tretinoin and Carcinoma--Embryonal

Retinoic acid (RA), the most potent natural form of vitamin A, plays an important role in many diverse biological processes such as embryogenesis and cellular differentiation. This chapter is a review of the mechanism of action of RA and the role of specific RA-regulated genes during the cellular differentiation of embryonal carcinoma (EC) and embryonic stem (ES) cells. RA acts by binding to its nuclear receptors and inducing transcription of specific target genes. The most studied mouse EC cell lines include F9 cells, which can be induced by RA to differentiate into primitive, parietal, and visceral endodermal cells; and P19 cells, which can differentiate to endodermal and neuronal cells upon RA treatment. ES cells can be induced to differentiate into a number of different cell types; many of which require RA treatment. Over the years, many RA-regulated genes have been discovered in EC and ES cells using a diverse set of techniques. Current research focuses on the elucidation how these genes affect differentiation in EC and ES cells using a variety of molecular biology approaches. However, the exact molecule events that lead from a pluripotent stem cell to a fully differentiated cell following RA treatment are yet to be determined.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Humans; Mice; Stem Cells; Transcription, Genetic; Tretinoin

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; DNA-Binding Proteins; Humans; Milk Proteins; Neurons; STAT5 Transcription Factor; Trans-Activators; Tretinoin

Mouse F9 embryocarcinoma (EC) cells constitute a well established cell-autonomous model system for investigating retinoid signaling in vitro as, depending on culture conditions, retinoic acid (RA) can induce their differentiation into either primitive, parietal or visceral extraembryonic endoderm-like cells. These RA-induced differentiations are accompanied by decreases in proliferation rates, modifications of expression of subsets of RA-target genes, and induction of apoptosis. To elucidate the roles played by the multiple retinoid receptors (RARs and RXRs) in response to RA treatments, F9 EC cells lacking one or several RARs or RXRs were engineered through homologous recombination. Mutated RARs and/or RXRs were then reexpressed in given RAR or RXR null backgrounds. WT and mutant cells were also treated with different combinations of ligands selective for RXRs and/or for each of the three RAR isotypes. These studies lead to the conclusion that most RA-induced events (e.g. primitive and visceral differentiation, growth arrest, apoptosis and activation of expression of a number of genes) are transduced by RARgamma/RXRalpha heterodimers, whereas some other events (e.g. parietal differentiation) are mediated by RARalpha/RXRalpha. heterodimers. They also demonstrate that both AF-1 and AF-2 activation functions of RARs and RXRs, as well as their phosphorylation, are differentially required in these RA-induced events. In RARgamma/RXRalpha heterodimers, the phosphorylation of RARgamma is necessary for triggering primitive differentiation, while that of RXRalpha is required for growth arrest. On the other hand, phosphorylation of RARalpha is necessary for parietal differentiation. Thus, retinoid receptors are sophisticated signal integrators that transduce not only the effects of their cognate ligands, but also those of ligands that bind to membrane receptors.

Topics: Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Carcinoma, Embryonal; Cell Differentiation; Cell Division; DNA, Neoplasm; Genetic Engineering; Mice; Models, Biological; Molecular Sequence Data; Phosphorylation; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Signal Transduction; Transcription Factors; Tretinoin; Tumor Cells, Cultured

The retinoids, natural and synthetic derivatives of vitamin A, are active in cancer therapy and prevention. Their biological effects are mediated through ligand-dependent interactions with retinoid receptors that associate with specific co-regulators. A better understanding of retinoid chemopreventive mechanisms is needed. Our prior work revealed that all-trans-retinoic acid (RA) prevented tobacco-specific carcinogenic transformation of cultured human bronchial epithelial cells. RA signaled G1 arrest that permitted repair of genomic DNA damage caused by these carcinogens. RA triggered G1 arrest at least partly through proteasome-dependent degradation of cyclin D1. Proteasomal inhibitors blocked RA-mediated cyclin D1 degradation. To confirm that a specific proteolysis pathway was induced by RA-treatment, a degradation assay was established using in vitro translated cyclin D1 and cellular extracts from RA-treated or untreated human bronchial epithelial cells. Incubation of RA-treated but not the control cellular extracts with in vitro translated cyclin D1 led to cyclin degradation. This degradation depended on the PEST domain of cyclin D1, implicating ubiquitination in this retinoid degradation. Retinoid receptor selective agonists demonstrated that retinoic acid receptor (RAR)beta and retinoid X receptor (RXR) but not RARalpha- or RARgamma-dependent pathways signaled this cyclin degradation. Findings were extended to the NT2/D1 human embryonal carcinoma differentiation model where a similar pathway was activated by RA-treatment. To determine whether G1 cyclins were involved directly in bronchial preneoplasia, immunohistochemical expression profiles for cyclins D1 and E were examined. Aberrant expression of these cyclins was frequent in bronchial preneoplasia. Taken together, these findings indicate that ubiquitin-dependent proteolysis of G1 cyclins is a retinoid chemoprevention mechanism. Whether the retinoids represent the optimal agents to activate this pathway is the subject of ongoing work. These findings provide a rationale for combining the retinoids in chemoprevention trials with other agents that do not activate this proteolysis pathway. What is now known about the retinoids as cancer prevention agents will be reviewed. Emphasis is placed on retinoid effects on cell cycle progression at G1.

Topics: Animals; Anticarcinogenic Agents; Bronchi; Bronchial Diseases; Carcinoma, Embryonal; Cell Differentiation; Cell Transformation, Neoplastic; Cyclins; Cysteine Endopeptidases; Endopeptidases; Epithelial Cells; G1 Phase; Gene Expression Profiling; Gene Expression Regulation; Humans; Metaplasia; Mice; Models, Biological; Multienzyme Complexes; Neoplasms; Precancerous Conditions; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein Structure, Tertiary; Receptors, Retinoic Acid; Retinoids; Tretinoin; Tumor Cells, Cultured; Ubiquitin; Vitamin A Deficiency

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; DNA-Binding Proteins; Genes, Neoplasm; Homeodomain Proteins; Octamer Transcription Factor-3; POU Domain Factors; Transcription Factors; Tretinoin

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; Base Sequence; CA-19-9 Antigen; Carcinoma, Embryonal; CCAAT-Binding Factor; Cell Line; Galactosyltransferases; Human Embryonic Stem Cells; Humans; Lamin Type A; Mice; Polysaccharides; Promoter Regions, Genetic; Signal Transduction; Sirtuin 1; Stage-Specific Embryonic Antigens; STAT3 Transcription Factor; Terminal Repeat Sequences; Tretinoin

The pluripotent mouse embryonal carcinoma cell line P19 is widely used as a model for research on all-trans-retinoid acid (RA)-induced neuronal differentiation; however, the signaling pathways involved in this process remain unclear. This study aimed to reveal the molecular mechanism underlying the RA-induced neuronal differentiation of P19 cells. Real-time quantitative polymerase chain reaction and Western blot analysis were used to determine the expression of neuronal-specific markers, whereas flow cytometry was used to analyze cell cycle and cell apoptosis. The expression profiles of messenger RNAs (mRNAs) in RA-induced neuronal differentiation of P19 cells were analyzed using high-throughput sequencing, and the functions of differentially expressed mRNAs (DEMs) were determined by bioinformatics analysis. RA induced an increase in both class III β-tubulin (TUBB3) and neurofilament medium (NEFM) mRNA expression, indicating that RA successfully induces neuronal differentiation of P19 cells. Cell apoptosis was not affected; however, cell proliferation decreased. We found 4117 DEMs, which were enriched in the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, Wnt signaling pathway, and cell cycle. Particularly, a few DEMs could be identified in the PI3K/Akt signaling pathway networks, such as PI3K, Akt, glycogen synthase kinase-3β (GSK3β), cyclin-dependent kinase 4 (CDK4), P21, and Bax. RA significantly increased the protein expression of PI3K, Akt, phosphorylated Akt, GSK3β, phosphorylated GSK3β, CDK4, and P21, but it reduced Bax protein expression. The Akt inhibitor affected the increase of TUBB3 and NEFM mRNA expression in RA-induced P19 cells. The molecular mechanism underlying the RA-induced neuronal differentiation of P19 cells is potentially involved in the PI3K/Akt/GSK3β signaling pathway. The decreased cell proliferation ability of neuronally differentiated P19 cells could be associated with the expression of cell cycle proteins.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Carcinoma, Embryonal; Cell Differentiation; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Mice; Neurons; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Tretinoin; Tumor Cells, Cultured

The protein phosphatase PPM1D (Wip1) was originally identified as a p53 target product. Activation of PPM1D through various mechanism promotes the tumorigenic potential of various cancers by suppressing p53 and other DNA damage response proteins. New functions of PPM1D have recently been revealed in physiological processes such as cell differentiation. However, the regulatory mechanisms of signalling pathway to maintain stemness and induce cell differentiation are still unclear. Here we report that PPM1D modulates retinoic acid (RA) signalling. PPM1D knockdown resulted in decreased alkaline phosphatase activity of the human teratocarcinoma cell line NT2/D1. Inhibition of PPM1D-induced cell differentiation and decreased gene expression of the stem cell marker Oct-4 (POU5F1). RA-induced cell differentiation was promoted by reducing PPM1D activity. RA treatment elicited activation of the MEK-ERK pathway and induced rapid and transient activation of the extracellular signal-regulated kinase 1/2 (ERK-1/2). PPM1D dephosphorylated a phosphopeptide with the TEY motif in ERK-1/2 in vitro. Moreover, phosphorylation of ERK-1/2 was facilitated by PPM1D inhibition. Our study shows that PPM1D plays an important role in maintaining the undifferentiation state and a new function in RA-induced ERK regulation and cell differentiation.

Topics: Antineoplastic Agents; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Protein Kinase Inhibitors; Protein Phosphatase 2C; Structure-Activity Relationship; Tretinoin

The ability to self-renew is one of the most important properties of embryonic stem (ES) cells. Pluripotin (SC1), a small molecule with high activity and low toxicity, promotes self-renewal in mouse ES cells. SC1 can noticeably change the morphology of retinoic acid (RA)-induced F9 embryonic carcinoma cells (F9 cells). However, in the long term, RA and SC1 together cause cell apoptosis. When being added after 18-24 h of RA-induced F9 cell differentiation, SC1 transitorily activated Nanog and Oct4. Both Nanog and Oct4 were downregulated when SC1 and RA were added simultaneously. On the other hand, Klf4 was continually activated when SC1 was added between 6 and 24 h. Phosphorylated Erk1/2 protein levels were reduced from 6 to 24 h, whereas unphosphorylated Erk1 protein levels remained unchanged. A higher concentration of SC1 promoted cell self-renewal by strengthening the inhibition of Erk1/2 protein phosphorylation in F9 cells. Furthermore, SC1 and RA affect global DNA methylation by influencing the expressions of methylation-associated proteins, including Dnmt3b, Dnmt3l, Tet1, Tet2, and Tet3. In conclusion, SC1 inhibits the differentiation of RA-induced F9 cells mainly by reducing the levels of phosphorylated Erk1/2 and enhancing the expression of Klf4, although it also reduces DNA methylation, which may have an additional effect on ES cell differentiation.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Kruppel-Like Factor 4; Mice; Nanog Homeobox Protein; Octamer Transcription Factor-3; Proto-Oncogene Proteins; Pyrazoles; Pyrimidines; Tretinoin

Transcription factor FOXM1 plays a critical role in maintenance of stem cell pluripotency through stimulating the transcription of pluripotency-related genes in mouse pluripotent stem cells. In this study, we have found that the repression of FOXM1 expression is mediated by FOXM1 3'UTR during retinoic acid-induced differentiation of human pluripotent NT2/D1 embryonal carcinoma cells. FOXM1 3'UTR contains a microRNA response element (MRE) for miR-134, which has been shown to attenuate the expression of pluripotency-related genes post-transcriptionally during mouse embryonic stem cell differentiation. We have determined that miR-134 is induced during RA-induced differentiation of NT2/D1 cells and the overexpression of miR-134 represses the expression of FOXM1 protein but not FOXM1 mRNA. Furthermore, the expression of OCT4 is diminished by FOXM1 knockdown and the OCT4 promoter is regulated directly by FOXM1, suggesting that FOXM1 is required for maintaining the expression of OCT4 in NT2/D1 cells. Together, our results suggest that FOXM1 is essential for human pluripotent stem cells and miR-134 attenuates its expression during differentiation.

Topics: Animals; Antineoplastic Agents; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; Embryonal Carcinoma Stem Cells; Forkhead Box Protein M1; Forkhead Transcription Factors; HEK293 Cells; Humans; Mice; MicroRNAs; Octamer Transcription Factor-3; Pluripotent Stem Cells; Promoter Regions, Genetic; Response Elements; RNA Interference; RNA, Messenger; RNA, Small Interfering; Tretinoin

Nuclear factor Y (NF-Y) is a histone substitute protein that specifically binds to the CCAAT box of the target genes and thereby promotes their regulation. NF-Y transcription factor, with defined CCAAT element-binding activities, target a gene family that encodes a group of basic helix-loop-helix ID factors (ID1-ID4), with or without CCAAT box at their promoter region. In this study, the expressions of NF-Y in mRNA and protein level were evaluated in a human embryonic carcinoma cell line, named NTera2, before and after 7 days induction of differentiation. We also looked into expression levels of ID genes in NTera2 cells during differentiation because of their critical role in development. By using chromatin immunoprecipitation coupled with real-time polymerase chain reaction, NF-Y incorporation and acetylation/dimethylation of histone H3 at lysine 9 (H3K9ac/me2) was quantitatively evaluated on the regulatory regions of considered genes to monitor the changes in epigenetic markers at ID gene promoters throughout differentiation. The results demonstrated a marked down-regulation of ID1, ID2, and ID3 genes, parallel to a loss of NF-Y binding to the promoters of these genes. The data show that although the genes encoding NF-Y complex remained expressed at mRNA level, NF-YC is lost at the protein level onset of differentiation. Additionally, the epigenetic marks of H3K9ac and H3K9me2 at the target gene promoters decreased and increased, respectively, after 1 day of differentiation. It is suggested that, in the absence of NF-Y binding, the corresponding regions adopt a heterochromatic nature, whereas when NF-Y comes back after 7 days of differentiation, the ID1-3 promoters become again converted into active chromatin. The ID4 gene, lacking a CCAAT box, behaves differently and does not show any incorporation. This experiment implies for the first time that the presence of NF-Y transcription factor plays a pivotal role in transcriptional regulation of ID genes in development.

Topics: Antigens, Differentiation; Carcinoma, Embryonal; CCAAT-Binding Factor; Cell Differentiation; Cell Line, Tumor; Epigenesis, Genetic; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Differentiation Proteins; Male; Testicular Neoplasms; Transcription, Genetic; Tretinoin

We analysed the effects of all-trans retinoic acid (ATRA) on proliferation and changes in the global proteome of the nullipotent human embryonal carcinoma cell line 2102Ep and the pluripotent cell line NTERA2 cl.D1 (NT2). Differentially expressed proteins were assessed by 2D-PAGE and mass spectrometry, followed by verification and analysis of protein modifications of proteins of the retinoid pathway. We established a proteome map of the germ cell tumor (GCT) cell line NT2 showing neuronal differentiation under ATRA treatment for 7days. Using bioinformatic analyses, we identified functional groups of altered proteins and potentially involved pathways, of which changes to the organization of the cytoskeleton and anti-apoptotic effects were the most prominent. Changes observed in the expression of factors involved in the retinoid pathway under ATRA, namely an upregulation of CRBP and CRABP2, were also reflected in GCT tissues of different histologies, providing further insight into factors involved in the differentiation of these pluripotent tumors.. Treatment of NT2 germ cell tumor cells with all-trans retinoic acid (ATRA) is a model to investigate differentiation. We analysed differentially expressed proteins by 2D-PAGE and mass spectrometry and provide a proteome map of NT2 cells under 7days of ATRA. By bioinformatic analyses, functional groups of proteins and involved pathways like changes to the cytoskeleton and anti-apoptotic effects were identified. Factors involved in the retinoid pathway, in particular upregulation of CRBP, CRABP1 and CRABP2, also showed differential expression in tumors with different histological subtypes, which provides insight into gene regulation under induced and spontaneous differentiation in germ cell tumors.

Topics: Antineoplastic Agents; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Protein Processing, Post-Translational; Proteome; Time Factors; Tretinoin; Up-Regulation

Male germ cell tumors (GCTs) are a model for a curable solid tumor. GCTs can differentiate into mature teratomas. Embryonal carcinomas (ECs) represent the stem cell compartment of GCTs and are the malignant counterpart to embryonic stem (ES) cells. GCTs and EC cells are useful to investigate differentiation therapy and chemotherapy response. This study explored mechanistic interactions between all-trans-retinoic acid (RA), which induces differentiation of EC and ES cells, and the Hedgehog (Hh) pathway, a regulator of self-renewal and proliferation. RA was found to induce mRNA and protein expression of Patched 1 (Ptch1), the Hh ligand receptor and negative regulator of this pathway. PTCH1 is also a target gene of Hh signaling through Smoothened (Smo) activation. Yet, this observed RA-mediated Ptch1 induction was independent of Smo. It occurred despite co-treatment with RA and Smo inhibitors. Retinoid induction of Ptch1 also occurred in other RA-responsive cancer cell lines and in normal ES cells. Notably, this enhanced Ptch1 expression was preceded by induction of the homeobox transcription factor Meis1, a direct RA target. Direct interaction between Meis1 and Ptch1 was confirmed using chromatin immunoprecipitation assays. To establish the translational relevance of this work, Ptch1 expression was shown to be deregulated in human ECs relative to mature teratoma and the normal seminiferous tubule. Taken together, these findings reveal a previously unrecognized mechanism through which RA can inhibit the Hh pathway via Ptch1 induction. Engaging this pathway is a new way to repress the Hh pathway that can be translated into the cancer clinic.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; Cells, Cultured; Embryonic Stem Cells; Hedgehog Proteins; Homeodomain Proteins; Humans; Male; Mice; Myeloid Ecotropic Viral Integration Site 1 Protein; Neoplasm Proteins; Patched Receptors; Patched-1 Receptor; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Seminiferous Tubules; Signal Transduction; Smoothened Receptor; Teratoma; Transcription Factors; Tretinoin; Zinc Finger Protein GLI1

E26 transformation-specific (ETS) transcription factors play important roles in normal and tumorigenic processes during development, differentiation, homeostasis, proliferation, and apoptosis. To identify critical ETS factor(s) in germ cell-derived cancer cells, we examined the expression patterns of the 27 ETS transcription factors in naive and differentiated NCCIT human embryonic carcinoma cells, which exhibit both pluripotent and tumorigenic characteristics. Overall, expression of ETS factors was relatively low in NCCIT cells. Among the 27 ETS factors, polyomavirus enhancer activator 3 (PEA3) and epithelium-specific ETS transcription factor-1 (ESE-1) exhibited the most significant changes in their expression levels. Western blot analysis confirmed these patterns, revealing reduced levels of PEA3 protein and elevated levels of ESE-1 protein in differentiated cells. PEA3 increased the proportion of cells in S-phase and promoted cell growth, whereas ESE-1 reduced proliferation potential. These data suggest that PEA3 and ESE-1 may play important roles in pluripotent and tumorigenic embryonic carcinoma cells. These findings contribute to our understanding of the functions of oncogenic ETS factors in germ cell-derived stem cells during processes related to tumorigenesis and pluripotency.

Topics: Carcinoma, Embryonal; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Size; DNA-Binding Proteins; Humans; Proto-Oncogene Proteins c-ets; Transcription Factors; Transcriptional Activation; Transfection; Tretinoin

Human Cripto-1 (CR-1) plays an important role in regulating embryonic development while also regulating various stages of tumor progression. However, mechanisms that regulate CR-1 expression during embryogenesis and tumorigenesis are still not well defined. In the present study, we investigated the effects of two nuclear receptors, liver receptor homolog (LRH)-1 and germ cell nuclear factor receptor (GCNF) and epigenetic modifications on CR-1 gene expression in NTERA-2 human embryonal carcinoma cells and in breast cancer cells. CR-1 expression in NTERA-2 cells was positively regulated by LRH-1 through direct binding to a DR0 element within the CR-1 promoter, while GCNF strongly suppressed CR-1 expression in these cells. In addition, the CR-1 promoter was unmethylated in NTERA-2 cells, while T47D, ZR75-1, and MCF7 breast cancer cells showed high levels of CR-1 promoter methylation and low CR-1 mRNA and protein expression. Treatment of breast cancer cells with a demethylating agent and histone deacetylase inhibitors reduced methylation of the CR-1 promoter and reactivated CR-1 mRNA and protein expression in these cells, promoting migration and invasion of breast cancer cells. Analysis of a breast cancer tissue array revealed that CR-1 was highly expressed in the majority of human breast tumors, suggesting that CR-1 expression in breast cancer cell lines might not be representative of in vivo expression. Collectively, these findings offer some insight into the transcriptional regulation of CR-1 gene expression and its critical role in the pathogenesis of human cancer.

Topics: Azacitidine; Binding Sites; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Embryonal; Cell Movement; Decitabine; DNA Methylation; DNA Modification Methylases; Dose-Response Relationship, Drug; Embryonal Carcinoma Stem Cells; Female; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Genes, Reporter; GPI-Linked Proteins; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Luciferases; MCF-7 Cells; Neoplasm Invasiveness; Neoplasm Proteins; Nuclear Receptor Subfamily 6, Group A, Member 1; Promoter Regions, Genetic; Receptors, Cytoplasmic and Nuclear; RNA Interference; RNA, Messenger; Time Factors; Tissue Array Analysis; Transcription, Genetic; Transfection; Tretinoin; Valproic Acid

P19 embryonal carcinoma (EC) cells are an invaluable tool for approximating the mechanisms that govern neuronal differentiation but with an enormous degree of simplification and have primarily been used to model the early stages of neurogenesis. However, they are often cultured under conditions that promote unrestricted non-neuronal growth that compromises neuronal viability. In this study we report an improved method to differentiate P19 EC cells that gives rise to high yields of functionally and morphologically mature neurons while significantly reducing the over-growth of non-neuronal cells in the cultures. In this protocol, P19 EC cells are induced in Minimum Essential Medium alpha supplemented with all-trans retinoic acid (RA) and 2.5% serum, and cultured as a monolayer. After RA-induction, cells are cultured on Matrigel coated-plates using defined media comprised of Neurobasal-A medium temporally supplemented with N2 and then B-27 for the remaining culture period. By treating the culture with Cytosine β-d-arabinofuranoside and 2'-Deoxycytidine for five days, the cultures are reliably promoted toward the neuronal differentiation vs non-neuronal differentiation, this accounting for a progressive neuronal enrichment of the cultures reaching 56% after 20 days of culture. P19-derived neural progenitor cells progressively expressed neuronal markers such as NeuN, Calretinin, Calbindin and Synapsin I in close resemblance to that occurring in vivo in the central nervous system (CNS). Furthermore, RA-induced P19 EC cells progressively acquired functional neuronal traits and after approximately 3 weeks in culture revealed mature neurophysiological properties, characteristics of CNS neurons. This protocol allows for a more specific assessment of the neuronal differentiation processes in vitro.

Topics: Animals; Carcinoma, Embryonal; Cell Culture Techniques; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Proliferation; Mice; Neurogenesis; Neurons; Tretinoin

Oct4 is a POU domain homeobox gene, expressed in undifferentiated embryonal carcinoma and embryonic stem cells and is quickly down-regulated upon induction of differentiation. Transcriptional repression of Oct4 is followed by pronounced epigenetic changes on the regulatory region of the gene. Oct4 has a long upstream regulatory region of about 2,600 bp, consisting of proximal enhancer (PE), distal enhancer (DE), and proximal promoter (PP). In this study, we induced differentiation of a human embryonic carcinoma cell line, NT2, under two different adherent and non-adherent culture conditions, and compared histone modifications as the epigenetic marks on the regulatory region of Oct4 gene after 3 days of differentiation. Using chromatin immunoprecipitation coupled with real-time PCR technique, it was shown that the after induction of differentiation the repressive epigenetic marks of hypoacetylation and methylation on lysine-9 of histone H3 occurred very effectively on the upstream of Oct4, especially in PP region. Also, comparing the two culturing systems it was shown that methylation of lysine-9 of H3 histone was more drastic in PE region of adherent cells rather than suspension cells. This epigenetic profile was in agreement with the difference observed in the expression level of Oct4 in these two culturing systems. The current study clearly shows the effective role of cell culture condition on the epigenetic regulation of gene expression.

Topics: Acetylation; Carcinoma, Embryonal; Cell Adhesion; Cell Culture Techniques; Cell Differentiation; Cell Line, Tumor; Chromatin Immunoprecipitation; Down-Regulation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histones; Humans; Lysine; Methylation; Octamer Transcription Factor-3; Real-Time Polymerase Chain Reaction; Regulatory Sequences, Nucleic Acid; Time Factors; Tretinoin

SOX3 is a member of the Sox gene family implicated in brain formation and cognitive function. It is considered to be one of the earliest neural markers in vertebrates, playing a role in specifying neuronal fate. Recently, we have established the first link between TALE (three-amino-acid loop extension) proteins, PBX1 (pre-B-cell leukemia homeobox 1) and MEIS1 (myeloid ecotropic viral integration site 1 homologue), and the expression of the human SOX3 gene. Here we present the evidence that TGIF (TG-interacting factor) is an additional TALE superfamily member involved in the regulation of human SOX3 gene expression in NT2/D1 cells by direct interaction with the consensus binding site that is conserved in primate orthologue promoters. Functional analysis demonstrated that mutation of the TGIF binding site resulted in the activation of SOX3 promoter. TGIF overexpression downregulates SOX3 promoter activity and decreases endogenous SOX3 protein expression in both uninduced and retinoic acid (RA)-induced NT2/D1 cells. Up to now, this is the first transcription factor identified as a negative regulator of SOX3 gene expression. The obtained results further underscore the significance of TALE proteins as important transcriptional regulators of SOX3 gene expression.

Topics: Animals; Base Sequence; Binding Sites; Carcinoma, Embryonal; Cell Line, Tumor; Down-Regulation; Embryonal Carcinoma Stem Cells; Homeodomain Proteins; Humans; Mice; Promoter Regions, Genetic; Rats; Repressor Proteins; SOXB1 Transcription Factors; Tretinoin

F9 embryonic carcinoma (EC) cells undergo extra-embryonic endodermal (ExE) differentiation in response to retinoic acid (RA) treatment, which induces the expression of two isoforms (p96 and p67) of the adaptor protein, Disabled-2 (Dab2). In the current study, constitutive and ectopic expression of the p96 isoform induced ExE differentiation in F9 EC cells in the absence of RA treatment via the activation of GATA-4 by p96. During the RA-induced differentiation process, Dab2 expression is induced by the GATA factors in a coherent feed-forward loop; on the other hand, we showed that p96 regulates GATA-4 in a positive feed-back manner in this study. Our results indicate that p96 Dab2 plays a key role in the ExE differentiation process.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Apoptosis Regulatory Proteins; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; Culture Techniques; Feedback, Physiological; GATA4 Transcription Factor; Mice; RNA, Small Interfering; Tretinoin

We investigated transactivation by NANOG in regulating growth and differentiation factor 3 (GDF3) expression in NCCIT cells. GDF3 expression was affected by shRNA-mediated downregulation and by exogenous overexpression of NANOG specifically, as well as by retinoic acid-mediated differentiation. GDF3 transcription was activated by NANOG, and the upstream region (-183 to -1) was sufficient to induce minimal transcriptional activity. Moreover, NANOG binds to the GDF3 minimal promoter in vivo and the transcriptional activity is mediated by NANOG transactivation domain. This study provides the first evidence that NANOG is a transcriptional activator of the expression of the oncogenic growth factor GDF3 in embryonic carcinoma cells.

Topics: Base Sequence; Binding Sites; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Growth Differentiation Factor 3; HEK293 Cells; Homeodomain Proteins; Humans; Molecular Sequence Data; Nanog Homeobox Protein; Neoplastic Stem Cells; Pluripotent Stem Cells; Promoter Regions, Genetic; Protein Structure, Tertiary; RNA, Small Interfering; Sequence Homology, Nucleic Acid; Trans-Activators; Tretinoin

We demonstrate the regulation of OCT4 gene expression mediated by liver receptor homolog-1 (LRH-1) in human embryonic carcinoma cells. LRH-1 and OCT4 are co-expressed in undifferentiated NCCIT cells and decreased during retinoic acid-induced differentiation. Dose-dependent overexpression of LRH-1 transactivated the OCT4 promoter activity and its dominant negative form with a deletion of activation function-2 motif reduced the activity even in the presence of LRH-1. The OCT4 promoter contains potent three LRH-1 binding sites; one within conserved region (CR) 1 and two within CR2. Mutagenesis of each binding site affected the decrease in OCT4 promoter activity and the 2nd binding site mutant most significantly reduced the transcriptional activity, compared to that of 1st and 3rd binding site mutants, respectively. Simultaneous disruption of 2nd and 3rd binding sites led to significant down-regulation of the activity even in the presence of 1st binding site-containing CR1. Moreover, mutation of each binding element within native or exogenous minimal promoter-driven CR1 or CR2 also decreased the promoter activity to some different extent, suggesting that three binding elements may be implicated in the induction of the full-activity of OCT4 promoter. In vivo binding assay revealed the 2nd and 3rd binding motifs within CR2 were more enriched than the 1st one within CR1. Taken together, our study indicates that LRH-1 acts as a transcriptional activator in the regulation of OCT4 gene expression through the cooperative interaction with three binding sites directly or/and indirectly.

Topics: Binding Sites; Carcinoma, Embryonal; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Octamer Transcription Factor-3; Promoter Regions, Genetic; Receptors, Cytoplasmic and Nuclear; Transcription, Genetic; Transcriptional Activation; Tretinoin

CADM1 is a multifunctional cell adhesion molecule expressed predominantly in the nerve system, testis and lung. The expression of the Cadm1 gene is induced during the neural differentiation of murine embryonal carcinoma P19 cells by treatment with retinoic acid (RA). Here, we show that the suppression of CADM1 expression using RNAi interfered with P19 cell aggregation and reduced cell populations expressing MAP2 after RA treatment. Nonaggregated P19 cells were not differentiated into neurons, suggesting that CADM1 participates in the aggregate formation and neuronal differentiation of P19 in vitro. A luciferase assay of a series of deletion mutants of the CADM1 promoter localized an RA-responsive cis-acting element to an approximately 90-bp fragment upstream of the translational start site. This element contains a putative binding site for transcription factor Sp1, named Sp1-binding site-1 (Sp1BS-1). Sp1BS-1 and adjacent Sp1-binding sites (Sp1BS-2 and Sp1BS-3) showed enhanced transcriptional activity by RA. Moreover, a chromatin immunoprecipitation showed that RA receptor (RAR)α was associated with a DNA fragment containing Sp1BS-1, whereas suppression of RARα expression using siRNA reduced the responsiveness of the CADM1 promoter to RA. These results suggest that Sp1 plays a critical role in RA-induced CADM1 expression through possible interaction with RARα in the neural differentiation of P19.

Topics: Animals; Binding Sites; Carcinoma, Embryonal; Cell Adhesion Molecule-1; Cell Adhesion Molecules; Cell Differentiation; Immunoglobulins; Mice; Neural Stem Cells; Reverse Transcriptase Polymerase Chain Reaction; Sp1 Transcription Factor; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Differentiation of human embryonic stem (ES) cells and embryonal carcinoma (EC) cells provides an in vitro model to study the process of neuronal differentiation. Retinoic acid (RA) is frequently used to promote neural differentiation of pluripotent cells under a wide variety of culture conditions. Through systematic comparison of differentiation conditions we demonstrate that RA induced neuronal differentiation of human ES and EC cells requires prolonged RA exposure and intercellular communication mediated by high cell density. These parameters are necessary for the up-regulation of neural gene expression (SOX2, PAX6 and NeuroD1) and the eventual appearance of neurons. Forced over-expression of neither SOX2 nor NEUROD1 was sufficient to overcome the density dependency of neuronal differentiation. Furthermore, inhibition of GSK3beta activity blocked the ability of RA to direct cell differentiation along the neural lineage, suggesting a role for appropriately regulated WNT signalling. These data indicate that RA mediated neuronal differentiation of human EC and ES cell lines is not a cell autonomous program but comprises of a multi-staged program that requires intercellular input.

Topics: Antineoplastic Agents; Blotting, Western; Carcinoma, Embryonal; Cell Cycle; Cell Differentiation; Cell Proliferation; Embryonal Carcinoma Stem Cells; Flow Cytometry; Fluorescent Antibody Technique; Humans; Neurons; Pluripotent Stem Cells; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tretinoin

Nestin is an intermediate filament protein expressed specifically in neural stem cells and progenitor cells of the central nervous system. DNA demethylation and histone modifications are two types of epigenetic modifications working in a coordinate or synergistic manner to regulate the expression of various genes. This study investigated and elucidated the epigenetic regulation of Nestin gene expression during embryonic differentiation along the neural cell lineage. Nestin exhibits differential DNA methylation and histone acetylation patterns in Nestin-expressing and nonexpressing cells. In P19 embryonic carcinoma cells, activation of Nestin expression is mediated by both trichostatin A and 5-aza-2'-deoxycytidine treatment, concomitant with histone acetylation, but not with DNA demethylation. Nestin transcription is also mediated by treatment with retinoic acid, again in the absence of DNA demethylation. Thus, histone acetylation is sufficient to mediate the activation of Nestin transcription. This study proposed that the regulation of Nestin gene expression can be used as a model to study the epigenetic regulation of gene expression mediated by histone acetylation, but not by DNA demethylation.

Topics: Acetylation; Animals; Azacitidine; Carcinoma, Embryonal; Cell Line; Chromatin; Chromatin Immunoprecipitation; Decitabine; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Embryonic Stem Cells; Enhancer Elements, Genetic; Epigenesis, Genetic; Histones; Hydroxamic Acids; Intermediate Filament Proteins; Introns; Mice; Nerve Tissue Proteins; Nestin; Promoter Regions, Genetic; Transcription, Genetic; Tretinoin

Ewing's sarcoma/primitive neuroectodermal tumor (EWS/PNET) has a characteristic chimeric oncogene EWS-FLI1, which results from chromosomal translocation t (11; 22), that is believed to initiate tumorigenesis of EWS/PNET. However, the specific details of EWS/PNET oncogenesis and exact role of EWS-FLI1 remain largely unknown. In this study we explored the role of EWS-FLI1 in tumor differentiation using an embryonal carcinoma cell line P19 as a model, with forced expression of EWS-FLI1 in these cells. EWS-FLI1 has been reported to promote neural differentiation in fibroblasts, mesenchymal stem cells and rhabdomyosarcoma cells. We show forced expression of EWS-FLI1 causes absence of retinoic acid-induced neural morphology, and decreases expression of neural-specific proteins MAPT and NCAM. Critical transcriptional factors for neural differentiation and stem cells are also altered in the presence of EWS-FLI1, including decreases in levels of Oct-3 and Pax-6, and an increase in the level of Id2, which is a target of EWS-FLI1. Increased proliferation and decreased apoptotic rates are also observed in P19 cells with forced expression of EWS-FLI1. Our results raise the possibility that arrest of neural differentiation by forced expression of EWS-FLI1 as observed in this study may result from dysregulation of the cell cycle and cell proliferation. Taken together, our results demonstrate that the modulation of neural differentiation in P19 cells which have a stem cell-like pluripotency in vitro can provide a novel model system to study the neural differentiation effects of EWS-FLI1 tumorigenesis of EWS/PNET.

Topics: Animals; Apoptosis; Carcinoma, Embryonal; Cell Cycle; Cell Differentiation; Cell Proliferation; Embryonal Carcinoma Stem Cells; Gene Expression Regulation, Neoplastic; Humans; Mice; Nerve Tissue Proteins; Neurons; Oncogene Proteins, Fusion; Proto-Oncogene Protein c-fli-1; RNA-Binding Protein EWS; Tetrazolium Salts; Thiazoles; Time Factors; Transfection; Tretinoin

Retinoids are dietary hormones acting through nuclear receptors for retinoic acid, important especially during embryonic development. This study focuses on the disruption of signaling pathways of retinoids by polycyclic aromatic hydrocarbons (PAHs) and their N-heterocyclic analogs (N-PAHs), important environmental contaminants with numerous biological effects. In vitro test with P19/A15 cell line stably transfected with luciferase reporter gene under control of retinoic acid-responsive elements was used to investigate both direct activation of retinoic acid receptors and modulation of response induced by natural ligand all-trans retinoic acid (ATRA) by 26 PAHs and N-PAHs. While none of individual compounds alone activated retinoic acid receptors, many of them modulated ATRA-mediated activity both after 6 h and 24 h exposure. Majority of compounds active after 6h downregulated ATRA-mediated activity (most effective were two analogs of dibenz[a,h]anthracene with LOECs about 185 nM), while most compounds active after 24h upregulated the effects of ATRA (most effective benz[a]acridine and dibenz[a,i]acridine caused 400% induction of ATRA response). Quantitative structure-activity relationship analysis identified molecular volume and dipole moment as the most important descriptors of inhibitory effects after 6h, while length, total molecular energy, gap-HOMO/LUMO and Van der Waals energy are important descriptors for stimulatory effects of PAHs and N-PAHs. This study demonstrates those abundant pollutants such as PAHs and their analogs interfere in vitro with retinoid signaling, which could play role in some in vivo effects of these organic contaminants such as teratogenicity.

Topics: Animals; Carcinoma, Embryonal; Cell Line, Tumor; Down-Regulation; Environmental Pollutants; Genes, Reporter; Luciferases; Mice; Polycyclic Aromatic Hydrocarbons; Quantitative Structure-Activity Relationship; Receptors, Retinoic Acid; Response Elements; Signal Transduction; Time Factors; Transfection; Tretinoin

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

Topics: Adult; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; Chromatin; Colonic Neoplasms; CpG Islands; DNA Methylation; DNA-Binding Proteins; Enhancer of Zeste Homolog 2 Protein; Epigenesis, Genetic; GATA4 Transcription Factor; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Nucleic Acid Conformation; Oxidoreductases, N-Demethylating; Polycomb Repressive Complex 2; Polycomb-Group Proteins; Repressor Proteins; Transcription Factors; Tretinoin

beta-Catenin-mediated Wnt signaling is essential in embryonic development and in adult tissues. Recent studies have demonstrated that Axin not only plays an important inhibitory role in coordinating beta-catenin degradation, but is itself degraded by the low-density-lipoprotein receptor-related protein (LRP)5/6 Wnt co-receptor. Here, we demonstrate that the endocytic adaptor molecule Disabled-2 (Dab2), which we have previously demonstrated to act as an inhibitor of beta-catenin signaling, interacts with Axin and prevents its interaction with and degradation by the LRP5 co-receptor, thereby increasing its half-life and stabilization. Dab2 levels induced during retinoic acid-induced differentiation of F9, or during transforming growth factor-beta-induced epithelial-mesenchymal transdifferentiation of mouse mammary epithelial cells result in the stabilization of Axin and concomitant inhibition of beta-catenin signaling. Ectopic expression of Dab2 in F9 cells as well as in transformed cell lines results in increased Axin expression and attenuation of Wnt-mediated signaling. We conclude that Dab2 may play an important role in the maintenance of the differentiated state and restrain Wnt-mediated proliferation through its association with and modulation of Axin.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Apoptosis Regulatory Proteins; Axin Protein; beta Catenin; Blotting, Western; Carcinoma, Embryonal; Cell Differentiation; Cell Proliferation; Epithelial Cells; Immunoprecipitation; Mesoderm; Mice; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta; Tretinoin; Wnt Proteins

Human embryonal carcinoma (EC) cell lines exhibit considerable heterogeneity in their levels of pluripotency. Thus, NT2/D1 cells differentiate into neural lineages upon exposure to all-trans retinoic acid (ATRA) and non-neural epithelial lineages upon exposure to bone morphogenetic protein-2 (BMP-2). In contrast, 27X-1 cells differentiate into extra-embryonic endodermal (ExE) cells upon treatment with either morphogen. To understand the molecular basis for the differential responses of the two cell lines, we performed gene expression profiling at the undifferentiated EC cell line state to identify constitutive differences in gene expression. NT2/D1 cells preferentially expressed transcripts associated with neurectodermal development, whereas 27X-1 cells expressed high levels of transcripts associated with mesendodermal characteristics. We then determined temporal expression profiles of 27X-1 cells during ExE differentiation upon treatment with ATRA and BMP-2 and compared the data with changes in gene expression observed during BMP-2- and ATRA-induced differentiation of NT2/D1 cells. ATRA and BMP-2 induced distinct sets of transcription factors and phenotypic markers in the two EC cell lines, underlying distinct lineage choices. Although 27X-1 differentiation yielded comprehensive gene expression profiles of parietal endodermal lineages, we were able to use the combined analysis of 27X-1 data with data derived from yolk sac tumors for the identification of transcripts associated with visceral endoderm formation. Our results demonstrate constitutive differences in the levels of pluripotency between NT2/D1 and 27X-1 cells that correlate with lineage potential. This study also demonstrates that EC cells can serve as robust models to investigate early lineage choices during both embryonic and extra-embryonic human development.

Topics: Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Morphogenesis; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Oligopeptides; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Tretinoin

Analyses of molecular events associated with reprogramming somatic nuclei to pluripotency are scarce. We previously reported the reprogramming of epithelial cells by extract of undifferentiated embryonal carcinoma (EC) cells. We now demonstrate reprogramming of DNA methylation and histone modifications on regulatory regions of the developmentally regulated OCT4 and NANOG genes by exposure of 293T cells to EC cell extract. OCT4 and NANOG are transcriptionally up-regulated and undergo mosaic cytosine-phosphate-guanosine demethylation. OCT4 demethylation occurs as early as week 1, is enhanced by week 2, and is most prominent in the proximal promoter and distal enhancer. Targeted OCT4 and NANOG demethylation does not occur in 293T extract-treated cells. Retinoic acid-mediated differentiation of reprogrammed cells elicits OCT4 promoter remethylation and transcriptional repression. Chromatin immunoprecipitation analyses of lysines K4, K9, and K27 of histone H3 on OCT4 and NANOG indicate that primary chromatin remodeling determinants are acetylation of H3K9 and demethylation of dimethylated H3K9. H3K4 remains di- and trimethylated. Demethylation of trimethylated H3K9 and H3K27 also occurs; however, trimethylation seems more stable than dimethylation. We conclude that a central epigenetic reprogramming event is relaxation of chromatin at loci associated with pluripotency to create a conformation compatible with transcriptional activation.

Topics: Acetylation; Carcinoma, Embryonal; Cell Line; Chromatin Assembly and Disassembly; DNA Methylation; DNA-Binding Proteins; Epigenesis, Genetic; Histones; Homeodomain Proteins; Humans; In Vitro Techniques; Lysine; Methylation; Nanog Homeobox Protein; Octamer Transcription Factor-3; Promoter Regions, Genetic; Protein Processing, Post-Translational; Regulatory Sequences, Nucleic Acid; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Embryonal carcinoma P19 cells provide an ideal model to study molecular programs along differentiation. Upon induction by retinoic acid (RA), the cells undergo a program of differentiation that generates functioning neurons within 60 h. RA induced cells that were plated as sparse (1000 cells/mm(2)) or dense (4000 cells/mm(2)) cultures showed a marked difference in the culture morphology with the dense cultures exhibiting rapid maturation and accelerated neurite outgrowth. The protein expression levels of the sparse and dense cultures were compared 48 h following RA. Cell extracts were separated by 1-DE and 2-DE and differential expression (>four-fold) proteins were identified by MS. Here, we focus on 20 proteins associated with cytoskeletal regulation and stress-dependent protein refolding. The first group includes drebrin, cofilin, alpha-internexin, vimentin, and nestin. Among the proteins in the second group are subunits of the TCP-1, and several chaperones of the Hsp70 and Hsp90 families. We show that coordinated remodeling of the cytoskeleton and modulations in chaperone activity underlie the change in neurite extension rate. Furthermore, a proteomics-based analysis applied on P19 neurons demonstrated pathways underlying neuronal outgrowth, suggesting that a malfunction of such pathways leads to neuropathological conditions.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Cytoskeletal Proteins; Cytoskeleton; Electrophoresis, Gel, Two-Dimensional; Mass Spectrometry; Mice; Molecular Sequence Data; Neurons; Protein Folding; Tretinoin; Tumor Cells, Cultured

Mutations in human angiogenin (hANG), an angiogenic member of the RNase A superfamily, have been recently reported in patients with amyotrophic lateral sclerosis (ALS), a progressive late-onset neurodegenerative disorder. However, very little is known about the expression and subcellular distribution of ANG in the nervous system or its role in differentiation. Here we report that mouse angiogenin-1 (mAng-1) is strongly expressed in the developing nervous system during mouse embryogenesis and neuroectodermal differentiation of pluripotent P19 embryonal carcinoma cells. mAng1 is strongly expressed in motor neurons (MNs) in the spinal cord and dorsal root ganglia as well as in post-mitotic MNs derived from P19 cells. We also show for the first time that ANG expression is in the growth cones and neurites. NCI 65828, an inhibitor of the ribonucleolytic activity of hANG, affected pathfinding by P19-derived neurons but not neuronal differentiation. Our findings clearly show that ANG plays an important role in neurite pathfinding and this has implications for ALS.

Topics: Amyotrophic Lateral Sclerosis; Animals; Carcinoma, Embryonal; Cell Differentiation; Embryo, Mammalian; Female; Fluorescent Antibody Technique; Male; Mice; Mice, Inbred C57BL; Naphthalenesulfonates; Neurites; Ribonuclease, Pancreatic; Tretinoin; Tumor Cells, Cultured

The epiblast, derived from the inner cell mass (ICM), represents the final embryonic founder cell population of mouse embryo and can give rise to all germ layer lineages including the neuroectoderm. The generation of neural stem cells from epiblast-like cells is of great value for studying the mechanism of neural determination during gastrulation stages of embryonic development. Mouse embryonic carcinoma (EC) P19 cells are equivalent to the epiblast of early post-implantation blastocysts. In this study, we establish a feasible induction system that allows rapid and efficient derivation of a high percentage ( approximately 95%) of neural stem cells from P19 EC cell in N2B27 serum-free medium. The induced neural stem cells bear anterior neuroectoderm characters, and can be efficiently caudalized by retinoic acid (RA). These neural stem cells have multilineage potential to differentiate into neurons, astrocytes, and oligodendrocytes. Mechanistic analysis indicates that inhibition of the bone morphogenetic protein (BMP) pathway may be the main reason for N2B27-neural induction, and that fibroblast growth factor (FGF) signaling is also involved in this process. This method will provide an in vitro system to dissect the molecular mechanisms involved in neural induction of early mouse embryos.

Topics: Animals; Carcinoma, Embryonal; Cell Culture Techniques; Cell Differentiation; Cell Line, Tumor; Cell Lineage; Clone Cells; Culture Media, Serum-Free; Ectoderm; Embryo, Mammalian; Feasibility Studies; Immunohistochemistry; Mice; Neurons; Stem Cells; Tretinoin

Retinoids are known to regulate important processes such as differentiation, development, and embryogenesis. Some effects, such as malformations in frogs or changes in metabolism of birds, could be related to disruption of the retinoid signaling pathway by exposure to organic contaminants. A new reporter gene assay has been established for evaluation of the modulation of retinoid signaling by individual chemicals or environmental samples. The bioassay is based on the pluripotent embryonic carcinoma cell line P19 stably transfected with the firefly luciferase gene under the control of a retinoic acid-responsive element (clone P19/ A15). The cell line was used to characterize the effects of individual chemicals and sediments extracts on retinoid signaling pathways. The extracts of sediments from the River Kymi, Finland, which contained polychlorinated dioxins and furans and polycyclic aromatic hydrocarbons (PAHs), significantly increased the potency of all-trans retinoic acid (ATRA), while no effect was observed with the extract of the sediment from reference locality. Considerable part of the effect was caused by the labile fraction of the sediment extracts. Also, several individual PAHs potentiated the effect of ATRA; on the other hand, 2,3,7,8-tetrachlorodibenzo-p-dioxin and several phthalates showed slightly inhibiting effect. These results suggest that PAHs could be able to modulate the retinoid signaling pathway and that they could be responsible for a part of the proretinoid activity observed in the sediment extracts. However, the effects of PAHs on the retinoic acid signaling pathways do not seem to be mediated directly by crosstalk with aryl hydrocarbon receptor.

Topics: Animals; Biological Assay; Carcinoma, Embryonal; Cell Line, Tumor; Dioxins; Environmental Pollutants; Finland; Furans; Genes, Reporter; Geologic Sediments; Luciferases, Firefly; Phthalic Acids; Polycyclic Aromatic Hydrocarbons; Retinoids; Risk Assessment; Signal Transduction; Tretinoin

The use of retinoids as anti-cancer agents has been limited due to resistance and low efficacy. The dynamics of nuclear receptor coregulation are incompletely understood. Cell-and context-specific activities of nuclear receptors may be in part due to distinct coregulator complexes recruited to distinct subsets of target genes. RIP140 (also called NRIP1) is a ligand-dependent corepressor that is inducible with retinoic acid (RA). We had previously shown that RIP140 limits RA induced tumor cell differentiation of embryonal carcinoma; the pluriopotent stem cells of testicular germ cell tumors. This implies that RIP140 represses key genes required for RA-mediated tumor cell differentiation. Identification of these genes would be of considerable interest.. To begin to address this issue, microarray technology was employed to elucidate in a de novo fashion the global role of RIP140 in RA target gene regulation of embryonal carcinoma. Subclasses of genes were affected by RIP140 in distinct manners.Interestingly, approximately half of the RA-dependent genes were unaffected by RIP140. Hence, RIP140 appears to discriminate between different classes of RA target genes. In general, RIP140-dependent gene expression was consistent with RIP140 functioning to limit RA signaling and tumor cell differentiation. Few if any genes were regulated in a manner to support a role for RIP140 in "active repression". We also demonstrated that RIP140 silencing sensitizes embryonal carcinoma cells to low doses of RA.. Together the data demonstrates that RIP140 has profound effects on RA-mediated gene expression in this cancer stem cell model. The RIP140-dependent RA target genes identified here may be particularly important in mediating RA-induced tumor cell differentiation and the findings suggest that RIP140 may be an attractive target to sensitize tumor cells to retinoid-based differentiation therapy. We discuss these data in the context of proposed models of RIP140-mediated repression.

Topics: Adaptor Proteins, Signal Transducing; Carcinoma, Embryonal; Cell Differentiation; Embryonal Carcinoma Stem Cells; Feedback, Physiological; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Neoplastic Stem Cells; Nuclear Proteins; Nuclear Receptor Interacting Protein 1; Oligonucleotide Array Sequence Analysis; Pluripotent Stem Cells; Receptors, Retinoic Acid; RNA Interference; RNA, Small Interfering; Signal Transduction; Transcription, Genetic; Tretinoin

LIF is a cytokine playing a key role in the regulation of self-renewal and maintenance of undifferentiated state in mouse ES cells. The response of pluripotent cells to LIF is mediated mainly by the STAT3 and ERK signalling pathways. Recently, we have shown that LIF potentiated retinoic acid-induced neural differentiation of pluripotent mouse embryonal carcinoma P19 cells. Here we demonstrate that pro-neural effects of LIF and partially also of retinoic acid are abolished by inhibition of the JAK2->STAT3 signalling pathway. In contrast, inhibition of the MEK1->ERK signalling pathway does not exhibit any effect. These results suggest that in neurogenic regions, cooperative action of LIF and other neuro-differentiation-inducing factors, such as retinoic acid, may be mediated by the STAT3 signalling pathway.

Topics: Animals; Biomarkers; Butadienes; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; Extracellular Signal-Regulated MAP Kinases; Janus Kinase 2; Leukemia Inhibitory Factor; MAP Kinase Signaling System; Mice; Neurons; Nitriles; Response Elements; Signal Transduction; STAT3 Transcription Factor; Transcription, Genetic; Transcriptional Activation; Tretinoin; Tyrphostins

Retinoic acid (RA) is a signaling molecule that plays a pivotal role in major cellular processes and vertebrate development. RA action is mediated by specialized transcription factors, the nuclear RA receptors (RARs), which regulate the transcription of genes containing a RA-responsive element (RARE). Here we demonstrate that the genes for the RA-receptor RARbeta2 and the cytochrome P450 RA-specific hydrolase Cyp26a1 involved in RA catabolism are coordinately regulated by RA. We found that both RARbeta2 and Cyp26a1 genes are epigenetically silenced in the absence of DNA methylation in RAC65, a P19 embryocarcinoma cell line derivative carrying a dominant-negative RARalpha mutant and resistant to the growth-inhibitory and differentiation effects of RA. In response to RA, RARbeta2 transcription is epigenetically regulated by RARalpha. Similarly, we found that Cyp26a1 transcription is epigenetically regulated by RARbeta2. Knocking down RARbeta2 transcription by RNA interference in wild-type P19 cells, with an intact RARalpha, induced Cyp26a1 transcriptional repression in the absence of DNA methylation. Concomitantly, cells developed RA resistance and did not undergo RA-induced neuron differentiation. Apparently, RARalpha, RARbeta2 and Cyp26a1 are components of a RA-regulated gene network. Factors affecting an upstream gene of the network can trigger repressive chromatin changes -- which are propagated in a domino fashion - at downstream genes of the network. This study also shows that chromatin inactivity, and consequent transcriptional silencing, can be achieved in the absence of DNA methylation.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Chromatin; Cytochrome P-450 Enzyme System; DNA Methylation; Epigenesis, Genetic; Gene Silencing; Mice; Neurons; Receptors, Retinoic Acid; Retinoic Acid 4-Hydroxylase; Tretinoin

Retinoic acid (RA) is a morphogen that induces endodermal differentiation of murine P19 embryonic carcinoma cells. RA-induced differentiation of P19 cells has been used as a model system to define the differentiation programs of pluripotent stem cells. Using this system it has been shown that G alpha13--the alpha-subunit of the heterotrimeric G protein G13--and its activation of JNK-module are critically required for the endodermal differentiation of P19 cells. However, the mechanism through which G alpha13 is linked to JNK-module is unknown. Here, we report that RA stimulates the expression of JNK-interacting leucine zipper protein (JLP), a newly identified JNK-scaffolding protein and its critical role in RA-mediated endodermal differentiation. Our results indicate that there is a physical association between JLP and G alpha13 in RA-stimulated P19 cells. More interestingly, silencing JLP abrogates RA-mediated endodermal differentiation of P19 cells analogous to the effects seen with the silencing of G alpha13 or JNK. Therefore, our studies presented here identify for the first time, a novel role for a newly identified scaffolding protein in RA-mediated endodermal differentiation, providing a new signaling conduit to transmit signals from RA to JNK module.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antineoplastic Agents; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; Endoderm; GTP-Binding Protein alpha Subunits, G12-G13; Mice; Models, Biological; Pluripotent Stem Cells; Signal Transduction; Tretinoin

Following the differentiation of cultured stem cells is often reliant on the expression of genes and proteins that provide information on the developmental status of the cell or culture system. There are few molecules, however, that show definitive expression exclusively in a specific cell type. Moreover, the reliance on a small number of molecules that are not entirely accurate biomarkers of particular tissues can lead to misinterpretation in the characterization of the direction of cell differentiation. Here we describe the use of technology that examines the mass spectrum of proteins expressed in cultured cells as a means to identify the developmental status of stem cells and their derivatives in vitro. This approach is rapid and reproducible and it examines the expression of several different biomarkers simultaneously, providing a profile of protein expression that more accurately corresponds to a particular type of cell differentiation.

Topics: Acetamides; Antigens, Surface; Antigens, Tumor-Associated, Carbohydrate; Biomarkers; Carcinoma, Embryonal; Cell Differentiation; Embryonal Carcinoma Stem Cells; Flow Cytometry; Gangliosides; Glycosphingolipids; Humans; Keratins; Neoplastic Stem Cells; Neurons; Peptides; Pluripotent Stem Cells; Proteoglycans; Proteome; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stage-Specific Embryonic Antigens; Tretinoin; Tubulin

Apoptosis is thought to be involved in the maintenance of cellular homeostasis, as well as various pathological processes. However, little information is available about the regulation of apoptosis during the aggregation stage of P19 embryonal carcinoma (EC) cells. Here we report that aggregation-induced apoptosis is markedly attenuated by treatment with retinoic acid (RA). PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression was down-regulated during the aggregation phase of P19 EC cells in the presence, but not in the absence, of RA. Suppression of PTEN expression during the aggregation was accompanied by increased phosphorylation of serine/threonine kinase Akt and glycogen synthase kinase-3beta (GSK-3beta). Our results suggest that RA attenuates the induction of apoptosis during the aggregation phase of P19 EC cells, probably by suppressing PTEN expression.

Topics: Animals; Apoptosis; Carcinoma, Embryonal; Cell Differentiation; Cell Shape; Down-Regulation; Enzyme Activation; Gene Expression Regulation, Neoplastic; Mice; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Transcription, Genetic; Tretinoin

Testicular germ cell tumors (TGCTs) arise despite possessing high levels of wild-type p53, suggesting p53 latency. We have previously shown that p53 repression in TGCT-derived human embryonal carcinoma (EC) is relieved upon treatment with all-trans retinoic acid (RA), resulting in enhanced p53 transactivation activity. To further investigate p53 repression in EC, a series of gal4-p53 truncation constructs were generated. Deletion of the core DNA-binding region, residues 117-274, had no effect on basal or RA-induced p53 activity. Progressively, larger truncations were made in the C- or N-terminal direction. Deletion of residues toward the C-terminus of p53 as far as residue 354 did not affect either the basal or RA-inducible activity of gal4-p53. When a small region in the N-terminus was deleted (residues 105-116), relief of the basal repression of p53 activity characteristic of EC was observed. Fusion of this region to the VP16 activation domain (VPAD) resulted in a 10-20-fold repression of VPAD activity in NT2/D1 human EC cells, indicating that this region acts as a heterologous repressor. Owing to its location in the N-terminal half of p53, we have named this region the p53 N-terminal Repression Domain (p53-NRD). The p53-NRD mediated repression in a variety of cell lines, with the most prominent repression observed in human EC cells. While RA alone had no effect on p53-NRD activity, cotreatment with RA and the histone deacetylase inhibitor trichostatin-A (TSA) completely relieved p53-NRD-mediated repression. In contrast, NRD-mediated repression was not sensitive to RA and TSA in a derived RA-resistant cell line with a retinoic acid receptor gamma (RARgamma) defect, but sensitivity could be restored with transfection of RARgamma. These data indicate that a unique repressor domain resides in p53 at residues 90-116 whose activity can be modulated in the presence of 'differentiation therapy' and 'transcription therapy' agents.

Topics: Amino Acid Sequence; Animals; Breast Neoplasms; Carcinoma, Embryonal; Cell Division; Cell Line, Tumor; Chlorocebus aethiops; CHO Cells; COS Cells; Cricetinae; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Transcription, Genetic; Transfection; Tretinoin; Tumor Suppressor Protein p53

Mouse F9 embryonal carcinoma cells have been widely used as a model for studying the mechanism of embryonic differentiation, because they are similar to the inner cell mass of early mouse embryos and can differentiate into primitive endoderm (PrE) following retinoic acid (RA) treatment. During F9 cell differentiation, the carbohydrate chains of glycoproteins and their corresponding glycosyltransferases are known to undergo rapid changes. However, there have been no corresponding reports on the expression of gangliosides. We have developed a custom cDNA array that is highly sensitive for the genes responsible for sphingolipid (SL) biosynthesis and metabolism. Using this, we found that, of the 28 selected genes, 26 exhibited increased expression during F9 differentiation into PrE. Although neutral glycosphingolipids (GSLs) were expressed at similar levels before and after differentiation, a greater than 20-fold increase in total ganglioside content was evident in PrE. Glucosylceramide synthase inhibitors (d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol [d-PDMP] and its analog) depleted gangliosides and this resulted in delayed expression of Disabled-2 (Dab-2), suggesting the involvement of gangliosides in F9 cell differentiation. Disruption of cholesterol-enriched membrane microdomains by methyl-beta-cyclodextrin (MbetaCD) also delayed differentiation. Both MbetaCD and d-PDMP blocked the accumulation of Src family kinases (SFKs) to microdomains. However, d-PDMP did not block flotillin accumulation, yet MbetaCD did. Additionally, confocal laser microscopy revealed the formation of distinct functional microdomains integrating SFKs with gangliosides and cholesterol during PrE differentiation. Thus, we demonstrate the outstanding up-regulation of ganglioside biosynthesis and its importance in the formation of distinct microdomains incorporating SFKs with gangliosides during RA-induced differentiation of F9 cells.

Topics: Animals; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; DNA Primers; Gangliosides; Mice; src-Family Kinases; Tretinoin; Up-Regulation

Embryonal carcinoma is a histologic subgroup of testicular germ cell tumors (TGCTs), and its cells may follow differentiation lineages in a manner similar to early embryogenesis. To acquire new knowledge about the transcriptional programs operating in this tumor development model, we used 22k oligo DNA microarrays to analyze normal and neoplastic tissue samples from human testis. Additionally, retinoic acid-induced in vitro differentiation was studied in relevant cell lines. We identified genes characterizing each of the known histologic subtypes, adding up to a total set of 687 differentially expressed genes. Among these, there was a significant overrepresentation of gene categories, such as genomic imprinting and gene transcripts associated to embryonic stem cells. Selection for genes highly expressed in the undifferentiated embryonal carcinomas resulted in the identification of 58 genes, including pluripotency markers, such as the homeobox genes NANOG and POU5F1 (OCT3/4), as well as GAL, DPPA4, and NALP7. Interestingly, abundant expression of several of the pluripotency genes was also detected in precursor lesions and seminomas. By use of tissue microarrays containing 510 clinical testicular samples, GAL and POU5F1 were up-regulated in TGCT also at the protein level and hence validated as diagnostic markers for undifferentiated tumor cells. The present study shows the unique gene expression profiles of each histologic subtype of TGCT from which we have identified deregulated components in selected processes operating in normal development, such as WNT signaling and DNA methylation.

Topics: Carcinoma in Situ; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; DNA Methylation; DNA-Binding Proteins; Galanin; Gene Expression Profiling; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Humans; Male; Octamer Transcription Factor-3; Oligonucleotide Array Sequence Analysis; Seminoma; Testicular Neoplasms; Tissue Array Analysis; Transcription Factors; Tretinoin; Up-Regulation

Embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, are the malignant counterparts of pluripotent embryonic stem (ES) cells, but commonly exhibit a reduced ability to differentiate, presumably because of continual selection for genetic changes that alter the balance between self-renewal, differentiation and apoptosis in favour of self-renewal. To explore the nature of the genetic changes that promote nullipotency, we have compared two human EC cell lines, a 'nullipotent' line, 2102Ep, and a 'pluripotent' line, NTERA2. A hybrid derived by fusion of these cells differentiates in response to retinoic acid but, unlike the parental NTERA2 line, does not form terminally differentiated neurons. This implies that the nullipotent EC cell line, 2102Ep, differs in expression of at least two functions in comparison with the NTERA2 pluripotent line, one affecting commitment to differentiation, and one affecting terminal neural differentiation. We have now investigated the possible role of the CDK inhibitor, p27kip1 (p27) in commitment and terminal differentiation. In NTERA2, but not in 2102Ep cells, retinoic acid induces up-regulation of p27 expression, suggesting that 2102Ep cells lack this capacity. However, constitutive expression of a p27 transgene does not overcome the block to differentiation in the 2102Ep parental cells; commitment to differentiation must be blocked elsewhere. On the other hand, constitutive over-expression of p27 from a transgene enhances the neural differentiation of NTERA2 cells. Our results suggest that p27 plays a role in terminal neuronal differentiation of human EC cells, but not in their initial commitment to differentiation, and that other factors, possibly Cyclin D2, specifically limit its ability to promote neural differentiation.

Topics: Base Sequence; Carcinoma, Embryonal; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; Cyclin D2; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Embryonal Carcinoma Stem Cells; Gene Expression; Humans; Neoplastic Stem Cells; Neurons; Plasmids; Pluripotent Stem Cells; Proteasome Inhibitors; Recombinant Proteins; Tretinoin; Tumor Suppressor Proteins

The P19 mouse embryonal carcinoma cell line was used as a model for a study of apoptosis accompanying differentiation induced by all-trans retinoic acid (ATRA). Apoptosis was detected both on the basis of morphological features (nuclear fragmentation, blebbing of plasma membrane, and formation of apoptotic bodies), and by using DNA electrophoresis and flow-cytometric measurement of DNA content. Actin cytoskeleton was studied both on morphological and submicroscopic levels. ATRA-treated cells manifested apoptosis-specific changes in the distribution of actin foremost in association with their entry into executive phase of apoptosis, when F-actin cables participated in cell disintegration into apoptotic bodies. Using immunogold labeling, actin was also identified in centers of fragmenting apoptotic nuclei, in the disintegration of which it is likely involved as well. At the same time, a cleavage of actin by active caspase-3 was proved, resulting in the emergence of 32 kDa fragment, termed fractin. Measurement of F-actin and fractin content using flow cytometry showed an unequivocal decrease of F-actin and synchronous increase of fractin in the apoptotic population as compared to non-treated cells. Therefore, our results proved both actin proteolysis and active involvement of specific actin structures in the final cell disintegration during apoptosis in the P19 cells.

Topics: Actins; Animals; Apoptosis; Blotting, Western; Carcinoma, Embryonal; Caspase 3; Caspases; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Cytoskeleton; DNA; DNA Fragmentation; Electrophoresis; Flow Cytometry; Fluorescent Antibody Technique, Indirect; HL-60 Cells; Humans; Immunohistochemistry; Light; Mice; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Peptide Fragments; Scattering, Radiation; Tretinoin

Testicular germ cell tumors (TGCTs) are the most common carcinomas of young men aged 15-35. The molecular events involved in TGCT genesis are poorly understood. TGCTs have near universal amplification of the short arm of chromosome 12, however positional cloning efforts have not identified causative genes on 12p involved in formation or progression of TGCTs. Human embryonal carcinoma (EC) are the stem cells of TGCTs and are pluripotent. EC cells terminally differentiate toward a neuronal lineage with all-trans retinoic acid (RA) treatment resulting in a concomitant G1 cell cycle arrest and loss of tumorigenicity. Our efforts to define the molecular mechanisms of RA-mediated tumor cell differentiation at a critical "commitment to differentiate" window has identified a cassette of genes on 12p that are repressed with RA precisely as EC cells lose tumorigenic potential. These are Nanog, CD9, EDR1 (PHC1), SCNN1A, GDF3, Glut3 and Stella. The master pluripotency regulator Oct4 is located on chromosome 6 and is also repressed by RA. Notably, knockdown of Oct4 with siRNA results in repression of basal Nanog, EDR1, GDF3 and Stella gene expression. Nanog has recently been identified to play a role in maintenance of the pluripotency of mouse embryonic stem cells and CD9, EDR1, GDF3, and Stella have each been implicated as stem cell markers. Since RA suppresses the tumorigenicity of EC cells, these genes may have a critical role in the etiology of TGCTs, suggesting a link between enforced pluripotency and transformation.

Topics: Adolescent; Adult; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; Chromosomes, Human, Pair 12; Humans; Male; Octamer Transcription Factor-3; Oligonucleotide Array Sequence Analysis; Pluripotent Stem Cells; RNA, Small Interfering; Testicular Neoplasms; Tretinoin

In response to retinoic acid, embryonic stem and carcinoma cells undergo differentiation to embryonic primitive endoderm cells, accompanied by a reduction in cell proliferation. Differentiation does not reduce the activation of cellular MAPK/Erk, but does uncouple mitogen-activated protein kinase (MAPK) activation from phosphorylation/activation of Elk-1 and results in inhibition of c-Fos expression, whereas phosphorylation of the cytoplasmic substrate p90RSK remains unaltered. Cell fractionation and confocal immunofluorescence microscopy demonstrated that activated MAPK is restricted to the cytoplasmic compartment after differentiation. An intact actin and microtubule cytoskeleton appears to be required for the restriction of MAPK nuclear entry induced by retinoic acid treatment because the cytoskeletal disrupting agents nocodazole, colchicine, and cytochalasin D are able to revert the suppression of c-Fos expression. Thus, suppression of cell proliferation after retinoic acid-induced endoderm differentiation of embryonic stem and carcinoma cells is achieved by restricting nuclear entry of activated MAPK, and an intact cytoskeleton is required for the restraint.

Topics: Active Transport, Cell Nucleus; Animals; Antineoplastic Agents; Carcinoma, Embryonal; Cell Differentiation; Cell Line; Cell Nucleus; Colchicine; Cytochalasin D; Cytoskeleton; Endoderm; Enzyme Activation; Fatty Acids, Unsaturated; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinases; Nocodazole; Nucleic Acid Synthesis Inhibitors; Proto-Oncogene Proteins c-fos; ras Proteins; Stem Cells; Subcellular Fractions; Tretinoin

The Sox6 gene is a member of the Sox gene family that encodes transcription factors. Previous studies have suggested that Sox6 plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of neurons, glia and fibroblast-like cells. In this report, we have shown that Sox6 mRNA increased rapidly in P19 cells during RA induction and then decreased during the differentiation of P19 into neuronal cells. To explore the possible roles of Sox6 during this process, stably Sox6-overexpressing P19 cell lines (P19[Sox6]) were established. These P19[Sox6] had acquired both characteristics of the wild-type P19 induced by RA. First, P19[Sox6] cells showed a marked cellular aggregation in the absence of RA. Second, P19[Sox6] could differentiate into microtubule-associated protein 2 (MAP2)-expressing neuronal cells in the absence of RA. Sox6 expression could cause the activation of endogenous genes including the neuronal transcription factor Mash-1, the neuronal development-related gene Wnt-1, the neuron-specific cell adhesion molecule N-cadherin, and the neuron-specific protein MAP2, resulting in neurogenesis. Moreover, E-cadherin, a major cell adhesion molecule of wild-type P19, was strongly induced by Sox6, resulting in cellular aggregation without RA. Thus Sox6 may play a critical role in cellular aggregation and neuronal differentiation of P19 cells.

Topics: Animals; Carcinoma, Embryonal; Cell Adhesion Molecules; Cell Aggregation; Cell Differentiation; Cell Line, Tumor; DNA-Binding Proteins; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; High Mobility Group Proteins; Mice; Microtubule-Associated Proteins; Neurons; RNA, Messenger; SOXD Transcription Factors; Transcription Factors; Transcriptional Activation; Transfection; Tretinoin

The microRNAs (miRNAs) are an extensive class of small noncoding RNAs (18 to 25 nucleotides) with probable roles in the regulation of gene expression. In Caenorhabditis elegans, lin-4 and let-7 miRNAs control the timing of fate specification of neuronal and hypodermal cells during larval development. lin-4, let-7 and other miRNA genes are conserved in mammals, and their potential functions in mammalian development are under active study.. In order to identify mammalian miRNAs that might function in development, we characterized the expression of 119 previously reported miRNAs in adult organs from mouse and human using northern blot analysis. Of these, 30 miRNAs were specifically expressed or greatly enriched in a particular organ (brain, lung, liver or skeletal muscle). This suggests organ- or tissue-specific functions for miRNAs. To test if any of the 66 brain-expressed miRNAs were present in neurons, embryonal carcinoma cells were treated with all-trans-retinoic acid to promote neuronal differentiation. A total of 19 brain-expressed miRNAs (including lin-4 and let-7 orthologs) were coordinately upregulated in both human and mouse embryonal carcinoma cells during neuronal differentiation. The mammalian ortholog of C. elegans lin-28, which is downregulated by lin-4 in worms via 3' untranslated region binding, was also repressed during neuronal differentiation of mammalian embryonal carcinoma cells. Mammalian lin-28 messenger RNAs contain conserved predicted binding sites in their 3' untranslated regions for neuron-expressed miR-125b (a lin-4 ortholog), let-7a, and miR-218.. The identification of a subset of brain-expressed miRNAs whose expression behavior is conserved in both mouse and human differentiating neurons implicates these miRNAs in mammalian neuronal development or function.

Topics: Adult; Animals; Brain; Caenorhabditis elegans Proteins; Carcinoma, Embryonal; Cell Differentiation; Cell Line; Cell Line, Tumor; Down-Regulation; Embryo, Mammalian; Gene Expression Profiling; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Genetic Linkage; Humans; Mice; Mice, Inbred C57BL; MicroRNAs; Neurons; Repressor Proteins; RNA, Messenger; Sequence Homology, Nucleic Acid; Stem Cells; Translocation, Genetic; Tretinoin

Pre-B cell leukemia transcription factors (PBXs) are important co-factors for the transcriptional regulation mediated by a number of Hox proteins during embryonic development. It was previously shown that the expression of several Pbx genes is elevated in mouse embryo limb buds and embryonal carcinoma P19 cells upon retinoic acid (RA) treatment although the mechanism of this induction is not well understood. In this report, we demonstrate that PBX1a, PBX1b, PBX2, and PBX3 mRNAs and PBX1/2/3 proteins are induced during endodermal and neuronal differentiation of P19 cells in a RAR-dependent subtype-unspecific manner following RA treatment. The increases in both PBX1 mRNA and PBX3 mRNA levels are secondary responses to RA treatment requiring new proteins synthesis while the increase in PBX2 mRNA is a primary response. The RA-dependent increases in PBX1 mRNA, PBX2 mRNA, and PBX3 mRNA levels are likely to be transcriptionally regulated since the stability of these mRNAs does not change. In addition, the half-lives of PBX1/2/3 proteins are significantly extended by RA treatment. Two possible mechanisms could contribute to the stabilization of PBX proteins: PBX proteins associate with RA-dependent increased levels of MEIS proteins, and RA may decrease the proteasome dependent degradation of PBX proteins.

