treatment of NTera-2 cells with 10 microM RA markedly increased the ACh-content of these cells, while dopamine levels were unchanged. Depolarisation with potassium (60 mM) enhanced ACh-outflow in the differentiated cells in a Ca(++) dependent way. Also neuropeptides like substance P and NPY were detectable in the undifferentiated NTera-2 cells, while vasointestinal peptide (VIP) could not be found in either precursor or RA-differentiated cells. Differentiation was accompanied by a marked reduction of neutral endopeptidase enzyme activity and aminopeptidase activity. From these observations it was concluded that RA induces a cholinergic neurochemical differentiation of this human teratocarcinoma cell line, and that these cells might provide a model system to investigate cholinergic properties of human origin.

Topics: Acetylcholine; Calcium; CD13 Antigens; Cell Differentiation; Cell Size; Dopamine; Humans; Neprilysin; Neuropeptide Y; Phenotype; Potassium Chloride; Substance P; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Epithelial and endothelial cells are susceptible to a subset of apoptosis known as anoikis. This type of programmed cell death is activated upon disruption of cell-substrate contacts. Here we demonstrate that mouse F9 embryonal carcinoma cell line acquires susceptibility to anoikis upon retinoic acid-induced differentiation towards non-malignant pariental endoderm-like cells. F9 cells survival becomes dependent on the substrate by the 4th day of retinoic acid treatment, when cells assume epithelial phenotype as revealed by actin, alpha-actinin and vinculin expression and distribution, and when focal adhesion contacts are formed. Differentiated F9 cells die in suspension by apoptosis as revealed by oligonucleosomal DNA laddering, DAPI staining and DNA flow cytometry analysis. On the contrary, undifferentiated F9 cells form large multicellular aggregates in suspension and survive. Thus, F9 cell line provides a new model to study pathways involved in both anoikis induction and inhibition.

Topics: Actinin; Actins; Animals; Anoikis; Cell Differentiation; Flow Cytometry; Mice; Phenotype; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured; Vinculin

Our previous study demonstrated the high incidence of non-induced DNA single strand breaks (SSB) in preimplantation mouse embryo genom (Patkin et al., 1994). F9 mouse teratocarcinoma cell line is an in vitro model for early embryonal differentiation, since F9 cells remind in many respects the inner cell mass cells of mouse blastocyst and are capable of differentiation under retinoic acid (RA) and dibutyryl cAMP (db-cAMP) treatment. Using gap filling reaction of F9 metaphase chromosomes and single-cell DNA electrophoresis, we have observed multiple SSB in undifferentiated F9 cells as well as in F9 cells at the early steps of RA-induced differentiation (days of RA treatment), but not in terminally differentiated F9 cells and in mouse embryonal fibroblasts. Rad51 nuclear protein that binds specifically single stranded DNA is highly expressed in all cells of undifferentiated F9 population and is not expressed in terminally differentiated F9 population. Multiple SSB could lead to enhanced rate of sister chromatid exchanges (SCE) in F9 cells. In undifferentiated F9 population the level of SCE was 9.6 +/- 0.44 per metaphase, that was not higher than in NIH 3T3 cell line. However, RA treatment for 48 h led to rising the SCE level up to 16.68 +/- 0.72 followed by its decrease to the initial rate by 72 h of RA treatment. Since the enhanced level of SSB in undifferentiated F9 cells and in mouse blastocyst does not normally lead to chromosomal instability, we consider SSB to be a natural consequence of fast-going DNA replication in these cells.

Topics: 3T3 Cells; Animals; Bucladesine; Cell Differentiation; DNA Damage; DNA Replication; DNA, Single-Stranded; Mice; Sister Chromatid Exchange; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

myo-Inositol plays a key role in signal transduction and osmotic regulation events in the CNS. Despite the known high concentrations of inositol in the human CNS, relatively little is known about its distribution within the different cell types. In this report, inositol homeostasis was studied in NT2-N cells, a unique cell culture model of human CNS neurons. Differentiation of precursor NT2 teratocarcinoma cells into NT2-N neurons by means of retinoic acid treatment resulted in an increase in inositol concentration from 24 to 195 nmol/mg of protein. After measurement of intracellular water spaces, inositol concentrations of 1.6 and 17.4 mM were calculated for NT2 and NT2-N cells, respectively. The high concentrations of inositol in NT2-N neurons could be explained by (1) an increased uptake of inositol (3.7 vs. 1.6 nmol/mg of protein/h, for NT2-N and NT2 cells, respectively) and (2) a decreased efflux of inositol (1.7%/h for NT2-N neurons vs. 9.0%/h for NT2 cells). Activity of inositol synthase, which mediates de novo synthesis of inositol, was not detected in either cell type. The observation that CNS neurons maintain a high intracellular concentration of inositol may be relevant to the regulation of both phosphoinositide signaling and osmotic stress events in the CNS.

Topics: Antineoplastic Agents; Astrocytoma; Biological Transport; Cell Differentiation; Cells, Cultured; Humans; Inositol; Myo-Inositol-1-Phosphate Synthase; Neuroblastoma; Neurons; Phlorhizin; Stem Cells; Teratocarcinoma; Tretinoin; Water; Water-Electrolyte Balance

Nuclear lamins are prominent elements of the nuclear matrix and are expressed in cell type-specific and differentiation state-specific patterns. A few observations have indicated that nervous tissue may display unusual patterns of lamin expression, in that some neurons appear to lack A-type lamins, which are generally prominently expressed in terminally differentiated, postmitotic cells. To investigate lamin expression patterns during the differentiation of a teratocarcinoma cell line into neurons, NT2/D1 cells were induced to differentiate with retinoic acid treatment. Lamin expression and organization during differentiation in vitro were examined by quantitative immunofluorescence and immunoblotting methods. Undifferentiated NT2/D1 cells were all strongly labeled with an anti-lamin B1 antibody, but displayed marked variation in A/C lamin immunoreactivity. After differentiation, neuronal nuclear envelopes were significantly more strongly labeled by anti-lamin B1 antibody than those of undifferentiated cells, but completely lacked A/C lamin immunoreactivity. In contrast, nonneuronal cells displayed a slight reduction in B1 lamin immunoreactivity, along with a distinct increase in A/C lamin levels. The loss of lamin A/C expression in NT2/D1 neurons is contrary to the pattern normally observed in most somatic cell types during early development and indicates that the nuclear matrix of some neurons, along with certain neuroendocrine and hematopoietic cells, is uniquely specialized in this regard.

Topics: Cell Differentiation; Humans; Immunohistochemistry; Kinetics; Lamin Type A; Lamin Type B; Lamins; Neurons; Nuclear Proteins; Teratocarcinoma; Time Factors; Tretinoin; Tumor Cells, Cultured

We reported two cases of chemotherapy-refractory testicular cancer treated with all trans-retinoic acid (ATRA). Case 1. A 21-year-old male patient underwent salvage surgery for lung metastasis which had developed after treatment with three different cisplatin-based chemotherapy regimens for malignant teratoma. After recovery from surgery, he was treated with oral ATRA at daily dose 80 mg/m2 for four weeks. Case 2. A-45-year-old patient suffered from lung metastasis after orchiectomy for teratocarcinoma. The patient failed to achieve a complete response despite two different cisplatin-based chemotherapy and high dose chemotherapy regimens with bone marrow rescue. He was treated with oral ATRA for five weeks. Both patients showed disease progression with increase in tumor size and elevation of tumor marker during ATRA therapy. Side effects were acceptable except the headache in Case 2, who needed a dose reduction of ATRA. In conclusion, oral ATRA with this dose failed to show clinical antitumor activity in patients with refractory testicular cancer.

Topics: Adult; Antineoplastic Agents; Combined Modality Therapy; Humans; Male; Middle Aged; Orchiectomy; Teratocarcinoma; Teratoma; Testicular Neoplasms; Tretinoin

Phospholipase D has been recognized as playing an important role in signal transduction in many types of cells. We investigated the expression of phospholipase D during the differentiation of F9 embryonal teratocarcinoma cells. The ADP ribosylation factor-dependent phospholipase D activity, as measured by an in vitro assay, and H2O2-induced phospholipase D activity and phospholipase D protein content in whole cells were decreased during the differentiation of F9 cells induced by a combination of dibutyryl cyclic AMP and all-trans retinoic acid. In contrast, these changes were not observed when cells were induced by retinoic acid. These results suggest that down-regulation of phospholipase D protein is associated with differentiation of F9 cells to a parietal endoderm lineage.

Topics: Animals; Antineoplastic Agents; Cell Differentiation; Cyclic AMP; Down-Regulation; Mice; Phospholipase D; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Zipper Protein Kinase (ZPK) is a leucine zipper protein localized to the nucleus which exhibits serine-threonine kinase activity and is associated with the stress dependent signal transduction pathway. ZPK forms heterodimers with leucine zipper containing transcription factors such as the cyclic AMP responsive element binding protein (CREB) and Myc. Furthermore ZPK phosphorylates both Myc and CREB. Overexpression of ZPK in NTera-2 human teratocarcinoma cells results in inhibition of PKA induced transcriptional activation by CREB and prevents retinoic acid induced differentiation of the cells to neurons. Our results suggest that ZPK stifles neural differentiation of NT-2 cells partly due to its inhibitory effect on CREB function.

Topics: 3T3 Cells; Animals; Cell Differentiation; Cloning, Molecular; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Dimerization; Humans; MAP Kinase Kinase Kinases; Mice; Neurons; Phosphorylation; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; Recombinant Proteins; Signal Transduction; Teratocarcinoma; Transcription, Genetic; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

Mutations in the presenilin-1 gene are linked to the majority of early-onset familial Alzheimer's disease cases. We have previously shown that the expression of transforming growth factor-beta is altered in Alzheimer's patients, compared to controls. Here we examine presenilin- expression in human post-mitotic neurons (hNT cells), normal human astrocytes, and human brain tumor cell lines following treatment with three isoforms of transforming growth factor-beta, or glial cell line-derived neurotrophic factor, a member of the transforming growth factor-beta superfamily. As the NT2/D1 teratocarcinoma cell line is treated with retinoic acid to induce differentiation to hNT cells, presenilin-1 messenger RNA expression is dramatically increased. Furthermore, there is a 2-3-fold increase in presenilin-1 messenger RNA expression following treatment of hNT cells with growth factors and similar results are found by Western blotting and with immunohistochemical staining for presenilin-1 protein. However, treatment of normal human astrocytes with cytokines results in minimal changes in presenilin-1 messenger RNA and protein. Interestingly, the expression of presenilin-1 in human U87 MG astrocytoma and human SK-N-SH neuroblastoma cells is only increased when cells are treated with glial cell line-derived neurotrophic factor or transforming growth factor-beta3. These findings suggest that endogenous presenilin-1 gene expression in human neurons can be induced by growth factors present in normal and diseased brain tissue. Cytokines may play a major role in regulating expression of presenilin-1 which may affect its biological actions in physiological and pathological conditions.

Topics: Astrocytes; Astrocytoma; Blotting, Western; Brain Neoplasms; Gene Expression Regulation; Glial Cell Line-Derived Neurotrophic Factor; Glioblastoma; Humans; Membrane Proteins; Neoplasm Proteins; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; Neurons; Presenilin-1; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Teratocarcinoma; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) plays a pivotal role during vertebrate development, both as morphogen and as potent teratogen. While RA function in axial development has been extensively studied, little is known about the genetic control of RA teratogenicity. The knockout of the homeobox gene goosecoid in the mouse revealed similarities to RA induced embryopathy. We show that RA treatment of mouse gastrula embryos in vitro and of E10.5 embryos in utero led to a rapid but transient down-regulation of goosecoid expression. Repression was dependent on retinoid X receptors (RXR). BMP-4 was repressed by RA-treatment as well, both in embryos and in F9 teratocarcinoma cells. Our data suggest that both goosecoid and BMP-4 function as mediators of RA teratogenicity in mouse embryos.

Topics: Animals; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Down-Regulation; Embryo, Mammalian; Female; Gastrula; Gene Expression Regulation, Developmental; Goosecoid Protein; Homeodomain Proteins; Mice; Organ Culture Techniques; Pregnancy; Protein Biosynthesis; Proteins; Receptors, Retinoic Acid; Repressor Proteins; Retinoid X Receptors; RNA, Messenger; Teratocarcinoma; Teratogens; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Retinoids signal biological effects through retinoic acid receptors (RAR) and retinoid X receptors (RXR) and their co-regulators. We previously reported that all-trans retinoic acid (RA) triggers terminal differentiation in the human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1), through an RARgamma dependent pathway. RARgamma repression in NT2/D1-R1 cells accounts for RA resistance in this line. This report finds RARgamma repression is due to selective repression of RARgamma but not RARbeta transcription in NT2/D1-R1 cells. The repression is neither due to mutations in RARgamma nor its promoter containing the RA response element. Prior work was confirmed and extended by demonstrating that an RARgamma selective agonist preferentially signals differentiation of NT2/D1 cells, while RARalpha/beta, RARbeta, RXR agonists and an RAR pan-antagonist do not even when NT2/D1 cells are treated with these retinoids at 10 microM dosages. None of these examined retinoids induced differentiation of the RA resistant NT2/D1-R1 cells. In contrast, N-(4-hydroxyphenyl)retinamide (4HPR), a reported transcriptional activator of RARgamma was shown to potently induce growth inhibition and apoptosis in both NT2/D1 and NT2/D1-R1 cells. 4HPR-induced apoptosis was unaffected by co-treatment of both cell lines with equimolar RAR antagonist. Semi-quantitative reverse transcription-polymerase chain reaction (RT - PCR) assays of total RNA from 4HPR-treated NT2/D1 and NT2/D1-R1 cells did not reveal RARgamma induction. Since 4HPR signals in RA-resistant NT2/D1-R1 cells having an RARgamma transcriptional block, these results indicate that 4HPR triggers apoptosis but not differentiation through an RARgamma independent pathway. Taken together, these findings implicate a therapeutic role for 4HPR mediated apoptosis in germ cell tumors even when a maturation block is present.

