Embryonal Carcinoma Cell Line F9 Research Articles

Involvement of PGC7 and UHRF1 in the regulation of DNA methylation of the IG-DMR in the imprinted Dlk1-Dio3 locus.

The gene dosage at the imprinted Dlk1-Dio3 locus is critical for cell growth and development. A relatively high gene expression within the Dlk1-Dio3 region, especially the active expression of Gtl2, has been identified as the only reliable marker for cell pluripotency. The DNA methylation state of the IG-DNA methylated regions (DMR), which is located upstream of the Gtl2 gene, dominantly contributes to the control of gene expression in the Dlk1-Dio3 locus. However, the precise mechanism underlying the regulation of DNA methylation in the IG-DMR remains largely unknown. Here, we use the F9 embryonal carcinoma cell line, a low pluripotent cell model, to identify the mechanism responsible for DNA methylation in the IG-DMR, and find that the interaction of PGC7 with UHRF1 is involved in maintaining DNA methylation and inducing DNA hypermethylation in the IG-DMR region. PGC7 and UHRF1 cooperatively bind in the IG-DMR to regulate the methylation of DNA and histones in this imprinted region. PGC7 promotes the recruitment of DNMT1 by UHRF1 to maintain DNA methylation in the IG-DMR locus. The interaction between PGC7 and UHRF1 strengthens their binding to H3K9me3 and leads to further enrichment of H3K9me3 in the IG-DMR by recruiting the specific histone methyltransferase SETDB1. Consequently, the abundance of H3K9me3 promotes DNMT3A to bind to the IG-DMR and increases DNA methylation level in this region. In summary, we propose a new mechanism of DNA methylation regulation in the IG-DMR locus and provide further insight into the understanding of the difference in Gtl2 expression levels between high and low pluripotent cells.

Asymmetric DNA methylation between sister chromatids of metaphase chromosomes in mouse embryos upon bisphenol A action

Earlier we showed that asymmetric methylation of sister chromatids (AMSC) was a specific characteristic of differentiation potency, and supposed that AMSC could be a useful marker of environmental impact connected with differentiation and/or dedifferentiation. Here we investigated the level of AMSC in chromosomes and the nuclei methylation in mouse preimplantation and postimplantation embryos, in comparison with the undifferentiated cells of mouse embryonal carcinoma cell line F9, and human differentiated HEK293 cells upon BPA influence. We found that exposure of mouse preimplantation embryos to BPA caused a significant decrease in the level of AMSC in chromosomes and the nuclei methylation. The BPA exposure of potentially differentiating F9 cells had no any influence on DNA methylation in nuclei but significantly decreased the number of AMSC. The level of DNA methylation and AMSC in HEK293 cells were not also changed. These data indicate that BPA exerts significant influence on differentiating and potentially differentiable cells. The most sensitive BPA targets are preimplantation embryos and stem cells.

Usefulness of a Non-invasive Reporter System for Monitoring Reprogramming State in Pig Cells: Results of a Cell Fusion Experiment

Dedifferentiation of differentiated cells such as fibroblasts into pluripotent stem cells, so-called iPS cells, was first reported by Yamanaka et al., who successfully employed retroviral gene delivery of four stem-cell-specific transcription factors (Oct-3/4, Klf4, Sox2 and c-myc). Despite the mouse system in which an Oct-3/4 or Nanog promoter-based reporter system has already been established, there is no useful system in pigs for reporting the reprogramming state of gene-engineered cells. In this study, we constructed a pOEIN plasmid carrying a ca. 5.4-kb mouse Oct-3/4 promoter linked to the EGFP cDNA and neomycin expression unit and produced a porcine embryonic cell line stably incorporating it in the genome. Cell fusion with mouse embryonal carcinoma cell line F9 resulted in generation of colonies with distinct EGFP-derived fluorescence around 14 days after fusion. RT-PCR using these colonies also confirmed expression of endogenous porcine pluripotency-specific Oct-3/4, Sox2 and Stat3 mRNA. These findings suggest that mouse-derived components are sufficient to induce dedifferentiation of differentiated pig cells and also that reprogramming proceeds gradually. The present non-invasive reporter system will be useful to better define the reprogramming mechanism and/or to identify novel reprogramming molecules in the pig.

Coordinate Gα13 and Wnt6-β-catenin signaling in F9 embryonal carcinoma cells is required for primitive endoderm differentiation

The mouse F9 embryonal carcinoma cell line is ideally suited to study the epithelial-to-mesenchymal transition accompanying the differentiation of primitive to parietal extraembryonic endoderm. In F9 cells, the application of exogenous agents including retinoic acid or activation of signal transduction cascades downstream of G-proteins triggers widespread changes in gene expression and leads to the formation of primitive endoderm. The epithelial-to-mesenchymal transition is completed and parietal endoderm develops as of result of increasing PKA activity in primitive endoderm cells. Expression of a constitutively active form of Galpha13(Q226L) is sufficient to induce F9 cells into parietal endoderm and a model is emerging that a signaling axis linking G-protein signaling to RhoA and the ERM protein moesin is required for differentiation. In this study, we found that expression of either p115RhoGEF or a constitutively active, GTPase-deficient form of RhoA(L63) promoted primitive, but not parietal, endoderm formation. The overexpression of Galpha13(Q226L) or p115RhoGEF, but not Rho(L63), caused beta-catenin to translocate to the nucleus. Surprisingly, the stimulation of the Wnt-beta-catenin pathway was accompanied by nuclear beta-catenin and primitive endoderm formation, even when a dominant negative was used to block the signaling axis at the level of p115RhoGEF or when ROCK activity was inhibited using the pharmacological agent Y-27632. Together, results indicate that the coordinate signaling by two independent pathways, one involving canonical Wnt-beta-catenin activation of target genes and the other with Galpha13 signaling to ERM proteins to modulate cytoarchitectural changes, is required during the retinoic acid induced differentiation of F9 cells to primitive endoderm.

Roles of the Nanog protein in murine F9 embryonal carcinoma cells and their endoderm-differentiated counterparts

Nanog is a recently discovered homeodomain transcription factor that sustains the pluripotency of embryonic stem (ES) cells and blocks their differentiation into endoderm. The murine F9 embryonal carcinoma cell line is a well-documented model system for endoderm cell lineage differentiation. Here, we examined the function of Nanog in F9 cell endoderm differentiation. Over-expression of Nanog returns the F9 cells to the early status of ES cells and represses the differentiation of primitive endoderm and parietal endoderm in F9 cells, whereas it has no effect on the differentiation of visceral endoderm. In contrast, the expression of C-terminal domain-truncated Nanog spontaneously promotes endoderm differentiation in F9 cells. These data suggest that Nanog is required to sustain the proper undifferentiated status of F9 cells, and the C-terminal domain of Nanog transduces the most effects in repressing primitive endoderm and parietal endoderm differentiation in F9 cells.

Laminin binding protein, 34/67 laminin receptor, carries stage-specific embryonic antigen-4 epitope defined by monoclonal antibody Raft.2

We previously produced monoclonal antibodies against the detergent-insoluble microdomain, i.e., the raft microdomain, of the human renal cancer cell line ACHN. Raft.2, one of these monoclonal antibodies, recognizes sialosyl globopentaosylceramide, which has the stage-specific embryonic antigen (SSEA)-4 epitope. Although the mouse embryonal carcinoma (EC) cell line F9 does not express SSEA-4, some F9 cells stained with Raft.2. Western analysis and matrix-assisted laser desorption ionization-time of flight mass spectrometry identified the Raft.2 binding molecule as laminin binding protein (LBP), i.e., 34/67 laminin receptor. Weak acid treatment or digestion with Clostridium perfringens sialidase reduced Raft.2 binding to LBP on nitrocellulose sheets and [ 14C]galactose was incorporated into LBP, indicating LBP to have a sialylated carbohydrate moiety. Subcellular localization analysis by sucrose density-gradient centrifugation and examination by confocal microscopy revealed LBP to be localized on the outer surface of the plasma membrane. An SSEA-4-positive human EC cell line, NCR-G3 cells, also expressed Raft.2-binding LBP.

Retinoids and retinoic acid receptor in cancer

Retinoids are vitamin A derivatives that exert major effects on growth control, epithelial differentiation and embryonic development and, accordingly, are highly teratogenic. In HL60 myelo-monocytic or F9 embryonal carcinoma cell lines, retinoic acid (RA) turns a transformed cell line into terminally differentiated non-proliferating cells. It was only in the mid 1980s that the cloning of the retinoic acid receptors gave a molecular basis to the pleiotropic action of these compound. Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are nuclear receptors, like those for steroids or thyroid hormones. They are hormone-activated transcription factors that bind to the promoters of their target genes as RAR/RXR heterodimers and repress or activate their transcription depending on the presence of the hormone. Both retinoic acid and its three RAR (α, β, γ) receptors have close links to cancer. Retinoids have significant antitumour activities in several clinical settings, in particular in cervical, skin, head and neck cancers, neuroblastoma and oral leukoplakia, where high doses have yielded significant improvements. A better understanding of the function targeted in the cancer cell and the receptor involved may lead to more specific drugs. Alterations of RARβ expression were reported in a variety of tumors and have suggested that it may act as a tumour suppressor. The clearest illustration of how alterations in RAR structure are linked to cancer is acute promyelocytic leukemia (APL), where a chromosome translocation fuses the PML gene to that of RARα, creating a PML/RARα fusion protein. Strikingly, in contrast, the previous malignancies that are RA-responsive, APL cells undergo terminal differentiation ex vivo or in vivo, similar to HL60 or F9 cells. APL therefore became the paradigm for differentiation therapy. Remarkably, APL is also exquisitely sensitive to arsenic trioxide and both RA and arsenic directly target the PML/RARα fusion protein, identifying the first example of oncogene-targeted therapies. Animal models have helped model the effects of these drugs and provide invaluable preclinical systems for one of the best understood malignancies to date.

Retinoic acid stimulates erythropoietin gene transcription in embryonal carcinoma cells through the direct repeat of a steroid/thyroid hormone receptor response element half-site in the hypoxia-response enhancer

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

Retinoic acid stimulates erythropoietin gene transcription in embryonal carcinoma cells through the direct repeat of a steroid/thyroid hormone receptor response element half-site in the hypoxia-response enhancer

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

Regulation of gp330/megalin expression by vitamins A and D.

A membrane-bound 550-kD Ca2+-binding glycoprotein belonging to the low-density lipoprotein (LDL) receptor superfamily has recently been identified as a putative calcium-sensing molecule. This molecule, known as gp330/megalin, is among several tissues present in the proximal tubule, parathyroid and placental cytotrophoblasts, in which a Ca2+-sensing function has been demonstrated. Regulation of mRNA and protein expression of gp330/megalin were studied in a recently established cell line derived from rat kidney proximal tubule cells (IRPTCs), in human JEG-3 cells and in the mouse embryonal carcinoma cell line F9. In IRPTCs, quantification of mRNA and protein expression demonstrated two- to five-fold increases after addition of 10(-6) mol L(-1) all-trans-retinoic acid, 9-cis-retinoic acid or 1,25-dihydroxyvitamin D3, alone or in combination. Similarly, an increase in gp330/megalin mRNA expression was seen in JEG-3 cells cultured with vitamin D and retinoids, as well as when F9 cells were differentiated by incubation with retinoic acid and cAMP. The IRPTCs were immortalized by viral infection with the SV40 genome preceded by a temperature-sensitive promoter. Thus, by culture of the cells at 41 degrees C, SV40 genome transcription is inhibited and the IRPTC phenotype is reversed towards non-infected proximal tubule cells. At 41 degrees C, gp330/megalin mRNA expression was significantly increased compared with cells incubated at 34 degrees C. The results indicate a correlation between exposure to retinoic acid or vitamin D or induction of cell differentiation (by retinoic acid/cAMP in F9 cells or inhibition of SV40 transcription in IRPTCs) and an increase in gp330/megalin protein and mRNA expression.

An alternative splice variant of the tissue specific transcription factor HNF4alpha predominates in undifferentiated murine cell types.

The transcription factor hepatocyte nuclear factor 4alpha (HNF4alpha) is a tissue specific transcription factor mainly expressed in the liver, kidney, intestine and the endocrine pancreas, but is also an essential regulator for early embryonic events. Based on its protein structure HNF4alpha is classified as an orphan member of the nuclear receptor superfamily. Comparing HNF4alpha transcription factors in the differentiated and dedifferentiated murine hepatocyte cell line MHSV-12 we identified in dedifferentiated cells the novel splice variant HNF4alpha7. This variant is characterized by an alternative first exon and has a lower transactivation potential in transient transfection assays using HNF4 dependent reporter genes. HNF4alpha7 mRNA and the corresponding protein are expressed in the undifferentiated pluripotent embryonal carcinoma cell line F9, whereas HNF4alpha1 only appears after differentiation of F9 cells to visceral endoderm. HNF4alpha7 mRNA is also found in totipotent embryonic stem cells. However, the function of HNF4alpha7 seems not to be restricted to embryonic cells as the HNF4alpha7 mRNA is also present in adult tissues, most notably the stomach. All these features suggest that the presence of distinct splice variants of HNF4alpha modulates the activity of HNF4alphain a cell type specific way.

A parallel association between differentiation and induction of galectin-1, and inhibition of galectin-3 by retinoic acid in mouse embryonal carcinoma F9 cells.

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

Modulation of α6/β1 Integrin Expression during Differentiation of F9 Murine Embryonal Carcinoma Cells to Parietal Endoderm

In vitrodifferentiation of the murine embryonal carcinoma (EC) cell line F9 parallels that of the early blastocyst, where visceral (VE) and parietal endoderm (PE) diverge from a common precursor, the primitive endoderm. This differentiation pathway is induced by retinoic acid (RA) and dibutyryl cyclicAMP (dcAMP) and is accompanied by progressive and dramatic changes in cell morphology and functions. Within 7 days of treatment the cells organize their cytoskeleton and synthesize large amounts of extracellular matrix proteins, becoming fully differentiated migratory cells; all these changes are likely to involve integrins expression and organization. We have investigated the changes in β1 integrin expression, its maturation, and organization on the cell surface in association with α6, during the transition from undifferentiated F9 stem cells to migrating PE cells. By Western blotting and immunoprecipitation we showed a gradual decrease in the amount of the β1 subunit on the cell surface and a parallel progressive accumulation of immature protein, indicating that the control of β1 expression during F9 cells differentiation occurs first at posttranslational level and then at the level of transcription. Moreover, the induction of differentiation produces a marked decrease of α6B and its association to a high molecular weight protein, while α6A level increases. By immunofluorescence we found that upon differentiation there is a relocation of the β1 and α6B integrin subunits from cell–cell contacts to focal contacts where they colocalize with vinculin. On the contrary α6A, weakly present in F9 stem cells, is present in the focal contacts of PE cells and along the stress fibers. We suggest different roles for the two α6 isoforms.

Positive regulation of the vHNF1 promoter by the orphan receptors COUP-TF1/Ear3 and COUP-TFII/Arp1.

vHNF1 (also termed HNF1 beta) is a member of the hepatocyte nuclear fa ctor 1 (HNF1; also termed HNF1 alpha) family of homeodomain-containing transcription factors that interact with a sequence motif found in the regulatory regions of a large number of genes expressed mainly in the liver. It has been suggested that vHNF1 plays a role in early differentiation of specialized epithelia of several endoderm- and mesoderm-derived organs, with HNF1 playing a role in later stages. In support of this idea, expression of vHNF1 but not HNF1 is induced upon treatment of the embryonal carcinoma cell line F9 with retinoic acid. We have cloned and analyzed the vHNF1 promoter to gain a better understanding of the regulation of vHNF1 expression and how it relates to the expression of HNF1. We have identified five sites of DNA-protein interaction within the first 260 bp upstream of the transcription start site, which involve at least three different families of transcription factors. Two sites, a distal DR-1 motif and a proximal octamer motif, are the most important for promoter activity. The DR-1 motif interacts with several members of the steroid hormone receptor superfamily including HNF4, COUP-TFI/Ear3, COUP-TFII/Arp1, and RAR alpha/RXR alpha heterodimers. The vHNF1 promoter is transactivated by COUP-TFI/Ear3 and COUP-TFII/Arp1 and, unlike the HNF1 promoter, is virtually unaffected by HNF4. Interestingly, the proximal octamer site and not the DR-1 site is required for COUP-TFI/Ear3 and COUP-TFII/Arp1 transactivation of the vHNF1 promoter. COUP-TFI/Ear3 does not bind directly to this proximal octamer site. We present evidence of an interaction between COUP-TFI/Ear3 and the octamer-binding proteins in vitro and in the cell, suggesting that COUP-TFI and COUP-TFII activate the vHNF1 promoter via an indirect mechanism.

Structural and biological consequences of increased vimentin expression in simple epithelial cell types

Cytoskeletal intermediate filaments (IFs) constitute a diverse family of proteins whose members are expressed in tissue-specific patterns. Although vimentin IFs are normally restricted to mesenchyme, a variety of cell types express vimentin alone or together with cell-specific IFs during growth, differentiation, and neoplasia. In this study, we have investigated the influence of increased vimentin expression on the simple epithelial cell phenotype. An expression vector encoding a human vimentin cDNA was transfected into murine HR9 endoderm and F9 embryonal carcinoma cell lines, which serve as models for early extraembryonic epithelial differentiation. Stable clones that expressed varying levels of human vimentin were characterized by human vimentin were characterized by immunofluorescence and biochemical analysis. A relatively high level of vimentin expression in HR9 and differentiated F9 epithelial cells resulted in aberrant vimentin structures with co-collapse of keratin K8/K18 filaments and lowered amounts of keratin protein. In F9 epithelial cells, the desmosomal proteins DP I/II did not appear to localize to cell surface desmosome s but rather but rather co-aggregated with the perturbed IFs. Although overall cell morphology was not dramatically altered, individual nuclei were distorted by excess intracellular vimentin. Furthermore, cell proliferation as well as the cell spreading response time were slowed. Ther appears to be a threshold effect regarding overall vimentin levels as cells that expressed lower amounts of the human vimentin exhibited no obvious structural nor biological effects. Our results demonstrate that wild-type vimentin can act as a "mutant" protein when present at high intracellular levels, inducing a variety of phenotypic changes.

Detection of Heat Shock Element-Binding Activities by Gel Shift Assay During Mouse Preimplantation Development

Heat shock gene expression is regulated by highly conserved sequence elements (HSE for "heat shock elements"). Some of heat shock genes display an atypical expression during preimplantation mouse development. We have examined the profile of HSE-binding activities (HSE-BA) in matured ovulated oocytes and during the preimplantation development by gel shift assay and quantified the data by PhosphorImager. In each of our experiments, the F9 embryonal carcinoma cell line that contains both constitutive and heat-induced activity has been used as a control. We determine the number of oocytes or embryos required to get reproducible signals and accurate quantification by PhosphorImager. Oocytes, one-cell, and two-cell embryos respond to heat shock by inducing a strong HSE-BA. At the four-cell stage, no HSE-BA can be induced by heat shock, which suggests that noninducibility of heat shock genes at this stage (when the general mechanism of transcription is well established) might result from a defect in HSF or in the mechanism of HSF activation. A progressive reappearance of the ability to induce HSE-BA by stress is observed between the eight-cell stage and the blastocyst stage, and this parallels the appearance of heat shock gene inducibility. Matured ovulated oocytes and the first cleavage stages of embryos do not contain any HSE-BA at normal temperature but we observed a HSE-BA at normal temperature at the morula stage, which is increased at the blastocyst stage. These data, which, to our knowlegde, for the first time describe the profile of a DNA-binding activity during the mouse preimplantation development, could serve as a basis for the study of other transcription factors during early embryogenesis.

Transcripts and CpG islands associated with the pro-opiomelanocortin gene and other neurally expressed genes

DNA sequences of vertebrate genes which code for neural or neuroendocrine peptides were analysed in terms of CpG dinucleotide distribution and G+C content. The vast majority of the genes were found to contain a region with the sequence characteristics of a CpG island surrounding the 5' end. In mammalian species, the gene which codes for the neuroendocrine polypeptide pro-opiomelanocortin (POMC) was shown to be associated with two separate CpG islands: a 5' CpG island which surrounds the POMC transcription start site and a 3' CpG island which lies approximately 5 kb downstream, encompassing the third exon of POMC. Short POMC-related transcripts, known to be transcribed in the germline, were found to initiate from a promoter within the 3' CpG island. The start sites of the short POMC-related transcripts in mouse testis were mapped to the region coding for gamma MSH in exon 3, in a similar location to transcription start sites identified in other mammalian POMC genes. Similar short POMC-related transcripts were identified in both the mouse F9 embryonal carcinoma cell line and mouse embryonic stem cells, suggesting that transcription initiating within the third exon may occur very early in development. No short transcripts were detected by Northern blot hybridization in either Xenopus laevis testis or oocyte poly(A)+ RNA extracts. The Xenopus laevis POMC genes, A and B, were associated with neither a 5' nor a 3' CpG island. Hence, the presence of a 5' CpG island is not required for production of full-length transcripts from the Xenopus laevis POMC gene, but the presence of a 3' CpG island may be required for transcription to occur from the third exon.

Application of poly(A) +RNA patterns method for searching of differentially expressed genes

Poly(A) +RNA composition differences for normal, fetal and cirrhotic human liver before and after retinoic acid-induced differentiation of the F9 embryonal carcinoma cell line were analyzed by a novel poly(A) +RNA patterns method. The method is based on the polyacrylamide gel electrophoretic analysis of short cDNA termination products, synthesized by reverse transcriptase using poly(A) +RNA as a template, a set of short 5'-end labeled primers, three natural and one terminator deoxyribonucleotide. A number of known differentially expressed genes and some unknown ones were then identified by direct sequencing of the differentially represented bands excised from a gel and searching a complementary mRNA target sites in GenBank database.

Expression of chicken vinculin complements the adhesion-defective phenotype of a mutant mouse F9 embryonal carcinoma cell.

A mutant cell line, derived from the mouse embryonal carcinoma cell line F9, is defective in cell-cell adhesion (compaction) and in cell-substrate adhesion. We have previously shown that neither uvomorulin (E-cadherin) nor integrins are responsible for the mutant phenotype (Calogero, A., M. Samuels, T. Darland, S. A. Edwards, R. Kemler, and E. D. Adamson. 1991. Dev. Biol. 146:499-508). Several cytoskeleton proteins were assayed and only vinculin was found to be absent in mutant (5.51) cells. A chicken vinculin expression vector was transfected into the 5.51 cells together with a neomycin-resistance vector. Clones that were adherent to the substrate were selected in medium containing G418. Two clones, 5.51Vin3 and Vin4, were analyzed by Nomarski differential interference contrast and laser confocal microscopy as well as by biochemical and molecular biological techniques. Both clones adhered well to substrates and both exhibited F-actin stress fibers with vinculin localized at stress fiber tips in focal contacts. This was in marked contrast to 5.51 parental cells, which had no stress fibers and no vinculin. The mutant and complemented F9 cell lines will be useful models for examining the complex interactions between cytoskeletal and cell adhesion proteins.

Induction of nuclear lamins A/C during in vitro-induced differentiation of F9 and P19 embryonal carcinoma cells

Lamin B is the major constituent of the nuclear lamina of undifferentiated mouse embryonal carcinoma cells. The full complement of the three major lamins A, B, and C, found in somatic mammalian cells, is acquired after induction of differentiation in vitro by certain drugs. In this study we have examined the time course of lamin A/C expression in the two embryonal carcinoma cell lines F9 and P19. We show here that lamins A/C are detectable in these cell lines, at the mRNA level and at the protein level, after 3 days of growth in media containing retinoic acid or retinoic acid + 3-isobutyl-1-methylxanthine. The data reported here indicate that the expression of lamins A/C is mainly regulated at the transcriptional level and occurs when the cells, by morphological and functional criteria, have differentiated along their developmental pathway.