Topics: Animals; Carcinoma, Embryonal; Cell Line, Tumor; Gene Expression Regulation; Homeodomain Proteins; Mice; Myeloid Ecotropic Viral Integration Site 1 Protein; Neoplasm Proteins; Pre-B-Cell Leukemia Transcription Factor 1; Proteasome Endopeptidase Complex; Protein Binding; Protein Processing, Post-Translational; Proto-Oncogene Proteins; RNA Stability; Transcription Factors; Transcription, Genetic; Tretinoin; Up-Regulation

P19 embryonic carcinoma cells, a model system for studying early development and differentiation, can differentiate into neurons and primitive endoderm-like cells depending on the culture conditions. We have previously reported that the activation of c-Jun amino-terminal kinase (JNK) is required for the retinoic acid-induced neural differentiation of P19 cells. However, the signaling pathway(s) responsible for the activation of JNK has not been known. In this study, we demonstrated that activities of MAPK kinase 4 (MKK4) and TAK1, one of the upstream kinases of MKK4, were enhanced in the neurally differentiating cells. Inhibition of the neural differentiation by an overexpression of protein phosphatase 2Cepsilon, an inactivator of TAK1, suggested a critical role of the TAK1 signaling pathway during the differentiation. Confocal microscopic analysis indicated that TAK1, phospho-MKK4, and phospho-JNK were colocalized with tubulin in the neurites and localized also in the nuclei of the differentiating cells. In contrast, two TAK1-binding proteins, TAB1 and TAB2, which are involved in the activation of TAK1, were localized in the neurites and the nuclei of the differentiating cells, respectively. These results suggest that two distinct TAK1-MKK4-JNK signaling pathways are independently activated at the different intracellular locations and may participate in the regulation of the neural differentiation of P19 cells.

Topics: Animals; Blotting, Northern; Carcinoma, Embryonal; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Nucleus; Enzyme Activation; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation, Developmental; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase Kinase 4; MAP Kinase Kinase Kinases; Mice; Microscopy, Confocal; Mitogen-Activated Protein Kinases; Neurons; Phosphoprotein Phosphatases; Precipitin Tests; Protein Phosphatase 2C; Signal Transduction; Transfection; Tretinoin; Tubulin

Regulation of tissue homeostasis is crucial to disease prevention; cell division, cell cycle arrest, differentiation and apoptosis have to be tightly controlled in order to maintain this homeostasis. Retinoic acid (RA) and the histone deacetylase inhibitors (HDACIs) have profound effects on these processes and thus may be critical regulators of homeostasis. Consequently, RA and/or histone deacetylase inhibitors are currently being tested in clinical trials for a variety of cancers. Unfortunately, little is known of the overall affect of these compounds on cellular gene expression. Therefore, we decided to compare the effects of all-trans retinoic acid (ATRA) and a particular HDACI-Trichostatin A (TSA)-on an embryonal carcinoma (EC) cell line (F9) using gene chip analysis. We have focused particular attention on those genes that may be differentially affected by these compounds. Within the parameters established for this study, only 116 of the 12,488 genes examined were similarly regulated by ATRA and TSA: 75 positively and 41 negatively. An additional 70 genes were affected by only one of the compounds and 19 genes were actually inversely regulated. The gene set inversely regulated by ATRA and TSA includes several important patterning genes as well as the crucial tumor suppressor/promoter, transforming growth factor beta 1 (TGFbeta1). Promoter analysis suggests a motif that may regulate one set of these genes. This study provides the first comprehensive comparison of global gene expression on EC cells as affected by ATRA and a HDAC inhibitor (TSA); reveals new targets for ATRA and HDAC inhibitors; identifies a new regulatory motif; demonstrates that ATRA and HDAC inhibitors do not always act synergistically on gene expression; and examines particular questions regarding their concurrent clinical application.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Embryonal; Embryonal Carcinoma Stem Cells; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Neoplastic Stem Cells; Oligonucleotide Array Sequence Analysis; Tretinoin; Tumor Cells, Cultured

MyoR is a bHLH protein whose expression was reported to be almost exclusively restricted to the precursors of the skeletal muscle lineage where it was postulated to function as a transcriptional repressor of myogenesis. However, previous studies in our laboratory suggested a much broader role for MyoR in embryonic cell differentiation. We demonstrated that, besides being expressed in several adult tissues of non-muscle lineage, MyoR was expressed at a much earlier stage in mammalian development than had previously been reported, that is, as early as the blastocyst stage, well before skeletal muscle specification. We also found that, as in skeletal muscle precursor cells, MyoR expression is inversely correlated with the cellular differentiative state of ectodermal, non-muscle embryonal carcinoma (EC) cells. Retinoic acid (RA) treatment of ectodermal EC or embryonal stem (ES) cells promotes their differentiation into primitive endoderm. However, in the present study, we show that the RA-induced expression of endodermal markers such as EndoA, collagen IV, and t-PA are inhibited by exogenous MyoR expression and that the level of inhibition of these markers correlates with the level of MyoR expressed. Conversely, knock-down of MyoR expression via RNA interference enhances RA-induced differentiation of EC cells, promoting earlier and much higher expression of the above-mentioned endodermal markers following RA treatment. Finally, we have narrowed the period of exogenous MyoR-induced embryonic lethality to between 3.5 and 5.5 days post-coitum (dpc), the period during which embryonic endoderm differentiates from the embryonic ectoderm. Our results suggest, therefore, that inhibition of endodermal differentiation between 3.5 and 5.5 dpc contributes to the embryonic death of mouse embryos overexpressing exogenous MyoR and consequently that MyoR may serve as a repressor of embryonal endoderm differentiation.

Topics: Animals; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; Collagen Type IV; Down-Regulation; Embryo, Mammalian; Endoderm; Helix-Loop-Helix Motifs; Mice; Mice, Transgenic; Molecular Sequence Data; Muscle Proteins; Muscle, Skeletal; RNA Interference; Stem Cells; Tissue Polypeptide Antigen; Transcription Factors; Transcriptional Activation; Tretinoin

Neural differentiation is controlled by complex molecular mechanisms that determine cell fate and diversity within the nervous system. Interactions between developing tissues play an important role in regulating this process. In vitro co-culture experiments offer a method to study cell differentiation and function under controlled conditions, with the additional benefit of investigating how interactions between populations of cells influence cell growth and behavior. However, it can often be difficult to distinguish between populations of co-cultured cells. Here we report the development of a human embryonal carcinoma (EC) stem cell line (named TERA2.cl.SP12-GFP) that expresses the genetic marker, green fluorescent protein (GFP). Here, we demonstrate that TERA2.cl.SP12-GFP stem cells stably express GFP and that this remains detectable during retinoic acid-induced differentiation. Regulated expression of neural markers during cell development correlated with the formation of morphologically identifiable neurons. Populations of post-mitotic GFP-positive neurons were readily purified and electrophysiological characterization confirmed that such neurons were functionally active. Thus, cultured TERA2.cl.SP12-GFP cells can be readily distinguished from alternative cell types in vitro and provide an amenable system for live cell imaging to study the development and function of human neurons in isolation, and in co-culture with other tissue types.

Topics: Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Dose-Response Relationship, Drug; Electrophysiology; Flow Cytometry; Gene Expression Regulation; Genes, Reporter; Genetic Vectors; Glycine; Green Fluorescent Proteins; Humans; Immunohistochemistry; Neurons; Patch-Clamp Techniques; Stem Cells; Time Factors; Tretinoin

In an earlier study, we showed that expressions of GD3, GT1b, and GQ1b gangliosides in P19 embryonic carcinoma (EC) cells were enhanced during their neural differentiation induced by retinoic acid. We now further demonstrated that this increase of the b-series gangliosides is due to an increase in their corresponding synthases (sialyltransferase-II, -IV, and -V) in the Golgi. Of the three gangliosides studied, GQ1b appeared to be the best candidate for monitoring such differentiation process. We also used fluorescence-labeled monoclonal antibodies and confocal fluorescence microscopy to obtain direct visual information about the relationship of gangliosides and neural specific proteins in neuron development. Again, GQ1b is the most interesting as it localizes with synaptophysin and neural cell adhesion molecules (NCAMs) on synaptic boutons or dendritic spines in RA-induced neurons (R/N). This suggests that GQ1b could be used as a marker for synapse formation during construction of the neural network.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Chromatography, Thin Layer; Gangliosides; Immunohistochemistry; Kinetics; Mice; Neurons; Organelles; Sialyltransferases; Tretinoin; Tumor Cells, Cultured

Embryonal carcinoma (EC) cells, the pluripotent stem cells of teratocarcinomas, show many similar-ities to embryonic stem (ES) cells. Since EC cells are malignant but their terminally differentiated derivatives are not, understanding the molecular mechanisms that regulate their differentiation maybe of value for diagnostic and therapeutic purposes. We have examined the expression of multiple components of two developmentally important cell-cell signalling pathways, Wnt and Notch, in the pluripotent human EC cell line, NTERA2, and the human ES cell line, H7. Both pathways have well-documented roles in controlling neurogenesis, a process that occurs largely in response to retinoicacid (RA) treatment of NTERA2 cultures and spontaneously in H7 cultures. In NTERA2, many ofthe genes tested showed altered transcriptional regulation following treatment with RA. These include members of the frizzled gene family (FZDI, FZD3, FZD4, FZD5, FZD6), encoding receptors forWnt proteins, the Frizzled Related Protein family (SFRPI, SFRP2, FRZB, SFRP4), encoding solubleWnt antagonists and also ligands and receptors of the Notch pathway (Dlkl, Jaggedl; Notchl, Notch2, Notch3). Few differences were found in the repertoire of Wnt and Notch pathway genes expressed by NTERA2 EC cells and H7 ES cells. We present a model in which interactions between and regulation of Wnt and Notch signalling are important in maintaining EC/ES stem cells and also controlling their differentiation.

Topics: Carcinoma, Embryonal; Cell Differentiation; Cell Line; Follistatin-Related Proteins; Frizzled Receptors; Gene Expression Regulation; Glycoproteins; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Morphogenesis; Pluripotent Stem Cells; Proto-Oncogene Proteins; Receptor, Notch1; Receptor, Notch2; Receptor, Notch3; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Notch; Signal Transduction; Stem Cells; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Wnt Proteins; Zebrafish Proteins

Pluripotent stem cell lines can be induced to differentiate into a range of somatic cell types in response to various stimuli. Such cell-based systems provide powerful tools for the investigation of molecules that modulate cellular development. For instance, the formation of the nervous system is a highly regulated process, controlled by molecular pathways that determine the expression of specific proteins involved in cell differentiation. To begin to decipher this mechanism in humans, we used oligonucleotide microarrays to profile the complex patterns of gene expression during the differentiation of neurons from pluripotent human stem cells. Samples of mRNA were isolated from cultured NTERA2 human embryonal carcinoma stem cells and their retinoic-acid-induced derivatives and were prepared for hybridization on custom microarrays designed to detect the expression of genes primarily associated with the neural lineage. In response to retinoic acid, human NTERA2 cells coordinately regulate the expression of large numbers of neural transcripts simultaneously. Transcriptional profiles of many individual genes aligned closely with expression patterns previously recorded by developing neural cells in vitro and in vivo, demonstrating that cultured human pluripotent stem cells appear to form neurons in a conserved manner. These experiments have produced many new expression data concerning neuronal differentiation from human stem cells in vitro. Of particular interest was the regulated expression of Pax6 and Nkx6.1 mRNA and the absence of Pax7 transcription, indicating that neurons derived from NTERA2 pluripotent stem cells are characteristic of neuroectodermal cells of the ventral phenotype.

Topics: Blotting, Northern; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; Cluster Analysis; Eye Proteins; Gene Expression; Homeodomain Proteins; Humans; Microscopy, Phase-Contrast; Neurons; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Paired Box Transcription Factors; PAX6 Transcription Factor; PAX7 Transcription Factor; Phenotype; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Complementary; RNA, Messenger; Stem Cells; Transcription, Genetic; Tretinoin

Nuclear receptor-mediated gene expression is proposed to be regulated by the ordered recruitment of large protein complexes in which activity depends on mutual interactions and posttranslational modifications. In contrast, relatively little attention has been given to mechanisms regulating the expression of the coregulator proteins themselves. Previously we have shown that the ligand-dependent corepressor, RIP140, is a direct transcriptional target of all-trans retinoic acid (RA). Here we demonstrate that RA induction of RIP140 constitutes a rate-limiting step in the regulation of retinoic acid receptor signaling. Silencing of the RA induction of RIP140 dramatically enhances and accelerates retinoid receptor transactivation, endogenous expression of other RA target genes, and RA-induced neuronal differentiation and cell cycle arrest in human embryonal carcinoma cells. The data suggest that RA induction of RIP140 constitutes a functional negative feedback loop that limits activation of retinoid receptors in the continued presence of RA and that acutely regulated expression of coregulators may be a general regulatory mechanism in hormonal signaling.

Topics: Adaptor Proteins, Signal Transducing; Carcinoma, Embryonal; Cell Cycle; Cell Differentiation; Cell Line; Cell Nucleus; Cytochrome P-450 Enzyme System; Feedback, Physiological; G1 Phase; Gene Expression Regulation; Humans; Left-Right Determination Factors; Neurons; Nuclear Proteins; Nuclear Receptor Interacting Protein 1; Receptors, Retinoic Acid; Retinoic Acid 4-Hydroxylase; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Transcriptional Activation; Transfection; Transforming Growth Factor beta; Tretinoin

Fenretinide (4-HPR) is a retinoid analogue with antitumor and chemopreventive activities. In addition to 4-HPR, there are several other new phenylretinamides bearing hydroxyl, carboxyl, or methoxyl residues on carbons 2, 3, and 4 of the terminal phenylamine ring [N-(2-hydroxyphenyl)retinamide (2-HPR), N-(3-hydroxyphenyl)retinamide, N-(2-carboxyphenyl)retinamide, N-(3-carboxyphenyl)retinamide, N-(4-carboxyphenyl)retinamide, and N-(4-methoxyphenyl)retinamide (4-MPR) ]. It is hypothesized that these agents can act independent of the nuclear retinoid receptor pathway. To test this hypothesis directly, we have analyzed the activity of these phenylretinamides in vitro on a panel of F9 murine embryonal carcinoma cell lines, which includes wild-type (F9-WT) and mutant cells that have disrupted genes for both retinoid X receptor alpha and retinoic acid receptor gamma retinoid receptors (F9-KO). The F9-KO cells lack almost all measurable response to all-trans-retinoic acid, the primary biologically active retinoid. Two distinct effects of retinamides were identified. The first is a rapid, dose-dependent induction of cell growth inhibition (reduced cell viability), and the second is a slower induction of differentiation and accumulation of cells in the G(1) phase of the cell cycle that was observed with a concentration of 1 micro M, for only those phenylretinamides bearing charged (hydroxyl or carboxyl) groups on the terminal phenylamine ring. The induction of differentiation and G(1) accumulation was only observed in the F9-WT cells, indicating that this effect is receptor-dependent. 4-MPR, a major metabolite of 4-HPR, lacks a charged group on the terminal phenylamine ring and did not induce retinoid receptor-dependent effects, but did induce cell growth inhibition. Thus, 4-MPR may play a role in the clinical activity of 4-HPR. This study further reveals the mechanism of action of these novel phenylretinamides and supports continued investigation into their development as chemopreventive drugs.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Carcinoma, Embryonal; Cell Death; Cell Differentiation; Fenretinide; G1 Phase; Immunoenzyme Techniques; Mice; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Retinoid X Receptors; S Phase; Signal Transduction; Transcription Factors; Tretinoin; Tumor Cells, Cultured

The formation of extraembryonic endoderm is one of the earliest steps in the differentiation of pluripotent cells of the inner cell mass during the early stages of embryonic development. The primitive endoderm cells and the derived parietal and visceral endoderm cells gain the capacity to produce collagen IV and laminin. The deposition of these components results in the formation of basement membrane and epithelium of the endoderm, with polarized cells covering the inner surface of the blastocoels. We used retinoic acid-induced endoderm differentiation of stem cell-like F9 embryonic carcinoma cells to study the role of the Ras pathway and its regulation in the formation of the visceral endoderm. Upon endoderm differentiation of F9 cells induced by retinoic acid, c-Fos expression, the downstream target of the Ras pathway, is suppressed by uncoupling Elk-1 phosphorylation/activation to MAPK activity. However, attachment to matrix gel greatly enhances the activation of MAPK in endoderm cells but not in undifferentiated F9 cells. Enhanced MAPK activation as a result of contact with basement membrane is able to compensate for reduced Elk-1 phosphorylation and c-Fos expression. We conclude that endoderm differentiation renders the activation of the Ras pathway basement membrane dependent, contributing to the epithelial organization of the visceral endoderm.

Topics: Animals; Basement Membrane; Carcinoma, Embryonal; Cell Differentiation; Cell Division; DNA-Binding Proteins; Endoderm; Enzyme Activation; ets-Domain Protein Elk-1; MAP Kinase Signaling System; Mice; Phosphorylation; Proto-Oncogene Proteins; ras Proteins; Transcription Factors; Tretinoin; Tumor Cells, Cultured

The nervous system is composed of many different types of neurons and glia cells. Differentiation of these cell types is regulated by various intrinsic transcriptional programs as well as extrinsic signals. Studies of neural differentiation have been focused on the roles of individual factors. Here we profiled global gene expression patterns during neural differentiation of P19 embryonic carcinoma cells. Grouping of the genes induced during P19 neural differentiation into functional categories reveals a set of important transcription factors and extracellular signaling pathways, many of which are also involved in neural development in vivo. In addition, clustering of the induced genes according to their temporal expression pattern reveals 6 groups of genes, each with distinct kinetics, suggesting the existence of different phases in P19 neural differentiation. Our studies provide a temporal array of global pictures of the gene expression patterns used during neural differentiation. The results of this study provide the framework for subsequent analysis of the effects of various intrinsic and extrinsic factors on neural differentiation.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Cluster Analysis; Gene Expression Profiling; Mice; Neurons; Oligonucleotide Array Sequence Analysis; Tretinoin

All-trans-retinoic acid receptors (RAR) and 9-cis-retinoic acid receptors (RXR) are nuclear receptors known to cooperatively activate transcription from retinoid-regulated promoters. By comparing the transactivating properties of RAR and RXR in P19 cells using either plasmid or chromosomal reporter genes containing the mRAR beta 2 gene promoter, we found contrasting patterns of transcriptional regulation in each setting. Cooperativity between RXR and RAR occurred at all times with transiently introduced promoters, but was restricted to a very early stage (<3 h) for chromosomal promoters. This time-dependent loss of cooperativity was specific for chromosomal templates containing two copies of a retinoid-responsive element (RARE) and was not influenced by the spacing between the two RAREs. This loss of cooperativity suggested a delayed acquisition of RAR full transcriptional competence because (i) cooperativity was maintained at RAR ligand subsaturating concentrations, (ii) overexpression of SRC-1 led to loss of cooperativity and even to strong repression of chromosomal templates activity, and (iii) loss of cooperativity was observed when additional cis-acting response elements were activated. Surprisingly, histone deacetylase inhibitors counteracted this loss of cooperativity by repressing partially RAR-mediated activation of chromosomal promoters. Loss of cooperativity was not correlated to local histone hyperacetylation or to alteration of constitutive RNA polymerase II (RNAP) loading at the promoter region. Unexpectedly, RNAP binding to transcribed regions was correlated to the RAR activation state as well as to acetylation levels of histones H3 and H4, suggesting that RAR acts at the mRAR beta promoter by triggering the switch from an RNA elongation-incompetent RNAP form towards an RNA elongation-competent RNAP.

Topics: Acetylation; Carcinoma, Embryonal; Chromosomes; Histone Deacetylase Inhibitors; Histones; Plasmids; Promoter Regions, Genetic; Receptors, Retinoic Acid; Recombination, Genetic; Response Elements; Retinoid X Receptors; RNA Polymerase II; Transcription Factors; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Embryonal carcinoma is a model of embryonic development as well as tumor cell differentiation. In response to all-trans retinoic acid (RA), the human embryonal carcinoma (EC) cell line, NT2/D1, differentiates toward a neuronal lineage with associated loss of cell growth and tumorigenicity. Through the use of cDNA-based microarrays we sought to identify the early downstream targets of RA during differentiation commitment of NT2/D1 cells. A total of 57 genes were induced and 37 genes repressed by RA. RA regulated genes were restricted at 8 h with 27 genes induced and five repressed. The total number of RA-responsive transcripts increased at 24 and 48 h and their pattern of expression was more symmetrical. For a given time point less than 1% of the 9128 cDNAs on the expression array were regulated by RA. Many of these gene products are associated with developmental pathways including those of TGF-beta (Lefty A, NMA, follistatin), homeo domain (HoxD1, Meis2, Meis1, Gbx2), IGF (IGFBP3, IGFBP6, CTGF), Notch (manic fringe, ADAM11), Hedgehog (patched) and Wnt (Frat2, secreted frizzled-related protein 1) signaling. In addition a large cassette of genes induced by RA at 24-48 h are associated with cell adhesion, cytoskeletal and matrix remodeling, growth suppression and intracellular signaling cascades. The majority of repressed genes are associated with protein/RNA processing, turnover or metabolism. The early induced genes identified may play a regulatory role in RA-mediated growth suppression and terminal differentiation and may have physiologic or pharmacologic importance during normal human development and retinoid-based cancer therapy or prevention.

Topics: Carcinoma, Embryonal; Cell Differentiation; Gene Expression Regulation, Developmental; Humans; Neurons; Oligonucleotide Array Sequence Analysis; Tretinoin; Tumor Cells, Cultured

Up-regulation of the c-jun gene is a critical event in the retinoic acid (RA)-mediated differentiation of embryonal carcinoma F9 cells. Activating transcription factor 2 (ATF-2) and p300 cooperate in the activation of transcription of the c-jun gene during the differentiation of F9 cells. We show here that the overexpression of Jun dimerization protein 2 (JDP2), a repressor of AP-1, inhibits the transactivation of the c-jun gene by ATF-2 and p300 by recruitment of the histone deacetylase 3 (HDAC3) complex, thereby repressing the RA-induced transcription of the c-jun gene and inhibiting the RA-mediated differentiation of F9 cells. Moreover, chromatin immunoprecipitation assays showed that the JDP2/HDAC3 complex, which binds to the differentiation response element within the c-jun promoter in undifferentiated F9 cells, was replaced by the p300 complex in response to RA, with an accompanying change in the histone acetylation status of the chromatin, the initiation of transcription of the c-jun gene, and the subsequent differentiation of F9 cells. These results suggest that JDP2 may be a key factor that controls the commitment of F9 cells to differentiation and shed new light on the mechanism by which an AP-1 repressor functions.

Topics: Activating Transcription Factor 2; Animals; Carcinoma, Embryonal; Cell Differentiation; Cyclic AMP Response Element-Binding Protein; E1A-Associated p300 Protein; Genes, jun; Histone Deacetylases; Mice; Nuclear Proteins; Recombinant Proteins; Repressor Proteins; Trans-Activators; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Retinoids are critical for differentiation of columnar epithelial cells and for preventing metaplasia of these cells into stratified squamous epithelial cells, in which tight junctions (TJs) are essentially absent. This implies that retinoids might play important roles in regulating the structures and functions of TJs of columnar epithelium. F9 murine embryonal carcinoma cells differentiate into epithelial cells resembling visceral endoderm bearing TJs, when grown in suspension as aggregates in the presence of retinoic acid (RA). We show that RA induces the TJ structure and expression of several TJ-associated molecules, such as ZO-1, occludin, claudin-6, and claudin-7, as well as a barrier function in the genetically engineered cell line F9:rtTA:Cre-ER(T) L32T2, which allows sophisticated genetic manipulations simply by addition of ligands (H. Chiba et al., 2000, Exp. Cell Res. 260, 334-339). Interestingly, our data indicate that a barrier for small substances is generated after that for large ones during de novo formation of TJs. We also compared the RA-induced expression of TJ components and barrier function in RXRalpha(-/-)-RARgamma(-/-) F9 cells with those in wild-type cells and show that the retinoid signals for transduction of these events are mediated by specific RXR-RAR pairs.

Topics: Animals; Blotting, Northern; Carcinoma, Embryonal; Cell Differentiation; Cell Polarity; Claudins; Endoderm; Freeze Fracturing; Immunohistochemistry; Keratolytic Agents; Kidney; Membrane Proteins; Mice; Occludin; Phosphoproteins; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Retinoid X Receptors; Reverse Transcriptase Polymerase Chain Reaction; RNA; Tight Junctions; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Zonula Occludens-1 Protein

Retinoic acid (RA), a derivative of vitamin A, is essential for the normal patterning and neurogenesis during development. RA treatment induces growth arrest and terminal differentiation of a human embryonal carcinoma cell line (NT2) into postmitotic central nervous system neurons. Using RNA fingerprinting by arbitrarily primed polymerase chain reaction, we identified a novel serine/threonine-rich protein, RA-regulated nuclear matrix-associated protein (Ramp), that was down-regulated during the RA-induced differentiation of NT2 cells. Prominent mRNA expression of ramp could be detected in adult placenta and testis as well as in all human fetal tissues examined. The genomic clone of ramp has been mapped to the telomere of chromosome arm 1q, corresponding to band 1q32.1-32.2. Associated with the nuclear matrix of NT2 cells, Ramp translocates from the interphase nucleus to the metaphase cytoplasm during mitosis. During the late stage of cytokinesis, Ramp concentrates at the midzone of the dividing daughter cells. The transcript expression of ramp is closely correlated with the cell proliferation rate of NT2 cells. Moreover, overexpression of Ramp induces a transient increase in the proliferation rate of NT2 cells. Taken together, our data suggest that Ramp plays a role in the proliferation of the human embryonal carcinoma cells.

Topics: Adult; Alkaline Phosphatase; Amino Acid Sequence; Carcinoma, Embryonal; Cell Differentiation; Cloning, Molecular; Embryo, Mammalian; Female; Gene Expression Regulation; Gene Library; Humans; Male; Molecular Sequence Data; Neurons; Nuclear Matrix; Nuclear Proteins; Organ Specificity; Placenta; Pregnancy; Promoter Regions, Genetic; Protein Conformation; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Ubiquitin-Protein Ligases

Retinoic acid induces cell differentiation and suppresses cell growth in a wide spectrum of cell lines, and down-regulation of activator protein-1 activity by retinoic acid contributes to these effects. In embryonic stem cell-like F9 teratocarcinoma cells, which are widely used to study retinoic acid actions on gene regulation and early embryonic differentiation, retinoic acid treatment for 4 days resulted in suppression of cell growth and differentiation into primitive and then visceral endoderm-like cells, accompanied by a suppression of serum-induced c-Fos expression. The MAPK (ERK) pathway was involved in mitogenic signaling in F9 cells stimulated with serum. Surprisingly, although c-Fos expression was reduced, the MAPK activity was not decreased by retinoic acid treatment. We found that retinoic acid treatment inhibited the phosphorylation of Elk-1, a target of activated MAPK required for c-Fos transcription. In F9 cells, the MAPK/MEK inhibitor PD98059 suppressed Elk-1 phosphorylation and c-Fos expression, indicating that MAPK activity is required for Elk-1 phosphorylation/activation. Phosphoprotein phosphatase 2B (calcineurin), the major phosphatase for activated Elk-1, is not the target in the disassociation of MAPK activation and c-Fos expression since its inhibition by cyclosporin A or activation by ionomycin had no significant effects on serum-stimulated c-Fos expression and Elk-1 phosphorylation. Thus, we conclude that retinoic acid treatment to induce F9 cell differentiation uncouples Ras/MAPK activation from c-Fos expression by reduction of Elk-1 phosphorylation through a mechanism not involving the activation of phosphoprotein phosphatase 2B.

Topics: Animals; Calcineurin; Carcinoma, Embryonal; Cell Differentiation; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; ets-Domain Protein Elk-1; Flavonoids; Mice; Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Although retinoids are known to regulate gene transcription by activating retinoid receptors, the targets of retinoid receptors are largely unknown. This study indicates effective all-trans retinoic acid (RA)-induced differentiation of human embryonal carcinoma cells engages p53. Unexpectedly, RA has been found to activate the transactivation function of p53 in the human embryonal carcinoma cell line, NT2/D1, in a retinoid receptor-dependent manner. A derived RA-resistant line, NT2/D1-R1, is deficient in this activity and is co-resistant to cisplatin. This indicates that RA and cisplatin responses may share a common pathway involving p53 in embryonal carcinomas. RA has no effect on p53 steady-state protein levels in either line. RA enhances endogenous p53 transactivation activity in NT2/D1 but not NT2/D1-R1 cells. In addition, RA induces transactivation activity of a gal4-p53 fusion protein, suggesting that RA activates p53 independent of increasing p53 levels or sequence-specific DNA binding. This activity is absent in retinoic acid receptor gamma (RARgamma)-deficient NT2/D1-R1 cells but can be restored upon co-transfection with specific RARs. Transient transfection of a dominant-negative p53 construct in NT2/D1 cells blocks the RA-mediated transcriptional decline of a differentiation-sensitive reporter plasmid and enhances survival of NT2/D1 cells following cisplatin treatment. Taken together, these findings indicate that RA activates the intrinsic activation function of p53 by a novel mechanism independent of effects on p53 stability or DNA binding and that this activation may be a general mechanism that contributes to RA-mediated G1 arrest.

Topics: Antineoplastic Agents; Carcinoma, Embryonal; Cell Differentiation; Cisplatin; Drug Resistance, Neoplasm; Fibroblast Growth Factors; Gene Expression Regulation, Neoplastic; Genes, p53; Germinoma; Humans; Male; Testicular Neoplasms; Transcriptional Activation; Tretinoin; Tumor Suppressor Protein p53

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

During development, many cells are specifically eliminated. Therefore, programmed cell death must be understood to fully elucidate embryogenesis. Retinoic acid (RA) and bone morphogenetic protein (BMP) 4 induce rapidly dividing P19 embryonal carcinoma cells to undergo apoptosis. RA alone minimally induces apoptosis, while BMP4 alone induces none. RA and BMP4 exposure also elevates the number of cells in the G1 phase of the cell cycle. Because many cell cycle proteins control both proliferation and apoptosis, we determined the role of these proteins in inducing apoptosis. Although the mRNA levels of cyclins D1 and D2 are reduced in cells undergoing apoptosis, the protein levels are not. In contrast, RA and BMP4 induce the Cdk inhibitor p27. This protein binds Cdk4 in RA- and BMP4-treated cells and inhibits Cdk4-dependent kinase activity. We used p27 antisense oligonucleotides to rescue the P19 cells from RA and BMP4 apoptosis thus proving that p27 is necessary. The Cdk4 substrate, retinoblastoma (Rb) protein, is also induced in apoptotic cells. Consistent with the decreased kinase activity of the apoptotic cells, this Rb protein is hypophosphorylated and presumably active. These data support the hypothesis that RA and BMP4 together induce the p27 protein leading to Rb activation and ultimately apoptosis.

Topics: Apoptosis; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Carcinoma, Embryonal; Cell Cycle; Cell Cycle Proteins; Cyclin D; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Ectoderm; Gene Expression Regulation, Neoplastic; Protein Binding; Proto-Oncogene Proteins; Retinoblastoma Protein; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

P19 embryonal carcinoma cells are known to differentiate into neurons and glia when treated with relatively high concentrations (>100 nM) of retinoic acid (RA). Concomitant with this RA-induced neural differentiation, we observed an activation of the c-Jun amino-terminal kinase (JNK). JNK was required for the RA-induced neural differentiation, because dominant-negative JNK blocked the differentiation. Studies using protein phosphatase inhibitors and protein kinase inhibitors suggested that both okadaic acid-sensitive protein phosphatase(s) and protein kinase C participate in the RA-induced activation of JNK.

Topics: Carcinoma, Embryonal; Cell Differentiation; Enzyme Activation; Enzyme Inhibitors; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Neurons; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C; Tretinoin; Tumor Cells, Cultured

Subcellular mRNA localization, a fundamental mechanism for regulating gene expression, leads to local protein translation that results in the generation of neuronal cell polarity. In this study, we have used P19 embryonic carcinoma cells, which are amenable to transfection, and selection of clonal stable cell lines that are not overexpressing the constructs. We identified the 3' untranslated region (3'UTR) tau axonal localization signal and examined its effect on tau protein localization in nondifferentiated and neuronally differentiated P19 cells. Using GFP-tagged tau constructs combined with in situ hybridization analysis, we demonstrated colocalization of the targeted tau mRNA and its translated protein in the axon and growth cone. Absence of or mutation in the 3'UTR axonal targeting region of tau mRNA resulted in suppression of tau mRNA localization, and both tau mRNA and tau protein remained in the cell body. Swapping between the 3'UTR tau mRNA axonal localization signal and the 3'UTR MAP2 mRNA dendritic targeting signal proved that the localization of the proteins into the axon or dendrites depends on the specific 3'UTR targeting signals. Moreover, the identification of ribosomal proteins in the axon lends further support to the presence of protein synthetic machinery in the axons, a prerequisite for local translation. It is suggested therefore that the P19 cell system can be used to analyze mutations that affect mRNA transport and local translation and that it has the potential of being used to examine the onset of the neuronal differentiation process.

Topics: 3' Untranslated Regions; Animals; Axons; Biological Transport; Carcinoma, Embryonal; Cell Differentiation; Cell Line; Green Fluorescent Proteins; Growth Cones; Immunohistochemistry; In Situ Hybridization; Luminescent Proteins; Mice; Microtubule-Associated Proteins; Neurons; Recombinant Fusion Proteins; Ribosomes; RNA, Messenger; tau Proteins; Transfection; Tretinoin

F9 embryonic stem cell-like teratocarcinoma cells are widely used to study early embryonic development and cell differentiation. The cells can be induced by retinoic acid to undergo endodermal differentiation. The retinoic acid-induced differentiation accompanies cell growth suppression, and thus, F9 cells are also often used as a model for analysis of retinoic acid biological activity. We have recently shown that MAPK activation and c-Fos expression are uncoupled in F9 cells upon retinoic acid-induced endodermal differentiation. The expression of the candidate tumor suppressor Disabled-2 is induced and correlates with cell growth suppression in F9 cells. We were not able to establish stable Disabled-2 expression by cDNA transfection in F9 cells without induction of spontaneous cell differentiation. Transient transfection of Dab2 by adenoviral vector nevertheless suppresses Elk-1 phosphorylation, c-Fos expression, and cell growth. In PA-1, another teratocarcinoma cell line of human origin that has no or very low levels of Disabled-2, retinoic acid fails to induce Disabled-2, correlating with a lack of growth suppression, although PA-1 is responsive to retinoic acid in morphological change. Transfection and expression of Disabled-2 in PA-1 cells mimic the effects of retinoic acid on growth suppression; the Disabled-2-expressing cells reach a much lower saturation density, and serum-stimulated c-Fos expression is greatly suppressed and disassociated from MAPK activation. Thus, Dab2 is one of the principal genes induced by retinoic acid involved in cell growth suppression, and expression of Dab2 alone is sufficient for uncoupling of MAPK activation and c-Fos expression. Resistance to retinoic acid regulation in PA-1 cells likely results from defects in retinoic acid up-regulation of Dab2 expression.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Adenoviridae; Animals; Apoptosis Regulatory Proteins; Blotting, Northern; Blotting, Western; Carcinoma, Embryonal; Cell Differentiation; DNA, Complementary; Dose-Response Relationship, Drug; Enzyme Activation; Flow Cytometry; Genes, Tumor Suppressor; Humans; MAP Kinase Signaling System; Mice; Models, Biological; Proteins; Proto-Oncogene Proteins c-fos; Signal Transduction; Time Factors; Transfection; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins; Up-Regulation

Embryonal carcinoma cells are pluripotent stem cells derived from germ cell tumors and can be used to study cell differentiation in vitro. This report describes an approach designed to isolate pluripotent stem cells from primary/parent stock cultures of explanted tumor material. Cells expressing the pluripotent stem cell marker, SSEA-3, were isolated from heterogeneous stock cultures of the human teratoma line, TERA2, using immunomagnetic isolation. Single cell selection was performed on isolated SSEA-3+ cells and clonal lines were established. Each line was ultimately grown as a homogeneous monolayer, independent of feeder cells and expressed high levels of markers for pluripotent stem cells. In response to retinoic acid, clone TERA2.cl.SP-12 cells displayed enhanced neural differentiation compared to previously isolated TERA2 sublines and formed both neurons and glia. Deriving human pluripotent stem cell lines that differentiate into a range of cell types will provide useful tools to understand the molecular mechanisms controlling cell differentiation in a manner pertinent to human embryonic development.

Topics: Carcinoma, Embryonal; Cell Differentiation; Clone Cells; DNA-Binding Proteins; Embryonal Carcinoma Stem Cells; Gene Expression Regulation; Humans; Immunomagnetic Separation; Neoplastic Stem Cells; Octamer Transcription Factor-3; RNA, Messenger; Time Factors; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Cell is a supramolecular dynamic network. Screening of tissue-specific cDNA library and results of Relative RT-PCR indicate that the relationship between genotype, (i.e., dynamic network of genes and their protein regulatory elements) and phenotype is non-bijective, and mendelian inheritance is a special case only. This implies non-linearity, complexity, and quasi-determinism, (i.e., co-existence of deterministic and non-deterministic events) of dynamic cellular network; prerequisite conditions for the existence of fractal structure. Indeed, the box counting method reveals that morphological patterns of the higher order, such as gland-like structures or populations of differentiating cancer cells possess fractal dimension and self-similarity. Since fractal space is not filled out randomly, a variety of morphological patterns of functional states arises. The expansion coefficient characterizes evolution of fractal dynamics. The coefficient indicates what kind of interactions occurs between cells, and how far from the limiting integer dimension of the Euclidean space the expanding population of cells is. We conclude that cellular phenomena occur in the fractal space; aggregation of cells is a supracollective phenomenon (expansion coefficient > 0), and differentiation is a collective one (expansion coefficient < 0). Fractal dimension or self-similarity are lost during tumor progression. The existence of fractal structure in a complex tissue system denotes that dynamic cellular phenomena generate an attractor with the appropriate organization of space-time. And vice versa, this attractor sets up physical limits for cellular phenomena during their interactions with various fields. This relationship can help to understand the emergence of extraterrestial forms of life. Although those forms can be composed of non-carbon molecules, fractal structure appears to be the common feature of all interactive biosystems.

Topics: Animals; Antineoplastic Agents; Biophysical Phenomena; Biophysics; Carcinoma, Embryonal; Cell Aggregation; Cell Differentiation; Colonic Neoplasms; Disease Progression; Exobiology; Fractals; Gallbladder Neoplasms; Humans; Image Processing, Computer-Assisted; Mice; Time Factors; Tretinoin; Tumor Cells, Cultured

Galectin-1 (gal-1), a galactoside-binding lectin, is found in many vertebrate tissues and its expression is regulated during development. We had found that gal-1 expression is increased in F9 murine embryonal carcinoma cells concurrently with induction of differentiation by all-trans retinoic acid (RA). In contrast, gal-1 expression was constitutively high in murine myoblastic C2C12 cells. Therefore, we used these two cell types as models to begin to understand the mechanisms underlying constitutive and RA-induced gal-1 expression. We transfected transiently into F9 cells a series of reporter constructs containing different deletions of the 5' upstream region of the gal-1 gene promoter placed upstream of the chloramphenicol acetyltransferase reporter cDNA and evaluated the activation of transcription by RA treatment. The results indicate that the induction of gal-1 by RA is regulated at least partially at the level of transcription. A strong RA responsiveness region was found within the sequence from -1578 to -1448 upstream of the transcription start site (+1). In contrast, the high constitutive gal-1 expression in C2C12 cells appeared to be mediated by a sequence within the promoter region from -62 to +1, which contains an Sp1 consensus sequence. A gel electrophoretic mobility shift assay indicated that the transcription factor SP1 bound to the gal-1 Sp1 site and mutagenesis of this Sp1 site abolished both the binding of nuclear proteins to the mutated Sp1 site and the high constitutive expression of the gal-1 gene. The results demonstrate that gal-1 expression is cell type-specific and suggest that different factors regulate constitutive and RA-induced gal-1 expression.

Topics: Animals; Binding Sites; Carcinoma, Embryonal; Cell Line; Galectin 1; Gene Expression Regulation; Genes, Reporter; Hemagglutinins; Mice; Promoter Regions, Genetic; Receptors, Retinoic Acid; Sp1 Transcription Factor; Transcription, Genetic; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

The P19 embryonal carcinoma-derived cell line consists of undifferentiated multipotential cells, which irreversibly differentiate into mature neurons after exposure to retinoic acid (RA). In the present study, the authors genetically engineered P19 cells to produce glial cell line-derived neurotrophic factor (GDNF), and grafted the cells in a rat model that had been rendered parkinsonian.. Undifferentiated P19 cells were grown in vitro and transduced with GDNF complementary DNA. The level of GDNF released from the transduced cells was measured using an enzyme-linked immunosorbent assay, and its neurotrophic activities were assessed by testing the effects on rat embryonic dopamine (DA) neurons in culture. After having been exposed to RA for 48 hours and allowed to differentiate into postmitotic neurons, the GDNF gene-transduced cells were implanted into the midbrain of immunosuppressed rats. A unilateral nigrostriatal lesion was then induced by intrastriatal infusions of 6-hydroxydopamine. Immunohistochemical analyses performed 4 weeks postgrafting revealed that the GDNF-producing cells expressed several neuronal markers without evidence of overgrowth. The grafts expressed GDNF protein and prevented the death of nigral DA neurons. Furthermore, the GDNF-producing cells implanted 4 weeks after nigrostriatal lesions restored the expression of tyrosine hydroxylase in injured DA neurons and induced their dendritic sprouting.. The results indicate that the P19 cell line transduced with the GDNF gene can stably secrete functional levels of GDNF, even after being converted to postmitotic neurons. Because it is has been established that GDNF exerts trophic effects on DA neurons, the means currently used to deliver GDNF into the brain could be a viable strategy to prevent the death of nigral DA neurons in cases of Parkinson's disease.

Topics: Analysis of Variance; Animals; Carcinoma, Embryonal; Cell Death; Cell Differentiation; Cell Survival; Cells, Cultured; Dendrites; Disease Models, Animal; Dopamine; Follow-Up Studies; Gene Expression Regulation, Enzymologic; Genetic Engineering; Male; Nerve Growth Factors; Neuroglia; Neurons; Parkinson Disease; Rats; Rats, Sprague-Dawley; Regeneration; Stem Cells; Substantia Nigra; Transduction, Genetic; Tretinoin; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

The MA-01 antigen, a thermolabile 210 kDa microtubule-interacting protein, is present in P19 embryonal carcinoma cells on microtubular structures as well as in cytosol. After aggregation of the cells and subsequent incubation with all-trans-retinoic acid (RA), the level of MA-01 expression increased approximately 10 times during 15 days. The increase started after 2 days of incubation with RA and preceded the appearance of neuron-specific tubulin betaIII, MAP2C and neurofilaments. Such elevated expression of MA-01 antigen was not detected in P19 cells treated with dimethylsulfoxide. These data indicate that enhanced expression of MA-01 antigen is one of the earliest events occurring in P19 cells during neuronal differentiation.

Topics: Animals; Antibodies; Biomarkers; Blotting, Western; Carcinoma, Embryonal; Cell Differentiation; Dimethyl Sulfoxide; Fluorescent Antibody Technique; Hot Temperature; Mice; Microtubule-Associated Proteins; Microtubules; Molecular Weight; Neurites; Precipitin Tests; Protein Denaturation; Time Factors; Tretinoin; Tumor Cells, Cultured

Differentiation of P19 embryonal carcinoma cells in response to the morphogen retinoic acid is regulated by Galpha(12/13) and is associated with activation of c-Jun N-terminal kinase. The role of MEKK1 and MEKK4 upstream of the c-Jun N-terminal kinase was investigated in P19 cells. P19 clones stably expressing constitutively active and dominant negative mutants of MEKK1 and MEKK4 were created and characterized. Expression of the constitutively active form of either MEKK1 or MEKK4 mimicked the action of retinoic acid, inducing these embryonal carcinoma cells to primitive endoderm. Expression of the dominant negative form of MEKK1 had no influence on the ability of retinoic acid to induce either JNK activation or primitive endoderm formation in P19 stem cells. Expression of the dominant negative form of MEKK4, in contrast, effectively blocks both morphogen-induced activation of JNK and cellular differentiation. These data identify MEKK4 as upstream of c-Jun N-terminal kinase in the pathway mediating differentiation of P19 stem cells to primitive endoderm.

Topics: Animals; Blastocyst; Carcinoma, Embryonal; Cell Differentiation; Endoderm; Enzyme Activation; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase Kinase 4; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinases; Mutation; Protein Serine-Threonine Kinases; Rats; Recombinant Proteins; Signal Transduction; Tretinoin; Tumor Cells, Cultured

Differentiation of the human embryonal carcinoma cell line NTERA-2 is characterized by changes in morphology, altered patterns of gene expression, reduced proliferative potential, and a loss of tumorigenicity. The cellular repressor of E1A-stimulated genes, CREG, was previously shown to antagonize transcriptional activation and cellular transformation by the Adenovirus E1A oncoprotein. These properties suggested that CREG may function to inhibit cell growth and/or promote differentiation. Here we show that CREG is a secreted glycoprotein which enhances differentiation of NTERA-2 cells. Northern blot analysis reveals that, although CREG mRNA is widely expressed in adult tissues, CREG mRNA is not significantly expressed in pluripotent mouse embryonic stem cells or NTERA-2 embryonal carcinoma cells. CREG mRNA is rapidly induced upon in vitro differentiation of both mouse embryonic stem cells and human NTERA-2 cells. We show that constitutive expression of CREG in NTERA-2 cells enhances neuronal differentiation upon treatment with retinoic acid. Media enriched in CREG was also found to promote NTERA-2 differentiation in the absence of an inducer such as retinoic acid. These studies suggest that secreted CREG protein participates in a signaling cascade important for differentiation of pluripotent stem cells such as those found in teratocarcinomas.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Glycosylation; Humans; Mice; Protein Processing, Post-Translational; Rabbits; Repressor Proteins; Tretinoin; Tumor Cells, Cultured

Gap junctional communication permits the direct intercellular exchange of small molecules and ions. In vertebrates, gap junctions are formed by the conjunction of two connexons, each consisting of a hexamer of connexin proteins, and are either established or degraded depending on the nature of the tissue formed. Gap junction function has been implicated in both directing developmental cell fate decisions and in tissue homeostasis/metabolite exchange. In mouse development, formation of the extra embryonal parietal endoderm from visceral endoderm is the first epithelial-mesenchyme transition to occur. This transition can be mimicked in vitro, by F9 embryonal carcinoma (EC) cells treated with retinoic acid, to form (epithelial) primitive or visceral endoderm, and then with parathyroid hormone-related peptide (PTHrP) to induce the transition to (mesenchymal) parietal endoderm. Here, we demonstrate that connexin43 mRNA and protein expression levels, protein phosphorylation and subcellular localization are dynamically regulated during F9 EC cell differentiation. Dye injection showed that this complex regulation of connexin43 is correlated with functional gap junctional communication. Similar patterns of connexin43 expression, localization and communication were found in visceral and parietal endoderm isolated ex vivo from mouse embryos at day 8.5 of gestation. However, in F9 cells this tightly regulated gap junctional communication does not appear to be required for the differentiation process as such.

Topics: Animals; Blotting, Northern; Blotting, Western; Bucladesine; Carcinoma, Embryonal; Cell Differentiation; Cells, Cultured; Connexin 43; Endoderm; Fluorescent Antibody Technique; Gap Junctions; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Parathyroid Hormone-Related Protein; Phosphorylation; Proteins; RNA, Messenger; Time Factors; Tretinoin; Tumor Cells, Cultured

In monolayers cultures, retinoic acid (RA) induces the differentiation of F9 embryonal carcinomal (EC) cells into primitive endoderm-like cells, while a combination of RA and dibutyryl cAMP leads to parietal endoderm-like differentiation. Knock out of all RARgamma isoforms (RARgamma(-/-) line) drastically impairs primitive and subsequent parietal endodermal differentiation and affects the induction of many endogenous RA-responsive genes. Using lines that reexpress RARgamma2 or overexpress RARalpha1 lacking their AF-2AD core (RARgammadeltaAF2 and RARalphadeltaAF2, respectively), we show that this conserved amphipatic alpha-helical motif (helix 12) of the ligand binding domain, and therefore the activation function AF-2 of both receptors, is required for the induction of differentiation and target gene expression upon RA treatment of F9 EC cells. We also show that these deletion mutants behave as dominant negatives.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Transformed; Conserved Sequence; Endoderm; Gene Deletion; Gene Expression Regulation, Neoplastic; Mutagenesis; Promoter Regions, Genetic; Protein Structure, Secondary; Protein Structure, Tertiary; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Transfection; Tretinoin; Tumor Cells, Cultured

Human embryonal carcinoma (EC) cells represent the stem cells of testicular germ cell tumours (TGCTs) and are morphologically, antigenically and functionally related to the stem cells of early mammalian embryos. Despite the large capacity for differentiation displayed by TGCT stem cells, little is known of the factors controlling their developmental potency. We have analyzed the differentiation elicited in NT2D1 human embryonal carcinoma (EC) cells by Bone Morphogenetic Proteins (BMPs) and compared it with that elicited by retinoic acid (RA). We have found that while RA induced expression of neuronal, endodermal and epithelial markers in NT2D1 human EC cells, treatment with BMPs resulted in a predominantly epithelial phenotype. We also provide evidence to suggest that at least some of the effects elicited by RA in human EC cells might be mediated through RA-induced expression of BMP-7. Thus BMPs may play an important role in specifying the type of differentiation arising from human multipotent stem cells. The manipulation of BMP signalling in human embryonic multipotent stem cells may therefore prove a useful approach in attempts to generate specific differentiated cell types in vitro, and loss of the malignant and/or transformed phenotype.

Topics: Blotting, Northern; Blotting, Western; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Carcinoma, Embryonal; Cell Differentiation; Cells, Cultured; Endoderm; Epithelium; Humans; Immunohistochemistry; Neurons; Phenotype; RNA; Time Factors; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tretinoin

We have previously reported that expression of the erythropoietin (Epo) gene in mouse embryonal cells was not induced by hypoxia, although hypoxia induced other hypoxia-inducible genes. This study identifies retinoic acid (RA) as an inducer for Epo production in the embryonal carcinoma cell lines P19 and F9. RA induced Epo production through the transcriptional activation of the Epo gene in an oxygen-independent manner. With the use of reporter assays in P19 cells, it is shown that a direct repeat of the nuclear hormone receptor-binding motif separated by a 2-bp spacer (DR-2) in the hypoxia-response enhancer was responsible for the transcriptional activation by RA. Electrophoretic mobility shift assays show that nuclear extracts from P19 cells contained RA receptor complexes that bound to DR-2. In human hepatoma Hep3B cells, an orphan receptor, hepatocyte nuclear factor-4, strongly augmented hypoxic induction of the Epo gene in cooperation with hypoxia-inducible factor-1 (HIF-1) by binding to DR-2, whereas in P19 cells, the interaction of RA receptors with DR-2 was sufficient for RA-induced transcriptional activation of the Epo gene without the requirement of the HIF-1 site. These results suggest that DR-2 regulates expression of the Epo gene by acting as the binding site for different transcription factors in different types of cells.

Topics: Animals; Base Sequence; Carcinoma, Embryonal; Cell Hypoxia; Enhancer Elements, Genetic; Erythropoietin; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Luciferases; Mice; Promoter Regions, Genetic; Receptors, Steroid; Receptors, Thyroid Hormone; Sequence Alignment; Sequence Homology, Nucleic Acid; Transfection; Tretinoin; Tumor Cells, Cultured

Gangliosides are constituents of the cell membrane and are known to have important functions in neuronal differentiation. We employed an embryonal carcinoma stem cell line P19 as an in vitro model to investigate the expression of gangliosides during neuronal development. After treatment with retinoic acid, these cells differentiate synchronously into neuron-like cells by a series of well-defined events of development. We examined several aspects of ganglioside metabolism, including the changes of ganglioside pattern, the activities and gene expression of several enzymes at different stages of differentiation, and the distribution of gangliosides in differentiating neurons. Undifferentiated P19 cells express mainly GM3 and GD3. After P19 cells were committed to differentiation, the synthesis of complex gangliosides was elevated more than 20-fold, coinciding with the stage of neurite outgrowth. During the maturation of differentiated cells, the expression of c-series gangliosides was downregulated concomitantly with upregulation of the expression of a- and b-series gangliosides. We also examined the distribution of gangliosides in differentiating neurons by confocal and transmission electron microscopy after cholera toxin B subunit and sialidase treatment. Confocal microscopic studies showed that gangliosides were distributed on the growth cones and exhibited a punctate localization on neurites and soma. Electron microscopic studies indicated that they also are enriched on the plasma membranes of neurites and the filopodia as well as on the lamellipodia of growth cones during the early stage of neurite outgrowth. Our data demonstrate that the expression of gangliosides in P19 cells during RA-induced neuronal differentiation resembles that of the in vivo development of the vertebrate brain, and hence validates it as an in vitro model for investigating the function of gangliosides in neuronal development.

Topics: Animals; Antigens, Differentiation; Carcinoma, Embryonal; Cell Differentiation; Gangliosides; Glycosyltransferases; Immunohistochemistry; Mice; Neuraminidase; Neurons; Patch-Clamp Techniques; RNA, Messenger; Stem Cells; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) treatment of embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) induces growth arrest and terminal differentiation along the neuronal pathway. In the present study, we provide a functional link between RA and p27 function in the control of neuronal differentiation in NT2/D1 cells. We report that RA enhances p27 expression, which results in increased association with cyclin E/cyclin-dependent kinase 2 complexes and suppression of their activity; however, antisense clones, which have greatly reduced RA-dependent p27 inducibility (NT2-p27AS), continue to synthesize DNA and are unable to differentiate properly in response to RA as determined by lack of neurite outgrowth and by the failure to modify surface antigens. As to the mechanism involved in RA-dependent p27 upregulation, our data support the concept that RA reduces p27 protein degradation through the ubiquitin/proteasome-dependent pathway. Taken together, these findings demonstrate that in embryonal carcinoma cells, p27 expression is required for growth arrest and proper neuronal differentiation.

Topics: Antineoplastic Agents; Carcinoma, Embryonal; Cell Cycle Proteins; Cell Differentiation; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cysteine Endopeptidases; Gene Expression Regulation, Neoplastic; Humans; Microtubule-Associated Proteins; Multienzyme Complexes; Neurons; Proteasome Endopeptidase Complex; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

The immunocytochemical localization of beta-citryl-L-glutamate (beta-CG) in primary neuronal cells and in the differentiation of P19 cells was examined. 1: Cells with the morphological features of neurons in the primary culture were specifically stained with the anti-beta-CG antibody both in neurites and in the cell body. 2: The neuronal cells differentiated from P19 cells were distinctly stained with the anti-beta-CG antibody both in neurites and in the cell body, while the non-neuronal cells were not. 3: The concentration of beta-CG was low in the P19 cells, but increased significantly with the differentiation of P19 cells into neurons. It was shown that beta-CG was localized exclusively in neurons. These findings suggest that beta-CG plays functional roles in the differentiation and growth of neuron.

Topics: Animals; Animals, Newborn; Brain; Carcinoma, Embryonal; Cell Differentiation; Cells, Cultured; Cerebral Cortex; Chick Embryo; Glutamates; Immunohistochemistry; Mice; Neurons; Tretinoin; Tumor Cells, Cultured

The developmental expression patterns of the nuclear orphan receptors COUP-TFs (chicken ovalbumin upstream promoter-transcription factors) have been correlated to neurogenesis in several animal species. Nevertheless, the role of COUP-TFs in neurogenesis remains unknown. We have studied the functional involvement of COUP-TFI in retinoic acid (RA)-induced neuronal differentiation of P19 embryonal carcinoma cells through two complementary approaches: 1) deregulated expression of COUP-TFI, and 2) inactivation of endogenous COUP-TFs by means of a dominant-negative COUP-TFI mutant. Low levels of wild-type (wt)COUP-TFI transgene expression did not inhibit neural cell fate and primarily enhanced neuron outgrowth from RA-treated P19 aggregates. In contrast, high COUP-TFI expression impeded the neuronal differentiation of P19 cells induced with RA, resulting in cell cultures lacking neurons. This morphological effect was correlated to an elevated level of E-cadherin mRNA. The dominant-negative COUP-TFI mutant induced cell packing after RA treatment and inhibited neurite extension and neuron outgrowth from aggregates. A RGD peptide interference assay indicated that endogenous COUP-TFs could favor migration of neurons through an integrin-dependent mechanism. Accordingly, vitronectin mRNA levels were shown to be up-regulated by COUP-TFI by RT-PCR analysis, and COUP-TFI stimulated the mouse vitronectin promoter activity in transient transfection assays. Taken together, these data indicate that COUP-TFI is not simply a global repressor of retinoid functions, but shows a high selectivity for regulating genes involved in cellular adhesion and migration processes that are particularly important for neuronal differentiation.

Topics: Animals; Axons; Base Sequence; Carcinoma, Embryonal; Cell Adhesion; Cell Differentiation; Cell Movement; COUP Transcription Factor I; DNA-Binding Proteins; Extracellular Matrix Proteins; Humans; Mice; Molecular Sequence Data; Neurons; Point Mutation; Promoter Regions, Genetic; Sequence Homology, Amino Acid; Transcription Factors; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured; Vitronectin

Bmp2, a highly conserved member of the transforming growth factor-beta gene family, is crucial for normal development. Retinoic acid, combined with cAMP analogs, sharply induces the Bmp2 mRNA during the differentiation of F9 embryonal carcinoma cells into parietal endoderm. Retinoic acid (RA) also induces the Bmp2 gene in chick limb buds. Since normal Bmp2 expression may require an endogenous retinoid signal and aberrant Bmp2 expression may cause some aspects of RA-induced teratogenesis, we studied the mechanism underlying the induction of Bmp2. Measurements of the Bmp2 mRNA half-life and nuclear run-on assays indicated that RA stimulated the transcription rate of the Bmp2 gene. The results of ribonuclease protection and primer extension assays indicated that Bmp2 transcription started 2,127 nucleotides upstream of the translation start site in F9 cells. To identify genetic elements controlling this transcription rate increase, upstream and downstream genomic sequences flanking the Bmp2 gene were screened using chloramphenicol acetyltransferase reporter genes in F9 cells and beta-galactosidase reporter genes in Saccharomyces cerevisiae that were cotransformed with retinoic acid receptor and retinoid X receptor expression plasmids. RA-dependent transcriptional activation was detected between base pairs -2,373 and -2,316 relative to the translation start site. We also identified a required Sp1 binding site between -2,308 and -2,298. The data indicate that Bmp2 is directly regulated by retinoic acid-bound receptors and Sp1.

Topics: Animals; Base Sequence; beta-Galactosidase; Binding Sites; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Carcinoma, Embryonal; Chloramphenicol O-Acetyltransferase; Drosophila; Gene Expression Regulation, Neoplastic; Genes, Reporter; Molecular Sequence Data; Plasmids; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Saccharomyces cerevisiae; Sp1 Transcription Factor; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

The formation of parietal endoderm (PE) is one of the first differentiation processes during mouse development and can be studied in vitro using F9 embryonal carcinoma (EC) cells. Treatment of F9 EC cells with retinoic acid (RA) induces differentiation toward primitive endoderm (PrE), while differentiation toward PE is induced by subsequent addition of parathyroid hormone (PTH) or PTH-related peptide (PTHrP). The signal transduction mechanisms involved in this two-step process are largely unclear. We show that the RA-induced differentiation toward PrE is accompanied by a sustained increase in Ras activity and that ectopic expression of oncogenic Ha-Ras is sufficient to induce PrE differentiation. Ras activity subsequently decreases upon PTH-induced differentiation toward PE. This is a necessary event, since expression of oncogenic Ha-Ras in PrE-like cells prevents PTH-induced PE differentiation. Expression of active PKA in PrE-like F9 cells mimics PTH-induced PE differentiation and is again prevented by oncogenic Ha-Ras. The effect of oncogenic Ras on both differentiation steps is abolished by the MEK inhibitor PD98059 and can be mimicked by constitutively active forms of Raf and MEK. In conclusion, our data suggest that activation of the Ras/Erk is sufficient to induce differentiation to PrE and to prevent subsequent differentiation toward PE. Activation of PKA down-regulates Ras activity, resulting in disappearance of this blockade and transmission of signal(s) triggering PE differentiation.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Embryonal; Cell Differentiation; Cell Transformation, Neoplastic; Cyclic AMP-Dependent Protein Kinases; Down-Regulation; Endoderm; Enzyme Activation; Mice; Parathyroid Hormone; ras Proteins; Rats; Tretinoin; Tumor Cells, Cultured

During embryonic development, cells not only increase in number, they also undergo specialization and differentiate into diverse cell types that are organized into different tissues and organs. Nervous system development, for example, involves a complex series of events such as neuronal and astroglial differentiation that are coordinated among adjacent cells. The organization of growth and differentiation may be mediated, at least partly, by exchange of small ions and molecules via intercellular gap junction channels. These structures are mode of connexons (hemichannels), which are hexameric assemblies of the gap junction proteins, connexins. We investigated the role of intercellular communication in neuronal and astroglial differentiation by using a gap junction blocking agent, carbenoxolone (CBX), in comparison to its inactive (control) analog, glycyrrhizic acid (GZA). We used the mouse P19 embryonal carcinoma cell line, which differentiates into neurons and astrocytes upon retinoic acid (RA) induction. Our results show that both GZA- and CBX-treated cells express alpha 1 connexin (connexin43). The level of alpha 1 connexin decreases upon RA induction. CBX treated cells show significant reduction in both neuronal (5-fold) and astrocytic (13-fold) differentiation compared with those of control. These results clearly indicate that the blockage of gap junction-mediated intercellular communication interferes with differentiation of P19 cells into neurons and astrocytes.

Topics: Animals; Astrocytes; Carbenoxolone; Carcinoma, Embryonal; Cell Communication; Cell Differentiation; Connexin 43; Fluoresceins; Gap Junctions; Glycyrrhizic Acid; Mice; Neurons; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

We studied the expression of the NO synthase isoforms in an in vitro model of neural development using RT-PCR, Western blot and immunohistochemistry. Murine PCC7-Mz1 cells (Jostock et al., Eur. J. Cell Biol. 76, 63-76, 1998) differentiate in the presence of all-trans retinoic acid and dibutyryl cAMP along the neural pathway into neuron-like, fibroblast-like and astroglia-like cells. Undifferentiated cells showed immunofluorescent staining for neuronal-type NOS I and endothelial-type NOS III. This expression pattern was retained in those cells differentiating into neurofilament- and tau protein-positive neuronal cells. Thymocyte alloantigen (Thy1.2/CD 90.2)-positive fibroblasts, appearing around day 3, and glial fibrillary acidic protein (GFAP)-positive astroglial cells, appearing after day 6 of differentiation, stained negative for any NOS isoform. Starting at day 6 of differentiation, expression of inducible-type NOS II could be stimulated with cytokines in a subset of cells, which may represent activated astrocytes. NOS II was always undetectable in non-induced cultures. These data indicate that the ability of stem cells to express NOS I and NOS III is only retained when the cells differentiate along the neuronal lineage, while a small subpopulation of cells acquires the ability to express NOS II in response to cytokines.

Topics: Animals; Astrocytes; Blotting, Western; Bucladesine; Carcinoma, Embryonal; Cell Differentiation; Cytokines; Enzyme Induction; Fibroblasts; Fluorescent Antibody Technique; Immunohistochemistry; Mice; Neurons; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Tumor Cells, Cultured

When treated with retinoic acid (RA), a human embryonal carcinoma (EC) cell line, NTera2 cl.D/1 (NT2), differentiates into several morphologically distinct cell types, which include terminally differentiated postmitotic central nervous system (CNS) neurons. Accumulating evidence has demonstrated the significant potential of NT2 cells in studies related to cancer therapy and neurodegenerative diseases. However, preparation of enriched NT2 neurons often requires a lengthy period (ca. five weeks) and depends largely on tedious techniques similar to those used for primary neuronal cultures. Here, we report a rapid protocol for the preparation of these human CNS neurons. Using the method of cell aggregation, enriched NT2 neurons can be obtained in approximately two weeks. We also demonstrated that cell aggregation reduced the time normally required for the induction of neuronal differentiation, as revealed by the early expression of neuronal markers. The period of RA treatment could also be reduced if NT2 cells were maintained as aggregates for a sufficient period of time. Taken together, our findings demonstrated that cell aggregation promoted RA-induced neuronal differentiation of NT2 cells and provided a rapid protocol for the efficient production of NT2 neurons. The ability to produce large quantities of human CNS neurons should facilitate future use of these neurons for basic research and applications in cell therapy.

Topics: Biomarkers; Biotechnology; Carcinoma, Embryonal; Cell Aggregation; Cell Differentiation; Central Nervous System; Humans; Nerve Tissue Proteins; Neurites; Neurons; Time Factors; Tretinoin; Tumor Cells, Cultured

Using a differential display PCR, we identified a differentially expressed cDNA fragment which was detectable in retinoic acid (RA) treated F9 embryonal carcinoma (EC) cells but not in untreated F9 cells. A homology search of the Gene Bank indicated that the cDNA fragment is part of the mouse homolog of the Drosophila Disabled (mDab2) gene. Aggregate cultures of F9 EC cells grown in the presence of the RA differentiated into nonmalignant cells resembling the visceral endoderm of the mouse embryo. Upon induction of endodermal differentiation with 10(-7) M RA, the gene expression of mDab2 was increased gradually during the first 96 h. Neither undifferentiated F9 cells, nor the undifferentiated aggregate cells without RA expressed mDab2. Whole-mount in situ hybridization and quantitative RT-PCR also showed that the temporal expression pattern of the mDab2 gene coincides with the initiation pattern of RA synthesis that occurs during mouse embryogenesis. Also, two alternative splicing messages of mDab2 were detected in a tissue specific manner. All the data indicate that mDab2 may play an important role in RA-induced signal transduction during mouse development.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Apoptosis Regulatory Proteins; Blotting, Northern; Carcinoma, Embryonal; Embryo, Mammalian; Embryonic and Fetal Development; Female; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Genes; Genes, Tumor Suppressor; In Situ Hybridization; Male; Mice; Phosphoproteins; Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tissue Distribution; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

The polyfunctional cytokine leukemia inhibitory factor (LIF) has been implicated in the maintenance of many stem and progenitor cell populations and as an autocrine growth factor for many tumor cell populations, including germ cell tumors. Studies of LIF transcript expression in germ cell tumor cell lines identified two novel human LIF transcripts, hLIF-M and hLIF-T, containing noncoding alternate first exons that are conserved among all reported LIF genes. Embryonal carcinoma (EC) cell lines expressed these transcripts at consistent levels and hLIF-M was generally the predominant LIF transcript in these cells. This expression pattern was characteristic of EC cells since variable independently regulated expression of these transcripts was evident in other cell lines. Overexpression analysis demonstrated that each alternate hLIF transcript generated different levels of extracellular LIF activity as a consequence of the translation of distinct but partially overlapping sets of proteins. Secreted LIF proteins translated from alternate initiation codons were expressed from the hLIF-D and hLIF-M transcripts. Intracellular, potentially cell-autonomous, proteins were encoded by the hLIF-M and hLIF-T transcripts. Since EC cell lines also expressed LIF receptor transcripts, the novel LIF transcription profiles and proteins identified here suggest a role for autocrine and/or cell-autonomous LIF signaling during germ cell tumorigenesis.

Topics: Alternative Splicing; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; Cells, Cultured; Exons; Extracellular Space; Gene Expression Regulation; Genes, Overlapping; Germinoma; Growth Inhibitors; Humans; Interleukin-6; Leukemia Inhibitory Factor; Lymphokines; Molecular Sequence Data; Protein Isoforms; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

We have shown previously that the PTH/PTHrP (PTH-related peptide) receptor mRNA becomes expressed very early in murine embryogenesis, i.e. during the formation of extraembryonic endoderm. Retinoic Acid (RA) is a potent inducer of extraembryonic endoderm formation and PTH/PTHrP-receptor expression in embryonal carcinoma (EC) and embryonal stem (ES) cells. Using the P19 EC cell line, we have characterized promoter elements of the murine PTH/PTHrP-receptor gene that are involved in this RA-induced expression. The data show that RA-induced expression of the PTH/ PTHrP-receptor gene is mediated by the downstream P2 promoter. Analysis of promoter reporter constructs in transiently transfected P19 cells treated with RA identified an enhancer region between nucleotides -2714 and -2702 upstream of the P2 transcription start site that is involved in the RA effect. This region matches a consensus hormone response element consisting of a direct repeat with an interspacing of 1 bp (R-DR1). The R-DR1 efficiently binds retinoic acid receptor-alpha (RARalpha)-retinoid X receptor-alpha (RXRalpha) and chicken ovalbumin upstream promoter (COUP)-transcription factor I (TFI)-RXRalpha heterodimers and RXRalpha and COUP-TFI homodimers in a bandshift assay using extracts of transiently transfected COS-7 cells. RA differentiation of P19 EC cells strongly increases protein binding to the R-DR1 in a band-shift assay. This is caused by increased expression of RXR (alpha, beta, or gamma) and by the induction of expression of RARbeta and COUP TFI/TFII, which bind to the R-DR1 as shown by supershifting antibodies. The presence of RXR (alpha, beta, or gamma) in the complexes binding to the R-DR1 suggests that RXR homodimers are involved in RA-induced expression of the PTH/PTHrP-receptor gene. The importance of the R-DR1 for RA-induced expression of PTH/ PTHrP-receptor was shown by an inactivating mutation of the R-DR1, which severely impairs RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs. Since this mutation does not completely abolish RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs, sequences other than the R-DR1 might also be involved in the RA effect. Finally, we show that the RA-responsive promoter region is also able to induce expression of a reporter gene in extraembryonic endoderm of 7.5 day-old transgenic mouse embryos.

Topics: Animals; Base Sequence; beta-Galactosidase; Carcinoma, Embryonal; Cell Differentiation; COUP Transcription Factors; DNA-Binding Proteins; Endoderm; Gene Expression Regulation, Developmental; Mice; Molecular Sequence Data; Mutation; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Promoter Regions, Genetic; Proteins; Receptors, Retinoic Acid; Receptors, Steroid; Recombinant Proteins; Repetitive Sequences, Nucleic Acid; Response Elements; Retinoid X Receptors; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Embryonal carcinoma cell lines (F9 EC and P19 EC) were stably transfected with 1.8 kb promoter sequence of RARbeta2 coupled to the lacZ gene as a system for measuring active retinoids. These stable transfectants, designated F9-1.8 and P19-1.8, were used as reporter cell lines to investigate different retinoids for their ability to activate the reporter gene. F9-1.8 cells showed similar EC(50) values for the acidic retinoids all-trans retinoic acid (RA), 4-oxo RA, 9-cis RA, and 13-cis RA, in the range of 1-7 nM, while P19-1.8 cells were less sensitive. Retinal showed decreased activity compared to the RA isomers in both lines. However, P19-1.8 cells hardly showed beta-gal activity after treatment with retinol, while the lacZ reporter in F9-1.8 cells was still inducible by this retinoid. In addition, the reporter system was used to investigate RA metabolism and its inhibition by P450 inhibitors. A combination of RA and liarozole showed a 10 times greater induction of the RARbeta2-lacZ reporter in P19-1.8 cells, but not in F9-1.8 cells. The EC(50) value for 4-oxo RA, however, was not altered, indicating that metabolic conversion of RA to 4-oxo RA is the target for inhibition by liarozole in P19-1.8 cells. HPLC analysis revealed nearly complete inhibition of RA metabolism after liarozole treatment in P19-1.8 cells, resulting in higher levels of RA. Finally, the F9-1.8 cells were used to detect active retinoids during different stages of chick limb bud development, demonstrating that it is the limb bud mesenchyme which generates RA and not the epidermis, with a twofold higher level of RA in the posterior half than in the anterior half.

Topics: Animals; Carcinoma, Embryonal; Chick Embryo; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Gene Expression Regulation, Developmental; Genes, Reporter; Imidazoles; Isomerism; Ketoconazole; Limb Buds; Mesoderm; Mice; Promoter Regions, Genetic; Receptors, Retinoic Acid; Retinoids; Time Factors; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

Mechanisms by which differentiation programs engage the cell cycle are poorly understood. This study demonstrates that retinoids promote ubiquitination and degradation of cyclin D1 during retinoid-induced differentiation of human embryonal carcinoma cells. In response to all-trans-retinoic acid (RA) treatment, the human embryonal carcinoma cell line NT2/D1 exhibits a progressive decline in cyclin D1 expression beginning when the cells are committed to differentiate, but before onset of terminal neuronal differentiation. The decrease in cyclin D1 protein is tightly associated with the accumulation of hypophosphorylated forms of the retinoblastoma protein and G(1) arrest. In contrast, retinoic acid receptor gamma-deficient NT2/D1-R1 cells do not growth-arrest or accumulate in G(1) and have persistent cyclin D1 overexpression despite RA treatment. Notably, stable transfection of retinoic acid receptor gamma restores RA-mediated growth suppression and differentiation to NT2/D1-R1 cells and restores the decline of cyclin D1. The proteasome inhibitor LLnL blocks this RA-mediated decline in cyclin D1. RA treatment markedly accelerates ubiquitination of wild-type cyclin D1, but not a cyclin D1 (T286A) mutant. Transient expression of cyclin D1 (T286A) in NT2/D1 cells blocks RA-mediated transcriptional decline of a differentiation-sensitive reporter plasmid and represses induction of immunophenotypic neuronal markers. Taken together, these findings strongly implicate RA-mediated degradation of cyclin D1 as a means of coupling induced differentiation and cell cycle control of human embryonal carcinoma cells.

Topics: Carcinoma, Embryonal; Cell Cycle; Cell Differentiation; Cyclin D1; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Protein Processing, Post-Translational; Tretinoin; Tumor Cells, Cultured; Ubiquitins

The rae28/mph1 gene is the mouse homologue of the Drosophila polyhomeotic gene, which plays a crucial role in the maintenance of the transcriptional repression state of Hox genes. Expression of the rae28/mph1 gene is induced during retinoic acid (RA)-mediated differentiation of embryonal carcinoma F9 cells. By transient-transfection experiments, we identified a pair of inverted differentiation response sequences (DRS(s)) in the 5' flanking region. Each of the DRS(s) contained the consensus sequence [5'-CCTCCCCXCXGCCCCCTCCXCXC-3'], which is also conserved in the human counterpart of the rae28/mph1 gene. Electrophoretic mobility shift assay and DNase I foot printing with nuclear extracts derived from F9 cells demonstrated the presence of novel DNA-binding factors which specifically interact with DRS(s). Nucleotide substitutions in the 3' DRS abrogated the factor binding and the transcriptional activation, suggesting that DRS(s) and DRS-binding factors play an important role in the transcriptional regulation of the rae28/mph1 gene.

Topics: Animals; Base Sequence; Carcinoma, Embryonal; Carrier Proteins; Cell Differentiation; Conserved Sequence; DNA Footprinting; DNA-Binding Proteins; Homeodomain Proteins; Humans; Mice; Nuclear Proteins; Polycomb Repressive Complex 1; Protein Binding; Response Elements; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

When retinoic acid-primed F9 cells are allowed to aggregate, they form embryoid bodies with an outer layer of (&agr;)-fetoprotein-producing visceral endoderm cells and an internal cavity. I show that maturation of the visceral endoderm is dependent on the size of F9 aggregates. Size fractionation of aggregates of retinoic acid-primed F9 cells on Percoll density gradients revealed that only aggregates with diameters larger than 180 microm developed into embryoid bodies with an endoderm layer secreting (&agr;)-fetoprotein. Size dependent alpha-fetoprotein-secretion was also observed when retinoic acid-primed F9 cells were cultured on porous microcarrier beads larger than 185 microm. Retinoic acid-primed F9 cells on flat microporous membranes did not differentiate and secrete alpha-fetoprotein unless exposed to a limited volume of medium at their basolateral surface. This suggested that maturation of the visceral endoderm is signaled by the volume of liquid phase below the epithelium. I postulate that the epithelial layer of an F9 aggregate encloses liquid and forms a barrier to diffusion of some critical factor(s). The concentration of such a factor may reach a threshold due to enlargement of the liquid phase during growth of the F9 aggregate and thereby signal maturation of the outer layer of cells into visceral endoderm.

Topics: alpha-Fetoproteins; Animals; Carcinoma, Embryonal; Cell Aggregation; Cell Differentiation; Endoderm; Epithelial Cells; Mice; Signal Transduction; Stem Cells; Tretinoin; Tumor Cells, Cultured

The two alleles of the 30 kDa TATA-binding protein associated factor (TAF(II)30) gene, have been targeted by homologous recombination in murine F9 embryonal carcinoma cells and subsequently disrupted using a Cre recombinase-loxP strategy. The TAF(II)30-null cells are not viable, but are rescued by the expression of human TAF(II)30. Cells lacking TAF(II)30 are blocked in G(1)/G(0) phase of the cell cycle and undergo apoptosis. In agreement with the G(1) arrest phenotype, the expression of cyclin E is impaired and the retinoblastoma protein is hypophosphorylated in the TAF(II)30-null cells. Interestingly, retinoic acid (RA) treatment prevented TAF(II)30-null cell death and induced primitive endodermal differentiation. In contrast, the RA- and cAMP-induced parietal endodermal differentiation was impaired in the TAF(II)30-null cells. Thus, TAF(II)30 is not indispensable for class II gene transcription in general, but seems to be required for the expression of a subset of genes.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Embryonal; Cell Cycle; Cell Differentiation; Cell Division; DNA-Binding Proteins; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Integrases; Mice; Mice, Knockout; Models, Genetic; Mutagenesis, Insertional; Stem Cells; TATA-Binding Protein Associated Factors; Time Factors; Transcription Factor TFIID; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Viral Proteins

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

The aberrant expression or processing of the amyloid precursor protein (APP) is the only known genetic basis for presenile familial Alzheimer's disease, and the molecular connection between APP and tau has been perplexing. Attention has focused on proline-directed serine/threonine kinases as mediating the cytoskeletal modifications of Alzheimer's disease, and we show that overexpression of APP can influence the activation of a candidate kinase, the mitogen-activated protein kinase (MAPK). In murine embryonal carcinoma cells stably transfected with the human 751 isoform of APP, we observed steady-state hyperactivation of p42(MAPK) concomitant with APP overexpression 3 days after neuroectodermal differentiation. In more mature differentiated cells, immunocytochemical analysis revealed enhanced basal somatic and nuclear immunoreactivity for phosphorylated MAPK coupled with an attenuated phosphorylation response to growth factor stimulation. Our results suggest that APP can influence the MAPK signaling pathway in such a way that the absolute and time-dependent activation required for discrimination of the appropriate downstream response are compromised. Such an effect would have important consequences for the functioning of cells coincidentally expressing both proteins, a situation that occurs in neuronal populations vulnerable to Alzheimer's disease pathology.

Topics: Amyloid beta-Protein Precursor; Animals; Carcinoma, Embryonal; Cell Differentiation; Ectoderm; Fibroblast Growth Factor 1; Heparin; Humans; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase 1; Phosphorylation; Protein Isoforms; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Transfection; Tretinoin; Tumor Cells, Cultured

The growth rate of malignant F9 embryonal carcinoma cells slows considerably following all-trans-retinoic acid-induced differentiation into benign parietal endoderm. To determine the mechanism of this process, we examined the expression of cyclins D1, D2, and D3 and the activity of their associated kinases. Cyclin D1 and D3 mRNA levels decreased during complete differentiation induced by all-trans-retinoic acid and dibutyryl cAMP, while the levels of cyclin D2 and the cyclin-dependent kinase (Cdk) inhibitor p27 mRNAs increased. Ultimately, terminally differentiated cells possessed 50% of the Cdk4-associated kinase activity observed in undifferentiated cells. Since numerous genes are differentially regulated during parietal endoderm differentiation, it is difficult to determine whether retinoic acid affects cell cycle gene expression directly or if these changes are caused by differentiation. We found that the retinoid X receptor (RXR)-selective agonists LG100153 and LG100268 significantly inhibited F9 cell growth without causing overt terminal differentiation as assessed by anchorage-independent growth and differentiation-associated gene expression. As seen in cells induced to differentiate by the RAR agonist all-trans-retinoic acid, RXR activation led to an increase in the number of cells in G1 phase. RXR agonists also sharply induced the levels of the Cdk regulatory subunits, cyclin D2 and D3. However, Cdk4-dependent kinase activity was reduced by RXR-selective retinoid treatment. These observations suggest that some retinoids can directly inhibit proliferation and regulate Cdk4-dependent kinase activity without inducing terminal differentiation.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Benzoates; Biomarkers; Carcinoma, Embryonal; Cell Adhesion; Cell Differentiation; Cell Division; Cyclin D1; Cyclin D2; Cyclin D3; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; Endoderm; Gene Expression Regulation, Neoplastic; Nicotinic Acids; Proto-Oncogene Proteins; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Stem Cells; Tetrahydronaphthalenes; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The vimentin gene encodes an intermediate filament protein expressed in the parietal endoderm, mesodermal, and early neural cells in vivo but by most in vitro-cultured cells regardless of their embryonic origin. Here we show that the vimentin gene promoter is very active in F9 embryonal carcinoma cells and increases in activity during differentiation. Using a series of 5'-deletion mutants, we provide evidence that the regions of the promoter involved in F9 cell activity are different from those previously demonstrated to be active in differentiated cell lines. Furthermore, we show that in differentiating F9 cells the activities of two different regions of the promoter are significantly enhanced. A distal region (-1710/-957) appears to contain functional binding sites for the murine Hox-A5 homeoprotein as demonstrated by band shift and footprinting experiments. A proximal region (-140/-78) contains a 30-bp repetitive sequence found in other genes activated during differentiation of F9 cells. Using band shift assays and methylation interference, we present evidence that a sequence-specific single-stranded DNA-binding protein(s) specifically interacts with the minus strand of the 30-bp sequence.

Topics: Animals; Binding Sites; Biomarkers; Bucladesine; Carcinoma, Embryonal; Cell Differentiation; Chloramphenicol O-Acetyltransferase; Consensus Sequence; Cyclic AMP; DNA Methylation; DNA-Binding Proteins; DNA, Single-Stranded; Endoderm; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Genes, Reporter; Homeodomain Proteins; Mice; Neoplasm Proteins; Phosphoproteins; Promoter Regions, Genetic; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Second Messenger Systems; Sequence Deletion; Transcription Factors; Transfection; Tretinoin; Vimentin

Undifferentiated human embryonal carcinoma cells are characterized by high expression of lactoneotetraosylceramide (nLc4), globoside (Gb4), and extended globo-series glycosphingolipids (GSLs) termed "stage-specific embryonic antigens 3 and 4" (SSEA-3 and -4). Expression of these GSLs declines in association with a decline of homotypic adhesion during the differentiation process. Therefore, these GSLs may play an essential role in adhesion among these cells. As an example, human embryonal carcinoma 2102 cells display strong adhesion to plates coated with Gb4 ("Gb4-dependent cell adhesion"). This adhesion, which simulates homotypic 2102 cell aggregation, is based on interaction between Gb4 and nLc4, or between Gb4 and GalGb4 (IV3GalGb4; the major SSEA-3 epitope), as indicated by the following observations: (i) adhesion of 2102 cells or GSL-liposomes to GSL-coated plates in various combinations; (ii) inhibition of Gb4-dependent 2102 cell adhesion by preincubation of cells with anti-SSEA-3 or anti-nLc4 antibodies, or by pretreatment of Gb4-coated plates with aqueous micellar solution of nLc4 or GalGb4; (iii) decline of the cell adhesion in association with retinoic acid-induced differentiation, whereby SSEA-3 and nLc4 levels are reduced. Since cell adhesion is an essential prerequisite for induction of differentiation, as observed at each step of embryogenesis, expression of seven transcription factors following adhesion of 2102 cells to Gb4-coated plates, and to detergent-insoluble substrate adhesion matrix prepared from 2102 cells, were studied. In both types of adhesion, a strong enhancement of AP1 and CREB site binding activity was observed during the early stage (15-60 min following initial adhesion). Although 2102 cells showed strong adhesion to Gg3-coated plates, based on interaction between Gg3 and Gb4, adhesion of the cells to Gg3 did not cause changes of AP1 and CREB activity. No other transcription factors showed changes induced by Gg3- or Gb4-dependent adhesion.

Topics: Antigens, Tumor-Associated, Carbohydrate; Bromodeoxyuridine; Carbohydrate Metabolism; Carbohydrate Sequence; Carcinoma, Embryonal; Cell Adhesion; Cell Differentiation; Cyclic AMP Response Element-Binding Protein; Embryonic and Fetal Development; Epitopes; Globosides; Glycosphingolipids; Humans; Models, Biological; Molecular Sequence Data; Signal Transduction; Stage-Specific Embryonic Antigens; Transcription Factor AP-1; Tretinoin; Tumor Cells, Cultured

We have previously cloned a novel guanine nucleotide-binding protein (G-protein)-coupled receptor, H218, that has sequence similarity to a lysophosphatidic acid receptor, edg2. We present here Northern analysis indicating that the H218 mRNA is expressed in undifferentiated F9 embryonal carcinoma cells. The H218 message is down-regulated and its stability is decreased during retinoic acid- and dibutyryl cAMP-induced differentiation. Treatment by various receptor-selective retinoids indicated that retinoic acid receptor beta or gamma signaling, but not retinoid X receptor activation, is required for the down-regulation of H218 mRNA. Activation of the H218 receptor may contribute to the phenotype of undifferentiated F9 embryonal carcinoma cells.

Topics: Animals; Benzoates; Bucladesine; Carcinoma, Embryonal; Cell Differentiation; Chromans; Down-Regulation; Gene Expression Regulation, Neoplastic; GTP-Binding Proteins; Mice; Naphthalenes; Neoplasm Proteins; Phenotype; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Receptors, Retinoic Acid; Retinoids; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Tetrahydronaphthalenes; Tretinoin; Tumor Cells, Cultured

Apart from modulation of tumor-cell drug sensitivity, induction of differentiation might be an alternative in the treatment of tumors resistant to cytotoxic drugs. In this report the capacity to induce differentiation and to modulate the cis-diamminedichloroplatinum(II) (CDDP) sensitivity of all-trans-retinoic acid (RA), docosahexaenoic acid (DCHA), and hexadecylphosphocholine (HePC) is examined in human germ-cell tumor cell lines. In the embryonal carcinoma cell line Tera-2 and its 3.7-fold CDDP-resistant subline Tera2-CP, we evaluated the effects of 96 h of pretreatment with RA (0.1 microM), DCHA (23 microM), and HePC (25 microM) on differentiation induction and on CDDP-induced cytotoxicity, DNA platination (4-h incubation), and apoptosis (continuous incubation). Without drug treatment, Tera2-CP showed less apoptosis than Tera-2. Pretreatment with RA decreased the cytotoxicity and apoptosis induced by CDDP without resulting in decreased DNA platination and increased differentiation in both cell lines. DCHA enhanced CDDP-induced cytotoxicity and apoptosis and did not affect the embryonal character of either cell line. HePC did not affect CDDP cytotoxicity or differentiation in either cell lines. Effects of the modulators on differentiation and on CDDP-induced cytotoxicity, DNA platination, and apoptosis did not differ between Tera-2 and Tera2-CP. RA can be applied for differentiation induction in CDDP-resistant germ-cell tumor models. However, in this model, RA reduced the apoptotic susceptibility. DCHA potentiated CDDP cytotoxicity in vitro; its in vivo modulatory capacity in germ-cell tumor cells deserves further study.

Topics: Apoptosis; Carcinoma, Embryonal; Cell Differentiation; Cell Survival; Cisplatin; DNA Damage; DNA Fragmentation; Docosahexaenoic Acids; Drug Resistance, Neoplasm; Humans; Male; Phosphorylcholine; Tretinoin; Tumor Cells, Cultured

The Drosophila protein Groucho is involved in the regulation of cell-determination events during insect neurogenesis and segmentation. A group of mammalian proteins, referred to as transducin-like Enhancer of split (TLE) 1 through 4, share with Groucho identical structures and molecular properties. The aim was to determine whether individual TLE proteins participate in the regulation of cell determination in mammals like their Drosophila counterpart. It is here reported that TLE family members are expressed in combinatorial ways during the in vitro differentiation of mouse P19 embryonic carcinoma cells (a model for neural determination) and rat CFK2 cells (a model for chondrocytic determination). TLE1 is up-regulated and TLE2 and TLE4 are down-regulated to different extents during early stages of differentiation. In contrast, later stages correlate with up-regulation of TLE2 and TLE4, and decreased expression of TLE1. Individual TLE proteins are also expressed in combinatorial as well as complementary patterns during the development of the cerebral cortex and spinal cord of mouse embryos. In particular, TLE1 is robustly expressed in both neural progenitor cells and postmitotic neurons of the outer layers of the cortical plate, whereas TLE4 expression marks preferentially postmitotic neurons of the inner layers. Taken together, these results strongly suggest non-redundant roles for individual TLE proteins during both cell-determination and cell-differentiation events.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Embryonal; Cartilage; Cell Differentiation; DNA-Binding Proteins; Drosophila melanogaster; Fetal Proteins; Gene Expression Regulation, Developmental; Glutathione Transferase; HeLa Cells; Humans; Jurkat Cells; Mice; Nervous System; Neuropeptides; Rats; Recombinant Fusion Proteins; Repressor Proteins; Tretinoin; Tumor Cells, Cultured

We have identified a novel type II activin receptor, called type IIA-N, the expression of which was induced during the neural differentiation of murine P19 embryonal carcinoma cells (P19 cells). P19 cells differentiate into several cell types dependent on the culture conditions. The induction of type IIA-N mRNA occurred predominantly in conjunction with neural differentiation. Sequence analysis of a cDNA clone for type IIA-N indicated that type IIA-N had a 24 bp insertion in the juxtamembrane region of the type IIA activin receptor suggesting that it is an alternative splicing product of the type IIA gene. Type IIA-N was also identified in human and Xenopus, and the amino acid sequences of three species were completely conserved. The expression of type IIA-N mRNA was specifically detected in neuroblastoma cells among several activin responsive cell lines. In vivo expression of type IIA-N mRNA was detected only in the neural tissues such as brain and spinal cord in adult mouse, by RT-PCR. Furthermore, its expression in developing Xenopus embryos was restricted to the neurula and later stages. These results suggest that the expression of type IIA-N is specific to neural cells and mediates neural differentiation-specific activin signaling.

Topics: Activin Receptors; Alternative Splicing; Amino Acid Sequence; Animals; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; DNA Primers; DNA, Complementary; Gene Expression; Humans; Mice; Neurons; Polymerase Chain Reaction; Receptors, Growth Factor; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Xenopus

Tuberous sclerosis is an autosomal dominant disorder. Besides the development of benign growths (hamartomas) in different tissues, one hallmark of this disease is the presence of highly epileptogenic dysplastic lesions in the cerebral cortex (tubers) composed of abnormal shaped neurones. Patients often show evidence of severe mental retardation. Linkage analysis revealed two disease-determining genes on chromosome 9 and chromosome 16. The TSC2 gene on chromosome 16 encodes a 1784-amino acid putative tumour suppressor protein, tuberin, that functions as a GTPase-activating protein. Here we show that tuberin expression is upregulated upon induction of neuronal differentiation in the neuroblastoma cell lines SK-N-SH and LAN-1. This upregulation occurs at post-transcriptional level and is independent of the proliferation status. TSC2 expression is unaffected during differentiation of C2C12 myoblasts into myotubes and of F9 embryonal carcinoma cells into cells resembling parietal endoderm. Antisense inhibition of tuberin expression in SK-N-SH or LAN-1 cells inhibits neuronal differentiation, but does not affect the differentiation of F9 cells. Ectopic overexpression of TSC2 not only reverts the antisense-associated phenotype but furthermore accelerates the neuronal differentiation process. Our data show for the first time that tuberin plays a critical role in neuronal differentiation. Such role is consistent with the phenotype of tuberous sclerosis patients, who inherit one defective TSC2 allele, and frequently lose the remaining normal allele in many of the tubers/hamartomas which develop in the central nervous system of these patients.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Cell Line; Humans; Mice; Neuroblastoma; Neurons; Oligonucleotides, Antisense; Repressor Proteins; Transfection; Tretinoin; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Cells, Cultured; Tumor Suppressor Proteins

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

This study addresses the contributions of specific retinoid receptors during all-trans-retinoic acid (RA)-mediated differentiation and growth suppression of human embryonal carcinoma cells. The pleiotropic effects of RA are mediated by retinoic acid receptors (RARs) and retinoid X receptors (RXRs), members of the nuclear receptor family of transcription factors. After RA-treatment the multipotent human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) displays limited proliferative potential, reduced tumorigenicity, and morphologic and immunophenotypic neuronal maturation. RARgamma over-expression in NT2/D1 cells signals mesenchymal NT2/D1 terminal differentiation while RARalpha and RARbeta do not and RARgamma overcomes retinoid resistance in an NT2/D1 clone (NT2/D1-RI) having deregulated RARgamma expression. Since RARgamma transfectants do not display neuronal maturation, this study sought to identify cooperating retinoid receptors engaged in NT2/D1 differentiation. Through gain of function experiments, this report highlights RXRbeta as playing an important role along with RARgamma in signaling differentiation of NT2/D1 cells. Stable over-expression of RXRbeta, but not RXRalpha or RXRgamma, was found to signal NT2/D1 growth suppression and to induce a non-neuronal morphology and immunophenotype. Notably, co-transfection of RARgamma and RXRbeta resulted in marked growth suppression and for the first time, expression of typical neuronal markers of NT2/D1 differentiation. To clarify the role of RXRbeta and RARgamma in this differentiation program, a modified transient fibroblast growth factor-4 (FGF4) promoter-enhancer reporter assay that reflects effective RA-mediated differentiation of NT2/D1 cells was employed. Transfection of RARgamma or RXRbeta in NT2/D1 cells augments transcriptional repression of the FGF4 reporter and RARgamma and RXRbeta co-transfection markedly repressed reporter activity, indicating the combined role of these receptors in RA-induced NT2/D1 differentiation. Taken together, these findings reveal specific retinoid receptors must cooperate to signal terminal growth suppression and maturation of NT2/D1 cells. Since the transcriptional repression of FGF4 is coupled to the effective maturation of human embryonal carcinoma cells, the described co-transfection strategy should prove useful to identify genes with positive or negative effects on the differentiation program of these tumor cells.

Topics: Carcinoma, Embryonal; Cell Differentiation; Cell Division; Dimerization; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Gene Expression Regulation, Neoplastic; Genes, Reporter; Growth Inhibitors; Humans; Proto-Oncogene Proteins; Receptors, Retinoic Acid; Recombinant Proteins; Retinoid X Receptors; Signal Transduction; Transcription Factors; Tretinoin

P19 cells are pluripotent murine embryonic carcinoma (EC) cells that can be induced by all-trans-retinoic acid (RA) to differentiate into cells that are biochemically and morphologically similar to cells of the central nervous system. We have established these cells as a reproducible cell system to evaluate potential effects of agents disrupting neuronal differentiation. The viability of P19 cells was assessed using a neutral red assay. Uptake of [3H]-gamma-amino butyric acid ([3H]GABA) was assessed as a marker of neuronal differentiation. We observed significant increases in [3H]GABA over time, corresponding with the appearance of cells with neuronal morphologies. 2,4-Diaminobutyric acid, a specific inhibitor of high affinity neuronal GABA uptake, reduced [3H]GABA uptake by approximately 75%. Additionally, [3H]GABA uptake in cells treated with dimethylsulfoxide (DMSO), which differentiate into mesodermal derivatives, was approximately 25% of uptake observed in RA-exposed, neuronally differentiated P19 cells. The morphology of P19 cell cultures correlated with [3H]GABA uptake: high [3H]GABA uptake was only observed in cultures with distinct neuronal morphologies. These results suggest that [3H]GABA uptake is a good indicator of neuronal differentiation in P19 cells. The responsiveness of P19 cells to developmental toxicants was assessed by comparing effects in P19 cells with effects observed previously in primary cultures of differentiating rat embryo midbrain (CNS) cells. Alkylating agents chosen for this investigation include methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS). The rank order of potency of these alkylating agents in the CNS cells was MMS > MNU > ENU > EMS. With the exception of ENU, concentrations that caused effects on growth and differentiation in the P19 cells were very comparable to those causing similar effects in CNS cell cultures. Our results with P19 cells suggest that this EC cell line may also be a useful in vitro cell system for studying mechanisms of developmental toxicity, with the advantages of being an established cell line.

Topics: Alkylating Agents; Aminobutyrates; Animals; Antineoplastic Agents, Alkylating; Carcinoma, Embryonal; Cell Differentiation; Cell Division; Dimethyl Sulfoxide; Ethyl Methanesulfonate; Ethylnitrosourea; gamma-Aminobutyric Acid; Male; Mesencephalon; Methyl Methanesulfonate; Methylnitrosourea; Mice; Neurons; Tretinoin; Tumor Cells, Cultured

A subset of male germ cell cancers presenting with advanced stage abundantly express the fibroblast growth factor-4 (FGF4). FGF4 expression is restricted in vitro to undifferentiated embryonal carcinomas (ECs). During induced differentiation, FGF4 expression is repressed in maturation sensitive but not resistant human ECs, suggesting FGF4 plays an important role in malignant growth or differentiation of ECs. To explore these FGF4 signals in male germ cell cancers, the multipotent human EC NTERA-2 clone D1 (NT2/D1) cell line was studied. All-trans-retinoic acid (RA)-treatment of these cells induces a neuronal phenotype and represses tumorigenicity and FGF4 expression. In contrast, RA-treatment of retinoid resistant lines derived from NT2/D1 cells failed to repress FGF4 expression. This implicated FGF4 directly in regulating human EC growth or differentiation. To evaluate further this FGF4 role, FGF4 was constitutively over-expressed in NT2/D1 cells using a CMV-driven expression vector containing the neomycin resistance gene. Three stable transfectants expressing exogenous FGF4 were studied as was a control transfectant only expressing the neomycin resistance gene. RA-treatment repressed endogenous but not exogenous FGF4 expression. RA-treatment of these transfectants induced morphologic and immunophenotypic maturation, changes in RA-regulated genes, and a G1 cell cycle arrest in a manner similar to parental NT2/D1 cells. This indicated FGF4 over-expression did not block RA-mediated differentiation. As expected, RA-treatment repressed tumorigenicity of the control transfectant after subcutaneous injection into athymic mice. Despite RA-treatment, this repressed tumorigenicity was overcome in all the transfectants over-expressing FGF4. The histopathology and neovascularization did not appreciably differ between xenograft tumors derived from FGF4 over-expressing versus control transfectants. FGF4 expression studies were extended to patient-derived germ cell tumors using total cellular RNA Northern analysis and an immunohistochemical assay developed to detect FGF4 protein expression. Germ cell tumors with EC components were significantly more likely to express FGF4 mRNA (P < or = 0.0179) than other examined germ cell tumors without EC components. Immunohistochemical results from 43 germ cell tumors demonstrated increased FGF4 expression especially in non-seminomas having EC components. Thus, FGF4 promotes directly malignant growth of cultured ECs, overcomes the

Topics: Antineoplastic Agents; Carcinoma, Embryonal; Cell Cycle; Cell Differentiation; Drug Interactions; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Humans; Proto-Oncogene Proteins; Recombinant Proteins; Signal Transduction; Transfection; Tretinoin

The purpose of this study was to evaluate the impact of modulating the repair of O6-alkylguanine adducts on the developmental toxicity of alkylating agents. Alkylating agents that have been shown to induce developmental toxicity following either in vitro or in vivo exposure were chosen for this investigation, and include methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS). P19 cells are pluripotent murine embryonic carcinoma cells that can be induced by all trans retinoic acid (RA) to differentiate into cells that are biochemically and morphologically very similar to cells of the central nervous system. These cells are useful for studying the ability of chemicals to affect neuronal viability and differentiation. Neuronally differentiating P19 cells were pretreated with O6-benzylguanine (O6-Bg), a potent and specific inhibitor of the O6-alkylguanine-DNA-alkyltransferase (AT) protein that repairs lesions at the O6-position of guanine. In previous studies using micromass rat embryo midbrain cells, O6-Bg greatly potentiated the ability of MNU but not ENU to inhibit differentiation, and did not significantly alter the effects of either MNU or ENU on viability. In the P19 cells, we found that AT inhibition potentiated the effects of MMS, MNU, and EMS to inhibit both viability and differentiation. Additionally, AT inhibition had a much greater effect on toxicity of the methylating agents, as compared to the ethylating agents. These results suggest that O6-alkylguanine adducts can inhibit both viability and differentiation in P19 cells treated with alkylating agents.

Topics: Alkylating Agents; Animals; Antineoplastic Agents; Carcinogens; Carcinoma, Embryonal; Cell Differentiation; Cell Division; Cell Survival; DNA Repair; Ethyl Methanesulfonate; Ethylnitrosourea; Guanine; Mesencephalon; Methyl Methanesulfonate; Methylnitrosourea; Mice; Neurons; O(6)-Methylguanine-DNA Methyltransferase; Rats; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) and dibutyryl cAMP plus theophilline (CT) trigger F9 cells to differentiate into parietal endoderm. The differentiation induces a 9-fold increase in total heparan sulfate (HStotal) biosynthesis and a 170-fold increase in anticoagulantly active HS (HSact) biosynthesis. Measurement of 3-O-sulfotransferase-1 mRNA and enzymatic activity demonstrated an increase of over 100-fold whereas determination of N-, 2-O, and 6-O-sulfotransferase enzymatic activities showed elevations of 2-, 3. 5-, and 3.7-fold, respectively. HSact precursor pool measurements reveal that 30% of control F9 HStotal can be converted into HSact while only an additional 10% of RACT F9 HStotal can be transformed into HSact. Disaccharide analysis of metabolic labeled HS indicated that 32% 3-O-sulfate containing disaccharides, i.e. GlcA-anManR3S and GlcA-anManR3S6S, are present in HSact and 68% GlcA-anManR3S and GlcA-anManR3S6S are found in anticoagulantly inactive HS (HSinact). By using adenosine 3'-phosphate 5'-phosphosulfate and purified 3-O-sulfotransferase-1, 30% of 3-O-sulfation occurs in HSact and 70% of 3-O-sulfation occurs in HSinact. The similar ratio of 3-O-sulfate distribution in HSact versus HSinact suggests that HSact production in the F9 system is determined by the abundance of 3-O-sulfotransferase-1 as well as the size of the HSact precursor pool. Extensively 3-O-sulfated HSinact may play an important functional role under in vivo conditions within the murine placenta.

Topics: Animals; Anticoagulants; Bucladesine; Carcinoma, Embryonal; Cell Differentiation; Disaccharides; Endoderm; Female; Heparitin Sulfate; Mice; Placenta; Pregnancy; Sulfotransferases; Tretinoin; Tumor Cells, Cultured; Up-Regulation

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

Soluble endogenous lactoside-binding lectins, galectins, have been implicated in cell adhesion, growth, differentiation, neoplastic transformation, and metastasis. Two major classes of these lectins, galectin-1 and galectin-3, are developmentally regulated. To explore the mechanisms by which the expression of the galectins is regulated and to examine their association with the differentiation processes induced by all-trans retinoic acid (RA), dibutyryl cyclic AMP (Bt2cAMP) and their combination, we used the murine embryonal carcinoma (EC) cell line F9 and its RA-resistant mutant, RA-3-10. RA induced endodermal differentiation and a concurrent induction of galectin-1 and its complementary glycoconjugates (laminin and lysosomal-associated membrane protein, LAMP) in the F9 wild-type (wt) line, but failed to induce differentiation and had no effects on or even reduced the expression of galectin-1, laminin, and LAMP in the RA-3-10 line. On the other hand, RA inhibited expression of galectin-3 in the wild-type line but had no effect on the RA-3-10 line. The galectin-1 gene is at least partially regulated at the transcriptional level. These results demonstrate a parallel association between differentiation and induction of galectin-1, and inhibition of galectin-3 in F9 cells by RA. The study suggests that a regulated expression of galectins and their complementary glycoconjugates is involved in the differentiation pathway induced by RA in F9 cells.

Topics: Animals; Antigens, Differentiation; Bucladesine; Carcinoma, Embryonal; Cell Differentiation; Galectin 1; Galectin 3; Gene Expression Regulation; Glycoconjugates; Hemagglutinins; Mice; RNA, Messenger; Tissue Plasminogen Activator; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Wnt signals have been shown to play an important role in the development of the central nervous system (CNS) of mouse. In our previous work, it was demonstrated that Wnt signal could initiate differentiation of P19 EC cells. In the present investigation, it was examined with RT-PCR whether expression of beta-catenin, a downstream gene of Wnt in its signal transduction pathway, is regulated. It was found that the level of protein or transcript beta-catenin during P19 neuronal differentiation was not changed. However, immunostaining data showed that beta-catenin was translocalized into nuclei after retinoic acid induced P19 cell aggregates were trypsinized and cultured in serum free N2 medium for 2 and 4 d. In this period, transcription of En-2, a downstream target gene of Wnt signal, increased evidently. The above data suggest that Wnt signals are involved in the early stage of neuronal differentiation process of P19 cell. Meanwhile, the distribution of beta-catenin on the neurites indicates that this protein may also be involved in neuritis outgrowth process.

Topics: Animals; beta Catenin; Carcinoma, Embryonal; Cell Differentiation; Cytoskeletal Proteins; Mice; Neurons; Proto-Oncogene Proteins; RNA, Messenger; Signal Transduction; Trans-Activators; Tretinoin; Tumor Cells, Cultured; Wnt Proteins; Zebrafish Proteins

Retinoic acid (RA) treatment of embryonal carcinoma (EC) cells initiates a cascade of alterations in gene regulation, leading to their differentiation into various cell types. In P19 EC cells RA treatment stimulates induction of the RAR beta gene, while it represses Oct3/4 gene expression. Here we present dimethylsulfate-based genomic footprinting analyses of these two genes. We found that the RAR beta promoter is not occupied prior to RA treatment, but following RA treatment all regulatory elements in this promoter become occupied. On the other hand, the Oct3/4 promoter is occupied at all three known elements before RA treatment, but this occupancy is coordinately lost following the treatment. Thus, factor occupancy coincides with expression of the genes. It is likely that the presence of factor binding or its absence revealed here represents a mechanism of the regulated expression of these genes in vivo. Our results demonstrate the power of genomic footprinting for studying regulatory events for transcription in vivo. In contrast, with in vitro protein-DNA binding assay, factors for both promoters are present in these cells regardless of RA treatment. It has been shown that RA receptor (RAR) and retinoid X receptor (RXR), by heterodimerization, mediate the RA action in EC cells. To elucidate the role of RAR/RXR heterodimers in the RAR beta promoter occupancy in vivo, genomic footprinting has been performed in P19 cells stably expressing dominant negative mutants of RXR. Two such mutants, lacking either the DNA binding domain or the C-terminal activation domain, inhibit RA induction of the RAR beta gene in these cells. RA-induced factor occupancy is also markedly inhibited at all elements in the RAR beta promoter in these cells. Our results show that binding of liganded RAR/RXR heterodimers to RARE is required for other factors to gain access to their respective elements in the promoter.

Topics: Animals; Carcinoma, Embryonal; Cell Line; Dimerization; DNA Footprinting; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Mice; Models, Genetic; Octamer Transcription Factor-3; Promoter Regions, Genetic; Receptors, Retinoic Acid; Regulatory Sequences, Nucleic Acid; Retinoid X Receptors; Transcription Factors; Tretinoin; Tumor Cells, Cultured

The expression pattern of the Wilms' tumor suppressor gene, WT1, during embryonal development suggests a role for the WT1 proteins in the differentiation of specific tissues. This notion is supported by the observation that WT1 knock-out mice fall to develop kidneys and gonads. We describe here the changes in the expression and DNA binding activity of the WT1 gene product in P19 embryonal carcinoma cells and embryonic stem cells triggered to differentiate by either retinoic acid (RA) or DMSO. In exponentially growing P19 embryonal carcinoma (EC) cells, WT1 mRNA and proteins were undetectable. During RA-induced but not DMSO-induced differentiation of P19 EC cells, WT1 expression and DNA binding are strongly activated. Treatment of embryonic stem cells with RA resulted in a similar activation of WT1. Immunohistochemical analysis showed that WT1 is expressed in endodermal, glial, and epithelial cell types. In addition, DNA binding by EGR-1, a transcription factor structurally related to WT1, increased during differentiation of P19 EC and embryonic stem cells. To investigate the possible functional consequences of DNA binding by WT1, we examined the expression levels of two putative transcriptional targets of WT1, the insulin-like growth factor 1 receptor and epidermal growth factor receptor. We found that after an initial induction, decreasing expression of the insulin-like growth factor I receptor is correlated with increasing WT1 expression. Our results demonstrate that expression of WT1 is induced in specific cell types during RA-induced differentiation of P19 EC cells, reflecting the tissue-specific expression of WT1 in vivo. Therefore, we believe that P19 EC cells are a suitable system to study activation and function of WT1 during differentiation.

Topics: Blotting, Western; Carcinoma, Embryonal; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; DNA-Binding Proteins; Early Growth Response Protein 1; Gene Expression Regulation, Developmental; Genes, Wilms Tumor; Humans; Immediate-Early Proteins; Neuroglia; Receptor, IGF Type 1; RNA, Messenger; Stem Cells; Transcription Factors; Tretinoin; WT1 Proteins

Retinoic acid (RA) induced the terminal differentiation of a human embryonal carcinoma cell line (NT2/D1) into several morphologically distinct cell types, including the postmitotic CNS neurons. Although RA has been suggested to play an important role in brain development, little is known about the molecular mechanism by which RA induces neuronal differentiation. In the present study, RNA fingerprinting by arbitrarily primed PCR (RAP-PCR) was used to identify the transcripts in NT2/D1 cells that were differentially regulated by RA. Northern blot analysis of the differentially amplified PCR fragments revealed 11 genes that were regulated by RA. Of these, seven were up-regulated and four were down-regulated along the course of RA treatment. More importantly, four of the RA-regulated genes that were identified in the present study are novel. Our findings suggested that there are a number of RA-regulated genes that have yet to be identified. RAP-PCR provides a useful tool for studying the patterns of transcript expression during the course of RA treatment and allows the cloning of novel genes involved in the process of neuronal differentiation. Furthermore, it provides a basis for the selection of genes that are involved in the RA-induced signaling pathway in the human CNS.

Topics: Blotting, Northern; Carcinoma, Embryonal; Cell Differentiation; DNA Fragmentation; DNA, Complementary; Gene Expression Regulation; Genes; Genetic Techniques; Humans; Neurons; Polymerase Chain Reaction; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

Intratubular malignant germ cells (ITMGC), as assessed by clinicopathologic or cytogenetic studies, are regarded as a preinvasive lesion of all human testicular germ cell tumors with the exception of yolk sac tumors (in infants) and spermatocytic seminomas. To characterize specific surface molecules of ITMGC, we raised three mouse monoclonal antibodies (mAb) against NCR-G3 (G3), a multipotent, human embryonal carcinoma (EC) cell line, and screened cryostat sections of human testicular tissue containing ITMGC. These three mAb (HB5, IgG1; HF2, IgG1; HE11, IgG1) reacted to the surface of ITMGC, seminomas, and EC in vivo as well as to human EC cell lines in vitro. Expression of HB5 and HF2 antigens was down-regulated during cellular differentiation of G3 cells by retinoic acid or N,N'-hexamethylene-bis-acetamide treatment, whereas that of HE11 antigen was up-regulated with cellular differentiation by retinoic acid. Furthermore, these three mAb reacted to stage-specific prespermatogonia in the human fetus but not in human adults. HB5, HF2, and HE11 antigens were shown to be glycoproteins with molecular weights of approximately 80, 80, and 70 kd, respectively, and could be immunoprecipitated after deglycosylation treatment. Peptide mapping with Staphylococcus aureus V8 protease suggested that the HB5 and HF2 antigens were identical. We concluded that HB5/HF2 and HE11 antigens are oncodevelopmental antigens in testicular germ cell tumors and human spermatogenesis that may play a significant role in tumorigenesis and the development of human germ cells.

Topics: Adult; Aging; Animals; Antibodies, Monoclonal; Antibody Specificity; Antigens, Neoplasm; Antigens, Surface; Carcinoma, Embryonal; Cell Differentiation; Embryonic and Fetal Development; Female; Fetus; Germinoma; Gestational Age; Humans; Infant; Infant, Newborn; Male; Mice; Mice, Inbred BALB C; Organ Specificity; Precancerous Conditions; Reference Values; Spermatogonia; Testicular Neoplasms; Testis; Tretinoin; Tumor Cells, Cultured

P19 EC cells undergoes apoptosis during neuronal differentiation induced by retinoic acid. Two CPP32-like proteases, CPP32 and Mch-3, are expressed in untreated and retinoic acid-treated P19 EC cells. CPP32-like activity is remarkably increased in apoptosis during neuronal differentiation of P19 EC cells. Inhibition of CPP32-like proteases prevents apoptosis, suggesting that activation of CPP32-like proteases play central roles in the apoptosis during neuronal differentiation of P19 EC cells. Wortmannin, PI-3K inhibitor, enhances the CPP32-like activity of the retinoic acid-treated P19 EC cells. PI-3K may be involved in the apoptosis during neuronal differentiation as negative regulator.

Topics: Androstadienes; Apoptosis; Carcinoma, Embryonal; Caspase 3; Caspases; Cell Differentiation; Cysteine Endopeptidases; DNA Fragmentation; Molecular Sequence Data; Neurons; Tretinoin; Tumor Cells, Cultured; Wortmannin

Retinoic acid (RA) induces expression of genes encoding the Hox family of transcription factors, whose differential expression orchestrates developmental programs specifying anterior-posterior structures during embryogenesis. Hox proteins bind DNA as monomers and heterodimers with Pbx proteins. Here we show that RA upregulates Pbx protein abundance coincident with transcriptional activation of Hox genes in P19 embryonal carcinoma cells undergoing neuronal differentiation. However, in contrast to Hox induction, Pbx upregulation is predominantly a result of post-transcriptional mechanisms. Interestingly, Pbx1, Pbx2, and Pbx3 exhibit different profiles of upregulation, suggesting possible functional divergence. The parallel upregulation of Pbx and Hox proteins in this model suggests an important role for transcriptional control by Pbx-Hox heterodimers during neurogenesis, and argues for precise control by RA.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Cell Nucleus; Dexamethasone; DNA-Binding Proteins; Estrogens; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Mice; Neurons; PC12 Cells; Pre-B-Cell Leukemia Transcription Factor 1; Proto-Oncogene Proteins; Rats; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoic acid plays important roles in development, growth and differentiation by regulating the expression of target genes. A new retinoic acid-inducible gene, Stra6, has been identified in P19 embryonal carcinoma cells using a subtractive hybridization cDNA cloning technique. Stra6 codes for a very hydrophobic membrane protein of a new type, which does not display similarities with previously characterized integral membrane proteins. Stra6, which exhibits a specific pattern of expression during development and in the adult, is strongly expressed at the level of blood-organ barriers. Interestingly, in testis Sertoli cells, Stra6 has a spermatogenic cycle-dependent expression which is lost in testes of RAR alpha null mutants where Stra6 is expressed in all tubules. We suggest that the Stra6 protein may be a component of an as yet unidentified transport machinery.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carcinoma, Embryonal; Choroid Plexus; Cloning, Molecular; Gene Expression Regulation, Developmental; In Situ Hybridization; Male; Membrane Proteins; Mice; Mice, Knockout; Molecular Sequence Data; Pigment Epithelium of Eye; Placenta; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Sertoli Cells; Time Factors; Tissue Distribution; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Yolk Sac

Apolipoprotein E has been shown to be a risk factor for late-onset Alzheimer's disease, with the apolipoprotein epsilon 4 allele conferring the risk. Apolipoprotein E is found in neurofibrillary tangles and senile plaques, the pathological characteristics of Alzheimer's disease. To date there is no direct evidence that human neurons can take up exogenous apolipoprotein E, which is necessary if apolipoprotein E is involved in the formation of neurofibrillary tangles. To examine apolipoprotein E uptake we employed the human NTera2/D1 cell line, which can be induced by retinoic acid to differentiate into postmitotic NTera2-N neurons, which have the characteristics and morphology of human central nervous system neurons. We defined the cell line as genotype apolipoprotein epsilon 3/3 and demonstrated that the cells do not synthesize apolipoprotein E but can take up and internalize exogenous recombinant apolipoprotein E3. We also confirmed the expression of the low-density lipoprotein receptor-related protein, a known receptor for apolipoprotein E. The NTera2/D1 cell line therefore provides a useful human cell model for examining the effects of other apolipoprotein E isoforms with a view to defining intraneuronal interactions of apolipoprotein E.

Topics: Alzheimer Disease; Apolipoprotein E3; Apolipoproteins E; Biological Transport; Carcinoma, Embryonal; Cell Differentiation; Humans; Low Density Lipoprotein Receptor-Related Protein-1; Mitosis; Models, Neurological; Neurons; Receptors, Immunologic; Receptors, LDL; Recombinant Proteins; Tretinoin; Tumor Cells, Cultured

We prepared 2,132 expressed sequence tags (ESTs) from undifferentiated mouse embryonal carcinoma F9 cells and found that 1,416 match known gene and/or protein sequences [Nishiguchi et al. (1996) J. Biochem. 119, 749-767]. To obtain information on the functions of the remaining 716 unidentified ESTs and to develop a system for characterizing ESTs matching no known genes, we analyzed their sequences by (i) repeated database searches, using the BLASTN, BLASTX, TBLASTX, and FASTA programs, (ii) using computer programs developed or modified for this work, such as the WFASTA, ORFTRNS, and MFASTA programs, together with the DBPROSITE and GRAIL programs, and (iii) examining the expression patterns of the corresponding mRNAs in F9 cells and several organs of adult mice, using the digoxigenin-labeled dot-blot method. We found that 216 of the 716 ESTs match known gene and/or protein sequences, and 307 show significant similarities to these sequences, with a Poisson p-value < 0.01. The strategy and usefulness of such analysis for characterizing unidentified ESTs are discussed.

Topics: Animals; Blotting, Northern; Carcinoma, Embryonal; Cell Differentiation; Databases, Factual; Gene Expression Regulation, Developmental; Mice; Molecular Sequence Data; Open Reading Frames; Organ Specificity; RNA, Messenger; Sequence Homology, Amino Acid; Sequence Tagged Sites; Software; Tissue Distribution; Tretinoin; Tumor Cells, Cultured

P19 mouse embryonal carcinoma cells can be stimulated to differentiate into endodermal-like, mesodermal-like, and neuronal-like phenotypes in response to specific morphogens. At low concentrations, retinoic acid stimulates P19 embryonal cells to differentiate to cells displaying an endodermal phenotype, whereas at higher concentrations it stimulates differentiation to neuroectoderm. The Galpha12 and Galpha13 subunits of heterotrimeric G-proteins are expressed in the embryonal P19 cells and stimulated in response to retinoic acid as the cells differentiate to endodermal or neuroectodermal phenotypes. Suppression of the expression of either Galpha12 or Galpha13 by antisense RNA is shown to promote cell detachment from substratum and apoptosis. Expression of the constitutively active, mutant form of Galpha12 (Q229L), in contrast, stimulates loss of the embryonal phenotype. Expression of the constitutively active form of Galpha13 (Q226L) stimulates differentiation of the cells from embryonal to endodermal, in the absence of retinoic acid. Thus, both Galpha12 and Galpha13 are essential to stimulation of cell differentiation by retinoic acid. Deficiency of either Galpha12 or Galpha13 increases programmed cell death.

Topics: Animals; Apoptosis; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Embryonal; Cell Differentiation; GTP-Binding Proteins; Mice; RNA, Messenger; Signal Transduction; Tretinoin

Retinoic acid induces P19 mouse embryonal carcinoma cells to differentiate to endoderm and increases expression of the heterotrimeric G-protein subunits Galpha12 and Galpha13. Retinoic acid was found to induce differentiation and sustained activation of c-Jun amino-terminal kinase, but not of ERK1,2 or of p38 mitogen-activated protein kinases. Much like retinoic acid, expression of constitutively active forms of Galpha12 and Galpha13 induced differentiation and constitutive activation of c-Jun amino-terminal kinase. Expression of the dominant negative form of c-Jun amino-terminal kinase 1 blocked both the activation of c-Jun amino-terminal kinase and the induction of endodermal differentiation in the presence of retinoic acid. These data implicate c-Jun amino-terminal kinase as a downstream element of activation of Galpha12 or Galpha13 obligate for retinoic acid-induced differentiation.

Topics: Animals; Anisomycin; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Embryonal; Cell Differentiation; Embryonic and Fetal Development; Enzyme Activation; GTP-Binding Proteins; JNK Mitogen-Activated Protein Kinases; Lewis X Antigen; Mice; Mitogen-Activated Protein Kinases; Tretinoin

Lithium is one of the most widely used drugs for treating bipolar (manic-depressive) disorder. Despite its efficacy, the molecular mechanism underlying its action has not been elucidated. One recent study has proposed that lithium inhibits glycogen synthase kinase-3 and thereby affects multiple cellular functions. Because glycogen synthase kinase-3 regulates the phosphorylation of tau (microtubule-binding protein that forms paired helical filaments in neurons of the Alzheimer's disease brain), we hypothesized that lithium could affect tau phosphorylation by inhibiting glycogen synthase kinase-3. Using cultured human NT2N neurons, we demonstrate that lithium reduces the phosphorylation of tau, enhances the binding of tau to microtubules, and promotes microtubule assembly through direct and reversible inhibition of glycogen synthase kinase-3. These results provide new insights into how lithium mediates its effects in the central nervous system, and these findings could be exploited to develop a novel intervention for Alzheimer's disease.

Topics: Alanine; Alzheimer Disease; Amino Acid Sequence; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Embryonal; Cell Differentiation; Glycogen Synthase Kinase 3; Glycogen Synthase Kinases; Humans; Kinetics; Lithium Chloride; Microtubules; Mutagenesis, Site-Directed; Neurons; Phosphorylation; Point Mutation; Recombinant Proteins; Sequence Tagged Sites; Serine; tau Proteins; Transfection; Tretinoin; Tumor Cells, Cultured

Histone acetylation is thought to have a role in transcription. To gain insight into the role of histone acetylation in retinoid-dependent transcription, we studied the effects of trichostatin A (TSA), a specific inhibitor of histone deacetylase, on P19 embryonal carcinoma cells. We show that coaddition of TSA and retinoic acid (RA) markedly enhances neuronal differentiation in these cells, although TSA alone does not induce differentiation but causes extensive apoptosis. Consistent with the cooperative effect of TSA and RA, coaddition of the two agents synergistically enhanced transcription from stably integrated RA-responsive promoters. The transcriptional synergy by TSA and RA required the RA-responsive element and a functional retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimer, both obligatory for RA-dependent transcription. Furthermore, TSA led to promoter activation by an RXR-selective ligand that was otherwise inactive in transcription. In addition, TSA enhanced transcription from a minimum basal promoter, independently of the RA-responsive element. Finally, we show that TSA alone or in combination with RA increases in vivo endonuclease sensitivity within the RA-responsive promoter, suggesting that TSA treatment might alter a local chromatin environment to enhance RXR/RAR heterodimer action. Thus, these results indicate that histone acetylation influences activity of the heterodimer, which is in line with the observed interaction between the RXR/RAR heterodimer and a histone acetylase presented elsewhere.

Topics: Animals; Apoptosis; Carcinoma, Embryonal; Cell Cycle; Cell Differentiation; Dimerization; Enzyme Inhibitors; Genes, Reporter; Histone Deacetylase Inhibitors; Hydroxamic Acids; Kinetics; Luciferases; Mice; Neurons; Promoter Regions, Genetic; Receptors, Retinoic Acid; Recombinant Proteins; Retinoid X Receptors; Time Factors; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

The role of RAR alpha 1 and RAR gamma 2 AF-1 and AF-2 activation functions and of their phosphorylation was investigated during RA-induced primitive and parietal differentiation of F9 cells. We found that: (i) primitive endodermal differentiation requires RAR gamma 2, whereas parietal endodermal differentiation requires both RAR gamma 2 and RAR alpha 1, and in all cases AF-1 and AF-2 must synergize; (ii) primitive endodermal differentiation requires the proline-directed kinase site of RAR gamma 2-AF-1, whereas parietal endodermal differentiation additionally requires that of RAR alpha 1-AF-1; (iii) the cAMP-induced parietal endodermal differentiation also requires the protein kinase A site of RAR alpha-AF-2, but not that of RAR gamma; and (iv) the AF-1-AF-2 synergism and AF-1 phosphorylation site requirements for RA-responsive gene induction are promoter context-dependent. Thus, AF-1 and AF-2 of distinct RARs exert specific cellular and molecular functions in a cell-autonomous system mimicking physiological situations, and their phosphorylation by kinases belonging to two main signalling pathways is required to enable RARs to transduce the RA signal during F9 cell differentiation.

Topics: Animals; Binding Sites; Carcinoma, Embryonal; Cell Differentiation; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Endoderm; Ligands; Mice; Phosphorylation; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Tretinoin; Tumor Cells, Cultured

Convertases of the subtilisin/kexin family are responsible for the biological activation of a variety of pro-proteins, pro-hormones, and pro-trophic factors, and thus can modulate various aspects of embryonic development. We investigated the expression of each convertase by Northern hybridization during cell differentiation in vitro, using the mouse embryonal carcinoma cell line P19 as a model. The neuroendocrine convertase PC2 and 7B2, its specific binding protein, are co-induced during neuronal differentiation of P19 cells with retinoic acid, whereas the other convertases are not or follow different patterns of temporal expression. The mature forms of PC2 and 7B2 proteins are detected together by immunoblotting following induction of mRNA expression, indicating that these proteins are processed early during brain development. These results demonstrate that PC2 and 7B2 gene expression and protein processing are in a close temporal association during neuronal differentiation and point to the value of the P19 cell model to study the significance and the regulation of this relationship in mammalian brain development.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Enzyme Induction; Furin; Gene Expression Regulation, Developmental; Lewis X Antigen; Mesoderm; Mice; Nerve Tissue Proteins; Neuroendocrine Secretory Protein 7B2; Neurofilament Proteins; Neurons; Pituitary Hormones; Proprotein Convertase 2; Proprotein Convertase 5; Proprotein Convertases; Protein Processing, Post-Translational; RNA, Messenger; Serine Endopeptidases; Subtilisins; Tretinoin; Tumor Cells, Cultured

A unique structure and in situ localization of E proteins were demonstrated in cultured neurons infected with neurovirulent and aneurovirulent strains of local Japanese encephalitis virus (JEV). Dilated rough endoplasmic reticulum (rER) containing smooth membrane structures (SMS) was continuous with the outer membrane of the nuclear envelope. These membranes were found to be connected to unique dense bodies, membrane vesicle structures (MVS). The de novo formation of SMS, annulate lamellae, and the appearance of MVS indicated proliferation of the membranous system in response to JEV infection. E proteins were possibly assembled in the virions in the nuclear envelope or rER or on the plasma membrane. The interconnections between MVS, rER, and the nuclear envelope and immunogold labeling of E proteins on the MVS provided strong evidence that MVS serve as a reservoir of JEV components during virus assembly.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Cells, Cultured; Encephalitis Virus, Japanese; Humans; Mice; Microscopy, Immunoelectron; Neuroblastoma; Neurons; Tretinoin; Tumor Cells, Cultured; Viral Envelope Proteins; Virus Replication

We have investigated the regulation of neurofilament gene expression during retinoic acid (RA)-induced neural differentiation of P19 embryonal carcinoma (EC) cells. Western blot analysis demonstrated that P19 EC cells contain significant levels of NF-L protein in the insoluble fraction but undetectable levels of NF-M and NF-H protein in either the insoluble or total cell fractions. However, immunocytochemical detection of NF-L protein in P19 EC cells showed diffuse staining within the majority of cells, rather than association with intermediate filament-like structures or staining within a subpopulation of differentiated neurons. Detectable levels of both NF-L and NF-M mRNA were present in P19 EC cells whereas NF-H mRNA remained below levels of detection, even by RT-PCR analysis. When RA-treated aggregates of P19 cells were cultured under conditions permissive for neurite outgrowth, we observed a significant increase in the amount of detectable NF-L protein localized within morphologically distinct neurons. Differentiation was also accompanied by the appearance of both the NF-M and NF-H subunits. Northern analysis revealed that this differentiation was accompanied by coincident increase in the steady-state levels of the mRNA for all three subunits and that the temporal pattern of increase was similar to what has been observed in the fetal and neonatal brain. The increase in NF-L and NF-M mRNA levels were accompanied by a concomitant increase in the rate of transcription, however, our results suggest that additional post-transcriptional mechanisms may be involved in regulating NF gene expression during the differentiation of pluripotent P19 cells.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Gene Expression Regulation, Neoplastic; Immunohistochemistry; Intermediate Filaments; Mice; Neurofilament Proteins; Neurons; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The P19 embryonal carcinoma cell line represents a pluripotential stem cell that can differentiate along the neural or muscle cell lineage when exposed to different environments. Exposure to retinoic acid induces P19 cells to differentiate into neurons and astrocytes that express similar developmental markers as their embryonic counterparts. We examined the expression of gap junction genes during differentiation of these stem cells into neurons and astrocytes. Untreated P19 cells express at least two gap junction proteins, connexins 26 and 43. Connexin32 could not be detected in these cells. Treatment for 96 hr with 0.3 mM retinoic acid induced the P19 cells to differentiate first into neurons followed by astrocytes. Retinoic acid produced a decrease in connexin43 mRNA, protein, and functional gap junctions. Connexin26 message was not affected by retinoic acid treatment. The neurons that developed consisted of small round cell bodies extending two to three neurites and expressed MAP2. Connexin26 was detected at sites of cell-cell and cell-neurite contact within 3 days following differentiation with retinoic acid. The astrocytes were examined for production of their intermediate filament marker, glial fibrillary acidic protein (GFAP). GFAP was first detected at 8 days by Western blotting. In culture, astrocytes co-expressed GFAP and connexin43 similar to primary cultures of mouse brain astrocytes. These results suggest that differentiation of neurons and glial cells involves specific connexin expression in each cell type. The P19 cell line will provide a valuable model with which to examine the role gap junctions play during differentiation events of developing neurons and astrocytes.

Topics: Animals; Astrocytes; Blotting, Western; Carcinoma, Embryonal; Cell Differentiation; Connexins; Gap Junctions; Keratolytic Agents; Mice; Microscopy, Confocal; Neurons; Tretinoin; Tumor Cells, Cultured

We have studied ganglioside alterations and their enzymatic basis during the course of neural differentiation of mouse embryonic carcinoma cell line P19. This cell line can differentiate into neurons and astrocytes on cell aggregation after treatment with retinoic acid (RA) or into muscle cells on dimethyl sulfoxide (DMSO) treatment. GD3, detected on immunostaining after thin-layer chromatography (TLC) with monoclonal antibody (MAb) R24, was markedly present in aggregates treated with RA. GM3 synthase (alpha 2,3-sialyltransferase, SAT-I) in neurons was found to exhibit the highest activity. GD3 synthase (alpha 2,8-sialyltransferase, SAT-II) and GD3 synthase mRNA, as analyzed by Northern blotting, were also markedly present in aggregates and neurons induced by RA. However, on treatment with DMSO, which induces muscle cells, there was no change in the level of GD3 synthase activity, and its transcript was hardly detected during the course of muscle differentiation. GT1b synthase (alpha 2,3-sialyltransferase, SAT-IV) was present at similar levels in undifferentiated cells and aggregates treated with RA, but a higher level was observed in neurons. On the other hand, the level of GQ1b synthase (alpha 2,8-sialyltransferase, SAT-V) in RA-induced aggregates was significantly higher than that in neurons. These results show that RA but not DMSO induces the expression of GM3, GD3, GT1b and GQ1b synthases, and particularly GD3 synthase mRNA, in the ganglioside biosynthetic pathway during the neural differentiation of embryonic carcinoma P19 cells.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Dimethyl Sulfoxide; Gangliosides; Gene Expression Regulation, Enzymologic; Mice; Muscle Development; Muscles; Nerve Tissue; Nerve Tissue Proteins; Neurons; RNA, Messenger; Sialyltransferases; Tretinoin; Tumor Cells, Cultured

P19 embryonic carcinoma (EC) stem cells are pluripotent and are efficiently induced to differentiate into neurons and glia with retinoic acid (RA) treatment. Within 5 days, a substantial number of differentiating P19 cells express gene products that are characteristic of a neuronal phenotype. P19 neurons were used as a model to explore the relationship between neuronal "differentiation" in vitro and the acquisition of gamma-aminobutyric acid (GABAA) receptors and functional GABA responses. Pulse-labeling experiments using bromodeoxyuridine indicated that all neurons had become postmitotic within 3-4 days after treatment with RA. This was confirmed by a reduction in the immunocytochemical detection of the undifferentiated stem cell antigen SSEA-1. Subsequently, a transient expression of nestin was observed during the first 5 days in vitro (DIV) after exposure to RA. By 5-10 DIV after RA, a significant number of neurons (approximately 80-90%) expressed immunocytochemically detectable glutamate decarboxylase and GABA coincident with the acquisition of membrane binding sites for tetanus toxin. These phenotypic markers were maintained for > 30 DIV after RA. Under current-clamp conditions, random, low-amplitude, spontaneous electrical activity appeared in neurons within the first few days after RA treatment and this was blocked by the specific GABAA receptor antagonist bicuculline. Thereafter, the appearance and progressive increases in the frequency of spontaneous action potentials in P19 neurons were observed that were similarly attenuated by bicuculline. In neurons > 5 DIV after RA, exogenous application of GABA elicited similar action potentials. The onset of excitatory responses to GABA or muscimol in voltage-clamped neurons appeared immediately after the cessation of neuronal mitosis and before the previously reported acquisition of responses to glutamate. In fura-2 imaging studies, the exogenous application of GABA resulted in neuron-specific increases in intracellular Ca2+. Thus, P19 neurons provide an in vitro model for the study of the early acquisition and properties of electrical excitability to GABA and the expression of functional GABAA receptors.

Topics: Action Potentials; Animals; Biomarkers; Calcium; Carcinoma, Embryonal; Cell Differentiation; gamma-Aminobutyric Acid; Mice; Neuroglia; Neurons; Receptors, GABA-A; Stem Cells; Tretinoin; Tumor Cells, Cultured

A number of different cell lines that exhibit a partial neuronal phenotype have been identified, but in many cases the full extent of their neuronal differentiation has not been directly addressed by functional studies. We have used electrophysiology and immunofluorescence to examine the formation of synapses and the development of neuronal polarity by murine embryonic stem (ES) cells and the mouse P19 embryonic carcinoma cell line. Within 2-3 weeks after induction by retinoic acid, subsets of P19 and ES cells formed excitatory synapses, mediated by glutamate receptors, or inhibitory synapses, mediated by receptors for GABA or glycine. In ES-cell cultures, both NMDA and non-NMDA receptors contributed to the excitatory postsynaptic response. Staining with antibodies to growth-associated protein-43 and microtubule-associated protein-2 revealed segregation of immunoreactivity into separate axonal and somato-dendritic compartments, respectively. Consistent with our physiological evidence for synapse formation, intense punctate staining was observed with antibodies to the synaptic vesicle proteins synapsin, SV2, and synaptophysin. These results demonstrate the in vitro acquisition by pluri-potent cell lines of neuronal polarity and functional synaptic transmission that is characteristic of CNS neurons.

Topics: Action Potentials; Animals; Carcinoma, Embryonal; Cell Differentiation; Cell Polarity; Cells, Cultured; Embryonal Carcinoma Stem Cells; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Mice; Neoplastic Stem Cells; Nerve Tissue Proteins; Neurites; Neurons; Receptors, GABA; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Stem Cells; Synapses; Synaptic Transmission; Tretinoin; Tumor Cells, Cultured

Meprins, metalloendopeptidases of the astacin family, are composed of alpha and/or beta subunits and are expressed at high levels in mammalian renal and intestinal brushborder membranes. Only one mRNA has been identified previously for each of the subunits in adult human and rodent tissues; a 3.6-kilobase message for the alpha subunit and a 2.5-kilobase message for the beta subunit. The present study reports that a larger beta subunit message (2.7 kilobases, referred to as beta'), and no alpha subunit message, is expressed in embryonal carcinoma cell lines, F9 and Nulli-SSC1, and in human colon adenocarcinoma cells, HT-28-18-C1. Furthermore, in Nulli-SSC1 cells, the beta isoform is induced by the morphogen retinoic acid. The beta' isoform differs from beta only in a portion of the 5'-coding (corresponding to the signal and prosequence domains of the protein) and noncoding region. Only one gene was found for the beta subunit in the mouse and human genome. The deduced amino acid sequence of beta' has no homology with beta in the first 35 NH2-terminal residues, but the two sequences are identical after that. In vitro translation experiments indicated that the size of the protein product of beta' cDNA was similar to that of the beta cDNA protein product, and, in the presence of microsomal membranes, both were glycosylated. These studies indicate that the messages for the meprin beta and beta' subunit result from differential promoter usage and alternate splicing. Expression of the two isoforms may be regulated differentially depending on cell type and/or differentiation state of the cell.

Topics: Alternative Splicing; Amino Acid Sequence; Animals; Base Sequence; Carcinoma, Embryonal; Colonic Neoplasms; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Genes; Humans; Isoenzymes; Membrane Proteins; Metalloendopeptidases; Mice; Molecular Sequence Data; RNA, Messenger; Sequence Alignment; Sequence Homology, Amino Acid; Solubility; Tretinoin; Tumor Cells, Cultured

Investigation of the molecular mechanisms that control smooth muscle cell (SMC) development and differentiation is a prerequisite in understanding the regulatory mechanisms of physiological and pathological SMC-associated vascular processes. The pluripotent murine embryonal carcinoma P19 cell, whose developmental potential resembles that of early embryonic cells, can develop into cell types derived from the neuroectoderm, mesoderm, and endoderm. In the present study, we have shown a unique strategy to enhance SMC differentiation in P19 cells. Under chemical induction of high concentrations of retinoic acid (1 micromol/L), P19 cells showed optimum differentiation into SMCs. Because the P19 cells thus induced also showed differentiation into neuronal cells, a strategy to block neuronal lineage differentiation was developed using a stable transformant antisense RNA construct against Brn-2, a neuronal lineage-specific POU-domain transcription factor; thus, by specifically inhibiting neuronal differentiation, enhanced SMC differentiation by P19 cells was attained. SMC expression was confirmed by immunohistochemical staining, RNA analysis (RNase protection assay), and protein analysis (Western blot) using SMC-specific markers (eg, SM1 and calponin) and alpha-smooth muscle actin. Our results show that the pathway of SMC differentiation may provide an in vitro system useful in the investigation of SMC regulatory mechanisms (eg, transcriptional regulation) and in the further understanding of SMC development and differentiation.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; Fluorescent Antibody Technique, Indirect; Gene Expression; Homeodomain Proteins; Mice; Molecular Sequence Data; Muscle, Smooth; POU Domain Factors; RNA, Antisense; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Testicular germ cell tumors are the most common form of cancer in young adult males. They result from a derangement of primordial germ cells, and they grow out from a noninvasive carcinoma-in-situ precursor. Since carcinoma in situ can readily be cured by low-dose irradiation, there is a great incentive for non- or minimally invasive methods for detection of carcinoma in situ. We have recently shown that human Tera-2 embryonal carcinoma cells, obtained from a nonseminomatous testicular germ cell tumor, show alternative splicing and alternative promoter use of the platelet-derived growth factor alpha-receptor gene, giving rise to a unique 1.5-kb transcript. In this study we have set up a reverse transcriptase-polymerase chain reaction strategy for characterization of the various transcripts for this receptor. Using this technique, we show that a panel of 18 seminomas and II nonseminomatous testicular germ cell tumors all express the 1.5-kb transcript. In addition, a panel of 27 samples of testis parenchyma with established carcinoma in situ were all found to be positive for the 1.5-kb transcript, while parenchyma lacking carcinoma in situ, placenta, and control semen were all negative. These data show that the 1.5-kb platelet-derived growth factor alpha-receptor transcript can be used as a highly selective marker for detection of early stages of human testicular germ cell tumors.

Topics: Adult; Alkaline Phosphatase; Base Sequence; Biomarkers, Tumor; Carcinoma, Embryonal; Choriocarcinoma; Clone Cells; DNA Primers; Gene Expression; Germinoma; Humans; Male; Molecular Sequence Data; Polymerase Chain Reaction; Receptor, Platelet-Derived Growth Factor alpha; Receptors, Platelet-Derived Growth Factor; Seminiferous Tubules; Seminoma; Teratoma; Testicular Neoplasms; Testis; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

NCR-G3 cells were established from a testicular embryonal carcinoma and are highly multipotential, differentiating into trophectoderm cells upon exposure to retinoic acid. Differentiated NCR-G3 cells begin to produce human chorionic gonadotropin (hCG), a trophectoderm-specific hormone. We have previously isolated the up-regulated genes at the early stage of differentiation. One of them was found to be a heat shock protein gene. The heat shock protein gene (HSP90) is induced at the early stage of differentiation and decreases to the basal level or under the basal level at the later stage. We speculate that heat shock per se induces the differentiation of human EC cells. With exposure to heat, NCR-G3 cells began to express a series of differentiation markers such as cytokeratin and hCG. Heat, which is classically known to induce heat shock proteins, is able to differentiate an embryonal cell line into trophectoderm lineages, implying a new recognized function of a heat-like event in early differentiation.

Topics: Carcinoma, Embryonal; Cell Differentiation; Chorionic Gonadotropin; Ectoderm; Gene Expression Regulation, Developmental; Heat-Shock Response; HSP90 Heat-Shock Proteins; Humans; Immunohistochemistry; Keratins; Male; Testicular Neoplasms; Tretinoin; Trophoblasts; Tumor Cells, Cultured

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

Patterning across the anteroposterior axis of the vertebrate limb bud involves a signal from the polarizing region, a small group of cells at the posterior margin of the bud. Retinoic acid (RA; Tickle, C., Alberts, B., Wolpert, L. and Lee, J. (1982) Nature 296, 554-566) and Sonic hedgehog (Shh; Riddle, R. D. Johnson, R. L., Laufer, E. and Tabin, C. J. (1993) Cell 25, 1401-1416; Chang, D. T., Lopez, A., von Kessler, D. P., Chiang, C., Simandl, B. K., Zhao, R., Seldin, M. F., Fallon, J. F. and Beachy, P. A. (1994 Development 120, 3339-3353) have been independently postulated as such signals because they can mimic the mirror image digit duplication obtained after grafting polarizing cells to the anterior of limb buds. Here we show that a embryonal carcinoma cell line, P19, transfected with a Shh expression vector shows low polarizing activity, but when cultured with retinoic acid, duplications like those induced by the polarizing region (ZPA) arise. Complete duplications are also obtained by cotransfecting P19 Shh cells with a constitutively active human retinoic acid receptor (VP16-hRARalpha). These data suggest that Shh and RA cooperate in generating ZPA activity and that Shh, while essential, may not act alone in this process.

Topics: Animals; Blotting, Western; Carcinoma, Embryonal; Cell Line; Cells, Cultured; Chick Embryo; Egg Proteins; Embryonic Induction; Hedgehog Proteins; Kinetics; Limb Buds; Membrane Glycoproteins; Mice; Protein Biosynthesis; Proteins; Receptors, Cell Surface; Recombinant Proteins; Signal Transduction; Time Factors; Trans-Activators; Transfection; Tretinoin; Tumor Cells, Cultured; Zona Pellucida Glycoproteins

We have examined the developmental expression of acetylcholinesterase (AChE) during the process of neuronal differentiation from a pluripotent stem cell. P19 embryonic carcinoma cells form embryoid bodies, which, when cultured with retinoic acid, are induced to differentiate into neurons and glia. No AChE activity is present in the undifferentiated stem cells, and mRNA protection analyses do not detect AChE mRNA. Commitment to a neuronal differentiation pathway results in increased levels of AChE mRNA, production of a tetrameric form of the enzyme, and secretion of AChE into the culture medium. Concomitant with subsequent morphological differentiation into neurons, enzyme secretion diminishes and AChE becomes largely tethered to the neuronal cell membranes. The enzyme is attached to the cell surface as a globular tetramer. Its hydrodynamic properties are consistent with association through a noncatalytic hydrophobic subunit rather than anchorage by a glycophospholipid tail. No change in the rate of transcription of the Ache gene was detected during the course of differentiation, suggesting that the gene is actively transcribed at very early stages of development. Results suggest that stabilization of a labile mRNA governs the increase in AChE mRNA and gene product. The studies presented indicate that an early event in neuronal differentiation is the stabilization of the mRNA leading to expression of a secreted form of AChE. A subsequent step associated with neurite outgrowth results in a transition from secretion of the tetrameric enzyme to its localization on the cell membrane.

Topics: Acetylcholinesterase; Animals; Carcinoma, Embryonal; Cell Differentiation; Cell Nucleus; Gene Expression Regulation, Enzymologic; Immunohistochemistry; Isoenzymes; Kinetics; Mice; Neurons; RNA, Messenger; Stem Cells; Time Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

COUP-TF family orphan receptors regulate activity of ligand-activated nuclear hormone receptors or function independently in the regulation of gene expression. COUP-TF II has a complex expression pattern suggesting that different mechanisms are involved in the regulation of its expression. We isolated the 5' regulatory region of the mouse COUP-TF II gene and demonstrated that the basal promoter is localized in a -200 bp region 5' from the transcription start site. All-trans retinoic acid and dibutyryl cyclic AMP have cell type specific effects on COUP-TF II promoter activity. The effect of cyclic AMP is mediated by the cyclic AMP response element that is localized 74 nucleotides upstream from the major transcriptional start. In vitro promoter analyses also demonstrated that the effect of all-trans RA is not directly mediated by the binding of RARs or RXRs to the promoter sequence.

Topics: Animals; Base Sequence; Bucladesine; Carcinoma, Embryonal; Chloramphenicol O-Acetyltransferase; COUP Transcription Factor I; COUP Transcription Factors; DNA Probes; DNA-Binding Proteins; DNA, Complementary; Gene Expression Regulation; Genomic Library; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Promoter Regions, Genetic; Receptors, Cell Surface; Receptors, Steroid; Recombinant Proteins; Regulatory Sequences, Nucleic Acid; Restriction Mapping; Transcription Factors; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured

The expression of the gamma 2 subunit into functional GABAA receptors has been examined in the embryonal carcinoma (EC) cell line P19, a pluripotent cell line which differentiates into a neuronal phenotype after exposure to retinoic acid. Whole-cell voltage-clamp recordings were used to examine the characteristics of the GABA receptors expressed in P19 cells at different times after exposure to retinoic acid. Messenger RNA for both the gamma 2L and gamma 2S splice variants of the GABAA receptor increased dramatically following differentiation of P19 EC cells with retinoic acid. By 12 days after retinoic acid treatment, while both mRNAs were present, there was an approximately 10-fold greater abundance of gamma 2S mRNA compared to gamma 2L. However, at this same time point neurons derived from P19 cells stained intensely with a polyclonal antibody raised against a peptide fragment specific for the gamma 2L subunit. A significant increase in both the affinity for GABA and the maximum current amplitude elicited by GABA occurred between 7 and 12 days after retinoic acid treatment. In contrast, the ability of the benzodiazepine agonist flurazepam to potentiate GABA-induced membrane current was the same at 7 and 12 days after retinoic acid treatment. These data suggest that the gamma 2 subunit of the GABAA receptor is expressed early following differentation of P19 cells into a neuronal phenotype, and that this subunit is incorporated into functional GABAA receptors. Moreover, the gamma 2S and gamma 2L splice variants of this subunit may be co-expressed in neurons derived from P19 cells. The observed affinity change for GABA may reflect a time-dependent change in the expression of alpha and/or beta subunits of the GABAA receptor, as occurs in developing neuronal tissue both in vitro and in vivo.

Topics: Amino Acid Sequence; Animals; Antibodies; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; Cell Line; DNA Primers; Flurazepam; gamma-Aminobutyric Acid; Gene Expression; Kinetics; Macromolecular Substances; Membrane Potentials; Mice; Molecular Sequence Data; Neurons; Patch-Clamp Techniques; Peptide Fragments; Polymerase Chain Reaction; Receptors, GABA-A; RNA, Messenger; Stem Cells; Time Factors; Tretinoin; Tumor Cells, Cultured

An in vitro model of apoptosis and differentiation in human embryonal carcinoma (EC) cells was developed to study the mode of cell death and mechanisms of cell death in early development. Death of these cells was induced by treatment with retinoic acid (RA) under the same conditions as those for induction of differentiation. The manner of this cell death was apoptosis, not necrosis, with the morphological criterion for apoptosis. serum deprivation likewise caused apoptosis in both undifferentiated and differentiated EC cells. In differentiated EC cells, DNA fragmentation was observed in a smear pattern lacking the ladder pattern typically associated with apoptosis. However, in differentiated EC cells, DNA fragmentation occurred in various sizes. The expression of a carbohydrate antigen, LeY, a reported marker of apoptotic cancer cells, was increased by the treatment with RA. However, two-color analysis by flow cytometry with nick end labelling method revealed that LeY expression was closely correlated with cellular differentiation but not apoptosis after RA treatment in the human EC cell system. Collection of LeY positive cells by the magnetic bead method demonstrated that this expression was not due to apoptosis but rather to differentiation. On the other hand, LeY expression associated with apoptosis was induced by serum deprivation in both undifferentiated and differentiated EC cells. These results show that a subpopulation of undifferentiated EC cells takes the apoptotic pathway by induction of differentiation. The results also suggest that the population of cells taking an apoptotic pathway differs from a population of cells taking a differentiation pathway. This in vitro system for apoptosis in human EC cells will be useful for studies concerning apoptosis or programmed cell death in human developmental biology.

Topics: Apoptosis; Carcinoma, Embryonal; Cell Differentiation; Cell Separation; Cycloheximide; DNA Damage; Humans; Lewis Blood Group Antigens; Male; Protein Synthesis Inhibitors; Tretinoin; Tumor Cells, Cultured

Topics: Alkaline Phosphatase; Animals; Carcinoma, Embryonal; Cell Differentiation; Cell Division; Cell Line; Collagen; Culture Media; Extracellular Matrix; Fibronectins; Gelatin; Laminin; Mice; Tretinoin; Tumor Cells, Cultured

The retinoids exert potent growth and differentiation effects on normal and neoplastic cells through two families of nuclear receptors. These are the retinoic acid receptors (RAR alpha, RAR beta, RAR gamma) and the retinoid-X receptors (RXR alpha, RXR beta, RXR gamma). All-trans retinoic acid (RA) induces terminal neuronal differentiation and represses tumorigenicity of the multipotent human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1). Hexamethylene bisacetamide (HMBA) induces a phenotype distinct from RA-treated NT2/D1 cells. This study reports the derivation and characterization of RA- and HMBA-resistant NT2/D1 clones. Nine RA-resistant (NT2/D1-R1 through NT2/D1-R9) and one HMBA-resistant (NT2/D1-H1) clones were derived after mutagen treatment of NT2/D1 cells and selection in RA or HMBA. NT2/D1-R cells were cross-resistant to 9-cis retinoic acid (9-cis RA), a ligand activating the RAR and RXR pathways, but retained maturation response to HMBA. A representative RA-resistant clone, NT2/D1-R1, overcame the antitumorigenic actions of RA as assessed in athymic mice. NT2/D1-H1 cells were dually resistant to RA and 9-cis RA. All these retinoid resistant cells exhibit deregulated expression of RAR gamma but not RAR alpha or RAR beta. Southern analysis using RAR gamma probes shows no apparent structural differences in genomic DNA between NT2/D1 cells and the RA-resistant subclones. Pulsed-field gel electrophoresis (PFGE) with RAR gamma probes demonstrated an Mlu-I restriction fragment length polymorphism, but no other structural abnormalities in these cells or a panel of germ cell tumor (GCT) cell lines. Full-length RAR gamma 1 coding region cDNAs were cloned from NT2/D1 and NT2/D1-R1 cells and these sequences were identical, suggesting RA resistance in these cells is due to altered regulation of RAR gamma. These differentiation-resistant cells are useful to study RAR gamma target genes or mechanisms engaged by these differentiation inducing agents in human embryonal carcinomas.

Topics: Acetamides; Animals; Antineoplastic Agents; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; Clone Cells; Drug Resistance, Neoplasm; Germinoma; Humans; Immunophenotyping; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Transplantation; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

Retinoic acid induced the differentiation of human embryonal carcinoma cells (NT2/D1) into several morphologically distinct cell types, including those resembling terminally differentiated postmitotic CNS neurones. The mechanism by which retinoic acid influences the process of neuronal differentiation in the CNS, however, remains unknown. In the present study, we have examined the ability of retinoic acid to induce the expression of the receptors that mediate the actions of the neurotrophins, using reverse transcription-polymerase chain reaction and Northern blot analysis. Our study demonstrated that the expression of mRNAs for three human trk receptors was significantly induced after treatment with retinoic acid. These findings suggest that the actions of retinoic acid on neuronal differentiation in the human CNS may potentially be mediated by the neurotrophins.

Topics: Antineoplastic Agents; Carcinoma, Embryonal; Cell Differentiation; Enzyme Induction; Humans; Immunoblotting; Nerve Growth Factors; Polymerase Chain Reaction; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Receptor, Ciliary Neurotrophic Factor; Receptor, trkA; Receptor, trkC; Receptors, Nerve Growth Factor; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) plays a critical role in normal development, growth and differentiation by modulating the expression of target genes. Using substractive hybridization cloning, we isolated two cDNAs, whose corresponding mRNAs are repressed upon RA treatment of P19 embryonal carcinoma (EC) cells. The cDNAs correspond to the serine hydroxymethyltransferase (shmt) gene and the early transposon, ETnMG1. RA appears to reduce the stability of ETnMG1 transcript. We also report the sequence of two different isoforms of mouse SHMT. Since SHMT activity is increased when cells are stimulated to proliferate and during the S phase of the cell cycle, we suggest that repression of shmt expression is an important step in RA-induced cell growth arrest and differentiation.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carcinoma, Embryonal; Cloning, Molecular; DNA, Complementary; Female; Gene Expression Regulation, Neoplastic; Male; Methyltransferases; Mice; Molecular Sequence Data; Neoplasms, Experimental; Tretinoin; Tumor Cells, Cultured

The product of the Wilms tumor suppressor gene, WT1, is thought to be a tissue specific transcription factor regulating cell growth and differentiation. To elucidate the function of WT1 in cellular differentiation, we examined the changes in the level of WT1 expression during retinoic acid induced-differentiation of embryonal carcinoma F9 cells into parietal endoderm cells. We found that, in response to retinoic acid addition, the expression of WT1 increased significantly after 12--24 h of incubation, then decreased and finally disappeared after 4 days, by which time most of the cells had differentiated into primitive endoderm cells. To examine the significance of these changes in WT1 expression, we established cell lines constitutively expressing one of the WT1 splicing variants. These cell lines showed a phenotype very similar to parental F9 cells in the absence of retinoic acid. However, in the presence of retinoic acid, they failed to differentiate into primitive endoderm cells and underwent apoptotic death 36 h after the addition of retinoic acid. These results suggest that downregulation of WT1 expression is necessary for normal differentiation of F9 cells into parietal endoderm cells.

Topics: 3T3 Cells; Animals; Apoptosis; Carcinoma, Embryonal; Cell Differentiation; Cell Line; DNA; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Mice; Transcription Factors; Transfection; Tretinoin; WT1 Proteins

Interferon regulatory factors (IRFs) bind to the interferon-stimulated response element (ISRE) and regulate interferon- and virus-mediated gene expression. IRF-1 acts as a transcriptional activator, while IRF-2 acts as a repressor. Here we show that IRF-1 and IRF-2 bind to both cellular TFIIB, a component of the basal transcription machinery, and recombinant TFIIB (rTFIIB) and that this protein-protein interaction facilitates binding of IRFs to the ISRE. A functional interaction between TFIIB and IRF was assessed by a newly established in vitro transcription assay in which recombinant IRF-1 (rIRF-1) stimulated transcription specifically from an ISRE-containing template. With this assay we show that rIRF-1 and rTFIIB cooperatively enhance the ISRE promoter in vitro. We found that the activity of an ISRE-containing promoter was cooperatively enhanced upon cotransfection of TFIIB and IRF-1 cDNAs into P19 embryonal carcinoma cells, further demonstrating functional interactions in vivo. The cooperative enhancement by TFIIB and IRF-1 was independent of the TATA sequence in the ISRE promoter but dependent on the initiator sequence (Inr) and was abolished when P19 cells were induced to differentiate by retinoic acid treatment. In contrast, cotransfection of TFIIB and IRF-1 into NIH 3T3 cells resulted in a dose-dependent repression of promoter activation which occurred in a TATA-dependent manner. Our results indicate the presence of a cell type-specific factor that mediates the functional interaction between IRFs and TFIIB and that acts in conjunction with the requirement of TATA and Inr for promoter activation.

Topics: 3T3 Cells; Animals; Base Sequence; Binding Sites; Carcinoma, Embryonal; Cell Differentiation; Cell Line; DNA-Binding Proteins; Gene Expression Regulation; Gene Expression Regulation, Viral; Genes, Reporter; Humans; Interferon Regulatory Factor-1; Interferon Regulatory Factor-2; Kinetics; Luciferases; Mice; Models, Biological; Phosphoproteins; Promoter Regions, Genetic; Recombinant Proteins; Repressor Proteins; TATA Box; Trans-Activators; Transcription Factor TFIIB; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

P19 mouse embryonal carcinoma cells differentiate into neurons and glial cells when treated with retinoic acid. In contrast, a subline of the P19 cells, RAC65, is known to show little sign of differentiation with the treatment. We treated the two embryonal carcinoma (EC) cell lines with 9-cis-retinoic acid and investigated its neuron-inducing activity. In P19 cells, 9-cis-retinoic acid showed an activity equal to that of all-trans-retinoic acid. However in RAC65 cells, 9-cis-retinoic acid induced neurons 10-fold more effectively than all-trans-retinoic acid. The order in which various retinoids appeared in P19 cells corresponded to that of retinoic acid receptors, and the order in RAC65 cells to that of retinoid X receptors (RXRs). Furthermore we found that the down-regulation of retinoid X receptor-gamma mRNA expression was associated with neuronal differentiation in both embryonal carcinoma cell lines. In addition, a synthetic RXR-selective retinoid induced neurons from both EC cells. Our findings support an intriguing possibility that the 9-cis-retinoic acid/retinoid X receptor system may play an important role in neural differentiation.

Topics: Animals; Bexarotene; Carcinoma, Embryonal; Cell Differentiation; Dose-Response Relationship, Drug; Down-Regulation; Mice; Neurons; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; RNA, Messenger; Stereoisomerism; Tetrahydronaphthalenes; Transcription Factors; Tretinoin; Tumor Cells, Cultured

The full-length cDNA corresponding to Stra8, a novel gene inducible by retinoic acid (RA) in P19 embryonal carcinoma cells, has been isolated and shown to encode a 45-kD protein. Both Stra8 mRNA and protein were induced in cells treated by all-trans and 9-cis retinoic acids. Two-dimensional gel analysis and dephosphorylation experiments revealed that the two stereoisomers of RA differentially regulate the phosphorylation status of the Stra8 protein, which was shown to exist in differently phosphorylated forms. Subcellular fractionation and immunocytochemistry studies showed that the Stra8 protein is cytoplasmic. During mouse embryogenesis, Stra8 expression was restricted to the male developing gonads, and in adult mice, the expression of Stra8 was restricted to the premeiotic germ cells. Thus, Stra8 protein may play a role in the premeiotic phase of spermatogenesis.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Animals; Animals, Newborn; Base Sequence; Carcinoma, Embryonal; Cell Line; Cytoplasm; DNA, Complementary; Gene Expression Regulation, Neoplastic; Gene Library; In Situ Hybridization; Male; Meiosis; Mice; Molecular Sequence Data; Organ Specificity; Protein Biosynthesis; Proteins; RNA, Messenger; Spermatogenesis; Stem Cells; Testis; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) affects the response of many cells to growth factors, including the bone morphogenetic proteins (BMPs). The BMPs are members of the TGF-beta, family of growth factors, originally identified by their bone-inducing activities. Their widespread expression suggests many roles other than that in osteogenesis. Because RA modulates the cell's response to growth factors, this may be a means by which the retinoids exert some of their known teratogenic effects. One such cellular response may be apoptosis. While apoptosis is required for normal development, the location and timing of its induction must be carefully controlled. Recently, several TGF-beta family members have been implicated in the induction of apoptosis in certain cell types. We show here, using P19 embryonal carcinoma cells, that the combination of RA and BMP2 or BMP4 synergistically induces apoptosis in 40% of the population within 24 hr. In contrast, RA alone induces apoptosis in only 10-15% of the population and each of the BMPs alone minimally induces apoptosis. Apoptosis depends on the dose of both the RA and the BMP as well as on new protein synthesis. Further, the induction of apoptosis prevents the formation of fully differentiated neurons and glial cells and instead leads to primarily smooth muscle cell differentiation. These results suggest that some of the malformations caused by retinoids may be due to the induction of inappropriate apoptosis in cells exposed to BMPs.

Topics: Apoptosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Carcinoma, Embryonal; Drug Synergism; Flow Cytometry; Keratolytic Agents; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

We examined changes in proteinase activities in P19 embryonal carcinoma cells during retinoic acid-induced differentiation. The interleukin-1 beta converting enzyme (ICE)-like Ac-YVAD-MCA hydrolytic activity was increased about 6-fold by treatment with retinoic acid. This activity was inhibited by N-ethylmaleimide and Ac-YVAD-H but not by E-64, EDTA, PMSF, or amastatin. The ICE-like activity in P19 cells eluted as a single peak just after the void volume on gel filtration. No ICE-like activity was observed at a molecular mass of 30-50 kDa. Enzymatic purification, Western blot analysis, and an immunoabsorption study demonstrated that the ICE-like activity in P19 cells is caused by the proteasome, and is stimulated during retinoic acid-induced differentiation. The proteasome purified from mouse liver also cleaved Ac-YVAD-MCA. These results strongly suggest that the proteasome is a major ICE-like proteinase in P19 cells and may be involved in the neural differentiation and the apoptotic pathway.

Topics: Animals; Apoptosis; Carcinoma, Embryonal; Caspase 1; Cell Differentiation; Coumarins; Cysteine Endopeptidases; Edetic Acid; Ethylmaleimide; Liver; Mice; Multienzyme Complexes; Oligopeptides; Proteasome Endopeptidase Complex; Substrate Specificity; Tretinoin; Tumor Cells, Cultured

Topics: Actins; Animals; Apoptosis; Carcinoma, Embryonal; Cell Differentiation; Cytoskeletal Proteins; Mice; Microscopy, Electron; Tretinoin; Tumor Cells, Cultured

Mouse P19 embryonal carcinoma cells can be reproducibly differentiated into neurons and glial cells upon treatment with high concentrations of retinoic acid (RA). To understand the molecular mechanisms that control early neural differentiation, we constructed P19 cell lines carrying an insertion of a gene-trap vector containing lacZ as the reporter gene and a G418 resistance gene. We tested expression of the lacZ gene during the RA-induced differentiation process of 300 clones selected with G418. Ten of these clones were stained with X-gal, and five of these ten clones showed up- or down-regulation of lacZ expression. We analyzed one clone, GT1, in which expression of the lacZ gene was markedly up-regulated. The 5'-flanking genomic DNA of the GT1 gene present at the site of integration was isolated by the plasmid rescue method, and we screened a cDNA library using this DNA gene as a probe. The GT1 cDNA is about 9000 bp long, with an open reading frame encoding 1840 amino acids. This amino acid sequence has a potential glycosaminoglycan attachment site (Ser-Gly-Gly-Gly) and three N-linked glycosylation sites, but no signal peptide. The sequence of GT1 does not show significant homology with any other known proteins, suggesting that GT1 may be a novel proteoglycan core protein. In situ hybridization revealed that GT1 mRNA was expressed ubiquitiously in the adult mouse brain. This expression was specifically localized in neurons but not in glial cells. Immunohistochemistry revealed that GT1 protein was also localized in neurons. These results suggest that this protein may play a fundamental role in neurons.

Topics: Amino Acid Sequence; Animals; Base Sequence; Brain Neoplasms; Carcinoma, Embryonal; Cell Differentiation; Cloning, Molecular; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Mice; Mice, Inbred ICR; Molecular Sequence Data; Neurons; Tretinoin; Tumor Cells, Cultured

Cellular retinoic acid-binding protein-I (CRABP-I) gene expression is induced in mouse embryonal carcinoma P19 cells specifically by retinoic acid (RA) and the induction is enhanced by sphinganine. The effects of retinoic acid and sphinganine on CRABP-I gene expression can be accounted for by a stimulation of its transcription rate. Using a lacZ reporter system, it was determined that a DNA fragment containing a putative AP-1 binding site in the promoter region of CRABP-I gene is required for the up-regulation of CRABP-I gene transcription.

Topics: Animals; beta-Galactosidase; Carcinoma, Embryonal; DNA; Gene Expression; Gene Transfer Techniques; Mice; Promoter Regions, Genetic; Receptors, Retinoic Acid; Sphingosine; Transcription Factor AP-1; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Midkine is a growth/differentiation factor which is found as a product of a retinoic acid-responsive gene. The 2.3-kb upstream sequence of the human MK gene has cis acting elements which confer retinoic acid-induced expression of fused chloramphenicol acetyl-transferase (CAT) gene in F9 embryonal carcinoma cells. In the 5'-region of the human gene, a sequence resembling the DR5-type retinoic acid-responsive element (AGGTCA-related direct repeats separated by 5 nucleotides) was present in a small block of highly homologous 5'-sequences shared by the human and mouse genes. Deletion of this direct repeat reduced retinoic acid-induced CAT gene expression. The core element in the human gene differs from the consensus sequence of retinoic acid-responsive element in two nucleotides and from the retinoic acid responsive element of the mouse MK gene in one nucleotide.

Topics: Animals; Base Sequence; Carcinoma, Embryonal; Carrier Proteins; Chloramphenicol O-Acetyltransferase; Cytokines; DNA; Gene Expression; Humans; Mice; Midkine; Molecular Sequence Data; Placenta; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; Sequence Homology, Nucleic Acid; Transfection; Tretinoin

Disruption of retinoic acid receptor (RAR) gamma in F9 embryonal carcinoma cells leads to aberrent differentiation and reduced activation of expression of several all-trans-retinoic acid (RA)-induced genes. We have analyzed the expression of several additional RA-responsive genes in RAR alpha- and RAR gamma-null F9 cells. The RA-induced activation of Cdx1, Gap43, Stra4, and Stra6 was specifically impaired in RAR gamma-null cells, supporting the idea that each RAR may regulate distinct subsets of target genes. To further investigate the role of RAR gamma in F9 cell differentiation, "rescue" cell lines reexpressing RAR gamma 2 or overexpressing either RAR alpha 1 or RAR beta 2 were established in RAR gamma-null cells. Reexpression of RAR gamma or overexpression of RAR alpha restored both target-gene activation and the differentiation potential. In contrast, over-expression of RAR beta only poorly restored differentiation, although it could replace RAR gamma for the activation of target genes. Functional redundancy between the various RARs is discussed.

Topics: Animals; Antibodies, Monoclonal; Avian Proteins; Blotting, Northern; Carcinoma, Embryonal; Cell Line; DNA-Binding Proteins; GAP-43 Protein; Gene Expression; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Homeodomain Proteins; Membrane Glycoproteins; Mice; Nerve Tissue Proteins; Phosphoproteins; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Transcription, Genetic; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Embryonal carcinoma (EC) cells and embryonic stem (ES) cells provide useful model systems for studying differentiation during early mammalian development. Previous studies have demonstrated that differentiation of two restricted mouse EC cell lines is accompanied by activation of the TGF-beta 2 gene. Moreover, one negative and two positive regulatory regions upstream of the transcription start site were identified, which appear to play key roles in the transcriptional regulation of the human TGF-beta 2 gene. In this report, we demonstrate that the same three regulatory regions strongly influence the activity of the TGF-beta 2 promoter in differentiated cells derived from the multipotent human EC cell line, NT2/D1, and from the murine totipotent ES cell line, CCE. We also determined that the same three regions are active in the regulation of the TGF-beta 2 gene in the murine parietal endoderm-like cell line, PYS-2. However, an additional negative regulatory region appears to contribute to the regulation of the TGF-beta 2 gene in PYS-2 cells. Last, mutation of a CRE/ATF element located just upstream of the transcription start site of the TGF-beta 2 gene reduces significantly the activity of the TGF-beta 2 promoter in the differentiated cells. However, in contrast to our previous findings, our gel mobility shift analyses demonstrate that this CRE/ATF element is bound by similar proteins in nuclear extracts prepared from undifferentiated and differentiated mouse EC cells as well as from undifferentiated human EC cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; Gene Expression Regulation, Developmental; Genes, Regulator; Genes, Reporter; Humans; Mice; Molecular Sequence Data; Point Mutation; Promoter Regions, Genetic; Stem Cells; Transcription Factors; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

We have found that the gene expression of the ninth member of the fibroblast growth factor (FGF) family, FGF9 was induced during retinoic acid(RA)-induced neuronal differentiation of murine embryonal carcinoma P19 cells. We have reported here the nucleotide sequence of the mouse FGF9 cDNA. The murine cDNA showed 92.4% nucleotide sequence homology to the human FGF9 cDNA and 98.2% homology to that of rats. This mouse FGF9 cDNA encoded a polypeptide consisting of 208 amino acids with amino acid sequence identical to that of rats. Only one amino acid was replaced compared to the human homolog. The highly conserved sequence homology of FGF9 suggests its functional importance. FGF9 was originally isolated from a culture medium of a human glioma cell line as a growth-promoting factor for glial cells [5]. Upon induction of neuronal differentiation by forming cell aggregates with 10(-6) M RA, the gene expression of FGF9 was increased biphasically during the first 96 hours when cells were aggregating and from 168 hours to 192 hours followed by plating onto a tissue culture dish as glia-like cells proliferated. Neither undifferentiated P19 cells nor the cells aggregated without RA remaining undifferentiated expressed FGF9. This indicates that RA regulates the gene expression of FGF9 that may play an important role in neuronal differentiation in both early and late developmental process.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carcinoma, Embryonal; Cattle; Cell Differentiation; DNA, Complementary; Fibroblast Growth Factors; Gene Expression Regulation, Developmental; Humans; Mice; Molecular Sequence Data; Neurons; Rats; RNA, Messenger; Sequence Analysis, DNA; Tretinoin; Tumor Cells, Cultured

Retinoic acid-induced differentiation of mouse F9 embryonal carcinoma cells into primitive endoderm is accompanied by increased transcription of the gene for J6, a heat shock protein implicated in collagen biosynthesis. In this paper we present evidence that transcription factor GATA-4, a retinoic acid-inducible GATA-binding protein, is involved in the regulation of J6 gene expression in F9 cells. Northern-blot analysis indicates that transcripts encoding GATA-4 and J6 increase in parallel during retinoic acid-induced differentiation of F9 cells. Gel-shift experiments and antibody binding studies demonstrate that: (1) GATA-4 is the major GATA-binding protein activity in differentiated F9 cells, and (2) GATA-4 binds to consensus GATA motifs in the retinoic acid-responsive portion of the J6 promoter. Co-transfection studies using NIH 3T3 cells show that GATA-4 is a potent trans-activator of the J6 promoter. These lines of evidence suggest that expression of J6 in F9 cells is regulated by GATA-4. We speculate that transcription factor GATA-4 may also control other genes involved in extracellular matrix formation in the yolk sac.

Topics: 3T3 Cells; Animals; Base Sequence; Bucladesine; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Transformed; Chlorocebus aethiops; DNA-Binding Proteins; Endoderm; GATA4 Transcription Factor; Gene Expression Regulation; Glycoproteins; HSP47 Heat-Shock Proteins; Isoantibodies; Mice; Molecular Sequence Data; Neoplasm Proteins; Promoter Regions, Genetic; Recombinant Proteins; Serpins; Transcription Factors; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

Despite intense interest in understanding the differentiation of vascular smooth muscle, very little is known about the cellular and molecular mechanisms that control differentiation of this cell type. Progress in this field has been hampered by the lack of an inducible in vitro system for study of the early steps of smooth muscle differentiation. In this study, we describe a model system in which multipotential mouse P19 embryonal carcinoma cells (P19s) can be induced to express multiple characteristics of differentiated smooth muscle. Treatment of P19s with retinoic acid was associated with profound changes in cell morphology and with the appearance at high frequency of smooth muscle alpha-actin-positive cells that were absent or present at extremely low frequency in parental P19s. A clonal line derived from retinoic acid-treated P19s (9E11G) stably expressed multiple characteristics of differentiated smooth muscle, including smooth muscle-specific isoforms of alpha-actin and myosin heavy chain, as well as functional responses to the contractile agonists phenylephrine, angiotensin II, ATP, bradykinin, histamine, platelet-derived growth factor (PDGF)-AA, and PDGF-BB. Additionally, 9E11G cells expressed transcripts for MHox, a muscle homeobox gene expressed in smooth, cardiac, and skeletal muscles, but not the skeletal muscle-specific regulatory factors, MyoD and myogenin. Results demonstrate that retinoic acid treatment of multipotential P19 cells is associated with formation of cell lines that stably express multiple properties of differentiated smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Actins; Animals; Biomarkers; Carcinoma, Embryonal; Cell Differentiation; Embryonal Carcinoma Stem Cells; Homeodomain Proteins; Mice; Muscle, Smooth; Myosins; Neoplastic Stem Cells; Tretinoin; Tumor Cells, Cultured

The retinoblastoma (RB) protein is present at low levels in early mouse embryos and in pluripotent P19 embryonal carcinoma cells; however, the levels of RB rise dramatically in neuroectoderm formed both in embryos and in differentiating cultures of P19 cells. To investigate the effect of inactivating RB and related proteins p107 and p130, we transfected P19 cells with genes encoding mutated versions of the adenovirus E1A protein that bind RB and related proteins. When these E1A-expressing P19 cells were induced to differentiate into neuroectoderm, there was a striking rise in the expression of c-fos and extensive cell death. The ultrastructural and biochemical characteristics of the dying cells were indicative of apoptosis. The dying cells were those committed to the neural lineages because neurons and astrocytes were lost from differentiating cultures. Cell death was dependent on the ability of the E1A protein to bind RB and related proteins. Our results suggest that proteins of the RB family are essential for the development of the neural lineages and that the absence of functional RB activity triggers apoptosis of differentiating neuroectodermal cells.

Topics: Adenovirus E1A Proteins; Animals; Apoptosis; Astrocytes; Carcinoma, Embryonal; Cell Differentiation; DNA Damage; Ectoderm; Gene Expression Regulation; Genes, Viral; Mice; Muscles; Nervous System; Neurons; Nuclear Proteins; Phosphoproteins; Protein Binding; Proteins; Proto-Oncogene Proteins c-fos; Retinoblastoma Protein; Retinoblastoma-Like Protein p107; Retinoblastoma-Like Protein p130; Sequence Deletion; Stem Cells; Tretinoin; Tumor Cells, Cultured

We examined the regulation of the Na+/H+ exchanger gene during differentiation of the P19 mouse embryonal carcinoma cells. Treatment of P19 cells with retinoic acid induces the development of neurons, astroglia, and microglia cells. Upon retinoic acid-induced differentiation of P19 cells, there was an early and rapid 10-fold increase in NHE1 transcription. A proximal cis-acting AP-2 site of the NHE1 promoter was sufficient for stimulation of transcription of the gene by differentiation. Bandshift experiments demonstrated that in retinoic acid-treated cells there was an elevated level of AP-2 transcription factor binding to the AP-2 consensus site of the Na+/H+ exchanger gene. In the differentiation defective mutant RAC65, the effect of differentiation on Na+/H+ exchanger gene expression was reduced by 60%. Examination of Na+/H+ exchanger activity showed that retinoic acid-treated P19 cells recovered from an acid load at a rate approximately three times greater than untreated cells. The increases in gene expression and protein activity preceded major changes in cell morphology, suggesting that the initiation of differentiation is linked to NHE1 gene expression. Our findings show for the first time that the NHE1 gene is activated early in cell differentiation and that this activation may play an important role in the process of neuronal cell differentiation.

Topics: Animals; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; DNA Primers; DNA-Binding Proteins; Gene Expression Regulation, Developmental; Mice; Molecular Sequence Data; Nervous System; Regulatory Sequences, Nucleic Acid; Sodium-Hydrogen Exchangers; Transcription Factor AP-2; Transcription Factors; Tretinoin; Tubulin; Tumor Cells, Cultured

The murine Pgk-1 gene is driven by a strong promoter that is regulated by a 304 bp upstream activator sequence (UAS). The activity of the UAS is high in undifferentiated embryonal carcinoma cells but declines when these cells are induced to differentiate with retinoic acid. The effect of the UAS on promoter activity is particularly striking when the activity of the Pgk-1 promoter is assayed following its integration into the genome, suggesting that it may function by regulating chromatin structure in the region of the core promoter. Three sites on the UAS bind nuclear proteins. Two of these sites bind factors present in both embryonal carcinoma cells and their differentiated derivatives whereas one site binds factors present only in differentiated cells. There appears to be both cooperation and antagonism in the binding of proteins to different sites in the UAS, suggesting that the activity of the Pgk-1 promoter is determined by the constellation of proteins assembled upstream of its transcription start site.

Topics: Animals; Base Sequence; beta-Galactosidase; Carcinoma, Embryonal; Cell Differentiation; Chromatin; Chromosome Mapping; DNA-Binding Proteins; Gene Expression Regulation; Mice; Molecular Sequence Data; Mutagenesis; Nuclear Proteins; Phosphoglycerate Kinase; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; Sequence Analysis, DNA; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Endothelins (ETs) were initially characterized as potent vasoactive peptides acting through at least two distinct receptors, ETA and ETB. Subsequently, their significant growth- and hypertrophy-promoting properties in cardiac and other cells were recognized. We investigated the expression of endothelin receptors during differentiation of a pluripotential embryonal carcinoma cell line (P19) to a cardiomyocyte or a neural lineage. These cells resemble those of the inner cell mass of the blastocyst, and their differentiation is believed to closely mimic critical events in early embryogenesis. Differentiation of P19 to a cardiomyocyte lineage, by aggregation and exposure to dimethyl sulfoxide resulted in induction of ETA receptors as demonstrated by radioligand binding studies, Northern blotting, and reporter gene analysis. Moreover, the P19 differentiated to a cardiac lineage responded to ET-1 with a 3-fold increase in the secretion of atrial natriuretic peptide. In contrast, differentiation to a neural lineage, by aggregation and exposure to retinoic acid, was associated with the induction of predominantly ETB. Therefore, selective differentiation of the P19 led to the differential expression of endothelin receptors in a pattern consistent with that observed in normal myocardial and neural tissue. The induction of endothelin receptors in a model system of early embryogenesis provides strong support for the critical role of this peptide/receptor family in differentiation and development. As well, this model system is well suited for the study of the mechanisms controlling endothelin receptor expression during differentiation.

Topics: Animals; Base Sequence; Blastocyst; Blotting, Northern; Carcinoma, Embryonal; Cell Aggregation; Cell Differentiation; Cell Division; Cell Line; Dimethyl Sulfoxide; DNA Primers; Endothelins; Gene Expression; Humans; Kinetics; Luciferases; Mice; Molecular Sequence Data; Myocardium; Neurons; Polymerase Chain Reaction; Promoter Regions, Genetic; Radioligand Assay; Rats; Receptors, Endothelin; RNA, Messenger; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoic acid receptor (RAR)-retinoid X receptor (RXR) heterodimers bind to cognate response elements in vitro more efficiently than do RAR or RXR homodimers, and both RAR and RXR partners have been shown to activate various promoters in transiently transfected cells. We have now investigated whether ligand-dependent activation of both heterodimeric partners is involved in induced expression of endogenous RA-responsive genes and in P19 and F9 cell differentiation. On their own, low concentrations of retinoids selective for either RAR alpha, RAR beta, or RAR gamma did not induce or very inefficiently induced the expression of several RA target genes or triggered differentiation. An RXR-specific synthetic retinoid was similarly inefficient at any concentration. In contrast, at the same concentrations, various combinations of RAR (RAR alpha, RAR beta, or RAR gamma) and RXR selective retinoids resulted in synergistic induction of all retinoic acid (RA) target genes examined, as well as in cell differentiation. However, the magnitude of this synergistic activation varied depending on both the RAR-RXR combination and the promoter context of the responsive genes. Promiscuous activation of the three RARs, or concomitant activation of RAR alpha and RAR gamma, at selective retinoid concentrations also resulted in induction of gene expression and cell differentiation. Taken together, our results are consistent with the conclusion that the RAR and RXR partners of RAR-RXR heterodimers can synergistically activate transcription of RA-responsive genes and can induce differentiation of P19 and F9 cells. Our results also indicate that there is a significant degree of functional redundancy between the three RAR types which, however, varies with the nature of the RA target genes.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Cell Line; Drug Synergism; Gene Expression; Gene Expression Regulation, Neoplastic; Ligands; Mice; Polymerase Chain Reaction; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Retinoids; RNA, Neoplasm; Structure-Activity Relationship; Transcription Factors; Tretinoin; Tumor Cells, Cultured

To access a wide a variety of expressed sequence from human chromosome 21 we have placed this chromosome into undifferentiated P19 mouse embryonic carcinoma cells. Cell lines resulting from these experiments have a range of morphologies and a wide variety of karyotypes. We have studied the retinoic acid response of five cell lines, compared to P19 cells, by observing three markers of retinoic acid induced P19 differentiation--cell morphology, RAR alpha and Wnt1 transcription. We see an 'early' retinoic acid response effect, however this response breaks down by the time the 'late' gene. Wnt1 would be transcribed in P19 cells. A highly responsive cell line will be useful for cloning expressed sequences from human chromosome 21 which are produced by early genes in retinoic acid inducible pathways, such as those involved in neurogenesis.

Topics: Animals; Base Sequence; Carcinoma, Embryonal; Cell Fusion; Cell Line; Chromosomes, Human, Pair 17; DNA Primers; Gene Expression; Humans; Hybrid Cells; In Situ Hybridization, Fluorescence; Karyotyping; Mice; Molecular Sequence Data; Polymerase Chain Reaction; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Retinoic Acid; Recombinant Proteins; Retinoic Acid Receptor alpha; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Wnt Proteins; Wnt1 Protein; Zebrafish Proteins

Cells of the P19 line of embryonal carcinoma cells differentiate into neurons, astrocytes and oligodendrocytes following treatment with retinoic acid. The neurons from these differentiating P19 cultures synthesize a pattern of neurotransmitters that resembles that of neurons of the forebrain. We treated P19 cells with retinoic acid and then implanted them into the striatum of adult rats. After times ranging from 1 to 15 weeks post-implantation, brain slices containing the implanted tissue were prepared and used for intracellular recording of electrical activity and responsiveness to application of neurotransmitters. Within 2 weeks of implantation, the P19-derived neurons had developed responsiveness to the excitatory neurotransmitter glutamate and the inhibitory transmitters gamma-aminobutyric acid and glycine. These neurons also exhibited spontaneous synaptic potentials. The responses to glutamate appear to be mediated by N-methyl-D-aspartic acid as well as non-N-methyl-D-aspartic acid receptor subtypes. Gamma-aminobutyric acid evoked bicuculline-sensitive depolarizing responses in the younger grafts and biphasic depolarizing/hyperpolarizing responses in older ones. Responses to glycine were strychnine sensitive and also showed age-related changes from depolarizing to biphasic character. Synaptic potentials in the younger grafts were exclusively depolarizing, but in older ones both depolarizing and hyperpolarizing events were observed. The synaptic potentials appear to arise from synaptic connections between P19-derived neurons within the grafts. Many of the features of P19-derived neurons are similar to those of neurons in the developing forebrain.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Cell Survival; In Vitro Techniques; Membrane Potentials; Neoplasm Transplantation; Nerve Net; Neurons; Neurotransmitter Agents; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Tretinoin; Tumor Cells, Cultured

Polar solvents such as N,N-dimethyl acetamide (DMA) are known inducers of tumor cell differentiation in vitro. Nothing is known about their ability to induce differentiation in vivo. Using PCC4 AZArL murine embryonal carcinoma (EC) cells we examined the time course and dose response relationship of DMA induced EC differentiation in vitro. The effective continuous dose range of 0.1% to 0.15% gave a linear increase in differentiation index with the probability of attaining complete differentiation. EC tumors were raised subcutaneously in the flanks of strain 129 mice and DMA injected intraperitoneally at a dose calculated to attain tissue levels of between 0.1% and 0.15%. DMA induced significant differentiation primarily into neuroepithelium when compared to negative controls, but DMA was not as effective as retinoic acid. The extent of differentiation was dosage dependent, but the maximal dose of DMA was limited by toxicity mainly to the liver and lymphoid tissues. A graduated dosage schedule of DMA treatment reduced toxicity. These preliminary studies suggest that "differentiation therapy" with polar solvents such as DMA may be an effective adjunct to standard therapies.

Topics: Acetamides; Animals; Carcinoma, Embryonal; Cell Differentiation; Dose-Response Relationship, Drug; Humans; Mice; Neoplasm Transplantation; Tretinoin; Tumor Cells, Cultured

Osteogenic protein-1 (OP-1) is a member of the transforming growth factor-beta super family closely related to the bone morphogenetic proteins and also known as bone morphogenetic protein-7. Other members of this family of growth factors influence cell differentiation as well as cell growth in a number of systems. The Drosophila homolog encoded by the decapentaplegic locus is involved in dorsal-ventral pattern formation during embryogenesis, whereas the expression of several bone morphogenetic proteins including OP-1 is developmentally regulated in mammalian embryos.. The effect of recombinant human OP-1 on the proliferation and differentiation of an established pluripotent human embryonal carcinoma (EC) cell line, NTERA2, and three nullipotent human EC cell lines, 2102Ep, 833KE and TERA-1, was investigated. These cells were grown under reduced serum conditions, and differentiation was monitored by morphology and expression of marker antigens.. OP-1 inhibited proliferation of NTERA2 and induced their differentiation, marked by changes in cellular morphology, the loss of EC cell antigens (SSEA3, SSEA4, the liver isozyme of alkaline phosphatase), and the appearance of new antigens, notably SSEA1 and class 1 major histocompatibility complex antigens. These changes were irreversible and did not involve significant cell degeneration or cell death. The OP-1-induced differentiation of NTERA2 appeared distinct from that induced by either retinoic acid or hexamethylene bisacetamide. Nevertheless, OP-1 did induce the homeobox gene, HOXA1. By contrast, OP-1 elicited only a limited and partial response from the nullipotent EC cell lines.. Our results suggest that pluripotent human EC cells differentiate in response to OP-1 and that this factor can modulate the differentiation induced by retinoic acid. Like other members of the transforming growth factor-beta super family, OP-1 might play an inductive role in the early embryo. The results also suggest a possible therapeutic value for OP-1 in the treatment of some germ cell tumors.

Topics: Acetamides; Antineoplastic Agents; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Carcinoma, Embryonal; Cell Differentiation; Cell Division; Gene Expression; Genes, Homeobox; Humans; Proteins; Recombinant Proteins; RNA; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

The mouse cellular retinoic acid binding protein-I (CRABP-I) gene is specifically up-regulated by retinoic acid (RA) in P19 mouse embryonal carcinoma cells, and its expression in animals is spatially and temporally restricted to RA-sensitive tissues during embryonic development. This study demonstrates that, in adult mouse tissues and P19 cells where the expression of CRABP-I is detected at the basal level, the 5'-flanking region of the CRABP-I gene is hypermethylated at the C residues of all the Hpa II sites. Conversely, in mouse embryos during early stages of development when the expression of CRABP-I gene is detected at a much higher level, this region is demethylated at these Hpa II sites. In P19, enhancement on the RA-induced up-regulation of CRABP-I can be observed in cells treated with 5-azacytidine (5-AzaC) in conjunction with RA, where partial demethylation in the 5'-flanking region of CRABP-I gene is observed. Nuclear run-on experiments indicate that increased message levels of CRABP-I in P19 cells can be accounted for, at least partially, by increases in its transcription rates. The induction of retinoic acid receptor (RAR) beta by RA can also be enhanced by 5-AzaC, but to a much lesser degree. In contrast, all the Hpa II sites in the structural gene portion, at least in the first two exons, are fully demethylated at the C residues.

Topics: Animals; Azacitidine; Base Sequence; Carcinoma, Embryonal; Deoxyribonuclease HpaII; Deoxyribonucleases, Type II Site-Specific; DNA; Embryonic and Fetal Development; Gene Expression Regulation, Developmental; Methylation; Mice; Molecular Sequence Data; Promoter Regions, Genetic; Receptors, Retinoic Acid; Restriction Mapping; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Treatment of P19 embryonal carcinoma cells with retinoic acid induces their differentiation into a population of cells consisting of neurons and other cell types normally derived from neuroectoderm. We used immunohistological and histochemical techniques to identify some of the neurotransmitters in the P19-derived neurons. The majority of neurons contained GABA, glutamic acid decarboxylase, and GABA-transaminase. Neuropeptide Y and somatostatin were less frequently found and both were partially co-expressed with GABA and with one another. Smaller numbers of cells were positive for tyrosine hydroxylase, DOPA decarboxylase, serotonin, calcitonin gene-related peptide, galanin and substance P. The variety and proportions of cells with different transmitter types were reproducible from one experiment to the next and varied very little over 40 days in culture except for cells containing enkephalin, which were abundant only in mature cultures of 32 days or more. Synapses formed between neurons and some contained both small clear and large dense-core vesicles within the presynaptic bouton. Because GABA, neuropeptide Y and somatostatin are abundant in P19-derived neurons as well as in embryonic neurons in rostral regions of the mammalian CNS, we suggest that the developmental events occurring in P19 cell cultures closely resemble those of the embryonic neuroectoderm.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Cell Line; Fluorescent Antibody Technique; Immunohistochemistry; Male; Mice; Microscopy, Electron; Neurons; Neuropeptides; Neurotransmitter Agents; Synapses; Tretinoin; Tumor Cells, Cultured

P19 embryonal carcinoma cells are pluripotent and can be efficiently induced to differentiate in culture into neurons and astroglia by brief treatment with retinoic acid. Retinoic acid-treated P19 cells survive after grafting into the adult rat striatum and differentiate into neurons and glia within the transplantation site. No tumours develop from the grafted cells which continue to express foreign genes that had been transfected into the parental P19 cells. The neurons in these grafts express a variety of neurotransmitters similar to those formed in retinoic acid-treated P19 cell cultures and they mature to acquire the electrophysiological properties expected of fully developed neurons. These results suggest that P19 cells may be used for studies related to neuronal cell development and maturation and that P19 cells may be considered for cell replacement strategies in neurodegenerative disorders of the central nervous system.

Topics: Animals; Brain Tissue Transplantation; Carcinoma, Embryonal; Cell Line; Cell Survival; Cell Transplantation; Dopamine; Electrophysiology; Histocytochemistry; Huntington Disease; Male; Mice; Neostriatum; Neurons; Neurotransmitter Agents; Parkinson Disease; Rats; Rats, Sprague-Dawley; Substantia Nigra; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

Rex-1 is a zinc finger-containing gene that is expressed in embryonal carcinoma (EC) cells and embryonic stem (ES) cells. Upon differentiation with retinoic acid (RA), transcription of the Rex-1 gene decreases rapidly. Analysis of the 5'-flanking region of the Rex-1 gene identified a consensus motif for the octamer family of transcription factors that stimulates expression from the Rex-1 promoter. In this report, we utilized gel mobility shift analysis to examine the binding of transcription factors to the Rex-1 octamer motif. F9 EC cells, D3 ES cells, and human NT2/D1 EC cells each from at least two prominent DNA/protein complexes with the octamer motif. Supershift analysis identifies Oct-1 and Oct-3 in these complexes. When F9 EC cells are induced to differentiate by treatment with RA for 48 h, there is a complete loss of the DNA/protein complex containing Oct-3. In contrast, the other DNA/protein complexes, including the DNA/protein complex containing Oct-1, can still be detected. These results provide support for a role of Oct-3 in the transcription of the Rex-1 gene.

Topics: Animals; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; Cell Line; Cell Nucleus; DNA-Binding Proteins; Embryo, Mammalian; Gene Expression; Genes, pX; Host Cell Factor C1; Mice; Molecular Sequence Data; Nuclear Proteins; Octamer Transcription Factor-1; Octamer Transcription Factor-3; Promoter Regions, Genetic; Stem Cells; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

We have isolated the murine homologs of the members of the COUP-family of steroid hormone receptors, COUP-TF1, ARP-1 and EAR2. The proteins encoded by the murine genes appeared to be highly conserved when compared to their human counterparts. The expression of COUP-TF1 and ARP-1 was induced during differentiation of P19 embryonal carcinoma (EC) cells into derivatives of all three germ layers. Retinoic acid (RA) treatment rapidly induced expression of both genes, while other methods of differentiation were less effective. Undifferentiated P19 cells were found to express EAR2 mRNA and the expression level was only slightly elevated by RA-treatment. In addition, we analyzed the expression in P19 cells of three members of the retinoid X receptor (RXR) family, which have been shown to heterodimerize with members of the COUP-family. During RA mediated differentiation of P19 cells, RXR alpha expression was induced while RXR beta expression was not modulated and RXR gamma expression was down regulated. Gel shift analysis revealed that in P19 cells the members of the COUP-family comprise the major portion of proteins binding to a RA-responsive direct repeat of the consensus steroid hormone receptor binding half site (AGGTCA) spaced by one nucleotide (DR + 1). The members of the COUP-family appeared to down regulate RA-induced activation of RA-response element-containing reporter constructs in a promoter context-dependent manner. The expression patterns of COUP-TF1, ARP-1 and EAR2 during development were investigated by in situ hybridization. In agreement with the results obtained in vitro, the three genes appeared to be expressed in tissues derived from all three germ layers. However, COUP-TF1 and ARP-1 were found to be expressed predominantly in the developing central nervous system in mutually exclusive domains. Furthermore, strong ARP-1 expression was detected in lung and kidney. Our data strongly suggest an important role for the members of the COUP-family in the hormonal control of gene expression regulating embryogenesis.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carcinoma, Embryonal; Cloning, Molecular; COUP Transcription Factor I; COUP Transcription Factor II; COUP Transcription Factors; DNA-Binding Proteins; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; In Situ Hybridization; Mice; Molecular Sequence Data; Receptors, Retinoic Acid; Receptors, Steroid; Retinoid X Receptors; RNA, Messenger; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Retinoic acid treatment of the multipotent human embryonal carcinoma cell line GCT 27 X-1 induced differentiation into an epithelial cell type which morphologically resembled rodent visceral endoderm in vitro. The differentiated cells expressed some markers characteristic of yolk sac, such as cytokeratin 19 and extracellular matrix proteins, but none of the secreted serum proteins produced by normal yolk sac or liver. HNF-3 alpha expression increased upon retinoic acid-induced GCT 27 X-1 differentiation. This increase in HNF-3 alpha expression may characterize an early stage in the differentiation of extraembryonic endoderm. In support of this hypothesis, we found that a yolk sac carcinoma cell line with a phenotype similar to rodent visceral endoderm expressed transcripts for HNF-1 alpha, -1 beta, -3 alpha, -3 beta, -3 gamma, and 4, whereas yolk sac carcinoma cell lines resembling rodent parietal endoderm expressed transcripts for HNF-3 alpha. The results support the conclusion that hepatocytic transcription factors, particularly HNF-3 alpha, may play an important role in early endodermal differentiation in man.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Antibody Specificity; Antigens, Surface; Biomarkers; Blotting, Northern; Carbohydrate Sequence; Carcinoma, Embryonal; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line; Cytoskeletal Proteins; DNA-Binding Proteins; Endoderm; Endodermal Sinus Tumor; Extracellular Matrix Proteins; Fluorescent Antibody Technique; Gene Expression; Hepatocyte Nuclear Factor 3-alpha; Humans; Kinetics; Laminin; Liver Neoplasms; Models, Biological; Molecular Sequence Data; Nuclear Proteins; Restriction Mapping; RNA, Messenger; Rodentia; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The P19 embryonal carcinoma (EC) cell line represents a useful model system for analysis of neural development and differentiation processes that are difficult to study in mammalian embryos. Since many members of the Wnt family of signaling molecules are expressed in the developing as well as adult nervous system, we have examined expression of these genes in P19 cells. Analysis of the mRNA accumulation profiles for Wnt genes during retinoic acid (RA)-induced neural differentiation of P19 cells showed that nine Wnt family members were expressed in a regulated manner during this process. Most were induced by RA treatment, and some were also expressed in undifferentiated P19 cells. Since Wnt-1 is not expressed in undifferentiated P19 cells but is induced during neuroectodermal differentiation we have generated P19 cell lines that overexpress Wnt-1 in the absence of RA treatment, in order to address the role of Wnt-1 in P19 differentiation. In the presence of ectopic Wnt-1, expression of other endogenous Wnt genes, which serve as early differentiation markers in this system, were induced without RA, which is normally required for appearance of these gene products. Furthermore, ectopic expression of Wnt-1 resulted in a loss of SSEA-1 antigen expression, a marker of undifferentiated P19 cells. Similarly to the parental cell line, addition of RA to P19 cells overexpressing Wnt-1 induced the neuroectodermal pathway, but expression of cell type-specific markers such as MASH-1, HNK-1, and GAP-43 was diminished and the morphology of neuronal processes, stained with an antibody to neurofilament, was abnormal. These data suggest that Wnt-1 itself can induce some aspects of early neuroectodermal differentiation and, furthermore, that the correct timing of Wnt-1 expression is necessary for proper RA-induced expression of the neural phenotype.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Blotting, Northern; Carcinoma, Embryonal; Cell Differentiation; Cell Line; Cloning, Molecular; DNA-Binding Proteins; Ectoderm; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Mice; Neurons; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogenes; RNA, Messenger; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Wnt Proteins; Wnt1 Protein; Zebrafish Proteins

Mouse P19 embryonal carcinoma cells can be reproducibly differentiated into neurons and glial cells upon treatment with high concentration of retinoic acid (RA). In order to understand the molecular mechanisms that control early neural differentiation, we screened a cDNA library made from 24-h RA-treated P19 cells with subtracted cDNA probes. One clone was positive in the secondary screening and was designated as p205. This clone (1.1 kb) has an open reading frame of 317 amino acids with homology to G-protein beta subunit. This protein sequence was identical to chicken and human genes previously identified as a major histocompatibility complex-associated gene. The complete conservation of its amino acid sequence between mouse, human and chicken provides strong evidence that the p205 protein fulfills a fundamental function. Developmental Northern blot analysis revealed that a p205 mRNA is expressed at high levels in the embryonic mouse brain, decreasing as development proceeds. In situ hybridization revealed that p205 mRNA is strongly and ubiquitously expressed in the embryonic and early postnatal mouse brain. This expression decreased during postnatal development and was localized in the dentate gyrus, habenula, piriform cortex, paraventricular nucleus of the hypothalamus and supraoptic nucleus of the adult brain. These results suggest that this protein plays an important role in the developing brain and neuronal differentiation.

Topics: Aging; Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Brain; Carcinoma, Embryonal; Cell Differentiation; Cell Line; Cloning, Molecular; DNA Probes; Embryonic and Fetal Development; Gene Library; Gestational Age; Mice; Molecular Sequence Data; Neurons; Neuropeptides; Organ Specificity; Receptors for Activated C Kinase; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

MK is a gene that is activated by retinoic acid in embryonal carcinoma (EC) cells and is expressed temporarily during the mid-gestation period of mouse embryogenesis. Midkine, the product of the gene is a novel heparin-binding growth/differentiation factor with neurite outgrowth and neurotrophic activities. The regulatory DNA element in the retinoic acid-induced expression of the MK gene has been investigated. The 1.9 kb 5'-flanking region of the MK gene can mediate retinoic acid-responsive gene expression in F9 and HM-1 EC cells. Analysis of this region by deletion mutagenesis in F9 EC cells shows that there is a retinoic acid-responsive enhancer (designated as REM1) around 900 bp upstream from the transcription start site. This enhancer is composed of two sequence elements, which are located between -1006 and -895 and between -901 and -794. The core element of the upstream region (-971 to -955), whose deletion abolished the retinoic acid responsiveness, contained a sequence highly homologous to a binding site for retinoic acid receptors. Binding of a retinoic acid receptor heterodimer to this core element was verified by gel shift assay. Thus, retinoic acid and the receptor complex can directly induce the expression of a growth/differentiation factor gene.

Topics: Animals; Base Sequence; Carcinoma, Embryonal; Carrier Proteins; Cell Differentiation; Chromosome Mapping; Cytokines; Enhancer Elements, Genetic; Exons; Gene Expression Regulation, Neoplastic; Genetic Code; Mice; Midkine; Molecular Sequence Data; Nerve Growth Factors; Protein Biosynthesis; Tretinoin; Tumor Cells, Cultured

We have investigated the pattern of PTPase transcript expression during in vitro differentiation of mouse embryonal carcinoma (F9) cells. While the transcripts of most PTPases were unchanged or undetected during embryonal differentiation induced by retinoic acid, several PTPase transcripts exhibited distinct patterns of induction. Mutant cells defective in differentiation did not display the induction of some of these PTPase transcripts. Interestingly, three out of the four PTPase transcripts induced were the same PTPase transcripts induced during in vitro erythroid differentiation of mouse erythroleukemia (MEL) cells [(1994) J. Biol. Chem. 269, 4709-4712] [corrected]. The possible role played by specific PTPases in cell differentiation is discussed.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; DNA Probes; Enzyme Induction; Mice; Plasminogen Activators; Protein Tyrosine Phosphatases; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

We present evidence demonstrating that the liver-enriched transcription factor HNF-3 alpha is activated upon retinoic acid-induced differentiation of mouse F9 embryonal carcinoma cells. We have detected increases in the DNA binding activity and mRNA level of HNF-3 alpha. Both are reflections of the actual activation mechanism at the level of transcriptional initiation, which we showed with the help of HNF-3 alpha promoter constructs. Time course studies clearly show that HNF-3 alpha activation is a transient event. Employing Northern blots, HNF-3 alpha mRNA can be detected between 16 and 24 hours post-differentiation, reaches its zenith at approximately 1 day, and then declines to virtually undetectable levels. F9 cells can give rise to three distinct differentiated cell types; visceral endoderm, parietal endoderm, and primitive endoderm. We have clearly shown that HNF-3 alpha stimulation occurs upon primitive endoderm formation. In addition, the transcription factor is also activated during the induction of cell lineages that give rise to parietal and visceral endoderm. HNF-3 alpha stimulation upon visceral endoderm differentiation is accompanied by the activation of HNF-3 target genes such as transthyretin, suggesting that HNF-3 alpha is involved in the developmental activation of this gene. In contrast, HNF-3 alpha target genes in parietal and primitive endoderm have yet to be identified. However, the stimulation of HNF-3 alpha during primitive endoderm formation, which is an extremely early event during murine embryogenesis, points towards a role for the factor in crucial determination processes that occur early during development.

Topics: Animals; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; DNA; DNA-Binding Proteins; Gene Expression Regulation; Hepatocyte Nuclear Factor 3-alpha; Mice; Molecular Sequence Data; Nuclear Proteins; Promoter Regions, Genetic; Protein Binding; RNA, Messenger; Stem Cells; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Rae-30, one of the retinoic acid (RA)-inducible cDNA clones in mouse embryonal carcinoma F9 cells, was sequenced and the deduced RAE-30 protein showed about a 70% homology to mammalian fructose 1,6-bisphosphatase (EC 3.1.3.11) (FBPase), in comparison to over 85% homology observed among the previously documented rat liver, pig kidney and human leukemic HL-60 cell FBPases. The Rae-30 mRNAs were not detected in various tissues of adult mice, including the liver and kidney, but were detected in a placenta and predominantly in the intestine of adult mice. These findings indicate that the Rae-30 cDNA encodes a novel isoenzyme of FBPase, which is likely to be involved in early differentiation in mammalian cells.

Topics: Amino Acid Sequence; Animals; Base Sequence; Biological Evolution; Carcinoma, Embryonal; Cloning, Molecular; DNA, Complementary; Fructose-Bisphosphatase; Humans; Isoenzymes; Mice; Molecular Sequence Data; RNA, Messenger; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Tretinoin; Tumor Cells, Cultured

The establishment of the parietal endoderm (PE) layer within the rodent blastocyst requires extensive outward migration of inner cell mass (ICM)-derived primitive endoderm (PrE) cells over the blastocoelic surface of the trophectoderm. While a role for Reichert's membrane in this process has been proposed, the identity of the basement membrane component(s) involved and the mechanism(s) by which they might promote and/or guide this movement remain to be determined. This study was a comparison of the gradient-associated influences of three components of Reichert's membrane--fibronectin, laminin, and collagen IV--on the migration of embryonic endoderm-like cells obtained from the F9 embryocarcinoma cell line. Gradients (positive or negative) or even coatings of each of these glycoproteins were established across polycarbonate filters; the filters were then placed in blind-well chambers. F9 cells, either undifferentiated or pretreated with retinoic acid (RA) alone or RA+dibutyryl cAMP to resemble PrE and PE, respectively, were then loaded into the upper chamber compartments. At the end of the migration assay (4-24 h), the number of cells that had moved through the filter pores and attached to the lower filter surface was determined for each treatment group. While even coatings of either fibronectin or laminin supported some cell migration through the filters, these levels were increased 13- and 20-fold (fibronectin and laminin, respectively) by application of the glycoproteins as positive gradients. In contrast, no migration occurred in response to negative gradients of either fibronectin or laminin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Bucladesine; Carcinoma, Embryonal; Cell Count; Cell Differentiation; Cell Movement; Collagen; Endoderm; Extracellular Matrix; Fibronectins; Kinetics; Laminin; Mice; Tretinoin; Tumor Cells, Cultured

We evaluated SR11237, a retinoid X receptor (RXR)-specific compound, for its pharmacologic effects on cell differentiation in F9 embryonal carcinoma cells and rhino mouse epidermis. SR11237 can cause RXR/RXR homodimers to form and transactivate a reporter gene containing a RXR-response element. We confirmed, using nuclear receptor co-transfection assays in COS-1 cells, that SR11237 is effective at transactivating a chloramphenicol acetyltransferase reporter gene through RXRs but not retinoic acid receptors. When SR11237 was tested for its ability to modulate cell differentiation, it was inactive on F9 embryonal carcinoma cells and rhino mouse skin. Because differentiation in these systems is known to be regulated by RAR-specific compounds, such as all-trans-retinoic acid and (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-prope nyl benzoic acid], our results with SR11237 are compatible with the concept that classical retinoid pleiotropic responses are mediated by RXR/RAR heterodimeric nuclear receptors rather than through RXR/RXR homodimers.

Topics: Animals; Benzoates; Carcinoma, Embryonal; Cell Differentiation; Female; Male; Mice; Mice, Hairless; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Skin; Transcription Factors; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

The interactions between 1,25-dihydroxyvitamin D3 [1,25(OH)2D] and retinoic acid (RA) on alkaline phosphatase (ALP) activity and vitamin D receptors (VDR) were determined in the SV40 immortalized osteoblastic cell line C1. Biochemical and cytochemical analysis showed that ALP activity increased with C1 cell density and that 1,25(OH)2D inhibited ALP activity at high density, whereas RA increased ALP activity at low density. The interactions of the two hormones were also cell density dependent, with a predominant stimulatory effect of RA at low density and a predominant inhibitory effect of 1,25(OH)2D at high density. In contrast, the two hormones inhibited C1 cell proliferation independently of cell density. Scatchard analysis and immunocytochemical staining showed that nuclear VDR levels increased with cell density and that RA, 1,25(OH)2D, and their combination upregulated VDR levels mainly at high cell density. Although RA increased VDR levels, RA did not potentiate the effect of 1,25(OH)2D on ALP activity. The results indicate that the effects and interactions of 1,25(OH)2D and RA on ALP activity in osteoblastic C1 cells depend on the state of phenotypic maturation.

Topics: Alkaline Phosphatase; Animals; Calcitriol; Carcinoma, Embryonal; Cell Division; Cell Line, Transformed; DNA Replication; Dose-Response Relationship, Drug; Drug Interactions; Genes, Viral; Immunohistochemistry; Kinetics; Mice; Osteoblasts; Receptors, Calcitriol; Simian virus 40; Tretinoin; Tumor Cells, Cultured

Retinoid X receptors (RXRs) heterodimerize with multiple nuclear hormone receptors and are thought to exert pleiotropic functions. To address the role of RXRs in retinoic acid- (RA) mediated gene regulation, we designed a dominant negative RXR beta. This mutated receptor, termed DBD-, lacked the DNA binding domain but retained the ability to dimerize with partner receptors, resulting in formation of nonfunctional dimers. DBD- was transfected into P19 murine embryonal carcinoma (EC) cells, in which reporters containing the RA-responsive elements (RAREs) were activated by RA through the activity of endogenous RXR-RA receptor (RAR) heterodimers. We found that DBD- had a dominant negative activity on the RARE reporter activity in these cells. P19 clones stably expressing DBD- were established; these clones also failed to activate RARE-driven reporters in response to RA. Further, these cells were defective in RA-induced mRNA expression of Hox-1.3 and RAR beta, as well as in RA-induced down-regulation of Oct3 mRNA. Gel mobility shift assays demonstrated that RA treatment of control P19 cells induces RARE-binding activity, of which RXR beta is a major component. However, the RA-induced binding activity was greatly reduced in cells expressing DBD-. By genomic footprinting, we show that RA treatment induces in vivo occupancy of the RARE in the endogenous RAR beta gene in control P19 cells but that this occupancy is not observed with the DBD- cells. These data provide evidence that the dominant negative activity of DBD- is caused by the lack of receptor binding to target DNA. Finally, we show that in F9 EC cells expression of DBD- leads to inhibition of the growth arrest that accompanies RA-induced differentiation. Taken together, these results demonstrate that RXR beta and partner receptors play a central role in RA-mediated gene regulation and in the control of growth and differentiation in EC cells.

Topics: Animals; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; Cell Division; DNA Primers; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Genes, Reporter; Mice; Molecular Sequence Data; Mutation; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) has been known to play an important role in cellular growth and differentiation as well as in vertebrate development. Many in vitro cell cultures also respond to RA by differentiating. Perhaps the most widely studied of these cultures are embryonal carcinoma (EC) cells. We have used an RA-hypersensitive EC cell mutant, created by retroviral insertion, to analyze the activity of the identifiable components in the RA response pathway. We have analyzed the mRNA expression patterns of the retinoic acid receptors (RARs) alpha, beta, and gamma, the retinoid X receptors (RXRs) alpha, beta, and gamma, and the cellular retinoic acid binding proteins (CRABPs) I and II. Our results indicate that CRABP I, RAR beta, and RAR gamma mRNAs are expressed differentially between parent and RA-hypersensitive mutant cells. All three messages are present at higher basal levels and at earlier times after RA addition in the mutant relative to parental cells. All other elements examined are equivalently expressed. Therefore analyses of the expression patterns of CRABPs, RARs, and RXRs in these RA-hypersensitive cells point to the probable importance of CRABP I, RAR beta, and RAR gamma in the RA induction pathway and also indicate that CRABP II and RXR gamma are not likely to be critical elements in the early differentiative response of cells to RA.

Topics: Animals; Biological Evolution; Carcinoma, Embryonal; Cell Differentiation; Cell Line; Gene Expression; Kinetics; Mice; Multigene Family; Mutagenesis, Insertional; Neoplasm Proteins; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; RNA, Messenger; Time Factors; Transcription Factors; Tretinoin; Tumor Cells, Cultured

While primary neuronal cell cultures have been used to investigate excitotoxicity, development of cell lines exhibiting glutamate receptor-mediated death is desirable. P19 mouse embryonal carcinoma cells, exposed to retinoic acid and plated onto a layer of cultured mouse cortical glial cells, differentiated into neuron-like elements immunoreactive for neurofilaments and neuron-specific enolase. Whole-cell recordings revealed inward currents in response to extracellular application of either NMDA or kainate. The NMDA-induced currents exhibited a voltage-dependent blockade by magnesium, required glycine for maximal activation, and were blocked by the NMDA antagonist dizocilpine. Kainate-induced currents were blocked by the AMPA/kainate receptor antagonist CNQX. Exposure to 500 microM NMDA for 24 h destroyed most P19 cells (EC50 approximately 70 microM); death was prevented by dizocilpine or D-APV. Exposure to 500 microM kainate also resulted in widespread death reduced by CNQX. Thus differentiated P19 cells exhibited both excitatory amino acid responses and vulnerability to excitotoxicity, characteristic of CNS neurons. These cells may provide a genetically open system useful for studying glutamate receptor-mediated phenomena at a molecular level.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amino Acids; Animals; Carcinoma, Embryonal; Cell Differentiation; Dizocilpine Maleate; Electrophysiology; Female; Immunohistochemistry; Indicators and Reagents; Ion Channels; Kainic Acid; Mice; N-Methylaspartate; Neuroglia; Neurons; Pregnancy; Quinoxalines; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) signal transduction is believed to be mediated through several high-affinity nuclear receptors [RA receptors (RARs) and retinoid X receptors], which are members of the steroid/thyroid/vitamin D superfamily and function as transcription factors. Why multiple RARs exist and what gene targets are regulated by each of the three receptors remain compelling questions in developmental biology. Through targeted disruption of both RAR gamma alleles, we have identified several differentiation-specific genes that are regulated either directly or indirectly by RAR gamma in F9 embryonal carcinoma cells. These include genes encoding Hoxa-1 (Hox-1.6) and the extracellular matrix proteins laminin B1 and collagen type IV (alpha 1), all of which are RA inducible in wild-type F9 embryonal carcinoma cells but are not significantly induced in the RAR gamma-/- lines. In contrast, transcripts encoding Hoxb-1 (Hox-2.9) and cellular RA binding protein II (CRABPII) are activated by RA for a longer period of time in the RAR gamma-/- lines compared to the wild-type F9 line. Not all RA-responsive genes are aberrantly expressed; Rex-1, RAR beta, and SPARC transcripts are regulated in the RAR gamma-/- lines as they are in F9 wild-type cells. Our results support the idea that each RAR may regulate different subsets of RA-responsive genes, which may explain, in part, the complex regulation of developmental processes by retinoids.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Gene Expression Regulation; Genes, Homeobox; In Vitro Techniques; Mice; Mutagenesis, Insertional; Receptors, Retinoic Acid; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

1. Cells from a pluripotent murine embryonal carcinoma cell line (P19) were differentiated in vitro into cells with neurone- and cardiac-like phenotypes. Cells treated with 0.5 microM retinoic acid developed into neurone-like cells possessing extensive neurites. Dimethyl sulphoxide treatment (0.5%) produced large, spontaneously contracting cell aggregates with many properties of cardiac cells. 2. The neurone- and cardiac-like cells contained voltage-sensitive Na+ channels with properties similar to those of native neuronal and cardiac cells. 3. We used whole-cell patch clamp techniques to measure inward currents from the neurone- and cardiac-like cells. Undifferentiated (untreated) cells had only small inward currents (peak of -0.15 nA in 150 mM external Na+). The peak inward current in the neurone-like and cardiac-like cells was -1.2 nA (in 154 mM external Na+) and -2.8 nA (in only 46 mM Na+), respectively. These large currents were absent when the external solution contained no Na+. 4. Tetrodotoxin (TTX) blocked the Na+ currents in the neurone- and cardiac-like cells in a dose-dependent manner. The Kd for TTX block of the Na+ current in the neurone-like cells was 6.7 nM. The Na+ current in the cardiac-like cells was much more resistant to TTX; the half-blocking concentration was two orders of magnitude higher, 710 nM. 5. The kinetic properties of the Na+ channel currents in the neurone- and cardiac-like cells were similar but developed over somewhat different voltage ranges. The voltage sensitivity of activation was similar in both cell types but the activation mid-point voltage was different: -12 mV in the neuronal cells and -34 mV for cardiac cells. Inactivation of the neuronal Na+ channels had a mid-point near -47 mV and was more sensitive to the membrane voltage than inactivation of the cardiac channels. The mid-point of inactivation for the cardiac Na+ channels was -80 mV.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Dimethyl Sulfoxide; Electrophysiology; Kinetics; Membrane Potentials; Mice; Myocardium; Neurons; Phenotype; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Tretinoin; Tumor Cells, Cultured

F9 embryonal cells can be induced to differentiate and synthesize basement membrane proteins. Perlecan and laminin are two basement membrane constituents that have extensive regions of homology. Expression of perlecan and laminin B1 genes was followed during differentiation of F9 cells by measurements of transcription rate and mRNA abundance using nuclear run on assays and Northern hybridizations, respectively. The rate of precursor protein synthesis was determined by immunoprecipitation from lysates of pulse-labeled F9 cells. The results showed that perlecan gene expression responds more rapidly after induction than does laminin B1 gene expression but is ultimately expressed at a substantially lower level than laminin. Thus, the perlecan and laminin genes appear to be regulated by different mechanisms and their gene products are not made in stoichiometric amounts.

Topics: Animals; Blotting, Northern; Bucladesine; Carcinoma, Embryonal; Cell Transformation, Neoplastic; Gene Expression; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Laminin; Mice; Proteoglycans; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The proto-oncogene Fgf-3 has been implicated as an important signalling molecule in vertebrate development. In the mouse, it is expressed for a limited time at a multitude of sites from embryonic day 7 to birth. Transcription of Fgf-3 initiates at three promoter regions resulting in the generation of various mRNAs which nevertheless all encode the same protein products. A 1.7kb DNA fragment which encompasses these regions was joined to the CAT reporter gene and shown to function as a promoter in embryonal carcinoma cells. In stable transfectants the promoter retains its retinoic acid inducibility, initiating transcription at the same cap-sites as the endogenous gene. In differentiated F9 cells, transient transfection of progressive and targeted deletion mutants of the promoter region has revealed at least two positive and three negative regulatory elements. With one exception, loss of these elements was shown to dramatically affect promoter activity in stable transfectants of F9 cells. However the promoter remained inducible by retinoic acid to differing degrees, apart from deletions encompassing PS-4A which essentially abolished promoter activity in both undifferentiated and differentiated cells. The sequences of these potential regulatory regions were further defined using DNase-I footprinting, revealing some similarities to consensus binding sites for known transcription factors.

Topics: Animals; Base Sequence; Carcinoma, Embryonal; Cyclic AMP; DNA Mutational Analysis; Fibroblast Growth Factor 3; Fibroblast Growth Factors; Gene Expression Regulation; In Vitro Techniques; Mice; Molecular Sequence Data; Promoter Regions, Genetic; Proto-Oncogene Proteins; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

P19 embryonal carcinoma cells can be induced to differentiate into neuron-like cells by retinoic acid. P19 neurons were recently shown to express both NMDA and non-NMDA type glutamate receptor-mediated currents and be susceptible to glutamate excitotoxicity. In this study, we used RT-PCR to survey differentiated P19 cultures for glutamate receptor transcript expression. The following transcripts were detected: at least one member of the GluR1-4 family, GluR5, GluR6, GluR7, KA1, KA2, NMDAR1, and NMDAR2B. Nuclease protection assays revealed a large quantitative induction of GluR6 transcripts following retinoic acid treatment. Inotropic glutamate receptors are a fundamental and major feature of CNS neurons which are not expressed by the cell lines commonly used as experimental models for mammalian neurons. The present results show that P19 cells express multiple genes involved in glutamate receptor biology. Since the stem cells can be manipulated genetically, the system has the basic requirements for analyzing mechanisms involved in glutamate receptor gene expression.

Topics: Animals; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; Cell Line; DNA Primers; Gene Expression; Mice; Molecular Sequence Data; Neurons; Polymerase Chain Reaction; Receptors, Glutamate; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

F9 embryonal carcinoma cells differentiate in response to retinoic acid (RA). To investigate the regulation of RA receptors (RARs) expression during this process, cDNA probes specific for the major RAR isoforms were used. In contrast to the level of RAR beta 2 mRNA which was high in cells treated 5 days with RA and below detection in untreated cells, as previously described, the steady state levels of RAR alpha 1, alpha 2, gamma 1, and gamma 2 mRNAs were markedly decreased in the RA-differentiated cells as compared to untreated cells. The down-regulation of the RA-responsive system in differentiated cells was also evident in gel shift assays as a marked decrease in binding capacity to a retinoid acid response element (beta 2RARE), as well as in chloramphenicol acetyltransferase (CAT) assays as a sixfold decrease in RA-mediated transacting activity via this element. The down-regulation of RAR DNA-binding and transacting activity coincided with the burst in tissue plasminogen activator secretion and thus, occurred at the hinge between early and late differentiation. The down-regulation of RA responsiveness may constitute an important event in the transition between early and late differentiation stage in F9 cells.

Topics: Animals; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; Chloramphenicol O-Acetyltransferase; DNA; Down-Regulation; Isomerism; Molecular Sequence Data; Oligonucleotide Probes; Promoter Regions, Genetic; Receptors, Retinoic Acid; Tissue Plasminogen Activator; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured