tretinoin and Teratoma

A number of genes containing sequences coding for DNA-binding domains homologues to homeobox sequences in Drosophila have been isolated in vertebrate and their mechanism of action have been studied. In Particular Hox family genes share with Drosophila homeotic genes a genomic organization in gene clusters and an expression pattern that is similar in a number of important respects. In situ hybridization experiment have shown that there is a strict correspondence, or colinearity, between the order of the Hox genes (3' to 5') within the chromosomal cluster and that of their expression domains (anterior to posterior) in the embryo. Recent data obtained in embryonal carcinoma (EC) cells induced to differentiate by retinoic acid (RA) cast some light on the molecular mechanisms underlying the colinear expression of the Hox genes.

Topics: Amino Acid Sequence; Animals; Cell Differentiation; Central Nervous System; Chromosome Mapping; Consensus Sequence; Drosophila; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity; Teratoma; Tretinoin; Tumor Cells, Cultured; Vertebrates

Topics: Animals; Cell Differentiation; Clone Cells; DNA; Gene Expression Regulation, Neoplastic; Gene Library; Human Genome Project; Humans; Mice; Oncogenes; Teratoma; Tretinoin; Tumor Cells, Cultured

P19 cells are a line of pluripotent embryonal carcinoma able to grow continuously in serum-supplemented media. The differentiation of these cells can be controlled by nontoxic drugs. Retinoic acid effectively induces the development of neurons, astroglia and microglia--cell types normally derived from the neuroectoderm. Aggregates of P19 cells exposed to dimethyl sulfoxide differentiate into endodermal and mesodermal derivatives including cardiac and skeletal muscle. P19 cells can be effectively transfected with DNA encoding recombinant genes and stable lines expressing these genes can be readily isolated. These manipulations make P19 cells suitable material for investigating the molecular mechanisms governing developmental decision made by differentiating pluripotent cells.

Topics: Animals; Cell Differentiation; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Ectoderm; Mesoderm; Mice; Teratoma; Transfection; Tretinoin

Topics: Animals; Butyrates; Butyric Acid; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cholecalciferol; Cyclic AMP; Humans; Melanoma; Protein Kinases; Retinoids; Teratoma; Tretinoin; Vitamin A

Topics: Cell Differentiation; Cyclic AMP; Neoplasms, Experimental; Protein Kinases; Teratoma; Tretinoin

EGF-Rs are cell membrane glycoproteins of wide distribution. They have not yet been fully characterized or purified but are probably molecules of 170-190,000 mol. wt. in most cells. The growth factor EGF binds and will saturate cell surface receptors with a KA of about 5 X 10(9) M-1 although a receptor class with an affinity in excess of 10(10) M-1 has been detected in some cells. The number of receptors on a cell does not determine the level of its response. Some cell types have receptors which bind EGF, but with no mitogenic response. The ways in which receptor affinity and/or number is modulated are described. This and other evidence is reviewed in a search for a suitable model of a mechanism of action on the cell, which best fits the current data. There is ample evidence that EGF binds to the receptor; that ligand-receptor complexes cluster or aggregate; and then are internalized and degraded, but evidence for a direct connection between internalization and the subsequent mitogenic response is lacking. Good correlations between internalization and mitogenic responses have been observed and developed into a theory of endocytic activation, but there is a body of evidence which cannot be accommodated by this theory. Instead, an alternative model is suggested.

Topics: Animals; Binding Sites; Cell Membrane; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Models, Biological; Molecular Weight; Organ Specificity; Peptides; Pregnancy; Receptors, Cell Surface; Species Specificity; Teratoma; Tissue Distribution; Tretinoin

The combination of non-human primate animals and their induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) provides not only transplantation models for cell-based therapy of heart diseases, but also opportunities for heart-related drug research on both cellular and animal levels. However, the subtypes and electrophysiology properties of non-human primate iPSC-CMs hadn't been detailed characterized. In this study, we generated rhesus monkey induced pluripotent stem cells (riPSCs), and efficiently differentiated them into ventricular or atrial cardiomyocytes by modulating retinoic acid (RA) pathways. Our results revealed that the electrophysiological characteristics and response to canonical drugs of riPSC-CMs were similar with those of human pluripotent stem cell derived CMs. Therefore, rhesus monkeys and their iPSC-CMs provide a powerful and practicable system for heart related biomedical research.

Topics: Animals; Calcium Signaling; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cellular Reprogramming; COUP Transcription Factor II; Fibroblasts; Heart Ventricles; Induced Pluripotent Stem Cells; Macaca mulatta; Mice; Mice, Inbred NOD; Mice, SCID; Microscopy, Fluorescence; Myocytes, Cardiac; Patch-Clamp Techniques; Teratoma; Transcription Factors; Tretinoin

Topics: Adenocarcinoma; Adult; Antineoplastic Agents; Cell Transformation, Neoplastic; Humans; Maintenance Chemotherapy; Male; Radiography; Retroperitoneal Neoplasms; Teratoma; Treatment Outcome; Tretinoin

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

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

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

Historically, our understanding of molecular genetic aspects of germ cell development has been limited. Recently, results demonstrated that the derivation of pluripotent stem cells may provide the necessary genetic system to study germ cell development. Here, we characterized an induced pluripotent stem cell (iPSC) line, which can spontaneously differentiate into embryonic bodies (EBs) after 3 days of suspension culture, expressing specific markers of three germ layers. Then, we induced the iPSCs to differentiate into germ cells by culturing adherent EBs in retinoic acid (RA) and porcine follicular fluid (PFF) differentiation medium or seminiferous tubule transplantation. Our results indicated that RA and PFF were beneficial for the derivation of germ cells and oocyte-like cells from iPSCs, and iPSCs transplantation could make a contribution to repairing the testis of infertile mice. Our study offers an approach for further study on the development and the differentiation of germ cells derived from iPSCs.

Topics: Animals; Busulfan; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Culture Media; Embryoid Bodies; Female; Follicular Fluid; Gene Expression Profiling; Germ Cells; Induced Pluripotent Stem Cells; Infertility, Male; Male; Mice; Seminiferous Tubules; Suspensions; Swine; Teratoma; Transplantation, Heterotopic; Tretinoin

Polycomb Repressive Complex (PRC) 1 and PRC2 regulate genes involved in differentiation and development. However, the mechanism for how PRC1 and PRC2 are recruited to genes in mammalian cells is unclear. Here we present evidence for an interaction between the transcription factor REST, PRC1, and PRC2 and show that RNF2 and REST co-regulate a number of neuronal genes in human teratocarcinoma cells (NT2-D1). Using NT2-D1 cells as a model of neuronal differentiation, we furthermore showed that retinoic-acid stimulation led to displacement of PRC1 at REST binding sites, reduced H3K27Me3, and increased gene expression. Genome-wide analysis of Polycomb binding in Rest⁻/⁻ and Eed⁻/⁻ mouse embryonic stem (mES) cells showed that Rest was required for PRC1 recruitment to a subset of Polycomb regulated neuronal genes. Furthermore, we found that PRC1 can be recruited to Rest binding sites independently of CpG islands and the H3K27Me3 mark. Surprisingly, PRC2 was frequently increased around Rest binding sites located in CpG-rich regions in the Rest⁻/⁻ mES cells, indicating a more complex interplay where Rest also can limit PRC2 recruitment. Therefore, we propose that Rest has context-dependent functions for PRC1- and PRC2- recruitment, which allows this transcription factor to act both as a recruiter of Polycomb as well as a limiting factor for PRC2 recruitment at CpG islands.

Topics: Animals; Binding Sites; Cell Differentiation; CpG Islands; Embryonic Stem Cells; Gene Expression Regulation, Developmental; HEK293 Cells; Humans; Jumonji Domain-Containing Histone Demethylases; Mice; Neurons; Polycomb-Group Proteins; Protein Binding; Repressor Proteins; Teratoma; Tretinoin

We assessed whether imaging α(v)β(3) integrin could distinguish mature teratoma from necrosis in human non-seminomatous germ cell tumour (NSGCT) post-chemotherapy residual masses.. Human embryonal carcinoma xenografts (six/rat) were untreated (controls) or treated to form mature teratomas with low-dose cisplatin and all-trans retinoic acid (ATRA) over a period of 8 weeks. In another group, necrosis was induced in xenografts with high-dose cisplatin plus etoposide (two cycles). (18)F-Fluorodeoxyglucose ((18)F-FDG) small animal positron emission tomography (SA PET) imaging was performed in three rats (one control and two treated for 4 and 8 weeks with cisplatin+ATRA). Imaging of α(v)β(3) expression was performed in six rats bearing mature teratomas and two rats with necrotic lesions on a microSPECT/CT device after injection of the tracer [(99m)Tc]HYNIC-RGD [6-hydrazinonicotinic acid conjugated to cyclo(Arg-Gly-Asp-D-Phe-Lys)]. Correlative immunohistochemistry studies of human and mouse α(v)β(3) expression were performed.. Cisplatin+ATRA induced differentiation of the xenografts. After 8 weeks, some glandular structures and mesenchymal cells were visible; in contrast, control tumours showed undifferentiated tissues. SA PET imaging showed that mature teratoma had very low avidity for (18)F-FDG [mean standardised uptake value (SUV(mean)) = 0.48 ± 0.05] compared to untreated embryonal carcinoma (SUV(mean) = 0.92 ± 0.13) (p = 0.005). α(v)β(3) imaging accurately distinguished mature teratoma (tumour to muscle ratio = 4.29 ± 1.57) from necrosis (tumour to muscle ratio = 1.3 ± 0.26) (p = 0.0002). Immunohistochemistry studies showed that α(v)β(3) integrin expression was strong in the glandular structures of mature teratoma lesions and negative in host stroma.. Imaging α(v)β(3) integrin accurately distinguished mature teratoma from necrosis following cisplatin-based treatment in human NSGCT xenografts.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Cisplatin; Diagnosis, Differential; Fluorodeoxyglucose F18; Humans; Integrin alphaVbeta3; Male; Molecular Imaging; Necrosis; Neoplasm, Residual; Rats; Teratoma; Testicular Neoplasms; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed; Tretinoin

Recent breakthroughs in the generation of induced pluripotent stem cells (iPSCs) have provided a novel renewable source of cells with embryonic stem cell-like properties, which may potentially be used for gene therapy and tissue engineering. Although iPSCs have been differentiated into various cell types, iPSC-derived keratinocytes have not yet been obtained. In this study, we report the in vitro differentiation of mouse iPSCs into a keratinocyte lineage through sequential applications of retinoic acid and bone-morphogenetic protein-4 and growth on collagen IV-coated plates. We show that iPSCs can be differentiated into functional keratinocytes capable of regenerating a fully differentiated epidermis, hair follicles, and sebaceous glands in an in vivo environment. Keratinocytes derived from iPSCs displayed characteristics similar to those of primary keratinocytes with respect to gene and protein expression, as well as their ability to differentiate in vitro and to reconstitute normal skin and its appendages in an in vivo assay. At present, no effective therapeutic treatments are available for many genetic skin diseases. The development of methods for the efficient differentiation of iPSCs into a keratinocyte lineage will enable us to determine whether genetically corrected autologous iPSCs can be used to generate a permanent corrective therapy for these diseases.

Topics: Animals; Calcium; Cell Differentiation; Cell Lineage; Cells, Cultured; Dermis; Epidermal Cells; Epidermis; Fibroblasts; Hair Follicle; Keratinocytes; Keratolytic Agents; Mice; Mice, Inbred ICR; Mice, Nude; Multipotent Stem Cells; Neoplasm Transplantation; Pluripotent Stem Cells; Sebaceous Glands; Teratoma; Tretinoin

This study evaluated the essentiality of glial cell line-derived neurotrophic factor (GDNF) for in vitro culture of established mouse multipotent adult germline stem (maGS) cell lines by culturing them in the presence of GDNF, leukemia inhibitory factor (LIF) or both. We show that, in the absence of LIF, GDNF slows the proliferation of maGS cells and result in smaller sized colonies without any change in distribution of cells to different cell-cycle stages, expression of pluripotency genes and in vitro differentiation potential. Furthermore, in the absence of LIF, GDNF increased the expression of male germ-line genes and repopulated the empty seminiferous tubule of W/W(v) mutant mouse without the formation of teratoma. GDNF also altered the genomic imprinting of Igf2, Peg1, and H19 genes but had no effect on DNA methylation of Oct4, Nanog and Stra8 genes. However, these effects of GDNF were masked in the presence of LIF. GDNF also did not interfere with the multipotency of maGS cells if they are cultured in the presence of LIF. In conclusion, our results suggest that, in the absence of LIF, GDNF alters the growth characteristics of maGS cells and partially impart them some of the germline stem (GS) cell-like characteristics.

Topics: Adult Stem Cells; Animals; Antineoplastic Agents; Cell Differentiation; Cell Line; Cell Proliferation; Cell Shape; Coculture Techniques; DNA Methylation; Gene Expression; Genomic Imprinting; Germ Cells; Glial Cell Line-Derived Neurotrophic Factor; Leukemia Inhibitory Factor; Male; Mice; Mice, Inbred DBA; Multipotent Stem Cells; Seminiferous Tubules; Stem Cell Transplantation; Teratoma; Tretinoin

Neonatal stroke presents with seizures and results in neurologic morbidity, including epilepsy, hemiparesis, and cognitive deficits. Stem cell-based therapy offers a possible therapeutic strategy for neonatal stroke. We developed an immature mouse model of stroke with acute seizures and ischemic brain injury. Postnatal day 12 CD1 mice received right-sided carotid ligation. Two or 7 days after ligation, mice received an intrastriatal injection of B5 embryonic stem cell-derived neural stem cells. Four weeks after ligation, hemispheric brain atrophy was measured. Pups receiving stem cells 2 days after ligation had less severe hemispheric brain atrophy compared with either noninjected or vehicle-injected ligated controls. Transplanted cells survived, but 3 out of 10 pups injected with stem cells developed local tumors. No difference in hemispheric brain atrophy was seen in mice injected with stem cells 7 days after ligation. Neural stem cells have the potential to ameliorate ischemic injury in the immature brain, although tumor development is a serious concern.

Topics: Animals; Atrophy; Brain Ischemia; Brain Neoplasms; Carotid Arteries; Cell Survival; Ligation; Mice; Neurons; Seizures; Stem Cell Transplantation; Stem Cells; Stereotaxic Techniques; Stroke; Teratoma; Tretinoin

The mechanism of retinal ganglion cell loss in Leber's hereditary optic neuropathy (LHON) is still uncertain, and a role of enhanced superoxide production by the mutant mitochondrial complex I has been hypothesized. In the present study, it was shown that LHON cybrids, carrying the np11778 mutation, became selectively more H(2)O(2) sensitive compared with the parental cell line only following short-term retinoic acid differentiation. They contained a decreased cellular glutathione pool (49%, p< or =0.05), despite 1.5-fold enhanced expression of the regulatory subunit of gamma-glutamylcysteine synthetase (p< or =0.05). This points to a reduction of the capacity to detoxify H(2)O(2) and to changes in thiol redox potential. The activity of the H(2)O(2) degrading enzyme glutathione peroxidase (GPx) and the activities of glutathione reductase (GR) and superoxide dismutase (SOD) were unaffected.

Topics: Antineoplastic Agents; Antioxidants; Cell Differentiation; Cell Line, Tumor; Electron Transport Complex I; Genotype; Glutathione; Glutathione Peroxidase; Glutathione Reductase; Humans; Hydrogen Peroxide; Mitochondria; Optic Atrophy, Hereditary, Leber; Oxidants; Point Mutation; Superoxide Dismutase; Teratoma; Tretinoin

Cells isolated from pancreas have a remarkable potential for self-renewal and multilineage differentiation. We here present a comprehensive characterisation of stem/progenitor cells derived from exocrine parts of the adult rat pancreas. Using purified cells from either single colonies or even single-cell clones, we specifically demonstrate: (i) the cells contain the typical stem/progenitor cell markers alkaline phophatase, SSEA-1, Oct-4, CD9, Nestin, Pax6, CD44, a-Fetoprotein and Brachyury, demonstrated by immunocytochemistry and RT-PCR; (ii) the cells have the potential to differentiate into lineages of all three germ layers in vitro; (iii) a clonal analysis revealed that even cell lines derived from a single cell have stem/progenitor cell properties such as self-renewal and spontaneous differentiation into various cell lineages; (iv) the cells have the propensity to form three-dimensional, teratoma-like structures in vitro, which contain cells of different lineages; and (v) external stimuli can activate the generation of certain cell types. For instance, cells treated with retinoic acid show an increased expression of alpha-smooth muscle actin. These results suggest that exocrine glands, such as pancreas may be a potential source of adult stem/progenitor cells, suitable for cell therapy of degenerative diseases.

Topics: Actins; Animals; Cell Culture Techniques; Cell Differentiation; Clone Cells; Male; Pancreas; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Teratoma; Tretinoin

A nerve cell line designated NC-HIMT was established from a HIMT cell line derived from a benign ovarian, three germ layer immature teratoma removed from a 21-year-old Japanese female. The HIMT cells were elongated, ellipsoid or spherical in shape, whose karyotype was on the high side of normal diploidy. Small amounts of retinoic acid enhanced differentiation and maturation of the HIMT cells into nervous tissue, and the NC-HIMT cell line was established by the colony isolating technique when the HIMT cell line was cultured in the presence of retinoic acid-supplemented medium. After establishment, the NC-HIMT cell line was cultured and maintained in retinoic acid-free growth medium. Even though these cells were cultured without retinoic acid, the phenotype of nerve cells remained and the cells were also maintained in a state of high normal diploidy. The nerve cells contacted each other with their long cell projections and formed networks. Immunocytochemical observations using anti-bovine NSE, alpha-internexin, neurofilament 200kD, peripherin and GFAP confirmed that the cells were either nerve cells or glia cells. These results assume that HIMT cells, which were derived from an immature teratoma, have progenitor and/or stem cells which can differentiate into nerve and/or glial cells.

Topics: Adult; Animals; Cell Differentiation; Cell Division; Cell Line; Diploidy; Embryonal Carcinoma Stem Cells; Female; Humans; Karyotyping; Mice; Mice, Nude; Neoplasm Transplantation; Neoplastic Stem Cells; Nerve Tissue Proteins; Neurons; Ovarian Neoplasms; Stimulation, Chemical; Teratoma; Tretinoin

Rcd1, initially identified as a factor essential for the commitment to nitrogen starvation-invoked differentiation in fission yeast, is one of the most conserved proteins found across eukaryotes, and its mammalian homolog is expressed in a variety of differentiating tissues. Here we show that mammalian Rcd1 is a novel transcriptional cofactor and is critically involved in the commitment step in the retinoic acid-induced differentiation of F9 mouse teratocarcinoma cells, at least in part, via forming complexes with retinoic acid receptor and activation transcription factor-2 (ATF-2). In addition, antisense oligonucleotide treatment of embryonic mouse lung explants suggests that Rcd1 also plays a role in retinoic acid-controlled lung development.

Topics: Animals; Cell Differentiation; HL-60 Cells; Humans; K562 Cells; Mice; Polymerase Chain Reaction; Recombinant Proteins; Teratoma; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

A patient with nonseminomatous germ cell cancer, treated with standard chemotherapy, subsequently developed a pathologically confirmed metastatic undifferentiated adenocarcinoma (non-germ-cell elements) arising from residual teratoma. Disease was present in both lobes of the liver and was deemed unresectable at the time of presentation. The patient was treated on a National Cancer Institute-sponsored institutional protocol with all-trans retinoic acid. After 60 days of oral therapy at a dose of 150 mg/m2/d (50 mg/m2 three times daily), the patient was found to have complete radiologic resolution of his hepatic metastases. He subsequently underwent surgery and his complete response was pathologically confirmed.

Topics: Adult; Antineoplastic Agents; Germinoma; Humans; Male; Neoplasms, Second Primary; Teratoma; Testicular Neoplasms; Tretinoin

NAT1/p97/DAP5 is a newly identified protein that shares homology with the translation initiation factor eIF4G. Studies in vitro and in transfected cells indicated that NAT1 might suppress global translation, thereby repressing cellular proliferation. Here we studied the functions of NAT1 in vivo by disrupting its gene in mice. NAT1(-/-) embryos died during gastrulation, indicating a crucial role for NAT1 in embryogenesis. Undifferentiated NAT1(-/-) embryonic stem cells were normal in morphology, proliferation, global translation and gene expression profile. However, NAT1(-/-) cells exhibited an impaired ability to differentiate: they were resistant to differentiation induced by retinoic acid, and teratomas derived from them consisted of undifferentiated and poorly differentiated tissues. The expression of retinoic acid-responsive genes, such as the cell-cycle inhibitor p21(WAF1), was selectively impaired in NAT1(-/-) cells. Transcription from synthetic retinoic acid-responsive elements was also impaired. These data demonstrated that this translation initiation factor homolog controls specific gene expression pathways required for cellular differentiation.

Topics: Animals; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Embryo, Mammalian; Embryonic and Fetal Development; Embryonic Induction; Fetal Death; Gene Deletion; Gene Expression Regulation, Developmental; Gene Targeting; Genes, Essential; Genes, Reporter; Genotype; Mice; Mice, Knockout; Peptide Initiation Factors; Protein Biosynthesis; Response Elements; Signal Transduction; Stem Cells; Teratoma; Transcription, Genetic; Tretinoin

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

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

Retinoic acid (RA) up-regulates retinoic acid receptor beta (RAR beta) gene expression in a variety of cell lines. Whether up-regulation of the RAR beta gene reflects increased activity in a RAR beta-mediated biological process is unclear since RAR beta tends to heterodimerize with retinoid x receptor (RXR). In F9 teratocarcinoma cell line, RA-induced differentiation is accompanied by increased expression of the RAR beta, RXR alpha, and alpha-fetoprotein (AFP) genes. Previously, we have shown that the RA-mediated regulation of the AFP gene is through RXR alpha homodimers. In contrast to F9 cells, Hep3B is unique in that the AFP gene is down-regulated by RA in a manner reminiscent of down-regulation of AFP in postfetal liver. In this paper, we have examined the RA-mediated regulation of the RAR, RXR, peroxisome proliferator-activated receptor (PPAR), and AFP genes in Hep3B cells. RA induced the expression of RAR alpha, beta, and gamma mRNA in Hep3B cells. However, the expression of RXR alpha mRNA was down-regulated, and the levels of RXR beta and RXR gamma mRNA remained unchanged after RA treatment. In addition, the expression of the PPAR alpha, beta, and gamma genes was also unchanged. Gel retardation assays demonstrated that RA decreased the overall binding of nuclear receptors to the RA and PPAR response elements. By super-shift assays using specific anti-RAR and -RXR antibodies, RA treatment decreased the amount of RXR alpha while increasing the amount RAR beta bound to retinoic acid response element-DR1 (direct repeat with spacer of one nucleotide), indicating the levels of RAR/RXR heterodimer, RXR/RXR homodimer, or RAR/RAR homodimers were altered upon RA treatment of Hep3B cells. In addition, the RA-mediated reduction of RXR alpha in part results in down-regulation of the AFP gene. Our data indicates that RA exerts its effects by differentially regulating its own receptor gene expression.

Topics: Animals; Carcinoma, Hepatocellular; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Retinoid X Receptors; RNA, Messenger; Teratoma; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Up-Regulation

We describe the cloning and initial characterization of a novel cDNA from human embryonal carcinoma (EC) cells. This cDNA, which we named human growth differentiation factor 3 (hGDF3), encodes the homologue of mouse GDF3, a TGFbeta superfamily member belonging to the Growth/Differentiation Factors. We have analysed the expression of hGDF3 in human embryonal carcinoma cell lines and in primary testicular germ cell tumours of adolescents and adults (TGCTs). Expression of hGDF3 in human EC cell lines is stem cell-specific, is down-regulated upon RA-mediated differentiation and is increased upon culture of the cells in the presence of activin A. In TGCTs, hGDF3 expression is low in seminomas, while expression in non-seminomas is readily detectable and appears to be associated with the EC and yolk sac components in the tumours. We have also mapped the hGDF3 locus to the short arm of human chromosome 12, a region consistently overrepresented in human testicular germ cell tumours. Thus, hGDF3 represents an embryonal carcinoma stem cell-associated marker both in vitro and in vivo.

Topics: Activins; Amino Acid Sequence; Base Sequence; DNA Fragmentation; DNA, Complementary; Gene Expression Regulation, Developmental; Growth Differentiation Factor 3; Growth Substances; Humans; Inhibins; Intercellular Signaling Peptides and Proteins; Male; Molecular Sequence Data; RNA, Messenger; Sequence Homology, Amino Acid; Teratoma; Testicular Neoplasms; 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

Mouse F9 teratocarcinoma cells converted into primitive endoderm and parietal endoderm-like cells when treated with retinoic acid (RA) and RA plus dibutyryl cyclic AMP (dbtcAMP), respectively. The carbohydrate chains of glycoconjugates are known to undergo rapid changes during F9 cell differentiation. The mechanism of gene regulation of beta 1,4-galactosyltransferase (beta 1,4GalT), one of the glycosyltransferases involved in the synthesis of carbohydrate structures, was explored during the differentiation of F9 cells. Northern blot analysis revealed that the amount of beta 1,4GalT mRNA increased approximately 1.5- and 6.5-fold in response to treatment with RA alone and RA plus dbtcAMP (RA/dbtcAMP), respectively, for 8 days. beta 1,4GalT specific activity also gradually increased up to 21-fold in response to treatment with RA/dbtcAMP for 8 days. The reason for the different rates of increase in mRNA and enzyme activity remains to be determined. The transcriptional activity of the beta 1,4GalT gene was measured during the course of RA/dbtcAMP-induced F9 cell differentiation in transient transfection experiments using 5'-upstream region DNA (1.8 kb) of the mouse beta 1,4GalT gene combined with luciferase cDNA. Although activity was slightly enhanced on the first day after induction, no significant rise in transcriptional activity was observed in the late stage of induction (3-6 days), when mRNA levels were greatly increased. This was further supported by the nuclear run-off assay which indicated that the rate of de novo synthesis of the beta 1,4GalT gene transcript in the RA/dbtcAMP-induced cells was almost the same as in undifferentiated F9 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Base Sequence; Bucladesine; Cell Differentiation; Drug Stability; Gene Expression Regulation, Neoplastic; Kinetics; Mice; Molecular Sequence Data; N-Acetyllactosamine Synthase; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

We have investigated the role that cellular retinoic acid binding protein I (CRABP-I) may play in the development of the murine hindbrain. Since the central nervous system (CNS) represents a major site of the teratogenic action of retinoic acid (RA), we have also determined the effects of exposure of high levels of RA on CRABP-I expression within the CNS. Expression of CRABP-I can first be detected within the presumptive hindbrain of presomitic mouse embryos and later also appears in neural crest cells and neural crest derivatives; it is thus tissue specific at these early stages. Exposure of 7.75-day mouse embryos to RA induces two phenotypes: one is externally normal and the other is exencephalic. In the exencephalic embryos we show that there is abnormal crest migration, a fusion of the trigeminal and facial-acoustic ganglia, a rostral and lateral shift of the otic vesicle, and a loss of hindbrain rhombomeres. Furthermore, and in contrast to in vitro studies, we demonstrate that CRABP-I appears to be up-regulated in both phenotypes of mouse embryos treated with RA and that this up-regulation is accompanied by an anteriorization of its expression within the nervous system. This new CRABP-I expression domain thus retains its tissue specificity. The role that CRABP-I may play in normal development of the hindbrain and in teratogenesis and the similarity of these results to those obtained with various Hox genes are discussed.

Topics: Animals; Central Nervous System; Embryonic and Fetal Development; Mice; Receptors, Retinoic Acid; RNA, Messenger; Teratoma; Tretinoin; Up-Regulation

Retinoic acid (RA) is widely involved in the control of cell proliferation and differentiation, as well as embryo pattern formation. Transcription of the oncodevelopmental protein, alpha-fetoprotein (AFP), is stimulated by retinoic acid (RA) in neoplastic cells. To study RA regulation of AFP gene expression, the 5'-flanking region of AFP gene was cloned and analyzed. In the present study, transfection of deletion mutants and sequence analysis revealed a retinoid X receptor response element (AFP-RXRE) located at position -139 to -127 of the AFP promoter. Synthetic AFP-RXRE was ligated into a reporter construct with the heterologous promoter and chloramphenicol acetyltransferase (CAT). AFP-RXRE conferred a marked RA responsiveness in the cotransfection with retinoid X receptor (RXR), but not with retinoic acid receptors (RARs). Consistent with these data, only RXR bound to AFP-RXRE with high affinity in the mobility shift assays. Chicken ovalbumin upstream promoter transcription factor (COUP-TF), an orphan member of the steroid/thyroid hormone superfamily, also demonstrated specific binding activity to AFP-RXRE in vitro. In cotransfection assays, COUP-TF dramatically repressed the transactivation of RXR on AFP-RXRE. The mechanism of repression by COUP-TF may involve the mutual occupancy of the AFP-RXRE binding site between RXR and COUP-TF.

Topics: alpha-Fetoproteins; Animals; Base Sequence; Binding Sites; Binding, Competitive; Cell Line; Chloramphenicol O-Acetyltransferase; COUP Transcription Factor I; DNA-Binding Proteins; Gene Expression Regulation; Haplorhini; Kidney; Liver Neoplasms, Experimental; Molecular Sequence Data; Promoter Regions, Genetic; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Teratoma; Transcription Factors; Transcriptional Activation; Transfection; Tretinoin

The identification and molecular characterization of Brn-3.2 has revealed a family of Brn-3-related mammalian POU proteins that share homology with the C. elegans developmental regulator Unc-86 and extended similarity with the Drosophila neurodevelopmental gene I-POU, which defines a novel POU-IV box. Brn-3.2 exhibits DNA binding properties similar to those of Brn-3.0, but its expression is uniquely regulated by retinoic acid in teratocarcinoma and neuroblastoma cells. In the developing PNS and retina, the expression pattern of Brn-3.2 is similar to that of Brn-3.0. In the caudal CNS (spinal cord, hindbrain, and midbrain) Brn-3.2 and Brn-3.0 are initially coexpressed, but diverge later in development. Rostral to the midbrain, Brn-3.2 and Brn-3.0 exhibit nonoverlapping patterns of expression, suggesting divergence of gene function in more recently evolved structures. Our analysis suggests that in the CNS Brn-3.2 is selectively expressed in postmitotic neurons, implying a role in specifying terminally differentiated neuronal phenotypes.

Topics: Amino Acid Sequence; Animals; Base Sequence; Brain; Cell Differentiation; Cell Line; DNA-Binding Proteins; DNA, Complementary; Drosophila; Embryo, Mammalian; Embryo, Nonmammalian; Gene Expression; Gene Expression Regulation; Gene Library; Genes, Homeobox; Homeodomain Proteins; In Situ Hybridization; Mesencephalon; Mice; Mitosis; Molecular Sequence Data; Neuroblastoma; Neurons; Organ Specificity; Rhombencephalon; Sequence Homology, Amino Acid; Spinal Cord; Teratoma; Transcription Factor Brn-3; Transcription Factor Brn-3A; Transcription Factor Brn-3B; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

We previously reported the isolation of a cDNA clone for a homeobox-containing gene designated Pem, shown by Northern analysis of Day 7 through Day 16 mouse embryos to be expressed in extraembryonic tissues. In this study, Pem gene expression was further examined using in situ hybridization and immunocytochemistry to determine the spatial distribution of Pem transcripts and protein in peri-implantation embryos and in embryoid bodies (EBs). Low amounts of Pem mRNA were detected in undifferentiated EBs. When EBs were induced to differentiate, the outer cell layer of visceral or parietal endoderm expressed both Pem mRNA and protein. In developing embryos, no Pem protein was detectable in the uncompacted morula; 12% of the nuclei in compacted morulae were Pem positive, while 25% of the blastocyst trophectoderm and 15% of inner cell mass cells expressed Pem protein. Shortly after implantation, in 5.5 and 6.5 d.p.c. embryos, Pem expression was limited to extraembryonic tissues and was present in distal and proximal visceral endoderm, parietal endoderm, and ectoplacental cone. By 7.5-8.5 d.p.c. neither Pem RNA nor protein was found in the distal squamous visceral endoderm, which surrounds the embryonic region of the egg cylinder, nor in the parietal endoderm. Expression was retained in the proximal columnar epithelium of the visceral endoderm. Prominent Pem expression was observed in the chorion, in trophoblast-derived cells of the ectoplacental cone, and in secondary giant cells, localized in the nuclear compartment. Pem was localized to the X chromosome and found to be expressed in cell lineages where only the maternal X chromosome is active. The data indicate a possible role for Pem in regulating genes involved in the differentiation of extraembryonic tissues.

Topics: Animals; Cell Differentiation; Cricetinae; Cricetulus; DNA-Binding Proteins; Embryo, Mammalian; Embryonic and Fetal Development; Gene Expression; Genes, Homeobox; Homeodomain Proteins; Hybrid Cells; Immunohistochemistry; In Situ Hybridization; Mice; Oligonucleotide Probes; Oligonucleotides, Antisense; Recombination, Genetic; Teratoma; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; X Chromosome

All-trans-retinoic acid (RA) treatment of the multipotent human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1) induces a neuronal phenotype. Compared to untreated NT2/D1 cells, RA treated cells have reduced proliferative potential. To identify which retinoic acid receptor (RAR) is directly linked to RA response in these cells, nine RA resistant subclones were derived and characterized. Unlike parental NT2/D1 cells, all these subclones and a de novo RA resistant human TC cell line, N2102Ep clone 2AG (abbreviated N2102ep), exhibited reduced RAR gamma expression. The RAR gamma gene was studied within NT2/D1 cells and a representative RA resistant NT2/D1 subclone called NT2/D1-R1 by Southern analysis and by the transcriptional properties of cloned RAR gamma cDNAs. No structural or functional differences between these RAR gamma species were found suggesting that RA resistance is due to reduced levels of RAR gamma expressed in NT2/D1-R1 cells. To explore this possibility an RAR gamma cDNA was stably transfected into NT2/D1-R1 cells. Expression of this cDNA partially restored the response to RA in NT2/D1-R1 cells. The role of RAR gamma in parental NT2/D1 cells was studied in transient transfection assays using an FGF4 promoter-enhancer reporter construct that is transcriptionally active in undifferentiated but not in differentiated TC cells. The dose dependent co-transfection of an expressed RAR gamma cDNA with this reporter more efficiently repressed its transcriptional activity in the presence of RA than transfection of the reporter without expressed RAR gamma. Co-transfection also decreased reporter activity in the absence of exogenously added ligand. Together, these findings reveal that RAR gamma expression is tightly coupled to RA response and resistance in human TC cells. These data implicate an important role for RAR gamma in the RA-mediated differentiation of these TCs.

Topics: Base Sequence; Cloning, Molecular; DNA Primers; Drug Resistance; Humans; Molecular Sequence Data; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Teratoma; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured

We raised an antibody against a synthetic peptide corresponding to amino acids 155-174 of human retinoic acid receptor alpha (RAR-alpha). The sequence is highly homologous in all RARs and their isoforms. When mouse and human RARs (alpha, beta and gamma) expressed in Cos cell were analysed with immunoblot, all receptors gave a specific 51 K signal. Mouse RAR-gamma gave an additional signal corresponding to 58 K. In human teratocarcinoma cells (F9) both 51 and 58K molecule sizes were detected. The RAR expression in F9 cells was slightly down-regulated in charcoal-stripped culture medium and returned to normal level after retinoic acid treatment. The 51 K protein was found in all ovarian and uterine samples, but the quantity of the 58 K protein varied in different species and organs, being highest in the mouse uterus and the rat and human ovary. Using immunohistochemistry the RARs were found in the nuclear compartment. In the rat uterus, positive immunoreaction was found mainly in the nuclei of epithelial, uterine glandular and stromal cells. In the rat ovary, positive reaction was found in the nuclei of germinal epithelial, follicular and stromal cells.

Topics: Amino Acid Sequence; Animals; Antibodies; Blotting, Western; Cells, Cultured; Escherichia coli; Female; Humans; Immunohistochemistry; Mice; Molecular Sequence Data; Ovary; Rats; Receptors, Retinoic Acid; Sequence Homology, Amino Acid; Teratoma; Tretinoin; Tumor Cells, Cultured; Uterus; Vitamin A

The Oct-3/4 gene product, which belongs to the POU family of transcription factors, is a good candidate for regulating initial differentiation decisions. It is expressed in the earliest stages of embryogenesis and repressed in subsequent stages. Retinoic acid (RA)-induced differentiation of embryonal carcinoma (EC) cells is accompanied by decreased expression of the Oct-3/4 gene. Previous findings show that sequences in the Oct-3/4 enhancer region (designated RARE1) are targets for RA-mediated repression (H. Okazawa, K. Okamoto, F. Ishino, T. Ishino-Kaneko, S. Takeda, Y. Toyoda, M. Muramatsu, and H. Hamada, EMBO J. 10:2997-3005, 1991). Our present results demonstrate conclusively that the TATA-less Oct-3/4 promoter is also a target for RA-induced repression. We identified a novel cis element in the Oct-3/4 promoter harbors a putative Sp1 binding site and a RA-responsive element (designated RAREoct), which are juxtaposed to one another. Protein binding to the Sp1 site is independent of protein binding to the RAREoct sequence. Unlike the RARE1 situated in the Oct-3/4 enhancer which does not contain a typical RAR recognition site, the RAREoct identified in this study consists of three directly repeated motifs that exhibit extensive homology to RARE sequences located in RA-responsive genes. Moreover, the RAREoct shows different DNA-binding characteristics and DNase I footprint patterns with nuclear proteins isolated from undifferentiated versus RA-differentiated EC cells. This suggests that the RAREoct element binds different nuclear proteins in RA-treated and untreated EC cells which most probably belong to the RA receptor, retinoid X receptor, or orphan receptor families of transcription factors. Using site-directed mutagenesis, we show that the RAREoct contributes to the transcriptional activation of Oct-3/4 promoter in P19 cells and, most interestingly, mediates the RA-induced repression in RA-differentiated EC cells. Thus, the RAREoct element could be one of the points of integration of several signalling pathways influencing Oct-3/4 expression. In accordance with the suggestion that suppression of Oct-3/4 expression is a crucial step during embryogenesis, the Oct-3/4 upstream region contains multiple targets for RA-induced repression, probably to ensure accurate and prompt repression of Oct-3/4 expression. It is possible that these repressors are differentially used at specific stages of development in response to various signals.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Line; Chloramphenicol O-Acetyltransferase; DNA-Binding Proteins; Enhancer Elements, Genetic; Exons; Gene Expression; Genomic Library; Introns; Kinetics; Liver; Mice; Molecular Sequence Data; Mutagenesis, Insertional; Mutagenesis, Site-Directed; Octamer Transcription Factor-3; Oligodeoxyribonucleotides; Point Mutation; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; Teratoma; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

High intracellular levels of heat shock proteins and enhanced protein glycosylation are two phenomena closely associated with the cellular stress response. GP50 is the major heat-induced glycoprotein in Chinese hamster ovary (CHO) cells; however, GP50 is not well characterized, and its function is unknown. J6 is a gene originally identified in F9 murine teratocarcinoma cells after exposure to retinoic acid. In this study we show that J6 is heat-inducible and codes for a protein that shares characteristics with GP50. Western blotting of CHO cell homogenates, using a J6 polyclonal antibody, showed a single band with a molecular weight identical to that of GP50. Thermotolerant cells showed increased levels of the J6/GP50 protein. Heat-shocked CHO cells also accumulated transiently high levels of J6 mRNA between 2 and 7 h following 10 min at 45 degrees C. These induction kinetics are similar to those for GP50 labeling with D-[3H]mannose and to the activation of major heat shock genes, e.g., hsp70. Hybrid selection of J6 mRNA from CHO cells, followed by in vitro translation, produced a single band on SDS-PAGE with a molecular weight identical to that of deglycosylated GP50. Neither cellular proliferation (exponential growth versus plateau phase) nor the specific heat shock temperature (41.5 degrees C versus 45 degrees C) had significant effects on J6 induction by heat stress. Stress conditions other than hyperthermia, including ethanol, arsenite, and hypoxia, increased J6 mRNA levels. Conversely, J6 mRNA was reduced by quercetin, brefeldin A, okadaic acid, uv, and hydrogen peroxide. Our data support the hypothesis that J6 is a heat shock gene with a gene product identical to the polypeptide moiety of GP50.

Topics: Animals; Arsenites; Brefeldin A; Cell Division; Cell Hypoxia; CHO Cells; Cricetinae; Cyclopentanes; Ethers, Cyclic; Gene Expression Regulation; Heat-Shock Proteins; Hot Temperature; Hydrogen Peroxide; Kinetics; Mice; Molecular Weight; Nerve Tissue Proteins; Okadaic Acid; Protein Synthesis Inhibitors; Protein Tyrosine Phosphatases; Quercetin; RNA, Messenger; Sodium Compounds; Teratoma; Transcription, Genetic; Tretinoin; Ultraviolet Rays; X-Rays

FGFs have been implicated in the induction of mesoderm in amphibian development and are present in the mouse embryo at stages that would be appropriate for a similar function in mammals. Primitive ectoderm would then be the target tissue. We have now changes in the expression of receptors for FGFs during the differentiation of embryonal carcinoma (EC) and embryonic stem (ES) cells from the mouse. These cells resemble those of the inner cell mass and later primitive ectoderm. On Northern blots of mRNA from undifferentiated cells, transcripts for FGF R1, R2 and R3 are expressed. All are upregulated during differentiation of ES cells and are upregulated or remain constant as EC cells differentiate. FGF R4 is only expressed after differentiation to derivatives resembling parietal endoderm. By contrast in human EC cells, FGF R2 is downregulated during differentiation, FGF R1 and FGF R3 are unchanged and FGF R4 is expressed before and after differentiation. In both human and mouse EC cells three members of the FGF family (a FGF, b FGF and k FGF, also known as FGFs 1,2 and 4) are mitogenic in serum-free medium and one (KGF or FGF 7) appears to have no effect on growth although cellular morphology is altered. Differences between human and mouse cells are primarily in the effects of heparin on the FGF-induced response.

Topics: Animals; Blotting, Northern; Bucladesine; Cell Differentiation; Cell Division; Cell Line; Culture Media, Conditioned; Down-Regulation; Embryo, Mammalian; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Laminin; Liver; Mice; Poly A; Proto-Oncogene Proteins; Rats; Rats, Inbred BUF; Receptors, Fibroblast Growth Factor; RNA; RNA, Messenger; Stem Cells; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

We have analyzed the regulation of fibroblast growth factor receptors (FGFRs) during retinoic acid (RA) induced differentiation of Tera-2 human embryonal carcinoma cells. Undifferentiated Tera-2 cells expressed mRNAs for all four known FGFRs. Their differentiation led to loss of FGFR-4 mRNA expression and mRNA levels for FGFR-2 and FGFR-3 were considerably downregulated, whereas the mRNA levels for FGFR-1 remained unaltered. A substantial decrease in binding of K-FGF was found to occur upon RA-induced differentiation of the cells. In undifferentiated Tera-2 cells FGF stimulation caused an increase of c-fos mRNA, and c-jun mRNAs, but no increase of junB mRNA, whereas in the differentiated cells, FGFs strongly stimulated the expression of all three genes. Thus differentiation of the Tera-2 cells leads to marked changes in FGFR gene expression as well as to complex alterations in their responses to exogenous FGFs.

Topics: Cell Differentiation; Clone Cells; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Genes, fos; Genes, jun; Humans; Poly A; Receptors, Fibroblast Growth Factor; RNA; RNA, Messenger; Signal Transduction; Teratoma; Thymidine; Tretinoin; Tritium; Tumor Cells, Cultured

We observed that decline of thymidine kinase (TK) enzyme activity was severalfold faster than the decay of full length TK mRNA during growth arrest of 3T6 mouse fibroblasts or during differentiation of myoblasts (C2Cl12) or F9 embryonal carcinoma cells. In order to study the molecular mechanism of this disparate behavior, a polyclonal antiserum against mouse TK was raised in rabbit. High level expression of mouse TK polypeptide in Escherichia coli was achieved with a T7 RNA polymerase-directed expression system. Using the antiserum in immunoblotting, no indication for a pool of inactive enzyme was found during differentiation of F9 or growth arrest of 3T6 cells. Pulse labeling of these cells in vivo with [35S]methionine showed a more than 6-fold decrease in the rate of TK-protein synthesis of in F9 cells after 3 days of treatment with retinoic acid as well as in 3T6 cells after 16 h under low serum. This was not due to increased turnover of the protein as measured in pulse chase experiments. In addition, full length TK mRNA stayed associated with polysomes under these conditions in F9 as well as 3T6 cells. Taken together the results suggest that endogenous TK mRNA becomes translationally repressed under a variety of conditions when mouse cells cease to grow.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Division; Cell Line; Cloning, Molecular; Enzyme Repression; Enzyme Stability; Escherichia coli; Kinetics; Methionine; Mice; Molecular Sequence Data; Oligonucleotide Probes; Polyribosomes; Protein Biosynthesis; Recombinant Proteins; RNA; RNA, Messenger; Sulfur Radioisotopes; Teratoma; Thymidine; Thymidine Kinase; Tretinoin; Tumor Cells, Cultured

A replication-defective retrovirus was used to introduce the marker gene nlsLacZ into the murine embryonal carcinoma (EC) cell line PCC7-S-aza-R-1009. Undifferentiated EC cells were implanted into the central nervous system of adult rats. One month later, the grafted cells continued to express the nlsLacZ gene. Immunohistochemical analysis demonstrated the presence of EC-derived neurons. These neurons were capable of expressing tyrosine hydroxylase and extended neurites into the host parenchyma. EC-derived glial cells could not be detected. There was no evidence of tumorigenicity. These results demonstrate the utility of EC cells for introduction of exogenous gene products into the central nervous system in experimental models of gene therapy.

Topics: Animals; beta-Galactosidase; Brain; Catecholamines; Cell Differentiation; Female; Genetic Markers; Glial Fibrillary Acidic Protein; Immunohistochemistry; Male; Mice; Mice, Inbred Strains; Neoplasm Transplantation; Neurites; Neurons; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; Retroviridae; Teratoma; Testicular Neoplasms; Thalamus; Transplantation, Heterotopic; Tretinoin; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

Murine P19 embryonal carcinoma cells irreversibly differentiate into neuroectoderm following brief exposure to retinoic acid (RA). We compared the expression of RA-responsive genes in P19 cells and in a mutant cell line from mouse, RAC65, which fails to differentiate in RA. Some RA-responsive genes were normally regulated by RA in RAC65 cells while others were not. Amongst the latter were Oct-3 and PEA-3, whose transcripts rapidly disappeared following RA treatment of P19 cells but which were lost only slowly and incompletely from RAC65 cells. Expression of the Hox 1.6, 1.4, and 1.3 transcripts was induced by RA in P19 cells but not in RAC65 cells. Nuclear run-on and transfection assays indicated that transcription of the Hox 1.6 gene was regulated by a previously identified [26] DNA sequence located 3' of the Hox 1.6 gene, probably through interaction with the alpha RA receptor (RAR alpha). Results of nuclear run-on analysis suggested that expression of the Hox 1.6 gene may also be regulated post-transcriptionally. Constitutive expression of Hox 1.6 from a heterologous promoter did not induce differentiation indicating that expression of this gene is insufficient to initiate the cascade of events that culminates in cell differentiation.

Topics: Animals; Carrier Proteins; Cell Differentiation; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Mice; Mutation; Neoplasm Proteins; Octamer Transcription Factor-3; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Receptors, Retinoic Acid; Teratoma; Transcription Factors; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured

We wish to identify genes involved in mediating early lineage decisions in the mouse embryo. F9 teratocarcinoma cells treated with retinoic acid (RA) in suspension culture develop into embryoid bodies (EBs) with an outer layer of visceral endoderm. In order to identify genes that are involved in establishing this extraembryonic endoderm lineage we have employed a PCR-based approach using cDNAs from early EBs as templates. PCR reactions were performed with degenerate oligonucleotide primers coding for the highly conserved regions of the homeodomains of the Drosophila Antennapedia, bicoid, and zerknüllt proteins. Among the PCR products were representatives of previously identified mouse genes, including Hox-A5 (1.3), A1 (1.6), A9 (1.7), B8 (2.4), B2 (2.8), C8 (3.1), and D12 (4.7). Whole mount in situ hybridization analysis, performed to examine the temporal and spatial distribution of transcripts, suggests a possible role for the Hox-D12 gene during endoderm differentiation in F9 EBs. Whereas the expression patterns of several other homeobox genes are essentially uniform throughout the aggregates, Hox-D12 expression is restricted to the outer surface of early EBs at a time when lineage decisions may be occurring. In order to establish the relationship between the Hox-D12 expression pattern and the role of RA in inducing F9 EB differentiation, we examined PSA-1 EBs that differentiate in the absence of added RA. PSA-1 EBs show similar temporal and spatial localization of Hox-D12 when compared to F9 EBs. These data suggest that the pattern of Hox-D12 expression correlates with endoderm differentiation and not with RA treatment and point to a possible role for homeobox-containing genes during the early stages of mouse embryogenesis.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Differentiation; DNA; Endoderm; Genes, Homeobox; In Situ Hybridization; Mice; Molecular Sequence Data; Polymerase Chain Reaction; Sequence Homology, Amino Acid; Teratoma; Tretinoin; Tumor Cells, Cultured

Although F9 cells labelled with [3H]glucosamine synthesize many glycoproteins that bind to Datura stramonium agglutinin-agarose, only a small proportion of these were immunoprecipitated with monoclonal antibodies to lamp-1 and lamp-2 (lamp = lysosomal membrane protein). Differentiation of F9 cells by retinoic acid increased labelling of all Datura stramonium-bound glycoproteins, including lamp-1 and lamp-2. Although the large polylactosaminoglycans excluded from Bio-Gel P-6 that are characteristic of F9 cells were obtained from total glycoproteins, little of these large polylactosaminoglycans was found on lamp-1 and lamp-2. There was no increase in large polylactosaminoglycans of lamp-1 and lamp-2 after retinoic acid treatment, but an increase in the size of small polylactosaminoglycans (included on Bio-Gel P-6) and tri- and tetra-antennary complex oligosaccharides. Therefore, other factors besides the expression of specific glycosyltransferases determine the extent of elongation of polylactosaminoglycans on lysosomal membrane proteins.

Topics: Amino Sugars; Animals; Antibodies, Monoclonal; Antigens, CD; Carbohydrate Sequence; Cell Differentiation; Cell Line; Chromatography, Affinity; Datura stramonium; Electrophoresis, Polyacrylamide Gel; Glucosamine; Glycopeptides; Glycoproteins; Lectins; Lysosomal Membrane Proteins; Lysosomes; Membrane Glycoproteins; Mice; Molecular Sequence Data; Plant Lectins; Plants, Medicinal; Plants, Toxic; Teratoma; Tretinoin; Tritium; Tumor Cells, Cultured

Induction of differentiation as a treatment modality for nonseminomatous germ cell tumors (NSGCTs) may promote the development of residual mature teratoma (RMT), which is usually associated with primary tumors that are capable of spontaneous somatic differentiation. Therefore, we studied the combination of a cytotoxic drug and a differentiation-inducing agent in vivo in three murine teratocarcinoma models with different levels of spontaneous somatic differentiation: E86-379 (moderate differentiation); NF-1 (poor differentiation); and MH-15 (no differentiation). We used retinoic acid (RA) as differentiation-inducing agent and cisdiaminodichloroplatinum (CDDP) as cytotoxic drug, plus a combination of both. In four separate experiments, the combination of RA and CDDP gave a significant further reduction of tumor size as compared with treatment with either RA or CDDP alone. Morphologically intact tumor after treatment with combined RA-CDDP contained a smaller proportion of undifferentiated tissue (embryonal carcinoma) than after CDDP alone. However, somatic differentiation was not induced in the tumor model lacking spontaneous somatic differentiation. Toxicity was reflected in loss of body weight and death of some animals and closely paralleled the degree of tumor reduction in all experiments.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; Cisplatin; Disease Models, Animal; Female; Mice; Mice, Inbred BALB C; Teratoma; Tretinoin

The growth inhibitory effects of all-trans and 13-cis retinoic acid (RA) and of the synthetic retinoids TTNPB, TTNPB-ethylester and TTNN were studied on seven human epithelial ovarian cancer cell lines and one ovarian teratocarcinoma cell line. Six of seven ovarian adenocarcinoma cell lines were inhibited in their growth by RA and by synthetic retinoids in a dose dependent manner. No response to these substances was observed for the ovarian teratocarcinoma cell line. The knowledge that RA and retinoids exert their action on the cells via nuclear receptors led us to examine the expression of RAR-alpha, -beta and -gamma mRNA by these cell lines by polymerase chain reaction following reverse transcription. All cell lines expressed RAR-alpha and -gamma mRNA and six of the eight cell lines were found to express additionally RAR-beta mRNA, among them the ovarian teratocarcinoma cell line. Our data indicate that there was no direct association between the presence of RAR subtype transcripts and the response to retinoids in ovarian cancer cell lines.

Topics: Adenocarcinoma; Base Sequence; Dose-Response Relationship, Drug; Female; Humans; Molecular Sequence Data; Ovarian Neoplasms; Polymerase Chain Reaction; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured

The S-type lactose-binding lectins are found in a variety of animal tissues and their primary sequences are highly conserved between species. Little is known, however, about their functions and endogenous ligands. We report here our studies on the carbohydrate binding specificity of one S-type lectin designated L-14, using glycopeptides and glycoproteins from Chinese hamster ovary cells and mouse teratocarcinoma F9 cells. Our studies demonstrate that L-14 binds with high affinity to oligosaccharides containing multiple repeating units (approximately 4) of the disaccharide [3Gal beta 1-4GlcNAc beta 1]n or poly-N-acetyllactosamine (PL) sequence. Interestingly, terminal beta-galactosyl residues are not necessary for high affinity binding of long PL-containing oligosaccharides to L-14. To characterize the glycoprotein ligands for L-14, we applied total [3H]galactose-labeled glycoproteins from differentiated F9 cells to L-14-Sepharose. Laminin was one of the major glycoproteins in both the cells and the media bound by the lectin. The possible functional significance of this interaction is suggested by the fact that in the absence of Ca2+ L-14 can mediate the binding in vitro of both CHO and F9 cells to immobilized laminin. Taken together, these studies demonstrate that L-14 binds with high affinity to laminin and to relatively long PL chains and indicate that L-14 can promote cell adhesion to laminin.

Topics: Animals; Bucladesine; Carbohydrate Sequence; Cell Adhesion; Cell Differentiation; CHO Cells; Chromatography, Affinity; Cricetinae; Cysteine; Disaccharides; Enzyme-Linked Immunosorbent Assay; Galactose; Galactosides; Galectins; Glycopeptides; Glycoproteins; Hemagglutinins; Laminin; Methionine; Mice; Molecular Sequence Data; Polysaccharides; Sulfur Radioisotopes; Teratoma; Tretinoin; Tritium; Tumor Cells, Cultured

The expression of glutamic acid decarboxylase (GAD) is a basic characteristic of a wide array of inhibitory neurons the use gamma-aminobutyric acid as a neurotransmitter. Clonal cell models will be essential for investigating the mechanisms which are responsible for the selective expression of GAD. P19 embryonal carcinoma cells are an important model for the analysis of neuronal gene expression. Depending on culture conditions, undifferentiated cells can be induced to form cells as widely divergent as cardiac muscle-like cells and neuron-like and glial-like cells. P19 cells are amendable to a number of powerful genetic manipulations including transformation with foreign DNA and selection of mutants. In this study we used nuclease protection assays and Northern blot analysis to determine if P19 cells express the GAD1 and GAD2 genes. The results show that uninduced P19 cells express these genes at very low but easily detectable levels. When the cells are induced to differentiate along the neuronal pathway with retinoic acid, the levels of transcripts for both GAD genes rise dramatically. At least some RNA transcripts of both genes from induced cells comigrate with the corresponding mRNA from the brain and thus probably represent processed mRNA. The expression of GAD genes in undifferentiated cultures of embryonal stem (ES) cells was also investigated. These cultures express levels of GAD1 transcripts that are higher than uninduced P19 cells. In contrast, expression of the GAD2 gene was barely detectable. These results indicate that P19 EC cells and ES cells will be useful for the investigation of the mechanisms that regulate the expression of the GAD1 and GAD2 genes.

Topics: Animals; Base Sequence; Cell Differentiation; Embryonal Carcinoma Stem Cells; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes; Glutamate Decarboxylase; Isoenzymes; Mice; Molecular Sequence Data; Neoplasm Proteins; Neoplastic Stem Cells; Nerve Tissue Proteins; Neurons; Rats; Teratoma; Tretinoin; Tumor Cells, Cultured

A nuclear protein that recognizes u.v.-damaged DNA was detected in extracts from murine F9 embryonic stem cells using a DNA-binding assay. The nuclear-protein-binding activity was increased in cells after treatment with retinoic acid/dibutyryl cyclic AMP (dbcAMP), with optimum induction at 6 days. In vitro treatment of nuclear extracts with agents that affect protein conformation (such as urea, Nonidet P40 and Ca2+) slightly modulated the damage-recognition activity. Furthermore, treatment of nuclear extracts with phosphatase dramatically inhibited the binding activity. In addition, damaged-DNA recognition of the nuclear extracts was effectively inhibited by damaged double- and single-stranded DNA. The expression of the nuclear protein with similar characteristics was abundant in HeLa cells and was increased in drug- or u.v.-resistant cells. The findings suggest that the recognition of a u.v.-DNA adduct is modulated, at least in part, by an activity that is induced during retinoic acid/dbcAMP-induced differentiation. These results also imply that the identified damage-recognition protein may be important for the sensitivity or resistance of mammalian cells to DNA damage.

Topics: Animals; Avian Sarcoma Viruses; Bucladesine; Cell Differentiation; Cell Nucleus; Cyclic AMP; DNA; DNA Damage; HeLa Cells; Humans; Mice; Nuclear Proteins; Promoter Regions, Genetic; Teratoma; Transfection; Tretinoin; Tumor Cells, Cultured; Ultraviolet Rays

Expression of phosphoinositide-specific phospholipase C (PLC)-gamma during the retinoic acid (RA)-induced differentiation of F9 teratocarcinoma stem cells was investigated. PLC-gamma activity and PLC-gamma protein content were decreased during the differentiation of F9 teratocarcinoma stem cells induced by a combination (RACT) of RA, dibutyryl cyclic AMP (C) and theophylline (T). However, PLC-gamma activity and protein levels in RA- or C- and T-treated F9 cells remained the same as that of the F9 stem cells. Northern analysis of the 5.4-kb PLC-gamma transcript in differentiated F9 cells by RACT revealed that RACT decreased the PLC-gamma gene expression. These results suggest that the expression of PLC-gamma is negatively controlled by RACT treatment in the differentiation of F9 teratocarcinoma stem cells.

Topics: Animals; Blotting, Northern; Blotting, Western; Bucladesine; Cell Differentiation; Gene Expression Regulation, Neoplastic; Mice; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoric Diester Hydrolases; Teratoma; Theophylline; Tretinoin; Tumor Cells, Cultured

The expression of mRNAs for inhibin subunits was studied in the human teratocarcinoma cell line Tera-2 clone 13 before and after differentiation with retinoic acid (RA). Both alpha- and beta B-subunits of inhibin were expressed. Subsequently, inhibin bio- and immunoactivity in the conditioned media of the Tera-2 cells were determined by studying the release of follicle-stimulating hormone from rat pituitary cells, by immunoassay and by immunoprecipitation using inhibin alpha- and beta B-subunit specific antibodies. Strikingly dissimilar high bio- and low immuno-activities were found. The ensuing hypothesis that the high bioactivity might be due to the presence of the activin-binding protein follistatin was confirmed by immunoprecipitation of 34 and 37 kDa labelled proteins, using a polyclonal anti-follistatin antiserum after culture of the Tera-2 cells with [35S]-methionine. Furthermore, expression of activin receptor types II and IIB mRNA was found in the cells. Addition of 5 microM RA to monolayer cultures of Tera-2 cells resulted in differentiation to flat endoderm-like cells and caused a slight suppression of the expression of the mRNA encoding the inhibin alpha- and beta B-subunits. The expression of follistatin and activin receptor type IIB was strongly suppressed, whereas the expression of the activin receptor type II was not affected. We conclude that Tera-2 cells secrete follistatin and express inhibin subunits and activin receptors differentially during RA-induced differentiation. The role of the decreased expression of follistatin and activin receptor IIB mRNA after addition of RA in the mechanism of RA-induced differentiation remains to be elucidated.

Topics: Activin Receptors; Activins; Biological Assay; Cell Differentiation; Cells, Cultured; Clone Cells; Culture Media, Conditioned; DNA Probes; Follicle Stimulating Hormone; Follistatin; Glycoproteins; Humans; Inhibins; Luteinizing Hormone; Macromolecular Substances; Pituitary Gland; Receptors, Cell Surface; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured

beta-All-trans-retinoic acid (RA)-induced endodermal differentiation of mouse F9 teratocarcinoma cells is accompanied by changes in glycoprotein glycosylation, including expression of i antigen (i.e. polylactosamine) and leukophytohemagglutinin-reactive oligosaccharides (i.e. -GlcNAc beta 1-6Man alpha 1-6-branched N-linked). We have used the F9 teratocarcinoma cells as a model to study developmental regulation of glycosyltransferase activities which are responsible for the biosynthesis of beta 1-6GlcNAc-branched N- and O-linked oligosaccharides and polylactosamine. Growth of F9 cells in the presence of 10(-6) M RA for 4 days increased core 2 GlcNAc transferase and GlcNAc transferase V activities by 13- and 6-fold, respectively, whereas the activities of GlcNAc transferase I, beta 1-3GlcNAc transferase (i), beta 1-4Gal transferase, and beta 1-3Gal transferase increased 2-4-fold. Induction of glycosyltransferase activities by RA was dose-dependent and showed a biphasic response with approximately half of the increase observed 3 days after RA treatment and the remainder occurred by day 4. PYS-2, a parietal endoderm cell line, showed levels of glycosyltransferase activities similar to those of RA-treated F9 cells. Glycosyltransferase activities in the RA-resistant F9 cell line (RA-3-10) were low and showed only a small induction by RA. These observations suggest that differentiation of F9 cells is closely associated with induction of multiple glycosyltransferase activities, with most pronounced increases in GlcNAc transferase V and 2',5'-tetradenylate (core 2) GlcNAc transferase. The increase in GlcNAc transferase V was also reflected by the 4-6-fold increase in the binding of 125I-leukophytohemagglutinin to several cellular glycoproteins, which occurred after 3 days of RA treatment. The endo-beta-galactosidase-sensitive polylactosamine content of membrane glycoproteins and, in particular, the LAMP-1 glycoprotein was markedly increased after RA treatment of F9 cells. Consistent with these observations, fucosylated polylactosamine (i.e. dimeric Lex) was also increased in RA-treated cells. Analysis of the aryl oligosaccharides produced by F9 cells cultured in the presence of aryl alpha-D-GalNAc showed that RA treatment enhanced the synthesis of disialyl core 2 O-linked oligosaccharides and increased the polylactosamine content of the aryl oligosaccharides by > 20-fold. The results suggest that differentiation of F9 cells into endoderm is closely associated with

Topics: Amino Sugars; Animals; beta-Galactosidase; Carbohydrate Conformation; Carbohydrate Sequence; Cell Differentiation; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Enzyme-Linked Immunosorbent Assay; Hexosyltransferases; Kinetics; Mice; Microsomes; Molecular Sequence Data; N-Acetylglucosaminyltransferases; Oligosaccharides; Polysaccharides; Teratoma; Tretinoin; Tumor Cells, Cultured

The nuclear signaling by the pleiotropic cytokine interleukin-6 (IL-6) has been investigated in human embryonal carcinoma cells and T cells. We show that Oct-1, a ubiquitously expressed octamer-binding protein known to be regulated posttranslationally, can also be regulated at the levels of mRNA and protein synthesis by IL-6 and by retinoic acid (RA) in human embryonal carcinoma cells. NF-IL6, an IL-6-inducible transcription factor of the C/EBP family, can confer this regulation and is itself regulated by both signals. The abundance and the molar ratios of the three forms of NF-IL6, corresponding to peptides initiated in frame from different AUGs of the same NF-IL6 mRNA species, are regulated by IL-6 and by RA. These results suggest that the two signal transduction pathways overlap in human embryonal carcinoma cells and that Oct-1 may be downstream of NF-IL6 in the shared regulatory cascade. Enhanced Oct-1 synthesis correlates with one of the functions of Oct-1, i.e., stimulation of adenovirus DNA replication. This provides an example of a possible functional consequence of IL-6 and RA signaling that is mediated by NF-IL6 and Oct-1 regulation.

Topics: Adenoviruses, Human; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; DNA Replication; DNA-Binding Proteins; Gene Expression Regulation; Host Cell Factor C1; Humans; In Vitro Techniques; Interleukin-6; Nuclear Proteins; Octamer Transcription Factor-1; Octamer Transcription Factor-3; Signal Transduction; Teratoma; Transcription Factors; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured; Virus Replication

We have shown previously that (i) retinoic acid (RA), an anti-neoplastic agent, activates the midkine (MK) gene in mammalian embryonic carcinoma cells, and that (ii) the MK of 118 amino acids, purified from L cells, induces neurite outgrowth of mammalian embryonic brain cells. In this paper, we describe an unconventional strategy for the purification of a fully active MK from E. coli with a high yield. The MK was overproduced in E. coli as a glutathione S-transferase (GST) fusion protein. The MK fusion protein extracted from the bacterial inclusion bodies with guanidine-HCl was renatured, refolded slowly and cleaved by thrombin at the site where the GST links to the MK. The purified free MK, like RA, induced neurite outgrowth from central neurons of the mouse spinal cord, and suppressed the growth of human HL60 leukemia cells in vitro. Unlike RA, however, the MK did not induce granulocytic differentiation of HL60 cells. Furthermore, the MK supported the survival of an NGF-insensitive sensory neuron subpopulation(s) from chicken embryo dorsal root ganglion. Thus, the actions of the MK and leukemia inhibitory factor (LIF) are surprisingly similar. There is no sequence similarity between MK and LIF, however, and unlike MK, LIF production does not appear to be RA-inducible.

Topics: Amino Acid Sequence; Animals; Carrier Proteins; Cell Differentiation; Cell Division; Cells, Cultured; Cloning, Molecular; Cytokines; Embryo, Mammalian; Escherichia coli; Glutathione Transferase; Humans; Leukemia, Promyelocytic, Acute; Mice; Midkine; Molecular Sequence Data; Nerve Growth Factors; Neurites; Neurons, Afferent; Protein Folding; Protein Sorting Signals; Recombinant Fusion Proteins; Recombinant Proteins; Sequence Homology, Amino Acid; Spinal Cord; Teratoma; Thrombin; Tretinoin; Tumor Cells, Cultured

The message for the zinc finger gene Rex-1 (Zfp-42) is expressed in undifferentiated murine F9 teratocarcinoma cells and embryonic stem cells. Expression of Rex-1 is reduced at the transcriptional level when F9 cells are induced by the addition of retinoic acid (RA) to differentiate. We have isolated genomic DNA for the Rex-1 gene (Zfp-42), characterized the gene's structure, and mapped the gene to mouse chromosome 8. Promoter elements contributing to the regulation of the Rex-1 promoter in F9 cells have been identified. A region required for Rex-1 promoter activity in F9 stem cells contains an octamer motif (ATTTGCAT) which is a binding site for octamer transcription factor members of the POU domain family of DNA-binding proteins. Rex-1 reporter plasmids including this octamer site also exhibited reduced expression in F9 cells treated with RA. Thus, the octamer motif is a regulatory element required for the activity of the Rex-1 promoter in F9 stem cells, and this motif contributes to the negative regulation by RA of the transcription of the Rex-1 gene. As an initial confirmation of the in vivo relevance of the isolated fragment, a larger Rex-1 promoter fragment, also containing the octamer site, was able to promote expression of the bacterial lacZ gene in mouse embryos at the morula stage.

Topics: Animals; Base Sequence; Binding Sites; Chloramphenicol O-Acetyltransferase; DNA; DNA-Binding Proteins; Exons; Gene Expression Regulation, Neoplastic; Genomic Library; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Promoter Regions, Genetic; Spleen; Teratoma; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured; Zinc Fingers

We have recently shown that the adenovirus E1A gene products block interferon-alpha-induced signal transduction and transcription factor NF-kappa B-mediated gene induction. Here we report that the same responses are also blocked in undifferentiated F9 teratocarcinoma cells. The block was removed upon cellular differentiation and regained upon the introduction of viral E1A into the differentiated cells. In undifferentiated cells, interferon-beta failed to induce the transcription of interferon-responsive genes because of a lack of activation of the cognate trans-acting factors. As a result, in these cells, virus replication was not inhibited by interferon. Similarly, in undifferentiated but not in differentiated F9 cells, tumor necrosis factor alpha failed to stimulate NF-kappa B-mediated transcription of a reporter gene because of a failure in the activation of NF-kappa B trans-acting factor. These results suggest that a cellular E1A-like activity, present in undifferentiated F9 cells, and adenoviral E1A use similar mechanisms for repressing the expression of specific cellular genes.

Topics: 2',5'-Oligoadenylate Synthetase; Adenoviridae; Adenovirus E1A Proteins; Animals; Cell Differentiation; Chloramphenicol O-Acetyltransferase; Gene Expression Regulation, Neoplastic; Interferon-beta; Interferon-gamma; Mice; NF-kappa B; Recombinant Proteins; Teratoma; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The expression of the retinoblastoma susceptibility (RB) gene was investigated in P19 embryonal carcinoma cells and in these cells induced to differentiate with retinoic acid (RA) or with dimethyl sulfoxide (DMSO). In undifferentiated cells very low levels of RB mRNA and protein were present. DMSO-treated P19 cell cultures develop into mesodermal and endodermal cells and RB expression increased only slightly in these differentiating cells. RA-treated P19 cells develop into neuroectoderm and this differentiation was accompanied by a marked increase in RB expression with mRNA levels increasing 15 fold by 4-6 days following initiation of RA treatment. No such increase occurred in mutant cells that fail to respond to RA. The RB promoter did not appear to be directly activated by RA. Nevertheless, the increase in RB expression in RA-treated cells appeared to be due to enhanced initiation of transcription because cells transfected with a reporter gene driven by the RB promoter expressed the reporter gene with kinetics similar to that of the RB gene. Thus the activation of the RB gene appears to be achieved indirectly by RA-induced factor(s) in differentiating neuroectodermal cells. The post-mitotic neurons that developed in RA-treated cultures contained only the hypophosphorylated form of the RB protein. Recent studies (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992) have shown that mice lacking the RB gene have abnormalities in early brain development suggesting that the rapid rise in RB expression and the hypophosphorylation of the protein are essential for neuronal cell differentiation.

Topics: Animals; Blotting, Western; Cell Differentiation; Chloramphenicol O-Acetyltransferase; Dimethyl Sulfoxide; Gene Expression Regulation, Neoplastic; Genes, Retinoblastoma; Humans; Mice; Neurons; Promoter Regions, Genetic; Retinoblastoma Protein; RNA, Messenger; Teratoma; Transfection; Tretinoin; Tumor Cells, Cultured

Midkine (MK) is a novel growth factor and is the product of a retinoic acid responsive gene. When P19 murine embryonal carcinoma (EC) cells were exposed to MK, they differentiated into neurons, and the neuronal differentiation was accompanied by expression of choline acetyltransferase activity. Synthesis and release of MK in the EC cells treated with retinoic acid were shown by Western blot analysis, and rabbit anti-MK antibody attenuated the action of retinoic acid to induce the neuronal differentiation. These results indicate that MK is one of the mediators of retinoic acid action to induce the neuronal differentiation in EC cells.

Topics: Animals; Antibodies; Blotting, Western; Carrier Proteins; Cell Differentiation; Choline O-Acetyltransferase; Cytokines; Enzyme Induction; Glutathione Transferase; Growth Substances; Kinetics; L Cells; Mice; Midkine; Neurons; Recombinant Fusion Proteins; Teratoma; Transfection; Tretinoin; Tumor Cells, Cultured

A P19 embryonal carcinoma stem cell line carrying an insertion of the E. coli LacZ gene in an endogenous copy of the Pax-3 gene was identified. Expression of the Pax-3/LacZ fusion gene in neuroectodermal and mesodermal lineages following induction of differentiation by chemical treatments (retinoic acid and dimethylsulfoxide) was characterized using this line and is consistent with the previous localization of Pax-3 expression in the embryo to mitotically active cells of the dorsal neuroectoderm and the adjacent segmented dermomyotome. Pax-3/LacZ marked stem cells were also utilized as target cells in mixing experiments with unmarked P19 cells that had been differentiated by pretreatment with chemical inducers. Induction of beta-galactosidase and neuroectodermal markers in the target cells demonstrates that: (1) some differentiated P19 cell derivatives transiently express endogenous Pax-3- and neuroectoderm-inducing activities, (2) undifferentiated target stem cells respond to these activities even in the presence of leukemia inhibitory factor and (3) the endogenous activities can be distinguished from, and are more potent than, retinoic acid treatment in inducing neuroectoderm. These observations demonstrate that P19 embryonal carcinoma cells provide a useful in vitro system for analysis of the cellular interactions responsible for neuroectoderm induction in mammals.

Topics: Animals; Blotting, Northern; Cell Line; Dimethyl Sulfoxide; DNA-Binding Proteins; Ectoderm; Embryonic Induction; Fluorescent Antibody Technique; Gene Expression; Mammals; Mesoderm; Nervous System; Teratoma; Tretinoin; Tumor Cells, Cultured

Thrombomodulin (TM) expression was investigated during differentiation of F9 embryonal carcinoma cells into primitive or parietal endoderm. Exposure of F9 cells to retinoic acid (RA) triggers differentiation into primitive endoderm and induces the appearance of barely detectable amounts of TM mRNA, whereas treatment with dibutyryl cAMP plus theophylline (CT) augments the levels of TM mRNA to a 4-fold greater extent than RA. Exposure of F9 cells to RA plus CT initiates differentiation into parietal endoderm and synergistically increases the levels of TM mRNA by 10- to 12-fold compared with CT. The time-dependent establishment of cooperativity between RA and CT appears to be secondary to RA-induced differentiation to primitive endoderm. The above alterations in TM mRNA levels occur by a transcriptional mechanism as judged by nuclear run-on experiments. Transient gene expression experiments show that the human TM promoter is transactivated by coexpression of the human RA receptor beta. Thus, the mechanism of induction of TM expression in F9 cells undergoing differentiation to parietal endoderm appears to be similar, but not identical, to that noted for other late response genes.

Topics: Animals; Base Sequence; Bucladesine; Cell Line; Cell Nucleus; Cyclic AMP; Gene Expression Regulation, Neoplastic; Mice; Molecular Sequence Data; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Receptors, Cell Surface; Receptors, Thrombin; RNA, Neoplasm; Teratoma; Theophylline; Thrombin; Transcription, Genetic; Tretinoin

The cellular retinoic acid-binding protein type II (CRABP-II) is a member of the serum and cytoplasmic retinoid-binding protein family. It is expressed during embryonic development and in adult skin and is upregulated by retinoic acid (RA) in F9 teratocarcinoma cells. We have determined the genomic organization of the murine CRABP-II gene and performed a detailed analysis of its transcriptional unit. The CRABP-II gene, located on mouse chromosome 2, is approximately 4.6 kilobases long and divided into four exons in a structure common to other members of the family of serum and cellular retinoid-binding proteins. Primer extension analysis and S1 nuclease protection assay were used to identify the transcription initiation site which is located 27 base pairs downstream of a typical TATAA box. Sequence analysis of the promoter also revealed a GC-rich region with overlapping putative SP1-binding sites at nucleotides -61 and AP-1 and AP-2-binding sites at nucleotides -518 and -544, respectively. The 3'-untranslated region contains two copies of the pentanucleotide AUUUA shown to be involved in messenger RNA destabilization. Consensus sequence for retinoic acid response elements were not detected in the promoter region of the CRABP-II gene. Results of nuclear run on experiments show that the CRABP-II gene is not transcriptionally activated by RA in F9 teratocarcinoma cells. These results suggest that the up-regulation of CRABP-II mRNA levels by RA is mainly controlled by a post-transcriptional mechanism.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carrier Proteins; Chloramphenicol O-Acetyltransferase; Chromosome Mapping; Cloning, Molecular; Cycloheximide; DNA; Female; Gene Expression Regulation; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Promoter Regions, Genetic; Receptors, Retinoic Acid; RNA Processing, Post-Transcriptional; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

A 47-kDa heat-shock protein (HSP47) is a major collagen-binding stress protein residing in the endoplasmic reticulum, and is assumed to be a molecular chaperone specific to collagen. Two-dimensional gel electrophoresis and immunoprecipitation studies showed that the expression of HSP47 was significantly induced during the differentiation of mouse teratocarcinoma F9 cells by treatment with retinoic acid alone or with retinoic acid and dibutyryladenosine 3',5'-phosphate. The induction of type-IV collagen was also observed during F9-cell differentiation. For further analysis, we cloned cDNA encoding mouse HSP47 from a cDNA library of BALB/c 3T3 cells and performed Northern-blot analysis. The cDNA contained a signal sequence at the N-terminus and an endoplasmic-reticulum-retention signal, RDEL, at the C-terminus. An homology search revealed that mouse HSP47, as well as chick HSP47, belonged to the serine protease inhibitor superfamily. While chick HSP47 mRNA was 4.5 kb with a long (2-kb) 3' untranslated region, mouse and human HSP47 mRNA were 2.5 kb, with a 0.8-kb 3' untranslated region. Northern-blot analysis revealed that the concurrent induction of HSP47 and type-IV collagen during F9-cell differentiation, and the transient induction of HSP47 after heat shock was regulated at the level of mRNA accumulation. These results suggested that HSP47 was closely related to collagens in terms of its expression as well as in its functional relevance.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Bucladesine; Cell Differentiation; Cloning, Molecular; Collagen; DNA; Electrophoresis, Gel, Two-Dimensional; Heat-Shock Proteins; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Precipitin Tests; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured

P19 embryonal carcinoma (EC) cells differentiate when treated with retinoic acid (RA). The P19 EC-derived mutant cell line RAC65 is resistant to the differentiation-inducing activity of RA. We show that these cells express a truncated retinoic acid receptor alpha(mRAR alpha-RAC65), probably due to the integration of a transposon-like element in the RAR alpha gene. This receptor lacks 71 C-terminal amino acids and terminates in the ligand-binding domain. In CAT assays in RAC65 cells, mRAR alpha-RAC65 fails to trans-activate the RAR beta promoter, which contains a RA-response element. In wild-type P19 EC cells mRAR alpha-RAC65 functions as a dominant-negative repressor of RA-induced RAR beta activation. Gel retardation assays demonstrate that mRAR alpha-RAC65 is still able to bind to the RA-response element of the RAR beta promoter, indicating that competition with functional RARs for the same binding site leads to the observed dominant-negative effect. In addition, in two RAC65 clones in which wild-type hRAR alpha was stably transfected RA-sensitivity was restored and in one RAR beta expression could be induced by RA. Taken together, these data show that the primary cause of RA-resistance of RAC65 cells is the expression of a defective RAR alpha, which prevents the trans-activation of RA-responsive genes and results in a loss of the ability to differentiate.

Topics: Animals; Base Sequence; Blotting, Northern; Blotting, Southern; Carrier Proteins; Cell Differentiation; Cell Line; Cloning, Molecular; DNA; Drug Resistance; Gene Expression Regulation; Mice; Microscopy, Fluorescence; Molecular Sequence Data; Neoplasm Proteins; Receptors, Retinoic Acid; Restriction Mapping; RNA, Messenger; Sequence Homology, Nucleic Acid; Teratoma; Transfection; Tretinoin

The CRABP-I and CRABP-II proteins are high affinity cytoplasmic retinoic acid-binding proteins. In undifferentiated F9 teratocarcinoma stem cells, only the CRABP-I protein is expressed at detectable levels. We have previously shown that overexpression of the CRABP-I protein in stably transfected F9 stem cell lines results in a lower sensitivity to a given external concentration of retinoic acid relative to that of untransfected F9 cells; in contrast, reduced CRABP-I expression in CRABP-I cDNA anti-sense transfected lines is associated with increased sensitivity of these lines to retinoic acid. These three types of cell lines were cultured in the presence of 50 nM [3H]retinoic acid, and the metabolism of retinoic acid was followed over the next 24 h. The results demonstrate that CRABP-I has the ability to alter both the levels and types of RA metabolites produced in the cytoplasm of differentiating embryonic stem cells. Moreover, the level of CRABP-I determines the rate of RA metabolism to 4-oxo-RA such that the higher the CRABP-I level, the faster the metabolism of [3H]retinoic acid. This is the first reported connection between the level of CRABP-I expression and intracellular RA metabolism.

Topics: Carrier Proteins; Chromatography, High Pressure Liquid; DNA; Receptors, Retinoic Acid; Teratoma; Tretinoin; Tumor Cells, Cultured

When F9 teratocarcinoma cells are treated with retinoic acid plus cyclic AMP (RACF9) they differentiate into parietal endoderm. Differentiation is accompanied by the acquisition of substrate adhesion sites and a change in the pattern of gene expression, including the synthesis of tissue-type plasminogen activator (tPA). We demonstrate here that dihydrocytochalasin B (DHCB) treatment of differentiating F9 cells prevents the assembly of a structured actin cytoskeleton and generates a more rounded and stellate cell morphology. This morphological change is accompanied by the accumulation of the usually visceral endoderm-specific marker urokinase-type plasminogen activator (uPA) and an increase in tPA levels in comparison to untreated RACF9 controls. The increase in tPA accumulation is preceded by an increase in tPA mRNA levels. These effects are reversible, with a lag, when DHCB is removed, and PA accumulation can be stimulated within 24 h when differentiated cells are exposed to DHCB. Exposure to the microtubule disrupting agent colchicine has no effect on uPA or tPA accumulation. In addition, antibody directed against the beta 1 integrin subunit can also specifically elicit increased PA production. Thus disturbing the cytoskeleton and cytoskeleton associated substrate adhesions promotes PA accumulation.

Topics: Actins; Animals; Colchicine; Cyclic AMP; Cytochalasin B; Cytoskeleton; Electrophoresis, Polyacrylamide Gel; Endoderm; Extracellular Matrix; Fluorescent Antibody Technique; Gene Expression; Immune Sera; Integrins; Plasminogen Activators; RNA, Messenger; Teratoma; Time Factors; Tissue Plasminogen Activator; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

The regulatory effects of retinoic acid (RA) on retinoic acid receptor (RAR) alpha, beta and gamma mRNA were examined in the F9 mouse teratocarcinoma cells. Northern blot hybridization showed that RA regulated all three types of RAR gene expression. The different transcripts for each RAR alpha and beta were differentially regulated by RA. The maximum induction of the 2.8 kb RAR alpha and 3.3 kb RAR beta transcript levels by RA took longer than the induction of the 3.8 kb RAR alpha and 3.5 kb RAR beta transcript levels. The data suggest that these transcripts originated from different promoters. Short term treatment (< or = 24 hours) of RA induced both 3.1 and 3.3 kb RAR gamma transcripts. Long term treatment (> 24 hours) of RA resulted in the inhibition of 3.1 kb mRNA, whereas the 3.3 kb mRNA remained elevated. In addition, a new RAR gamma transcript of 2.9 kb was induced. In contrast to RAR alpha and beta, the effect on RAR gamma gene expression was irreversible. Cycloheximide did not prevent the effect of RA on RAR gene expression, whereas actinomycin D totally abolished the RA effect on the expression of all three receptor genes. The data suggest that the biological effects of RA may be constrained or augmented by differential regulation of its own receptor gene expression.

Topics: Animals; Carrier Proteins; Dactinomycin; Dose-Response Relationship, Drug; Gene Expression Regulation; Mice; Receptors, Retinoic Acid; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured

The expression of the genes in the human HOX2 locus has been studied during differentiation of two human neuroblastoma (SH-SY5Y and Kelly), a human glioblastoma (251-MG), and the murine F9 embryonal carcinoma cell lines. Cells were differentiated with retinoic acid (RA), or with RA together with dibutyral cyclic AMP (db-cAMP) and nerve growth factor (NGF) in order to assess the changes in the expression patterns of these homeobox genes during neuronal differentiation. We show that the genes of the HOX2 locus are expressed in a complex transcription pattern that varies with cell type. The two uninduced neuroblastoma cell lines show a similar pattern of expression for a number of HOX2 genes although the levels of expression are different for individual cell lines. The embryonal carcinoma cell line F9 expresses low levels of several HOX2 genes which is restricted to the 5' region of the HOX2 cluster. The glioblastoma cell line, 251-MG expresses almost all of the genes of the HOX2 locus. Differentiation of these cells modulates the expression of the HOX2 genes in a manner that is dependent upon the cell type as well as the differentiation factor. Differentiation affects both the level of HOX2 gene expression and the distribution of transcript sizes. In conclusion, our analysis reveals a complex pattern of expression for the genes of the HOX2 locus in neuronal and glial cells and suggests that the cell-specific expression of these genes may be correlated with the phenotypic differences that are observed between different neuronal and glial cell populations within the nervous system.

Topics: Animals; Blotting, Northern; Bucladesine; Cell Differentiation; Gene Expression; Genes, Homeobox; Glioma; Humans; Mice; Nerve Growth Factors; Neuroblastoma; Neurons; RNA, Neoplasm; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Protooncogene c-kit, a transmembrane tyrosine kinase receptor, was recently shown to map to the dominant white spotting locus (W) of the mouse. W mutations affect melanogenesis, gametogenesis, and hematopoiesis during development and in adult life. In order to determine the regulation of the c-kit gene in cell differentiation, we investigated its expression during the differentiation of F9 cells. Undifferentiated F9 cells and F9 cells treated with retinoic acid (RA) alone or dbcAMP alone showed little expression of c-kit mRNA if any. The subsequent addition of dbcAMP to F9 cells treated with RA markedly increased the expression of c-kit mRNA. Furthermore, the effect of dbcAMP on c-kit expression is reversible. In differentiated cells treated with RA, c-kit gene expression is induced by agents such as forskolin or theophylline, which are known to elevate cellular cAMP level. These results indicate that the expression of the c-kit gene is regulated by the level of intracellular cAMP in differentiated F9 cells induced by RA.

Topics: Animals; Blotting, Northern; Bucladesine; Cell Differentiation; Gene Expression Regulation, Neoplastic; Laminin; Mice; Precipitin Tests; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-kit; Proto-Oncogenes; Receptors, Cell Surface; Teratoma; Tissue Plasminogen Activator; Tretinoin; Tumor Cells, Cultured

Combination of chemotherapeutic drugs with agents that induce cell differentiation is a possible means of improving cancer chemotherapy. To explore this approach we used 4 cell lines established from the human teratocarcinoma-derived cell line PA-1; 2 retinoic acid (RA)-sensitive lines compared to 2 RA-resistant lines transformed by an activated N-ras oncogene. Equal numbers of colony-forming cells were exposed for 72 hr to 10(-6)M RA and subsequently to a range of concentrations of cisplatinum, etoposide or bleomycin. Enhanced cytotoxicity of cisplatin and etoposide (3- to 5-fold) was observed in the N-ras-transformed cell lines compared to the non-transformed lines. Treatment with RA caused an increase in the cytotoxicity of all 3 drugs to the 2 RA-sensitive cell lines. In contrast, a reduction of cytotoxicity was observed in the 2 N-ras-transformed lines. Our results indicate that sensitivity to cytotoxic agents can be increased by RA in RA-sensitive cells, but the opposite effect is seen in N-ras transformed, RA-resistant cells. Therefore, a general rationale for combination therapy with RA and cytotoxic drugs cannot be inferred.

Topics: Antineoplastic Combined Chemotherapy Protocols; Bleomycin; Cell Line, Transformed; Cisplatin; Drug Interactions; Drug Screening Assays, Antitumor; Etoposide; Female; Fibronectins; Genes, ras; Humans; Ovarian Neoplasms; Teratoma; Transfection; Tretinoin

Primitive endoderm in the peri-implantation mouse embryo differentiates into two separate lineages, visceral and parietal endoderm (VE and PE). F9 teratocarcinoma cells, when grown in suspension in the presence of retinoic acid (RA), differentiate into embryoid bodies (EBs) which can be used as a model system to study the spatially appropriate induction of VE- and PE-specific gene expression. We have used a whole mount in situ hybridization technique to follow the localization of VE-specific AFP and PE-specific tPA mRNAs during EB differentiation. We show that the putative endoderm-specific markers are localized to the endoderm in mature EBs, and that AFP mRNA is localized to the apical edge of the VE in RA EBs. Surprisingly, prior to localization of endoderm-specific markers at the periphery of EBs, these genes are expressed at low to moderate levels in all cells of the EB. Our data suggest that the establishment of endoderm in EBs is position dependent and not the result of sorting out of predetermined, randomly distributed cells. Our observations also suggest a two-step process for establishing endoderm-specific gene expression, involving up-regulation of transcripts throughout the EB prior to the restriction of their expression to the outer differentiating cell layer.

Topics: alpha-Fetoproteins; Animals; Blotting, Northern; Bucladesine; Cell Differentiation; Endoderm; Enzyme Induction; Enzyme-Linked Immunosorbent Assay; Mice; Neoplasm Proteins; Nucleic Acid Hybridization; Organoids; RNA, Messenger; RNA, Neoplasm; Teratoma; Tissue Plasminogen Activator; Tretinoin

Mouse embryonal carcinoma cell line PCC7-Mz1 can serve as a model of mammalian neural development [1989, J. Cell. Biol. 109, 2481-2493]. Upon exposure to all-trans retinoic acid (RA), Mz1 cells differentiate into a stable pattern of neurons, astroglia and fibroblasts whereas variants of the parental cell line either are restricted in their patterns of derivatives or do not respond at all to RA. Using gene probes specific for the alpha 1, alpha 2 and beta 2 isoforms of the retinoic acid nuclear receptor, we have studied by Northern blot analysis the expression of these transcription factors in uninduced and induced cells of clone Mz1 and in variants with different developmental potential. alpha 1-RAR is expressed constitutively in all variants independent of whether RA is present or not. Soon after addition of 10(-7) M RA, alpha 2-RAR is induced in RA-responsive cells reaching within a few hours a plateau level that remains unchanged throughout the developmental process. In contrast, the beta 2 isoform is expressed only transiently after RA-induction despite the continuous presence of RA. Other RAR isoforms are expressed only in trace amounts.

Topics: Animals; Blotting, Northern; Bucladesine; Carrier Proteins; Cell Differentiation; Cell Nucleus; Mice; Poly A; Receptors, Retinoic Acid; RNA; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

In most studies concerning organ-specific metastasis, different or selected lines are compared with one another. Here we report results with the same cell line (the F9 murine teratocarcinoma) which can be directed towards liver or lung, depending on whether or not it is treated with retinoic acid and cyclic AMP. Thus, organ-specific colonization by tail-vein-injected murine F9 teratocarcinoma cells shows a particular pattern: unselected, undifferentiated F9 cells preferentially colonize the liver of the syngeneic animal. The lungs, the first capillary bed encountered by cells thus injected, are only very rarely colonized. By contrast, the lungs become the main target organ of F9 cells induced to differentiate by treatment with retinoic acid and cyclic AMP. We have recently shown that liver colonization by undifferentiated F9 cells correlated with the adhesiveness of the cells (higher to fibronectin and liver-derived extracellular matrix than to laminin and lung-derived extracellular matrix) as well as with their growth response to organ-derived extracts (no response with lung extracts and good response with liver extracts). The data reported below indicate that induction of differentiation causes at most a decreased adhesiveness of F9 cells to all the substrata tested (laminin, fibronectin, type-IV collagen, organ-derived extracellular matrix), suggesting that the shift in organ colonization observed with differentiated F9 cells is not due to an enhancement of the specific adhesion to the lung matrix. On the other hand, differentiated cells, but not undifferentiated ones, were able to respond to growth stimulation mediated by lung-derived extracts, thereby implying a relevant role for organ-specific growth stimulation in lung colonization by differentiated F9 cells.

Topics: Animals; Cell Adhesion; Cell Division; Extracellular Matrix Proteins; Lung Neoplasms; Mice; Organ Specificity; Teratoma; Tretinoin; Tumor Cells, Cultured

It is well-established that fibroblast growth factors (FGFs) participate in mesoderm formation and patterning in the developing embryo. To identify cells in mammalian embryos that produce and/or respond to FGFs, we utilized the F9 teratocarcinoma cell system. Undifferentiated F9 cells resemble inner cell mass (ICM) cells of the mouse blastocyst by several criteria including having a characteristic high nuclear to cytoplasmic ratio and by their expression of stage-specific embryonic antigens. F9 stem cells differ from ICM cells by their low spontaneous rate of differentiation and their differentiation potential. ICM cells are heterogeneous with a proportion of the cells maintaining totipotency. In contrast, F9 stem cells appear capable of forming only endodermal derivatives. Retinoic acid (RA) treatment of F9 stem cells is required for them to differentiate, and under different culturing conditions the F9 cells will form either extraembryonic parietal or visceral endoderm. We have previously shown that FGF is synthesized by F9 parietal endoderm, but not by F9 stem cells. Our present study demonstrates that F9 aggregate cultures that contain visceral endoderm cells produce cell-associated-heparin-binding mitogens for 3T3 and endothelial cells, factors with characteristics of FGFs. Furthermore, our studies detect endothelial cell-mitogens within the extracellular matrix (ECM) of F9 parietal endoderm cells, not detected within F9 stem cell 'matrices'. Parietal endoderm cell matrix mitogens could be removed by prior treatment of the ECM with buffers containing heparin or 2 M NaCl, and could be neutralized by basic FGF antibodies.

Topics: Animals; Cell Differentiation; Embryo, Mammalian; Embryo, Nonmammalian; Endoderm; Extracellular Matrix; Fibroblast Growth Factors; Stem Cells; T-Lymphocytes; Teratoma; Tretinoin; Tumor Cells, Cultured

Retinoic acid-induced differentiation of mouse P19 teratocarcinoma cells is accompanied by alterations in the level of E2F transcription factor. P19 stem cells contain free, uncomplexed E2F and an E2F complex termed E2F/stem. This stem cell complex is a heterotrimeric protein aggregate consisting of E2F transcription factor, E2F-binding protein (E2F/bp1), and cyclin A. Retinoic acid treatment converts P19 stem cells into differentiated neurons, glial cells, and fibroblasts. The presented experiments clearly show that the level of uncomplexed E2F gradually decreases upon differentiation, and fully differentiated cells do not contain free E2F. In addition, the stem cell-specific E2F aggregate is converted into a smaller complex, termed E2F/diff. This smaller complex, which is specific for differentiated cells, does not contain cyclin A and consists of E2F transcription factor associated with E2F/bp1. Finally, the role of E2F complexes in the cessation of cell proliferation, which accompanies P19 cell differentiation, is discussed.

Topics: Animals; Base Sequence; Carrier Proteins; Cell Cycle Proteins; Cell Differentiation; Cell Division; Cyclins; DNA-Binding Proteins; E2F Transcription Factors; Gene Expression Regulation, Neoplastic; Mice; Molecular Sequence Data; Neoplasm Proteins; Protein Binding; Retinoblastoma-Binding Protein 1; Teratoma; Transcription Factor DP1; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The amount of the heterotrimeric G protein subunit G alpha i2 decreases after the induction of F9 teratocarcinoma cells to become primitive endoderm in the presence of retinoic acid (RA). The reduction of the G alpha i2 protein in F9 cells by antisense RNA expression was associated with (i) loss of receptor-mediated inhibition of adenylyl cyclase; (ii) decreased cell doubling time; (iii) induction of a primitive, endoderm-like phenotype in the absence of RA; and (iv) production of the differentiation marker tissue-type plasminogen activator. Expression of a constitutively active, mutant G alpha i2 blocked RA-induced differentiation. These data suggest the involvement of G alpha i2 in the control of stem cell differentiation and provide insight into the involvement of G proteins in growth regulation.

Topics: Adenylyl Cyclases; Animals; Base Sequence; Cell Differentiation; Gene Expression; GTP-Binding Proteins; Mice; Molecular Sequence Data; RNA, Antisense; Teratoma; Thrombin; Tretinoin; Tumor Cells, Cultured

Leukemia inhibitory factor (LIF) is a cytokine previously shown to maintain pluripotent embryonic stem cells in their undifferentiated state. We have examined the effects of LIF in nullipotent embryonal carcinoma cell lines, and have found that LIF blocks differentiation induced by retinoic acid and at low temperature in OTF9 cells. LIF did not block differentiation in a parent F9 cell line. For OTF9 cells, LIF acts early in differentiation, inhibiting the appearance of parietal endoderm-type product cells. However, it acts subsequent to retinoic acid, and at least one early retinoic acid-induced event is unaltered in the presence of LIF. This finding provides both a means of dissecting the cascade of events leading to EC cell differentiation, and a well-characterised target cell type for studying the mechanism of action of LIF.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Line; Growth Inhibitors; Interleukin-6; Kinetics; Leukemia Inhibitory Factor; Leukemia Inhibitory Factor Receptor alpha Subunit; Lymphokines; Mice; Molecular Sequence Data; Oligodeoxyribonucleotides; Oncogenes; Polymerase Chain Reaction; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myb; Receptors, Cytokine; Receptors, Immunologic; Receptors, OSM-LIF; Teratoma; Tretinoin

A mutant embryonal carcinoma cell line, NR1-6, was created through retroviral insertion. We have previously reported that due to a single insertional event the mutant cell line is altered in regard to both its morphology and its tumorigenic capacity. We now report that this same cell line is also aberrant in its differentiative potential following exposure to the morphogen retinoic acid (RA). Unlike the parental NR1-0 cells, the NR1-6 cells apparently do not respond to RA by elaborating primitive endodermal derivatives in monolayer culture but rather appear morphologically to differentiate into mesodermal cells. This hypothesis is substantiated by the observation that RA treatment induces the transcription of both Endo A and B mRNA in parental but not mutant cells. No differences have been observed in the transcription of other RA sensitive markers such as c-myc, tissue plasminogen activator, collagen type IV, and laminin. In addition, the mutant cells are quantitatively much more sensitive to RA induction than are the parental cells, achieving full differentiation within 72 h of treatment with 10(-10) M RA. The parental cells, in contrast, will only differentiate at concentrations of 10(-5) or 10(-6) M RA, following 5 to 7 days of treatment. A spontaneous revertant cell line, which was isolated from an NR1-6 population and lacks the retroviral insert, is identical to the parental population in all parameters. Therefore, these data indicate that, in this case at least, a single genetic locus is involved in regulating both the qualitative and quantitative response of EC cells to RA-induced differentiation, as well as their morphology and tumorigenic potential.

Topics: Animals; Blotting, Northern; Cell Differentiation; Collagen; Genes, Viral; Laminin; Mice; Mutagenesis, Insertional; Phenotype; Proto-Oncogene Proteins c-myc; Retroviridae; RNA, Messenger; Teratoma; Tissue Plasminogen Activator; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Activin and retinoids, which are involved in the induction and regulation of the early differentiation process in vertebrate embryogenesis, synergistically increased the amount of c-jun mRNA in P19 embryonal carcinoma cells, but activin alone had no effect. Among the retinoids, all-trans-retinoic acid most effectively increased c-jun mRNA. Activin (lng/ml) was sufficient to induce the synergistic increase of c-jun mRNA in P19 EC cells with all-trans-retinoic acid. The synergistic increase of the amount of c-jun mRNA by their cooperative action may be important in vertebrate development.

Topics: Activins; Animals; Cell Line; Drug Synergism; Gene Expression Regulation, Neoplastic; Genes, jun; Growth Substances; Inhibins; Kinetics; Mice; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin

It is well documented that activated macrophages, but not nonactivated ones, kill tumor cells in vitro without damaging normal cells. We, however, have previously shown that embryo-derived teratocarcinoma cells (F9, P19, PCC4) are efficiently killed by nonactivated macrophages as well as by activated ones. Whereas other tumor cells are killed extracellularly by macrophages, we found that F9 teratocarcinoma cells are phagocytosed alive by macrophages and subsequently killed intracellularly by a process dependent on intact lysosomal function. Neither the H-2 antigens nor the mRNAs for the alpha-chain and beta 2-microglobulin are detectable in embryo-derived teratocarcinoma cells. An obvious explanation for this unique killing is that the nonactivated macrophages recognize and kill these cells due to their lack of class I MHC antigen expression, assuming that class I MHC gene products on the target cells switch off the cytolytic machinery of nonactivated macrophages. Our present findings demonstrate that there is no correlation between H-2 antigen expression on tumor cells and their susceptibility to killing by macrophages. Retinoic acid-differentiated F9 cells and P19 cells expressing H-2 antigen after exposure to MAF (IFN-gamma) were sensitive to the killing by nonactivated macrophages. Hybrids that arose from fusion of P19 teratocarcinoma cells with embryonal normal fibroblasts (C57BL/6), which displayed the morphology of embryonal carcinoma stem cells and expressed H-2 antigens, were also sensitive to the killing by nonactivated macrophages. On the other hand, the H-2-negative testicular 402AX teratocarcinoma cells and K1735P melanoma cells were both resistant to the killing by nonactivated macrophages. We concluded that the unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not related to a lack of H-2 antigen expression.

Topics: Animals; Cell Death; Cell Differentiation; Cytotoxicity Tests, Immunologic; H-2 Antigens; Lysosomes; Macrophages; Mice; Mice, Inbred C57BL; Phagocytosis; Teratoma; Tretinoin; Tumor Cells, Cultured

We have recently reported a protein sequence deduced from the retinoic acid (RA)-inducible mRNA J6 as a novel serine protease inhibitor (serpin). In this study we have reported that the J6 serpin gene is 7.7 kilobases in length and consists of five exons with an additional option. Comparison of the organization of the J6 gene and other serpin genes reveals that the structure of the J6 gene is different from the reported four serpin gene groups. Nonetheless, intron B of the J6 gene and members of the alpha-antitrypsin gene group are at the equivalent positions, suggesting that the J6 gene is more closely related to the members of alpha-anti-trypsin gene group than other serpins. To identify the RA response region, we have further examined the nucleotide sequence of the 1-kilobase 5'-flanking region of the J6 gene. The DNA sequence from position -1050 to -738 is essential for the gene activation by RA as revealed by the stable transfection experiments. Within this region, present are four GA-GATAG motifs which are the known binding sites for GATA transcription factor family. Interestingly, there is a potential heat shock element with alternate arrays of blocks XGAAX and XTTCX spanning from -88 to -59, indicating that the J6 gene perhaps is heat-inducible.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Line; Cloning, Molecular; DNA, Neoplasm; Exons; Genes; Glycoproteins; HSP47 Heat-Shock Proteins; Introns; Mice; Molecular Sequence Data; Oligodeoxyribonucleotides; Promoter Regions, Genetic; Restriction Mapping; Sequence Homology, Nucleic Acid; Serpins; Teratoma; Tretinoin

Sodium butyrate (NaB) can induce teratocarcinoma cell differentiation as retinoic acid (RA). However, the function of these two agents seems to be a little different [Kosaka et al., Exp Cell Res, 192:46-51, 1991]. F9 cells treated with NaB synthesize both tissue-type (tPA) and urokinase-type (uPA) plasminogen activator, though RA induces only tPA production. Urokinase-type PA is demonstrated to exist in association with membrane and to localize its activity to the close environment of the cell surface. This may cause the specific cell morphology and characteristics of differentiated F9 cells induced with NaB.

Topics: Animals; Butyrates; Butyric Acid; Cell Differentiation; Cell Membrane; Kinetics; Mice; Teratoma; Tissue Plasminogen Activator; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

H-2RIIBP is a member of the nuclear hormone receptor superfamily that binds to the region II enhancer of major histocompatibility complex (MHC) class I genes. The binding occurs through the GG(T/A)CA motif present also in many other genes. The role of H-2RIIBP in developmental regulation of MHC class I genes has been studied in undifferentiated N-Tera2 embryonal carcinoma cells by transient cotransfection of an expressible H-2RIIBP plasmid and a chloramphenicol acetyltransferase reporter gene linked to the MHC class I promoter. Transfection of the expression plasmid led to production of H-2RIIBP transcripts and enhanced MHC class I promoter activity in cells that were treated with retinoic acid but not yet differentiated. Retinoic acid concentrations required for transactivation overlapped with those capable of inducing morphological differentiation and expression of endogenous MHC class I genes in these cells. This enhancement was mediated by region II, as a heterologous thymidine kinase promoter driven by region II also served as a target for H-2RIIBP transactivation. Deletion of the bulk of the DNA-binding domain or the ligand-binding domain of H-2RIIBP, but not of the N-terminal domain, abolished transactivation, indicating that the former two domains are critical for the enhancement. Moreover, H-2RIIBP transactivation exhibited a strict cell-type restriction. As observed in other cell lines, N-Tera2 cells that had undergone differentiation failed to elicit transactivation, suggesting that H-2RIIBP acts in concert with a cofactor expressed in undifferentiated N-Tera2 cells that requires retinoic acid for its function. These results suggest that H-2RIIBP can function as a developmentally specific transcription factor for MHC class I genes.

Topics: Base Sequence; Cell Differentiation; DNA Mutational Analysis; DNA-Binding Proteins; Enhancer Elements, Genetic; Gene Expression Regulation; Genes, MHC Class I; Humans; In Vitro Techniques; Molecular Sequence Data; Nuclear Proteins; Polymerase Chain Reaction; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; Structure-Activity Relationship; Teratoma; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Treatment of mouse embryonal carcinoma (F9) cells with retinoic acid, an inducer of F9 cell differentiation, greatly increased the level of mRNA specific to one of the heat-shock proteins (HSP86). Experiments including the one employing differentiation-resistant mutant F9 cells suggested that the increase represents early molecular events associated with the embryonal differentiation. The increased HSP86 mRNA declined to the original level during further incubation. The presence of cyclic AMP, which stimulates conversion of the retinoic acid-induced primitive endoderm cells to parietal endoderm cells, prevented the decline. These results suggest that not only the elevation of HSP86 mRNA level represents early molecular events in F9 cell differentiation but also that sustaining the elevated level (by cyclic AMP) is associated with further differentiation of the embryonal cells.

Topics: Animals; Blotting, Northern; Bucladesine; Cell Differentiation; Endoderm; Gene Expression; Heat-Shock Proteins; In Vitro Techniques; Mice; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

We used a series of cell clones from a human teratocarcinoma cell line, PA-1, to study the effect of transformation by an activated N-ras oncogene on the expression of genes involved in retinoic acid (RA)-induced differentiation and growth regulation. Recently, it has been shown that expression of human HOX 2 genes is sequentially activated by RA beginning from Hox 2.9 at the 3' end of the HOX 2 cluster (A. Simeone, D. Acampora, L. Arcioni, P. W. Andrews, E. Boncinelli, and F. Mavilio, Nature [London] 346:763-766, 1990). We now report that six different genes of the cluster HOX 1 are sequentially induced by RA in a similar temporal pattern, beginning with genes at the 3' end of the cluster. However, in N-ras-transformed cell clones, RA-induced expression of these homeobox genes is delayed. Hox 1.4 and Hox 1.3, genes abundantly induced in nontransformed clones after 3 days of RA treatment, are expressed in N-ras-transformed cells only after 10 days of RA treatment. At this time, the cells' growth is arrested at very high density, and no differentiated morphologic characteristics are observed. Constitutive expression of a transfected Hox 1.4 gene under the control of a simian virus 40 promotor leads to differentiated cell morphology similar to that of the RA-induced phenotype and restores the growth-inhibitory effects of RA in N-ras-transformed cells. These observations provide evidence that enhanced proliferation in N-ras-transformed cells compromises teratocarcinoma cell differentiation by a mechanism that transiently suppresses homeobox gene induction and implies a central role for homeobox genes in RA-induced cell differentiation. We conclude that stimulation of a putative growth factor signal pathway, associated with ras-induced proliferation, transiently suppresses the induction of transcription factors functionally involved in cell growth and differentiation.

Topics: Amino Acid Sequence; Base Sequence; Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cloning, Molecular; DNA, Recombinant; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Genes, ras; Humans; Molecular Sequence Data; Multigene Family; Oligonucleotide Probes; Polymerase Chain Reaction; RNA, Neoplasm; Teratoma; Transcriptional Activation; Transfection; Tretinoin

A pluripotent human embryonal carcinoma (EC) clone, NT2/D1, which was derived from the Tera-2 cell line, was induced to differentiate into cells with neuronal-like cell morphology by treatment with berberine, an alkaloid derived from a Chinese herbal medicine (Huang Lien). As early as one day after 24-hour treatment of cells with berberine at a nontoxic dose of 0.1 mg/ml in culture medium, the cells started to show morphologic changes, developing into terminally differentiated neuronal-like cells with long, inter-connecting network cellular structures. This process was much faster as compared with that induced by treatment with retinoic acid (RA), which took at least several days to develop. Unlike RA, berberine could not induce murine EC cell line, F9, to differentiate into endodermal cells. It was also found that although the NT2/D1 cell clone exhibited amplification and enhanced mRNA expression of c-Ki-ras2 gene as did the parent cell line, a marked down-regulation of c-Ki-ras2 mRNA expression was observed. However, there was no change in actin mRNA expression even after differentiation had occurred. Thus, morphologic differentiation of EC cells into neuronal-like cells was found to be associated with down-regulation of a protooncogene which plays some definite role in oncogenesis. The mechanism by which berberine induces differentiation in these cells needs further investigation.

Topics: Actins; Berberine; Cell Differentiation; Cell Division; Cell Line; Down-Regulation; Drugs, Chinese Herbal; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Male; Neurons; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured

To investigate the possible involvement of topoisomerases in embryonal differentiation, we examined the effect of topoisomerase inhibitors on the in vitro differentiation of mouse embryonal carcinoma F9 cells. We found that camptothecin, teniposide (VM-26), or genistein, specific inhibitors of topoisomerases, induced morphological as well as biochemical changes (production of tissue plasminogen activator, synthesis of laminin, and disappearance of stage-specific embryonic antigen 1) specific to F9 cell differentiation. Since these changes were indistinguishable from those observed in F9 differentiation induced by retinoic acid (plus dibutyryl cyclic AMP), it was suggested that inhibition of cellular topoisomerase activities triggered F9 cell differentiation into parietal endoderm-like cells in the same manner as retinoic acid (plus dibutyryl cyclic AMP). Experiments using differentiation-resistant mutant F9 cell lines, however, indicated that the molecular cascade involved in topoisomerase inhibitor-induced differentiation involves different steps from those functioning in the retinoic acid-induced differentiation cascade.

Topics: Animals; Blotting, Western; Camptothecin; Cell Differentiation; Cell Line; DNA Topoisomerases, Type I; Dose-Response Relationship, Drug; Genistein; In Vitro Techniques; Isoflavones; Laminin; Lewis X Antigen; Mice; Protein-Tyrosine Kinases; Teniposide; Teratoma; Tissue Plasminogen Activator; Topoisomerase I Inhibitors; Tretinoin

We have been studying the effect of oncogenes on differentiation using the human ovarian teratoma-derived cell line PA-1. From this study we have characterized variants representing four stages relevant to multistage carcinogenesis, two non-tumorigenic and two tumorigenic. The two non-tumorigenic cell variants differ in that one is resistant to transformation by ras oncogenes whereas the other can be transformed to tumorigenicity. When these non-tumorigenic PA-1 variants are treated with retinoic acid (RA), a morphogen, they stop dividing, begin to express homeobox genes, and change in morphology. Transfection of an activated N-ras oncogene into ras-resistant non-tumorigenic PA-1 cells does not alter the RA responsiveness of the cells, indicating that expression of the activated oncogene is not sufficient for blocking RA-induced differentiation. Spontaneous activation of an N-ras oncogene leading to tumorigenic transformants and gene transfer-induced N-ras transformants are resistant to these effects of RA. However, another spontaneous transformant of PA-1 cells that does not contain an activated N-ras is responsive to RA. We prepared somatic cell hybrids of the RA-non-responsive, N-ras-transformed and tumorigenic PA-1 cell and the RA-responsive, ras-resistant non-tumorigenic PA-1 cell; the hybrid cell lines continue to express the oncogene but are non-tumorigenic. These non-tumorigenic hybrids are responsive to RA with regard to morphological changes, growth arrest and induction of homeobox gene expression. Tumorigenic revertants of these hybrids arise as a result of the loss of some chromosomes; these hybrid cells express the oncogene but have lost RA responsiveness. These results indicate that tumorigenic transformation in general is not sufficient to induce RA resistance, and resistance to differentiation may be oncogene-specific. In addition, the expression of an activated N-ras oncogene alone is insufficient to induce resistance to RA and ras-induced tumorigenicity is necessary. Therefore, some feature of cellular metabolism that is altered by and discordantly segregates with tumorigenic transformation controls responsiveness to RA. This controlling element is presumably a tumor suppressor.

Topics: Blotting, Northern; Cell Differentiation; Cell Division; Chromosome Banding; Female; Gene Expression; Genes, Homeobox; Genes, ras; Humans; Hybrid Cells; Karyotyping; Ovarian Neoplasms; RNA, Neoplasm; Teratoma; Transfection; Tretinoin

Topics: Carrier Proteins; Cell Line; Dose-Response Relationship, Drug; Embryonal Carcinoma Stem Cells; Gene Expression Regulation; Genes, Homeobox; Humans; Neoplastic Stem Cells; Receptors, Retinoic Acid; Teratoma; Tretinoin

F9 teratocarcinoma cells differentiate into parietal endodermlike cells when treated with retinoic acid (RA) and cyclic AMP (cAMP). We have previously found that F9 cells can be induced to differentiate by treatment with cAMP in the absence of RA. In the course of determining why other investigators had failed to observe cAMP-induced differentiation, we found that the growth medium played an important role in determining the response of F9 cells to differentiating agents. When F9 cells were grown in minimal essential medium (MEM) and treated with either 8-bromo-cAMP (8BrcA) + 1-methyl, 3-isobutylxanthine (MIX), or dibutyryl cAMP (DBcA) + theophylline (T), they differentiated to parietal endodermlike cells without any requirement for exogenous RA. However, when F9 cells were grown in Dulbecco's modified Eagle's medium (DME), DBcA/T failed to induce differentiation alone and required exogenous RA to induce formation of parietal endoderm-like cells. 8BrcA/MIX alone was still able to induce some differentiation, although the extent was not as great as those cells grown in MEM. These results could not be explained by the different growth-promoting properties of the two culture media because there was no difference in the doubling time of F9 cells grown in either medium. Likewise, RA and cAMP both inhibited growth to the same extent in either medium. Inasmuch as almost all published reports on F9 cell differentiation have used DME as a growth medium, and RA with or without DBcA/T as the differentiating agents, these studies would not have had the appropriate conditions to detect the cAMP-induced differentiation of F9 cells.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Animals; Butyrates; Cell Division; Collagen; Culture Media; Cyclic AMP; Growth Substances; Humans; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured

When P19 embryonal carcinoma (EC) cells were cocultured with cells from one of several established visceral-endoderm-like cell lines, the EC cells were rapidly induced to aggregate and differentiate, into cell types including mesoderm-derived cardiac and skeletal muscle. Neither parietal-endoderm- nor mesoderm-like cell lines induced aggregation or differentiation of EC cells in coculture, although a cell line with both parietal and visceral endoderm characteristics induced aggregation but not differentiation. Also, without the feeder cells aggregates of P19 failed to differentiate, provided that serum in the culture medium had been previously passed over dextran-coated charcoal to remove lipophilic substances, which may include endogenous retinoids. All experiments were carried out using serum treated in this way. Taken together, the results demonstrated that aggregation was necessary, but not sufficient, to make P19 EC cells differentiate. Direct contact between the two cell types was not necessary, since even when separated by an agar layer in cocultures, aggregates of P19 still differentiated. Medium conditioned by cells of the END-2 line, a visceral-endoderm-like derivative of P19, was particularly potent in inducing endodermal and mesodermal differentiation of single P19 aggregates, confirming the involvement of a diffusible factor secreted specifically by visceral-endoderm-like cells in this process.

Topics: alpha-Fetoproteins; Animals; Antibodies; Antibodies, Monoclonal; Cell Aggregation; Cell Communication; Cell Differentiation; Cell Line; Endoderm; Fibronectins; Fluorescent Antibody Technique; Keratins; Laminin; Low Density Lipoprotein Receptor-Related Protein-2; Membrane Glycoproteins; Mice; Myosins; Teratoma; Tretinoin

All-trans retinoic acid (RA) induces alkaline phosphatase (ALP) activity by 3-8-fold in murine F9 teratocarcinoma cells, in parallel with their differentiation towards primitive endoderm. The elevation of ALP activity is associated with increases in the amounts of liver/bone/kidney-type ALP protein and the respective transcript. These effects of RA are due to activation of ALP gene transcription rather than to an increase in the half-life of the mRNA. Induction of ALP mRNA does not require de novo protein synthesis, since it is not blocked by treatment with cycloheximide. Dibutyryl cyclic AMP, which is known to induce further differentiation of F9 cells from the primitive to the parietal endoderm, blocks the induction of ALP mRNA by RA.

Topics: Alkaline Phosphatase; Animals; Blotting, Northern; Blotting, Western; Bone and Bones; Bucladesine; Cycloheximide; Dactinomycin; Enzyme Induction; Gene Expression Regulation, Enzymologic; Kidney; Liver; Mice; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Human Tera 2 embryonal carcinoma cells switch gradually from rapidly growing undifferentiated cells to almost nonproliferating cells during retinoic acid (RA)-induced neuronal differentiation. This process is associated with the increased expression of type 1 plasminogen activator inhibitor (PAI 1) mRNA, and the secreted inhibitor is immobilized to the pericellular area. Furthermore, the differentiation is accompanied by a decrease in the amount of both the secreted tissue-type PA (tPA) and the mainly cell-associated urokinase-type PA (uPA) activity. In RA-differentiated cells, uPA becomes localized at the vinculin-rich cell-substratum adhesion sites. Fibroblast growth factor activity has been associated with various events during embryonal growth and with the regulation of proteolytic enzymes. A short-term treatment of the undifferentiated Tera 2 cells with basic fibroblast growth factor (bFGF) increases uPA mRNA levels and the cell-associated uPA activity, whereas the secretory tPA activity decreases. bFGF induces PAI 1 mRNA expression in the undifferentiated cells, but unlike PAI 1 protein after RA-treatment, the inhibitor does not accumulate around the cells but is released in the medium. A similar exposure to bFGF has less effect on the RA-differentiated Tera 2 cells. Under these conditions bFGF treatment leads to an increase in the amounts of PAI 1 and uPA mRNAs, but no changes in the localization of these components can be seen. Differentiation of human embryonal carcinoma cells is thus connected with an altered response to bFGF.

Topics: Blotting, Northern; Cell Differentiation; Cytoskeletal Proteins; Electrophoresis, Polyacrylamide Gel; Fibrinolysis; Fibroblast Growth Factors; Humans; Immunohistochemistry; Plasminogen Activators; Plasminogen Inactivators; RNA, Messenger; Teratoma; Tissue Plasminogen Activator; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator; Vinculin

The Egr-1 gene (zfp-6) encodes a 'zinc finger'-type transcription factor that is one of the early growth response genes induced, together with c-fos proto-oncogene, in many cell types. Our earlier work indicated that Egr-1 and c-fos may also play roles in differentiation and we now present data to show some features of their regulation. Transcriptional regulation accounts at least partly for the increased steady-state levels of Egr-1 mRNA in differentiating teratocarcinoma cells; this rate increases threefold over the 7-10 days of differentiation of P19 embryonal carcinoma cells with both 0.5% DMSO (to give predominantly cardiac muscle) and 1 microM retinoic acid (to give nerve and glial cells). The stability of Egr-1 transcripts remains the same (T1/2 = 90 min) in undifferentiated EC and differentiated cell products. In contrast, transcripts for c-fos are barely detectable in EC cells and increase 20-fold during differentiation. The basis for this is a marked increase in stability of c-fos mRNA after differentiation. The protein products of both genes parallel the steady-state levels of their mRNAs, but both proteins become more stable in differentiated cells. This is particularly marked for c-Fos protein, which appears as a distinct 58 kDa species in terminally differentiated P19 cells. Both Egr-1 and c-Fos proteins remain at high constitutive levels in differentiated cells indicating a distinct role for these transcription factors, For instance, it appears that this form of Fos protein may not repress the synthesis of the Egr-1 gene as it does during transient expression of serum-stimulated genes.

Topics: Animals; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Gene Expression Regulation, Neoplastic; Mice; Precipitin Tests; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Up-Regulation; Zinc Fingers

Retinoic acid (RA), a developmental morphogen, causes activation of a transcript of an endogenous retrovirus-related element in the human teratocarcinoma-derived cell line PA-1. This provirus is defective, and the provirus-related sequences exist as multicopy elements (more than 20 copies) in human DNA. This is the first human endogenous retroviral mRNA that is known to be transcriptionally activated by RA. The nucleotide sequence of the 3,357 bp of this viral cDNA was determined and shows a strong homology to the type C-related human endogenous retroviral proviruses ERV3 and 4-1. This cDNA contains 'R-U5-delta pol-env-U3-R sequences of the provirus. Adjacent to the putative 5' long terminal repeat of this provirus there is an 18-bp sequence complementary to the 3' end of isoleucine tRNA. We named this RA-responsive virus RRHERV-I.

Topics: Base Sequence; Cell Line; DNA, Bacterial; DNA, Viral; Humans; Molecular Sequence Data; Proviruses; Reading Frames; Retroviridae; Teratoma; Transcription, Genetic; Tretinoin

Glycolipids synthesized by the mouse teratocarcinoma F9 cells and F9 cells (RA/F9 cells) induced to differentiate by a 3-day treatment with 0.1 microM all-trans-retinoic acid were analyzed. Both F9 cells and RA/F9 cells were incubated in media containing either D-[6-3H]galactose or D-[6-3H]glucosamine; the metabolically-radiolabeled glycolipids were isolated and the oligosaccharides were released from the glycolipids by ozonolysis and alkali fragmentation. From both cells, a single major pentasaccharide was isolated from the mixture of neutral [3H]oligosaccharides by affinity chromatography on a column of immobilized Helix pomatia agglutinin. The structure of this oligosaccharide was analyzed by methylation analysis and specific exoglycosidase treatments and identified as the Forssman pentasaccharide alpha-D-GalpNAc-(1----3)-beta-D-GalpNAc-(1----4)-alpha-D-Galp-(1----4)-b eta-D- Galp-(1----4)-D-Glc. There was a 3-4-fold decreased amount of the Forssman pentasaccharide from RA/F9 cells relative to F9 cells. In contrast, there were no major differences between these cells in the levels of globoside, the precursor to Forssman glycolipid. To investigate the basis for the decline in Forssman glycolipid synthesis upon differentiation, the activity of UDP-D-Gal-NAc:GbOse4Cer alpha-(1----3)-N-acetyl-D-galactosaminyltransferase (Forssman synthase) was determined in extracts of both the F9 and RA/F9 cells. The specific activity of Forssman synthase was approximately 70% lower in differentiated relative to the nondifferentiated cells. These data demonstrated that F9 cells synthesize authentic Forssman glycolipid, and that its expression and the activity of Forssman synthase were decreased following induced cellular differentiation.

Topics: Animals; Carbohydrate Sequence; Cell Differentiation; Chromatography, Affinity; Forssman Antigen; Globosides; Lectins; Mice; Molecular Sequence Data; Oligosaccharides; Teratoma; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA), the natural acid derivative of vitamin A, can induce the differentiation of some cell lines, such as the murine P19 teratocarcinoma cell line. RA can alter the expression of specific genes and the level of the corresponding protein. This report describes the effect of RA on the level of the alkaline phosphatase (ALP) mRNA and protein in P19 teratocarcinoma cells. RA caused a rapid, dose-dependent, and protein synthesis-dependent induction of ALP activity. The increased enzyme activity was detected 4 h after initiation of treatment and maximum induction of ALP activity required 48 h of RA exposure. Increased enzymatic activity was coincidental with increased levels of both a 67-kDa ALP protein and ALP mRNA. By Northern (RNA) blot analysis the increase of a 2.7-kilobase ALP mRNA was observed within 3 h of RA treatment. The RA-induced enhanced ALP mRNA level did not appear to be mainly due to the stabilization of preexisting mRNA, but rather to an increase in transcription of the ALP gene.

Topics: Alkaline Phosphatase; Animals; Blotting, Northern; Blotting, Western; Cell Differentiation; Cycloheximide; Dactinomycin; Dose-Response Relationship, Drug; Enzyme Induction; Gene Expression; In Vitro Techniques; Mice; RNA, Messenger; Teratoma; Time Factors; Transcription, Genetic; Tretinoin

All-trans-retinoic acid, an endogenous morphogen, induced neuronal differentiation of P19 murine embryonal carcinoma cells. Peak differentiation, as judged by the elaboration of neuronal processes, occurred 8 days after exposure of the cells to 0.5 mM retinoic acid, a concentration known to induce neuronal differentiation. An examination of the expression of the extracellular matrix receptors, integrins, during this retinoic acid-induced differentiation period, demonstrated a specific and strong induction of expression of two polypeptides (130 and 115 kDa) immunoprecipitated with an anti-human vitronectin receptor antiserum. The expression of a 90-kDa polypeptide, also immunoprecipitating with this antiserum was induced as well, but to a much smaller extent. The expression of a 96-kDa polypeptide immunoprecipitated by this antiserum and present in the untreated cells was not induced by retinoic acid. The increase in the expression of these polypeptides paralleled the neuronal differentiation of the P19 embryonal carcinoma cells. The expression of these integrins was not induced in a variant of the P19 cells, P19RAC65, which are resistant to differentiation induction by retinoic acid. Utilizing integrin subunit-specific anti-cytoplasmic peptide antibodies together with immunoprecipitation and Western blot analysis, the 130- and 115-kDa polypeptides were identified as the integrin alpha v and beta 1 subunits, respectively. The 90-kDa polypeptide, also induced by retinoic acid, was identified as beta 3, whereas the identity of the uninduced 96-kDa polypeptide remains unclear as yet. Peptide map analysis of deglycosylated polypeptides demonstrated that the 90- and 96-kDa polypeptides are distinct proteins and that the 115-kDa polypeptides immunoprecipitated with either anti-alpha v or anti-beta 1 antibodies are identical, further establishing that the 115-kDa polypeptide associating with alpha v is beta 1. The retinoic acid-induced expression of beta 1 occurred at the level of mRNA expression which also paralleled neuronal differentiation, but peaked slightly ahead of the cell surface expression of beta 1. The expression of other beta 1-associated alpha subunits was not induced by retinoic acid in these cells. These data demonstrate that retinoic acid strongly induces the expression of the integrin heterodimer alpha v beta 1 and also, to a smaller extent, the expression of alpha v beta 3. The retinoic acid-induced, high level surface expression of the alpha v

Topics: Amino Acid Sequence; Animals; Blotting, Western; Cell Line; Chromatography, Affinity; Gene Expression; Immunoblotting; Integrins; Macromolecular Substances; Mice; Molecular Sequence Data; Molecular Weight; Neurons; Oligopeptides; Peptide Mapping; RNA, Messenger; Teratoma; Tretinoin

The recombination activating genes, RAG-1 and RAG-2, are likely to encode components of the V(D)J site-specific recombination machinery. We report here the detection of low levels of the RAG-1 transcript in the murine central nervous system by polymerase chain reaction, in situ hybridization, and Northern blot analyses. In contrast, an authentic RAG-2 transcript could not be detected reproducibly in the central nervous system. The RAG-1 transcript was found to be widespread in embryonic and postnatal neurons, with transcription being most apparent in regions of the postnatal brain with a high neuronal cell density (the cerebellum and the hippocampal formation). The results suggest that RAG-1 functions in neurons, where its role might be to recombine elements of the neuronal genome site-specifically, or to prevent detrimental alterations of the genome in these long-lived cells.

Topics: Aging; Animals; Base Sequence; Brain; Cell Division; Cell Line; Chromosome Mapping; Cytarabine; Genes; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Neurons; Nucleic Acid Hybridization; Oligonucleotide Probes; Organ Specificity; Polymerase Chain Reaction; Recombination, Genetic; RNA Probes; Teratoma; Transcription, Genetic; Tretinoin

In HL60 cells a nuclear protein of Mr 55,000 is retinoylated, with the formation of a thioester bond. To gain further knowledge on the role of retinoylation we studied it in cell lines with varied responses to retinoic acid (RA). Compared to HL60 the extent of retinoylation (mol/cell) was about fivefold higher in HL60/MRI, a mutant which is more sensitive to RA than HL60. Retinoylation occurred to the same extent and at similar rates in HL60 and in HL60/RA-res, a mutant resistant to differentiation by RA. One-dimensional polyacrylamide gel electrophoresis patterns for the three HL60 cell lines were similar. However, two-dimensional polyacrylamide gel electrophoresis patterns of the three HL60 cell lines were distinct. While we saw the same major retinoylated protein of Mr 55,000 in the three cell lines, the HL60/RA-res cells also contained a high level of a protein with the same Mr and a lower pI. The extent of retinoylation was greater in the RA-sensitive embryonal carcinoma cell line, PCC4.aza1R, than in a RA-resistant cell line, PCC4.(RA)-2. One-dimensional polyacrylamide gel electrophoresis patterns of retinoylated proteins of the embryonal carcinoma cell lines were different from HL60 and from each other. The retinoylation pattern of the normal canine kidney cell line (MDCK) was different from either HL60 or the embryonal carcinoma cells. These results showed the retinoylation was widespread and that the response to RA of different cell types may depend on the retinoylation of specific proteins.

Topics: Cell Line; Cell Transformation, Neoplastic; Drug Resistance; Electrophoresis, Gel, Two-Dimensional; Humans; Kidney; Leukemia; Molecular Weight; Mutation; Neoplasm Proteins; Nuclear Proteins; Teratoma; Tretinoin; Tumor Cells, Cultured

In an attempt to study the regulatory properties of cells required for gene amplification, the capacity for amplification of the dehydrofolate reductase gene (dhfr) was determined in F9 teratocarcinoma stem cells during differentiation. By stepwise selection of surviving cells exposed to progressively increasing concentrations of methotrexate (MTX) up to 1,000 microM within 4 months, non-differentiated F9 cells reached a more than 40-fold amplification of their dhfr gene, elevated levels of dhfr mRNA and a 5-log increase in MTX resistance. Exposure to 5 Gy of 60Co-gamma-irradiation accelerated the process of amplification. In contrast, F9 cells that had been differentiated to endodermal cells by treatment with retinoic acid (RA) did not grow in MTX concentrations above 0.1 microM, either with or without radiation pretreatment and did not amplify the dhfr gene to any measurable extent over the same period of time. Upon treatment of methotrexate-resistant (F9-MTXr) cells with retinoic acid, loss of the amplified DNA in the absence of selection was significantly retarded. The ability to amplify the dhfr gene was correlated with the occurrence of origin binding activity in vitro (early domain of SV40 minimal origin of replication). These data indicate an increase in genomic stability and rigorous control of origin activity by differentiation.

Topics: Animals; Base Sequence; Cell Differentiation; Gene Amplification; Methotrexate; Mice; Molecular Sequence Data; Proto-Oncogene Proteins c-myc; Stem Cells; Teratoma; Tretinoin; Tumor Cells, Cultured

The human teratocarcinoma cell line Tera 2 can be induced to differentiate in vitro after exposure to retinoic acid. We show in this paper that whereas the K-FGF oncogene is expressed in undifferentiated cells, addition of retinoic acid rapidly (less than 60 min) downregulates the expression of this gene. However, when cells are cultured in RA for an extended period of time (greater than 15 days) K-FGF transcripts reappear. We also report that K-FGF is expressed in approximately one-third of primary human germ cell tumours but not in the corresponding normal testicular tissue.

Topics: Cell Differentiation; Cell Transformation, Neoplastic; Down-Regulation; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Gene Expression; Humans; Kinetics; Proto-Oncogene Proteins; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured

After retinoic acid treatment, a large percentage of cells of the human embryonal carcinoma cell line NT2/D1 differentiate into neuronal cells. We demonstrate here that the differentiated cells, but not the undifferentiated cells, contain high levels of neurofilament mRNA. We have also measured mRNA, protein, and activity levels of two kinases, cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), in order to explore the role of protein kinases in the establishment of the differentiated state. RNA levels for the catalytic (C alpha and C beta) subunits of PKA increased after differentiation. Total PKA activity levels increased 7-fold in the differentiated cells. Parallel with this, a rise in the level of catalytic subunit protein occurred. A 12-fold induction of Type 2 (beta) PKC mRNA levels was observed after neuronal differentiation. Increases in PKC activity and in Type 2 (beta) and Type 3 (alpha) PKC protein levels also accompanied differentiation. These changes in PKA- and PKC-specific RNA levels and enzyme activity may be necessary for production and maintenance of the differentiated state in these cells.

Topics: Cell Differentiation; Enzyme Induction; Humans; Neoplasm Proteins; Neurons; Protein Kinase C; Protein Kinases; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured

cDNA libraries have been generated from Nulli-SCCl murine embryonal carcinoma (EC) cells untreated or treated for 24 h with all-trans retinoic acid (RA) or hexamethylenebisacetamide (HMBA), two chemically unrelated inducers of differentiation of EC cells. The libraries were screened for gene sequences whose expression was differentially regulated by one or both compounds. Of 20,000 cDNA clones screened, only 12 showed reproducible quantitative differences. One of the latter clones (pH 34) has been studied in detail. pH 34 cDNA hybridizes with a polyadenylated RNA (650 nucleotides) which is abundant in untreated Nulli-SCCl EC cells but whose steady-state levels decrease within 6 h of exposure to HMBA, reaching a minimum at 24 h. RA has a less-marked effect on this mRNA. Addition of inducers to the cells in fresh medium produces an early (15 min) transient increase in pH 34 mRNA levels. Nuclear run-on experiments are consistent with the view that the decrease in pH 34 mRNA is due to post-transcriptional events. Subclones of pH 34 in pGEM-4 were used to synthesize mRNA which could be translated in vitro into a 14-kDa protein. DNA sequencing of the pH 34 cDNA revealed that it is 607 bp in length with a single open reading frame capable of encoding a protein of 118 amino acids. Primer extension experiments revealed that the insert contains the full 5' sequence. Comparison with known sequences failed to reveal significant homology with previously sequenced proteins.

Topics: Acetamides; Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Cell Differentiation; Cell Nucleus; DNA, Neoplasm; Gene Expression; Gene Library; Mice; Molecular Sequence Data; Protein Biosynthesis; Restriction Mapping; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin

A novel DNA sequence has been isolated from a subtraction cDNA library of P19 embryonal carcinoma cells treated with retinoic acid which induces neural differentiation of the stem cells. The cDNA insert (4B) hybridized with a single 1.7 kb mRNA, whose abundance was markedly increased in P19 cells after retinoic acid treatment. The 1.7 kb mRNA was also expressed in the brain, but not in other non-neuronal tissues. A 1.6 kb cDNA insert (4BFL), which was cloned by screening another cDNA library with the 4B probe, encodes a novel protein sequence of 325 amino acids (Mr 36,831). The protein expressed in 4BFL-transfected COS cells was translocated into the nuclei as detected with antibodies against subsequences of the predicted protein. The antibodies stained the nuclei of neurally differentiated P19 cells but not of the undifferentiated stem cells. This novel mRNA encoding the nuclear protein, termed necdin, may represent a useful marker for the differentiation and development of brain cells.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Brain; Cell Differentiation; Cell Line; Male; Mice; Molecular Sequence Data; Nerve Tissue Proteins; Nuclear Proteins; Organ Specificity; RNA, Messenger; Teratoma; Transfection; Tretinoin

P19 embryonal carcinoma cells can be induced to differentiate with a pulse of only 4 hr in retinoic acid (RA). The efficiency of RA-induced differentiation is independent of the position of P19 cells in the cell cycle but is critically dependent on the ratio between the number of cells and the number of moles of RA in the culture medium. P19 cultures at lower cell density are more efficiently induced to differentiate than cultures containing cells at higher cell densities. This effect is not mediated by cell-to-cell contact but may be related to the rapid metabolism of RA by the cells. Individual clones of differentiating P19 cells can develop into at least three different cell types suggesting that each cell in the population of embryonal carcinoma cells retains pluripotency.

Topics: Antigens, Differentiation; Antigens, Surface; CD57 Antigens; Cell Cycle; Cell Differentiation; Contact Inhibition; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; Glycolipids; In Vitro Techniques; Lewis X Antigen; Neurons; Teratoma; Tretinoin; Tumor Cells, Cultured

A newly identified gene MK is transiently expressed in early stages of retinoic acid-induced differentiation of embryonal carcinoma cells (Kadomatsu, K., M. Tomomura, and T. Muramatsu, 1988. Biochem. Biophys. Res. Commun. 151:1312-1318). MK gene has been predicted to code a polypeptide that is rich in basic amino acids and cysteine and is not related to any other peptides so far reported. In the present study, we investigated MK expression during mouse embryogenesis by in situ hybridization. The MK transcript was detected all over the embryo proper of the 7-d embryo, while it was not detectable in the 5-d embryo. The ubiquitous expression continued in the 9-d embryo proper. On the 11th-13th d of gestation, the sites where MK gene was intensely expressed became progressively restricted; these sites were the brain ectoderm around the lens and brain ventricles, the anterior lobe of the pituitary gland, the upper and lower jaw, the caudal sclerotomic half of vertebral column, the limbs, the stomach, and the epithelial tissues of the lung, the pancreas, the small intestine, and the metanephros. These areas include the region where secondary embryonic induction is prominent. In the 15-d embryo, only the kidney expressed MK significantly. These data suggest that MK gene plays a fundamental role in the differentiation of a wide variety of cells; MK gene may also play some specific roles in generation of epithelial tissues, and remodeling of mesoderm.

Topics: Animals; Cell Differentiation; Embryonic and Fetal Development; Gene Expression; Kidney; Mice; Mice, Inbred ICR; Nucleic Acid Hybridization; Organ Specificity; RNA; Teratoma; Transcription, Genetic; Tretinoin

F9 teratocarcinoma stem cells differentiate into parietal endoderm-like cells when given retinoic acid (RA) and dibutyryl cyclic adenosine monophosphate (DB-cAMP). It is generally accepted that the stem cells are resistant to the action of cAMP alone and need to be primed by RA in order to respond to cAMP. In this report, we demonstrate that F9 stem cells differentiate into parietal endoderm-like cells in the absence of exogenous RA when treated with cholera toxin and 1-methyl,3-isobutyl xanthine (CT/MIX) or 8-bromo-cAMP/MIX (8B2-cAMP/MIX). Cells treated with CT/MIX or 8B2-cAMP/MIX were morphologically similar to parietal endoderm-like cells, produced high amounts of plasminogen activator, and synthesized both type IV collagen and laminin mRNA. Conversely, markers made in abundance by stem cells such as stage-specific embryonic antigen (SSEA-1) and an mRNA species of 6.8 kb (pST6-135) were markedly reduced in CT/MIX-treated cells. To prove that cAMP alone could induce differentiation Lipidex-1000, a hydrophobic gel, was used to remove 80-90% of the endogenous serum retinoids. F9 cells grown in this retinoid-depleted serum and treated with 8B2-cAMP/MIX differentiated to parietal endoderm-like cells as shown by both dramatic changes in morphology and induction of type IV collagen mRNA. Our results indicate that the differentiation of F9 to parietal endoderm-like cells can be induced by increased intracellular cAMP and is not strictly dependent on the addition of RA.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Cell Differentiation; Collagen; Cyclic AMP; Fluorescent Antibody Technique; Glycolipids; Humans; Laminin; Lewis X Antigen; RNA, Messenger; Teratoma; Tissue Plasminogen Activator; Tretinoin

Mouse F9 teratocarcinoma stem cells differentiate in monolayer cultures in the presence of retinoic acid, dibutyryl cAMP, and isobutyl methylxanthine. This differentiation is associated with a marked increase in the synthesis rates and mRNA concentrations of basement membrane proteins such as type IV collagen. We report here that the differentiation also involves an increase of up to 50-fold in the concentrations of the mRNAs for the alpha and beta subunits of prolyl 4-hydroxylase, the enzyme required for the cotranslational and post-translational hydroxylation of proline residues in collagens. The time courses and magnitudes of increases in these two mRNA concentrations were similar to those observed in the same experiments for the mRNA of the alpha chain of type IV collagen. In the differentiated F9 cells the concentration of the alpha subunit mRNA was about 30% of the beta subunit mRNA concentration. Northern blot analyses indicated that the sizes of the alpha and beta subunit mRNAs in the differentiated mouse F9 cells are similar to those in human skin fibroblasts. The F9 cell differentiation system appears to provide a useful model for studies on the regulation of prolyl 4-hydroxylase synthesis.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Blotting, Northern; Bucladesine; Cell Differentiation; Cell Line; Collagen; DNA Probes; Gene Expression Regulation; Genes; Glyceraldehyde-3-Phosphate Dehydrogenases; Kinetics; Macromolecular Substances; Mice; Procollagen-Proline Dioxygenase; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured

The F9 cell is a mouse embryonal teratocarcinoma which can be induced to differentiate into visceral endoderm by treatment with retinoic acid (RA). Treatment with RA in conventional studies was carried out in the constant presence of RA. Here we demonstrate that treatment with RA can be as short as 3 hrs to induce differentiation of F9 cells. Morphology, alpha-fetoprotein gene activity, and temporal patterns of F9 cell differentiation are the same with both short- and long-term treatment with RA.

Topics: alpha-Fetoproteins; Animals; Blotting, Northern; Cell Differentiation; Cycloheximide; Gene Expression Regulation, Neoplastic; Mice; RNA, Neoplasm; Teratoma; Time Factors; Tretinoin; Tumor Cells, Cultured

Fetomodulin is a surface marker protein of differentiated F9 embryonal carcinoma cells. Gene cloning has recently identified it as thrombomodulin which binds thrombin and proteolytically activates protein C. Activity assays and RNA blotting were adopted to analyze F9 cell differentiation with specific reference to another well-characterized marker, tissue plasminogen activator. Retinoic acid induced primitive endoderm differentiation of F9 cells and simultaneously activated tissue plasminogen activator synthesis. This differentiation, however, did not result in fetomodulin expression. When primitive endoderm cells were exposed to 1 mM dibutyryl cyclic AMP, the tissue plasminogen activator level rose further within 6 hr. In contrast, the cofactor activity of fetomodulin stayed below a detectable level for as long as 15 hr and then increased with time. Expression of the two marker proteins appeared to be regulated differently.

Topics: Animals; Antibodies, Monoclonal; Bucladesine; Cell Differentiation; Cell Line; Endoderm; Gene Expression; Kinetics; Membrane Glycoproteins; Methionine; Mice; Nucleic Acid Hybridization; RNA; RNA, Messenger; Teratoma; Tissue Plasminogen Activator; Tretinoin

Topics: Animals; Blotting, Northern; Carrier Proteins; Cell Fractionation; Cell Line; Centrifugation, Density Gradient; Chromatography, Gel; Cloning, Molecular; Cytosol; Radioisotope Dilution Technique; Receptors, Retinoic Acid; Retinol-Binding Proteins; RNA; Teratoma; Tretinoin; Tritium; Ultracentrifugation

The murine keratins Endo B and Endo A, which are homologs of the human keratins K18 and K8, constitute the intermediate filaments (IFs) that are found in all simple epithelia of the adult and in the first epithelial derivatives of the early embryo. The cellular role of simple epithelial keratins in development and differentiation was investigated by inducing filament collapse in HR9 endoderm and F9 embryonal carcinoma cells in which mutant Endo B protein was constitutively expressed. By immunolocalization techniques a perturbation of the keratin network was revealed as well as concomitant disruption of vimentin IFs and displacement of surface desmosomal proteins, demonstrating an intimate structural association of Endo B/A filaments with these cellular components. In aggregates of differentiating F9 cells displaying altered Endo A/B IFs, the formation of a compact, polarized visceral endoderm layer was significantly compromised. These results indicate that an intact keratin network influences the three-dimensional formation of cell-cell or cell-substratum contacts in embryonic visceral endoderm.

Topics: Animals; Bucladesine; Cell Differentiation; Cell Line; Cytoskeleton; Desmosomes; Endoderm; Intermediate Filaments; Keratins; Mice; Morphogenesis; Recombinant Fusion Proteins; Teratoma; Tretinoin; Tumor Cells, Cultured; Vimentin

Topics: Animals; Bucladesine; Cell Differentiation; Cell Line; Culture Media; Culture Techniques; Mice; Teratoma; Transfection; Tretinoin

Topics: Animals; Carrier Proteins; Cell Line; Cell Nucleus; Collagen; Gene Expression Regulation, Neoplastic; Immunoblotting; Indicators and Reagents; Laminin; Mice; Nucleic Acid Hybridization; Receptors, Retinoic Acid; RNA, Messenger; RNA, Neoplasm; Teratoma; Transcription, Genetic; Transfection; Tretinoin; Ultracentrifugation

Retinoic acid (RA) induces differentiation of murine embryonal carcinoma PCC4.aza1R cells. In this study, the expression of nuclear retinoic acid receptors (RARs) in PCC4.aza1R cells is examined. Analyses of [3H]RA-labeled nuclear extracts prepared from PCC4.aza1R cells by size-exclusion high-performance liquid chromatography demonstrated the presence of a specific RA-binding activity that migrated with a molecular weight of approximately 50,000. More than 95% of this binding activity was associated with the nuclear fraction. In contrast to cytosolic retinoic acid-binding protein, the RARs bound RA analogues of the Ch-series very effectively. Northern blot analyses of total RNA with complementary DNA probes specific for RAR alpha, RAR beta, and RAR gamma showed that PCC4.aza1R cells contain predominantly transcripts encoding RAR alpha and RAR gamma; RAR beta transcripts were undetectable. Treatment of PCC4.aza1R cells with RA increased the levels of RAR beta mRNA in a dose- and time-dependent manner. The RA concentration for half-maximum induction of RAR beta mRNA was 1 nM. An increase in RAR beta mRNA was detectable as early as 2 h after the addition of RA. This increase was not abrogated by cycloheximide, suggesting that protein synthesis is not required for this response. The ability of several retinoids to increase RAR beta mRNA levels in PCC4.aza1R cells correlated well with their binding affinity to the RARs but not with their binding affinity to cytosolic retinoic acid-binding protein. Two mutant cell lines, PCC4(RA)-1 and (RA)-2, which do not undergo differentiation after RA treatment, contained levels of RAR-binding activity very similar to those of the parental cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blotting, Northern; Carrier Proteins; Cell Nucleus; Cycloheximide; Cytoplasm; DNA Probes; Gene Expression; In Vitro Techniques; Mice; Mutation; Receptors, Retinoic Acid; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured

Embigin is a member of the immunoglobulin superfamily found in embryonal carcinoma cells. The present study deals with embigin gene expression in adult and embryonic cells. By RNA blot analyses, high levels of embigin mRNA were detected in embryonal carcinoma cells with different differentiation potentials, namely F9 and PCC4 cells. Similar or slightly higher levels of the RNA were detected in the embryonic and extraembryonic portions of 9-day mouse embryos. When F9 cells were induced to differentiate by treatment with retinoic acid and dibutyryl cyclic AMP for 2-5 days, the RNA level increased significantly. On the other hand, embigin mRNA dramatically dropped to almost background levels in embryos, from 11 days postcoitus (p.c.). Many organs of adult mice expressed only low levels of embigin mRNA, but considerable amounts of the transcript were found in the ovary and also in the uterus of pregnant mice (4 days p.c. and 12 days p.c.). In situ hybridization experiments revealed large amounts of embigin RNA in the embryo from 7 to 9 days p.c. The visceral endoderm of 7-day embryos, and the brain, visceral yolk sac and presumptive foregut of 9-day embryos were the sites where intense signals of embigin RNA were localized.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bucladesine; DNA; Embryonic and Fetal Development; Endoderm; Female; Fibroblasts; Gene Expression; Glycoproteins; Immunoglobulins; Mice; Molecular Sequence Data; Neoplasm Transplantation; Ovary; Pregnancy; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured; Uterus

Effects of retinoic acid (RA), dibutyryl cyclic AMP (dBcAMP), and nerve growth factor (NGF) on the morphology and the NADPH-specific dihydropteridine reductase (NADPH-DPR) activity of teratoma 402AX cells were studied. The population of cells with flattened shape was increased by the treatment with RA+dBcAMP, or with RA+dBcAMP+NGF, while the treatment with NGF alone had almost no effect on the morphology. The NADPH-DPR activity was increased by the treatment of the cells with RA+dBcAMP+NGF and with RA+dBcAMP by about 2.2-fold and 1.8-fold, respectively, of that detected in the control (untreated) cells. Also in this case, NGF alone failed to increase the enzyme activity. On the other hand, no change in NADH-specific dihydropteridine reductase (NADH-DP) activity was detected by the treatment of the cells with either RA+dBcAMP, RA+dBcAMP+NGF, or NAF alone. These results suggest that the cells acquire responsiveness to NGF by the treatment with RA and dBcAMP and that NADPH-DPR may play some regulatory role in tetrahydrobiopterin regeneration from its quinonoid-dihydro form.

Topics: Biopterins; Bucladesine; Dihydropteridine Reductase; Humans; Nerve Growth Factors; Teratoma; Tretinoin; Tumor Cells, Cultured

Differentiation of the F9 cell line was induced by treating the cells with retinoic acid (10(-6) M) and dibutyryl cycloadenosinemonophosphate (10(-4) M). The population doubling time and the portion of cells in G1-phase increase and saturation density falls as the result of this treatment. Differentiated F9 cells demonstrate a decreased capacity of forming colonies in the soft agar, lose their capacity of proliferating at the clonal density, and acquire the limited life-span in culture after reseeding at a high density. Some cells in the differentiated population retain their capacity of forming colonies in the soft agar and (or) of binding antibodies against the stem cell marker SSEA-1. Cells with the stem cell morphology were found in the course of passaging of differentiated cells after reseeding at a high density. These cells were able to differentiate after the standard procedure of the induction of differentiation with retinoic acid and dibutyryl cAMP. Causes of the rising and supporting of heterogeneity of the differentiated F9 cells are discussed.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Surface; Biomarkers, Tumor; Bucladesine; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Glycolipids; Lewis X Antigen; Mice; Phenotype; Teratoma; Tretinoin; Tumor Cells, Cultured

RETINOIC acid had been implicated as a natural morphogen in chicken and frog embryogenesis, and is presumed to act through the gene regulatory activity of a family of nuclear receptors. Homeobox genes, which specify positional information in Drosophila and possibly in vertebrate embryogenesis, are among the candidate responsive genes. We previously reported that retinoic acid specifically induces human homeobox gene (HOX) expression in the embryonal carcinoma cell line NT2/D1. We now show that the nine genes of the HOX2 cluster are differentially activated in NT2/D1 cells exposed to retinoic acid concentrations ranging from 10(-8) to 10(-5) M. Genes located in the 3' half of the cluster are induced at peak levels by 10(-8) M retinoic acid, whereas a concentration of 10(-6) to 10(-5) M is required to fully activate 5' genes. At both high and low retinoic acid concentrations, HOX2 genes are sequentially activated in embryonal carcinoma cells in the 3' to 5' direction.

Topics: Antigens, Surface; Cell Line; Clone Cells; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Humans; Kinetics; Teratoma; Tretinoin; Tumor Cells, Cultured

Neural differentiation of the embryonal carcinoma P19 cell line markedly increased the abundance of mRNA encoding Alzheimer amyloid beta/A4-protein precursor (APP). In P19 cells treated with retinoic acid, the abundance of mRNA encoding APP695, which lacks the protease inhibitor domain, reached a maximum on days 2-4 and decreased thereafter, whereas the abundances of mRNAs encoding APP751 and APP770, both possessing the protease inhibitor domain, slowly increased to reach higher levels than APP695 mRNA at later stages of neural differentiation. The induction of APP695 mRNA was consistent with the appearance of neurons in the P19 cultures. A high abundance of APP695 mRNA was also detected in mouse brain at a stage of the period of neuroblast formation. Thus, neural differentiation of P19 cells may present a suitable model for studying the regulation of APP gene expression during early differentiation of brain cells in vivo.

Topics: Actins; Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Animals; Base Sequence; Blotting, Northern; Brain; Cell Differentiation; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Intermediate Filament Proteins; Mice; Molecular Sequence Data; Molecular Weight; Neurofilament Proteins; Neurons; Oligonucleotide Probes; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured

Cyclic AMP is known to enhance retinoic acid-induced differentiation of F9 mouse teratocarcinoma cells to parietal endoderm. Recently, we showed that a parathyroid hormone-related protein (PTHrP), by activating adenylate cyclase, can substitute for exogenous cAMP in promoting retinoic acid-induced differentiation of F9 cells. However, the mechanisms by which endogenous cAMP levels are regulated during F9 differentiation are poorly defined. We therefore assessed whether Gs alpha, a subunit of the stimulatory coupling protein of adenylate cyclase, is induced during this process. Treatment of F9 cells with retinoic acid (1 microM) for 5 days resulted in a 20-fold increase in steady-state levels of a 2.0-kilobase Gs alpha mRNA. This was accompanied by an increase in the expression of 52- and 45-kDa Gs alpha polypeptides. Gs alpha mRNA increases within 24 h of exposure to retinoic acid, whereas the expression of alpha 1 (IV) collagen, a marker for F9 differentiation, did not increase until 48 h of treatment. In the presence of retinoic acid, exogenous human PTHrP-(1-34)-amide (20 nM) produced a further 2-fold increase in Gs alpha mRNA. These effects of retinoic acid and PTHrP were exerted largely if not entirely at the level of Gs alpha gene transcription, as assessed by nuclear run-on assay. Bt2cAMP (1 mM) did not reproduce the stimulatory effects of PTHrP on Gs alpha transcription, mRNA, or protein. These data demonstrate that a marked increase in Gs alpha expression accompanies F9 differentiation induced by retinoic acid and PTHrP, and that the regulation is predominantly transcriptional. The resulting increase in adenylate cyclase responsiveness to PTHrP and perhaps other ligands may be a critical component of the differentiation process. The effect of PTHrP on the expression of Gs alpha appears to be mediated by signals other than cAMP.

Topics: Animals; Cell Line; Cell Nucleus; Gene Expression Regulation; GTP-Binding Proteins; Humans; Kinetics; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Peptide Fragments; Proteins; RNA, Neoplasm; Teratoma; Transcription, Genetic; Tretinoin

Mouse F9 teratocarcinoma cell lines can be induced to differentiate into either parietal endoderm or embryoid bodies which contain visceral endoderm-like cells. The nature of the early molecular events involved in these two differentiation pathways has not yet been fully elucidated. Moreover, since the process of differentiation is often accompanied by changes in cell growth, it is often difficult to determine which of the events that do occur during the early stages of differentiation are a direct result of the process of differentiation and which events are indirect results that occur as a consequence of altered cell growth. In the experiments reported here we have attempted to distinguish between these two possibilities by examining the patterns of expression of a representative group of growth-associated genes (i.e., c-myc, p53, and histone H3) when F9 cell aggregates are induced to differentiate into embryoid bodies containing visceral endoderm. By analysis of the patterns of growth-associated gene expression in both retinoic acid treated and nontreated F9 cell aggregates, we were able to classify early events as differentiation-specific events (events which occurred only following retinoic acid treatment of aggregates) or nondifferentiation-specific events caused by reduction in cell growth (events which occurred even when aggregates were not treated with retinoic acid). Our results show that F9 cells differentiated into embryoid bodies containing visceral endoderm-like cell exhibit an early reduction in both growth and c-myc mRNAs which is neither retinoic acid-specific nor differentiation-specific. However, following this initial response to aggregation, constant levels of c-myc mRNA are maintained despite continued reduction in growth. Thus, it appears that alteration in c-myc expression is a differentiation-specific event along the pathway to formation of visceral endoderm. Interestingly, however, the nature and time course of this alteration in c-myc expression in F9 cells' differentiation into visceral endoderm is different from that observed in F9 cells differentiated into parietal endoderm.

Topics: Animals; Cell Differentiation; Gene Expression Regulation; Histones; Mice; Oncogene Proteins; Phosphoproteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Retinol-Binding Proteins; RNA, Messenger; Teratoma; Time Factors; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Adenovirus infection was compared in F9 (OTF963) cells and cells induced to differentiate with retinoic acid, in order to study expression of early genes under the control of the reported "E1a-like factor" in F9 cells. However, not only was transcription of the viral E1a gene defective in undifferentiated cells but expression of all the other early genes was found to be reduced in OTF963 cells in comparison to differentiated cells. The defect in early gene expression was detected at the level of transcriptional initiation during the first 48 h of infection and resulted in similarly low levels of viral cytoplasmic mRNA and viral protein synthesis. Viral DNA replication was delayed and reduced. After 48 h of infection, the defect in transcription in OTF963 cells of E1a and other early genes was relieved, so that by 72 h postinfection the level of transcription was similar to that 16 h after infection of differentiated cells. At no time did adenovirus early gene expression occur independently of viral E1a. These results suggest limits to the generality and explanatory power of the hypothesis that F9 embryonal carcinoma cells contain an E1a-like factor.

Topics: Adenovirus Early Proteins; Adenoviruses, Human; Animals; Cell Differentiation; Cell Line; Cell Nucleus; Genes, Viral; Kinetics; Nucleic Acid Hybridization; Oncogene Proteins, Viral; Plasmids; RNA, Messenger; RNA, Viral; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) receptor alpha (RAR alpha) and RAR gamma steady-state mRNA levels remained relatively constant over time after the addition of RA to F9 teratocarcinoma stem cells. In contrast, the steady-state RAR beta mRNA level started to increase within 12 h after the addition of RA and reached a 20-fold-higher level by 48 h. This RA-associated RAR beta mRNA increase was not prevented by protein synthesis inhibitors but was prevented by the addition of cyclic AMP analogs. In the presence of RA, cyclic AMP analogs also greatly reduced the RAR alpha and RAR gamma mRNA levels, even though cyclic AMP analogs alone did not alter these mRNA levels. The addition of either RA or RA plus cyclic AMP analogs did not result in changes in the three RAR mRNA half-lives. These results suggest that agents which elevate the internal cyclic AMP concentration may also affect the cellular response to RA by altering the expression of the RARs.

Topics: Animals; Blotting, Northern; Bucladesine; Carrier Proteins; Cycloheximide; Dactinomycin; Dose-Response Relationship, Drug; Gene Expression Regulation; Mice; Receptors, Retinoic Acid; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Murine homologs of the PDGF A, PDGF B, and PDGF receptor alpha subunit genes were cloned. These were used, together with a mouse PDGF receptor beta subunit cDNA clone, to monitor gene expression in early postimplantation mouse embryos and in F9 embryonal carcinoma cells. RNAse protection analysis shows that PDGF A chain, but not B chain, mRNA is expressed in 6.5- to 8.5-day embryonic and extraembryonic tissues. Both alpha and beta receptor subunit mRNAs are expressed in early embryos, however, alpha subunit mRNA appears earlier and is more abundant than beta subunit mRNA. Undifferentiated F9 embryonal carcinoma stem cells express abundant levels of A chain, but not B chain, mRNA. Neither of the PDGF receptor genes is expressed in stem cells. Treatment with retinoic acid stimulates expression of both PDGF receptor genes. As in postimplantation mouse embryos, alpha receptor subunit mRNA appears earlier and is substantially more abundant than beta subunit mRNA. Collectively, these data demonstrate that the genes encoding the two chains of PDGF and their receptors are regulated independently during development and suggest that the two systems have some nonoverlapping functions in vivo. PDGF A, but not PDGF B, may be particularly important in modulating early events in mouse embryonic development.

Topics: Amino Acid Sequence; Animals; Cloning, Molecular; Embryo, Mammalian; Gene Expression; Mice; Molecular Sequence Data; Platelet-Derived Growth Factor; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-sis; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; RNA, Messenger; Teratoma; Tretinoin

Although undifferentiated F9 teratocarcinoma cells express low levels of mRNAs for collagen IV, previous transient transfection experiments using an alpha 1(IV) collagen gene promoter-enhancer-CAT construct revealed a high level of transcriptional activity in these cells. In this report, we find that when this construct is introduced stably into undifferentiated F9 teratocarcinoma cells, expression does not occur unless the cells are treated with retinoic acid and dibutyryl-cAMP. Such activation mimics the endogenous gene activity. Treatment of the cells containing the integrated construct with 5-azacytidine, an agent which prevents DNA methylation, also activates transcription and acts synergistically with retinoic acid and cAMP. Analysis of DNA isolated from F9 teratocarcinoma cells revealed that there was a specific demethylation of the DNA within the 5'-flanking region of the collagen IV genes following treatment with retinoic acid and cAMP. These results suggest that during differentiation, DNA demethylation may play an important role in transcriptional regulation of the collagen IV genes.

Topics: Animals; Bucladesine; Cell Differentiation; Cell Line; Clone Cells; Collagen; DNA, Neoplasm; Gene Expression; Methylation; Mice; RNA, Messenger; Teratoma; Transfection; Tretinoin; Tumor Cells, Cultured

The human teratocarcinoma cell NTERA-2 cl. D1 (NT2/D1) is a cloned embryonal cancer cell line that differentiates into a neuronal phenotype and other cellular lineages after treatment with retinoic acid (RA). We examined the regulated expression of growth factors and proto-oncogenes in NT2/D1 cells. We studied RNA levels after six days of RA treatment to assess gene expression coincident with observed morphologic differentiation. Three growth factors were markedly down-regulated following RA treatment: Hst-1/kFGF and TGF-alpha expression became undetectable by Northern analysis and bFGF expression was substantially reduced. Minimal decline was seen for c-myc, N-myc, c-fos, and c-myb. Increased expression with differentiation was seen for the human homeotic genes Hox 2.1 and Hox 2.2. Assay of RNA levels daily after one to six days of RA treatment showed that the growth factor down-regulation inversely correlated with the homeotic gene up-regulation. Nuclear run-on studies showed low transcriptional rates for these homeotic genes, Hst-1/kFGF, and TGF-alpha that did not measurably change with RA treatment. To explore whether these regulated genes in NT2/D1 play a role in human testicular cancer, we examined RNA levels in a panel of human germ cell cancer lines. Hst-1/kFGF and bFGF are commonly expressed in five of seven male germ cell cancer lines. These data show that specific proto-oncogenes and growth factors are down-regulated with RA treatment of the NT2/D1 cell and that some of these regulated genes are often expressed in human germ cell cancer lines.

Topics: Blotting, Northern; Cell Line; Cell Nucleus; Clone Cells; DNA Probes; Gene Expression; Humans; Proto-Oncogene Proteins; Proto-Oncogenes; Receptors, Cell Surface; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Expression of the retinoic acid receptors alpha and beta (RAR-alpha and RAR-beta) was examined in F9 cells, an embryonal carcinoma cell model established for the study of retinoid metabolism and function. Addition of retinoic acid to F9 cell medium caused a dose-dependent increase in RAR-beta mRNA within 3 hr that reached 5- to 30-fold greater than the constitutively expressed mRNA by 24 hr. The elevation in mRNA resulted from increased transcription, as demonstrated by nuclear run-on transcription, did not require protein synthesis, and required the constant presence of retinoic acid. N6,O2'-Dibutyryl-cAMP attenuated the retinoic acid-induced increase in RAR-beta mRNA by a post-transcriptional mechanism. In contrast, RAR-alpha mRNA in F9 stem cells was affected less (1.2- to 1.4-fold increase) by retinoic acid and decreased 3-fold transiently when fresh serum was added to the medium. Differentiation of F9 cells resulted in increased steady-state levels of RAR-beta mRNA in primitive (4-fold), parietal (3-fold), and visceral (8-fold) endoderm but decreased steady-state levels of RAR-alpha mRNA in primitive (2-fold), parietal (3-fold), and visceral (1.4-fold) endoderm. These data demonstrate that RAR-beta is a primary target gene for retinoic acid in a characterized model of retinoid function, indicate that constitutive expression of both RAR-beta and RAR-alpha is dependent upon the differentiation state, and suggest hormonal modulation of RAR-beta by cAMP and modulation of RAR-alpha by serum factors. These results distinguish the effects of serum, cAMP, and retinoic acid on the expression of RAR from the effects mediated by differentiation.

Topics: Animals; Bucladesine; Carrier Proteins; Cell Differentiation; Cell Line; Cell Nucleus; DNA Probes; Gene Expression; Kinetics; Nucleic Acid Hybridization; Receptors, Retinoic Acid; Restriction Mapping; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin

c-fos was studied in F9 cells to determine whether changes in its expression are an early and/or obligatory event in retinoic acid-induced F9 cell differentiation. Induction of c-fos transcripts was not observed at times early or late during retinoic acid-promoted differentiation, but a decrease in c-myc mRNA was noted as early as 1 h after retinoic acid dosing. Induction of a rapid and transient change in c-fos expression in F9 cells was observed only in response to serum stimulation. Therefore, although expression of c-fos may be involved in the cellular growth and proliferation of F9 cells, as indicated by the response to serum, an increase in c-fos is not required for retinoic acid-induced differentiation.

Topics: Blood; Bucladesine; Cell Differentiation; Gene Expression; Humans; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-myc; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

We have studied cell-type-specific expression by JC virus (JCV) DNA regulatory sequences using embryonal carcinoma (EC) cells as a model system. In transient transfection assays, JCV enhancer demonstrated activity in retinoic acid-differentiated neuronal type cells but not in undifferentiated or DMSO-differentiated muscle type cells. To correlate in vivo activity with the binding of transcription factors, we performed DNasel footprinting experiments. Retinoic acid-treated EC cell extracts provided three completely protected regions, each containing sequences with homology to nuclear factor 1 (NF1) binding motifs and the partially protected TATA box. Oligonucleotide competition studies suggest that all three NF1 binding motifs are bound by the same factors but with different affinities and that there are cooperative interactions between NF1 proteins binding to adjacent regions. No protected region other than the partially protected TATA box was detected in undifferentiated and DMSO-differentiated EC cells in which JC regulatory sequences were not expressed.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Line; Deoxyribonuclease I; Dimethyl Sulfoxide; DNA, Viral; Enhancer Elements, Genetic; JC Virus; Mice; Molecular Sequence Data; Oligonucleotide Probes; Plasmids; Polyomavirus; Regulatory Sequences, Nucleic Acid; Teratoma; Transcription Factors; Transfection; Tretinoin

F9 embryonal mouse teratocarcinoma cells were differentiated to a primitive endoderm-like phenotype by retinoic acid and to a parietal endoderm-like phenotype by retinoic acid in combination with dibutyryl cyclic AMP. The secretion of tissue plasminogen activator (tPA) is a characteristic of the cells displaying the differentiated phenotypes. The fundamental question of whether tPA secretion is regulated acutely by G-protein-mediated transmembrane signaling was explored. Cells differentiated to primitive and parietal endoderm demonstrated a rapid tPA response to stimulation by beta-adrenergic agonist (isoproterenol). Adenylyl cyclase activity in response to isoproterenol and GTP, but not forskolin, was greater in primitive and parietal endoderm than F9 stem cells. Both primitive and parietal endoderm cells, but not F9 stem cells, displayed beta-adrenergic stimulation of cyclic AMP accumulation. Retinoic acid induced F9 stem cells to the primitive endoderm phenotype and increased beta-adrenergic receptor levels 3-fold. Gi alpha 2 levels declined, G beta-subunits increased, and Gs alpha levels were unchanged following differentiation to primitive endoderm. In parietal endoderm cells beta-adrenergic receptors increased 2-fold over F9 stem cells, Gi alpha 2 levels declined even further than in primitive endoderm, G beta-subunits increased compared to F9 stem cells, and Gs alpha levels again were unchanged. The marked potentiation of short-term stimulation of tPA secretion in the differentiated state may be best explained by the retinoic acid-induced increase in expression of beta-adrenergic receptors coupled with a decline in Gi alpha 2 levels. Short-term regulation by G-protein-linked receptors represents a novel mode for the control of tPA secretion.

Topics: Animals; Bucladesine; Cell Differentiation; Cell Line; Endoderm; GTP-Binding Proteins; Isoproterenol; Kinetics; Mice; Receptors, Adrenergic, beta; Signal Transduction; Teratoma; Tissue Plasminogen Activator; Tretinoin

Differentiation of the murine embryonal carcinoma (EC) cell lines F-9 and PC-13, induced by beta-all transretinoic acid (RA) resulted in an increased level of two lysosomal-associated membrane glycoproteins (LAMP-1 and LAMP-2). After differentiation, the levels of both LAMPs in the EC cells were comparable to those found in visceral and parietal endoderm cell lines (PSA-5E and PYS-2, respectively). RA treatment of the EC cells also resulted in an increase in the apparent Mr of both LAMPs apparently due to increased glycosylation because the deglycosylated LAMP-1 from undifferentiated and from differentiated cells had a similar electrophoretic migration. Indeed, the binding of 125I-labeled L-phytohemagglutinin (L-PHA) to glycoproteins with Mr or 90,000-130,000 increased after differentiation and about 24 times more 125I-labeled L-PHA bound to LAMP-1 isolated by immunoprecipitation from extracts of RA-treated F-9 cells than to LAMP-1 from undifferentiated cells. The increased level of the LAMPs was detected in F-9 cells treated with greater than 10(-7) M RA and required greater than 48 h of treatment as did the increased expression of the B1 chain of laminin, an established marker for differentiation in this system. LAMP-1- and L-PHA-reactive glycoproteins were localized by fluorescence techniques to intracellular vesicles, presumably lysosomes, and to the cell surface and both increased after RA treatment. LAMP-2 was barely detectable intracellularly in undifferentiated cells but could be detected clearly after differentiation. In contrast, no LAMP-2 could be detected on the cell surface either before or after differentiation of F-9 cells. The increased level and glycosylation of both LAMP-1 and LAMP-2 was observed also in cells treated with a synthetic chalcone carboxylic acid analog of RA and by combination of either retinoid with dibutyryl cyclic AMP. These results demonstrate that differentiation of EC cells is accompanied by changes in the synthesis and glycosylation of LAMP glycoproteins and that these changes are specific for the cell type that results after differentiation.

Topics: Animals; Antigens, CD; Bucladesine; Cell Differentiation; Cell Line; Fluorescent Antibody Technique; Glycoside Hydrolases; Glycosylation; Immunoblotting; Kinetics; Laminin; Lectins; Lysosomal Membrane Proteins; Lysosomes; Membrane Glycoproteins; Mice; Molecular Weight; Protein Processing, Post-Translational; Teratoma; Tretinoin

The induced differentiation of F9 cells by retinoic acid (RA) and cyclic AMP (cAMP) activated transcription of the tissue plasminogen activator (t-PA) gene. This differentiation-responsive regulation of the t-PA promoter was also observed in transient assays. Multiple sequence elements within 243 bp of t-PA DNA contributed to the high level of transcription in retinoic acid- and cyclic AMP-differentiated cells. To investigate the factors involved in controlling t-PA transcription upon differentiation, we used F9 cell extracts to examine proteins that bind two proximal promoter elements. These elements (boxes 4 and 5) are homologous to GC boxes that are known binding sites for transcription factor Sp1. Mobility shift assays in the presence and absence of anti-Sp1 antibodies demonstrated that the proteins which bound to this region were immunologically related to human Sp1. The proteins also had a DNA-binding specificity similar to that of a truncated form of Sp1. Mutations of the GC motif within boxes 4 and 5 that interfered with Sp1 binding reduced in parallel the binding of the F9 cellular factors and lowered transcription in vitro as well as in vivo. Although this proximal region of the t-PA promoter was active in vivo only in differentiated cells, the Sp1-like binding proteins were present in equal concentrations and had similar properties in extracts of both stem and differentiated cells. These data suggest that other cellular elements participate with this Sp1-like factor in controlling differentiation-specific expression.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Line; Cyclic AMP; Gene Expression Regulation; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Promoter Regions, Genetic; Sp1 Transcription Factor; Teratoma; Tissue Plasminogen Activator; Tretinoin

Some undifferentiated F9 embryonal carcinoma cells allow adenovirus genes to be expressed independently of the E1a oncogene normally required for their activation; this has been attributed to a cellular equivalent of E1a in F9 cells. However, transcription of all early genes was low in undifferentiated OTF963 embryonic carcinoma cells during the first 48 hr after infection with adenovirus type 5 (Ad5). Transcription then increased to about the level seen 16 hr after infection of cells induced to differentiate by retinoic acid (RA) (referred to as RA-dF9 cells), but this increase did not occur in cells infected by the E1a deletion mutant dl312. Addition of E1a in trans, or of RA, had no immediate effect on viral transcription in OTF963 cells, but viral transcription increased about 48 hr after these additions. Ad5 induced transcription of several differentiation-specific genes in OTF963 cells with about the same kinetics as their induction by RA. These genes were superinduced in RA-dF9 cells by cAMP or infection by adenovirus. We suggest the small amount of E1a produced early in infection of OTF963 cells activates cellular genes, some of which are differentiation specific and required for efficient transcription of viral genes, so that E1a both induces and is induced by differentiation. The simple hypothesis of a cellular equivalent to E1a does not adequately explain the complex interactions between viral and cellular genes in OTF963 embryonic carcinoma cells.

Topics: Adenovirus Early Proteins; Adenoviruses, Human; Animals; Cell Differentiation; Cell Line; Cell Transformation, Viral; Cytomegalovirus; Gene Expression Regulation, Viral; Kinetics; Oncogene Proteins, Viral; Plasmids; Promoter Regions, Genetic; Restriction Mapping; Teratoma; Transcription, Genetic; Transfection; Tretinoin

Pluripotential embryonal carcinoma cells such as those of the P19 line differentiate when exposed to retinoic acid (RA). The RAC65 cell line is a mutant clone of P19 cells selected to be RA nonresponsive. RAC65 cells carry a rearrangement affecting one of the genes encoding a nuclear retinoic acid receptor (RAR alpha). The mutant gene encodes a protein, RAR alpha', that has lost its 70 C-terminal amino acids, thus truncating the RA-binding domain. The RAR alpha' was found to be a dominant repressor of transcription from an RA-responsive target gene; however, expression of RAR alpha' was insufficient to confer RA nonresponsiveness, suggesting that RAC65 cells carry an additional mutation(s) affecting RA-induced genes.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Southern; Carrier Proteins; Cell Differentiation; Cell Line; DNA, Neoplasm; Drug Resistance; Gene Library; Genes; Genes, Dominant; Mice; Molecular Sequence Data; Mutation; Receptors, Retinoic Acid; RNA, Messenger; Teratoma; Tretinoin

Retinoic acid together with dibutyryl cyclic AMP stimulated transcription of the platelet-derived growth factor alpha-receptor gene in embryonal carcinoma cells (line F9). Processed mRNA transcripts appeared within 4 h after exposure to these agents, and functional alpha:alpha homodimers appeared within 24 h.

Topics: Animals; Blotting, Northern; Cell Line; Cell Nucleus; Cycloheximide; Gene Expression; Kinetics; Macromolecular Substances; Mice; Platelet-Derived Growth Factor; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; Teratoma; Transcription, Genetic; Tretinoin

Differentiation of mouse embryonal carcinoma cells to the parietal endoderm phenotype is associated with expression of PTH-responsive adenylate cyclase. A PTH-like protein (PLP), which binds to PTH receptors and activates adenylate cyclase in classical PTH target cells was recently isolated and cloned. We assessed whether the parietal endoderm phenotype is associated with the expression of PLP or its receptor. A 1.4-kilobase PLP transcript was detected in the mouse parietal endoderm cell line PYS-2. No hybridizing transcripts were evident in undifferentiated mouse embryonal carcinoma cells PSA-1 or F9. However, differentiation of these cells to parietal endoderm, either spontaneously (PSA-1) or by treatment with retinoic acid and dibutyryl cAMP (F9), resulted in expression of the 1.4-kilobase PLP message. Undifferentiated F9 cells displayed negligible specific binding of [125I]PLP-(1-34)amide. When F9 cells were induced to differentiate to parietal endoderm, specific binding sites for [125I]PLP-(1-34)amide were expressed in parallel with PLP-responsive adenylate cyclase. These receptors, like those in classical PTH target tissues, displayed identical affinity (Kd = 5.2 nM) for bPTH-(1-34) and hPLP-(1-34)amide; with binding capacity (Bmax) of 6.6 x 10(4) sites/cell. In the presence of retinoic acid, exogenous PLP substituted for dibutyryl cAMP in a concentration-dependent fashion in promoting the differentiation of F9 cells to parietal endoderm. Thus, both PLP mRNA and PLP receptors coupled to adenylate cyclase are expressed during the differentiation of mouse embryonal carcinoma cells. Increased cAMP levels produced by autocrine stimulation of PLP receptors by PLP may contribute to differentiation of embryonal carcinoma cells into parietal endoderm.

Topics: Adenylyl Cyclases; Animals; Bucladesine; Cell Differentiation; Endoderm; Mice; Neoplasm Proteins; Parathyroid Hormone-Related Protein; Peptide Fragments; Protein Biosynthesis; Proteins; Receptors, Cell Surface; Receptors, Parathyroid Hormone; RNA, Messenger; RNA, Neoplasm; Teratoma; Tretinoin; Tumor Cells, Cultured

Activin/EDG, a stimulator of the secretion of follicle stimulating hormone (FSH) from pituitary gland and an inducer of erythroid differentiation for Friend leukemia cells, has since been implicated in a variety of biological roles. Here, we show some novel effects of activin on murine embryonal carcinoma cells (EC cells). First, activin acts as a growth factor on undifferentiated P19 cells, a well characterized EC cell line for the study of mammalian development. Second, activin inhibits the retinoic acid (RA) induced differentiation of P19 cells to neurons and glial cells. The inhibitory effect of activin on neural differentiation, which has yet to be proved in other physiological peptides, is confirmed also on the differentiation of various neuroblastoma cell lines. Our results suggest a possible role of activin as a negative regulator of neural differentiation in mammalian development.

Topics: Activins; Animals; Cell Differentiation; Cell Division; Cell Line; Inhibins; Kinetics; Neuroblastoma; Neurons; Teratoma; Tretinoin

The fluorescent dye Hoechst 33342 is able to differentiate F9 EC cells at low concentrations. This differentiation is accompanied by synthesis of large amounts of laminin, production of a well-developed cytoskeleton, disappearance of the SSEA-1 antigen, and synthesis of large amounts of fibronectin, all characteristics of the primitive endoderm. The dye immediately blocks the cells at the S/G2 phase of the cell cycle and produces a complete arrest in proliferation. This effect is not specific for the nullipotent F9 cell line, as multipotent EC cell lines like PCC3, P19, and PCC4 can also be easily differentiated into the same pathway by treatment with the Hoechst dye. In contrast, the dye has no remarkable effects on terminal differentiated, immortalized cells like NIH 3T3 or the parietal endoderm-like cell PYS-2.

Topics: Animals; Benzimidazoles; Cell Cycle; Cell Line; Cell Transformation, Neoplastic; Embryonal Carcinoma Stem Cells; Endoderm; Fibroblasts; Humans; Neoplastic Stem Cells; Teratoma; Tretinoin

The human teratocarcinoma NTERA-2 cl. D1 (NT2/D1) cell is a cloned multipotential embryonal cancer cell line that differentiates into a neuronal phenotype and other cellular lineages with retinoic acid (RA) treatment. Here we report that mRNA for the transforming growth factor-alpha is expressed in these RA-untreated cells and that RA-treatment results in a reduction of mRNA expression within 24 hr of treatment. In total cellular RNA, TGF-alpha mRNA is not detectable by Northern analysis at 6 days when there is increased expression of the human homeotic genes Hu-1 (Hox 2.1) and Hu-2 (Hox 2.2), known markers of RA response in NT2/D1 cells. RA treatment also causes a marked reduction in cloning efficiency and tumorigenicity of these cells. The addition of TGF-alpha or EGF (epidermal growth factor) protein to RA-untreated NT2/D1 cells augments soft agar cloning under limited fetal calf serum conditions. Blocking monoclonal antibodies directed against the EGF receptor (EGFr) can prevent this augmentation. We conclude that TGF-alpha expression inversely correlates with the state of RA-induced differentiation of this human teratocarcinoma cell and that TGF-alpha and EGF proteins are stimulatory growth factors in NT2/D1 cells under these culture conditions.

Topics: Cell Differentiation; Cell Line; Clone Cells; Gene Expression; Humans; Kinetics; RNA, Messenger; Teratoma; Transcription, Genetic; Transforming Growth Factors; Tretinoin; Tumor Cells, Cultured

Topics: Animals; Basement Membrane; Cell Differentiation; Chondroitin Sulfate Proteoglycans; Collagen; Gene Expression Regulation, Neoplastic; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Laminin; Mice; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

MK is a gene that is expressed temporarily during the early stages of retinoic acid-induced differentiation of embryonal carcinoma (EC) cells and during the mid-gestation period of mouse embryogenesis. The 5'-regions of MK cDNAs and their mRNAs are heterogeneous; so far three kinds of MK cDNAs (MK1, MK2, and MK3) have been isolated. The MK gene was cloned from a genomic DNA library of a BALB/c mouse, and its structure was elucidated. 5'-Region sequences specific for MK1, MK2, and MK3 were arranged in the order of MK3, MK2, and MK1. Then, there was a sequence common to all MK cDNAs consisting of four exons. The results indicate that different species of MK mRNA are generated by the use of alternative promoters and different modes of splicing.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Differentiation; Cloning, Molecular; DNA; Embryonic and Fetal Development; Exons; Gene Expression; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Promoter Regions, Genetic; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured

A newly identified gene, MK, is transiently expressed in the early stages of embryonal carcinoma cell differentiation and in the mid-gestation period of mouse embryogenesis (Kadomatsu, K., Tomomura, M., and Muramatsu, T. (1988) Biochem. Biophys. Res. Commun. 151, 1312-1318). Analysis of various MK cDNA clones revealed differences in the 5'-region. So far three classes of cDNA clones (MK1, MK2, and MK3) have been identified; they were different in the 5'-untranslated region but shared the rest of the sequence. Ribonuclease protection, RNA blotting, and primer extension revealed that MK2-type RNA was the major MK RNA in retinoic acid-treated embryonal carcinoma cells. In addition, the number of A residues in an oligo(A) stretch in the 5'-side of the common sequence differed from 9 to 29. The number was 9 in the most frequent cases, when the putative MK polypeptide had a molecular weight of about 15,500 and had a signal peptide-like sequence. Hybrid selected MK RNA yielded the predicted polypeptide upon in vitro translation. When pancreatic microsomal membranes were included in the translation system, the translation product of MK RNA was processed and entered into the lumen of the membranes. These results suggest that the product of the MK gene is an extracellular polypeptide.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Differentiation; Cell Line; Cloning, Molecular; DNA Probes; DNA, Neoplasm; Gene Expression; Gene Library; Genes; Molecular Sequence Data; Nucleic Acid Hybridization; Oligonucleotide Probes; Protein Biosynthesis; Restriction Mapping; RNA, Neoplasm; Teratoma; Transcription, Genetic; Tretinoin

B2 genes are short repeated sequences which are transcribed by RNA polymerase III. Abundant transcripts accumulate in embryonic and transformed cells, but transcripts are rare or absent from normal differentiated cell types. During retinoic acid-induced differentiation of P19 embryonal carcinoma cells, an early transient increase in B2 RNA levels is followed by a rapid drop in expression. The marked changes in B2 RNA levels are most likely due to transcriptional modulation since B2 RNA stabilities are unaffected by differentiation. At least four short-lived B2 RNAs with apparent lengths of 150, 180, 240, and 500 nucleotides were characterized. The two larger RNAs are polyadenylated and are more stable in cells. A cDNA of a B2 gene was isolated which was over 99% identical to the consensus sequence. This B2 cDNA can be transcribed in human cells and yields at least two distinct transcripts. We propose a model for B2 RNA metabolism which describes transcription, posttranscriptional modification and processing, and nucleocytoplasmic transport.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Line; Cloning, Molecular; DNA, Neoplasm; Gene Library; Humans; Mice; Molecular Sequence Data; Nucleic Acid Conformation; Nucleic Acid Hybridization; Plasmids; RNA, Neoplasm; Teratoma; Transcription, Genetic; Transfection; Tretinoin

We have previously isolated several cDNA clones specific for mRNA species that increase in abundance during the retinoic acid-associated differentiation of F9 teratocarcinoma stem cells. One of these mRNAs, J6, encodes a approximately 40 kDa protein as assayed by hybrid selection and in vitro translation (Wang, S.-Y., LaRosa, G., and Gudas, L. J. (1985) Dev. Biol. 107, 75-86). The time course of J6 mRNA expression is similar to those of both laminin B1 and collagen IV (alpha 1) messages following retinoic acid addition. To address the functional role of this protein, we have isolated a full-length cDNA clone complementary to this approximately 40-kDa protein mRNA. Sequence analysis reveals an open reading frame of 406 amino acids (Mr 45,652). The carboxyl-terminal portion of this predicted protein contains a region that is homologous to the reactive sites found among members of the serpin (serine protease inhibitor) family. The predicted reactive site (P1-P1') of this J6 protein is Arg-Ser, which is the same as that of antithrombin III. Like ovalbumin and human monocyte-derived plasminogen activator inhibitor (mPAI-2), which are members of the serpin gene family, the J6 protein appears to have no typical amino-terminal signal sequence.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Line; Cloning, Molecular; DNA, Neoplasm; Gene Library; Mice; Molecular Sequence Data; Protease Inhibitors; Restriction Mapping; RNA, Messenger; Sequence Homology, Nucleic Acid; Teratoma; Tretinoin

MK is a gene whose expression increases transiently during retinoic acid-induced differentiation of embryonal carcinoma cells. MK polypeptide was secreted by differentiating HM-1 embryonal carcinoma cells and by L-cells transfected with an MK cDNA under the control of the beta-actin promoter and Rous sarcoma virus enhancer. MK polypeptide was found to have heparin binding activity. Conditioned medium of the transfected L-cells promoted growth of PC-12 pheochromocytoma cells. These findings support the view that MK polypeptide is a secreted factor involved in regulation of growth and differentiation.

Topics: Animals; Carrier Proteins; Cell Differentiation; Cell Line; Chromatography, Affinity; Gene Expression; Heparin; Kinetics; L Cells; Promoter Regions, Genetic; Protein Biosynthesis; Rabbits; Reticulocytes; Teratoma; Transfection; Tretinoin

The level of expression of N-myc in mouse teratocarcinoma stem cells is very high. Previous studies have shown that N-myc expression significantly decreases when the stem cells are subjected to long-term induction for differentiation by retinoic acid (RA). We found that in a stem cell line, OTF9, a steep yet transient decrease of N-myc expression takes place much earlier, immediately after induction by RA. To examine whether this decrease is responsible for differentiation, we constructed a gene, miwNmyc, to express N-myc cDNA constitutively and transformed OTF9 cells with this gene construct. Transformants under the constitutive expression of miwNmyc differentiated normally, as judged by morphological changes and by modulation of c-myc, Hox1.1, and laminin B1 expression. Therefore, transient decrease of N-myc expression may be the consequence of RA-induced differentiation, even though it occurs very early in the process. Alternatively, in addition to N-myc decrease, there may be redundant mechanisms which lead to OTF9 differentiation after induction by RA, so that suppression of N-myc decrease is bypassed by at least one other mechanism.

Topics: Animals; Cell Differentiation; Cell Line; Gene Expression Regulation, Neoplastic; Genes, Regulator; Mice; Plasmids; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Proto-Oncogenes; Restriction Mapping; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The c-myc protooncogene is expressed in many tumor cells as well as during normal development. In order to study the role of c-myc in differentiation, proliferation and tumorigenicity of F9 mouse teratocarcinoma cells, the pSVmyc1 plasmid constitutively expressing an active c-myc oncogene was introduced into F9 stem cells by cotransfection with the selectable marker RSVneo. Enhanced expression of c-myc did not alter the properties of F9 stem cells. Prolonged proliferation during retinoic acid induced differentiation was observed in cell clones constitutively expressing c-myc. In contrast, as determined by morphology, by immunocytochemistry for markers specific for stem cells and differentiated derivatives, and by Northern hybridization for mRNAs specific for differentiated cells, differentiation was neither inhibited nor delayed by constitutive c-myc expression. Tumorigenicity of stem cells as well as retinoic acid-treated cells--as measured by soft agar cloning efficiency and tumor formation in syngenic mice--was not altered by SVmyc1. We conclude that in F9 teratocarcinoma cells down-regulation of c-myc is related to arrest of proliferation rather than differentiation.

Topics: Animals; Blotting, Northern; Cell Differentiation; Cell Division; Cell Line; Cloning, Molecular; Collagen; DNA; Gene Expression Regulation; Mice; Oncogenes; Plasmids; RNA; Teratoma; Transfection; Tretinoin

We have identified new repeated interspersed DNA sequences by analysis of homologous RNA transcripts from a human teratocarcinoma cell line (NTERA-2 clone D1). The abundance of transcripts varies upon retinoic acid induced differentiation of NTERA-2/D1 cells, and it is highest when the cells display the embryonal carcinoma phenotype. The expression of these novel repeated sequences appears to be tissue specific as no detectable expression was found in various cell lines of different embryological derivation. Characterization of the RNA transcripts by analysis of recombinant cDNA clones indicated that transcripts of different genomic units are present in undifferentiated embryonal teratocarcinoma cells. Nucleotide sequencing of the cloned cDNAs reveals a complex structure composed by unique and tandemly repeated sub-elements.

Topics: Base Sequence; Codon; DNA; Gene Expression Regulation; Humans; Molecular Sequence Data; Nucleic Acid Hybridization; Repetitive Sequences, Nucleic Acid; Restriction Mapping; RNA; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

A hybrid clone was developed by the fusion of a pluripotent mouse teratocarcinoma cell line PCC-4 AzaR to the Zajdela ascitic hepatoma (ZAH) of rat origin. This hybrid cell line, F2231A, possessed a predominantly teratocarcinoma morphology with a large nucleus and prominent nucleoli, and grew in nests. F2231A cells formed undifferentiated tumours in irradiated Sv/129 mice. It formed aggregates when subcultured at high densities in bacteriological Petri dishes. The hybrid cell line differentiated in response to retinoic acid and also underwent spontaneous differentiation upon overgrowth. Karyological analysis showed the presence of several rat chromosomes in the hybrid and upon isozyme analysis it was found that only the rat variant of the X-linked enzyme HGPRT was expressed. Analysis of the genomic DNA with a cloned probe, specific for rat repetitive sequences, gave strong positive signals in the hepatoma parent and F2231A cells while the parental embryonal carcinoma (EC) cells were negative. The hybrid cell line, like the PCC-4 cells, expressed the SSEA-1 surface marker but not SSEA-3, intercellular fibronectin and EGF receptors. Upon differentiation of F2231A cells there was a loss of expression of SSEA-1. The mRNA for alpha-fetoprotein was expressed by the hybrid cell line and in this respect it resembled the hepatoma parent. Albumin mRNA was not detectable in the hybrid cell line. The mRNA for the transformation-related protein, p53, was expressed at a high level in F2231A cells. The hybrid cell line F2231A retained several of the biochemical and immunological properties of the teratocarcinoma cells.

Topics: Animals; Antigens, Surface; Blotting, Southern; Cell Differentiation; DNA, Neoplasm; Epidermal Growth Factor; Hybrid Cells; Isoenzymes; Karyotyping; L-Lactate Dehydrogenase; Liver Neoplasms, Experimental; Mice; Molecular Weight; Phenotype; Rats; Repetitive Sequences, Nucleic Acid; RNA; Stem Cells; Teratoma; Tretinoin; Tumor Cells, Cultured

Thy-1 is a well characterized glycoprotein known to be variably expressed on the surface of different cell types. Serological analysis of a limited number of teratocarcinoma-derived cell lines suggested that mouse embryonal carcinoma cells do not express Thy-1 and that its expression is associated with the appearance of differentiated cells. In this report we show that monoclonal antibody 1aG4, recognizing Thy-1.2 epitope, binds specifically to P19 embryonal carcinoma cells and their undifferentiated subclones. A number of control experiments confirmed that 1aG4 antibody binds to the Thy-1.2 glycoprotein expressed on the surface of P19 embryonal carcinoma cells and not to the antigen expressed on differentiated derivatives of these cells or to a cross-reactive epitope. Transcriptional activity of the Thy-1 gene in undifferentiated P19 cells was shown by transfection experiments in which transfer of the Thy-1.1 gene into P19 cells resulted in stable expression of the Thy-1.1 antigen on the surface of recipient cells. Direct evidence for the presence of Thy 1 mRNA in P19 cells was obtained by Northern blot analysis with a Thy-1-specific cDNA probe. Treatment of P19 cells with retinoic acid resulted in a decrease in the expression of Thy-1 antigen which preceded changes in morphology of the cells. These data indicate that Thy-1 is a developmentally regulated surface marker of P19 embryonal carcinoma cells which is amenable to direct genetic analysis.

Topics: Animals; Antibodies, Monoclonal; Antigens, Surface; DNA Probes; Epitopes; Gene Expression Regulation, Neoplastic; Isoantibodies; Mice; RNA, Messenger; Teratoma; Thy-1 Antigens; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

A double immunofluorescence method was developed for the monitoring of proliferation and differentiation of F9 embryonal carcinoma cells. Cytokeratin filament expression was used as a marker for differentiation, and proliferating cell nuclear antigen (PCNA)/cyclin or bromodeoxyuridine labeling were used as markers for proliferation. F9 cells had a high proliferation rate and were cytokeratin-filament-negative. Upon treatment with retinoic acid and dibutyryl cyclic AMP, cytokeratin-filament-positive cells with differentiated phenotype appeared. After 3 days, the extent of proliferation of cytokeratin-filament-positive cells was comparable to, but after 5 days significantly lower than, that of cytokeratin-filament-negative cells in the same culture. In differentiating F9 cells, cytokeratin filament expression is associated with, and even slightly precedes, the dramatic decrease in the rate of proliferation.

Topics: Actin Cytoskeleton; Animals; Antigens, Neoplasm; Bucladesine; Cell Differentiation; Cell Division; Keratins; Mice; Nuclear Proteins; Proliferating Cell Nuclear Antigen; Teratoma; Tretinoin; Tumor Cells, Cultured

In the presence of retinoic acid (RA), cultured F9 murine teratocarcinoma stem cells differentiate into nontumorigenic cells resembling the extraembryonic endoderm of the early mouse embryo. By differential hybridization screening of an F9 cell cDNA library, we isolated a 1,745-nucleotide cDNA for a gene, REX-1 (for reduced expression), whose steady-state mRNA level began to decline in F9 cells in monolayer culture within 12 h after the addition of RA. By 48 to 96 h after RA treatment of F9 cells in monolayer culture, the REX-1 steady-state mRNA level was more than sevenfold lower than the level in undifferentiated F9 stem cells. The REX-1 mRNA decrease did not result from the reduction in cell growth rate associated with the differentiation process, since the REX-1 mRNA level did not decline in F9 cells that were partially growth arrested after 48 h of isoleucine deprivation. The RA-associated REX-1 mRNA decrease resulted primarily from a reduction in the transcription rate of the REX-1 gene in the presence of RA. In contrast to results in F9 cells, we have been unable thus far to detect REX-1 mRNA in day 7.5 to 12.5 mouse embryo RNA samples or in the P19 teratocarcinoma stem cell line. The putative REX-1 protein identified by DNA sequence analysis contains four repeats of the zinc finger nucleic acid-binding motif and a potential acidic activator domain, suggesting that REX-1 encodes a regulatory protein. The REX-1 gene is not identical to the previously reported murine genes that encode zinc finger-containing proteins.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Cell Line; Cloning, Molecular; DNA-Binding Proteins; Embryo, Mammalian; Gene Expression Regulation; Genes; Isoleucine; Metalloproteins; Mice; Molecular Sequence Data; Plasmids; RNA, Neoplasm; Sequence Homology, Nucleic Acid; Teratoma; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Zinc

Plasminogen activators are believed to play an important role in tissue remodeling and cell migration. During mouse embryogenesis, visceral endoderm secretes urokinase-type plasminogen activator (uPA) whereas parietal endoderm secretes tissue-type plasminogen activator (tPA). Visceral endoderm from F9 embryoid bodies can transdifferentiate into parietal endoderm under the appropriate culture conditions. We have examined at the protein and mRNA levels the type of plasminogen activator expressed in whole embryoid bodies, visceral endoderm and its parietal endoderm derivatives. Our experiments show that the visceral endoderm on F9 embryoid bodies synthesizes and secretes substantial amounts of both tPA and uPA. In contrast, the parietal endoderm derived directly from the visceral endoderm secretes dramatically increased levels of tPA and decreases production of uPA to low or below detectable levels. These data support the finding that visceral endoderm can transdifferentiate to parietal endoderm. In addition, this transition provides an excellent model for studying the molecular basis of the coincident down- and upregulation of the two plasminogen activators as well as their potential function during embryogenesis.

Topics: Animals; Cell Differentiation; Endoderm; Gene Expression; Mice; Molecular Weight; RNA, Messenger; Teratoma; Tissue Plasminogen Activator; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Retinoic acid, a natural derivative of vitamin A (retinol), induces mouse F9 teratocarcinoma stem cells to differentiate into nontumorigenic parietal endoderm cells. The mouse cellular retinoic acid binding protein (CRABP) has been implicated in the mechanism of action of retinoic acid (RA), since a mutant F9 cell line, RA-3-10, which possesses less than 5% of the wild type level of [3H]RA:CRABP binding activity, fails to differentiate in response to RA. In order to study the CRABP in this RA-induced differentiation process, we have cloned and sequenced the full-length mouse CRABP complementary DNA and have characterized its expression in wild type F9 and mutant cells. The mouse CRABP mRNA is a single, low abundant mRNA approximately 800 bases in length. The steady state level of the CRABP mRNA was measured in untreated stem cells and after the addition of RA alone, dibutyryl cyclic AMP plus theophylline (CT), or retinoic acid, dibutyryl cyclic AMP and theophylline (RACT) to F9 wild type and the mutant RA-3-10 cells. The CRABP mRNA was present in wild type F9 stem cells, and the level of its expression was changed by RA. When RA was added to F9 wild type cells, the steady state level of CRABP mRNA decreased 2- to 3-fold. When RACT was added to wild type cells, the level of CRABP mRNA increased and then decreased, resulting in a peak of CRABP mRNA expression between 24 and 48 h. In contrast, untreated mutant RA-3-10 cells had a lower level of CRABP mRNA than wild type stem cells, and the mutant cells responded quite differently to the addition of RA and RACT. The addition of RA caused an impressive 60-fold increase in the steady state level of CRABP mRNA in RA-3-10 cells by 120 h. One interpretation of this result is that there is negative regulation of CRABP mRNA expression, mediated directly or indirectly by the wild type functional CRABP protein, and that this regulation is aberrant in the RA-3-10 cells.

Topics: Base Sequence; Bucladesine; Carrier Proteins; Cell Differentiation; Cloning, Molecular; DNA; Humans; Molecular Sequence Data; Receptors, Retinoic Acid; RNA, Messenger; Teratoma; Theophylline; Tretinoin

F9 cells induced to differentiate with retinoic acid (RA) increase transcription of the tissue plasminogen activator (t-PA) gene. Further treatment of these cells with cyclic AMP (cAMP) results in an additional stimulation of t-PA gene transcription. To investigate the mechanism of this two-stage regulation, 4 kilobase pairs (kbp) of 5'-flanking sequence from the murine t-PA gene was isolated. Two major start sites for transcription were found, neither of which depended on a classical TATA motif for correct initiation. By using transient transfection assays, it was determined that 4-kbp of flanking sequence could confer on reporter genes the same two-stage differentiation-specific expression as was observed for the endogenous t-PA gene. Deletion analyses of this 4-kbp fragment showed that 190 bp of flanking sequence was sufficient to bestow the same degree of two-stage regulation on reporter gene constructs. Within this region of DNA, sequence analysis revealed a possible cAMP regulatory element, a CTF/NF-1 recognition sequence, two potential Sp1 sites, and five potential binding sites for transcription factor AP-2. The deletion experiments, coupled with the positions of these potential cis-acting elements, suggest that multiple transcription factors, including those that bind to cAMP regulatory element, CTF/NF-1, Sp1, and AP-2 sites, may be involved in regulation of the t-PA gene during F9 cell differentiation.

Topics: Animals; Base Sequence; Cell Differentiation; Chromosome Deletion; Chromosome Mapping; Cyclic AMP; DNA, Neoplasm; Gene Expression Regulation; Mice; Molecular Sequence Data; Promoter Regions, Genetic; Teratoma; Tissue Plasminogen Activator; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA)-induced differentiation of human teratocarcinoma (T2) cells results in a change from a normally non-permissive phenotype for human cytomegalovirus (HCMV) infection to cells which are fully permissive. We have used this system to analyse factors associated with differentiation which may regulate HCMV gene expression. Differentiation of T2 cells results in an increase of c-ras expression. Consequently, we have introduced ras expression vectors into T2 cells. We find that, as with RA induction, transfection of T2 cells with oncogenic human Ha-ras results in cells which are permissive for HCMV infection and gene expression. However, unlike RA which induces a cessation of cell proliferation and terminal differentiation, ras transfection only appears to result in changes associated with early events in RA-induced differentiation of T2 cells.

Topics: Cell Transformation, Neoplastic; Cytomegalovirus; Cytomegalovirus Infections; Gene Expression Regulation; Genes, ras; Humans; Phenotype; Teratoma; Transfection; Tretinoin; Tumor Cells, Cultured

F9 teratocarcinoma stem cells treated with retinoic acid (RA) and dibutyryl cAMP (but2 cAMP) differentiate into embryonic parietal endoderm. Using heparin-affinity chromatography, endothelial cell proliferation assays, immunoprecipitation, and Western analysis with antibodies specific for acidic and basic fibroblast growth factors (FGFs), we detected biologically active FGF in F9 cells only after differentiation. A bovine basic FGF cDNA probe hybridized to 2.2-kb mRNAs in both F9 stem and parietal endoderm cells and to a 3.8-kb mRNA in F9 stem cells. A genomic DNA probe for acidic FGF hybridized to a 5.8-6.0-kb mRNA in both F9 stem and parietal endoderm cells, and to a 6.0-6.3-kb mRNA only in parietal endoderm cells. Although these FGF mRNAs were present in the stem cells, we could find no evidence that F9 stem cells synthesized FGFs, whereas differentiated F9 cells synthesized both acidic and basic FGF-like proteins. We conclude that biologically active factors with properties characteristic of acidic and basic FGF are expressed by F9 parietal endoderm cells after differentiation. Differentiating embryonic parietal endoderm thus may serve as a source of FGF molecules in the developing blastocyst, where these factors appear to play a central role in subsequent embryogenesis.

Topics: Animals; Blotting, Northern; Blotting, Western; Bucladesine; Cell Differentiation; DNA Probes; Embryonal Carcinoma Stem Cells; Fibroblast Growth Factors; Heparin; Mice; Neoplastic Stem Cells; Teratoma; Tretinoin; Tumor Cells, Cultured

In addition to having profound effects on embryonic pattern formation, retinoic acid (RA) has striking effects on differentiation and maintenance of epithelial cells in vivo and in vitro Skin is a major target organ for retinoids both in its normal and pathological states. The discovery of two human nuclear receptors for RA (hRAR alpha and hRAR beta) acting as transcriptional RA-inducible enhancer factors has provided a basis for understanding how RA controls gene expression. To investigate the specific role that RARs might play during development and in adult tissues, we have cloned the mouse RAR alpha and RAR beta (mRAR alpha and mRAR beta). Their amino-acid sequences are much more homologous to those of hRAR alpha and hRAR beta, respectively, than to each other, which suggests strongly that RAR alpha- and beta-subtypes have different functions. Most interestingly we have discovered a novel RAR subtype (mRAR gamma) whose expression in adult mouse seems to be highly restricted to skin, whereas RAR alpha and RAR beta are expressed in a variety of adult tissues. Furthermore, both mRAR alpha and mRAR gamma RNAs are readily detected in undifferentiated F9 embryocarcinoma (EC) cells, whereas mRAR beta messenger RNA is induced at least 30-fold in RA-differentiated F9 cells.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carrier Proteins; Cell Line; Cloning, Molecular; Codon; Embryo, Mammalian; HeLa Cells; Humans; Mice; Molecular Sequence Data; Neoplasm Proteins; Receptors, Retinoic Acid; Skin; Teratoma; Transcription, Genetic; Transfection; Tretinoin

P19 embryonal carcinoma (EC) cells can be induced to differentiate in vitro into a variety of cell types by treatment with different concentrations of retinoic acid (RA). A study was conducted to explore the regulation of expression of the genes for cellular retinoic acid-binding protein (CRABP) and cellular retinol-binding protein (CRBP) in P19 cells induced to differentiate by RA. For each retinoid-binding protein, both the level of specific mRNA and of immunoreactive protein were measured, respectively, by RNase protection assay and by a specific RIA. Dramatic increases in CRABP and CRBP were seen, at both the mRNA and protein levels, during the RA-induced differentiation. CRBP induction differed from that of CRABP in several major ways. 1) Induction of CRBP occurred at lower concentrations of RA (10(-9) M) than did that of CRABP (10(-8)-10(-7) M). 2) CRBP induction was an early response (within 3 h) to RA treatment, whereas CRABP induction occurred at a later time (12-24 h). 3) Induction of CRABP mRNA by RA was blocked by the protein synthesis inhibitor cycloheximide, whereas induction of CRBP mRNA was not. 4) Several differentiation inducers were tested for their effects on the expression of CRABP and CRBP in P19 cells. CRBP induction occurred with a wider spectrum of inducers than did that of CRABP. 5) In addition, the induction of CRABP and CRBP mRNAs by RA was examined in six different cell lines, including three EC lines. CRBP induction occurred in a wider spectrum of cell lines than did that of CRABP. The induction of CRABP in EC cells seems, in general, to correlate with their differentiation into neuron-like cells. Taken together, our results suggest that CRBP induction may be a direct response to RA and represent a general event in RA-induced cell differentiation, whereas CRABP induction may be an indirect response and represent a later event restricted to only certain differentiation pathways. CRBP may be an early response gene induced by RA.

Topics: Animals; Base Sequence; Carrier Proteins; Cattle; Cell Differentiation; DNA Probes; Mice; Molecular Sequence Data; Radioimmunoassay; Receptors, Retinoic Acid; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; RNA, Messenger; Teratoma; Tretinoin

We have selected a mutant F9 teratocarcinoma stem cell line, RA-5-1, which does not exhibit normal differentiation into parietal endoderm in the presence of retinoic acid, dibutyryl cyclic AMP, and theophylline (RACT). In this report, we demonstrate that the RA-5-1 mutant possesses a prolyl-4-hydroxylase enzyme with a higher Km for a synthetic collagen substrate and that this alteration results in a 6-7-fold reduction in the amount of collagen IV in the medium of RACT-treated mutant cells, as compared to wild type F9 cells. In addition, the collagen IV that is secreted by RACT-treated RA-5-1 cells has an abnormally low molecular weight and contains 6-9-fold less 4-hydroxyproline than the collagen IV secreted by RACT-treated wild type F9 cells. A brief ascorbate treatment can increase the hydroxyproline content of the collagen IV secreted by RACT-treated RA-5-1 cells. A large reduction in the amount of laminin in the medium of RACT-treated RA-5-1 mutant cells is also observed. Concomitant with the reduction in collagen IV and laminin polypeptides in the medium, the expression of several other differentiation-specific mRNAs is delayed in the RACT-treated RA-5-1 cells relative to wild type F9 cells. Moreover, the mutant cells do not exhibit the morphology or the complete growth arrest of wild type terminally differentiated parietal endoderm cells in the presence of RACT. These results suggest that a defect in the post-translational modification of collagen IV in the mutant RA-5-1 prevents the complete expression of the differentiation program in response to RACT. These experiments also demonstrate that the expression of certain differentiation-specific genes is compatible with continued proliferation in the mutant line.

Topics: Animals; Ascorbic Acid; Bucladesine; Cell Differentiation; Cell Division; Cell Line; Collagen; Hydroxylation; Kinetics; Laminin; Mutation; Procollagen-Proline Dioxygenase; Protein Processing, Post-Translational; RNA, Messenger; Teratoma; Theophylline; Transcription, Genetic; Tretinoin

The individual blastomeres of the preimplantation mouse embryo become polarized during the 8-cell stage of development. This polarity forms as a result of a specific cell-cell interaction that has been termed induction. The ability of embryonal carcinoma (EC) cells to induce 8-cell blastomere polarization has been investigated by aggregating nonpolar 8-cell blastomeres with various types of EC cells. F9, a nullipotent stem cell, induced polarization of a nonpolar 8-cell companion in 80% of the aggregates. Stimulation of differentiation of F9 cells with retinoic acid (RA), with or without dibutyryl cAMP, caused a reduction in the polarity-inducing ability of these cells. Other EC cells, PSA-1, NULLI-SCC1, 3TDM, C3HNE, and P10, all displayed less polarity-inducing activity than F9. In addition, it was observed that when any of these cell types assumed a more differentiated phenotype, either spontaneously or in response to specific stimuli, they displayed a decrease in their ability to induce 8-cell polarization. As a control, the inducing ability of cells from normal mouse tissues was examined. It was found that neither STO mouse fibroblasts nor primary cultures of mouse lymphocytes were able to induce significant polarization of 8-cell stage blastomeres. These data support the hypothesis that while undifferentiated stem cell populations retain the ability to induce 8-cell blastomere polarization, it is apparently lost upon cellular differentiation.

Topics: Animals; Blastomeres; Bucladesine; Cell Adhesion; Cell Communication; Cell Differentiation; Embryonal Carcinoma Stem Cells; Embryonic and Fetal Development; Embryonic Induction; Female; Fibroblasts; Lymphocytes; Mice; Neoplastic Stem Cells; Stimulation, Chemical; Teratoma; Tretinoin; Tumor Cells, Cultured

Using iodinated insulin-like growth factors (IGFs) we have detected receptors for IGF-I at the cell surface of the clonally derived human embryonal carcinoma cell line Tera 2 clone 13. Affinity crosslinking of IGFs to Tera 2 clone 13-derived membrane preparations revealed the presence of proteins with features of both type-I and type-II IGF receptors. Treatment of Tera 2 clone 13 cells with retinoic acid to induce differentiation results in an increased number of cell surface receptors, apparently without altering the ratio of type-I and type-II receptors. In addition, Tera 2 clone 13 IGF-I receptors catalyze (auto)phosphorylation at tyrosine upon IGF-I and insulin binding. These findings suggest that type-I IGF receptors might be involved in mediating the effects of IGFs and insulin upon the proliferation of Tera 2 clone 13 cells.

Topics: Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Gene Expression; Humans; Insulin; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Neoplasms, Germ Cell and Embryonal; Phosphotransferases; Receptors, Cell Surface; Receptors, Somatomedin; Teratoma; Tretinoin; Tumor Cells, Cultured

The embryonal carcinoma cell line PCC7-S-AzaR1 (clone 1009) has been shown to differentiate in the presence of all-trans retinoic acid and dibutyryl cAMP into cells of predominantly neural properties (Paulin, D., H. Jakob, F. Jacob, K. Weber, and M. Osborn. 1982. Differentiation. 22:90-99). By analyzing the marker expression of derivatives in further detail, we characterized the two major cell phenotypes as neuron- and fibroblast-like and the two minor ones as astroglia- and endothelial-like. The stability of developmental commitment of clone 1009 was tested by recloning. The isolated subclones exhibited different patterns of chemically induced derivatives, with some of them (denoted N-clones) producing only a single (neuronal) cell type. As shown by long-term cultures in the absence of retinoic acid, the properties of isolated subclones remained essentially stable. In contrast to the clones producing neuron-like and other derivatives upon induced differentiation, the (exclusively neuronal) derivatives of N-clones detached and died within a few days in culture. If maintained in the presence of other neural cell types, however, their survival was dramatically extended indicating a requirement for specific interactions with other cells of the same tissue. The patterns of derivatives obtained from N-clones depended on the chemical nature of the substrate on which they were grown. Thus, when seeded on laminin-coated surfaces before induced differentiation, N-clones developed not only to neuron-like derivatives but rather to the same four derivatives observed with the original cell pool. These and further results suggest a common cell lineage of the identified phenotypes. The isolated subclones of uninduced cells probably represent different states of commitment within the same developmental pathway. Their stability offers the opportunity to analyze the nature of cellular commitment on the cellular, molecular, and genetic levels. This makes the family of clones derived from PCC7-S-AzaR1 (clone 1009) cells an advantageous in vitro model of mammalian brain early ontogenesis.

Topics: Animals; Antibody-Dependent Cell Cytotoxicity; Bucladesine; Cell Differentiation; Cell Line; Clone Cells; Complement System Proteins; Mice; Nerve Tissue Proteins; Neurons; Teratoma; Tretinoin

The retinoic acid (RA)-associated differentiation of murine F9 teratocarcinoma stem cells results in dramatic changes in gene expression. The cellular gene encoding the B1 subunit of the extracellular matrix protein laminin is transcriptionally activated by RA, and its transcription is further enhanced by N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) during the differentiation of F9 stem cells into extraembryonic parietal endoderm cells. We now report that expression vectors encoding the human RA receptors RAR-alpha, RAR-beta, and RAR-gamma can activate chloramphenicol acetyltransferase (CAT) expression from laminin B1 promoter/CAT expression vectors (e.g., p1.6LAMCAT) in RA-treated F9 cells, as measured in a transient transfection assay. Bt2cAMP does not further enhance the RA-associated increase in CAT activity. Through the use of deletion and mutation analyses, the RA-responsive element (RARE) of the murine laminin B1 gene has been defined as a 46-base-pair element between -477 and -432 of the laminin B1 5' flanking region. Insertion of a region of DNA containing this RARE in either orientation into a thymidine kinase promoter/CAT expression vector causes CAT expression to be activated 5- to 9-fold by the cotransfected human RAR-alpha or RAR-beta constructs in RA-treated F9 cells, and this RARE also functions in human HeLa cells. In contrast, this RARE in the p1.6LAMCAT vector does not activate CAT expression when cotransfected into F9 stem cells with the c-erbA gene in the presence of thyroid hormone. This suggests that the laminin B1 gene is activated by RA but not by thyroid hormone in vivo.

Topics: Animals; Base Sequence; Bucladesine; Cell Differentiation; Cell Line; Chloramphenicol O-Acetyltransferase; Gene Expression Regulation; Genes; Genetic Vectors; HeLa Cells; Humans; Laminin; Macromolecular Substances; Mice; Molecular Sequence Data; Promoter Regions, Genetic; Restriction Mapping; Teratoma; Thymidine Kinase; Transcription, Genetic; Transfection; Tretinoin

Most developmentally regulated epitopes identified on embryonal carcinoma cells and murine preimplantation embryos are associated with a glycoprotein-bound large glycan called embryoglycan. To prepare monoclonal antibodies recognizing other, less immunogenic stage-specific embryonic epitopes, we used embryoglycan-negative embryonal carcinoma cells P19XT.1.1 as immunogen. One monoclonal antibody prepared by this strategy was found to react specifically with mouse embryonal carcinoma and embryo-derived stem cell lines. The target epitope, TEC-4, was found to be expressed on eggs and two-cell embryos but was undetectable on later stages of mouse embryos and adult mouse tissues. NaDodSO4/PAGE of immunoaffinity-isolated antigen revealed that TEC-4 epitope is associated with glycoproteins of apparent Mr 120,000 and 240,000. The epitope was resistant to oxidation by sodium periodate and to digestion by endoglycosidase F but was sensitive to treatment with protein-denaturing agents and proteases, which suggested that the epitope is located in the protein moiety of the molecule. In the course of retinoic acid-induced differentiation of embryonal carcinoma cells the epitope disappeared before the onset of morphological differentiation. The combined data indicate that TEC-4 is an unusual stage-specific embryonic antigen that may be amenable to direct genetic analysis.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Cell Differentiation; Cell Line; Cytotoxicity, Immunologic; Fluorescent Antibody Technique; Gene Expression; Glycolipids; Lewis X Antigen; Mice; Molecular Weight; Polysaccharides; Teratoma; Tretinoin; Tumor Cells, Cultured

In mouse embryos, the int-1 proto-oncogene is transiently expressed in areas of the developing neural system. Retinoic acid-treated P19 embryonal carcinoma cells have often been used as an in vitro model for the molecular basis of neural development. We shown here that int-1 is transiently expressed in differentiated P19 cells. The time course and retinoic acid dose dependence of int-1 expression suggest that the gene is specifically expressed during early neural differentiation. P19 cells may be a useful model to assist in the study, at the cellular level, of the role of int-1 in neural development.

Topics: Animals; Cell Differentiation; Gene Expression Regulation; Neurons; Proto-Oncogene Proteins; Proto-Oncogenes; Teratoma; Tretinoin; Tumor Cells, Cultured; Wnt Proteins; Wnt1 Protein; Zebrafish Proteins

The effects of aggregation, retinoic acid, and medium conditioned by Buffalo rat liver (BRL) cells, alone and in combination, on the differentiation of PSA4TG12 embryonal carcinoma and E14 embryonal stem cells are reported. The observations indicate that BRL-conditioned medium has more than one effect on the differentiation process, that retinoic acid has at least two effects which operate in different concentration ranges, and that both agents influence the choice of differentiation pathway as well as the extent of differentiation.

Topics: Animals; Cell Aggregation; Cell Differentiation; Cell Line; Clone Cells; Culture Media; Embryo, Mammalian; Liver; Mice; Microscopy, Electron; Rats; Rats, Inbred BUF; Teratoma; Tretinoin

Proto-oncogene expression is altered in P19 embryonal carcinoma cells during retinoic acid-induced neuronal differentiation. A transient three- to four-fold increase in erbB proto-oncogene expression and a similar although smaller increase in src expression was observed during the period of time when events committing the cells to differentiate were occurring, but prior to the expression of the differentiated phenotype. During the differentiation phase, the only change was a decrease in myc proto-oncogene expression. These changes were not observed in untreated controls, cell treated with retinoic acid while growing as monolayer cultures or with mutants of P19 which did not undergo neuronal differentiation in response to retinoic acid treatment, suggesting some degree of specificity for neuronal differentiation.

Topics: Animals; Cell Differentiation; Mice; Mice, Inbred C3H; Proto-Oncogenes; Teratoma; Tretinoin; Tumor Cells, Cultured

A novel human gene, encoding a 188 amino acid polypeptide that contains a region similar to that of the epidermal growth factor, has been isolated. The gene, expressed in undifferentiated human and mouse teratocarcinoma cells, is shut off after inducing the cells to differentiate by treatment with retinoic acid. Introduction of the cDNA under the control of a viral LTR induces transformation of NIH3T3 cells.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Cell Differentiation; Cell Line; Cells, Cultured; DNA, Neoplasm; Epidermal Growth Factor; Genes; Humans; Mice; Molecular Sequence Data; Multigene Family; Sequence Homology, Nucleic Acid; Teratoma; Transcription, Genetic; Tretinoin

Cell interactions have been implicated in the differentiation of visceral and parietal endoderm in the developing mouse embryo. Embryoid bodies formed from F9 embryonal carcinoma cells have been useful in characterizing the events which lead to endoderm formation. As part of our effort to specify the interactions which may be involved in this process we have isolated visceral endoderm-like cells (VE) from F9 embryoid bodies and cultured them under various conditions. Using a combination of immunoprecipitation and enzyme-linked immunosorbent assay, we demonstrate that monolayer culture of these cells on a number of different substrates leads to a dramatic decrease in the level of alphafetoprotein (AFP), a VE-specific marker. Northern blot analysis of AFP mRNA indicates very low levels of this message are present after 48 hr in monolayer culture. Coincident with the drop in AFP levels is an increase in the levels of the cytokeratin Endo C and tissue plasminogen activator, both markers for parietal endoderm (PE). Morphological evidence at the ultrastructural level supports a transition from VE to PE. In contrast, the VE phenotype can be maintained in vitro by interaction with aggregates, but not monolayers, of stem cells. In addition, culturing the cells on the curved surface of gelatin-coated dextran beads, but not on a flat gelatin surface facilitates AFP expression and the cells are morphologically intermediate between VE and PE cells. The potential role of junctional complexes and cell shape are discussed.

Topics: alpha-Fetoproteins; Animals; Bucladesine; Cell Communication; Cell Differentiation; Ectoderm; Endoderm; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Immunosorbent Techniques; Keratins; Mice; Microscopy, Electron; RNA, Messenger; Stem Cells; Teratoma; Tissue Plasminogen Activator; Tretinoin; Tumor Cells, Cultured; Viscera

To test the putative role of c-fos in F9 differentiation, we have attempted to inhibit c-fos expression in these cells using an SV40-based expression vector (pSVneo-sof) that programs expression of c-fos antisense (sof) sequences as a 3' extension of a neo mRNA transcript. Of six G418-resistant clones isolated in transfection experiments, five expressed neo-sof transcripts. Two clones synthesized polyadenylated mRNA of the expected size (3.8 kb), two were smaller than expected, and one was larger. Two clones that expressed reduced levels of c-fos protein were inhibited in the induction of laminin, type IV collagen, and proteoglycan-19 RNA transcripts measured after 4 days of differentiation induction with RA and dibutyryl cyclic AMP. Also inhibited was the induction of the differentiation markers, TROMA-1 and TROMA-3. Antisense-expressing cells were not inhibited in the differentiation pathway to visceral endoderm since the alpha-fetoprotein gene was activated normally. We conclude that c-fos antisense expression inhibits some aspects of differentiation in F9 cells.

Topics: Bucladesine; Cell Differentiation; Collagen; DNA, Recombinant; Endoderm; Fluorescent Antibody Technique; Gene Expression Regulation; Immunoenzyme Techniques; Laminin; Nucleic Acid Hybridization; Plasmids; Proteoglycans; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; RNA; RNA, Antisense; RNA, Messenger; Simian virus 40; Teratoma; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured

Two tissue plasminogen activator (tPA) cDNA clones were isolated from mouse cDNA libraries and characterized. The sequence coding for mature mouse tPA protein including its 29-residue signal peptide, as well as that for the 3' and part of the 5'-nontranslated regions of the mRNA was determined. The mature protein consists of 530 amino acids and is encoded by a transcript of approximately 2800 nucleotides. The mouse tPA protein has 81% homology with its human counterpart, and amino acid sequence conservations suggest that the multidomain-like structure found for human tPA is maintained in the mouse enzyme. The tPA cDNA has been used as a probe to analyze tPA gene expression during F9 cell differentiation. tPA mRNA is not detected in F9 stem cells. When F9 cells are induced to differentiate with retinoic acid and dibutyryl cyclic AMP, tPA mRNA accumulates. We demonstrate that this induction occurs at least in part because of an increase in tPA gene transcription.

Topics: Animals; Bucladesine; Cell Differentiation; Cloning, Molecular; DNA; Gene Expression Regulation; Mice; Molecular Sequence Data; RNA, Messenger; Teratoma; Tissue Plasminogen Activator; Tretinoin; Tumor Cells, Cultured

Human pluripotential embryonic teratocarcinoma cells differentially expressed gene activity controlled by the human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) long terminal repeats (LTRs) when differentiation was induced by the morphogen all-trans retinoic acid. The alterations occurred after commitment and before the appearance of the multiple cell types characteristic of these pluripotential cells. After commitment, gene activity controlled by the HIV-1 LTR markedly increased, whereas that controlled by the HTLV-I LTR decreased. Steady-state mRNA levels and nuclear run-on transcription indicated that the increased HIV-1-directed activity during differentiation occurred posttranscriptionally, whereas the decreased HTLV-I activity was at the transcriptional level. Phorbol esters did not cause commitment but strongly enhanced expression by both viral LTRs at the transcriptional level. A specific inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, indicated that the enhanced activity involved the activation of protein kinase(s) C; altered cyclic nucleotide metabolism was apparently not involved. Differentiating cells gradually lost the ability to respond to phorbol ester stimulation. Experiments with a deletion mutant of the HIV-1 LTR suggested that this was due to imposition of negative regulation during differentiation that was not reversed by phorbol ester induction. Cycloheximide, with or without phorbol ester, slightly stimulated HIV-1-directed activity at the transcriptional level and massively increased the amounts of steady-state mRNA by posttranscriptional superinduction. It appeared, however, that new nuclear protein synthesis was required for maximal transcriptional stimulation by phorbol esters. Thus, changing cellular regulatory mechanisms influenced human retrovirus expression during human embryonic cell differentiation.

Topics: Cell Differentiation; Cycloheximide; Deltaretrovirus; Gene Expression Regulation; Genes, Viral; HIV; Humans; Plasmids; Promoter Regions, Genetic; Repetitive Sequences, Nucleic Acid; RNA, Messenger; RNA, Viral; Teratoma; Tetradecanoylphorbol Acetate; Transcription Factors; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured

Treatment of PYS cells with the tumor promoter (TPA) has been previously shown to enhance calcium- and phospholipid-dependent protein kinase (PK.C) in the membranes and to decrease its activity in the cytosol. Evidence is presented that 0.1 microM TPA treatment of PYS cells causes an opposite effect on the cyclic AMP-dependent protein kinases (PK.A). Within 10 min TPA led to an increase in PK.A in the cytosol and a concomitant decrease in the membranes, as measured by both the kemptide phosphorylation activity and photoaffinity labeling of RI and RH regulatory subunits of PK.A, with 8-azido-cyclic [32P]AMP. Moreover, the antitumor promoter retinoic acid (RA, 0.1 microM), when added simultaneously with TPA to the PYS cells, completely abolished the TPA effects on PK.A. When RA was added 25 min before TPA, the counteraction was not observed, indicating that RA was counteracting the TPA effect directly. These results suggest that TPA induces a rapid change in the compartmentalization of PK.A between the membrane and the soluble fraction. This possible translocation of PK.A seems to be blocked by RA, suggesting that the early antagonistic effects of RA toward TPA-mediated events occur at the plasma membranes.

Topics: Animals; Azides; Cell Membrane; Cyclic AMP; Mice; Molecular Weight; Protein Kinases; Teratoma; Tetradecanoylphorbol Acetate; Tretinoin

We have recently reported a mammalian cell plasmid (L factor) whose structure is related to that of polyomavirus (T. Kusano, H. Uehara, H. Saito, K. Segawa, and M. Oishi, Proc. Natl. Acad. Sci. USA 84:1789-1793, 1987). When composite DNA constructed from L factor and a foreign gene was introduced into mouse embryonal carcinoma (F9) cells by transfection, the DNA was reestablished in the cells as a plasmid. The reestablished plasmid DNA in F9 cells could be rescued in Escherichia coli. The plasmid-bearing cells underwent normal in vitro differentiation in response to retinoic acid. The efficiency of plasmid establishment of the L-factor-derived DNA and transcriptional and transient replicational activities were compared with those of similar composite DNA constructed from polyomavirus and an embryonal carcinoma mutant of polyomavirus which is permissive in F9 cells. The results suggest an inverse relationship between the efficiency of the plasmid establishment and the activity of gene expression controlled by the intrinsic enhancer-promoter of the DNA.

Topics: Animals; Cell Differentiation; DNA Replication; DNA, Recombinant; Escherichia coli; Gene Expression Regulation; Genetic Vectors; L Cells; Mice; Plasmids; Polyomavirus; Recombinant Fusion Proteins; Teratoma; Tretinoin; Tumor Cells, Cultured

Several differentiation-specific genes, including those for collagen IV and laminin, are induced by retinoic acid (RA) in mouse F9 teratocarcinoma cells. Dibutyryl cAMP can enhance the effect of RA in these cells, but dibutyryl cAMP alone does not induce these genes. Inhibition of RNA synthesis with 5-6-dichloro-1-B-D-ribofuranosylbenzimidazole prevents the induction of these genes by RA; inhibition of DNA synthesis with aphidicolin does not prevent the induction. In vitro transcription studies (Wang et al., Dev. Biol., 107:75-86, 1985) demonstrate that these differentiation-specific genes are regulated by RA at least partially at the level of transcription. To determine whether the regulation of transcription of these differentiation-specific genes is a primary effect of RA, we measured the sensitivity of the induction of mRNAs specific for these RA-inducible genes to inhibitors of protein synthesis. RNA was isolated from F9 cells that had been treated for 20 hr with RA (with or without dibutyryl cAMP) in the presence or absence of either cycloheximide or puromycin. We then hybridized the 32P-labeled recombinant plasmids collagen IV (alpha 1) (pcI5), laminin B1 (pcI56), and pcJ6 to RNA from the treated cells. Both cycloheximide and puromycin inhibited the RA induction of the collagen IV (alpha 1), laminin B1, and J6 mRNAs. In contrast, in a control experiment, a 20-hr treatment with cycloheximide did not inhibit the accumulation of metallothionein I-specific mRNA in response to zinc in F9 cells. Thus protein synthesis is required for the expression of the collagen IV (alpha 1), laminin B1, and J6 genes, and this result suggests that the transcriptional regulation of these genes by RA is indirect.

Topics: Animals; Aphidicolin; Bucladesine; Cell Line; Collagen; Dichlororibofuranosylbenzimidazole; Diterpenes; Gene Expression Regulation; Laminin; Protein Synthesis Inhibitors; Puromycin; Retinol-Binding Proteins; RNA, Messenger; Teratoma; Tretinoin

Down-regulation of Myc expression is the earliest documented change in gene expression in retinoic acid-induced differentiation of murine F9 teratocarcinoma cells. F9 cells transfected with plasmids expressing antisense Myc sequences under control of the simian virus 40 (SV40) early promoter exhibit a decrease in Myc protein. The result of this decrease is the spontaneous differentiation into cells that resemble retinoic acid-treated F9 cells as judged by plasminogen activator assays. In contrast, when F9 cells are transfected with a plasmid expressing Myc under control of the SV40 early promoter, resulting cell clones are resistant to differentiation by retinoic acid as shown by the lack of induction of plasminogen activator. These results suggest that down-regulation of Myc is sufficient and necessary for F9 cell differentiation.

Topics: Animals; Cell Differentiation; Cell Line; Oncogenes; Phenotype; Plasmids; Simian virus 40; Teratoma; Transfection; Tretinoin

We have isolated and characterized cDNA clones coding for the H1 histone subtype H1(0) in mouse teratocarcinoma cells. The mRNA is 2100 nt long and contains a coding sequence which is highly related to that of the human H1(0) gene. Using this cDNA as a probe, we have shown that, in comparison to undifferentiated F9 cells, differentiated F9 teratocarcinoma cells contain large amounts of H1(0) mRNA. This increase takes place very early during differentiation and does not correlate with changes in the rate of cell division. This indicates that the accumulation of H1(0) mRNA is not the result of reduced proliferation. Most likely on the contrary, the increase in the amount of H1(0) and the resulting effects on the formation of high order chromatin structures are parts of the differentiation program induced in F9 cells.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bucladesine; Cell Cycle; Cell Differentiation; Cloning, Molecular; Endoderm; Gene Expression Regulation; Histones; Mice; Molecular Sequence Data; RNA, Messenger; Teratoma; Tretinoin

Topics: 5'-Nucleotidase; Animals; Bucladesine; Cell Differentiation; Dimethyl Sulfoxide; Mice; Neoplasm Transplantation; Nucleotidases; Teratoma; Tretinoin

Embryonal carcinoma cells are useful in the study of embryogenesis and development, and their differentiation into neurons serves as a model of neuronal development. Retinoic acid was used to differentiate P19S18O1A1 embryonal carcinoma cells into neuronal, glial, and fibroblast-like cells and the phenotype of the neuronal population was examined. Neuron-specific enolase was present in the neuronal cells, suggesting that these neurons had reached some degree of maturity. A population (approximately 70%) of the neurons showed positive immunocytochemistry for tyrosine hydroxylase, dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase, three enzymes in the pathway of catecholamine synthesis. Therefore a population of the neurons appeared to be adrenergic. These neurons also showed a low level of histofluorescence for endogenous catecholamines and exhibited an exogenous catecholamine reuptake system. In order to determine the phenotype of other neuron-like cells found to be negative for the adrenergic properties examined, immunocytochemistry for neuropeptides and neurotransmitters known to coexist within central neurons was performed. Serotonin, vasoactive intestinal peptide, glutamic acid decarboxylase, and choline acetyltransferase were all absent from retinoic acid-treated P19S18O1A1 neuronal cultures. These studies, along with those that compare the effects of retinoic acid and other growth modulators on neuronal differentiation of embryonal carcinoma cells, should aid in the understanding of neuronal induction and development in vivo.

Topics: Animals; Catecholamines; Cell Differentiation; Intermediate Filament Proteins; Mice; Neoplasm Proteins; Neurofilament Proteins; Neurons; Neurotransmitter Agents; Phenotype; Teratoma; Tretinoin; Tumor Cells, Cultured

We have previously shown that retinoic acid-treated cultures of the P19 line of embryonal carcinoma cells differentiate into neurons, glia, and fibroblast-like cells (Jones-Villeneuve et al., 1982). We report here that the monoclonal antibody HNK-1 reacts with the neurons at a very early stage of their differentiation and is, therefore, an early marker of the neuronal lineage. Cells in differentiated P 19 cultures synthesized acetylcholine but not catecholamines, suggesting that at least some of the neurons are cholinergic. The neurons also carry high-affinity uptake sites for GABA but not for serotonin. In long-term cultures, neuronal processes differentiated into axons and dendrites, which formed synapses. This biological system should prove valuable for examining the development and maturation of cholinergic neurons, since their differentiation occurs in cell culture.

Topics: Acetylcholine; Animals; Antibodies, Monoclonal; Cell Line; Cell Transformation, Neoplastic; gamma-Aminobutyric Acid; Mice; Neurons; Neurotransmitter Agents; Teratoma; Tretinoin

We have studied the regulation of expression of the carbamoyl-phosphate synthetase II-aspartate transcarbamylase-dihydroorotase gene in F9 teratocarcinoma cells during their differentiation into parietal endoderm cells by induction with a combination of retinoic acid and dibutyryl cyclic AMP. Steady-state levels of CAD mRNA decreased by 7-fold in F9 cells following 120 h of retinoic acid and dibutyryl cyclic AMP induction as compared to levels in uninduced cells. Conversely, no apparent changes were found in the steady-state levels of beta-actin mRNA between induced and uninduced cells. Despite a 7-fold decrease in the steady-state levels of CAD mRNA, its rate of transcription remained the same between induced and uninduced cells, indicating a role for posttranscriptional mechanisms for its down regulation during retinoic acid- and dibutyryl cyclic AMP-induced differentiation of F9 cells. The cellular growth rate of F9 cells as determined by [3H]thymidine uptake and parallel cell counting decreased markedly during their induction with retinoic acid and dibutyryl cyclic AMP. Taken together, it is apparent that the expression of the CAD gene is cell-growth-dependent and its regulation in this system is at the posttranscriptional level.

Topics: Actins; Amidohydrolases; Animals; Aspartate Carbamoyltransferase; Bucladesine; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); Cell Differentiation; Cell Division; Dihydroorotase; DNA; Gene Expression Regulation; Ligases; Mice; Nucleic Acid Hybridization; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA), the natural acidic derivative of vitamin A, can modulate the expression of specific genes and can induce some cell types, such as the murine F9 teratocarcinoma stem cell line, to differentiate in culture. As an initial step toward understanding the molecular mechanism(s) by which RA exerts these effects, we previously isolated cDNA clones for a gene, ERA-1, which has the characteristics of an early, direct target for RA. We demonstrated that RA causes a rapid, dose-dependent, and protein synthesis-independent expression of the ERA-1 gene (G. J. LaRosa and L. J. Gudas, Proc. Natl. Acad. Sci. USA 85:329-333, 1988). We now report the full-length cDNA sequence and the further characterization of this gene. The data indicate that the RA-induced 2.2- to 2.4-kilobase ERA-1 RNA species that we previously detected consists of two alternately spliced messages. One mRNA encodes a protein with a predicted mass of about 36 kilodaltons (kDa) that possesses the Hox 1.6 homeobox domain. The other mRNA encodes a truncated protein of about 15 kDa which is identical to the 36-kDa protein for 114 amino acids at the amino-terminal end but which lacks the homeobox amino acid sequence. The RA-associated increase in the ERA-1 mRNA level does not appear to be due to message stabilization, suggesting that the response is at the level of transcription. By Northern (RNA) blot analysis, the usual 2.2- to 2.4-kilobase mRNA species was also rapidly expressed in P19 teratocarcinoma cells during their differentiation to fibroblastic cells in response to RA and was detected in day 10.5 and day 13.5 mouse embryos. This result indicates that the expression of this gene is not limited to the endodermal differentiation of F9 cells.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Line; Cloning, Molecular; DNA, Neoplasm; Genes; Genes, Homeobox; Molecular Sequence Data; Neoplasm Proteins; Nucleic Acid Hybridization; RNA Splicing; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin

Previous work has shown that the members of the myc protooncogene family are differentially regulated during murine development (Zimmerman et al., 1986). In this study, we have used the F9 mouse teratocarcinoma cell line to investigate the expression of two members of this family, the N- and c-myc genes. We demonstrate here that both N-myc and c-myc RNAs are unstable in these cells, but that they are clearly differentially regulated during a variety of cellular processes. Following retinoic acid addition, N-myc expression declines but then returns to initial levels as the cells undergo endodermal differentiation. c-myc RNA levels decrease more slowly and remain low in the differentiated cells. Additionally, we find that serum starvation and serum stimulation, treatments that alter c-myc RNA levels, do not have a significant effect on N-myc expression. These results provide further support for a role of c-myc expression in growth control but demonstrate that N-myc levels are not correlated with proliferative state of the cell.

Topics: Animals; Blood Physiological Phenomena; Cell Differentiation; Cell Division; Culture Media; Gene Expression Regulation; Growth Substances; Mice; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured

Immunofluorescence staining with antibodies to tubulin, neurofilaments and glial filaments was used to study the effects of methylmercury on the differentiation of retinoic acid-induced embryonal carcinoma cells into neurons and astroglia and on the cytoskeleton of these neuroectodermal derivatives. Methylmercury did not prevent undifferentiated embryonal carcinoma cells from developing into neurons and glia. Treatment of committed embryonal carcinoma cells with methylmercury doses exceeding 1 microM resulted in the formation of neurons with abnormal morphologies. In differentiated cultures, microtubules were the first cytoskeletal element to be affected. Their disassembly was time- and concentration-dependent. Microtubules in glial cells and in neuronal perikarya were more sensitive than those in neuronal processes. Neurofilaments and glial filaments appeared relatively insensitive to methylmercury treatment but showed reorganization after complete disassembly of the microtubules. The data demonstrate 1) the sensitivity of microtubules of both neurons and glia to methylmercury-induced depolymerization, and 2) the heterogeneous response of neuronal microtubules to methylmercury, presumably reflecting posttranslational modifications of different subpopulations of microtubules in the perikarya and neurite.

Topics: Animals; Cell Differentiation; Cells, Cultured; Cytoskeleton; Drug Interactions; Embryonal Carcinoma Stem Cells; Female; Fluorescent Antibody Technique; Humans; Methylmercury Compounds; Mice; Microtubules; Neoplastic Stem Cells; Neurons; Pregnancy; Teratoma; Tretinoin

The study of early human development is of great importance but has been limited by the lack of suitable reagents. Recently, however, the human embryonal carcinoma (EC) cell line NT2D1 has been isolated. This cell line will differentiate upon exposure to retinoic acid (RA). A cDNA library was constructed from poly(A)+ RNA derived from NT2D1 cells treated with 10(-5) M-RA for 7 days (delta NT2D1 cells). By differential cDNA screening, it was found that 1.12% of delta NT2D1 cDNA recombinants screened detected an increase in signal with 32P-cDNAs derived from delta NT2D1 as compared with NT2D1. To compare RA-induced differentiation of mouse and human EC cells, the delta NT2D1 cDNA library was rescreened with 32P-cDNAs derived from the mouse EC cell line F9 and the result compared with 32P-cDNA derived from F9 differentiated to parietalendoderm (F9PE)-like cells and visceral-endoderm (F9VE)-like cells. Approximately 1.2% of the delta NT2D1 cDNA recombinants detected a differential increase in signal following differentiation of mouse EC cells to F9VE and/or F9PE. Of these homologous regulated sequences, 0.3% were common to both mouse and human EC cell RA-induced differentiation. Five different cDNA clones were isolated that detect a marked increase (5- to 75-fold) in mRNA abundance following RA-induced differentiation of NT2D1. Of these five clones, three detect homologous mRNAs which also increase in abundance following differentiation of the mouse EC cell line F9 to PE- and/or VE-like cells; the other two clones do not detect sequences in the mouse mRNAs tested. One clone shows homology to SPARC, a gene known to be regulated during mouse embryonic development. While another clone, SO5A, has a limited range of expression, being detected in F9VE and in a human parietal-endoderm-like cell, but not in F9PE and a human visceral-endoderm-like cell. This work shows that there are both similarities and differences in mouse and human EC cell differentiation, and these cDNA clones provide some of the first reagents for studying the molecular biology of human development.

Topics: Animals; Autoradiography; Blotting, Northern; Cell Differentiation; Cell Line; Cloning, Molecular; DNA, Circular; Endoderm; Humans; Mice; Molecular Probe Techniques; Teratoma; Tretinoin; Tumor Cells, Cultured

Mouse embryonal carcinoma (EC) cells of the P19 line can be induced to differentiate into neurons, astrocytes and fibroblasts by exposure to retinoic acid (RA), whereas treatment of the EC cells with dimethyl sulfoxide (DMSO) leads to differentiation into mesodermal tissues, including cardiac and skeletal muscle. In RA- but not DMSO-treated cultures, the level of c-myc mRNA underwent two transient increases. The concentration of c-myc mRNA in RA-treated cultures increased 3-fold after 3 h in the presence of the drug, returned to normal by 9 h, increased again 5-fold from 48 to 96 h, and finally decreased below pre-treatment values by 144 h. Increased levels of c-myc protein were observed at the times of elevated c-myc mRNA. Nuclear run-on assays of the c-myc gene and measurements of c-myc mRNA stability indicated that both transcriptional and post-transcriptional mechanisms contribute to the modulated expression of the c-myc gene. The two transient increases in c-myc expression suggest a role for the c-myc protein in the differentiation of cells along neuroectodermal lineages.

Topics: Animals; Astrocytes; Cell Differentiation; Cell Line; Cell Nucleus; Dactinomycin; Dimethyl Sulfoxide; Gene Expression Regulation, Neoplastic; Introns; Mice; Neurons; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Proto-Oncogenes; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

We report here that both the mouse teratocarcinoma F9 cells and F9 cells induced to differentiate by treatment with retinoic acid contain cell surface glycoconjugates with terminal alpha-linked galactose residues, as shown by agglutination of cells with antisera to blood type B, but not to type A. In addition, both cell types contain high numbers of binding sites for Griffonia simplicifolia-I, a lectin which binds to terminal alpha-linked galactose residues, although differentiated F9 cells contain approximately 50% more binding sites/cell for this lectin. We have also confirmed that differentiation is accompanied by a decrease in the expression of the fucose-containing stage-specific embryonic antigen (SSEA)-1, as evidenced by the fact that F9 cells, but not differentiated F9 cells, are agglutinated by monoclonal antibody to this antigen. Since these results indicate that surface glycoconjugates contain terminal alpha-linked galactose residues, we assayed cell extracts for the enzyme UDP-Gal:beta-D-Gal-alpha 1,3-galactosyltransferase. We have found that F9 cell extracts contain this activity, and differentiation results in a significant increase in the specific activity of the enzyme, from approximately 2 nmol/mg h in F9 extracts to 7 nmol/mg h in RA/F9 extracts. It has been suggested that the loss of the SSEA-1 antigen upon differentiation of F9 cells is due to decreased activity of the enzyme GDP-Fuc:beta-D-GlcNAc-alpha 1, 3-fucosyltransferase. We therefore determined the activities of this fucosyltransferase and several other glycosyltransferases, which included UDP-GlcNAc:beta-D-Gal-beta 1,3-N-acetylglucosaminyltransferase, UDP-Gal:beta-D-GlcNAc-beta 1,4-galactosyltransferase, and GDP-Fuc:beta-D-GlcNAc-alpha 1,6-fucosyltransferase. We have found that extracts from both cell types contain these enzyme activities; differentiation, however, does not result in substantial changes in any of these activities.

Topics: Agglutination; Animals; Cell Differentiation; Cell Line; Galactosyltransferases; Hexosyltransferases; Kinetics; Mice; Teratoma; Tretinoin

We have used replication-defective selectable retroviruses to express inducibly or constitutively the v-src gene in the embryonal carcinoma (EC) cell lines F9, PC13 and P19. High v-src expression in F9 and PC13 cells does not induce or disrupt their differentiation. In contrast, P19 cells expressing high levels of v-src have a 'differentiated' morphology and are unable to respond to signals that normally induce differentiation along the neural or muscle pathways. Regulation of v-src transcription from the inducible human metallothionein (MT) promoter has shown that v-src expression above a threshold level induces differentiation of these cells, as defined by loss of the stem cell marker ECMA-7. These results suggest that v-src expression may be compatible with differentiation events occurring early in embryogenesis, represented by F9 and PC13 cell differentiation, but might disrupt the differentiation of cell types present at later developmental stages.

Topics: Animals; Cell Differentiation; Dimethyl Sulfoxide; Gene Expression Regulation; Mice; Muscles; Neurons; Oncogene Protein pp60(v-src); Retroviridae Proteins; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured

Chromosomal loci that are specifically active in embryonal carcinoma stem cells were cloned from the mouse genome by functional selection. P19 cells, a pluripotent embryonal carcinoma cell line, were transfected with an enhancer trap (a plasmid containing an enhancerless inactive neo gene), and NEO+ transformants were isolated. All of the NEO+ cell lines retained pluripotency and expressed the neo gene. When the cells were induced to differentiate, most of the cell lines continued to express the neo gene, while the neo gene in some cell lines became repressed. From the latter group of cell lines, we have cloned the integrated neo gene plus the flanking cellular DNA sequences. Three of the six cloned DNAs possessed a high NEO+-transforming activity in undifferentiated P19 cells. Among these three, two (015 and 052) were inactive in differentiated P19 cells and NIH 3T3 cells, while the remaining one was active in these differentiated cells. Deletion analysis suggested that both 015 and 052 contain two regulatory elements (promoter and enhancer) of cellular DNA origin. The putative enhancer and promoter are separated by at least 6 kilobases in 015 and 1 kilobase in 052. Therefore, 015 and 052 cloned fragments contain regulatory DNA elements that are specifically active in the embryonal carcinoma stem cells.

Topics: Animals; Cell Differentiation; Cell Line; Chromosome Mapping; Cloning, Molecular; Mice; Plasmids; Teratoma; Transfection; Tretinoin

Topics: Animals; Cell Differentiation; Mice; Neoplasm Proteins; Teratoma; Tretinoin; Tumor Cells, Cultured

F-9 teratocarcinoma stem cells differentiate into parietal endoderm when monolayer cultures are treated with retinoic acid. This change in phenotype is accompanied by increased accumulation and altered organization of fibronectin deposits. Although both stem cells and treated cells synthesize and accumulate fibronectin, only the treated cells deposit a fibrillar array of the protein. We have monitored the accumulation of fibronectin in nontreated and treated F-9 cells with indirect immunofluorescence and have biochemically analyzed the fibronectin synthesized by each cell type with one- and two-dimensional acrylamide gels and peptide maps. Our data suggest that no differences exist between these fibronectins to account for the observed changes in accumulation. Thus, another mechanism may regulate the organization of matrix deposition.

Topics: Cell Line; Collagen; Electrophoresis, Polyacrylamide Gel; Embryonal Carcinoma Stem Cells; Endoderm; Extracellular Matrix; Fibronectins; Fluorescent Antibody Technique; Laminin; Neoplastic Stem Cells; Phenotype; Protein Binding; Teratoma; Tretinoin

For study of the correlation between differentiation and organ colonization properties of tumor cells, F9 embryonal carcinoma (EC) cells were treated with retinoic acid, an inducer of differentiation; and their organ colonization pattern was assessed by the experimental metastasis assay. Untreated cells were found to colonize the liver, whereas treated cells colonized the lungs. This pattern held true when metastases were scored after spontaneous death or after a careful microscopic search for micrometastases. Histologic examination revealed that both the tumor nodules produced by the untreated and the treated cells had the characteristics of EC devoid of any evidence of differentiation. The immunohistochemical study of the expression of markers typical of embryonal carcinoma cells or of the extracellular matrix components laminin and collagen type IV, typical of differentiated cells, confirmed these results. However, the lack of expression of stage-specific embryonal antigen 1 (SSEA-1), a marker generally associated with the undifferentiated state, observed only in the tumors obtained after injection of treated cells, indicates that the lung nodules probably derive from cells that have responded to the induction in vitro but have dedifferentiated in vivo.

Topics: Animals; Antigens, Surface; Cell Differentiation; Cell Line; Collagen; Extracellular Matrix; Laminin; Liver Neoplasms; Lung Neoplasms; Neoplasm Metastasis; Phenotype; Skin Neoplasms; Teratoma; Tretinoin

Vitamin A and its derivatives (retinoids) exhibit profound effects on the proliferation and differentiation of many cell types. However, the molecular mechanism by which retinoids exert these effects is unknown. Cultured murine F9 teratocarcinoma stem cells, which differentiate into nontumorigenic endoderm cells in response to retinoic acid (RA), have been used to identify genes regulated by RA. A cDNA library synthesized from F9 cells treated with RA for 8 hr has been screened with a cDNA probe enriched for sequences rapidly induced by RA, and a gene that exhibits the characteristics of a primary target for RA has been identified. This gene, early retinoic acid-1 (Era-1), encodes a 2.2- to 2.4-kilobase polyadenylylated RNA; the level of Era-1 mRNA rapidly and transiently increases up to 35-fold, depending on the concentration of exogenous RA. The increase in Era-1 mRNA is dependent on the continuous presence of exogenous RA. The RA-associated increase in Era-1 mRNA is seen even in the presence of protein synthesis inhibitors, but the increase is prevented by inhibitors of RNA synthesis such as actinomycin D. This increase in the steady-state level of Era-1 mRNA in F9 cells is a very early effect of retinoic acid on gene expression in this differentiation system.

Topics: Animals; Cell Differentiation; Cell Line; Cloning, Molecular; Genes; Kinetics; Nucleic Acid Hybridization; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin

The expression and properties of mouse embryonic antigens, recognized by monoclonal antibody TEC-02, were analyzed in teratocarcinoma-derived cell lines. TEC-2 antigens were found in the majority of the parietal endoderm cells PYS-2 and in a fraction of cultured embryonal carcinoma cells but not in other cell lines tested. During the course of retinoic acid-induced differentiation of embryonal carcinoma cells F9, the expression of TEC-2 was transiently increased. Immunolabeling of extracts from F9 and PYS-2 cells separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that TEC-2 antigens are polydisperse glycoconjugates of high molecular weight (mostly greater than 100,000). The TEC-2 epitope was shown to be carbohydrate which in F9 cells might be attached to the same carrier as another developmentally regulated carbohydrate epitope TEC-1. The TEC-2 antigens, isolated by indirect immunoprecipitation, were degraded by extensive pronase digestion or mild alkaline treatment to mostly large products. Immunostaining of glycolipid standards suggested that TEC-2 epitope involves the GalNAc beta 1----4Gal beta 1----4R sequence. Combined data indicate that TEC-2 is a new developmentally regulated carbohydrate epitope carried in embryonal carcinoma cells predominantly on glycoprotein-bound large carbohydrates.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Carbohydrates; Carcinoembryonic Antigen; Cell Line; Chromatography, Gel; Detergents; Epitopes; Glycoconjugates; Mice; Molecular Weight; Precipitin Tests; Teratoma; Tretinoin

Murine F9 embryonal carcinoma cells exposed to retinoic acid and dibutyryl cyclic AMP gradually arborize and acquire a neuron-like morphology in monolayer culture. Whether F9 cells can be induced to differentiate into cells with features specific to neural cells is controversial. We analyzed the intermediate filament content and pericellular matrix proteins of F9 cells after exposing them to retinoic acid, dibutyryl cyclic AMP, and nerve growth factor. In long-term cultures, a great majority of the cells appeared neuron-like, but showed intra- and pericellular laminin and type IV collagen, and frequently cytokeratin filaments as well. Several monoclonal antibodies to neurofilaments did not react with these cells in immunofluorescence or immunoblotting, though they recognize either all or individual mouse neurofilament triplet proteins. Polyclonal antibodies to neurofilament proteins gave a diffuse, nonfibrillar, vinblastine-resistant fluorescence in the morphologically neuron-like cells, but in immunoblotting failed to reveal polypeptides compatible with neurofilament triplet proteins. In long-term cultures, most of the cells appeared to have partially or totally lost the intermediate filaments. This was confirmed with anti-IFA antibodies which normally react with all intermediate filament proteins. The F9-derived cells did not respond to nerve growth factor in any detectable way. We conclude that the morphologically neuron-like derivatives of F9 cells display characteristics of modified parietal endoderm-like cells and do not show unequivocal features of neural cells.

Topics: Animals; Antibodies, Monoclonal; Bucladesine; Cell Differentiation; Cell Line; Endoderm; Mice; Neoplasm Proteins; Nerve Growth Factors; Neurons; Teratoma; Tretinoin

When F9 murine-embryonal-carcinoma cells were incubated with all-trans-[3H]retinoic acid, approximately 10% of the tritium label taken up by the cells was recovered in the nuclei. Sonication or DNase I digestion followed by extraction with 0.6 M NaCl released 20-40% of the nuclear-associated retinoic acid. Analysis of these extracts showed that retinoic acid was bound to protein sedimenting at 4 S. This nuclear retinoic-acid-binding component bound all-trans- and 13-cis-retinoic acid with comparable affinity whereas retinol competed less efficiently for binding. These results suggest that F9 embryonal-carcinoma cells contain a nuclear binding protein for retinoic acid that is distinct from the cellular retinoic-acid-binding protein.

Topics: Animals; Binding Sites; Carrier Proteins; Cell Nucleus; Cells, Cultured; Centrifugation, Density Gradient; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Hydrolysis; Mice; Receptors, Retinoic Acid; Subcellular Fractions; Teratoma; Tretinoin; Tumor Cells, Cultured

The differentiation of retinoic acid-treated F9 cells (primitive endoderm-like F9 cells) into parietal endoderm-like F9 cells induced by dibutyryl cAMP was studied as a culture model of the morphogenesis of early mouse embryo. For this purpose, 6 cDNA clones coding for mRNAs specifically expressed in parietal endoderm-like F9 cells were selected. Northern hybridization of RNA extracted from variously treated F9 cells to nick-translated plasmid DNA of these clones demonstrated the reversible expression of many mRNAs depending on the presence of dibutyryl cAMP in the culture medium. This result suggested that the differentiated state of parietal endoderm, which is formed from primitive endoderm at a position adjacent to the trophectoderm in mouse embryo, can be reversed if the local signal is removed. One of the selected clones, pLAM, hybridized to an mRNA of 6.3 kb and selected mRNA producing a laminin B subunit in an in vitro translation system. This clone has an inserted sequence of 3.1 kb. Among the restriction sites in this sequence, six were consistent with those in a 1.7 kb inserted sequence of pPE 49 and pPE 386, which were isolated by Barlow et al. as laminin B1 clones. An XbaI site found in both pPE 49 and pPE 386 was, however, not found at the corresponding position of pLAM. Dot hybridization of RNA with pLAM showed that expression of laminin B in F9 cells is stimulated more than 100-fold during differentiation of F9 stem cells into parietal endoderm-like F9 cells.

Topics: Animals; Bucladesine; Cell Differentiation; Cell Line; Cloning, Molecular; DNA; Embryo, Mammalian; Endoderm; Laminin; Mice; Protein Biosynthesis; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin

Transcript of the c-myb gene, one of the proto-oncogenes, was clearly detected in undifferentiated mouse F9 teratocarcinoma stem cells, but not in terminally differentiated mouse parietal endoderm PYS-2 cells. As F9 cells can be induced to differentiate into parietal endoderm-like cells by the addition of retinoic acid and dibutyryl cAMP, we examined levels of the c-myb transcript under this experimental condition and found that the c-myb transcript was decreased significantly. Thus, a decrease in the c-myb transcript is probably related to early differentiation of mammalian cells.

Topics: Animals; Bucladesine; Cell Differentiation; Cell Line; DNA, Neoplasm; Embryonal Carcinoma Stem Cells; Mice; Neoplastic Stem Cells; Proto-Oncogenes; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin

The rate at which P19 embryonal carcinoma cells in monolayer culture become anchorage dependent during differentiation induced by retinoic acid (RA) was investigated. In both nonsynchronized cultures and cultures synchronized by mitotic selection, the ability to grow in semisolid medium, characteristic of the malignant stem cell, decreased after a lag period of about 12 hr in the continuous presence of RA, prior to an increase in cell generation time. However, striking differences between synchronized and nonsynchronized cultures were observed in their commitment to differentiation following RA removal. After only 2 hr of exposure to RA, synchronized cells continued a program of differentiation in which they became anchorage dependent, while at least 24 hr of exposure was required for exponentially growing cells to become similarly committed. Induction of anchorage dependence by RA was also strikingly cell cycle dependent; 2 or 4 hr of exposure of synchronized cells to RA in G1 phase, when the intrinsic capacity for soft agar growth is low, was sufficient to commit cells to anchorage dependence, but a similar exposure in S phase was not. Together, these results suggested that interactions between cells in different cell cycle phases in asynchronous cultures influenced commitment since exposure to RA for more than one cycle (13 hr) was required for all cells to become anchorage dependent. Increased plasminogen activator secretion and epidermal growth factor binding, markers of certain differentiated cell types, increased only 3 and 5 days after RA addition, respectively, and were not induced by pulsed exposure to RA of less than 24 hr, even in synchronized cells.

Topics: Animals; Antibodies, Monoclonal; Cell Adhesion; Cell Cycle; Cell Differentiation; Cell Line; Culture Media; Cytoskeleton; Epidermal Growth Factor; ErbB Receptors; Glycolipids; Laminin; Lewis X Antigen; Mice; Plasminogen Activators; Teratoma; Time Factors; Tretinoin

The mouse embryonal carcinoma cell line F9 differentiates into parietal endoderm cells after a 3-day exposure to retinoic acid and dibutyryl cyclic AMP. Using the experimental metastases assay, we investigated the organ colonization properties of RA-treated and -untreated populations of F9 cells. The results show that untreated F9 cells colonize the liver with a high degree of specificity while the treated populations colonize the lungs. Cells derived from a lung colony colonized only the liver unless they were treated with RA. However, removal of the inducer from culture of differentiated cells did not cause reversal of the lung colonization potential. Our observations also indicate that it is unlikely that lung colonization is due to cell aggregation or to interaction between differentiated and undifferentiated cells. Taken together, these results suggest that RA induces the observed changes of organ colonization properties of F9 cells.

Topics: Animals; Bucladesine; Cell Line; Fucose; Glycolipids; Glycosylation; Lewis X Antigen; Liver Neoplasms; Lung Neoplasms; Mice; Neoplasm Metastasis; Teratoma; Tretinoin

A 2.4 kb RNA encoded by the murine Hox 1.1 (m6) homeobox gene is induced when F9 stem cells are differentiated with retinoic acid and dibutyryl cyclic AMP. The regulation of Hox 1.1 expression was probed by using cycloheximide, an inhibitor of protein synthesis. Production of the Hox 1.1 RNA in differentiating F9 cells was not blocked by treatment with cycloheximide, indicating that new protein synthesis is not required for its induction. On the contrary, this transcript was detected in F9 stem cells treated with cycloheximide, anisomycin, or emetine alone. Nuclear transcription assays indicated that the Hox 1.1 gene was transcribed in F9 stem cells and that the rate of transcription did not change early in the differentiation of F9 cells. These observations indicate that the induction of Hox 1.1 transcripts in F9 stem cells during differentiation is not regulated at the level of transcription initiation but results from stabilization of the transcript.

Topics: Animals; Bucladesine; Carcinoma; Embryonal Carcinoma Stem Cells; Gene Expression Regulation; Genes, Homeobox; Mice; Neoplastic Stem Cells; RNA Processing, Post-Transcriptional; Teratoma; Tretinoin

The treatment of 311 cells, a pluripotent mouse teratocarcinoma cell line, with a new type of inducer, 3,5-di-tert-butylchalcone 4'-carboxylic acid (Ch55), results in the suppression of the c-mos gene, accompanied by early marker changes associated with cell differentiation, i.e., the enhanced secretion of plasminogen activator and the decrease in peanut agglutinin receptors and glucose transport. This indicates that Ch55 is a potent inducer of teratocarcinoma cells and suppresses c-mos expression.

Topics: 3-O-Methylglucose; Animals; Cell Differentiation; Chalcone; Chalcones; Gene Expression Regulation; Lectins; Methylglucosides; Mice; Molecular Weight; Peanut Agglutinin; Plasminogen Activators; Propiophenones; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mos; Proto-Oncogenes; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured

[3H]Retinoic acid (RA) and [3H]retinol bind in an unsaturable manner to isolated nuclei from Nulli-SCC1 and PCC4.aza1R embryonal carcinoma (EC) cells. When nuclei are challenged with the same labeled retinoids on their respective binding proteins (CRABP and CRBP), much less binding is observed and the binding is saturable. RA-CRABP does not compete with [3H]retinol-CRBP for binding to specific Nulli-SCC1 nuclear sites, whereas retinol-CRBP (but not apo-CRBP) actually potentiates the binding of [3H]RA-CRABP to these nuclei. The binding of [3H]RA-CRABP and [3H]retinol-CRBP is not dramatically affected by prior removal of the outer nuclear membrane with Triton X-100. However, treatment with the detergent after the binding reaction is complete removes about half of the bound [3H]RA-CRABP and almost all of the bound [3H]retinol-CRBP. We measured specific retinoid-binding activities in nucleoplasmic extracts of Nulli-SCC1 and PCC4.aza1R cells. The only readily detectable specific binding activity in nucleoplasmic extracts from untreated cells was for [3H]retinol in PCC4.aza1R preparations. Nucleoplasmic extracts from Nulli-SCC1 and PCC4.aza1R cells pretreated with RA had considerable levels of specific [3H]RA-binding activity with little or no increase in [3H]retinol binding. By contrast, similar extracts from Nulli-SCC1 cells treated with retinol bound large amounts of both [3H]retinol and [3H]RA. Under the same conditions, PCC4.aza1R extracts also contained [3H]RA-binding activity with no increase in [3H]retinol binding above the high endogenous levels. Although these results might reflect translocation of binding proteins from cytoplasm to nucleus, other interpretations must be considered since we often observed an increase, rather than the expected reduction, in cytoplasmic retinoid-binding protein levels.

Topics: Animals; Carrier Proteins; Cell Line; Cell Nucleus; Kinetics; Mice; Receptors, Retinoic Acid; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Teratoma; Tretinoin; Vitamin A

DNA competition studies have been used to investigate the presence of a repressor of viral enhancer function in F9 mouse embryonal carcinoma cells. The complete polyoma virus enhancer region, cotransfected into F9 cells with the SV40 promoter/enhancer attached to a chloramphenicol acetyl transferase marker gene, induced a small increase in pSV2CAT expression. This can be explained by preferential but weak binding by polyoma sequences of a molecule repressing pSV2CAT transcription. Repressor activity substantially disappeared when the cells were induced to differentiate by retinoic acid. Repressor binding was localised to one half of the polyoma enhancer, but was lost on further fragmentation of this region. It appears that multiple sequence elements may be required for repressor binding and that these are at least partially separable from the complement of elements binding enhancer activating molecules.

Topics: Animals; Cell Differentiation; Cell Line; Cyclic AMP; DNA, Bacterial; DNA, Recombinant; Enhancer Elements, Genetic; Gene Expression Regulation; Genes, Regulator; Genes, Synthetic; Genes, Viral; Mice; Polyomavirus; Promoter Regions, Genetic; Recombinant Fusion Proteins; Repressor Proteins; Simian virus 40; Teratoma; Transcription Factors; Transfection; Tretinoin

We have analyzed the glycolipid markers of a recently cloned human embryonal carcinoma (EC) cell line, NTERA-2, which differentiates extensively into a variety of somatic cell types when exposed to retinoic acid. These tumor cells provide a model system that can be used to study the ontogeny of glycolipid diversity during human embryonic development. Glycolipid antigens were identified by cell surface immunofluorescence and thin-layer chromatography immunostaining using a comprehensive set of anticarbohydrate monoclonal antibodies. Undifferentiated NTERA-2 cells were found to express predominantly globo-series glycolipids, including Gb3, Gb5 (IV3GalGb4), globo-ganglioside (IV3NeuAc alpha 2----3GalGb4), globo-H (IV3Fuc alpha 1----2GalGb4), and globo-A (IV3GalNAc alpha 1----3[Fuc alpha 1----2]GalGb4). When NTERA-2 cells were induced to differentiate by culturing in the presence of 10(-5) M retinoic acid, a remarkable shift of cellular glycolipids from globo-series to lacto- and ganglio-series was observed: Globo-series structures declined, particularly during the period 7-20 days after first exposure to retinoic acid, while lacto-series structures, including fucosyl alpha 1----3 type 2 chain (Lex) and sialosyl type 2 chain, and ganglio-series structures, including GM3, GD3, 9-O-acetyl-GD3, GM2, GD2, and GT3, increased. The presence of globo-A and globo-H as the major ABH blood group antigens in undifferentiated NTERA-2 cells suggests that globo-series blood group antigens are embryonic antigens, synthesis of which switches to lacto-series during human development. Two-color immunofluorescence analysis indicated preferential expression of several ganglio- and lacto-series antigens on different subsets of differentiated cells and permitted the relationship of these subsets to the development of neurons in NTERA-2 cultures to be determined. The results suggest that glycosyltransferase, particularly those involved in controlling glycoconjugate core structure assembly, are key enzymes regulated during the differentiation of human EC cells and, by implication, during human embryogenesis.

Topics: Antigens; Cell Differentiation; Cell Line; Cell Membrane; Chromatography, Thin Layer; Fluorescent Antibody Technique; Gangliosides; Globosides; Glycolipids; Glycosides; Humans; Immunologic Tests; Teratoma; Tretinoin

Human teratocarcinoma NT2/D1 cells undergo differentiation into a variety of cell types, including neurons, treated with retinoic acid. In the present study, the concentrations of alpha S-100 and beta S-100 proteins (alpha and beta subunits of S-100 proteins), and three subunits (alpha, beta and gamma) of enolase in NT2/D1 cells were measured using the sensitive enzyme immunoassay method. The concentration of beta S-100 was markedly increased in the cells after treatment with retinoic acid, whereas the concentration of alpha S-100 was undetectably low, indicating that the S-100b (beta beta) protein was induced by retinoic acid. On the other hand, the concentrations of the three forms of enolase isozymes did not change in the same culture. The induction of S-100b protein was not observed in the NT2/D1 cells after treatment with forskolin, dibutyryl cyclic AMP or cholera toxin. The indirect double-labeled immunofluorescence, using antibodies specific to beta S-100 and monoclonal antibodies specific to neurofilaments, revealed that both the S-100b protein and the neurofilaments were induced in the same subpopulation of cells which underwent neuronal differentiation.

Topics: Antibodies, Monoclonal; Biomarkers; Cell Differentiation; Cell Line; Fluorescent Antibody Technique; Humans; Immunoassay; Immunohistochemistry; Isoenzymes; Nerve Growth Factors; Nerve Tissue; Phosphopyruvate Hydratase; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Teratoma; Tretinoin

The quantity of c-myc mRNA was measured during the retinoic-acid-induced differentiation of the pluripotent human teratoma cell line, Tera-2 cl. 13. As the cells were exposed to retinoic acid for longer periods of time the duration of the cell cycle progressively increased (measured by the rate of S phase entry) until the cells were effectively quiescent and expressed characteristic differentiation markers. Under these circumstances steady-state levels of c-myc expression increased by up to 1.6-fold with respect to rapidly growing undifferentiated cells. Southern blot analysis of the c-myc genes in Tera-2 indicated no major rearrangement or amplification in the cell line.

Topics: Cell Differentiation; Cell Line; Humans; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured

A concanavalin-A(Con A)-resistant variant of the pluripotent mouse embryonal carcinoma cell line, PSA1-NG2, was isolated. This variant, designated NG2-2.16, fails to exhibit the extensive spontaneous differentiation displayed by PSA1-NG2 in colonies in vitro and in tumours in vivo. The molecular nature of the defect in NG2-2.16 cells was not revealed by quantitative studies of the binding, uptake and metabolism of tritiated Con A, or by Western blotting of membrane and whole cell homogenates, thus indicating the defect to be the result of a more subtle molecular alteration. Statistical evidence suggests that the same mutation is responsible for both the Con A resistance and the lack of spontaneous differentiation. NG2-2.16 cells were induced to differentiate by exposure to retinoic acid, suggesting that the mutation affects the regulation of differentiation rather than the potential for differentiation.

Topics: Animals; Cell Differentiation; Cell Line; Concanavalin A; Drug Resistance; Mice; Mutation; Teratoma; Tretinoin

We have stably transformed PCC4 and F9 teratocarcinoma stem cells with the human beta-globin gene. The transformed PCC4 cells retained their ability to produce in host mice tumours containing various differentiated tissues. Three of the five clones examined had human beta-globin transcripts, with correct 5' termini; however, some transcripts were also initiated upstream from the natural cap site in two of the three clones. The expression of the human beta-globin gene was maintained when the stem cell phenotype was changed, either into endodermal cell type by retinoic acid treatment of the F9 clones, or into fibroblastic cell type by cell hybridization of transformed PCC4 cells with L fibroblasts. Thus, the human beta-globin gene introduced into early embryonic cells can be expressed constitutively and its expression is maintained when the pattern of gene expression in the cells is changed.

Topics: Animals; Cell Differentiation; Endoderm; Gene Expression Regulation; Globins; Humans; Hybrid Cells; L Cells; Mice; Teratoma; Transcription, Genetic; Transfection; Tretinoin

Differentiation of F9 embryonal carcinoma cells by retinoic acid treatment results in extraembryonic endoderm-like cells. The effects of this process on the protein composition of the intermediate filaments were studied by two-dimensional gel electrophoresis and by immunoblotting. By this approach, two new proteins induced in differentiating cells, p57 and p54, were identified in cytoskeletal preparations enriched in intermediate filaments. The 57-kDa protein could be resolved into at least three components (pI 5.6-5.9), and the 54-kDa protein into at least two components (pI approximately 5.6). Both proteins reacted with a monoclonal antibody which recognizes an antigenic determinant common to all intermediate filaments. Based on these results, the two proteins were identified as members of the intermediate filament protein family. Partial digestion with V8 protease showed that p57 was different from vimentin, another intermediate filament protein present in these cells. p57 and p54 were also immunodetected by a polyclonal anti-keratin anti-serum, which suggests that these proteins share some homology with the keratins. These two proteins are different from the endodermal cytoskeletal protein A and B (endo A and endo B) keratins, which are known to be present in extraembryonic endoderm-like cells. They were also more abundant than endo A and endo B in differentiating F9 embryonal carcinoma cells, but almost undetectable in terminally differentiated extraembryonic endoderm-like cells, where endo A and endo B are readily detectable. This suggests that p57 and p54 have a different pattern of expression than endo A and endo B.

Topics: Animals; Cell Differentiation; Cell Line; Cytoskeleton; Immunochemistry; Intermediate Filament Proteins; Teratoma; Tretinoin; Vimentin

Many pluripotent embryonal carcinoma (EC) cell lines and all embryonic stem (ES) cell lines have hitherto been maintained in the undifferentiated state only by culture on feeder layers of mitomycin C-treated embryonic fibroblasts. We now demonstrate that medium conditioned by incubation with Buffalo rat liver (BRL) cells prevents the spontaneous differentiation of such cells which occurs when they are plated in the absence of feeders. This effect is not mediated via cell selection but represents a fully reversible inhibitory action ascribed to a differentiation-inhibiting activity (DIA). BRL-conditioned medium can therefore replace feeders in the propagation of homogeneous stem cell populations. Such medium also restricts differentiation in embryoid bodies formed via aggregation of EC cells and partially inhibits retinoic acid-induced differentiation. The PSA4 EC line gives rise only to extraembryonic endoderm-like cells when aggregated or exposed to retinoic acid in BRL-conditioned medium. This suggests that DIA may be lineage-specific. DIA is a dialysable, acid-stable entity of apparent molecular weight 20,000-35,000. Its actions are reproduced neither by insulin-like growth factor-II nor by transforming growth factor-beta. DIA thus appears to be a novel factor exerting a negative control over embryonic stem cell differentiation.

Topics: Animals; Cell Aggregation; Cell Differentiation; Cells, Cultured; Culture Media; Liver; Mice; Rats; Rats, Inbred BUF; Stem Cells; Teratoma; Tretinoin

Embryonal carcinoma (EC) cells are the pluripotent stem cells of teratocarcinoma. The aggregates of P19, a mouse EC cell line, undergo differentiation and rapidly lose its colony-forming ability in culture with retinoic acid (RA) or DMSO. Loss of plating efficiency is used to assess the rate of drug-induced differentiation. When exposed respectively to DMSO or RA, the isolated EC cell mutants (D3 and RAC65) did not differentiate but the gap junctions could be formed. In RA-treated co-aggregates of RAC65 and O1A1, each cell line gave responses independent of the presence of the other cell line. However, in DMSO-treated co-aggregates of P19 and D3, D3 cells remained undifferentiated and P19 cell differentiation was much poorer than that in the absence of D3 cells. The results suggest that cell-cell interaction be present in the early stage of DMSO-induced differentiation and absent in the early stage of RA-induced differentiation.

Topics: Animals; Cell Communication; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Mice; Teratoma; Tretinoin

Tumor-producing substances that promote lipolysis in vitro may also account for fat mobilization in cachectic cancer patients. Cachexia might improve if this lipolytic action of cancer cells could be halted. This study examined the lipolytic activities of media from four tumor cell lines after treatment with retinoic acid (RA), a cell differentiation inducer. An in vitro adipocyte bioassay measured lipolysis. All four tumor cell lines were intrinsically lipolytic, with elevated baseline lipolytic activities relative to fibroblast-conditioned controls (128% to 287% of control, p less than 0.05). After a 2-week exposure to RA in culture medium followed by 3 days of continued growth in fresh medium, two of four cell lines (both rat prostatic adenocarcinomas) showed significantly reduced lipolytic activities (16% and 61% of corresponding untreated controls, p less than 0.05). These reductions in lipolytic activity after RA treatment were not generalized phenomena; nor were they simply caused by cell differentiation, as the other cell lines (human malignant melanoma and human ovarian teratocarcinoma) showed no reductions despite evidence of cell differentiation. No effect on lipolytic activity was seen after only a 24-hour exposure to RA. We conclude that RA can affect the lipolytic activity of certain tumor cells in vitro, perhaps by influencing tumor-producing lipolytic factor(s).

Topics: Adenocarcinoma; Animals; Cell Line; Cell Transformation, Neoplastic; Female; Fibroblasts; Humans; Lipolysis; Male; Melanoma; Neoplasms; Rats; Teratoma; Time Factors; Tretinoin

The lamin complement of nuclear matrix isolated from F9 embryonal carcinoma cells was studied during retinoic acid-induced differentiation in culture. Differentiation of the original cells into parietal endoderm-like cells was accompanied by the gradual appearance of lamins A and C while lamin B was present throughout all stages. Lamins were identified by their molecular masses, isoelectric points, recognition by a monoclonal antibody and a polyclonal antiserum, and by peptide mapping. The increase in the amounts of lamins A and C found in the matrix was due to de novo synthesis as no extranuclear pools of these lamins were detected in the undifferentiated cells. These results provide biochemical evidence that, as in amphibian embryogenesis, there are variations in nuclear lamina composition during mammalian development.

Topics: Animals; Cell Differentiation; Cell Line; Cell Nucleus; Lamin Type B; Lamins; Mice; Nuclear Proteins; Peptide Mapping; Teratoma; Tretinoin

To determine what effect retinoic acid might have in modulating cyclic AMP-mediated events at the nucleus of teratocarcinoma cells, we have investigated the effect of retinoic acid treatment of F9 and PC13 cells on cyclic AMP-dependent protein kinase activity and the amounts of the RI and RII cyclic AMP binding proteins present in the nuclear fraction. Exposure of F9 cells to retinoic acid (0.1 microM) induces differentiation to parietal endoderm, while retinoic acid treatment (3 microM) of PC13 cells induces differentiation to visceral endoderm. In both cell types retinoic acid treatment causes a rapid (within 4 h) and pronounced (by 2-fold) decrease in nuclear cyclic AMP-dependent protein kinase activity. Conversely, as measured by cyclic [8-azido-32P]AMP photoaffinity labeling a similar rapid and pronounced decrease in the RI and RII regulatory subunits is observed at the nucleus. This decrease in nuclear cyclic AMP-dependent protein kinases in at least two cell types may be an early event of retinoid action important in the initiation of differentiation.

Topics: Animals; Cell Fractionation; Cell Line; Cell Membrane; Cell Nucleus; Cytosol; Kinetics; Mice; Protein Kinases; Teratoma; Tretinoin

F9 cells maintained in culture were shown to have a reduced ability to differentiate. The cells produced decreased amounts of alphafetoprotein when induced with retinoic acid. We show that consistent responses can be recovered after passage of F9 cells as a tumor. In addition, optimal differentiation of F9 cells to visceral endoderm may be achieved by the addition of very low concentrations of dibutyryl cyclic AMP (dbcAMP) to the medium.

Topics: alpha-Fetoproteins; Animals; Bucladesine; Cell Differentiation; Cell Line; Culture Media; Embryonal Carcinoma Stem Cells; Endoderm; Laminin; Mice; Neoplastic Stem Cells; Teratoma; Tretinoin

Human teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early human development, we have characterized the alterations that occur in the hormonal responsiveness of human embryonal carcinoma cell adenylate cyclase with differentiation in response to 10 microM retinoic acid. Two cell lines CL12 and CL13, cloned from Tera 2 cells by Dr. C. F. Graham, have been used in these studies. Adenylate cyclase of CL12 and CL13 cells is stimulated in the presence of 10 microM GTP by epinephrine and calcitonin, with calcitonin being the most potent stimulator of cyclic AMP production. Exposure of these cells to retinoic acid leads to an arrest in growth and within 6 days to a differentiated cell population with a stable nonreversible phenotype. No changes in basal, GTP- and fluoride-stimulated adenylate cyclase activities are observed with retinoic acid treatment, but the cyclase of differentiated cells exhibits a greater stimulation by calcitonin (7.5-fold) and the appearance of a somatostatin inhibitory effect. Somatostatin specifically inhibits, by 25%, the hormonal stimulation of adenylate cyclase of cells treated for 5 days with retinoic acid. The increase in calcitonin stimulation of adenylate cyclase activity of the differentiated cells is related to an increase (congruent to 3-fold) in the number of hormonal receptors and not to a significant change in receptor binding affinity (Kd 4.6 X 10(8) M-1). These alterations in calcitonin and somatostatin responsiveness suggest a possible regulatory role for these hormones during embryonic development. Furthermore, the results indicate that changes in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of human embryonal carcinoma cell differentiation.

Topics: Adenylyl Cyclases; Animals; Calcitonin; Cell Differentiation; Cell Line; Enzyme Activation; Humans; Mice; Somatostatin; Teratoma; Tretinoin

The expression of plasma membrane receptors for insulin-like growth factors (IGFs) by PC13 embryonal carcinoma (EC) cells, and their immediate differentiated progeny PC13END was examined by binding radiolabelled IGF-I to cell monolayers. Both cell types express high-affinity IGF receptors, but the apparent number of unoccupied receptors sites falls by about 60% upon differentiation. Crosslinking studies reveal that both type 1 and type 2 IGF receptors are expressed by PC13EC cells. PC13END-cell-conditioned medium contains developmentally regulated, separable activities, one of which reacts directly with IGF-II, and the other with IGF for plasma membrane receptors. The former activity represents a soluble secreted IGF-binding protein. The latter activity is structurally and functionally similar to rat IGF-II. Polyclonal antibodies raised against purified rat IGF-II specifically recognize multiple forms of IGF in radiolabelled culture supernatants and material which closely resembles the soluble IGF-binding protein. Immunoprecipitation of radiolabelled culture supernatants with anti-rat IGF-II reveals that the differentiation of PC13EC cells is accompanied by the coexpression of IGF-like molecules and the soluble binding protein, and that IGF-like molecules are expressed by extraembryonic tissues of mesodermal origin in the early postimplantation mouse embryo. These findings show that IGF-like molecules are expressed in early mammalian development and may act in an autocrine fashion in vivo.

Topics: Cell Differentiation; Cells, Cultured; Chromatography, High Pressure Liquid; Embryonal Carcinoma Stem Cells; Immunologic Techniques; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Neoplastic Stem Cells; Receptor, Insulin; Receptors, Somatomedin; Somatomedins; Teratoma; Tretinoin

Retinoic acid induces differentiation of embryonal carcinoma F9 cells into parietal endoderm. The surface proteins of F9 cells from induced and control cultures were labeled with the 125I-lactoperoxidase system and analyzed by two-dimensional gel electrophoresis. Their quantitative comparison has shown an 11-fold increase of protein p220 of apparent MW 220,000 and isoelectric point 5.6. Among other enhanced surface proteins, 3.5-fold increases of p50, p45, and p40 of MW 50,000-40,000 and isoelectric point 5.1-5.3 were observed. Simultaneously another surface protein, p70 of MW 70,000 and isoelectric point 6.1-6.3, disappeared. The quantitative changes of surface proteins produced after treatment with retinoic acid were enhanced in the presence of dibutyryl cAMP. Analysis of lectin-binding proteins demonstrated that increasing proteins p220, p50, p45, and p40 have an affinity for concanavalin A, whereas p70, which decreases, has an affinity for wheat germ agglutinin. Antibodies raised against p70 from undifferentiated cells have shown a specific immunoreaction with p220 from differentiated cells and also with the subunit B of purified laminin. The electrophoretic mobilities of p220 and of the B subunit of laminin are similar. It is suggested that p70, p220, and laminin B subunit share structural homology.

Topics: Animals; Bucladesine; Cell Differentiation; Cell Line; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Embryonal Carcinoma Stem Cells; Glycoproteins; Immunologic Techniques; Laminin; Lectins; Neoplastic Stem Cells; Receptors, Mitogen; Surface Properties; Teratoma; Tretinoin; Wheat Germ Agglutinins

Human teratocarcinoma stem cells are nonpermissive for human cytomegalovirus (HCMV) but become permissive after being induced to differentiate by treatment with retinoic acid. We show that in uninduced teratocarcinoma stem cells, and also in transformed human 293 cells expressing adenovirus E1a gene products, the HCMV immediate-early (IE) 68,000-molecular-weight polypeptide (68K polypeptide) was not expressed, and consequently input viral genomes were not replicated. However, after differentiation of the teratocarcinoma cells, synthesis of the HCMV IE 68K polypeptide was induced, and viral DNA replication occurred. In contrast to our observations for HCMV, simian cytomegalovirus (SCMV) displayed constitutive expression of its analogous IE 94K polypeptide, and the input SCMV genomes were replicated in both uninduced stem cells and 293 cells. Since little, if any, HCMV IE RNA was detectable in human teratocarcinoma or 293 cells after infection under IE conditions, we suggest that a direct transcriptional block to permissivity occurs in these cells. The presence of tandemly repeated sequences which bind nuclear factor I protein in the promoter for the SCMV IE 94K polypeptide gene but not in the promoter for the HCMV IE 68K polypeptide gene may allow the expression of the simian but not of the human IE gene product in transformed cells.

Topics: Adenoviruses, Human; Animals; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Cytomegalovirus; DNA Replication; Embryonal Carcinoma Stem Cells; Genes, Viral; Humans; Neoplastic Stem Cells; Promoter Regions, Genetic; Teratoma; Transcription, Genetic; Tretinoin; Viral Proteins; Virus Replication

The myeloproliferative sarcoma virus (MPSV) is a unique member of the Moloney murine sarcoma virus family. Due to mutations in the U3 region of its long terminal repeat, MPSV has an expanded host range that includes cells of the hematopoietic compartment. Using a MPSV recombinant containing the gene for neomycin-resistance (NeoR-MPSV), we demonstrate that the host range of MPSV also includes undifferentiated F9 embryonal carcinoma cells. Transfer of G418-resistance with NeoR-MPSV to F9 cells is almost as efficient as G418-resistance transfer to fibroblasts, in contrast to G418-resistance transfer to PCC4 embryonal carcinoma cells, which is at least 3 orders of magnitude lower. To isolate NeoR-MPSV mutants that are efficiently expressed in PCC4 cells, G418-resistant PCC4 cell lines were induced to differentiate, and the provirus was rescued by superinfection with murine leukemia virus. Viral isolates (PCMV-5 and -6; PCMV = PCC4 cell-passaged NeoR-MPSV) were obtained and assayed for expression in embryonal carcinoma cells. The efficiency of NeoR transfer was equally as high in both F9 and PCC4 as in fibroblasts. mos oncogene expression was unaltered as judged by transformation capability. No gross alteration in the coding region and in the long terminal repeat was detectable by restriction enzyme analysis. NeoR-MPSV and its mutants PCMV-5 and -6 can thus be utilized as vectors for the efficient transduction of genes into embryonic cells.

Topics: Animals; Cell Differentiation; Cell Line; Drug Resistance; Gene Expression Regulation; Mice; Moloney murine sarcoma virus; Mutation; Neomycin; Rats; Sarcoma Viruses, Murine; Teratoma; Transduction, Genetic; Tretinoin

Retinoic acid induces the differentiation of NTERA-2 cl. D1 human embryonal carcinoma (EC) cells into neurons, cells permissive for the replication of human cytomegalovirus (HCMV), and other cell types that cannot as yet be classified but are distinguishable from the stem cells. We tested several additional agents for their ability to induce the differentiation of these EC cells. No differentiation was induced by butyrate, cyclic AMP, cytosine arabinoside, the tumor promoter 12-0-tetradecanoylphorbol 13-acetate (TPA), or the chemotherapeutic agent cis-diaminedichloroplatinum, although morphological changes were detected at the highest concentrations of these agents that permitted cell survival. However, retinal, retinol, 5-bromouracil 2'deoxyribose (BUdR), 5-iodouracil 2'deoxyribose (IUdR), hexamethylene bisacetamide (HMBA), dimethylacetamide (DMA), and dimethylsulfoxide (DMSO) all induced some neuronal differentiation, but to a lesser extent than retinoic acid. Also, BUdR, IUdR, HMBA, and DMA induced the appearance of many cells permissive for the replication of HCMV. Differentiation was, in all cases, accompanied by the loss of SSEA-3, a globoseries glycolipid antigen characteristically expressed by human EC cells. However, another glycolipid antigen, A2B5, which appears in 60%-80% of differentiated cells 7 days following retinoic acid induction, was detected in less than 20% of the cells induced by the other agents studied. This implies that the HCMV-permissive cells induced by retinoic acid are not identical to those induced by BUdR, IUdR, and DMA.

Topics: Acetamides; Bromodeoxyuridine; Butyrates; Butyric Acid; Cell Differentiation; Cell Line; Cisplatin; Clone Cells; Cyclic AMP; Cytarabine; Dimethyl Sulfoxide; Humans; Idoxuridine; Kinetics; Neurons; Teratoma; Tetradecanoylphorbol Acetate; Tretinoin

Persistent infection with human cytomegalovirus (HCMV) can be established in cultures of human ovarian teratocarcinoma (PA1) cells, and maintained for more than 200 days. Infected cultures maintained at 34 degrees C (PA1CMV34) and 37 degrees C (PA1CMV37) entered crisis and subsequently displayed massive cytopathic effects (CPE), whereas infected cultures maintained at 32 degrees C (PA1CMV32) and 39 degrees C (PA1CMV39) continued to release small amounts of infectious virus until 240 or 151 days post-infection (p.i.) respectively. PA1CMV32 cultures shifted to 37 degrees C at 258 days p.i. resumed synthesis of infectious virus which resulted in cell destruction, indicating that latent infection with HCMV was maintained in PA1 cells at 32 degrees C. In contrast, PA1CMV39 cells did not produce infectious virus even when cultured at 37 degrees C for more than 100 days after the temperature shift.

Topics: Bromodeoxyuridine; Cell Differentiation; Cells, Cultured; Cytomegalovirus; Female; Humans; Ovarian Neoplasms; Teratoma; Tretinoin; Virus Replication

We found that monolayer cultures of F9 cells induced to differentiate with trans-retinoic acid (RA) contain two major subpopulations of cells. These two cell types can be distinguished by their cellular morphology, their pattern of laminin accumulation, and their ability to undergo further differentiation in response to N6-O2-dibutyryl adenosine 3':5' cyclic monophosphoric acid (dBcAMP). Furthermore, the developmental pathway induced by RA appears to lead to two alternative pathways, and differentiation at the branch point is either directly or indirectly controlled by cAMP. Differentiation along one branch of this pathway can be induced by 5-bromodeoxyuridine, whereas differentiation along an unrelated pathway is induced by N'-N'-dimethylacetamide. In all cases, differentiation is closely paralleled by suppression of the tumorigenic phenotype, indicating that these two processes are tightly linked and probably share a common step.

Topics: Acetamides; Bromodeoxyuridine; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Cyclic AMP; Fluorescent Antibody Technique; Laminin; Phenotype; Teratoma; Tretinoin

The goal of research into retinoic acid is to maintain or improve the pharmacologic activities of topically applied retinoic acid while decreasing both local and systemic toxicity and improving chemical stability. Data are presented from a wide range of in vitro and in vivo test systems for retinoic acid, its congeners such as etretinate, and recently synthesized derivatives. The in vitro tests involved cell culture and biochemical assays: rat testes cytosol (receptor binding), mouse embryo teratocarcinoma cells (morphologic evaluation of differentiation), transformed human keratinocytes (cross-linked envelope formation), and polymorphonuclear leukocytes (lipoxygenase enzyme). The in vivo tests were carried out in the hairless rat (ornithine decarboxylase inhibition), the rabbit (irritation), and the guinea pig (ultraviolet erythema). Results from these experiments were used to determine biologic indices for the test substances. Subsequently, these test systems were used to evaluate a series of new compounds designed to control the chemical nature of a key double bond in the retinoic acid structure. Results are presented to show that different actions on the proliferative, differentiating, and inflammatory processes can be obtained by progressive aromatization of the retinoic acid structure.

Topics: Administration, Topical; Animals; Binding Sites; Carrier Proteins; Cell Division; Cell Line; Cells, Cultured; Humans; Receptors, Retinoic Acid; Retinoids; Teratoma; Tretinoin

Murine embryonal carcinoma F9 cells, which do not express appreciable levels of major histocompatibility complex (MHC) class I mRNA, start to express the mRNA and proteins upon differentiation induced by retinoic acid (RA). To investigate the molecular mechanism of this regulation, we examined in F9 cells transient expression of the chloramphenicol acetyltransferase (CAT) gene directed by the 5' flanking region of a MHC class I gene, H-2Ld. The native 1.4-kilobase H-2Ld 5' upstream region gave very low CAT activity in undifferentiated F9 cells. Deletion between positions -210 and -135 relative to the cap site resulted in a 4- to 5-fold increase in CAT activity as compared with constructs containing the region. However, all of these constructs, regardless of the deletion, expressed comparable CAT activity in differentiated F9 cells. These data suggest the presence of a negative cis-acting element that is under developmental control. Further analysis revealed that the sequence conferring the negative regulation resides between positions -195 and -161. This region, highly conserved among the MHC class I genes, is found to be capable of increasing CAT activity in NIH 3T3 cells that express the class I genes constitutively. Further, this regulatory sequence, when connected to the simian virus 40 promoter, produced repressive and enhancing effects in F9 and NIH 3T3 cells, respectively. Based on these results, we suggest that the expression of MHC class I genes during development involves switching from negative to positive regulation dictated by the class I regulatory element located between positions -195 and -161.

Topics: Acetyltransferases; Animals; Base Sequence; Cell Differentiation; Cell Line; Chloramphenicol O-Acetyltransferase; Gene Expression Regulation; Genes, Regulator; H-2 Antigens; Mice; Promoter Regions, Genetic; Teratoma; Transcription, Genetic; Transformation, Genetic; Tretinoin

We have analysed immunocytochemically the differentiation in vitro of clones of the human teratoma cell line Tera-2. The proliferating stem cells expressed Thy-1 and N-CAM/D2-CAM antigens. On treatment with 5 X 10(-5) M retinoic acid in either monolayer or aggregate cultures they began to express receptors for tetanus toxin and McAb A2B5. Three weeks after initiating retinoic acid treatment, the cultures contained a variety of cell morphologies, including about 3% of cells with a neuron-like morphology. These cells were reactive with tetanus toxin, McAb A2B5, and antibodies against Thy-1 and N-CAM/D2-CAM. They also expressed 55 000 and 210 000 Da neurofilament subunits.

Topics: Animals; Antigens, Surface; Cell Adhesion Molecules; Cell Differentiation; Cell Line; Clone Cells; Fluorescent Antibody Technique; Humans; In Vitro Techniques; Mice; Neurons; Teratoma; Tetanus Toxin; Tretinoin

Retinoic acid-induced differentiation of mouse F9 teratocarcinoma cells is known to be accompanied by changes in gene expression. We examined the expression of the c-myc gene during retinoic acid-induced differentiation of F9 cells. Retinoic acid caused a 50% reduction in the level of c-myc mRNA after 3 hr of treatment and a 90% reduction after 12 hr. We have also examined several features of c-myc genomic structure in F9 cells, PYS2 (differentiated parietal yolk sac) cells, and liver--namely, methylation, amplification, and gross organization. Comparison of Hpa II and Msp I digests of DNAs from F9, PYS2, and liver showed that the c-myc gene in F9 cells is hypomethylated relative to that in PYS2 cells and in liver. The Hpa II sites that undergo methylation during differentiation were found to be in the second intron. Gross structural changes of the c-myc gene have not occurred in F9 or PYS2, and the c-myc gene is not amplified.

Topics: Animals; Cell Differentiation; Cell Line; Gene Amplification; Gene Expression Regulation; Methylation; Mice; Proto-Oncogene Proteins; Proto-Oncogenes; Teratoma; Tretinoin

Certain embryonal carcinoma (EC) cell lines can colonize the embryo following blastocyst injection or embryo aggregation, giving rise to EC-embryo chimaeras. However, such chimaeras often develop abnormally. For example, diploid P19 cells colonize the embryo readily but resulting chimaeras are usually abnormal, with persistence of tumour cells. Retinoic acid (RA) induces differentiation of EC cells to a variety of cell types in vitro but, in this study, it was shown that pretreatment of P19 cells with RA did not result in more normal development of P19-embryo chimaeras. The only significant effect of RA was to reduce the ability of P19 cells to participate in embryonic development at all after blastocyst injection. RA did not have a direct toxic or teratogenic effect on preimplantation mouse embryos and did not affect the ability of pluripotent embryo cells to colonize chimaeras. Therefore, RA may not be the normal inducer of differentiation in early embryogenesis.

Topics: Animals; Blastocyst; Cell Line; Chimera; Embryo Transfer; Embryonal Carcinoma Stem Cells; Embryonic Development; Female; Mice; Morula; Neoplastic Stem Cells; Pregnancy; Teratoma; Tretinoin

A teratocarcinoma stem cell line, P19S1801A1, differentiates after induction with drugs (retinoic acid or dimethyl sulfoxide) in aggregate cultures as outgrowths in vitro. We have used this model of murine embryonic differentiation to show that several homoeobox-containing genes are newly activated while others remain active throughout. Transcripts of 1.5, 1.7, 1.8, 3.9, and 5.0 kb were detected using two different DNA probes. Some of these transcripts were stage-specific but not cell-type specific. Affinity-purified antibodies to a synthetic peptide also showed the appearance of nuclear antigen in the embryonal carcinoma stem cells and in differentiated cell types in heterogeneous patterns. This study illustrates the usefulness of teratocarcinoma model systems to analyze homoeobox gene activation during embryonic development and differentiation.

Topics: Animals; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Gene Expression Regulation; Genes, Regulator; Mice; Myocardium; Neuroglia; Neurons; RNA, Messenger; Teratoma; Transcription, Genetic; Transcriptional Activation; Tretinoin

A novel selection for cell lines resistant to the blocking of metabolic cooperation by chemical inhibitors is described. The selection has been used to isolate a pluripotent embryonal carcinoma clone which cooperates in retinoic acid (RA), an inhibitor of junctional communication in all other cell lines tested to date. The selected cells require higher levels of RA to induce differentiation, but show little or no increase in resistance to toxic effects of RA.

Topics: Animals; Cell Communication; Cell Differentiation; Cell Survival; Drug Resistance; Mice; Selection, Genetic; Teratoma; Tretinoin; Uracil Nucleotides

Modulation of protein biosynthesis by retinoic acid during induction of differentiation of F9 teratocarcinoma stem cells was investigated by using computerized analysis of double label autoradiography of two-dimensional polyacrylamide gels. As early as 6 h after induction increased synthesis of 5 and decreased synthesis of 2 proteins occur. By 12 h after induction, synthesis of 13 proteins is elevated and by 24 h that of 17. At 24 h the range of stimulation is from two- to fourfold, as demonstrated by a 3H:14C ratio divided by the mode ratio. Examination of the Gaussian distributions of frequency of ratio indicates that many subtle changes in protein synthesis accompany the development of the new phenotype.

Topics: Animals; Autoradiography; Cell Differentiation; Isoelectric Point; Mice; Molecular Weight; Neoplasm Proteins; Teratoma; Time Factors; Tretinoin

We have shown that c-myc mRNA levels decrease more than 20-fold when F9 teratocarcinoma stem cells are induced to arrest growth and terminally differentiate to parietal endoderm after exposure to retinoic acid and cyclic AMP (Campisi et al., Cell 36:241-247, 1984). Here, we demonstrate that although growth arrest and full expression of the differentiated phenotype required about 3 days, c-myc mRNA declined abruptly between 8 and 16 h after the addition of retinoic acid and cyclic AMP. The decline was independent of cyclic AMP. We found little or no change in the level of c-myc transcription during differentiation, although two other genes showed marked transcriptional regulation. Thus, decreased c-myc mRNA is a consequence of very early posttranscriptional regulation directed by retinoic acid. Differentiation was not fundamental to this regulation. We have shown that sodium butyrate blocks expression of the differentiated phenotype if added within 8 h of retinoic acid and cyclic AMP (Levine et al., Dev. Biol. 105:443-450, 1984). However, butyrate did not inhibit the decrease in c-myc mRNA. Furthermore, F9 cells partially arrested growth without differentiating when grown in isoleucine-deficient medium. Under these conditions, c-myc mRNA levels also declined. Our results suggest that induction of differentiation-specific genes may be under retinoic acid-mediated control dissimilar from that responsible for the decay of c-myc mRNA. In addition, they raise the possibility that growth arrest may be initiated by reduced c-myc expression.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Genes, Regulator; Kinetics; Mice; Oncogenes; RNA Processing, Post-Transcriptional; RNA, Messenger; Teratoma; Tretinoin

Inhibition of DNA synthesis in F9 embryonal carcinoma cells with high thymidine induces differentiation similar to that induced with retinoic acid (RA). The presence of differentiated cells is evident after 15 h of treatment with 2 mM thymidine, during which period DNA synthesis is inhibited 99%. The addition of RA during the period of high thymidine treatment does not increase the amount of differentiation seen at the end of the 15-h treatment, but does increase the amount seen after thymidine is removed. The inhibition of proliferation by low serum concentration does not induce differentiation in the absence of RA. In partially synchronized cultures of F9 cells, the addition of RA alters the pattern of DNA replication during the first third of S phase. If RA is present during this part of S phase, differentiation is evident both morphologically and biochemically during the following cell cycle. Addition of RA during the second half of S phase does not lead to obvious differentiation until after the next cell cycle. These results suggest that particular events during the early replication period of F9 cells are targets for RA action in induction of differentiation of F9 cells.

Topics: Animals; Blood; Cell Differentiation; Cell Division; Cell Line; Culture Media; DNA; DNA Replication; Embryonal Carcinoma Stem Cells; Interphase; Laminin; Neoplastic Stem Cells; Phenotype; Teratoma; Thymidine; Tretinoin

Human embryonal carcinoma (EC) cells generally express the cell-surface, stage-specific embryonic antigens 3 and 4 (SSEA-3 and SSEA-4), the epitopes of which are defined by two monoclonal antibodies that recognize different portions of an extended globoseries oligosaccharide. To examine further the relationship between these epitopes and the human EC phenotype, we investigated the properties of two newly isolated clones from the human teratocarcinoma cell line, TERA-2. One clone expresses SSEA-3 and SSEA-4; the other does not. Nevertheless, these clones otherwise resemble one another, and based upon their morphology, their expression of other cell-surface antigens, and their ability to form xenograft tumors containing a variety of cell types, we conclude that both clones are composed of pluripotent human EC cells. When exposed to retinoic acid in vitro, neither clone differentiates as extensively as other clones that we have previously derived from TERA-2. These observations indicate heterogeneity among stem cells derived from a single human teratocarcinoma, and suggest that SSEA-3 and SSEA-4 are not necessarily integral features of the human EC phenotype. On the other hand, EC cells in xenograft tumors derived from the SSEA-3- and SSEA-4-negative clone re-express these epitopes. Further, this re-expression is stable, since EC cell lines that are SSEA-3- and SSEA-4-positive grow out when the tumors are explanted in vitro. We conclude that the expression of these globoseries epitopes can be modulated by environmental influences.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Surface; Cell Differentiation; Clone Cells; Cloning, Molecular; Embryonal Carcinoma Stem Cells; Epitopes; Humans; Karyotyping; Mice; Mice, Nude; Microscopy, Fluorescence; Neoplasm Transplantation; Neoplastic Stem Cells; Phenotype; Teratoma; Transplantation, Heterologous; Tretinoin

It has been shown that differentiated derivatives of retinoic acid (RA)-treated F9 embryonal carcinoma cells become non-malignant. In the present study it is asked whether this loss of malignancy is due to cellular differentiation. Because the ability of cells to grow in suspension correlates with in vivo tumorigenicity, we determined the time course of the loss of this property, after RA treatment, with relation to the differentiation to parietal endoderm and the acquisition of normalcy in several common transformation-specific properties of F9 cells. Our results show that pretreatment with RA for 24 h caused 80% inhibition of anchorage-independent growth in F9 cells, and this inhibition reached its highest level (98%) after pretreatment with RA for 48 h and longer. However, all other observed transformation-related properties, and the levels of plasminogen activator (marker for parietal endoderm) remained unaltered at this early post-treatment stage. These observations suggest that the loss of malignancy is a relatively early event in the biochemical pathways involved in the RA-induced differentiation of F9 cells. Furthermore, our data show that the presence of elevated levels of p53 alone may not be sufficient to maintain the anchorage-independent growth and the rapid proliferation of F9 cells.

Topics: Actins; Animals; Bucladesine; Cell Adhesion; Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Neoplastic; Deoxyglucose; Embryonal Carcinoma Stem Cells; Epidermal Growth Factor; ErbB Receptors; Fibronectins; Mice; Neoplasm Proteins; Neoplastic Stem Cells; Phosphoproteins; Plasminogen Activators; Receptors, Cell Surface; Teratoma; Tretinoin; Tumor Suppressor Protein p53

F9 mouse teratocarcinoma stem cells differentiate into primitive endoderm cells in response to retinoic acid (RA). A cDNA library, synthesized from RA and dibutyryl cyclic AMP-treated F9 teratocarcinoma cells, has been screened for gene sequences regulated by RA. By hybridization-selection and in vitro translation, the pcJ6, pcJ31, and pcF117 homologous mRNAs encode polypeptides of 40,000; 35,000-37,000; and 24,000 Da respectively. The pcI56 and pcI5 homologous mRNAs encode laminin B and collagen IV (S-Y. Wang and L. J. Gudas, 1983, Proc. Natl. Acad. Sci. USA 80, 5880-5884). Prior to RA addition, these gene sequences correspond to low-abundance mRNAs (less than 0.05% of total cellular mRNAs). Within 24 hr after the addition of RA (5 X 10(-7) M) to F9 cells, the expression of these sequences increases dramatically, and at 72 hr after drug addition, a 5- to 30-fold induction of these genes can be attained. Addition of lower concentrations of RA results in a smaller increase in the expression of these genes. If the F9 cells are treated with both RA (5 X 10(-7) M) and dibutyryl cyclic AMP (but2cAMP), the levels of mRNA specific for these five inducible genes are further increased by approximately 30- to 110-fold over those in the stem cells; but2cAMP alone does not induce the expression of these genes. The expression of J6 and J31, but not I56 (laminin) and 15 (collagen IV), is also regulated by RA in the P19 teratocarcinoma stem cell line, which differentiates into a fibroblastic cell type in response to RA. In vitro transcription experiments demonstrate that laminin and collagen IV are transcriptionally regulated by RA; but2cAMP also enhances the rate of transcription of these genes in F9 cells.

Topics: Animals; Bucladesine; Cell Differentiation; Cell Nucleus; Cells, Cultured; Cloning, Molecular; Dose-Response Relationship, Drug; Gene Expression Regulation; Genes; Mice; Nucleic Acid Hybridization; Protein Biosynthesis; Teratoma; Theophylline; Transcription, Genetic; Tretinoin

Whereas human cytomegalovirus (HCMV) did not replicate in human embryonal carcinoma (EC) cells, it did replicate in some of the differentiated cells arising following the exposure of TERA-2-derived human EC cells to retinoic acid. On the other hand, retinoic acid did not induce a permissive state in several other diverse human cell lines, including an EC line, 2102Ep, which did not differentiate in response to this agent. Also, both TERA-2 and 2102Ep EC cells differentiated to a limited extent when grown at low cell density and a few of these cells became permissive for HCMV. Thus, susceptibility is the result of differentiation and not due to a direct effect of retinoic acid on viral replication. The nature of the block to HCMV replication in human EC cells is unknown, but viral DNA could be detected in the nucleus within an hour of infection and there was an increased anchorage-independent growth of undifferentiated and differentiated cells following HCMV infection. Viral replication is not subject to a general block in these cells, since another herpesvirus, herpes simplex virus type 1, replicated well.

Topics: Cell Adhesion; Cell Differentiation; Cells, Cultured; Cytomegalovirus; Cytomegalovirus Infections; Humans; Teratoma; Tretinoin; Virus Replication

Topics: Acetylcholinesterase; Animals; Bucladesine; Cell Differentiation; Cell Line; Culture Techniques; Endoderm; Mice; Neurons; Phenotype; Teratoma; Tretinoin

F9 embryonal carcinoma cells treated with 1 microM retinoic acid undergo irreversible differentiation and simultaneously lose their tumorigenicity. Described here are the isolation and characterization of an F9 variant clone (5C), which undergoes partial differentiation in retinoic acid. The behavior of 5C cells indicates that retinoic acid successfully initiates the differentiation pathway but that complete differentiation is not achieved due to a subsequent block in the pathway. The fact that 5C cells do not undergo complete differentiation, or lose their tumorigenicity in response to retinoic acid, indicates that the lesion affects an element involved in the regulation of both these events. Therefore, further genetic and biochemical characterization of this variant should provide information concerning the relationship between the regulation of differentiation and tumorigenicity. Furthermore, the isolation of this variant establishes the feasibility of genetically dissecting the various steps of the differentiation pathway.

Topics: Alkaline Phosphatase; Animals; Antigens, Surface; Carrier Proteins; Cell Differentiation; Cell Line; Fibronectins; Karyotyping; Laminin; Mice; Phenotype; Receptors, Retinoic Acid; Teratoma; Tretinoin

Using immunological probes, we have detected expression of the receptor for epidermal growth factor (EGF) at the cell surface of the clonally-derived human embryonal carcinoma (EC) cell lines, 2102Ep cl. 4D3 and NTERA-2 cl. D1. By this method, the level of receptor expression by these cells is estimated to be 3- to 5-fold less than for the human diploid fibroblast cell line, WI38, and our results indicate that it is the EC stem cells that display this receptor and not a subpopulation of differentiated cells. The human EC cell receptor binds ligand and catalyses autophosphorylation at tyrosine in a normal fashion. Treatment of NTERA-2 cl. D1 cells with retinoic acid (RA) for 7 days to induce differentiation results in decreased levels of receptor expression, and a subpopulation of differentiated cells possessing a markedly higher level of the EGF receptor was not detected among the cultures exposed to RA for longer periods.

Topics: Cell Line; Cell Membrane; ErbB Receptors; Fibroblasts; Fluorescent Antibody Technique; Humans; Receptors, Cell Surface; Teratoma; Tretinoin

Nidogen and laminin were localized at preimplantation stages of mouse development by immunofluorescence. Laminin was already present on the cell surface at the 2-cell stage, while nidogen was first detectable on compacted 8- to 16-cell stage morulae. Nidogen and laminin colocalized at the blastocyst stage and in postimplantation basement membranes. Immunoblot analyses of tissue extracts and cell culture media indicated the 150-kDa form of nidogen as the largest and predominant form in all tissues examined. Radiolabeled nidogen and laminin synthesized by Reichert's membrane were coprecipitated by antibodies against each antigen, indicating complex formation in situ. Equimolar amounts of laminin and nidogen were determined in 6 M guanidine X HCl extracts of tissues by radioimmunoassays, further indicating stoichiometric complexes. However, lower levels of nidogen than laminin were found in tissue and cell culture media. A less than 2-fold increase in nidogen was found when F9 cells were stimulated to differentiate with retinoic acid and dibutyryl cAMP, compared to a 30-fold increase in laminin secretion.

Topics: Animals; Basement Membrane; Bucladesine; Electrophoresis, Polyacrylamide Gel; Embryonic Development; Female; Fluorescent Antibody Technique; Immunosorbent Techniques; Laminin; Membrane Glycoproteins; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred Strains; Pregnancy; Radioimmunoassay; Teratoma; Tretinoin

Mouse teratocarcinoma cells in culture offer an in vitro system to study the initial steps of embryogenesis. It has been suggested that, at such early stages, cell functions are regulated by an autocrine process in which embryonic cells produce factors that in turn act on themselves. F.9 cells possess specific membrane receptors for calcitonin (CT) (120 fmol/mg of protein, Ka, = 3.5 X 10(8) M-1). These cells produce CT detected by heterologous radioimmunoassay in serum-free culture-conditioned medium (75 pg/10(7) cells/12 h). When F.9 cells are incubated in serum-free medium, CT binding and secretion concomitantly drop by 50% within the first 2 h, then increase progressively to an upper plateau after the sixth hour. Preincubation with 10(-6) M CT leads to disappearance of CT receptors and CT secretion in the culture medium up to 6 h. Avoiding accumulation of CT in the medium by a continuous flow rate for 6 h leads to a progressive decrease of CT receptors. In addition, retinoic acid treatment of cells induces a parallel progressive decrease of CT receptor number and of total CT synthesis. These results suggest a reciprocal regulation of CT receptors and CT secretion, or a close relationship between their regulations.

Topics: Animals; Calcitonin; Cell Line; Culture Media; Female; Kinetics; Mice; Pregnancy; Radioimmunoassay; Receptors, Calcitonin; Receptors, Cell Surface; Teratoma; Time Factors; Tretinoin

Monolayer cultures of F9 teratocarcinoma stem cells and P19 stem cells differentiate into endoderm, and fibroblast-like cells, respectively, when treated with retinoic acid. We demonstrate that this differentiation is associated with a large increase (greater than 40-fold) in the activity of an enzyme, prolyl-4-hydroxylase, involved in the posttranslational modification of collagens. This large increase in prolyl-4-hydroxylase activity occurs between 42 and 72 h after retinoic acid addition, and is associated with an increased amount of immunoprecipitable prolyl hydroxylase enzyme. This enzyme should be a useful marker for certain differentiated cell types produced during differentiation of teratocarcinoma stem cell lines.

Topics: Animals; Bucladesine; Cell Differentiation; Cell Line; Collagen; Endoderm; Fibroblasts; Immunosorbent Techniques; Kinetics; Mice; Procollagen-Proline Dioxygenase; Protein Processing, Post-Translational; Stem Cells; Teratoma; Theophylline; Tretinoin

The effects of dibutyryl cAMP on the differentiation of embryonal carcinoma F9 cells were studied mainly using the secretion of laminin and type IV collagen as the marker. For this purpose, F9 cells were labeled with 35S-methionine and radioactive proteins in the medium were analyzed by SDS-polyacrylamide gel electrophoresis. Treatment of F9 cells with retinoic acid alone induced differentiation into cells secreting type IV collagen. The combination of retinoic acid and dibutyryl cAMP stimulated laminin secretion in addition to type IV collagen secretion. This effect of dibutyryl cAMP was observed only 16 h after adding dibutyryl cAMP. Immunofluorescence staining demonstrated that the majority of the cells in culture were converted into cells secreting laminin under these conditions. In contrast to the irreversible effect of retinoic acid, the effect of dibutyryl cAMP on laminin and type IV collagen secretion was reversible at least during the first 5 days of maintaining cells in the medium containing retinoic acid plus dibutyryl cAMP. Removal of dibutyryl cAMP from the culture medium decreased the protein secretion to the basal levels within 2 days. This reversibility was not due to a change in cell number. An in vitro translation assay also suggested the reversible effect of dibutyryl cAMP on the levels of laminin mRNA. Coinciding with variations of the protein secretion, a reversible and homogeneous change in the morphology of retinoic acid generated F9 cells was observed by dibutyryl cAMP.

Topics: Animals; Bucladesine; Cell Count; Cell Line; Collagen; Endoderm; Laminin; Methionine; Mice; Protein Biosynthesis; RNA, Messenger; Teratoma; Tretinoin

The transient expression vector pSV2CAT, which carries the bacterial chloramphenicol acetyl transferase (CAT) gene under the control of the SV40 early promoter, was used to transfect the murine embryonal carcinoma cell line F9 at various times during the retinoic acid-induced differentiation of these cells. Expression of the CAT gene under SV40 promoter control was found to increase markedly on F9 cell differentiation, measured relative to expression from the thymidine kinase promoter in the same cells. A series of constructs was prepared to identify the features of the SV40 early promoter required for transcription in differentiated and undifferentiated cells, as well as the factors limiting transcription in each case. The increased transcription seen on F9 cell differentiation was not observed when cells were transfected with molecules lacking a functional enhancer. It appears that as embryonal carcinoma cells differentiate, increased SV40 transcription results from enhancer sequence activation. In both differentiated and undifferentiated cell types the level of transcription was found to be limited by the availability and/or activity of cellular factors necessary for enhancer function.

Topics: Acetyltransferases; Animals; beta-Galactosidase; Cell Differentiation; Cell Line; Chloramphenicol O-Acetyltransferase; Enhancer Elements, Genetic; Genes; Genes, Regulator; Genes, Viral; Kinetics; Mice; Plasmids; Simian virus 40; Teratoma; Transfection; Tretinoin; Virus Activation

We investigated changes in the activity and subcellular distribution of cyclic-AMP-dependent protein kinases (cAMP-PKs) in response to treatment with retinoic acid in three different embryonal carcinoma cell lines derived from the same teratoma 6050. After retinoic-acid treatment, F9 and PCC4 cells gave rise to parietal-like endoderm, while PC13 cells differentiated into visceral endoderm. Retinoid treatment of F9 and PCC4 cells caused an increase in cAMP-PK activity as measured by histone phosphorylation, as well as increases in the amount of the RI and RII regulatory subunits of the cAMP-PKs, as quantitated by photoaffinity labeling with 8-azido-cyclic-32P-AMP, in both the soluble and plasma-membrane fractions. The increases in membrane cAMP-PK activity and RI and RII levels reached their maximum within 18 h of retinoid treatment, and then dropped to intermediate levels after 3 days of treatment. The cytosolic activity and the levels of the regulatory subunits exhibited a progressive increase during the 3 days of exposure to retinoic acid. The relative RI/RII ratios in the cytosol and membrane fractions of the treated cells were comparable to those found in established PYS-2 parietal-endoderm cells. PC13 stem cells had high levels of cAMP-PK activity and cAMP binding to the regulatory subunits in both the cytosol and plasma membranes, while also exhibiting very low levels of type-II cAMP-PK. Retinoid treatment induced a progressive increase in cAMP-PK activity in the cytosol, and a decrease in activity at the membrane level.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Affinity Labels; Animals; Azides; Cell Line; Cell Transformation, Neoplastic; Cyclic AMP; Embryonal Carcinoma Stem Cells; Mice; Neoplastic Stem Cells; Protein Kinases; Subcellular Fractions; Teratoma; Tretinoin

Retinol and retinoic acid dose-response curves were obtained for promotion of the differentiation of F9 murine embryonal carcinoma cells with an enzyme-linked immunoadsorbent assay for laminin, a product of differentiated F9 cells. Retinoic acid produced a half-maximum response at 1.3 nM and a maximum response at about 30 nM; retinol was 1/175th as potent. Maximum differentiation required 48 hr of continuous exposure to retinoic acid, whereas retinol required 72 hr of exposure. The half-time of retinoic acid conversion into polar metabolites was 3.5 hr; metabolism was accelerated by pretreating F9 cells with retinoic acid. An inhibitor of oxidative metabolism, ketoconazole, decreased the rate of retinoic acid metabolism and decreased the concentration of retinoic acid required to produce a half-maximum response. Unchanged retinoic acid was the sole compound isolated from nuclei of F9 cells incubated with retinoic acid. Retinol had a half-life approximately 5-fold longer than retinoic acid, attained greater cell concentrations, and was converted into retinoic acid by F9 cells. These data indicate that retinoic acid itself directs the differentiation of F9 cells and may mediate differentiation induced by retinol.

Topics: Animals; Cell Differentiation; Cell Line; Chromatography, High Pressure Liquid; Kinetics; Mice; Teratoma; Tretinoin; Tritium; Vitamin A

When cultured in the presence of either retinoic acid (RA) or dimethyl sulfoxide (DMSO), aggregates of the P19 line of mouse embryonal carcinoma (EC) cells differentiate and the spectrum of cell types formed depends on the drug dose. It is shown here the EC cells rapidly lose their colony-forming ability when cultured as aggregates in the presence of DMSO. This loss of plating efficiency (PE) also occurs rapidly following RA treatment. Loss of PE has been used as a quantitative procedure for assessing the rate of drug-induced differentiation. The relationship between drug dose and loss of PE is much steeper for DMSO than for RA, suggesting that these two drugs affect different stages of the differentiation decision-making apparatus. Mutant EC cell lines (D3 and RAC65) do not differentiate in the presence of drug-inducers (DMSO and RA, respectively). Neither differentiation-deficient mutant has an altered ability to form gap junctions. When D3 and P19 cells were mixed within the same DMSO-treated aggregates, the D3 cells remained undifferentiated and the P19 cells differentiated much less efficiently than if they were cultured in the absence of the D3 cells. When RAC65 and P19 cells were mixed in RA-treated aggregates, each cell responded to the drug as though the other were absent. Thus RA behaves as a cell-autonomous inducer of differentiation, whereas DMSO-induced differentiation seems to be mediated by interactions between neighboring cells.

Topics: Animals; Cell Communication; Cell Transformation, Neoplastic; Cells, Cultured; Cytological Techniques; Dimethyl Sulfoxide; Intercellular Junctions; Mice; Mutation; Phenotype; Teratoma; Tretinoin

A murine embryonal carcinoma cell line (F9) was used to examine the effect of a pulsed electromagnetic field on the growth and differentiation of malignant cells. The cells can be induced to differentiate into parietal endodermal cells by treatment with retinoic acid. The pulsed electromagnetic field (1 Gauss and 10 Gauss) promoted the growth of embryonal carcinoma cells in both the presence and absence of retinoic acid. The pulsed electromagnetic field was also found to inhibit retinoic acid-induced differentiation, when the degree of differentiation was based on morphological criteria or on the production of plasminogen activator.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Electromagnetic Phenomena; Endoderm; Fibrin; Fucose; Glycopeptides; Mice; Microscopy, Electron, Scanning; Microscopy, Phase-Contrast; Teratoma; Tretinoin

Murine embryonal carcinoma F9 cells can be induced to differentiate by 2-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC). The differentiated phenotype is similar to that of retinoic acid (RA)-treated F9 cells. In contrast to F9 cells the differentiated cells secrete plasminogen activator and express keratin intermediate filaments. Both DFMO and RA reduce ornithine decarboxylase activity, polyamine levels and inhibit cell proliferation of F9 cells. These compounds also reduce ODC, polyamine levels and proliferation of mouse BALB/c 3T6 fibroblasts. RA inhibits the induction of ODC by insulin, serum and to a lesser extent that of epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The action of DFMO and RA can be distinguished by their response to putrescine. The induction of differentiation and the inhibition of cell proliferation by DFMO can be totally abolished upon the addition of putrescine, whereas the actions of RA are not affected at all. These results suggest that the inhibition of ODC and reduction of polyamines are not causal in the induction of differentiation and the inhibition of proliferation by RA.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Cells, Cultured; Eflornithine; Fibroblasts; Kinetics; Mice; Mice, Inbred BALB C; Ornithine; Ornithine Decarboxylase Inhibitors; Polyamines; Putrescine; Teratoma; Tretinoin

alpha-Difluoromethylornithine (DFMO), a highly selective inhibitor of ornithine decarboxylase (ODC), induced terminal differentiation of F9 mouse embryonal carcinoma cells in culture. Differentiation was assessed using morphological criteria and the level of plasminogen activator activity. The observed phenotypic changes and the fact that the cells did not synthesize alpha-fetoprotein, indicate that they were parietal endoderm cells. The putrescine, spermidine and spermine content of untreated control cells increased during exponential growth and then decreased gradually with continued time in culture. The increases in putrescine and spermidine contents were prevented by DFMO treatment. In fact, the putrescine and spermidine content decreased below the limits of detection after only one day of treatment. The addition of putrescine to the culture medium at any time within 4 days of DFMO treatment, prevented the DFMO-induced differentiation, suggesting that the effects observed were indeed caused by polyamine depletion. The phenotypic changes induced by DFMO were similar to those induced by retinoic acid, a very potent inducer of embryonal carcinoma differentiation. Although retinoic acid can inhibit ODC activity and putrescine accumulation, it is unlikely that this mechanism of action is responsible for retinoic acid-induced F9 cell differentiation, inasmuch as putrescine addition did not prevent the expression of the differentiated phenotype. Undifferentiated F9 embryonal carcinoma cells exhibited a very short G1 phase, and in this respect they are similar to the cells of the preimplantation mouse embryo. In control (exponentially growing) cultures a majority of the F9 cells were in the S phase, but in DFMO-treated cultures they accumulated in the G1 phase and showed no further proliferative potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antineoplastic Agents; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Chromatography, Thin Layer; Eflornithine; Embryonal Carcinoma Stem Cells; Mice; Neoplastic Stem Cells; Ornithine; Ornithine Decarboxylase; Plasminogen Activators; Polyamines; Putrescine; Teratoma; Tretinoin

Murine embryonal carcinoma (EC) cells can be stimulated to differentiate by several chemical inducers. Since the response of EC cells to induction is likely to occur shortly after exposure to the inducer, we report here the changes that occur in polyamine levels in a number of EC cell lines shortly after exposure to two chemical stimuli, alpha-difluoromethylornithine (DFMO) and retinoic acid (RA). Our results suggest that polyamine levels are important in determining the state of EC cell differentiation, but that reduction in these levels alone is not sufficient to induce differentiation in all the EC cell lines tested. Also, it is apparent that RA does influence levels of polyamines. However, this influence does not seem to be mediated through direct interaction with ODCase.

Topics: Animals; Cell Differentiation; Cell Line; Eflornithine; Kinetics; Mice; Ornithine; Ornithine Decarboxylase; Polyamines; Teratoma; Tretinoin

Murine embryonal carcinoma (EC) cells are induced to differentiate when cultured in the presence of retinoic acid (RA). Whereas the EC cells have a high plating efficiency, the differentiated cells have little or no colony-forming ability under the same conditions. We have assumed that the loss of colony-forming ability following exposure of EC cells to RA corresponds to the irreversible commitment of EC cells to differentiate. We found that uncommitted EC cells persist in RA-treated aggregates of EC cells and that the proportion of EC cells stabilizes at a level inversely related to the RA concentration. Both experimental evidence and mathematical modelling results are consistent with the interpretation that there is a dynamic equilibrium achieved by a balance between the processes of EC cell proliferation and differentiation. Since different cell types are induced by different RA concentrations, our results suggest that the commitment to differentiate is not related in any simple way to the developmental program which ensues.

Topics: Animals; Cell Aggregation; Cell Cycle; Cell Differentiation; Cell Division; Cell Line; Embryonal Carcinoma Stem Cells; Mathematics; Mice; Models, Biological; Neoplastic Stem Cells; Stem Cells; Teratoma; Tretinoin

The electrophysiological properties of differentiated cells which were derived from teratocarcinoma OTT6050 in culture showed two different types of spike generation: (1) In the differentiated cells treated by retinoic acid, the action potential of cells involved Ca2+; (2) in the differentiated cells treated by both retinoic acid and nerve growth factor (NGF), the action potential of cells involved Na+ and CA2+. The results indicate that the effects of drugs appear to be important factors in the induction as well as in the regulation of cellular functions in the differentiated cells.

Topics: Action Potentials; Animals; Calcium; Cell Differentiation; Cell Line; Cell Membrane; Electric Stimulation; Electrophysiology; Nerve Growth Factors; Sodium; Teratoma; Tretinoin

Several embryonal carcinoma (EC) cell lines were tested in culture for their ability to metabolize all-trans-[3H]retinol, all-trans-[3H]retinyl acetate, and all-trans-[3H]retinoic acid. There was little, if any, metabolism of all-trans-retinol to more polar compounds; we failed to detect conversion to acidic retinoids by reverse-phase high performance liquid chromatography and derivatization. We also did not observe [3H]retinoic acid when EC cells were incubated with [3H]retinyl acetate. Unlike the other retinoids, all-trans-[3H]retinoic acid, even at micromolar levels, was almost totally modified by cells from several EC lines within 24 h. Most of the labeled products were secreted into the medium. Some EC lines metabolized retinoic acid constitutively, whereas others had an inducible enzyme system. A differentiation-defective line, which contains little or no cellular retinoic acid-binding protein activity, metabolized retinoic acid poorly, even after exposure to inducers. At least eight retinoic acid metabolites were generated; many contain hydroxyl residues. Our data lead us to propose that retinol does not induce differentiation of EC cells in vitro via conversion to retinoic acid. Also, the relatively rapid metabolism of retinoic acid by EC cells suggests either that the induction of differentiation need involve only a transient exposure to this retinoid or that one or more of the retinoic acid metabolites can also promote differentiation.

Topics: Animals; Cell Differentiation; Cell Line; Chromatography, High Pressure Liquid; Diterpenes; Isomerism; Neoplasms, Experimental; Retinoids; Retinyl Esters; Teratoma; Tretinoin; Vitamin A

F9 embryonal carcinoma cells treated with 5 X 10(-8) M retinoic acid and cultured in suspension for 8 days form aggregates consisting of an outer epithelial layer of alpha-fetoprotein-producing visceral endoderm cells. We have previously shown (Grover, A., Oshima, R. G., and Adamson, E. D. (1983) J. Cell Biol. 96, 1690-1696) that the differentiation of F9 cells to visceral endoderm is accompanied by the activation of several genes, and increased laminin synthesis is one of the earliest events. Here we analyze in detail the syntheses and secretion of fibronectin, type IV collagen, and laminin during the 8-day process. Employing immunoprecipitation and enzyme-linked immunosorbent assay, we show that the levels of all three components change with different patterns. Unstimulated F9 cells synthesize and secrete relatively high levels of fibronectin and low levels of type IV collagen. Fibronectin synthesis and secretion decreases to 10% of its original level whereas type IV collagen synthesis rises approximately 3-fold during the differentiation process. Laminin synthesis also rises at least 2-fold, and the proportions of its subunits change as the syntheses of B1 and A accelerate starting on day 2. However, unlike fibronectin and type IV collagen, laminin is largely accumulated in the aggregates. The data suggest that fibronectin has a role in aggregation whereas laminin is important in the differentiation process.

Topics: Animals; Cell Differentiation; Collagen; Extracellular Matrix; Fibronectins; Kinetics; Laminin; Mice; Teratoma; Tretinoin

P10 is a line of embryonal carcinoma cells with a euploid female karyotype. By making use of variant alleles of X-linked genes and of cytogenetic procedures, we have previously shown that the P10 cells have two genetically active X chromosomes. We show here that P10 cells rapidly differentiate into a cell type resembling extraembryonic endoderm when cultured in the presence of retinoic acid. This differentiation is accompanied by X chromosome inactivation as determined by the appearance of a late-replicating X chromosome. Analysis of the X-linked variant alleles indicated that the P10-derived endoderm did not preferentially inactivate paternally derived X chromosomes. This result is in contrast to the situation in normal extraembryonic endoderm, and suggests that the X inactivation process in differentiating P10 cultures resembles that which occurs in normal embryonic rather than extraembryonic tissues.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Electrophoresis; Female; Kinetics; Mice; Microscopy, Fluorescence; Teratoma; Tretinoin; X Chromosome

N-Acetylneuraminic acid lyase (NAN-lyase) activity has been found to be much higher in the differentiated, murine parietal endodermal cell (PYS-2) in culture than in the related, undifferentiated embryonal teratocarcinoma cell (F9). The level of the enzyme rapidly increases in F9 cells exposed to an inducer of differentiation such as retinoic acid (RA) (10(-7) M). The level of the enzyme increases during log phase of growth of PYS-2 cells and decreases after the cells reach confluence. NAN-lyase from PYS-2 cells has been purified 365-fold and has been partially characterized. While most of the enzyme is freely soluble, at least 16% of the enzyme in PYS-2 cells is associated with the nucleus. The possible function of NAN-lyase in the cell and the significance of its marked elevation during growth and differentiation are discussed in view of the fact that the levels of NAN, neuraminidase, NAN transferases and the enzymes that synthesize and activate NAN, remain essentially unchanged during differentiation.

Topics: Animals; Cell Differentiation; Cell Line; Cell Nucleus; Hydrogen-Ion Concentration; Mice; Neoplasm Proteins; Oxo-Acid-Lyases; Sialic Acids; Teratoma; Tretinoin

Human chromosome 17 contains a cluster of at least three distinct homeo box regions, Hu1, Hu2, and Hu5, within a 20 kb stretch of DNA. A mouse homeo box region, Mu1, which maps to chromosome 11, was isolated and found to contain extensive nucleotide homology with a 4 kb region encompassing the Hu1 homeo box. The conservation of these chromosomal regions between man and mouse was confirmed by nucleotide sequence analysis: approximately 380 bp of DNA are more than 90% homologous and the 61 amino acids of the homeo box domain are perfectly conserved. We found that a human teratocarcinoma cell line expresses high levels of Hu1 homeo-box-containing mRNA only after differentiation of the cells following treatment with retinoic acid. In contrast, mouse teratocarcinoma cells did not express Mu1 homeo-box-containing mRNA at any stage of differentiation, whereas the expression of such transcripts was detected in mouse embryos from 10 to 17 days of gestation.

Topics: Amino Acid Sequence; Animals; Cell Differentiation; Cell Line; Chromosome Mapping; Chromosomes, Human, 16-18; Embryo, Mammalian; Embryonal Carcinoma Stem Cells; Gene Expression Regulation; Genes; Humans; Mice; Neoplastic Stem Cells; Nucleic Acid Hybridization; RNA, Messenger; Sequence Homology, Nucleic Acid; Teratoma; Transcription, Genetic; Tretinoin

Analysis of two-dimensional gel electrophoretic patterns of proteins labeled with [35S]methionine was undertaken to investigate changes in gene expression in embryonal carcinoma cell line Nulli-SCC1 during the induction of differentiation in vitro by retinoic acid. The results indicate that changes in protein profiles occur long before overt morphological differentiation is observed. Studies utilizing the antimetabolite, alpha-amanitin, coupled with those involving the cell-free translation of poly(A)+ -RNA, suggest that gene expression is controlled post-transcriptionally as well as transcriptionally. In this respect, embryonal carcinoma cells appear to resemble early embryonic cells, which show evidence of multiple levels of control of gene expression during the initial stages of development and differentiation.

Topics: Amanitins; Animals; Cell Differentiation; Cell Line; Cell-Free System; Embryonal Carcinoma Stem Cells; Mice; Neoplasm Proteins; Neoplastic Stem Cells; Poly A; Protein Biosynthesis; Reticulocytes; RNA; RNA, Messenger; Stem Cells; Teratoma; Transcription, Genetic; Tretinoin

Single cell clones were isolated from the human teratoma line, Tera-2. The cells of three of these clones were studied. The progressively growing cells were shown to be tumorigenic, and they were characterized by the lack of expression of beta 2-microglobulin and HLA-A,B,C determinants on the cell surface. The majority of the cells expressed Thy-1 antigen and a 90 X 10(3) molecular weight protein recognized by the monoclonal antibody F10.44.2; between a third and half of the cells expressed the sugar specificities detected by the anti-SSEA-1 monoclonal antibody. In response to 5 X 10(-5) M-retinoic acid applied to cells in monolayer culture, the cells differentiated into a population of flat static cells arrested in the G1 phase of the cell cycle. A substantial proportion of these differentiated cells expressed beta 2-microglobulin and 43 X 10(3) molecular weight HLA-A,B,C polypeptides, Thy-1, SSEA-1 sugar determinants, and the 90 X 10(3) Mr protein recognized by F10.44.2. The apparent molecular weight of fibronectin secreted by the cells decreased by about 5 X 10(3) Mr to 235 X 10(3) Mr after differentiation. The progressively growing cells lacked reactivity with reagents that mark cells in the nervous system. Following aggregation and retinoic acid treatment, neuron-like cells were formed. These cells reacted with reagents that also react with human neurons in culture: they reacted with tetanus toxin, the anti-neurofilament antibodies BF10 and RT97, the anti-ganglioside, GQ1c antibody F12 A2B5, and anti-Thy-1. The progressively growing cells of these Tera-2 clones are therefore capable of forming at least two types of cell: the flat cells in monolayer cultures and the neuron-like cells. None of the cell populations reacted with the monoclonal antibody against SSEA-3 and these cloned cells are therefore distinct from previous isolates from Tera-2.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Epitopes; Fibronectins; Humans; Karyotyping; Mice; Mice, Nude; Neoplasm Proteins; Neurons; Phenotype; Teratoma; Tretinoin

We have established conditions to efficiently differentiate embryonic carcinoma stem cells of the line P19 into myogenic cells. As inducers for differentiation, a combination of embryoid body formation in conjunction with treatment with dimethyl sulfoxide and retinoic acid proved to be most efficient. Under these conditions we detected an accumulation of myosin- and actin-specific RNA. Also, large amounts of type IV collagen RNA were produced. Type IV collagen is a component of the muscle basement membrane. In analogy to the F-9 system, we found a drastic decrease in stable p53 mRNA under the differentiation conditions used.

Topics: Cell Line; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Gene Expression Regulation; Muscles; RNA; RNA, Messenger; Teratoma; Tretinoin

F9 mouse teratocarcinoma stem cells differentiate into parietal endoderm cells in the presence of retinoic acid, dibutyryl cyclic AMP, and theophylline (RACT). When F9 cells are exposed to 2-5 mM sodium butyrate plus RACT, they fail to differentiate. Differentiation is assessed by induction of laminin and collagen IV mRNA, the synthesis of laminin, collagen IV and plasminogen activator proteins, and alterations in cell morphology. Butyrate inhibits differentiation only when added within 8 hr after retinoic acid addition. Thus an early event in retinoid action on F9 cells is butyrate-sensitive. The population doubling time and cell cycle distribution of F9 cells are not altered within the first 24 hr after butyrate addition, suggesting that butyrate does not inhibit differentiation by inhibition of growth or normal cycling. However, butyrate does inhibit histone deacetylation in F9 cells, and this could be the mechanism by which butyrate inhibits differentiation.

Topics: Acetates; Animals; Bucladesine; Butyrates; Butyric Acid; Cell Differentiation; Cell Line; Flow Cytometry; Mice; Plasminogen Activators; Teratoma; Theophylline; Tretinoin

Poly(ADP-ribose) synthesizing activity in mouse teratocarcinoma EC-A1 cells decreased markedly during differentiation induced by retinoic acid; the activities assayed in permeabilized cells decreased to 25% and 10% of the activity of control (uninduced cells) 2 and 3 days, respectively, after the addition of 0.1 microM retinoic acid to the culture medium. This change preceded changes in morphology and DNA synthesis, which became prominent after 4 days. The decrease in poly(ADP-ribose) synthesizing activity appeared to be caused by a diminution of the synthetase protein and not by a decrease in its catalytic activity, because the full activity disclosed by DNase I treatment decreased in parallel, albeit at about 20 times higher levels. When 8 mM 3-aminobenzamide or 10 mM nicotinamide, specific inhibitors of poly(ADP-ribose) synthetase, was added to the culture medium, the cells underwent differentiation after 7-9 days. An analogue, 3-aminobenzoic acid, which is not inhibitory to the synthetase, induced differentiation much less efficiently than did 3-aminobenzamide, and the effect of 3-aminobenzoic acid appeared to be ascribable to its potent cytotoxicity. Immunohistochemical analysis using anti-poly(ADP-ribose) antibody confirmed the marked reduction in poly(ADP-ribose) synthesizing activity in nuclei of the cells treated with retinoic acid or 3-aminobenzamide but not with 3-aminobenzoic acid. These results suggest that a decrease in poly(ADP-ribose) synthesis triggers differentiation of teratocarcinoma cells.

Topics: Aminobenzoates; Animals; Benzamides; Cell Differentiation; Cell Division; Cell Survival; Cells, Cultured; DNA Replication; Mice; NAD+ Nucleosidase; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Teratoma; Tretinoin

F9 line embryonal carcinoma cells were induced to differentiate into neural direction by long-term treatment of monolayer cultures with retinoic acid and dibutyryl cyclic AMP. Bi- and multi-polar cells appeared, expressing acetylcholinesterase and neurofilament proteins but not markers of glial differentiation including GFA-protein. Nerve growth factor combined with both retinoic acid and dibutyryl cyclic AMP greatly enhanced the development of neuron-like morphology and induced expression of immunoreactivity to tyrosine hydroxylase as well as to Leu-encephalin-like peptides. Similarly, serotonin-like immunofluorescence but not substance P-like immunoreactivity was demonstrable in such cultures. In addition, synaptic-like vesicles were often found in the processes. Analysis of matrix expression in neuronally differentiated F9 cells revealed marked increase in laminin production, as judged by immunofluorescence and immuno-electron microscopy, but no demonstrable intracellular staining for fibronectin or type IV collagen. The results with neuronal cells contrast with the expression of all the three matrix components in endodermally differentiating F9 cells in the same cultures.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cyclic AMP; Laminin; Mice; Neurons; Teratoma; Tretinoin

Cells from embryonal carcinoma (EC) lines 6050AJ and PCC4.aza 1R differentiate in response to treatment with sodium butyrate as well as retinoic acid (RA) or hexamethylenebisacetamide (HMBA). Murine 6050AJ EC cells exposed to sodium butyrate possess hyperacetylated forms of histones H4 and altered forms of histones H2a and H2b, whereas histones from cells treated with other inducers appear to be unaffected. These results might indicate that the mechanism by which sodium butyrate promotes differentiation of EC cells is different from the ways in which RA and HMBA act. Differentiation-defective PCC4(RA)-1 EC cells fail to respond to RA, presumably because they possess minimal amounts of active binding protein for RA (cRABP). Sodium butyrate treatment of these cells results in only a modest level of differentiation. On the other hand, exposure to sodium butyrate plus RA leads to extensive differentiation. As is the case with 6050AJ cells, PCC4(RA)-1 cells treated with sodium butyrate also contain hyperacetylated histones. Furthermore, these cells now possess high levels of cRABP. The latter observations suggest that sodium butyrate has the ability to reactivate a silent cRABP gene in PCC4(RA)-1 cells and thereby lead to extensive differentiation via the retinoid pathway when RA is added.

Topics: Acetylation; Animals; Antigens, Neoplasm; Butyrates; Butyric Acid; Carrier Proteins; Cell Differentiation; Cell Line; Embryonal Carcinoma Stem Cells; Glycolipids; Histones; Lewis X Antigen; Mice; Neoplastic Stem Cells; Receptors, Retinoic Acid; Stem Cells; Teratoma; Tretinoin

The human embryonal carcinoma cell lines NT2 /D1 and NT2 /B9, clonally derived from Tera-2, differentiate extensively in vitro when exposed to retinoic acid. This differentiation is marked by the appearance of several morphologically distinct cell types and by changes in cell surface phenotype, particularly by the disappearance of stage-specific embryonic antigen-3 (SSEA-3), which is characteristically expressed by human EC cells. Among the differentiated cells are neurons, which form clusters interconnected by extended networks of axon bundles, and which express tetanus toxin receptors and neurofilament proteins. These observations constitute the first instance of extensive somatic differentiation of a clonal human EC cell line in vitro.

Topics: Antigens, Surface; Cell Differentiation; Cell Line; Clone Cells; Embryonal Carcinoma Stem Cells; Glycolipids; Humans; Intermediate Filament Proteins; Lewis X Antigen; Membrane Proteins; Neoplastic Stem Cells; Neurons; Receptors, Cholinergic; Stem Cells; Teratoma; Tretinoin

We have generated cell hybrids by fusing embryonal carcinoma (EC) cells which fail to differentiate in response to retinoic acid (RA) and/or hexamethylenebisacetamide (HMBA). The first two classes of hybrids were between an RA- line (also unresponsive to HMBA) that lacks cellular RA binding protein (cRABP) activity and HMBA- lines which possess cRABP and differentiate in the presence of RA. All of the hybrid clones possessed cRABP and differentiated normally upon exposure to either RA or HMBA. When the aforementioned RA- mutant was fused with a second mutant which was refractory to RA and HMBA but possessed cRABP activity, the resultant hybrid clones were responsive to both RA and HMBA and had cRABP activity. These results suggest that all of these mutants were recessive and complementary. Tumors from these hybrid lines differentiated extensively, in some instances much more so than the mutant parental lines and even the wild-type lines from which the mutants were derived. Based upon these observations, we propose that various EC lines might differentiate poorly in tumor form for different reasons. Hybrids between two differentiation-defective, cRABP- lines appeared to be at least partially complemented for responsiveness to RA and HMBA. These hybrids contained low but detectable levels of cRABP. This is not a consequence of tetraploidy since fusions between cells from the same mutant line retained their differentiation-defective phenotype and possessed little or no cRABP activity. Unlike tumors from the other hybrids described above, tumors from these hybrid lines expressed a very restricted pattern of differentiated cell types. This might be because the mutant lines in the latter hybrids originally derived from the same wild-type EC line.

Topics: Acetamides; Animals; Antigens, Surface; Carrier Proteins; Cell Differentiation; Cell Line; Embryonal Carcinoma Stem Cells; Glycolipids; Hybrid Cells; Lewis X Antigen; Male; Mice; Mutation; Neoplastic Stem Cells; Plasminogen Activators; Receptors, Retinoic Acid; Stem Cells; Teratoma; Tretinoin

Murine embryonal carcinoma tumors were induced to differentiate in vivo by administration of retinoic acid. Six long-term surviving animals had seven slowly growing tumors which were transplanted s.c. into strain 129 mice. Untreated embryonal carcinomas were transplanted as controls. All of the 16 control transplants grew rapidly and killed their hosts within 25 days. All of the 24 transplants of retinoic acid-differentiated tumor survived. Sixteen experimental transplants originating from five original tumors showed no or slow growth for up to 16 weeks and were found to be histologically benign cystic teratomas. Two original tumors gave rise to eight relatively rapidly growing transplants. One tumor resulted in four histologically similar solid tumors which resembled chondrosarcomas, and the second tumor gave rise to four histologically similar solid tumors which proved to be a mixture of glioma and chondrosarcoma. Examination of the tumor sources of these latter transplants showed benign cystic teratomas with focal solid, mitotically active cellular areas which were histologically similar to the transplants. These data confirm that retinoic acid-induced differentiation of murine embryonal carcinoma cells results in altered biological potential of these cells and usually the formation of a benign teratoma. Rarely (about 1 per 2 X 10(8], the resulting differentiated cells will give rise to rapidly growing, histologically malignant tumors. One can predict such biological propensity when solid, mitotically active areas in the original tumor are found.

Topics: Animals; Cell Differentiation; Cell Line; Cell Survival; Clone Cells; Mice; Neoplasm Transplantation; Staining and Labeling; Teratoma; Tretinoin

A human immature teratoma cell line was established from tumor tissue of the ovary. The cell line, designated YK , synthesized and secreted alpha-fetoprotein, but there was a gradual decrease in the synthesis of this protein during serial passage. The doubling time at passage 43 was about 20.3 hr, and the plating efficiency was 30 to 40%. Tumorigenicity of the cell line in athymic nude mice was 100% even when 10(4) cells were inoculated s.c. When the cell line was cultivated for 5 days in the presence of 80 microM retinoic acid, proliferation was completely inhibited, while alpha-fetoprotein secretion into the medium was increased. Specific lactate dehydrogenase activity in the tumor grown in nude mice as well as in the cultured cells was 8 times higher than that in the original tumor. The activity of alpha-hydroxybutyrate dehydrogenase in the original tumor was similar to that in the tumor grown in nude mice, while the activity in the cultured cells was about 2.5 times higher than those in both original tumor and the tumor grown in nude mice. The original tumor had all five ordinary bands of lactate dehydrogenase isoenzymes. In contrast, both the cultured cells and the tumor grown in nude mice had a characteristic band which was considered to have similar electrophoretic mobility as testicular lactate dehydrogenase X isoenzyme in humans.

Topics: alpha-Fetoproteins; Animals; Cell Division; Cell Line; Female; Humans; Kinetics; Mice; Mice, Nude; Neoplasm Transplantation; Ovarian Neoplasms; Teratoma; Transplantation, Heterologous; Tretinoin

F9 teratocarcinoma cells can be grown as monolayers or aggregates, and upon treatment with retinoic acid they will differentiate into parietal or visceral endoderm, respectively. Visceral endoderm specifically synthesizes alpha-fetoprotein and albumin mRNAs, which are not found in parietal endoderm. In contrast, both endoderms produce enhanced levels of the major histocompatibility antigen (H2) mRNA compared with F9 cells. F9 cells contain highly methylated DNA as judged by restriction enzyme digestion. However, upon differentiation into visceral endoderm, there is a genome-wide loss of methylation in induced, silent, and constitutively expressed genes. Experiments in which methylation loss is induced via the methyltransferase inhibitor 5-azacytidine result in no induction of alpha-fetoprotein mRNA and no morphological differentiation, suggesting that methylation loss alone is not sufficient to induce the visceral endoderm phenotype. Likewise, 5-azacytidine treatment of differentiated cells does not result in enhanced expression of alpha-fetoprotein mRNA. However, the patterns of loss of DNA methylation at all sites examined after differentiation or 5-azacytidine treatment were remarkably similar, suggesting that the two occur by a similar mechanism, the inhibition of DNA methyltransferase activity. These results argue that the specificity for methylation loss at a given site is an inherent property of aggregated F9 cell chromatin. This system provides a model for studying a tissue-specific change in DNA methylation upon differentiation.

Topics: alpha-Fetoproteins; Animals; Cell Differentiation; Cell Line; DNA, Neoplasm; Genes; Major Histocompatibility Complex; Methylation; Mice; Nucleic Acid Hybridization; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin

Expression of wild-type polyomavirus (Py) is restricted in murine embryonal carcinoma (EC) cells. The block appears to be located at the level of early transcription. Since no T antigen is produced, we investigated the fate of viral DNA upon infection of these cells; we showed that wild-type Py DNA replicates efficiently in all EC cells, probably via a T-antigen-independent mechanism. Furthermore, we studied, at permissive and restrictive temperatures, the replication of tsa (thermosensitive for T antigen) viral DNA of an in vitro-constructed deletion mutant lacking part of the early region coding sequences and of a double mutant carrying both the tsa mutation and the PyEC F9 mutation (allowing expression of early and late viral functions in EC cells). Our results imply that replication of wild-type A2 strain Py DNA can occur in EC cells in the absence of a functional T antigen. However, this protein clearly enhances viral DNA replication and is absolutely required in differentiated cells.

Topics: Animals; Antigens, Viral, Tumor; Cell Line; DNA Replication; DNA, Viral; Embryonal Carcinoma Stem Cells; Mice; Mutation; Neoplastic Stem Cells; Polyomavirus; Teratoma; Tretinoin; Virus Replication

To study the mode of action of human cytomegalovirus, an important teratogenic agent in human populations, the susceptibility of a pluripotent human embryonal carcinoma cell line to the virus was investigated. Viral antigens were not expressed nor was infectious virus produced by human embryonal carcinoma cells after infection, although the virus was able to penetrate these cells. In contrast, retinoic acid-induced differentiated derivatives of embryonal carcinoma cells were permissive for antigen expression and infectious virus production. Replication of human cytomegalovirus in human teratocarcinoma cells may therefore depend on cellular functions associated with differentiation.

Topics: Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cytomegalovirus; Embryonal Carcinoma Stem Cells; Humans; Neoplastic Stem Cells; Stem Cells; Teratoma; Tretinoin; Virus Replication

Retinoic acid induces the differentiation of many murine teratocarcinoma stem cell lines. To elucidate the molecular mechanism of action of retinoic acid, we have selected a series of mutants which exhibit altered differentiation responses to retinoic acid. All of the mutants display abnormal morphology following addition of 5 X 10(-7) M retinoic acid (RA) and dibutyryl cAMP. In addition, none of the mutants are resistant to the cytotoxic effects of higher concentrations of retinoic acid (greater than 75 microM). After the addition of retinoic acid, one mutant, RA-3-10, does not differentiate by any of the biochemical criteria we have used; this mutant also possesses less than 5% of the wild type level of cellular retinoic acid binding protein (CRABP). Other mutants, such as RA-3-3, RA-3-4, and RA-5-1, contain the same amount of CRABP as wild type F9 cells. However, the mutants RA-3-3 and RA-3-4 exhibit lower levels of plasminogen activator activity, and RA-3-4 also exhibits only 10-20% of the wild type synthesis and secretion of laminin and collagen IV following treatment with RA. After RA treatment of the mutant RA-5-1, laminin and collagen IV are synthesized and secreted at reduced rates relative to wild type cells, and the secreted collagen IV has a lower molecular weight than that of wild type; this suggests that RA-5-1 cells have a mutation in one of the enzymes responsible for post-translational modification of collagen IV. None of the mutants tested exhibits alterations in either cytosolic or membrane bound cAMP-dependent protein kinase activity. These studies provide genetic evidence that the CRABP is required for the differentiation of F9 teratocarcinoma stem cells by retinoic acid. However, even in the presence of CRABP, other types of alterations, such as synthesis of collagen IV with an abnormal molecular weight, appear to cause alterations in the differentiation response of cells to retinoic acid.

Topics: Animals; Bucladesine; Cell Differentiation; Cell Division; Cell Line; Kinetics; Mice; Mutation; Neoplasm Proteins; Teratoma; Theophylline; Tretinoin

Retinoic acid (RA) has previously been shown to induce the differentiation of mouse embryonal carcinoma (EC) cells to endoderm-like cells that have a slower rate of proliferation and are nontumorigenic. These cells also acquire the ability to respond to a range of exogenous growth factors. We have analysed the change in growth phenotype for PC13 EC cells using video recordings and autoradiography. We have shown that the endoderm-like cells have a longer cell cycle time than their undifferentiated counterparts (five cell divisions after exposure to RA the differentiated cells had a median cell cycle time of 1800 min compared to 800 min for control cells). The endoderm-like cells also have a progressively decreasing probability of dividing again and this indicates that the differentiation process is accompanied by the acquisition of a limited life-span. The characteristics of mortal cells are well documented, and the endoderm-like cells demonstrate the properties of such cells. In addition, we have confirmed the observation that epidermal growth factor (EGF) can stimulate the proliferation of the endoderm-like cells and have shown, using autoradiography, that 92% of these cells express EGF receptors. Using video recordings, we have demonstrated that the effect of EGF is to shorten the cell cycle of the differentiating cells. We have also shown that EGF can enhance the survival of the endoderm-like cells and thereby prolong their life-span. It is known that EGF and other growth factors can prolong the life-span of mortal cells derived from normal tissues, but we have demonstrated that EGF can have this effect on the differentiated derivatives of a tumour cell.

Topics: Animals; Cell Cycle; Cell Division; Cell Survival; Cell Transformation, Neoplastic; Clone Cells; Culture Media; Epidermal Growth Factor; Mice; Neoplasms, Experimental; Teratoma; Tretinoin; Video Recording

Exposure of F9 cells to all-trans-retinoic acid over a period of 6 days resulted in 4-fold induction of cell surface N-acetylglucosaminide beta (1----4)galactosyltransferase (GT) activity. The retinoic acid-induced GT activity was further enhanced by treatment of the cells with 8-bromo cyclic AMP. The ability of retinoic acid alone, or retinoic acid in combination with 8-bromo cyclic AMP, to induce GT activity was inhibited by both actinomycin D and cycloheximide. These findings indicate that the induction of galactosyltransferase activity noted with differentiation of F9 cells involves de novo synthesis of new enzyme protein.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase; Cell Differentiation; Cell Line; Cell Membrane; Cycloheximide; Dactinomycin; Embryonal Carcinoma Stem Cells; Enzyme Induction; Galactosyltransferases; Neoplastic Stem Cells; Stem Cells; Teratoma; Tretinoin

Topics: Acetylcholinesterase; Animals; Cell Differentiation; Cell Line; Cells, Cultured; Dibutyryl Cyclic GMP; Embryonal Carcinoma Stem Cells; Endoderm; Extracellular Matrix; Fibronectins; Intermediate Filament Proteins; Laminin; Mice; Neoplastic Stem Cells; Neurons; Stem Cells; Teratoma; Tretinoin

In monolayer cultures, embryonal carcinoma (EC) cells from the cell line Nulli-SCCl can be induced to differentiate at high efficiency by exposure to either retinoic acid or hexamethylenebisacetamide. Depending on which inducing agent is used, two distinct differentiated phenotypes result. These phenotypes resemble two of the earliest differentiated derivatives of the cells of the inner cell mass of a mouse blastocyst, i.e., parietal and visceral endoderm. Both differ from EC cells as well as from each other on the basis of their morphology, antigenic expression, secretion of plasminogen activator, and protein-synthetic profiles.

Topics: Acetamides; Animals; Antigens, Neoplasm; Blastocyst; Cell Differentiation; Cell Line; Mice; Phenotype; Teratoma; Tretinoin

Murine embryonal carcinoma tumors were induced to differentiate in vivo using retinoic acid. Six mice bearing seven tumors survived more than 100 days after treatment. Histological samples of these tumors showed no residual embryonal carcinoma cells, and, for the most part, they were benign cystic teratomas. Three tumors, in addition to the benign tissue, had solid, mitotically active areas. Two of these tumors upon transplantation gave rise to progressively growing, potentially lethal tumors which have proven to be permanently transplantable cell lines. Using techniques of light and electron microscopy, immunohistochemistry, flow microfluorometry, and cytogenetics, we have characterized these lines. One is a chondrosarcoma, and one is a glioma:chondrosarcoma mixture. Both are chromosomally different from the parent embryonal carcinoma stem cell line, but both were clearly derived from it.

Topics: Animals; Cell Differentiation; Cell Line; Chondrosarcoma; DNA Replication; Glioma; Karyotyping; Mice; Teratoma; Tretinoin

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

Topics: Animals; Cell Differentiation; Cell Line; Cells, Cultured; Embryonal Carcinoma Stem Cells; Mice; Neoplastic Stem Cells; Stem Cells; Teratoma; Time Factors; Tretinoin

Time-lapse films were made of PC13 embryonal carcinoma cells, synchronized by mitotic shake off, in the absence and presence of retinoic acid. Using a method based on the transition probability model, cell cycle parameters were determined during the first five generations following synchronization. In undifferentiated cells, cell cycle parameters remained identical for the first four generations, the generation time being 11-12 hr. In differentiating cells, with retinoic acid added at the beginning of the first cycle, the first two generations were the same as controls. The duration of the third generation, however, was increased to 15.7 hr while the fourth and fifth generation were approximately 20 hr, the same as in exponentially growing, fully differentiated cells. The increase in generation time of dividing cells was principally due to an increase in the length of S phase. Cell death induced by retinoic acid also occurred principally in the third and subsequent generations. Cell population growth was then significantly less than that expected from the generation time derived from cycle analysis of dividing cells. Cells lysed frequently as sister pairs suggesting susceptibility to retinoic acid toxicity determined in a generation prior to death. Morphological differentiation, as estimated by the area of substrate occupied by cells, was shown to begin in the second cell cycle after retinoic acid addition. These results demonstrate that as in the early mammalian embryo, differentiation of embryonal carcinoma cells to an endoderm-like cell is also accompanied by a decrease in growth rate but that this is preceded by acquisition of the morphology characteristic of the differentiated progeny.

Topics: Animals; Cell Cycle; Cell Differentiation; Cell Line; Cell Membrane; Cell Survival; Embryonal Carcinoma Stem Cells; Kinetics; Mice; Models, Biological; Neoplastic Stem Cells; Probability; Stem Cells; Teratoma; Tretinoin

To determine possible ectopic production of, and altered responsiveness to, specific hormones and growth factors which may be involved in mediating embryonic differentiation and development embryonal carcinoma cells in culture have been employed to serve as an in vitro system of embryogenesis. Exposure of F9 embryonal carcinoma cells to all-trans-retinoic acid previously has been shown to induce differentiation of these undifferentiated stem cells to parietal endoderm and to markedly alter the ability of calcitonin and parathyroid hormone to stimulate adenylate cyclase activity. Evidence is presented that F9 cells secrete immunoreactive calcitonin into the culture medium (200 pg/12 hr/10(7) cells) while parietal yolk sac (PYS) cells secrete immunoreactive parathyroid hormone (800 pg/12 hr/10(7) cells). Retinoic-induced differentiation of F9 cells to endoderm results in a progressive reduction in immunoreactive calcitonin production, while there is an increase in the level of immunoreactive parathyroid hormone found in the conditioned medium. After exposure of F9 cells to retinoic acid for 5 days, little calcitonin is detectable in 12-hr conditioned medium. Changes in the intracellular levels of immunoreactive calcitonin and PTH follow a pattern similar to that noted for changes in the amount of secreted hormones. Thus, immunoreactive calcitonin is produced by undifferentiated F9 cells which possess a calcitonin responsive adenylate cyclase system, while parathyroid hormone is produced by parietal endoderm cells which respond to parathyroid hormone with increased cyclic AMP synthesis. Sephadex G50 gel filtration of F9-conditioned medium shows two peaks of immunoreactive calcitonin with Mr of 3500 and 20,000. Immunoprecipitation of calcitonin from 35S-labeled F9 cells reveals a specific band of 20,000 Mr. Likewise, two peaks of parathyroid hormone immunoreactive material of Mr 8000 and 39,000 are noted after gel filtration of PYS cell-conditioned medium, whereas parathyroid hormone immunoprecipitation from the same cells reveals a specific band of 39,000 Mr. These results raise the possibility that embryo production of these two hormones at specific stages in development may contribute to the regulation of subsequent steps of differentiation.

Topics: Animals; Calcitonin; Cell Differentiation; Cell Line; Embryonal Carcinoma Stem Cells; Kinetics; Mice; Neoplastic Stem Cells; Parathyroid Hormone; Radioimmunoassay; Stem Cells; Teratoma; Tretinoin

Topics: Animals; Carrier Proteins; Cell Differentiation; Cell Line; Clone Cells; Glycolipids; Humans; Lewis X Antigen; Mice; Receptors, Retinoic Acid; Teratoma; Tretinoin

Expression and DNA methylation of the Moloney murine leukemia virus (M-MuLV) genome were investigated in murine teratocarcinoma cells after virus infection. The newly acquired viral genome was devoid of methylation, yet its expression was repressed. The integrated viral genome in undifferentiated teratocarcinoma cells was methylated within 15 days after infection. Although 5-azacytidine decreased the level of DNA methylation, it did not activate M-MuLV in undifferentiated cells. Activation by 5-azacytidine occurred only in differentiated teratocarcinoma cells. Thus two independent mechanisms seem to regulate gene expression during the course of differentiation. The first mechanism operates in undifferentiated cells to block expression of M-MuLV and other exogeneously acquired viral genes, such as SV40 and polyoma virus, and does not depend on DNA methylation. The second mechanism relates only to differentiated cells and represses expression of genes in which DNA is methylated.

Topics: Animals; Azacitidine; Bromodeoxyuridine; Cell Cycle; Cell Differentiation; Cell Line; DNA, Viral; Gene Expression Regulation; Genes, Viral; Methylation; Mice; Moloney murine leukemia virus; Recombination, Genetic; Teratoma; Transcription, Genetic; Transfection; Tretinoin

Immunofluorescence and immunoblotting techniques were used to study the presence and distribution of vimentin and keratin type intermediate filaments, actin, and vinculin (130 kD protein) during retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma (EC) cells. The undifferentiated F9 cells regularly expressed vimentin, usually concentrated close to the nucleus, but not keratin. Actin appeared as short intracellular filaments and as spikes at the edges of the colonies, together with some diffuse cytoplasmic staining. F9 cells also showed a weak, diffuse cytoplasmic vinculin-specific fluorescence in addition to occasional small focal vinculin patches at the edges of the cell colonies. RA treatment led into a series of changes in the cytoskeletal organization of F9 cells. These changes were initiated by the appearance of distinct vinculin plaques and followed by formation of actin stress fibers and by profound changes in the organization of vimentin in the flattening cells. RA treatment finally led to the appearance and co-expression of keratin fibrils in many of the vimentin-containing F9 cells. This sequence of changes suggests that the vinculin-containing adhesion plaques may be important in the mechanism of RA-induced differentiation of EC cells.

Topics: Actins; Animals; Cell Differentiation; Cell Line; Cytoskeleton; Intermediate Filament Proteins; Keratins; Muscle Proteins; Teratoma; Tretinoin; Vimentin; Vinculin

Treatment of F9 teratocarcinoma cells with all trans retinoic acid (RA) causes them to differentiate into two or three morphologically distinct cell types. Whereas the majority of these retinoid-derived cells exhibit properties resembling parietal endoderm, a small percentage of this differentiated cell population manifests properties distinct from the parietal endoderm cell type. The isolation and partial characterization of such a non-parietal endoderm cell line (Dif 5) derived from F9 cells following prolonged (44 days) exposure to 1 microM retinoic acid are described. Unlike the retinoid-induced parietal endoderm-like cell population, which exhibits a dramatic, characteristic morphological change upon treatment with 8-bromo cAMP, Dif 5 cells do not show any morphological change with exposure to this cAMP analog. Dif 5 cells synthesize and deposit an extracellular matrix consisting of several components of Reichert's membrane (fibronectin, laminin, and type IV collagen). This new cell line does not synthesize alpha-fetoprotein but does secrete plasminogen activator. An interesting property of these cells is their ability to grow in the absence of serum or other hormonal supplements. Yet the Dif 5 cells do exhibit density-dependent inhibition of growth. Unlike the parent F9 cells or parietal yolk sac (PYS-2) cells, these cells do possess specific cell surface receptors for epidermal growth factor (EGF). The growth-arrested Dif 5 cells can be reinitiated to proliferate by the addition of fetal calf serum (FCS) or EGF. The properties of Dif 5 cells determined fail to fulfill all the characteristics described for either parietal or visceral endodermal cells. This raises the possibility that Dif 5 cells might represent an endodermal cell type which is intermediate in differentiation to either parietal or visceral endoderm but which lacks the biochemical signal to complete this stage of differentiation. This new Dif 5 cell line should be of considerable value in studying the modulation of growth requirements and extracellular matrix formation during early embryonic development.

Topics: alpha-Fetoproteins; Animals; Blood; Bucladesine; Cell Division; Cell Line; Cell Separation; Culture Media; Endoderm; Epidermal Growth Factor; Extracellular Matrix; Myristic Acids; Neoplasms, Experimental; Plasminogen Activators; Teratoma; Tretinoin

The synthesis of laminin A and B chains, and of entactin, has been measured in murine F9 embryonal carcinoma cells differentiating in response to retinoic acid and cyclic AMP. Undifferentiated cells synthesis low levels of laminin, amounting to approximately 0.02% of the [35S]methionine incorporated into cytoplasmic protein during a 15-min pulse. After 6 days induction, laminin synthesis has increased 15- to 20-fold. Undifferentiated F9 cells synthesise more intracellular laminin B2 chains (Mr 225,000) than B1 chains (Mr 225,000), but the excess B2 chains are apparently not assembled into the secreted laminin molecule. Indirect immunofluorescence shows faint cytoplasmic staining and short fibrils of laminin between the undifferentiated F9 cells.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Basement Membrane; Cell Differentiation; Cyclic AMP; Fluorescent Antibody Technique; Glycoproteins; Kinetics; Laminin; Membrane Glycoproteins; Mice; Molecular Weight; Teratoma; Tretinoin

We have generated by mutagenesis eight differentiation-defective sublines from three murine embryonal carcinoma (EC) cell lines. These mutants grossly resemble parental cells in the absence of inducers of differentiation. Based upon response to retinoic acid (RA) or hexamethylenebisacetamide (HMBA), the mutants can be grouped into three types: (a) RA-selected cells that lack cellular RA binding protein (cRABP) activity and fail to differentiate in response to RA or HMBA; (b) RA- or HMBA-selected cells that possess cRABP but differentiate poorly, if at all, in the presence of RA or HMBA; and (c) cells originally selected for lack of response to HMBA but which retain cRABP and the ability to differentiate in response to RA.

Topics: Acetamides; Animals; Carrier Proteins; Cell Differentiation; Cell Line; Mice; Mutation; Plasminogen Activators; Receptors, Retinoic Acid; Teratoma; Tretinoin

Antibodies reactive with a murine teratocarcinoma cell line (F9) were detected by immunofluorescence staining in sera from patients bearing ovarian germ cell tumour. Immunochemical studies revealed that antibodies binding to the cell surface of F9 cells react with large glycopeptides which are known to be components of F9 antigens defined by murine anti-F9 antibodies. In addition, treatment of F9 cells with retinoic acid, which induces differentiation of embryonal carcinoma cells, distinctly reduced both the ability of these antibodies to stain F9 cells and the biosynthesis of large glycopeptides by the cells. These findings indicate that the large glycopeptides precipitable by the patients' antibodies are differentiation associated antigens on characteristic embryonic cells.

Topics: Antibodies, Neoplasm; Antigens, Neoplasm; Cell Differentiation; Cell Line; Female; Fluorescent Antibody Technique; Glycopeptides; Humans; Neoplasms, Germ Cell and Embryonal; Ovarian Neoplasms; Teratoma; Tretinoin

It has been proposed that cellular retinoic acid binding protein is essential for retinoid-induced differentiation of embryonal carcinoma line PCC4.aza1R. To assess the generality of this proposal, we have tested for the presence of cellular retinoic acid binding protein activities in several other embryonal carcinoma lines. Cytosolic extracts from all cells were found to possess binding proteins for retinoic acid and also for retinol, although levels varied widely among the different lines. There was no clear quantitative relationship between binding protein activities and the propensity of the cells for differentiation in tumor form or under various in vitro conditions. Our results suggest that other factors might modulate the response of embryonal carcinoma cells to retinoids and/or that alternate pathways for differentiation which do not involve retinoids and retinoid binding proteins exist in these cells. When embryonal carcinoma cells are stimulated to differentiate, the derivatives can possess higher, lower, or similar levels of retinoic acid binding protein activity. These levels appear to reflect the phenotype of the differentiated cells rather than the conversion from a tumorigenic to a nontumorigenic state.

Topics: Animals; Carrier Proteins; Cell Differentiation; Cell Line; Mice; Receptors, Retinoic Acid; Retinol-Binding Proteins; Teratoma; Tretinoin; Vitamin A

Mouse embryonal carcinoma F9 cells were exposed to retinoic acid and dibutyryl cyclic AMP. The treated cells synthesized and secreted into the culture medium the basal lamina components, laminin (GP-1 and GP-2) and entactin. The time course of secretion of the basal lamina components was examined by electron microscopic and immunochemical procedures. The induction of the cells resulted in major morphological changes and the deposition of both laminin and entactin at the cell surface and cell junctions. Intracellular deposits of laminin could be localized to the endoplasmic reticulum and membrane-bound intracytoplasmic vacuoles. Concomitant with the appearance of laminin and entactin, there was a loss of fibronectin synthesis and a marked decrease in a 190,000-Da sulfated glycoprotein that appeared to be related to entactin. In the induced cells, laminin and entactin were associated in a complex that could be dissociated with low concentrations of sodium dodecyl sulfate. The induction of laminin and entactin seem to be independent. The enhanced synthesis of laminin appeared to be under transcriptional regulation since it was found that induced F9 cells contained translatable mRNA for GP-2 when tested in a rabbit reticulocyte lysate system. The uninduced cells did not contain detectable quantities of translatable GP-2 mRNA.

Topics: Animals; Basement Membrane; Bucladesine; Cell Line; Fluorescent Antibody Technique; Glycoproteins; Kinetics; Laminin; Membrane Glycoproteins; Membrane Proteins; Mice; Neoplasms, Experimental; Teratoma; Tretinoin

Five arotinoids have been compared with all-trans- and 13-cisretinoic acids for their ability to promote differentiation of cells from murine embryonal carcinoma line Nulli-SCC1. Ro-13-7410, which contains a terminal carboxylic acid residue, and Ro-14-9572, the sodium sulfinate derivative, are potent inducers of differentiation. The sodium sulfonate derivative, Ro-14-3899, is somewhat less active, whereas the ethyl sulfone (Ro-15-1570) and Ro-15-0778, an arotinoid lacking a terminal group, have little or no effect on embryonal carcinoma cell differentiation. Competition by the arotinoids with all-trans-retinoic acid for sites on the cellular retinoic acid-binding protein is qualitatively consistent with their capacity for promoting differentiation. This relationship and the response of differentiation-defective embryonal carcinoma cells to Ro-13-7410 support the view that arotinoids and retinoids promote differentiation of embryonal carcinoma cells via the same mechanism.

Topics: Animals; Benzoates; Carrier Proteins; Cell Division; Cell Line; Isomerism; Kinetics; Mice; Neoplasm Proteins; Neoplasms, Experimental; Receptors, Retinoic Acid; Retinoids; Structure-Activity Relationship; Teratoma; Tretinoin; Vitamin A

The synthesis of the proteins laminin and collagen IV is stimulated approximately equal to 20-fold in F9 mouse teratocarcinoma stem cells after treatment of the cells with retinoic acid and N6, O2'-dibutyryl-cAMP (Bt2cAMP). A cDNA library from F9 cells treated with retinoic acid, Bt2cAMP, and theophylline (F9-R + DBC cells) was constructed to isolate cDNA coding for collagen IV or laminin. The recombinant plasmids were screened by differential colony hybridization to cDNA synthesized from poly(A)+ RNA isolated from F9 stem and F9-R + DBC cells. Differentially hybridizing plasmids were then used as probes to hybridize to RNA transfer blots to determine the size of their specific mRNA. Only plasmids containing cDNA sequences specific for high molecular weight mRNA were further analyzed. Studies by hybridization-selection, in vitro translation, and immunoprecipitation showed that a plasmid clone, pc15, contains cDNA homologous to collagen IV (alpha 2) mRNA, and another plasmid clone, pc156, contains cDNA homologous to laminin B mRNA. By RNA blot analyses, the size of mRNA coding for collagen IV (alpha 2) is 7.6 kilobases; the size of mRNA for laminin is 6.8 kilobases. Using the technique of RNA blot hybridization, we studied the time course of the increase in mRNA coding for collagen IV (alpha 2) and laminin B in F9 cells after retinoic acid and Bt2cAMP treatment. Both collagen IV (alpha 2) and laminin B mRNAs are present in F9 stem cells. Collagen IV (alpha 2) mRNA and laminin B mRNA levels increase slightly at approximately 12 hr after retinoic acid and Bt2cAMP addition, with a dramatic increase between 12 and 24 hr after drug treatment.

Topics: Animals; Bucladesine; Cell Line; Cloning, Molecular; Collagen; DNA; Genes; Glycoproteins; Laminin; Mice; Neoplasms, Experimental; Nucleic Acid Hybridization; Protein Biosynthesis; RNA, Neoplasm; Teratoma; Theophylline; Tretinoin

We have isolated cDNA clones for mouse type IV procollagen from a library constructed from total poly A+RNA of 13.5 day mouse embryo parietal endoderm (PE) cells. In Northern analysis these clones hybridise to a 6.8 kb RNA which is abundant in embryonic PE cells and in differentiated F9 teratocarcinoma cells. Hybrid selection and in vitro translation of the cDNA specific mRNA produced a single polypeptide of Mr = 165 000. This polypeptide was specifically immunoprecipitated with mouse type IV procollagen antisera and comigrated on SDS-gel electrophoresis with one of the two in vitro synthesised chains of type IV procollagen. Undifferentiated F9 teratocarcinoma cells can be induced by retinoic acid and dibutyryl cAMP to differentiate in vitro into endoderm-like cells which resemble mouse PE cells in synthesising large amounts of basement membrane proteins, including type IV procollagen. Here we show, using one of the cDNA clones as a probe for type IV procollagen, that an increase in cellular concentration of type IV procollagen mRNA occurs within 24 to 48 hours of induction, reaching a constant high level by 72 hours.

Topics: Animals; Bucladesine; Cell Differentiation; Cell Line; Cloning, Molecular; Collagen; DNA; Embryo, Mammalian; Endoderm; Female; Mice; Nucleic Acid Hybridization; Pregnancy; Procollagen; Protein Biosynthesis; Rabbits; Reticulocytes; RNA, Messenger; Teratoma; Tretinoin

Teratocarcinoma stem cell F9 expressed a potent fucosyltransferase activity acting on asialofetuin. A majority of the product was susceptible to alpha-L-fucosidase I from almond emulsin, indicating that the linkage formed was mainly Fuc alpha 1 leads to 3GlcNAc. The specific activity of the transferase decreased when the stem cells were induced to differentiate into parietal endoderm cells by retinoic acid and dibutyryl cyclic AMP. Furthermore, PYS-2 cell, a parietal endoderm cell line virtually lacked the transferase. The change in the fucosyltransferase activity could be correlated with cell surface changes occurring during differentiation.

Topics: Bucladesine; Cell Differentiation; Cell Line; Embryonal Carcinoma Stem Cells; Fucosyltransferases; Hexosyltransferases; Neoplastic Stem Cells; Stem Cells; Surface Properties; Teratoma; Tretinoin

F9 embryonal carcinoma (EC) cells, cultured in suspension in medium containing 5 X 10(-8) M retinoic acid, aggregate and differentiate into embryoid bodies with an outer layer of visceral endoderm cells that synthesize and secrete alphafetoprotein (AFP) (Hogan, B. L. M., A. Taylor, and E. Adamson, 1981, Nature (Lond.). 291:235-237). Here we analyze the formation of the outer layer of cells as a model for epithelial differentiation. Three morphological phases are described, but analyses of cell numbers and the synthetic rates of some proteins, as well as the appearance of markers of visceral endoderm and basement membrane, show that the formation of the outer layer occurs as an orderly progression of multiple events. The markers used to follow the ontogeny of epithelial layer formation include SSEA-1, l, and i blood group antigens, laminin, fibronectin, type IV collagen, cytoskeletal intermediate filament proteins (vimentin, Endo A, and B), and AFP. The onset of epithelium formation occurs between the third and fourth day of culture, but its function is maximally expressed only when it is well organized. We found the rate of AFP secretion to be a measure of the proper alignment and maturity of the epithelium which occurs at the seventh or eighth day. This model of epithelium formation may help to explain how similar processes occur during embryogenesis.

Topics: Animals; Basement Membrane; Cell Aggregation; Cell Differentiation; Cell Division; Cell Line; Epithelial Cells; Fluorescent Antibody Technique; Glycoproteins; Laminin; Teratoma; Tretinoin

Retinoic acid (RA) can induce the differentiation of teratocarcinoma cells in culture (S. Strickland and V. Mahdavi, 1978, Cell 15, 393-403; E. M. V. Jones-Villeneuve, M. W. McBurney, K. A. Rogers, and V. I. Kalnins, 1982, J. Cell. Biol. 94, 253-262). With the teratocarcinoma cell line, P19S1801A1 (O1A1), the differentiated cell types formed from cell aggregates exposed to RA are dependent on the concentration of drug used in the treatment. Cultures exposed to low concentrations (10(-9)M) are characterized by an abundance of cardiac muscle. Skeletal muscle becomes abundant at higher drug concentrations (10(-8)M) with neurons and astroglia appearing at very high concentrations (10(-7) to 10(-5) M). These results suggest that during normal embryogenesis, the commitment of pluripotent cells to particular developmental avenues may be determined in part by concentration gradients of substances such as retinoids.

Topics: Animals; Cell Differentiation; Cell Line; Dose-Response Relationship, Drug; Fluorescent Antibody Technique; Teratoma; Tretinoin

We have previously shown that the P19 line of embryonal carcinoma cells develops into neurons, astroglia, and fibroblasts after aggregation and exposure to retinoic acid. The neurons were initially identified by their morphology and by the presence of neurofilaments within their cytoplasm. We have more fully documented the neuronal nature of these cells by showing that their cell surfaces display tetanus toxin receptors, a neuronal cell marker, and that choline acetyl-transferase and acetyl cholinesterase activities appear coordinately in neuron-containing cultures. Several days before the appearance of neurons, there is a marked decrease in the amount of an embryonal carcinoma surface antigen, and at the same time there is a substantial decrease in the volumes of individual cells. Various retinoids were able to induce the development of neurons in cultures of aggregated P19 cells, but it did not appear that polyamine metabolism was involved in the effect. We have isolated a mutant clone which does not differentiate in the presence of any of the drugs which are normally effective in inducing differentiation of P19 cells. This mutant and others may help to elucidate the chain of events triggered by retinoic acid and other differentiation-inducing drugs.

Topics: Animals; Cell Differentiation; Cell Line; Embryonal Carcinoma Stem Cells; Mice; Mice, Inbred C3H; Mutation; Neoplastic Stem Cells; Neuroglia; Neurons; Stem Cells; Teratoma; Tetanus Toxin; Tretinoin

Non-dividing STO mouse fibroblasts have been used for some time as feeder cells for maintaining certain embryonal carcinoma cells in an undifferentiated state. We report here that medium conditioned by these feeders can inhibit embryonal carcinoma (ec) cell differentiation induced either by removal from feeders, or, in the case of cells not normally requiring a feeder layer, by retinoic acid treatment.

Topics: Animals; Cell Differentiation; Cell Line; Culture Media; Embryo, Mammalian; Fibroblasts; Mice; Teratoma; Tretinoin

Pluripotent teratocarcinoma cell line, 311, was cultured in the presence of retinoic acid (RA) and studied for the processes of early marker changes associated with cell differentiation. The cell populations that have lost peanut agglutinin (PNA), Lotus tetragonolobus agglutinin (LTA) or wheat germ agglutinin (WGA) receptor increased in proportion to the period since the start of RA treatment. The kinetics of the appearance of these receptor-negative cell populations suggests that the differentiating cells lose lectin receptors in the order of PNA, LTA and WGA. However, the changes in F9 antigen(s) and LTA receptor occurred at an equal frequency in PNA+ and PNA- cells, indicating that, although the loss of lectin receptors takes place in a distinct order, the change in each receptor itself proceeds independently of the state of other lectin receptors.

Topics: Animals; Cell Differentiation; Cell Line; Cell Membrane; Kinetics; Lectins; Mice; Peanut Agglutinin; Receptors, Mitogen; Teratoma; Tretinoin

We have addressed the question of the possible presence of calcium-activated phospholipid-dependent protein kinase (Ca2+-PL protein kinase) activity in undifferentiated embryonal carcinoma cells, and if this activity might be altered during differentiation to a parietal endoderm cell type. Undifferentiated nullipotent F9 embryonal carcinoma cells, as well as differentiated parietal endoderm cells (PYS-2), were utilized. Using an in vitro assay with histone H1 as phosphate acceptor, Ca2+-PL protein kinase activity could not be found in the 100,000 X g supernatant prepared from either cell type. However, passage of 100,000 X g supernatant from PYS cells over a DEAE-cellulose column revealed Ca2+-PL protein kinase activity which eluted with 0.045 M NaCl. The partially purified PYS enzyme has an approximate Mr = 70,000 as determined by Sephadex G-150 gel filtration, and exhibits an apparent Ka for Ca2+ of 32 microM. The PYS Ca2+-PL protein kinase also exhibits a requirement for Mg2+, with maximal activity noted at 10 mM Mg2+. This enzyme is stimulated by acidic phospholipids, while neutral phospholipids such as phosphatidylcholine have little effect. Diacylglycerol markedly increased histone H1 phosphorylation in the presence of Ca2+ and phospholipid. Unlike that of PYS cells, when the 100,000 X g supernatant prepared from F9 cells was passed over a DEAE-cellulose column no Ca2+-PL protein kinase activity could be found in the eluted fractions. Previously it has been reported that exposure of F9 cells to all-trans-retinoic acid induces differentiation to a parietal endoderm cell type. Treatment of F9 cells with 0.1 microM retinoic acid provoked a time-dependent increase in cytosolic Ca2+-PL protein kinase activity as measured after DEAE-cellulose chromatography of the 100,000 X g supernatant. This increase in Ca2+-PL protein kinase activity correlates with differentiation to the parietal endoderm cell type. These findings indicate that cytosolic Ca2+-PL protein kinase activity is very low, or nonexistent, in undifferentiated embryonal carcinoma stem cells. With differentiation to a parietal endoderm cell type there is a marked increase in soluble Ca2+-PL protein kinase activity which exhibits properties similar to those described for this enzyme in other differentiated tissues.

Topics: Animals; Calcium; Cell Differentiation; Cell Line; Cytosol; Molecular Weight; Phospholipids; Protein Kinases; Teratoma; Tretinoin

Murine extra-embryonic endodermal cell lines derived from either teratocarcinomas or mouse embryos contain a cytoskeletal protein (Endo A) of Mr = 55,000. Endo A was immunoprecipitated from [35S]methionine-labeled lysates of three parietal endodermal cell lines, A presumptive visceral endodermal cell line, and a fetal hepatoma cell line, but not from fibroblasts, myoblasts, erythroleukemic cells, neuroblastoma cells, keratinocytes, or embryonal carcinoma cells. Embryonal carcinoma cells induced to differentiate by exposure to retinoic acid synthesized increased amounts of Endo A approximately 48 h after exposure to the inducer. Two-dimensional gel analysis of immunoprecipitated samples confirmed that Endo A is distinct from vimentin and murine keratinocyte proteins recognized by two different keratin antisera. Comparison by two-dimensional gel electrophoresis of immunoprecipitated Endo A labeled with either [35S]methionine or [32P]orthophosphate indicated that the multiple forms of Endo A resolved by isoelectric focusing were due, at least in part, to phosphorylation. Serine was identified as the phosphorylated amino acid. Endo A was the only major antigenic protein found in a parietal endodermal cell line which was recognized by a monoclonal antibody prepared by other investigators against trophoblast cytoskeletons. The results indicate that Endo A, like the previously described Endo B protein, is distinct from other cytoskeletal proteins and will be useful as a marker of the differentiation of murine embryonal carcinoma cells to extra-embryonic endoderm.

Topics: Animals; Antibodies, Monoclonal; Cell Line; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Embryo, Mammalian; HeLa Cells; Keratins; Mice; Molecular Weight; Protein Biosynthesis; Proteins; Teratoma; Tretinoin

Teratocarcinoma differentiation has been studied using sera specific for each of the five intermediate filament (IF) classes. These antibodies distinguish cells of epithelial, muscle, neural, astrocytic, and mesenchymal origin. In embryoid bodies, derived from embryo transplants and obtained in the ascitic fluid by transplantation of teratocarcinoma, the cells of the inner cellular mass did not express any of these intermediate filament types while the outer cells expressed cytokeratin. Intermediate filament expression in the embryoid body thus appears analogous to that in the blastocyst and differs from that in embryonal carcinoma (EC) lines. Twelve EC lines have now been shown to express vimentin although in some EC lines not all cells express vimentin. Other established permanent differentiated cell lines, derived from EC lines in vitro or from tumors in vivo, have been characterized with respect to the type of IF they contain. The distribution of different IF types has been examined in EC cells induced to differentiate by addition of retinoic acid. The proportion of cells expressing each type of intermediate filament appears to depend on the EC cell line used, on the inducing agent, and on the length of treatment. Thus, for instance, F9 cells express cytokeratin, PCC3 derivatives express vimentin, many 1009 derivatives express either glial fibrillar acidic protein (GFA) or neurofilament proteins. Overall the results obtained are in excellent agreement with emerging principles of intermediate filament expression during embryonic differentiation, thus emphasizing the potential use of the various EC lines to study differentiation in culture.

Topics: Animals; Cell Differentiation; Cell Line; Cytoskeleton; Desmin; Female; Fluorescent Antibody Technique; Intermediate Filament Proteins; Keratins; Male; Mice; Neoplasms, Experimental; Protein Precursors; Teratoma; Tretinoin; Vimentin

Topics: Affinity Labels; Animals; Azides; Cell Membrane; Cyclic AMP; Cytosol; Kinetics; Mice; Protein Binding; Protein Kinases; Teratoma; Tretinoin

PCC4azal embryonal carcinoma tumors were grown in strain 129 mice by s.c. transplantation. When palpable, the tumors were treated with a combination of retinoic acid and dimethylacetamide. In vitro, this embryonal carcinoma cell line shows minimal spontaneous differentiation and is exquisitely sensitive to retinoic acid and/or dimethylacetamide induction of differentiation. Ten daily 20-microliter intratumor injections of a solution of 10 mg retinoic acid per ml of dimethylacetamide resulted in nearly complete induction of morphological differentiation mainly into neuroepithelial and glandular derivatives. Control tumors showed minor spontaneous differentiation. Differentiation was associated with decreased tumor growth rate, decreased mitotic index, decreased extent of necrosis, and increased survival time of the hosts. In 4 of 18 cases, long-term survival of the hosts was effected by a complete differentiation of the malignant embryonal carcinoma tumors into benign teratomas. Retinoic acid:dimethylacetamide was also effective in inducing differentiation with the same dosage and schedule when administered systemically, i.e., i.p. or s.c.

Topics: Acetamides; Animals; Cell Differentiation; Drug Evaluation, Preclinical; Mice; Mitotic Index; Neoplasm Transplantation; Neoplasms, Experimental; Neoplasms, Germ Cell and Embryonal; Teratoma; Tretinoin

Topics: Animals; Bucladesine; Cell Differentiation; Cell Line; Fucose; Galactose; Glycopeptides; Mice; Teratoma; Tretinoin

The effect of retinoic acid on the synthesis and degradation of basement membrane components by endoderm cells derived from mouse embryonal carcinoma (EC) cells was studied in a serum-free, defined medium. By immunofluorescence these cells accumulate type IV collagen, laminin, and fibronectin after growth in media containing epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin, transferrin, and Pedersen fetuin. Collagen accounted for 2 to 4% of the newly synthesized proteins, of which 90% were found in the culture media. This collagen was identified as Pro-type IV be gel electrophoresis and enzymatic susceptibility. The EC cells preferentially attached to type IV collagen in vitro and such attachment was mediated by laminin. Treatment of EC cells with retinoic acid caused an increased accumulation of collagen (10 to 15% of secreted proteins) and also stimulated the elaboration of latent protease which degraded laminin and type IV collagen. The laminin-degrading activity was plasminogen dependent. The type IV collagen-degrading activity was a metal protease which could be activated by trypsin or plasmin. It is likely that at least part of the laminin degrading activity is plasmin (mediated through plasminogen activator), since highly purified plasmin is shown to degrade native laminin.

Topics: Animals; Basement Membrane; Cells, Cultured; Collagen; Culture Media; Endoderm; Fibronectins; Fluorescent Antibody Technique; Glycoproteins; Laminin; Mice; Microbial Collagenase; Teratoma; Tretinoin

Embryonal carcinoma (EC) cells are resistant to infection with polyoma virus and become permissive when allowed to differentiate. Polyoma host-range (PyEC) mutants have been selected on two embryonal carcinoma cell lines, PCC4 and F9, which differ in their differentiation potential, both in vitro and in vivo. Since PyEC mutants selected on one line failed to develop, or developed only poorly on the other, we used these two classes of mutants as probes towards several EC lines which differed in their origin and differentiation properties. From their susceptibility to either mutant, and from the effect of temperature upon the efficiency of infection, we inferred a classification of these teratocarcinoma cell lines, which is an agreement with a previous one based upon metabolic coupling. We also discussed results indicating that different EC cell lines might present steps in the process of cell determination at the embryonic level.

Topics: Animals; Cell Differentiation; Gene Expression Regulation; Genetic Variation; Mice; Neoplasms, Experimental; Polyomavirus; Teratoma; Tretinoin

F9 embryonal carcinoma cells express high levels of a 53,000-molecular-weight cellular tumor antigen called p53. When F9 cell cultures are treated with retinoic acid and dibutyryl adenosine 3',5'-phosphate, they differentiate, predominantly into endoderm-like cells. This differentiation is accompanied by a marked decrease in the levels of p53. The mechanism(s) responsible for this decline in the level of p53 in differentiated cells was investigated. The results demonstrate that the high levels of p53 in F9 cells relative to their differentiated progeny were not due to alterations in the stability or turnover of this protein. Rather, the regulation during differentiation involved a marked decrease in the amount of in vitro translatable p53 mRNA detected in the differentiated cell cultures. This mechanism is unlike the one operating during the simian virus 40 infection or transformation, where the increased levels of p53 are largely due to the increased stability of the p53 protein.

Topics: Animals; Bucladesine; Cell Differentiation; Cell Transformation, Viral; Cells, Cultured; Fibroblasts; Mice; Mice, Inbred BALB C; Phosphoproteins; RNA, Messenger; Simian virus 40; Teratoma; Tretinoin; Tumor Suppressor Protein p53

The embryonal carcinoma mouse cell line F-9 was used as a convenient model for a quantitative study of the production of the basement membrane proteins laminin and type IV collagen. Both proteins could be identified in the culture medium and cell layer by radioimmuno assays, metabolic labeling and immunofluorescence. More than 95% of the material is secreted into the medium. Lack of ascorbic acid inhibits secretion of type IV collagen but not of laminin. Induction of differentiation into endoderm-like cells by retinoic acid consistently caused after a lag period of 2-3 days a 5-10 fold increase in the production of basement membrane proteins but not of total protein. Dibutyryl cyclic AMP further potentiated this specific effect particularly with respect to type IV collagen synthesis. Insulin, epidermal growth factor and nerve growth factor produced only moderate increases (10-60%) in the amount of laminin and type IV collagen. Effects of these hormones were only observed with certain doses and were quite variable between different experiments.

Topics: Animals; Ascorbic Acid; Basement Membrane; Bucladesine; Cell Line; Collagen; Electrophoresis, Polyacrylamide Gel; Fluorescent Antibody Technique; Glycoproteins; Growth Substances; Kinetics; Laminin; Mice; Teratoma; Tretinoin

Murine embryonal carcinoma (EC) cells fail to express viral T antigen following infection with polyoma (Py) virus, while some differentiated derivatives are fully susceptible to viral infection. The block to expression in EC cells apparently occurs after adsorption and penetration but prior to the synthesis of early viral proteins. The F9 EC cell line was employed to further investigate the failure of Py gene expression in EC cells as well as the viral susceptibility of certain differentiated cell types. F9 cultures treated with retinoic acid (RA) or RA in combination with dibutyryl cAMP (dBcAMP) are quantitatively induced to differentiate to endodermal cell types. When the fate of the viral DNA was examined in F9 EC cells, free viral DNA was observed early after infection but was eventually lost with continued cell growth. Cultures induced to differentiate in the presence of RA demonstrated limited viral susceptibility which increased with prolonged RA exposure. Polyoma sensitivity did not directly parallel basement membrane antigen production, a marker used to distinguish this differentiated endodermal cell type. Viral gene expression could be obtained if Py DNA was introduced into the cells prior to RA induction. In contrast, the endodermal progeny derived from a dual treatment with RA plus dBcAMP appeared highly refractory to Py infection.

Topics: Animals; Antigens, Viral; Bucladesine; Cell Differentiation; Cell Line; DNA, Viral; Polyomavirus; Teratoma; Transfection; Tretinoin; Tumor Virus Infections

Undifferentiated F-9 teratocarcinoma cells derived from 129/J mice are induced in vitro to express several differentiation markers. Neither undifferentiated nor differentiated F-9 cells express endogenous retroviral glycoprotein (gp70), although the latter can be productively infected with exogenous retroviruses. This is discussed in context with previous findings that all antigen-activated lymphocytes of all mice express endogenous retroviral gp70.

Topics: Animals; Bucladesine; Cell Differentiation; Cell Line; Mice; Neoplasms, Experimental; Retroviridae; Teratoma; Tretinoin; Viral Envelope Proteins; Viral Proteins

In five lines of mouse embryonal carcinoma cells, PCC3/A1, PCC4, PCC4/Aza-R1, and F9, collagen synthesis was examined by immunofluorescence reaction using specific antibodies directed against collagen. All the embryonal carcinoma cell lines showed type IV collagen, and PCC7-S/Aza-R1 revealed the additional presence of type III collagen. When the F9 and PCC3/A1 EC cells were treated with retinoic acid and dibutyryl-cAMP, they differentiated into morphologically different cellular types. These cellular types showed new types of collagen. Thus, in treated F9 cells, type I, type III, and type V collagen were detected and in treated PCC3/A1 cells, type III and type V collagen were detected. In two established cellular strains, PYS-2 corresponding to parietal endoderm and 3TDM-1 corresponding to trophoblastoma, collagen was identified by immunological reaction and electrophoretic mobility. The trophoblastoma cell line was characterized by the production of type I, type III, and type IV collagen, whereas endodermal PYS-2 revealed type IV collagen.

Topics: Animals; Antigens; Cell Line; Collagen; Electrophoresis, Polyacrylamide Gel; Fluorescent Antibody Technique; Neoplasms, Experimental; Teratoma; Tretinoin

Cell surface glycosyltransferases are thought to participate in a variety of cellular interactions, but their specific glycoside acceptors have received little attention. In this paper, poly(N)-acetyllactosamine glycoconjugates are shown to be the endogenous substrates for embryonal carcinoma (EC) cell surface galactosyltransferases. All controls have been performed to ensure a surface localization for the galactosyltransferase activity. The galactosylated product(s) is relatively insoluble in organic solvents, is larger than conventional glycopeptides following pronase digestion, and is highly sensitive to endo-beta-galactosidase degradation. Solubilized polylactosaminyl glycoconjugates serve as competitive exogenous acceptors for the surface galactosyltransferase. In addition, the endogenous galactosyl acceptor(s) reacts with antiserum directed against EC cell poly(N)-acetyllactosamines. Anti-EC antiserum inhibits galactosylation of endogenous acceptors, simultaneously stimulates galactosylation of an exogenous acceptor, and immunoprecipitates 74% of the reaction product. Differentiated EC cells no longer react with anti-EC antiserum and no longer show anti-EC antiserum effects on surface galactosyltransferase activity. Interestingly, forced galactosylation with UDPGal releases polylactosaminyl substrates from the cell surface. In the absence of UDPGal, glycoconjugate release is dramatically reduced. GDPMan cannot substitute for UDPGal, and a galactosyltransferase inhibitor prevents glycoside release from the cell surface. Thus, surface galactosyltransferase preferentially binds poly(N)-acetyllactosamine glycoconjugates and serves as at least one of their surface receptors on EC cells.

Topics: Animals; Antigens, Surface; Cell Line; Cell Membrane; Fucosyltransferases; Galactosyltransferases; Hydrolases; Kinetics; Mice; Receptors, Antigen; Substrate Specificity; Teratoma; Tretinoin

A new clonal CBA mouse teratocarcinoma cell line called H6 grows rapidly without calcium in suspension culture. When attached to a gelatin-coated surface in the presence of calcium, differentiation to large flat cells, which secrete plasminogen activator, occurs after exposure to retinoic acid. Differentiation is terminal after about six cell divisions. H6 variants resistant to 6-thioguanine, 5-bromodeoxyuridine, ouabain, chloramphenicol, retinoic acid, or combinations thereof, have been isolated. Cell hybridizations were made between undifferentiated stem cells and the differentiated derivatives of H6. These experiments indicate that the undifferentiated cells can "rescue" cells in the initial stages of differentiation and that the signs of differentiation are soon lost in such hybrids. Hybridization without "rescue" may also occur, as suggested by small abortive colonies of large cells.

Topics: Cell Differentiation; Cell Fusion; Cells, Cultured; Plasminogen Activators; Teratoma; Tretinoin

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Kinetics; Lectins; Male; Mice; Peanut Agglutinin; Teratoma; Testicular Neoplasms; Tretinoin

Topics: Animals; Cell Cycle; Cell Differentiation; Cell Line; Clone Cells; Interphase; Mice; Teratoma; Time Factors; Tretinoin

Topics: Antineoplastic Agents; Brain Neoplasms; Etretinate; Female; Humans; Male; Melanoma; Ovarian Neoplasms; Teratoma; Testicular Neoplasms; Tretinoin

Murine embryonal carcinoma cells can differentiate into a varied spectrum of cell types. We observed the abundant and precocious development of neuronlike cells when embryonal carcinoma cells of various pluripotent lines were aggregated and cultured in the presence of nontoxic concentrations of retinoic acid. Neuronlike cells were also formed in retinoic acid-treated cultures of the embryonal carcinoma line, P19, which does not differentiate into neurons in the absence of the drug. The neuronal nature of these cells was confirmed by their staining with antiserum directed against neurofilament protein in indirect immunofluorescence experiments. Retinoic acid-treated cultures also contained elevated acetylcholinesterase activity. Glial cells, identified by immunofluorescence analysis of their intermediate filaments, and a population of fibroblastlike cells were also present in retinoic acid-treated cultures of P19 cells. We did not observe embryonal carcinoma, muscle, or epithelial cells in these cultures. Neurons and glial cells appeared in cultures exposed to retinoic acid for as little as 48 h. We found no evidence for retinoic acid toxicity, suggesting that the effect of the drug was to induce the development of neurons and glia rather than to select against cells differentiating along other developmental pathways.

Topics: Animals; Cell Differentiation; Cell Line; Dose-Response Relationship, Drug; Mice; Neuroglia; Neurons; Teratoma; Tretinoin

Retinoic acid has been shown to induce the differentiation of mouse embryonal carcinoma cells. Previous workers have reported that bulk cultures of the differentiated derivatives have a slower growth rate and a reduced capacity to form tumours. We have analysed this change in growth rate for a sub-tetraploid EC cell line, PC13 clone 5 MA2, at a clonal level and have shown that the production of cells with a slower growth rate is not a result of cell selection. We have also demonstrated that the action of retinoic acid on growth rate is delayed for approximately 48 h and that the new growth phenotype, once attained, is stable. Finally we have confirmed at a clonal level that the differentiated derivatives of EC cells exposed to retinoic acid have a reduced capacity to form tumours. Clones of EC cells exposed to retinoic acid for longer than 96 h are unable to form tumours in a 30-day period, whilst 87% of their untreated counterparts are able to do so.

Topics: Animals; Cell Differentiation; Cell Survival; Clone Cells; Dose-Response Relationship, Drug; Male; Mice; Neoplasm Transplantation; Neoplasms, Experimental; Phenotype; Teratoma; Time Factors; Tretinoin

The messenger RNA species that code for two extra-embryonic endodermal cytoskeletal proteins (Endo A and Endo B) have been identified. Endo A and Endo B messenger RNA species are differentially expressed. They are absent, or at basal levels, in undifferentiated embryonal carcinoma cells (F9.22 cell line) and relatively abundant in parietal endoderm (PFHR9 cell line) or in embryonal carcinoma cells that have been induced, by retinoid acid, to differentiate to parietal endoderm. Endo A and Endo B messenger RNA can be detected in F9.22 cells 48 to 72 h after exposure to retinoic acid, which is coincident with the expression of Endo A and Endo B proteins. The size of Endo A and Endo B messenger RNA has been determined by denaturing methyl mercury hydroxide agarose gels to be 2.0 +/- 0.1 and 1.5 +/- 0.2 kilobases, respectively.

Topics: Animals; Cell Differentiation; Cell Line; Cytoskeleton; Female; Mice; Molecular Weight; Neoplasm Proteins; Pregnancy; RNA, Messenger; Teratoma; Time Factors; Tretinoin

Topics: Acetamides; Animals; Autoradiography; Bone Marrow; Cell Cycle; Cell Differentiation; Cell Line; Cell Nucleolus; Embryo, Mammalian; Fibroblasts; Mice; Mitotic Index; Neoplasms, Experimental; RNA, Ribosomal; Silver Nitrate; Staining and Labeling; Teratoma; Tretinoin

Topics: alpha-Fetoproteins; Animals; Bucladesine; Cell Aggregation; Cell Differentiation; Cell Line; Clone Cells; Embryo, Mammalian; Mice; Teratoma; Tretinoin

The mouse teratocarcinoma stem cell line, F9, becomes permissive for productive polyoma infection upon treatment with retinoic acid. Through the use of M13-polyoma recombinant single-stranded DNA probes, spliced and unspliced early viral RNA were detected after polyoma infection of retinoic acid-treated and untreated F9 cultures.

Topics: Animals; Antigens, Neoplasm; Antigens, Viral; Antigens, Viral, Tumor; Cell Differentiation; Cell Line; Mice; Plasminogen Activators; Polyomavirus; RNA, Viral; Teratoma; Tretinoin

Topics: Adenylyl Cyclases; Animals; Bucladesine; Calcitonin; Cell Differentiation; Cell Division; Cell Line; Female; Isoproterenol; Mice; Parathyroid Hormone; Pregnancy; Teratoma; Tretinoin

Murine embryonal carcinoma cells from line PCC4.aza1R differentiate readily in response to retinoic acid. By treating PCC4.aza1R cells with the mutagen N-methyl-N' -nitro-N-nitrosoguanidine, we derived two embryonal carcinoma lines in which the cells failed to differentiate during exposure to retinoic acid. Although these dif(RA)- cells maintained the tumorigenic potential of the parental cells, they differentiated poorly in tumor form. Similarly, the tendency of dif(RA)- cells to differentiate when aggregated in vitro was diminished relative to that of PCC4.zaz1R cells. The rate of retinoic acid uptake in cells from the two mutant lines did not appear to be reduced compared with the rate in cells from the parental line; however, specific cytoplasmic retinoic acid-binding protein activity was virtually absent in both mutants. These results strengthen the view that differentiation of embryonal carcinoma cells in response to retinoic acid requires formation of retinoic acid-cytoplasmic retinoic acid-binding protein complexes.

Topics: Animals; Biological Transport; Cell Differentiation; Cell Division; Cell Line; Kinetics; Mice; Mutation; Neoplasms, Experimental; Teratoma; Tretinoin

The insulin-receptor binding activity and insulin-stimulated growth response of PC13 clone 5 cells were investigated for both the embryo carcinoma (EC) and retinoic acid-induced differentiated derivatives of this cell line. Whereas the EC cell was found to have very few, if any, receptors and showed no demonstrable dependence on insulin for growth, the differentiated derivative cell expressed a large number of insulin receptors and, when challenged with the hormone, showed stimulation of both DNA synthesis and cell division. The same data were obtained for five independent PC13 clones. These results, coupled with previous observations, lend weight to the suggestion that the appearance of specific receptors for growth regulatory substances may be a manifestation of a general change in growth-regulatory mechanisms accompanying EC cell differentiation and loss of malignancy.

Topics: Cell Differentiation; Cell Division; Cells, Cultured; Insulin; Receptor, Insulin; Teratoma; Tretinoin

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Cell Membrane; DNA; Fibronectins; Mice; Muscle Proteins; Peptide Biosynthesis; Protein Biosynthesis; Teratoma; Tretinoin; Tropomyosin; Vimentin

Topics: Animals; Carcinoembryonic Antigen; Cell Line; Cell Transformation, Neoplastic; Electrophoresis, Polyacrylamide Gel; Endoderm; Female; Fibroblasts; Lactoperoxidase; Membrane Proteins; Mice; Neoplasms, Experimental; Pregnancy; Surface Properties; Teratoma; Tretinoin

Topics: alpha-Fetoproteins; Animals; Cell Differentiation; Cell Line; Endoderm; Kinetics; Mice; Structure-Activity Relationship; Teratoma; Tretinoin

This paper reports the growth and differentiation of the mouse embryonal carcinoma cell line F9 in completely defined culture media. The defined growth medium, referred to as EM-3, contains plasma fibronectin, insulin, and transferrin in place of serum. F9 cells cultured in EM-3 for over 15 generations retain their ability to form tumors and to differentiate. Fibronectin is essential for the attachment of F9 cells in defined media and its effect can be blocked with affinity-purified anti-fibronectin. When retinoic acid was added to EM-3, the F9 cells differentiated. The majority of the the newly formed cells differed from patient F9 cell two major respects: (i) they were morphologically different; and (ii) they secreted plasminogen activator, and the secretion was stimulated by dibutyrlyl adenosine cyclic monophosphate.

Topics: Animals; Bucladesine; Cell Differentiation; Cell Division; Cell Line; Culture Media; Fibronectins; Insulin; Mice; Neoplasms, Experimental; Plasminogen Activators; Teratoma; Transferrin; Tretinoin

Topics: Acetylcholinesterase; Animals; Bucladesine; Cell Differentiation; Culture Techniques; Endoderm; Enzyme Induction; Mice; Neoplasms, Experimental; Nervous System; Teratoma; Tretinoin

It has previously been shown that retinoic acid induces multiple phenotypic changes in cultures of F9 teratocarcinoma stem cells. In this paper we demonstrate that these retinoid-generated cells can be converted to yet another cell type by compounds that elevate cAMP concentrations. The phenotype of the new cell type is characterized by the synthesis of plasminogen activator, laminin and type IV collagen, and by very low levels of alkaline phosphatase and lactate dehydrogenase. The secretion of plasminogen activator and type IV collagen, and low levels of alkaline phosphatase and lactate dehydrogenase, have been previously shown to be properties of parietal endoderm, an extraembryonic cell which is generated early in mouse embryonesis. We show here that parietal endoderm also synthesizes laminin. The cell type generated by retinoic acid and dibutyryl cAMP treatment is therefore indistinguishable from definitive parietal endoderm. Analysis of the final phenotype indicates that it is not dependent upon the continued presence of either compound, and that cAMP agents are active only on cells that have been treated with retinoic acid.

Topics: Animals; Bucladesine; Cell Differentiation; Cell Line; Collagen; Endoderm; Mice; Plasminogen Activators; Proteins; Teratoma; Tretinoin

Murine embryonal carcinoma cells (ECCs) do not express antigens of the major histocompatibility complex (H-2), but do express cell-surface molecules shared with early embryos. ECCs are also characterized by their insusceptibility to infection by various oncogenic viruses, and their ability to differentiate into a variety of adult cell types. Differentiation of ECCs in vitro can occur spontaneously or can be induced. On exposure to retinoic acid the ECC line F9 (ref. 13) differentiates into cells which have the characteristics of parietal endoderm. When ECCs are exposed to simian virus 40 (SV40), the SV40 tumour (T) antigen is not expressed, although the virus genome reaches the nucleus, and a primary transcript of the SV40 A gene is made. However, following exposure to retinoic acid, the differentiated cells, like most mouse somatic cells, are susceptible to SV40 abortive infection and synthesize large T and small t antigens. To monitor the molecular events associated with the expression of the SV40 A gene on differentiation, we have constructed an ECC line (F9 12-1) containing a single integrated copy of the SV40 genome. This was accomplished by introducing a recombinant plasmid consisting of pBR322 linked to the herpes simplex type 1 thymidine kinase gene and SV40 genome into a thymidine kinase-deficient F9 cell line. We report here that in F9 12-1 cells exposed to retinoic acid, synthesis of the SV40 A gene product(s), T and tumour-associated specific antigens (TASA), parallels the appearance of the normal hallmarks of differentiation in this cell line, H-2 antigens and the basement membrane protein laminin.

Topics: Animals; Antigens, Neoplasm; Antigens, Viral; Cell Differentiation; Genes, Viral; Glycoproteins; H-2 Antigens; Laminin; Mice; Neoplasms, Experimental; Simian virus 40; Teratoma; Tretinoin

Topics: Animals; Basement Membrane; Cell Division; Cell Line; Endoderm; Immune Sera; Mice; Plasminogen Activators; Teratoma; Tretinoin

Topics: Animals; Cell Line; Glycosaminoglycans; Hyaluronic Acid; Kinetics; Teratoma; Tretinoin

Topics: Animals; Basement Membrane; Cell Differentiation; Cell Line; Endoderm; Glycopeptides; Isoelectric Point; Mice; Molecular Weight; Teratoma; Tretinoin; Yolk Sac

Topics: Animals; Antigens, Neoplasm; Cell Line; Epitopes; Mice; Teratoma; Tretinoin

Infection of differentiated mouse embryo cells by simian virus 40 (SV40) leads to the production of the early mRNAs and the tumor (T) antigens that they encode. In contrast, undifferentiated F-9 murine teratocarcinoma cells do not support these early stages of the SV40 cycle. This block results from the inability to accumulate stable processed early SV40 mRNAs. It has recently been shown that vitamin A and its derivatives can induce in vitro differentiation of stem cells. Undifferentiated F-9 cells, upon treatment with a low concentration of retinoic acid, exhibited pronounced morphologic changes as well as the appearance of the H-2 surface antigens. After differentiation, the susceptibility of F9 cells to SV40 infection could be demonstrated by the appearance of large T and small T antigens, as shown by immunofluorescence and immunoprecipitation. Furthermore, SI nuclease mapping of early SV40 transcripts confirmed the presence of the two spliced early mRNAs. These results indicate that the undifferentiated F-9 stem cells contain the genetic information needed for generating stable processed early SV40 mRNAs but are blocked in the production of functional species.

Topics: Animals; Antigens, Neoplasm; Antigens, Viral; Cell Differentiation; Cell Line; Genes, Viral; Mice; RNA, Messenger; RNA, Viral; Simian virus 40; Teratoma; Tretinoin; Viral Proteins

Mouse teratocarcinoma stem cells (embryonal carcinoma, or EC cells) bind very small amounts of mouse epidermal growth factor (EGF) and the latter hormone seems to have no stimulatory effect on the growth of two cloned lines of EC cells. However, when EC cells are induced to differentiate into large flat endodern-like cells (END cells), EGF receptors increase in number reaching a plateau in 6 to 8 days. At 8 to 10 days after induction, END cells multiply very slowly, but when EGF is added (3 x 10(-10) M) to the medium, cell division is stimulated and a further change in morphology occurs. This letter describes the binding characteristics and numbers of the EGF receptors on EC and END cells and shows that exogenous retinoic acid increases the numbers of EGF receptors on END cells. We were unable to find endogenous competing factors produced by EC cells. Such factors could account for the lack of detectable binding of EGF on these cells. As EC cells differentiate to END cells, so the ability of the cells to form tumours is reduced. Since this change is accompanied by an increase in the number of EGF receptors there may be a relationship between these two events.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Epidermal Growth Factor; Mice; Neoplasms, Experimental; Peptides; Receptors, Cell Surface; Teratoma; Tretinoin

Topics: Animals; Cell Adhesion; Cell Aggregation; Cell Differentiation; Cell Line; Fibronectins; Glucosamine; Membrane Proteins; Mice; Plasminogen Activators; Teratoma; Tretinoin

Topics: Carrier Proteins; Cell Differentiation; Cell Line; Cytosol; Structure-Activity Relationship; Teratoma; Tretinoin; Vitamin A

Embryonal carcinoma cells, the stem cells of teratocarcinomas, usually undergo extensive differentiation in vivo and in vitro to a wide variety of cell types. There exist, however, several embryonal carcinoma cell lines that have almost completely lost the capacity to differentiate, so that the cells are propagated primarily as the stem cells. Using one such cell line, F9, we have found that retinoic acid at concentrations as low as 10(-9) M induces multiple phenotypic changes in the cultures in vitro. These changes include morphological alteration at the resolution of the light microscope, elevated levels of plasminogen activator production, sensitivity to cyclic AMP compounds and increased synthesis of collagen-like proteins. The nature of these changes, as well as their independence of the continued presence of retinoic acid, are consistent with the proposition that retinoic acid induces differentiation of embryonal carcinoma cells into endoderm.

Topics: Acetylglucosaminidase; Alkaline Phosphatase; Bucladesine; Cell Differentiation; Cell Line; Collagen; Endoderm; Enzyme Induction; Neoplasms, Experimental; Plasminogen Activators; Teratoma; Tretinoin; Vitamin A