Topics: Anticarcinogenic Agents; Apoptosis; Carcinoma, Embryonal; Cell Differentiation; DNA Fragmentation; Fenretinide; Humans; Neurons; Promoter Regions, Genetic; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Retinoids; RNA, Messenger; Teratocarcinoma; Time Factors; Tretinoin; Tumor Cells, Cultured

In contrast to most other human endogenous retroviral families, various HERV-K members have open reading frames that code for functional viral proteins which can form noninfectious particles in some germ cell tumors. The HERV-K viral genes are highly transcribed in germ cell tumors but are transcribed to lower or undetectable levels in most other tissue and tumor types. To further analyze the expression patterns of these proviruses, long terminal repeats (LTRs) were isolated from the human genome and used in reporter gene assays. Expression of some HERV-K LTRs was found to be high in human and murine germ cell tumors (testicular teratocarcinomas) and low in non-germ-cell tumors. Furthermore, upon differentiation of a teratocarcinoma cell line, the expression of an active LTR dropped dramatically, suggesting developmental regulation of these proviral LTRs. Transgenic mice harboring an active LTR driving lacZ expression were generated and analyzed. Adult mouse testes showed the highest levels of expression, and the transgene staining appeared to be restricted primarily to the more undifferentiated spermatocytes. Most other tissues analyzed revealed very low or undetectable levels of expression both by reverse transcription-PCR and by Northern blot analysis. Whether the restricted expression of HERV-K in germ cells and in germ cell-derived tumors is of significant importance during development or tumorigenesis remains to be elucidated. Germ line expression of these viruses would allow for their expansion and movement, while somatic repression would ensure limited insertional mutagenesis and misexpression in an individual.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Line, Transformed; DNA, Viral; Endogenous Retroviruses; Gene Expression; Genes, Reporter; Humans; Lac Operon; Male; Mice; Mice, Transgenic; Molecular Sequence Data; Spermatocytes; Teratocarcinoma; Terminal Repeat Sequences; Testis; Tretinoin; Tumor Cells, Cultured

We examined neuronal differentiation of F9 teratocarcinoma cells using retinoic acid (RA) and cyclic AMP (cAMP) as inducing agents. Neuronal differentiation was monitored using (1) cDNA probes for the rat 68-kDa neurofilament gene, (2) RT-PCR for neurofilament genes and (3) antibodies against several neuronal differentiation markers. We found by Northern blotting that the uninduced F9 cells, grown in 10% serum, expressed mRNA for the 68-kDa neurofilament protein whereas the control cells, grown in 3% serum, failed to express detectable levels of the 68-kDa neurofilament transcripts. However, RT-PCR allowed detection of both the 68- and 200-kDa neurofilament gene transcripts in F9 cells with or without the inducing agents. Under serum deprivation, a prolonged (> 10-15 days) cultivation of the F9 cells in the presence of RA and cAMP was required for the expression of detectable levels of the 68-kDa neurofilament transcripts and immunocytochemically detectable neurofilament proteins. Treatment of the F9 cells with RA and cAMP was also required for induction of their neuronal phenotype. Immunocytochemically, the uninduced F9 cells expressed several neuronal antigens including the 68-kDa neurofilament protein, the 200-kDa neurofilament protein, neural cell adhesion molecule (N-CAM) and a neuronal specific tubulin isoform (TUJI). The control cells expressed N-CAM and TUJI, but failed to express the neurofilament proteins. A subclone, D9L2, derived from a single F9 parent cell, expressed both TUJI and neurofilament proteins, but no N-CAM molecule. The present results indicate that both the 68- and the 200-kDa neurofilament genes are constitutively active in uninduced F9 teratocarcinoma cells. Under serum deprivation both RA and cAMP are required for expression of detectable levels of neurofilament mRNA and protein. Thus, serum deprivation of the F9 cells either down-regulates the NF gene expression, stability of mRNA or degradation of the NF-proteins. Importantly, expression of a neuronal phenotype by a subpopulation of F9 cells appears to require administration of RA and cAMP, although expression of neuronal marker proteins is not dependent on these agents. Lastly, we demonstrate cloning of a novel cell line (D9L2), derived from a single F9 parent cells, capable of extending neurites and expressing several neuronal antigens under serum deprivation without the requirement of RA and cAMP. We propose that the D9L2 cell line may offer a simplified F9 cell model sys

Topics: Animals; Antineoplastic Agents; Biomarkers; Blotting, Northern; Bucladesine; Cell Differentiation; Cloning, Molecular; DNA Primers; Gene Expression Regulation, Neoplastic; Neural Cell Adhesion Molecules; Neurofilament Proteins; Neurons; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Incubation with all-trans retinoic acid (RA) induces PCC7-Mz1 embryonic carcinoma cells to cease proliferation and to develop into a tissue-like pattern of neuronal, astroglial, and fibroblast-like derivatives over a period of several days. Concomitant with the induction of differentiation by RA, a sizable fraction of the Mz1 stem cells detaches and dies, with the maximal level of cell death achieved after 10 h of RA treatment. This RA-induced cell death fulfills all criteria of apoptosis, including nuclear condensation, intranucleosomal DNA degradation, expression of cysteine aspases (caspases), and the formation of apoptotic bodies. Apoptosis could be suppressed by the pan-caspase inhibitor zVAD-fmk (benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethyl ketone). Induction of apoptosis required at least 2 h of incubation with RA and followed the same RA concentration (EC50 = 10(-7) M RA) and time dependence as the induction of differentiation as delineated by the expression of the neuron-specific protein kinase C substrate GAP-43. RA-induced apoptosis increased with the plating density of PCC7-Mz1 cells. This effect was not due to deprivation of an essential nutrient or factor from the medium because apoptosis was not significantly affected by an increase of the concentration of fetal calf serum. In addition to RA, apoptosis could be induced by DNA-damaging treatment (UV light, cisplatin, methanesulfonic acid methyl ester) and cell cycle-arresting agents (hydroxyurea) as well as by serum depletion. Because inhibition of transcription and translation caused cell death efficiently even in the presence of serum, the synthesis of apoptosis-inhibiting factors by the cultured cells is indicated. Neither ApoI/Fas antibody nor glutamate induced apoptosis. Mz1 cells that have entered a differentiation pathway in response to RA treatment become increasingly less sensitive to apoptosis. This may be due in part to the expression of the bcl-2 proto-oncogene, which was detectable on the mRNA and protein level beginning 4 days after the addition of RA. The intracellular signaling pathway leading to apoptosis does not involve conventional or novel members of the protein kinase C gene family. Neither activation of protein kinase C by phorbol esters (phorbol 12,13-dibutyrate) nor inhibition by specific inhibitors (GF109203X, Gö 6976) and long-term treatment with phorbol 12,13-dibutyrate, in the presence or absence of RA, significantly influenced the amount or rate of a

Topics: Animals; Apoptosis; Cell Differentiation; GAP-43 Protein; Gene Expression Regulation; Genes, bcl-2; Mice; Neurons; Protein Kinase C; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

The density of beta 1-adrenergic receptors (beta 1-AR) is up-regulated upon differentiation of embryonic F9 teratocarcinoma cells by retinoic acid (RA) to the primitive endodermal phenotype. To identify the domains involved in RA-mediated activation of beta 1-AR gene transcription, three kb of 5'-flanking sequence of the beta 1-AR gene were ligated to a luciferase reporter gene and transiently transfected into F9 cells that were pre-exposed to 100 nM RA for 2 days. By generating deletions in the beta 1-AR promoter, a region between -125 and -100 was found to mediate a 3-fold induction in cells exposed to RA for an additional 2 days. Through site-directed mutagenesis of this region, it was determined that the RA responsive element (RARE) was organized as a direct repeat separated by 5 nucleotides in which the 5'-most AGGTCG half-site was between nucleotides -106 and -101 and the 3'-most AGGTCA half-site was between nucleotides -117 and -112. The RA receptor alpha (RAR alpha) isoform bound to the oligomer representing the sequences between -125 and -100 as a heterodimer complex with the retinoid X receptor alpha (RXR alpha). In a separate study, it was determined that the nucleotides between -125 and -100 are involved in thyroid hormone-mediated activation of the beta 1-AR gene in ventricular myocytes. Therefore, transcriptional activation of the beta 1-AR gene by thyroid hormone or RA involves a single binding site in the promoter.

Topics: Animals; Base Sequence; Binding Sites; Gene Expression Regulation, Neoplastic; Luciferases; Mice; Mutagenesis, Site-Directed; Nuclear Proteins; Oligodeoxyribonucleotides; Promoter Regions, Genetic; Receptors, Adrenergic, beta-1; Recombinant Fusion Proteins; Sequence Deletion; Teratocarcinoma; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured; Up-Regulation

We investigated the expression of protein disulfide isomerase family proteins (PDI, ERp61, and ERp72) in mouse F9 teratocarcinoma cells during differentiation induced by treatment with retinoic acid and dibutyryl cAMP. Each member of this family was expressed at a constitutive level in undifferentiated F9 cells. During differentiation of F9 cells to parietal or visceral endodermal cells the protein level of all these enzymes increased, although the extent of this increase in both protein and mRNA levels varied among the enzymes. Certain proteins were found to be coimmunoprecipitated with PDI, ERp61, and ERp72 in the presence of a chemical crosslinker. Type IV collagen was significantly coprecipitated with PDI whereas laminin was equally coprecipitated with the three proteins. Furthermore, 210 kDa protein characteristically coprecipitated with ERp72. Thus, the induction of PDI family proteins during the differentiation of F9 cells and their association with different proteins may implicate specific functions of each member of this family despite the common redox activity capable of catalyzing the disulfide bond formation.

Topics: Animals; Blotting, Northern; Blotting, Western; Bucladesine; Cell Differentiation; Electrophoresis, Polyacrylamide Gel; Endoderm; Enzyme Induction; Fluorescent Antibody Technique; Heat-Shock Proteins; Humans; Isomerases; Membrane Glycoproteins; Mice; Precipitin Tests; Protein Disulfide-Isomerases; Stem Cells; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

The antiepileptic drug valproic acid (VPA) is an established human teratogen that affects neural development in human fetuses exposed to the drug during early pregnancy. The development of simple cell culture models that reflect normal neuronal development will be useful for the screening of neural developmental toxicity of new drugs and for the identification of the specific molecular targets for the teratogenic action. The present study tests a human NT2 cell line for its suitability to serve as such a model. Treatment with VPA, but not with its non-teratogenic-analogue, 2-isopropylpentanoic acid (IPPA), inhibited cell growth, had a cytotoxic effect and blocked retinoic acid-induced neuronal differentiation of NT2 cells. Differentiation was evaluated by immunostaining for neurofilament proteins, microtubule associated proteins-2 (MAP-2), tau and neural cell adhesion molecule, NCAM. However once differentiation had taken place, VPA did not revert this process, although it did affect neuronal aggregation and neurite fasciculation. These results suggest that the human NT2 cell line might be useful for identifying drugs affecting neuronal development in humans.

Topics: Anticonvulsants; Cell Aggregation; Cell Differentiation; Cell Division; Cell Survival; Humans; Mitosis; Molecular Weight; Neurites; Neurons; Pentanoic Acids; Teratocarcinoma; Teratogens; Tretinoin; Valproic Acid

The Wnt gene family encodes a series of conserved glycoproteins that regulate pattern formation during embryogenesis, in a variety of tissues including the nervous system. As with other genes that control embryonic cell differentiation, members of the Wnt family have also been implicated in tumourigenesis. To search for Wnt genes involved in human teratocarcinomas, with a possible role in human embryogenesis, we used RT-PCR primed with degenerate oligonucleotides to analyse mRNA from differentiating cultures of the pluripotent human embryonal carcinoma (EC) cell line NTERA-2. NTERA-2 EC cells differentiate into neurons and other cell types when induced with retinoic acid. Wnt gene expression was not detected in the undifferentiated EC cells, but Wnt-related PCR fragments were amplified from differentiating cultures, 4-14 days after induction with retinoic acid. The RT-PCR products were composed primarily of DNA fragments corresponding to the recently identified human Wnt-13 gene. No other Wnt-related genes were identified. Northern analysis confirmed induction of Wnt-13 as a 2.4 kb mRNA during the early phases of retinoic acid-induced differentiation, and during differentiation along a non-neural pathway induced by hexamethylene bisacetamide (HMBA), but not in the terminally differentiated neurons. Wnt-13 remained expressed in non-neural differentiated NTERA-2 cells, even several weeks after the induction of differentiation. The time course of induction, its induction by HMBA, and its persistence in differentiated cells indicate that Wnt-13 expression is not dependent upon direct activation by retinoic acid. Wnt-13 was not detected, or only detected at low levels, in other human EC cells. However, it was found to be expressed at a high level in one malignant teratoma cell line, 577MF, that does not exhibit an EC phenotype although it was derived from a testicular teratocarcinoma. At least two members of the human frizzled gene family, thought to encode receptors for Wnt proteins, were also expressed in the NTERA-2 cells, suggesting the presence of a mechanism by which endogenously expressed Wnt-13 could modulate the histogenesis of teratocarcinomas by mediating interactions between sub-populations of differentiating EC cells. We note that Wnt-13 maps to chromosome 1p13, a region reported to be subject to relatively frequent loss of heterozygosity in germ cell tumours. Further analysis indicated that 465 bp of the published Wnt-13 sequence, within the pred

Topics: Acetamides; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; Drosophila Proteins; Frizzled Receptors; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Male; Membrane Proteins; Molecular Sequence Data; Neoplasms, Germ Cell and Embryonal; Neurons; Receptors, G-Protein-Coupled; Sequence Homology, Nucleic Acid; Teratocarcinoma; Testicular Neoplasms; Tretinoin; Wnt Proteins

In the presence of retinoic acid (RA), F9 murine teratocarcinoma cells differentiate into cells resembling the extra-embryonic endoderm of the early mouse embryo. Using differential hybridization, we have cloned and characterized six cDNAs corresponding to mRNAs that exhibit reduced expression in F9 cells following RA treatment. Two of these cDNAs encode novel genes (REX-2 and REX-3). The other isolated cDNAs encode genes that have been previously described in other contexts: 1-4 (cyclin D3); 2-10 (pyruvate kinase); 2-12 (glutathione S-transferase); and 2-17 (GLUT 3). The mRNA levels of these genes are reduced by RA or RA plus theophylline and cAMP (RACT) only after 48 h of treatment, and continue to decrease at 96 h. The half-lives of these mRNAs are not changed by RA treatment, indicating that these mRNAs may be regulated through a transcriptional mechanism. In isoleucine-deprived cells, which are growth arrested but do not differentiate, the steady state mRNA levels of genes Rex 2, Rex 3, pyruvate kinase and GLUT 3 are not reduced, in contrast to cyclin D3 and glutathione S-transferase. The expression of the REX-2, REX-3, pyruvate kinase, glutathione S-transferase and GLUT 3 genes is reduced by RACT to the same extent in F9 RARgamma-/- and RARalpha-/- lines as in F9-Wt. In contrast, cyclin D3 exhibits lower mRNA expression in F9 RARgamma-/- and RARalpha-/- stem cells, and this mRNA is not decreased by RACT treatment. Overexpression of cyclin D3 blocks the RA-induced growth arrest of F9 cells, indicating that the downregulation of this gene following RA treatment may constitute a necessary step in the cascade of events leading to growth inhibition by RA.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Cell Differentiation; Cell Division; Cyclin D3; Cyclins; DNA-Binding Proteins; DNA, Complementary; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Mice; Molecular Sequence Data; Sequence Alignment; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

The zinc-finger transcription factor Krox24 was analysed for its role in differentiation in P19 embryonal carcinoma cells. Reciprocal dominant negative mutants consisting of Krox24 deleted for a crucial region of the zinc-finger domain (delta Krox24) or of the zinc-finger region alone (delta Krox24Zf) abolished the activation of transcription by Krox24 in P19 cells. Expression of Krox24 led to spontaneous differentiation of P19 cells in a lineage-independent fashion. Krox24 transfected populations, as well as individual clones randomly picked from them, displayed a wide array of diverse morphologies and expressed markers characteristic of a variety of differentiated cells. The dominant negative mutants blocked differentiation of P19 cells. We conclude that expression of Krox24 is sufficient for pluripotent differentiation of embryonal carcinoma cells, and that expression of Krox24 or other egr family members is essential to this process.

Topics: Amino Acid Substitution; Animals; Carcinoma, Embryonal; Cell Differentiation; DNA-Binding Proteins; Early Growth Response Protein 1; Immediate-Early Proteins; Mice; Mutation; Neoplasm Proteins; Phenotype; Recombinant Fusion Proteins; Sequence Deletion; Teratocarcinoma; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured; Zinc Fingers

Treatment with all-trans-retinoic acid (ATRA) induces complete remission in many acute promyelocytic leukemia patients. However, plasma drug levels progressively decrease following prolonged treatment with oral ATRA. This decrease is due, at least in part, to the induced cytochrome P-450-dependent metabolism of ATRA. To investigate if incorporation of ATRA in liposomes could alter its metabolism, we compared the cellular metabolism of liposomal-ATRA (L-ATRA) with free drug. Microsomes isolated from the rat liver metabolized L-ATRA to a significantly lower extent than they did free-ATRA. Similarly, in F9 cells, L-ATRA was metabolized at a slower rate than the free drug. These results suggest that L-ATRA may have important clinical implications in terms of slowing down the rate of ATRA metabolism and producing long-term remission in APL patients.

Topics: Animals; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Drug Carriers; Enzyme Inhibitors; Imidazoles; Liposomes; Male; Microsomes, Liver; Rats; Teratocarcinoma; Testicular Neoplasms; Tretinoin; Tumor Cells, Cultured

Calcitonin gene-related peptide (CGRP), expressed predominantly in F9 embryonal carcinoma cells, is both a potent chemotactic agent and an autocrine growth factor for these cells. We analyzed the effect of retinoic acid (RA)-induced differentiation of F9 cells into primitive parietal endoderm-like cells, on CGRP production and the CGRP responsiveness of these cells. Poly(A) RNA extracted from F9 cells and analysed by Northern blotting and hybridization with a CGRP probe showed a specific band of about 1200 bases corresponding to mature CGRP mRNA. This band was not detected in F9 cells treated for 6 days with RA (differentiated primitive parietal endoderm-like cells) or in PYS cells (established parietal endoderm-like cell line). During RA-induced differentiation of F9 cells, CGRP mRNA levels fell within 24 h after treatment and were almost undetectable after 2 days. RA treatment also reduced CGRP secretion by F9 cells; the effect was maximal at 3 days and remained stable thereafter. Similarly, RA rapidly reduced adenylate cyclase responsiveness to chicken CGRP (cCGRP) and human CGRP (hCGRP). An 80% fall in cAMP release into the culture medium in the presence of CGRP was observed after 24 h of RA treatment. These results demonstrate that RA rapidly abolishes the CGRP autocrine system involved in the proliferation of F9 cells, at the same time inducing their differentiation into primitive parietal endoderm. They point to the interaction between retinoic acid and growth factors in the regulation of cell proliferation and differentiation.

Topics: Adenylyl Cyclases; Blotting, Northern; Calcitonin Gene-Related Peptide; Cyclic AMP; DNA Primers; Factor Analysis, Statistical; Humans; Male; Polymerase Chain Reaction; Radioimmunoassay; RNA, Messenger; Teratocarcinoma; Testicular Neoplasms; Tretinoin; Tumor Cells, Cultured

Studies of sister chromatid exchanges (SCE) and recombination rate of certain minisatellite DNAs have demonstrated that their levels are considerably higher during the preimplantation stage than in latest developmental stages of embryos. It appeared likely that single-strand DNA breaks (SSB) may be relevant to both events during early development. With this in mind, we estimated SSB during in vitro retinoic acid (RA)-induced and spontaneous differentiation of mouse teratocarcinoma (EC) and embryonic stem (ES) cells. Using the method of nucleoid sedimentation and single-cell DNA electrophoresis, we have observed a dramatic increase in the SSB during the first 2-4 mitoses after beginning of differentiation of EC cells, followed by a gradual return to the basal level characteristic of undifferentiated cells. The increase in the SSB was manifested as the appearance of mass nucleoids with slow sedimentation rates, as well as the low-weight mass fragments in DNA patterns of most cells. We concluded that not less than half of genomic DNA has been nicked at the early steps of differentiation. The decrease in SSB level was observed in spite of continuing differentiation, as judged by embryonic antigens and morphological criteria. Also, the increase in the SCE level coincided with that of SSB, possibly being its consequence. The scheduled "surge" of SSB may be the earliest event in commencing differentiation at steps without a phenotypic manifestation.

Topics: Animals; Antigens; Cell Differentiation; Centrifugation, Density Gradient; DNA, Single-Stranded; Electrophoresis, Agar Gel; Embryo, Mammalian; Fluorescent Antibody Technique; Karyotyping; Mice; Mitosis; Sister Chromatid Exchange; Stem Cells; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Specific signaling molecules play a pivotal role in the induction and specification of tissues during early vertebrate embryogenesis. BMP-4 specifies ventral mesoderm differentiation and inhibits neural induction in Xenopus, whereas three molecules secreted from the organizer, noggin, follistatin and chordin dorsalize mesoderm and promote neural induction. Here we report that follistatin antagonizes the activities of BMP-4 in frog embryos and mouse teratocarcinoma cells. In Xenopus embryos follistatin blocks the ventralizing effect of BMP-4. In mouse P19 cells follistatin promotes neural differentiation. BMP-4 antagonizes the action of follistatin and prevents neural differentiation. In addition we show that the follistatin and BMP-4 proteins can interact directly in vitro. These data provide evidence that follistatin might play a role in modulating BMP-4 activity in vivo.

Topics: Animals; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Cell Aggregation; Cell Differentiation; Embryo, Nonmammalian; Embryonic Induction; Follistatin; Gene Expression Regulation, Developmental; Glycoproteins; Mesoderm; Mice; Nervous System; Polymerase Chain Reaction; Prolactin; RNA, Antisense; RNA, Messenger; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured; Xenopus; Xenopus Proteins

Gap junctions are plasma membrane specializations that allow direct communication among adjoining cells. We used a human pluripotential teratocarcinoma cell line, NTera-2/clone D1 (NT2/D1), as a model to study gap junctions in CNS neurons and their neuronal precursors. These cells were differentiated following retinoic acid (RA) treatment for 4 weeks and antiproliferative agents for 3 weeks, respectively, to yield post-mitotic CNS neuronal (NT2-N) cells. The cytoplasmic RNA was isolated from NT2/D1 cells both before and during RA treatment and from differentiated neurons (NT2-N cells). These RNA samples were examined using Northern blot analysis with cDNA probes specific for connexin26, -32, and -43. Connexin26 and -32 mRNAs were absent in NT2/D1 and NT2-N cells. Connexin43 mRNA was expressed at high levels in NT2/D1 cells before RA treatment, but it decreased significantly during RA induction. There was no detectable connexin43 mRNA in NT2-N cells. Western blot analysis confirmed the expression of connexin43 protein in NT2/D1 cells before and during RA treatment. The protein profile detected in Western blot analysis indicated two bands representing different phosphorylation states of connexin43. Our immunocytochemistry results did not show connexin26 and -32 immunoreactivity in NT2/D1 and NT2-N cells. However, we detected connexin43 immunoreactivity in NT2/D1 cells with a decreasing pattern upon RA induction. Both Western blotting and immunocytochemistry confirmed the absence of connexin43 protein in NT2-N cells. NT2/D1 cells passed calcein readily to an average of 18 cells, confirming the functionality of gap junctions in these cells. The extent of dye-coupling decreased about 78% when NT2/D1 cells were RA treated for 4 weeks. NT2-N differentiated neurons did not pass dye to the adjacent cells. We conclude that both connexin43 expression and dye coupling capacity decrease during neuronal differentiation of NT2/D1 cells.

Topics: Cell Communication; Cell Differentiation; Connexin 26; Connexin 43; Connexins; Fluoresceins; Fluorescent Dyes; Gap Junctions; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Microscopy, Fluorescence; Neoplasm Proteins; Nerve Tissue Proteins; Neurons; RNA, Messenger; RNA, Neoplasm; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Photoisomerization of all-trans-retinoic acid and the geometrical isomers [9-cis-retinoic acid, 11-cis-retinoic acid, 13-cis-retinoic acid and 9,13-di-cis-retinoic acid] in ethanol and their biological effects on F9 teratocarcinoma cells were analyzed. The rates of photoisomerization of the retinoic acids illuminated by fluorescent lamps (1,200 lx) increased in inverse proportion to their concentrations. When the ethanolic solution of all-trans-retinoic acid (10(-5) M) was kept under illuminated condition, the equilibrium mixture of the geometrical isomers of retinoic acid [all-trans-retinoic acid 25%, 9-cis-retinoic acid 10%, 11-cis-retinoic acid 10%, 13-cis-retinoic acid 30%, 9,13-di-cis-retinoic acid 5% and unidentified compound 20%] formed at around 30 min. The apparent velocity of the photoisomerization was approximately 8 x 10(-7) mol/L.min. Equilibrium mixtures with similar compositions were obtained by the photoisomerization of other geometrical isomers. The geometrical isomers produced by the photoisomerization possessed significantly different biological effects in the induction of differentiation of F9 cells into parietal endoderm-like cells: activities of 9-cis-retinoic acid (ED50, 8 x 10(-7) M), 11-cis-retinoic acid (ED50, 8 x 10(-7) M), and 13-cis-retinoic acid (ED50, 8 x 10(-7) M) were approximately 1/10 of all-trans-retinoic acid (ED50, 8 x 10(-8) M), and activity of 9,13-di-cis-retinoic acid (ED50, 1 x 10(-5) M) was 1/100 of the level of all-trans-retinoic acid. Further, the retinoic acids acted with each other additively on F9 cells.

Topics: Animals; Ethanol; Fluorescence; Isomerism; Light; Mice; Photochemistry; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

NT2 cells, a human teratocarcinoma cell line, are shown to be differentiated in neuron-like cells (NT2-N cells) by treatment with retinoic acid. The present study identified the neurotransmitter receptors expressed in NT2-N cells using patch-clamp recording. Voltage-sensitive Na+ currents, which are specific for neurons, were observed in NT2-N cells but not in NT2 cells, suggesting that NT2-N cells actually function as neurons. Glutamate receptor agonists, N-methyl-D-aspartate (NMDA) and kainate, evoked whole-cell currents. In addition, gamma-aminobutyric acid (GABA) evoked currents and the currents were inhibited by the selective GABAA receptor antagonist, bicuculline. In outside-out patches, GABA elicited single channel currents with two classes of the slope conductance (26 and 50 pS). No current, however, was induced by ACh, serotonin, or dopamine NT2-N cells, thus, express at least two types of the major excitatory and inhibitory neurotransmitter receptor in the central nervous system, the glutamate and GAGAA receptors, suggesting that these receptors have a crucial role in neurotransmission from the earlier stage of the brain development.

Topics: Bicuculline; Cell Differentiation; Electric Conductivity; Evoked Potentials; gamma-Aminobutyric Acid; Humans; Kainic Acid; Membrane Potentials; N-Methylaspartate; Neurons; Patch-Clamp Techniques; Receptors, GABA-A; Sodium Channels; Teratocarcinoma; Tetrodotoxin; Tretinoin; Tumor Cells, Cultured

Topics: Animals; beta-Galactosidase; Escherichia coli; Genes, Reporter; L Cells; Lac Operon; Luciferases; Mice; Receptors, Retinoic Acid; Recombinant Proteins; Retinaldehyde; Retinoid X Receptors; Teratocarcinoma; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured; Vitamin A

We have investigated the adenylyl cyclase (AC) activity and gene expression in retinoic acid (RA)-primed murine P19 teratocarcinoma cells, which recapitulate in vitro the first stages of neuroectodermal formation. Here we show that the P19 stem cells possess a basal Ca2+/CaM-stimulated AC activity, which increases about 10-fold after RA induction. The rise of AC activity is associated with a stage-specific up-regulation of AC2, AC5 and AC8 mRNAs and a down-regulation of AC3 mRNA. P19 cells provide a powerful model to investigate the role and specific regulation of AC isoforms during neuronal differentiation.

Topics: Adenylyl Cyclases; Animals; Blotting, Northern; Calcium; Calmodulin; Cell Differentiation; DNA Probes; Down-Regulation; Gene Expression Regulation, Enzymologic; Isoenzymes; Mice; Neurons; RNA, Messenger; Stem Cells; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured; Up-Regulation

alpha-Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, and all-trans-retinoic acid (RA) are known to induce F9 teratocarcinoma stem cell differentiation. Both compounds induce the formation of the same cell type, i.e., parietal endoderm-like cells expressing tissue plasminogen activator and collagen type IV alpha-1. The present study shows that DFMO and RA induce terminal differentiation of F9 cells through different pathways. Thus, retinoic acid receptor (RAR) alpha mRNA is weakly expressed during DFMO treatment, but strongly induced during an early phase of RA treatment. RAR beta mRNA is not detectable in DFMO-treated cells, but very strongly induced by RA and maintained at a high level throughout the differentiative process. RAR gamma mRNA is relatively strongly expressed in untreated control cells and remains at approximately the same level during DFMO-induced differentiation. In RA-treated cells, however, RAR gamma mRNA is rapidly down-regulated and becomes nondetectable during the final course of differentiation. These experiments show that the differentiation of F9 cells into parietal endoderm-like cells does not necessarily involve changes in any of the RAR mRNA subtypes. Even though the steady-state levels of the RAR alpha and RAR gamma transcripts may be sufficient to support the differentiative process, our data clearly show that induction of RAR beta mRNA transcription is neither a prerequisite for F9 cell differentiation, nor an absolute consequence of the elevated c-jun mRNA expression that is consistently observed during the course of parietal endoderm differentiation.

Topics: Animals; Cell Differentiation; Eflornithine; Embryonal Carcinoma Stem Cells; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, jun; Mice; Neoplastic Stem Cells; Ornithine Decarboxylase Inhibitors; Polyamines; Proto-Oncogene Proteins c-jun; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; RNA, Messenger; RNA, Neoplasm; Teratocarcinoma; Tissue Plasminogen Activator; Tretinoin; Tumor Cells, Cultured

We stably transfected a gene encoding a dominant negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA) into F9 cells and generated cell lines partially deficient in PKA activity (DN16 and DN19). In these cell lines, the retinoic acid (RA) receptor beta and laminin beta(1) chain (LAMB1) genes were regulated normally by RA alone, indicating that in the absence of exogenous modulation of cAMP levels, the PKA signaling pathway does not seem to play a major role in the RA-associated regulation of these genes. However, alterations in gene regulation were observed when the mutant cell lines were treated with a combination of RA and cAMP analogues. Moreover, in the DN16 cell line, which exhibits the lowest PKA activity among the mutant cell lines [22% of wild type (WT) at 1 microM cAMP], there was a significant decrease in the cAMP-associated activation of the LAMB1 gene DNase I hypersensitivity site 2 enhancer, as measured by chloramphenicol acetyl transferase assays. Using electrophoretic mobility shift assays, less protein binding was observed at one of the motifs (C2) within this enhancer region in the DN16 cells as compared to the F9 WT cells after treatment of the cells with RA and cAMP analogues for 24 h. Furthermore, no increase in C2 binding was observed when extracts from RA-treated F9 ST or DN16 cells were subjected to in vitro phosphorylation, suggesting that PKA is involved in the induction of the C2-binding protein in RA-treated cells. In contrast to the results with RA receptor beta and LAMB1, the effects of cAMP analogues on the RA-associated regulation of the bone morphogenetic protein 2 gene were not altered in the cell lines that exhibited reduced PKA activity. These results suggest that a partial reduction in PKA activity is not sufficient to abrogate the effects of cAMP analogues on all of the genes regulated by RA.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Gene Expression Regulation, Neoplastic; Laminin; Mice; Receptors, Retinoic Acid; Signal Transduction; Teratocarcinoma; Transfection; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

The linkage between Gialpha2 and morphogen-induced promotion of F9 embryonic teratocarcinoma stem (F9 stem) cells to primitive endoderm was explored using probes of the mitogen-activated protein (MAP) kinase network. The morphogen-induced decline in Gialpha2 is shown to trigger activation of phospholipase C, thereby activating protein kinase C, MAP kinase, and cell progression to primitive endoderm. In the absence of retinoic acid, reduction-of-function mutants (Gialpha2-deficient) display the effects of morphogen, i.e. activation of phospholipase C, protein kinase C, MAP kinase, and progression to primitive endoderm. Gain-of-function mutants (expressing the Q205L activating-mutation of Gialpha2) displayed no activation of phospholipase C, protein kinase C, MAP kinase and no progression to primitive endoderm, even in the presence of retinoic acid. Selective inhibitors of protein kinase C, like the gain-of-function mutations, effectively block morphogen-induced progression to primitive endoderm. Morphogen triggers F9 stem cell progression by triggering Gialpha2 loss and thereby activation of downstream elements, including protein kinase C and MAP kinase.

Topics: Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cell Differentiation; Diglycerides; Embryonal Carcinoma Stem Cells; Endoderm; Enzyme Activation; Gene Expression Regulation, Enzymologic; GTP-Binding Proteins; Mice; Mitogen-Activated Protein Kinase Kinases; Neoplastic Stem Cells; Protein Kinase C; Protein Kinases; RNA, Messenger; Signal Transduction; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured; Type C Phospholipases

Treatment of mouse teratocarcinoma F9 cells with all-trans-retinoic acid (RA) causes a 9-fold increase in steady-state levels of mRNA for UDP-Gal:beta-D-Gal alpha1,3-galactosyltransferase (alpha1,3GT) beginning at 36 h. Enzyme activity rises in a similar fashion, which also parallels the induction of laminin and type IV collagen. Nuclear run-on assays indicate that this increase in alpha1,3GT in RA-treated F9 cells, like that of type IV collagen, is transcriptionally regulated. Differentiation also results in increased secretion of soluble alpha1,3GT activity into the growth media. The major alpha-galactosylated glycoprotein present in the media of RA-treated F9 cells, but not of untreated cells, was identified as laminin. Differentiation of F9 cells is accompanied by an increase in alpha-galactosylation of membrane glycoproteins and a decrease in expression of the stage-specific embryonic antigen, SSEA-1 (also known as the Lewis X antigen or LeX), which has the structure Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-R. However, flow cytometric analyses with specific antibodies and lectins, following treatment of cells with alpha-galactosidase, demonstrate that differentiated cells contain LeX antigens that are masked by alpha-galactosylation. Thus, RA induces alpha1,3GT at the transcriptional level, resulting in major alterations in the surface phenotype of the cells and masking of LeX antigens.

Topics: Animals; Bucladesine; Carbohydrate Conformation; Carbohydrate Sequence; Carcinoma, Embryonal; Cell Differentiation; Cell Nucleus; Flow Cytometry; Galactosyltransferases; Gene Expression Regulation, Neoplastic; Glycosylation; Kinetics; Laminin; Lewis X Antigen; Membrane Glycoproteins; Mice; Molecular Sequence Data; Raffinose; RNA, Messenger; Teratocarcinoma; Time Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The mammalian achaete-scute homolog 1 (MASH1) protein is required for the early development of the nervous system. However, the molecular and biochemical mechanism by which MASH1 acts to determine neurogenesis are still unknown. The myocyte enhancer factor 2A (MEF2A) is a MADS transcription factor that is essential for the specification and differentiation of the muscle lineage. Here we show that MEF2A and MASH1 are coordinately induced during the differentiation of the teratocarcinoma cell line P19 along a neuronal lineage and that in transient transfection assays, MEF2A and MASH1 cooperatively activate gene expression. This cooperativity appears to be due to a specific physical interaction between MEF2A and MASH1. Taken together, these findings suggest that MASH1 via a cooperative interaction with MEF2A may regulate the expression of specific genes that are critical for neuronal differentiation.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Binding Sites; Cell Differentiation; Cell Line; Chloramphenicol O-Acetyltransferase; Chlorocebus aethiops; DNA-Binding Proteins; Mammals; MEF2 Transcription Factors; Mice; Mutagenesis; Myogenic Regulatory Factors; Neurons; Protein Binding; Recombinant Proteins; Sequence Deletion; Teratocarcinoma; Transcription Factors; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA)-induced endodermal differentiation of F9 teratocarcinoma cells is accompanied by altered of many genes, including the retinoic acid receptor (RAR) alpha, beta, and gamma and retinoic x receptor (RXR) alpha and gamma genes. In addition, RA enhances the binding of nuclear receptors to the RA responsive elements and increases the trans-activation of the RA-responsive genes in F9 cells. These data suggest that RA increases the overall function of RA receptors in F9 cells. However, the cell line F9E, established by long-term exposure of F9 cells to RA, is insensitive to RA. These cells expressed the genes for keratin 8 and tissue specific plasminogen activator (t-PA), which are characteristic of endoderm cells. Northern blot analysis showed that F9E cells expressed high basal levels of RAR and RXR mRNAs, but genes including the RARs and RXRs, which are normally regulated by RA in F9 cells, were no longer regulated in F9E cells. Further, F9E cells were not sensitive to the antiproliferative effect of RA. In these cells, high levels of RA receptors constitutively bound to RA responsive elements, but RA could neither increase the amount of RA receptor binding to the RA responsive elements, nor enhance the transcription of the RA target genes. These findings provide a possible explanation for insensitivity of endodermal cells to the actions of RA. Our data also indicate that the levels of endogenous RA receptors do not predict the degree of RA sensitivity, and that RA responsiveness might be a function of the cell's ability to regulate RA receptor genes.

Topics: Animals; Basal Metabolism; Base Sequence; Cell Differentiation; Cell Line; Endoderm; Gene Expression Regulation; Mice; Molecular Sequence Data; Receptors, Retinoic Acid; Teratocarcinoma; Tretinoin

Retinoic acid, a developmental signal implicated in the formation of the neural axis, is present at high levels in the early embryonic trunk region, where it is synthesized by a novel dehydrogenase. Here we show that the same enzyme is inducible by retinoic acid in P19 teratocarcinoma cells, and we report the cloning from P19 cells of a cDNA encoding a novel dehydrogenase, named retinaldehyde dehydrogenase-2 (RALDH-2). Expression in COS cells shows RALDH-2 to be highly effective in oxidation of retinaldehyde, with no detectable activity on any other aldehyde tested. In situ hybridization histochemistry on the embryonic trunk reveals RALDH-2 mRNA both in mesoderm and neuroectoderm, with highest neuroectodermal expression in the ventral horn of the spinal cord at two restricted locations along the anteroposterior axis, presumably the subpopulation of motoneurons that innervate the limbs.

Topics: Aging; Aldehyde Oxidoreductases; Amino Acid Sequence; Animals; Antisense Elements (Genetics); Base Sequence; Brain; Cell Line; Chlorocebus aethiops; Cloning, Molecular; DNA Primers; Embryo, Mammalian; Embryonic and Fetal Development; Enzyme Induction; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; In Situ Hybridization; Liver; Male; Mice; Molecular Sequence Data; Organ Specificity; Polymerase Chain Reaction; Recombinant Proteins; Retina; Retinal Dehydrogenase; RNA, Messenger; Teratocarcinoma; Testis; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured

Tera-2 is a human teratocarcinoma cell line, which is induced to differentiate into neuronal direction by retinoic acid. Once differentiated, the cells form an almost nondividing population that can be maintained for weeks under conventional culture conditions. If differentiation by retinoic acid is induced while the cells are growing on type I collagen or if the already-differentiated cells are transferred onto collagen, they survive only a few days unless the cultures are repeatedly supplied with FGF-2. Lack of this growth factor induces programmed cell death (apoptosis) detectable after 24-48 h, as marked by DNA cleavage and nuclear fragmentation. The undifferentiated stem cells survive and proliferate readily on collagen without addition of FGF-2. Tera-2 cells express two members of the FGF family, FGF-2 and FGF-4. The expression of both FGFs is turned off during differentiation on collagen substratum, whereas when cultivated on plain tissue culture dish, the expression of only FGF-4 becomes undetectable. The results indicate that signaling through cell surface FGF receptors is vital for the cells, and differentiation on collagen substratum results in complete extinction of the autocrine stimulatory loop. In vivo, such induction of growth factor dependency upon differentiation would result in apoptotic death of those cells which fail to find adequate conditions for continuing FGF stimulation.

Topics: Apoptosis; Cell Differentiation; Cell Line; Cell Nucleus; Collagen; Fibroblast Growth Factor 2; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Humans; Kinetics; Neurons; Proto-Oncogene Proteins; Recombinant Proteins; RNA, Messenger; Teratocarcinoma; Time Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Topics: Animals; Bucladesine; Cell Differentiation; Endoderm; Fibronectins; Mice; Microscopy, Electron; Spheroids, Cellular; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

The retinoic acid receptors (RARs) transduce retinoid dependant gene regulation, and many biological effects of retinoids are mediated through binding and activation of three closely related receptor subtypes (RAR alpha, RAR beta, and RAR gamma). In order to investigate the role of receptor subtypes, we have carried out a chemical synthesis program to seek selective retinoids for these receptors. We measured receptor binding affinity using recombinant RAR alpha, -beta, and -gamma proteins and assessed cellular differentiating activity in F9 murine teratocarcinoma cells (F9 cells). This research has identified the 4-substituted-3-(1-adamantyl)phenyl moiety as a new pharmacophore which can replace the beta-cyclogeranylidene ring of the naturally occurring all-trans-retinoic acid. Two chemical series derived from the general structures 6-(3-tertioalkylphenyl)-2-naphthoic acid (series I) and 4-[(E)-2-(3-tertioalkylphenyl)propenyl]benzoic acid (series II) were developed. In particular, we have obtained the RAR gamma selective derivatives 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthoic acid (7) [Ki(RAR alpha) = 6500 nM, Ki(RAR beta) = 2480 nM, Ki(RAR gamma) = 77 nM] and 4-[(E)-2-[3-(1-adamantyl)-4-hydroxyphenyl]propenyl]benzoic acid (19) [Ki(RAR alpha) = 1,144 nM, Ki(RAR beta) = 1245 nM, Ki(RAR gamma) = 53 nM]. In series I, the presence of a phenol group, irrespective of the nature of tertioalkyl group, imparted at least partial RAR gamma selectivity, whereas in series II, the presence of both adamantyl and phenol groups is needed to confer RAR gamma selectivity. The RAR gamma selective ligands induce differentiation in F9 cells (7, AC50 = 33 nM; 19, AC50 = 66 nM). From series I, a mixed RAR beta-gamma agonist with potent cellular differentiating activity was selected for development as a topical antiacne agent, 6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid (5, CD 271) [Ki(RAR alpha) = 1100 nM, Ki-(RAR beta) = 34 nM, Ki(RAR gamma) = 130 nM, AC50(F9) = 37 nM]. Finally, from series II, we have obtained a weak antagonist in the F9 cellular differentiation assay, 4-[(E)-2-(3-tert-butyl-4-hydroxyphenyl)propenyl]benzoic acid (15, IC50 = 700 nM).

Topics: Animals; Cell Differentiation; Magnetic Resonance Spectroscopy; Mice; Receptors, Retinoic Acid; Recombinant Proteins; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Retinoids; Structure-Activity Relationship; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA), a well-known inducer of differentiation, has been shown to regulate its own receptor gene expression in F9 teratocarcinoma cells. The homologous regulation of receptors by RA might be critical for RA-induced F9 cell differentiation. F9 cell lines from two different laboratories, named F9-1 and F9-2, were compared for retinoic acid receptor (RAR) and retinoid x receptor (RXR) gene expression in response to RA. The data show that both F9-1 and F9-2 cell lines are embryonal carcinoma cells, but of different phenotypes and different sensitivity to RA. In F9-1 cells, RA regulates all three RARs (alpha, beta, and gamma), two RXRs (alpha and gamma), two activin receptors (ActR II and IIB), and tissue-specific plasminogen activator (t-PA) gene expression. In F9-2 cells RA regulates only the RAR beta, RXR alpha, and t-PA genes. The induction of mRNA levels was much higher in F9-1 than in F9-2 cells. Different basal RAR gamma and RXR gamma mRNA levels were also noted. In these two cell lines F9-2 cells expressed greater amounts of RAR gamma 1, gamma 2, and gamma 3 mRNA isoforms, but lacked RXR gamma mRNA compared with F9-1 cells. Since RAR gamma 1 has been shown to exert an antagonistic effect on other types of RA receptors, the decreased sensitivity of F9-2 cells to RA might be due to its high level of RAR gamma 1 and/or low level of RXR gamma. This notion was in part supported by gel shift assay which demonstrated constitutive binding of RAR gamma to a RA responsive element (RAR beta E) in F9-2 cells. Further, the binding of nuclear protein to RAR beta E was increased upon RA treatment in F9-1 cells, but not in F9-2 cells. These differences in the regulation of RA receptors might determine the sensitivity of the two substrains of F9 cells to RA.

Topics: Animals; Cell Differentiation; Keratins; Lewis X Antigen; Mice; Receptors, Retinoic Acid; Retinoid X Receptors; Teratocarcinoma; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Mouse embryonal carcinoma F9 cells are pluripotent stem cells and differentiate into primitive endodermal cells upon treatment with retinoic acid (RA). We have recently shown that in F9 cells RA regulates gene expression of activin receptor type II (ActR-II), whose ligand is a potent differentiation agent. The present study examined the regulation of the newly cloned activin receptor type IIB (ActR-IIB) gene by RA. F9 cells expressed equal amounts of three ActR-IIB transcripts of 8.0, 7.5 and 4.0 kb. Both 9-cis-RA (c-RA) and all-trans-RA (t-RA) induced ActR-IIB gene expression in a dose-dependent manner. At 10(-9) M c-RA exerted no effect, while 10(-5) M c-RA increased the 8.0 kb ActR-IIB transcript about sevenfold. In contrast, t-RA induced the 8.0 kb ActR-IIB transcript fivefold at 10(-9) M and up to eightfold at 10(-5) M. The inductive effect on the 8.0 kb transcript was greater than that on the 7.5 kb transcript, and was least effective on the 4.0 kb transcript, suggesting that these three mRNA isoforms may originate from different promoters. Both cycloheximide and actinomycin D inhibited the inductive effect of t-RA on ActR-IIB gene expression, in contrast to ActR-II whose gene expression was not suppressed by cycloheximide but abolished by actinomycin D. Thus, endodermal differentiation of F9 cells is associated with activation of ActR-IIB gene and the mechanisms involved in the regulation of ActR-II and IIB gene expression are different.

Topics: Activin Receptors; Animals; Cell Differentiation; Endoderm; Gene Expression Regulation, Neoplastic; Mice; Neoplasm Proteins; Receptors, Growth Factor; Stem Cells; Teratocarcinoma; Tretinoin

Homeobox genes code for transcription factors and are arranged in clusters, named A, B, C and D, found on four separate chromosomes in vertebrates. They contain a homeobox DNA sequence which codes for the homeodomain, a region of amino acids responsible for the DNA binding exhibited by these proteins. During embryonic development, the homeobox genes are both spatially and temporally regulated. In teratocarcinoma cell cultures, homeobox genes are regulated by retinoic acid (RA). The cDNAs from the first gene in the human HOX A cluster, HOX A1 (1.6), were cloned and the nucleotide sequence of a full-length cDNA was determined. It is highly homologous to its murine counterpart. Another HOX A1 cDNA was cloned, corresponding to an alternatively spliced form. In vitro translation of the full-length cDNA clone gave rise to a protein of 36 kDa. In PA-1 human teratocarcinoma cells HOX A1 is the earliest HOX A gene to be expressed after treatment with RA. To test whether HOX A1 could function as a early regulator of other HOX A cluster genes, we cotransfected into PA-1 human teratocarcinoma cells sense and antisense HOX A1 cDNAs expressed from an SV40 promoter with a 5.4-kb RA-sensitive HOX A4 (1.4) promoter-cat reporter. We found no effect of HOX A1 on the HOX A4 promoter. However, cotransfection of HOX A5 (1.3) was able to inhibit the HOX A4 promoter activity.

Topics: Alternative Splicing; Amino Acid Sequence; Base Sequence; Cloning, Molecular; DNA, Complementary; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Homeodomain Proteins; Humans; Models, Genetic; Molecular Sequence Data; Multigene Family; RNA, Messenger; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Species Specificity; Teratocarcinoma; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Chicken ovalbumin upstream promoter-transcription factors (COUP-TF) are expressed in the developing nervous system and interact with nuclear hormone receptors to regulate expression of different genes. The role of COUP-TF orphan receptors in neurogenesis is virtually unknown. To study the possible function of COUP-TF I during neuronal differentiation, we generated COUP-TF I overexpressing teratocarcinoma PCC7 cell lines and analyzed retinoic acid (RA)-induced neuronal differentiation of these cells. COUP-TF I overexpression results in the blockade of morphological differentiation after induction to differentiate. COUP-TF I represses expression of microtubule-associated protein 2 (MAP2) gene and delays induction of growth-associated protein 43 (GAP43) gene expression. In contrast, expression of the neurofilament light subunit (NF-L) gene is not affected by COUP-TF I overexpression during neuronal differentiation. Also, cells overexpressing COUP-TF I do not stop proliferating after RA and dBcAMP treatment and possess suppressed transcriptional activation from different RA response elements. These results suggest that COUP-TF I plays an important role in regulating RA-induced neuronal differentiation.

Topics: Animals; Base Sequence; Biomarkers; Bucladesine; Cell Cycle; Cell Differentiation; COUP Transcription Factor I; DNA-Binding Proteins; Enhancer Elements, Genetic; GAP-43 Protein; Gene Expression; Membrane Glycoproteins; Mice; Microtubule-Associated Proteins; Molecular Sequence Data; Nerve Tissue Proteins; Neurofilament Proteins; Receptors, Glucocorticoid; Teratocarcinoma; Transcription Factors; Tretinoin; Tumor Cells, Cultured

F9 embryonic teratocarcinoma stem cells differentiate into an epithelial cell type called extraembryonic endoderm when treated with retinoic acid (RA), a derivative of retinol (vitamin A). This differentiation is presumably mediated through the actions of retinoid receptors, the RARs and RXRs. To delineate the functions of each of the different retinoid receptors in this model system, we have generated F9 cell lines in which both copies of either the RAR alpha gene or the RAR gamma gene are disrupted by homologous recombination. The absence of RAR alpha is associated with a reduction in the RA-induced expression of both the CRABP-II and Hoxb-1 (formerly 2.9) genes. The absence of RAR gamma is associated with a loss of the RA-inducible expression of the Hoxa-1 (formerly Hox-1.6), Hoxa-3 (formerly Hox-1.5), laminin B1, collagen IV (alpha 1), GATA-4, and BMP-2 genes. Furthermore, the loss of RAR gamma is associated with a reduction in the metabolism of all-trans-RA to more polar derivatives, while the loss of RAR alpha is associated with an increase in metabolism of RA relative to wild-type F9 cells. Thus, each of these RARs exhibits some specificity with respect to the regulation of differentiation-specific gene expression. These results provide an explanation for the expression of multiple RAR types within one cell type and suggest that each RAR has specific functions.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Division; Cell Line; Chromatography, High Pressure Liquid; DNA Primers; Gene Expression; Kinetics; Mice; Molecular Sequence Data; Mutagenesis; Polymerase Chain Reaction; Receptors, Retinoic Acid; Recombinant Proteins; Recombination, Genetic; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Retinoids; Teratocarcinoma; Transfection; Tretinoin; Tumor Cells, Cultured

A reporter cell line was established from F9 mouse teratocarcinoma cells containing the RAR beta 2 promoter coupled to the lacZ (beta-galactosidase) reporter gene. All-trans-, 9-cis-, and all-trans-4-oxoretinoic acid were equipotent in inducing cell differentiation at 1 microM, determined by induction of collagen IV mRNA expression, of morphological changes, as well as of beta-galactosidase enzyme activity. By the same criteria, beta-carotene at 10 microM also induced differentiation, but less strongly and more slowly than the retinoic acids. In contrast, the oxocarotenoid (or xanthophyll) canthaxanthin, at 10 microM, had little effect on differentiation, unless preincubated in culture medium, from which 4-oxoretinoic acid was recovered and identified as a decomposition product. This indicates that canthaxanthin can act as an effective inducer of differentiation only after breakdown to active metabolites. Likewise, beta-carotene probably also acts subsequent to breakdown to retinoic acid. Throughout these experiments the response of the RAR beta promoter-lacZ reporter gene correlated well with other parameters of differentiation, making this cell line a useful system for examination of inducers of embryonal carcinoma cell differentiation.

Topics: Animals; beta Carotene; Canthaxanthin; Carotenoids; Cell Differentiation; Collagen; Gene Expression Regulation, Neoplastic; Genes, Reporter; Lac Operon; Mice; Promoter Regions, Genetic; Receptors, Retinoic Acid; RNA, Messenger; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

The differentiation of F9 teratocarcinoma cells mimics the formation of a mouse embryonic tissue, the primitive endoderm. In vitro, small aggregates of F9 cells, termed embryoid bodies, differentiate in response to retinoic acid and develop a surface epithelium that is characterized by the production of alpha-fetoprotein. In the present study, cellular autofluorescence profiles obtained by fluorescence-activated embryoid bodies were composed of a single type of cell. In contrast, retinoic acid-induced embryoid bodies were composed of two cell types: a major population displaying autofluorescence levels similar to those of cells from undifferentiated embryoid bodies and a second population displaying higher autofluorescence. RNA analyses demonstrated that the transcription of alpha-fetoprotein was associated only with the more highly autofluorescent population, indicating that flow cytometry provides a novel mechanism for the separation of undifferentiated cells from differentiated endoderm cells in F9 embryoid bodies.

Topics: Animals; Antibodies; Cell Differentiation; Cell Line; Cell Separation; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Immunohistochemistry; Mice; Rabbits; RNA; RNA, Neoplasm; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured; Wheat Germ Agglutinins

Changes in Hox1.6 and c-jun gene expression were examined upon F9 cell differentiation that was induced by three independent methods: a drug treatment with retinoic acid (RA), that with sodium butyrate (NaB), and a genetic approach using the ts mutant. To obtain further information on the mechanism of teratocarcinoma cell differentiation we have examined the kinetics of the induction of Hox1.6 and c-jun mRNA whose gene products have been demonstrated to have specific roles in gene regulation. Expression of Hox1.6 mRNA was induced more rapidly than c-jun mRNA by all the above three inducing methods. Furthermore, protein synthesis was not required for the induction of Hox1.6 mRNA as well as of c-jun mRNA synthesis in all three methods. The data suggested that the transcriptional increase in the Hox1.6 mRNA was a primary response and could play an important role in F9 cell differentiation.

Topics: Butyrates; Butyric Acid; Cell Differentiation; Cycloheximide; Gene Expression Regulation; Homeodomain Proteins; Humans; Kinetics; Mutation; Proto-Oncogene Proteins c-jun; RNA, Messenger; Teratocarcinoma; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Telomerase, a ribonucleic acid-protein complex, adds hexameric repeats of 5'-TTAGGG-3' to the ends of mammalian chromosomal DNA (telomeres) to compensate for the progressive loss that occurs with successive rounds of DNA replication. Although somatic cells do not express telomerase, germ cells and immortalized cells, including neoplastic cells, express this activity. To determine whether the phenotypic differentiation of immortalized cells is linked to the regulation of telomerase activity, terminal differentiation was induced in leukemic cell lines by diverse agents. A pronounced downregulation of telomerase activity was produced as a consequence of the differentiated status. The differentiation-inducing agents did not directly inhibit telomerase activity, suggesting that the inhibition of telomerase activity is in response to induction of differentiation. The loss of telomerase activity was not due to the production of an inhibitor, since extracts from differentiated cells did not cause inhibition of telomerase activity. By using additional cell lineages including epithelial and embryonal stem cells, down-regulation of telomerase activity was found to be a general response to the induction of differentiation. These findings provide the first direct link between telomerase activity and terminal differentiation and may provide a model to study regulation of telomerase activity.

Topics: Animals; Base Sequence; Butyrates; Butyric Acid; Calcitriol; Cell Differentiation; Cell Line; Cell Nucleus; Colonic Neoplasms; Cytoplasm; Dimethyl Sulfoxide; Growth Inhibitors; HL-60 Cells; Humans; Interleukin-6; Kidney; Leukemia Inhibitory Factor; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphokines; Mice; Repetitive Sequences, Nucleic Acid; Stem Cells; Telomerase; Teratocarcinoma; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

PA-1 cell line is derived from human ovary teratocarcinoma. When it grows in 10% fetal calf serum, it can be induced to differentiate by 10(-5) mol/L retinoic acid (RA). Some morphological changes can be observed after RA induction. By immunostaining of cultured cells, we found that the expression and distribution pattern of desmin and some extracellular matrix molecules, such as fibronectin, laminin and tenascin, had been changed after induction, and these changes were associated with the morphological changes. Cell growth study showed that RA treatment had no effects on growth and autocrine activities of PA-1 cells. These results suggest that some PA-1 cells were induced to differentiate along muscle cells pathway by RA.

Topics: Cell Transformation, Neoplastic; Child; Desmin; Extracellular Matrix; Female; Fibronectins; Humans; Ovarian Neoplasms; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Mitogenic stimulation of protein synthesis is accompanied by an increase in eIF-4E phosphorylation. The effect on protein synthesis by induction of differentiation is less well known. We treated P19 embryonal carcinoma cells with the differentiating agent retinoic acid and found that protein synthesis increased during the first hour of addition. However, the phosphorylation state, as well as the turnover of phosphate on eIF-4E, remained unchanged. Apparently, the change in protein synthesis after RA addition is regulated by another mechanism than eIF-4E phosphorylation. By using P19 cells overexpressing the EGF receptor, we show that the signal transduction pathway that leads to phosphorylation of eIF-4E is present in P19 cells; the EGF-induced change in phosphorylation of eIF-4E in these cells is likely to be regulated by a change in eIF-4E phosphatase activity. These results suggest that the onset of retinoic acid-induced differentiation is triggered by a signal transduction pathway which involves changes in protein synthesis, but not eIF-4E phosphorylation.

Topics: Amino Acid Sequence; Cell Differentiation; Embryonal Carcinoma Stem Cells; Epidermal Growth Factor; ErbB Receptors; Eukaryotic Initiation Factor-4E; Molecular Sequence Data; Neoplasm Proteins; Neoplastic Stem Cells; Peptide Initiation Factors; Phosphates; Phosphorylation; Signal Transduction; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

The teratocarcinoma-derived F9 cells respond to retinoic acid (RA) and RA plus dibutyrylcyclic adenosine monophosphate (dcAMP) by differentiating into endoderm cells, which elaborate a laminin and type IV collagen-rich matrix. We found that the induction of differentiation is accompanied by a small but consistent increase in cell adhesiveness to a variety of substrates, including laminin. Therefore we investigated biochemical mechanisms involved in this phenomenon. Endoglycosidase treatment showed that laminin contains complex and hybrid oligosaccharide structures. RA enhanced general biosynthesis of laminin without a specific increase in galactose incorporation: this sugar was mainly in polylactosamine structures in the A chain of laminin and as terminal galactose alpha 1,3 galactose in the B chain. Laminin receptor analysis showed that RA decreased laminin binding protein-37 (LBP-37) but increased the amount of beta 1 integrin, suggesting the involvement of beta 1 integrin in the attachment process. Northern blot analysis showed increased expression of retinoid receptors within hours of RA exposure. These studies demonstrate that RA increases cell to substrate interactions by increasing the biosynthesis of laminin and beta 1 integrin. These effects are most likely subsequent to the RA-induced biosynthesis of the retinoid receptors.

Topics: Cell Adhesion; Cell Differentiation; Galactose; Integrins; Laminin; Lectins; Oligosaccharides; Receptors, Laminin; Receptors, Retinoic Acid; Staining and Labeling; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Treatment of the human teratocarcinoma line NTera2/c1.D1 (NT2) with retinoic acid induces terminal neuronal differentiation. In a previous study, we found that the neurons obtained in this way express functional N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor channels. We now show by reverse transcriptase-polymerase chain reaction and Southern blotting that these neurons transcribe each of the nine known non-NMDA glutamate receptor genes (GluR1-7, Ka-1, and Ka-2) and that four of these genes (GluR2, GluR6, GluR7, and Ka-1) are also transcribed by undifferentiated NT2 cells. Patch clamp studies demonstrate that individual non-NMDA glutamate receptor channels are readily isolated from NT2-derived neurons and that these channels are potently modulated by the desensitization blocker cyclothiazide. NT2-derived neurons are susceptible to kainate excitotoxicity but are not injured by prolonged exposure to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. We expect that the NT2-derived human neuronal culture system will facilitate studies of human neuronal non-NMDA glutamate receptor channels and of the pathophysiology of neuronal excitotoxicity.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Base Sequence; Blotting, Southern; Cell Differentiation; Clone Cells; DNA Primers; Gene Expression; Humans; Ion Channels; Kainic Acid; L-Lactate Dehydrogenase; Molecular Sequence Data; Neurons; Polymerase Chain Reaction; Quinoxalines; Receptors, Glutamate; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Induction of differentiation of F9 teratocarcinoma stem cells by retinoic acid and cAMP has been shown to involve the activation of the transcription factor AP-1 (a heterodimer of the proto-oncogene products c-Fos and c-Jun); moreover, stable expression of either Fos or Jun drives F9 cells into differentiation. Phorbol ester tumor promoters and short-wave-length ultraviolet (uv) irradiation are efficient inducers of AP-1 activity in various differentiated cells, but it has been shown that phorbol esters do not induce AP-1 activity in undifferentiated F9 cells. We examine here whether uv irradiation induces AP-1 activity in these cells and drives F9 cells into differentiation. We show that uv induces, in contrast to phorbol esters, the formation of active AP-1 by activating transcription from the c-jun gene. Ultraviolet-induced AP-1 drives transcription from AP-1-dependent promoters coding for differentiation-associated proteins (such as urokinase and keratin 18). However, in uv-treated cells, these genes are activated earlier and to a greater extent than in cells treated with retinoic acid and cAMP. More importantly, uv, in contrast to retinoic acid and cAMP, does not induce the accumulation of collagen alpha 1 (IV) and laminin B1 RNA. Our data suggest that the c-jun gene in F9 cells is accessible to immediate activation, but that uv-induced AP-1 activation does not suffice to induce the full program of F9 cell differentiation.

Topics: Animals; Cell Differentiation; Collagen; Cyclic AMP; Gene Expression Regulation, Neoplastic; Genes, jun; Keratins; Laminin; Mice; Phorbol Esters; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; RNA, Messenger; Stem Cells; Teratocarcinoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Ultraviolet Rays; Urokinase-Type Plasminogen Activator

Retinoic acid (RA) has striking effects on vertebrate development and induces differentiation of several lines of cells including embryonal carcinoma F9 cells. It is generally accepted that the actions of RA are mediated by nuclear receptors for RA. However, we now provide evidence that F9 cells can differentiate in response to RA without trans-activation by nuclear receptors. Irreversible differentiation of F9 cells was induced by 18 h of exposure to RA with subsequent incubation in the absence of RA. This induction of differentiation was not blocked after inhibition of protein synthesis and mRNA synthesis during the 18-h treatment with RA, but the endogenous RA receptors failed to activate transcription from their target genes that contain the receptor-binding sequences. During the commitment to RA-induced differentiation, at least five sets of four phosphorylated proteins underwent changes in the absence of protein synthesis de novo. These results suggest that there is a novel pathway for the action of RA that is independent of nuclear receptor-mediated trans-activation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Cell Differentiation; Genes, jun; Isoquinolines; Mice; Phosphoproteins; Piperazines; Protein Kinase Inhibitors; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Staurosporine; Teratocarcinoma; Time Factors; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

We have investigated the retinoic acid-mediated activation of the transcriptional regulator HNF-3 beta during differentiation of mouse F9 embryonal carcinoma cells. Using gel shifts, HNF-3 beta DNA binding activity was clearly detected in differentiated cells, while F9 stem cells were devoid of this activity. We also demonstrated that HNF-3 beta mRNA is specific for differentiated cells. Addition of retinoic acid to F9 stem cells results in delayed activation of HNF-3 beta mRNA which can be detected 1-2 days after the initiation of differentiation. HNF-3 beta mRNA concentrations are maximal at approximately 4 days postdifferentiation and stay at elevated levels for at least 4 additional days. Nuclear run-on experiments clearly show that HNF-3 beta is activated at the level of transcriptional initiation, suggesting that the increases of beta-specific DNA binding activity and mRNA concentration are merely a reflection of this activation mechanism. F9 cells can give rise to three distinct differentiated cell types, visceral endoderm, parietal endoderm, and primitive endoderm, and we have observed HNF-3 beta stimulation during the formation of all three tissues. HNF-3 beta stimulation upon visceral endoderm differentiation is accompanied by the activation of HNF-3 target genes such as transthyretin, suggesting that HNF-3 beta is involved in the developmental activation of this gene. In contrast, HNF-3 beta target genes in parietal and primitive endoderm have yet to be identified. However, the stimulation of HNF-3 beta during primitive endoderm formation, which is an extremely early event during murine embryogenesis, points toward a role for the factor in crucial determination processes that occur early during development.

Topics: Animals; Base Sequence; Cell Differentiation; DNA-Binding Proteins; Endoderm; Gene Expression Regulation; Hepatocyte Nuclear Factor 3-beta; Mice; Molecular Sequence Data; Nuclear Proteins; RNA, Messenger; Teratocarcinoma; Time Factors; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

In this work we have characterized a process of cell death by apoptosis occurring in retinoic acid-induced differentiation of F9 embryonal carcinoma cells. Morphological changes corresponding to apoptosis were observed concomitantly with those of differentiated phenotype, in cell cultures treated for 48 h with physiological concentrations of all-trans-retinoic acid. Internucleosomal DNA fragmentation was also detected using agarose gels. These observations show that retinoic acid can induce both differentiation and apoptosis in an experimental system--the teratocarcinoma stem cell lines--widely used so far in the study of differentiation. We present this system as a tool for a better understanding of the role of retinoic acid in the regulation of cell differentiation and programmed cell death.

Topics: Animals; Apoptosis; Cell Differentiation; DNA; DNA Damage; Flow Cytometry; Mice; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Genetic alterations in elements of normal signal transduction mechanisms are known to be oncogenic events often resulting in aberrant activation of programs of gene transcription. We have investigated the effect of N-ras oncogene-induced tumorigenic transformation on the transcription factor AP-2. N-ras oncogene-induced transformation of human teratocarcinoma cells PA-1 results in sixfold elevated AP-2 mRNA levels. However, the level of AP-2-mediated trans-activation is dramatically inhibited in these cells. We show here that the high-level expression of AP-2 ultimately results in transcriptional "self-interference". The activation domain of AP-2, when fused to the DNA-binding domain of GAL4, is sufficient for self-interference. Non-N-ras PA-1 cells constitutively expressing AP-2 or GAL4-AP-2 fusion protein from an SV40 promoter exhibit reduced AP-2-mediated transcriptional activation, inhibition of differentiation, and promotion of anchorage-independent growth, properties that are similar to N-ras-transformed PA-1 cells. Thus, AP-2 is placed in the N-ras signal transduction pathway, and many of the biological effects of N-ras can be accomplished by overexpression of AP-2. This is the first evidence that inhibition of the activity of a transcription factor by self-interference contributes to a physiological process.

Topics: Base Sequence; Binding Sites; Cell Transformation, Neoplastic; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Molecular Sequence Data; Peptide Fragments; Protein Binding; Recombinant Fusion Proteins; Teratocarcinoma; Transcription Factor AP-2; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transformation, Genetic; Tretinoin; Tumor Cells, Cultured

We constructed cDNA libraries from a clonal human teratocarcinoma-derived cell line and two retinoic acid-induced derivatives: a homogeneous population of neurons and a FACS-isolated, non-neuronal population. These libraries are large and representative of the cells from which they were derived, as determined by colony hybridization. PCR analysis indicates that the transcripts encoding P- and E-cadherin are down-regulated whereas the the prion protein (PrP) transcript is up-regulated in neurons. These cells offer a promising system for investigations of human prion infection and the cDNA libraries provide a source of neuron-specific genes.

Topics: Antibodies, Monoclonal; Base Sequence; Cell Differentiation; Cell Separation; Embryonal Carcinoma Stem Cells; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genomic Library; Humans; Molecular Sequence Data; Neoplastic Stem Cells; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

The regulation of cell surface fibroblast growth factor (FGF) receptors during the differentiation of F9 teratocarcinoma cells was investigated. The capacity of F9 cells to bind 125I-basic FGF (FGF-2) increased upon induction of differentiation with dibutyryl cAMP and retinoic acid. No change in binding capacity was observed in the first 24 h after addition of differentiating agents, but a sixfold increase in binding capacity was observed after 48 h and a fivefold increase after 72 h. Scatchard analysis of the binding data indicated that the increased binding of 125I-FGF-2 was due to an increase in the number of receptors with no change in their affinity. When 125I-FGF-2 was cross-linked to cell surface receptors, an increase in FGF-2-receptor complexes with molecular weights of 140,000-160,000 was also observed in the differentiated F9 cells. Undifferentiated F9 cells are known to secrete FGF-4 and cease expression of this molecule upon differentiation. To determine whether the low level of receptors in undifferentiated cells might be related to their production of FGF ligands, the ability of suramin, a drug that can disrupt FGF-receptor interactions, to modulate receptor number on F9 cells was investigated. Suramin treatment increased the 125I-FGF-2 binding capacity of undifferentiated F9 cells threefold but had little effect on the binding capacity of differentiated cells. In addition, antibodies to FGF-4 increased the 125I-FGF-2 binding capacity of undifferentiated F9 cells by 58%. These results suggest that undifferentiated F9 cells might be responding in an autocrine manner to their own FGF ligands resulting in downregulation of cell surface FGF receptors. The increased number of receptors observed in differentiated cells may partly result from the decreased production of FGF ligands by these cells.

Topics: Animals; Bucladesine; Cell Differentiation; Cross-Linking Reagents; Down-Regulation; Fibroblast Growth Factor 2; Mice; Molecular Weight; Receptors, Fibroblast Growth Factor; Suramin; Teratocarcinoma; Time Factors; Tretinoin; Tumor Cells, Cultured

Mouse teratocarcinoma F9 cells were induced to primitive endoderm differentiation with retinoic acid, and poly-N-acetyllactosamine-containing surface glycoproteins were identified by radiolabelling endo-beta-galactosidase-cleavable glycans with galactosyltransferase and radiolabelled UDP-galactose. One major radiolabelled band with an apparent size of 250-500 kDa was identified which differed from the known poly-N-acetyllactosamine-containing glycoproteins laminin, fibronectin, lysosome-associated membrane protein (LAMP)-1 and LAMP-2. This acidic glycoprotein, resistant to glycosaminoglycan-degrading enzymes and proteases, was purified by extraction and phase partition with Triton X-114, octyl Sepharose and Helix pomatia lectin chromatography. The purified glycoprotein could be digested by endo-beta-galactosidase and glycopeptide N-glycosidase F to an apparent size of 160-240 kDa. During retinoic-acid-induced differentiation into primitive endoderm cells, the glycoprotein showed a several-fold increase and a broadening to an apparent size of 200- > 700 kDa. The glycoprotein was no longer detected in retinoic-acid and dibutyryl-cAMP-treated cells which had undergone further differentiation to parietal endoderm cells, nor in the permanently differentiated parietal endoderm line F9-AC. The results suggest that the glycoprotein is a major carrier of poly-N-acetyllactosamine chains on differentiating teratocarcinoma F9 cells, and that its expression as revealed by the poly-N-acetyllactosamine labelling method is regulated by the stage of cellular differentiation.

Topics: Animals; Bucladesine; Cell Differentiation; Cell Line; Cell Membrane; Chromatography, Affinity; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Endoderm; Kinetics; Laminin; Lectins; Membrane Glycoproteins; Mice; Polysaccharides; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

The multipotent human teratocarcinoma (TC) cell NTera-2 clone D1 (abbreviated NT2/D1) differentiates into a neuronal lineage after retinoic acid (RA) treatment and a distinct phenotype after hexamethylene bisacetamide (HMBA) treatment. We previously reported that RA treatment of NT2/D1 cells reduces cellular cloning efficiency and nude mouse tumorigenicity. This accompanied a loss of mRNA expression of transforming growth factor-alpha (TGF-alpha) and the fibroblast growth factor kFGF, also known as hst-1 (abbreviated hst-1/kFGF). This study extends prior work by reporting that the distinct phenotype induced by HMBA also decreases cloning efficiency, tumorigenicity, and TGF-alpha and hst-1/kFGF mRNA expression in NT2/D1 cells. These RNA findings were confirmed by measurements of growth factor protein in the conditioned media of inducer-treated and untreated NT2/D1 cells. In two established human TC lines refractory to the actions of RA, N2102ep and Tera-1, RA fails to decrease expression of either growth factor despite induction of its nuclear receptor, RAR-beta. However, HMBA induces morphologic maturation and down-regulation of these growth factors in N2102ep cells. This indicates that the loss of TGF-alpha and hst-1/kFGF expression serves as a new marker of differentiation in human TCs. To explore the effects of these growth factors on growth and differentiation of NT2/D1 cells, TGF-alpha or hst-1/kFGF protein was added following inducer treatment or no treatment. Neither growth factor blocked immunophenotypic differentiation, but both promoted the growth of uninduced NT2/D1 cells in cloning assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetamides; Antineoplastic Agents; Cell Differentiation; Cell Division; Cell Line; Clone Cells; Drug Resistance; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Gene Expression; Humans; Proto-Oncogene Proteins; Teratocarcinoma; Transforming Growth Factor alpha; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) treatment of F9 murine teratocarcinoma (TC) cells reduces the expression of the protein kinase A (PKA)-associated G protein, G alpha i2. The present study reveals interactions between the RA and PKA pathways during differentiation of the multipotent human TC cell line NTERA-2 clone D1 (abbreviated NT2/D1) which differ from prior reports in F9 TC cells. Compared to untreated NT2/D1 cells, differentiated NT2/D1 cells expressed increased levels of G alpha s and G alpha i1,2 proteins as shown by both immunoblot analysis and cholera toxin- and pertussis toxin-induced ADP ribosylation. To further explore cooperation between these pathways during human TC differentiation, we examined the effects of cyclic adenosine monophosphate (cAMP) on RA-responsive genes and of RA treatment on the transcriptional activation of a cAMP response element (CRE). Compared to RA alone, combined treatment with RA and cAMP augmented the expression of the RA nuclear receptor-beta (RAR-beta). Also, transient transfection assays revealed that cAMP and RA cooperated to enhance CRE transcriptional activation. The cAMP-induced enhancement of RA actions in NT2/D1 cells extended to immunophenotypic changes typical of the neuronal differentiation program induced by RA. In contrast to these findings in NT2/D1 cells, prior work in F9 TC cells showed that cAMP inhibits the RA-mediated augmentation of RAR-beta expression and switches the differentiation program from visceral to parietal endoderm. Thus, unlike murine TC cells, in human NT2/D1 cells RA stimulates PKA-associated G proteins and PKA pathway activation enhances RA-mediated TC differentiation.

Topics: Cell Differentiation; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Drug Synergism; GTP-Binding Proteins; Humans; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Two classes of nuclear receptors for retinoic acid (RA) have been identified--retinoic acid receptor (RAR) and retinoid x receptor (RXR). Previously, we demonstrated that all-trans-retinoic acid (t-RA) differentially self-regulated the expression of RAR alpha, -beta, and -gamma transcripts. In the present study, we examined the effect of t-RA and 9-cis-RA (c-RA) on the expression of RXR genes in F9 cells by Northern blot analyses. The results showed that t-RA increased the levels of both the 5.6-kb RXR alpha and 3.8-kb RXR gamma mRNAs, decreased the amounts of 2.3-kb RXR gamma mRNA, but had no significant effect on the levels of RXR beta mRNA. Addition of a cyclic AMP analog along with t-RA further induced the differentiation of F9 cells to become parietal endodermal cells, but did not change the regulatory patterns of RXR mRNAs. The RNA synthesis inhibitor, actinomycin D, blocked the induction of 5.6-kb RXR alpha and 3.8-kb RXR gamma mRNA by t-RA, suggesting that the regulations at least in part were at the transcriptional levels. The protein synthesis inhibitor, cycloheximide, induced the expression of 5.6-kb RXR alpha mRNA and further enhanced the inductive effect of t-RA. In contrast, cycloheximide prevented the t-RA-regulated expression of both 3.8- and 2.3-kb RXR gamma mRNA, suggesting that ongoing protein synthesis was required for the regulation of RXR gamma gene. In addition, c-RA exerted effects similar to those of t-RA on RXR gene expression. A concentration of 10(-8) M was required for c-RA to regulate the expression of RXR genes, while 10(-9) M of t-RA was effective in regulating RXR genes. Addition of t-RA and c-RA simultaneously had neither synergistic nor additive effects in regulating RXR gene expression. These data suggest that RAR may play an important role in RA-regulated RXR gene expression.

Topics: Animals; Cyclic AMP; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; In Vitro Techniques; Mice; RNA, Messenger; RNA, Neoplasm; Stereoisomerism; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Rapidly growing human teratocarcinoma cells (Tera-2) can be induced to differentiate into quiescent, nontumorigenic cells expressing neuronal markers. To more closely mimic the in vivo conditions for tumor growth, we grew Tera-2 cells in three-dimensional collagen gel cultures. The undifferentiated cells proliferated in the gel, forming tight colonies. Addition of soluble fibroblast growth factor 1 or 2 (FGF1 or FGF2) into the gel resulted in scattering of single cells throughout the collagen gel. In a FGF gradient the cells moved rapidly toward a higher concentration. On the contrary, cells first differentiated for 8 days in retinoic acid died within a few days after transfer into the collagen gel. Alternatively, if retinoic acid was included in the collagen gel, the proliferating undifferentiated cells died after 4-5 days in the gel. This differentiation-related cell death was completely opposed by including FGF in the collagen gel. When placed in the FGF gradient, the fully differentiated cells survived at the areas of higher FGF concentration, but no more migrated. The survival of retinoic acid-differentiated Tera-2 cells in collagen was also mediated by direct contact with glioma cells or the heparan sulfate-rich portion of glioma or endothelial cell matrix. These effects on differentiated cells were sensitive to inhibition by affinity-purified anti-FGF2 IgG. Thus, FGF has the potential to act as a migration-inducing factor either in solution or, more likely, in vivo, as an immobilized, matrix-bound growth factor directing the movement of responsive cells. The development of differentiation-associated FGF dependency allows survival of the cells only at places where they are in close contact with either FGF-synthesizing cells or FGF-rich extracellular structures such as basement membranes.

Topics: Animals; Cattle; Cell Communication; Cell Differentiation; Cell Survival; Embryonal Carcinoma Stem Cells; Epidermal Growth Factor; Humans; Neoplastic Stem Cells; RNA, Messenger; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Expression of ALP in F9 teratocarcinoma cells is induced by all-trans retinoic acid (ATRA) (Gianni' et al., Biochem. J. 274: 673-678, 1991). The specific ligand for retinoic acid related receptors (RXRs), 9-cis retinoic acid (9-cis RA), and three synthetic analogs binding to the alpha, beta and gamma forms of the retinoic acid receptors (RARs), AM580, CD2019, and CD437, were used to study their effects on alkaline phosphatase (ALP) enzymatic activity and mRNA levels. At concentrations close to the Kd for their respective receptors, 9-cis RA, AM580 (the RAR alpha agonist) and CD437 (the RAR gamma agonist) clearly upregulate the expression of the ALP gene, whereas the effect of CD2019 (the RAR beta agonist) is very modest. A specific inhibitor of the RAR alpha, Ro 41-5253, completely blocks the induction of ALP triggered by AM580, while it has minor effects on the upregulation caused by ATRA, 9-cis RA, CD437 and CD2019. The induction of ALP observed with the various retinoids is inhibited by the contemporaneous treatment with dibutyryl cAMP. The levels of the RAR alpha and gamma transcripts are unaltered, while RAR beta mRNAs are induced by ATRA, AM580, CD437 and to a lower extent by 9-cis RA and CD2019.

Topics: Alkaline Phosphatase; Animals; Dose-Response Relationship, Drug; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Isomerism; Mice; Receptors, Retinoic Acid; Retinoids; RNA, Messenger; Structure-Activity Relationship; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

We previously reported that protein kinase C (PKC) stimulation through phorbol ester (TPA) treatment enhances the effects of all-trans retinoic acid (RA) on immunophenotypic differentiation and RA nuclear receptor (RAR) activation in the multipotential human teratocarcinoma (TC) cell line NTera-2/clone D1 (abbreviated NT2/D1). This study extends prior work in NT2/D1 cells by demonstrating that PKC pathway activation is an early effect of RA treatment which regulates RAR transcriptional activity. RA activated the PKC pathway prior to induction of RAR-beta expression at 6 h, which is an established early marker of RAR activation in NT2/D1 cells. RA caused a transient 1.3-fold increase in intracellular diacylglycerol (DG) at 2 min and a translocation of the gamma isozyme of PKC (PKC-gamma) within 5 min. Transient co-transfection studies provided evidence that PKC pathway activation plays a role in the regulation of RAR-beta expression. In these studies a constitutively active PKC-gamma augmented the RA-mediated transactivation of a luciferase reporter containing the native RAR-beta promoter which has a retinoic-acid-response element (RARE). These findings reveal that PKC pathway activation is an early step in RA-mediated human TC differentiation and that PKC-gamma can potentiate the effects of RA on RAR transcriptional activation.

Topics: Cell Differentiation; Cell Membrane; Cytosol; Diglycerides; Enzyme Activation; Humans; Protein Kinase C; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

This study explored cooperation between the retinoic acid (RA) and protein kinase C (PKC) pathways during differentiation of the multipotential human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1). We report here that, compared to RA treatment alone, RA combined with the PKC stimulator 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced the regulated expression of the immunophenotypic differentiation markers SSEA-3, a globo-series carbohydrate, and the ganglio-series carbohydrate antigens GD2 and GD3. Northern analysis and transient transfection assays revealed that TPA co-treatment augmented the RA-induced expression and activation of the RA nuclear receptor-beta (RAR-beta), one early marker of RA response in NT2/D1 cells. This finding was extended with transient co-transfection experiments using a PKC-alpha expression vector which revealed that the PKC pathway can augment the activation of RAR-beta by RA. These experiments establish PKC as a modulator of RAR-beta expression in NT2/D1 cells. Similarly, experiments showed that RA can modulate activation of the PKC-responsive AP-1 complex, a transcription factor rapidly activated by TPA. Northern analysis and transient transfection assays revealed that, compared to TPA treatment alone, RA and TPA augmented the expression and transcriptional activity of AP-1 in NT2/D1 cells. In contrast, transient transfection assays revealed no cooperative effect between RA and TPA in HeLa cells, indicating that this effect in NT2/D1 cells is cell type-specific. In summary, these studies show that stimulation of the PKC second messenger pathway can modulate tumor differentiation and transcriptional activation of a retinoid receptor associated with RA response.

Topics: Biomarkers; Cell Differentiation; Clone Cells; Drug Synergism; Humans; Immunophenotyping; Protein Kinase C; Proto-Oncogene Proteins c-jun; Receptors, Retinoic Acid; Teratocarcinoma; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Prior work has shown that all-trans retinoic acid (t-RA) treatment of the human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1) induces a neuronal phenotype and other cell lineages. This study sought to explore the potential of 9-cis retinoic acid (9-cis RA) as a differentiation-inducing agent of this multipotent cell. Findings reported here show that 9-cis RA induced a phenotype similar to t-RA treatment of NT2/D1 cells. This similarity extended to their effects on the nuclear receptors retinoic acid receptor-beta (RAR-beta) and retinoid X receptor-alpha (RXR-alpha). Both retinoids prominently augmented RAR-beta expression and transactivated a reporter plasmid containing putative RAR response elements (RAREs) with direct repeats separated by five nucleotides (DR5). Both retinoids had no appreciable effect on RXR-alpha expression and both minimally transactivated a reporter plasmid containing putative RXR response elements (RXREs) with direct repeats separated by one nucleotide (DR1). These studies suggest that 9-cis RA and t-RA activate common events during retinoid-mediated NT2/D1 differentiation. This hypothesis was supported by the finding that NT2/D1 cells rendered refractory to t-RA (NT2/D1-R1) were also resistant to 9-cis RA. To discover alterations that could confer retinoid-refractoriness, retinoid receptor expression was examined in NT2/D1-R1 cells. In contrast to NT2/D1, the NT2/D1-R1 cell was found to have reduced RXR-alpha expression at the level of total cellular RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cell Differentiation; Drug Resistance; Humans; Neurons; Phenotype; Receptors, Retinoic Acid; Stereoisomerism; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

All-trans retinoic acid (RA) treatment of the multipotent human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1) induces a neuronal phenotype and other cell lineages. NT2/D1 cells basally express transforming growth factor alpha (TGF-alpha) mRNA and secreted protein. After RA-treatment TGF-alpha expression is markedly reduced. This decline in TGF-alpha expression accompanies the induction of the neuronal phenotype and a marked reduction of tumorigenicity in athymic mice. This suggested a causal link between reduced TGF-alpha expression and the induced differentiation or loss of tumorigenicity of these RA-treated TC cells. To evaluate this possibility, an RA-refractory NT2/D1 subclone was analysed. This subclone, designated NT2/D1-R1, failed to induce differentiation or to decrease TGF-alpha expression despite RA treatment. To further explore the relationship between TGF-alpha expression and RA actions in this human TC cell, a TGF-alpha cDNA was stably transfected and expressed in NT2/D1 cells. RA-treatment of independently obtained TGF-alpha over-expressing clones and a representative control transfectant only expressing the neomycin resistance gene produced a neuronal phenotype similar to parental NT2/D1 cells as assessed by morphologic, immunophenotypic, and gene expression markers of differentiation. RA-treatment of these clones also induced a G1 arrest similar to parental cells. However, only the TGF-alpha over-expressing clones that secreted high levels of TGF-alpha protein into the conditioned media before and after RA treatment still developed tumors in athymic mice despite prior exposure to these cells to RA. This finding demonstrates that TGF-alpha can inhibit the anti-tumorigenic effects of RA in human TCs. Thus, over-expression of a single growth factor that normally declines with RA treatment antagonizes the anti-tumorigenic but not the differentiation actions of RA in this human tumor cell.

Topics: Animals; Blotting, Northern; Cell Transformation, Neoplastic; Culture Media, Conditioned; DNA, Neoplasm; Flow Cytometry; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Immunophenotyping; Mice; Mice, Nude; RNA, Messenger; Teratocarcinoma; Transfection; Transforming Growth Factor alpha; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

We have monitored production of platelet-derived growth factor (PDGF) in F9 teratocarcinoma cells. We show that undifferentiated F9 cells express PDGF A mRNA and produce biologically active PDGF AA homodimers. When differentiation is induced by treatment with retinoic acid and cyclic AMP, the production of PDGF AA protein is terminated. Contrary to expectation, inhibition of PDGF synthesis is exerted at a posttranscriptional level. Both undifferentiated and differentiated (1 day) F9 cell cultures contain comparable amounts of PDGF A mRNA. However, this mRNA becomes dissociated from polysomes during F9 cell differentiation.

Topics: 3T3 Cells; Animals; Cell Differentiation; Mice; Mice, Inbred BALB C; Platelet-Derived Growth Factor; Protein Biosynthesis; RNA, Messenger; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

The beta-amyloid or beta/A4 peptides that accumulate as filamentous aggregates in the extracellular space of Alzheimer disease (AD) brains are derived from one or more alternatively spliced amyloid precursor proteins (APPs). The more abundant APPs in the central nervous system are the 695-(APP695), 751- (APP751), and 770- (APP770) amino acid isoforms, and each could be the source of beta/A4 peptide that accumulates in the AD brain. It is plausible that altered metabolism of these APPs by central nervous system neurons could lead to the release and deposition of beta/A4 peptide in brain parenchyma. Thus, we examined the expression and processing of the three major brain APPs in nearly pure human neurons (NT2N cells) derived from a teratocarcinoma cell line (NTera2/c1.D1 or NT2 cells) after retinoic acid treatment. NT2N neurons expressed almost exclusively APP695, whereas NT2 cells expressed predominantly APP751/770. Furthermore, the processing of the APPs in NT2N cells was distinct from NT2 and nonneuronal cells. Most significantly, the NT2N neurons but not the NT2 cells constitutively generated intracellular beta/A4 peptide and released it into the culture medium. This work demonstrates the intracellular production of beta/A4 peptide and suggests that cultured NT2N cells may provide a unique model system for understanding the contribution of neurons and APP695 to amyloidogenesis in the AD brain.

Topics: Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Cell Differentiation; Gene Expression; Glycosylation; Humans; Neurons; Protein Processing, Post-Translational; RNA, Messenger; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured