Differentiation Of F9 Cells Research Articles

The Promoter of the H1° Histone Gene contains a DNA Element bound by Retinoic Acid Receptors

Retinoic-acid mediated differentiation of F9 cells is accompanied by an increased transcription of the histone H1° gene. This increase is an early response after addition of retinoic acid, suggesting a direct effect of the hormone on transcription of the gene. We show now that the promoter of histone H1° contains a DNA element, localized 531 base-pairs upstream of the cap site, that is composed of a direct repeat of the sequence PuGGTCA separated by eight base-pairs. This element confers retinoic acid responsiveness to a heterologous thymidine kinase promoter in F9 and HeLa cells. Furthermore, the element forms retarded complexes not only with bacterially expressed retinoic acid receptors (RARs) and retinoid X receptors (RXRs), but also with endogenous F9 receptors. Our results suggest therefore that retinoic acid receptors can control the expression of a chromatin structural gene the expression of which is associated with a differentiated phenotype.

Ultraviolet Irradiation, although It Activates the Transcription Factor AP-1 in F9 Teratocarcinoma Stem Cells, Does Not Induce the Full Complement of Differentiation-Associated Genes

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

Differentiation‐induced increase in Clostridium botulinum C3 exoenzyme‐catalyzed ADP‐ribosylation of the small GTP‐binding protein Rho

The specific [32P]ADP-ribosylation by Clostridium botulinum exoenzyme C3 was used to study differentiation-dependent changes in the regulation of the low-molecular-mass GTP-binding protein Rho. Differentiation of F9 teratocarcinoma cells to neuronal-like cells by treatment with retinoic acid and dibutyryl-adenosine 3',5'-monophosphate [(Bt)2cAMP] increased the C3-catalyzed ADP-ribosylation of RhoA proteins in cytosolic and membrane fractions by about threefold and sixfold, respectively. Phenotypical differentiation of F9 cells was not required for increase in ADP-ribosylation. Increase in ADP-ribosylation after (Bt)2cAMP and retinoic acid treatments was blocked by cycloheximide, indicating the requirement of protein biosynthesis. As deduced from specific rho mRNA amounts and from Western analysis with a monoclonal RhoA antibody, the stimulation in the [32P]ADP-ribosylation of Rho was not caused by an increased de-novo synthesis of Rho proteins. GDP increased the ADP-ribosylation of membrane-associated Rho from non-differentiated, but not from differentiated F9 cells. GTP[S] decreased ADP-ribosylation of membranous Rho from differentiated and much less from non-differentiated F9 cells. Differentiation-dependent increase in ADP-ribosylation of cytosolic Rho was reversed by protein phosphatase type-1. Treatment with SDS (0.01%) which releases Rho from complexation with guanine nucleotide dissociation inhibitor, increased ADP-ribosylation both in differentiated and non-differentiated cells, indicating no differentiation-specific change of such complexes. In total, our data indicate that the induction of the differentiation process in F9 cells is accompanied by changes in the regulation of cytosolic and membrane-associated Rho proteins.

Perturbing cell surface beta-(1,4)-galactosyltransferase on F9 embryonal carcinoma cells arrests cell growth and induces laminin synthesis.

Cell growth and differentiation are influenced by intercellular contact, suggesting that cell adhesion molecules may be instrumental in triggering these events. F9 embryonal carcinoma cells are an ideal system in which to examine the function of cell adhesion molecules in growth and differentiation, since the relevant cell adhesion molecules and differentiation markers are well defined. Intercellular adhesion in F9 cells is mediated by uvomorulin, or E-cadherin, and cell surface beta-(1,4)-galactosyltransferase. Since previous studies suggested that neither F9 cell growth nor differentiation is directly dependent on uvomorulin function, in this study we examined whether cell surface galactosyltransferase plays any role in F9 cell growth or differentiation. A variety of galactosyltransferase perturbants, including anti-galactosyltransferase antibodies, UDPgalactose, and the substrate modifier protein alpha-lactalbumin, inhibited the growth of F9 cells, whereas control reagents did not. To examine this in more detail, we analyzed the effects of perturbing surface galactosyltransferase on progression through the F9 cell cycle. Anti-galactosyltransferase IgG treatment inhibited ornithine decarboxylase activity and lengthened the F9 cell cycle during G1 and G2, the latter mimicking the effects of retinoic acid, a reagent known to prolong the F9 cell cycle and induce differentiation. In contrast, anti-uvomorulin antibodies had no effect on F9 cell growth, ornithine decarboxylase activity, or progression through the cell cycle. Furthermore, perturbation of surface galactosyltransferase adhesions in F9 cell aggregates induced precocious F9 cell differentiation, as assayed by increased laminin synthesis, whereas control reagents had no effect. Thus, perturbing surface galactosyltransferase adhesions in F9 cells both decreases growth and stimulates synthesis of laminin. These results imply that interactions between surface galactosyltransferase and its oligosaccharide ligand during cell adhesion may affect the normal growth-regulatory and differentiation-inducing signals, as is seen, in part, during treatment with retinoic acid.

Isolation and characterization of retinoic acid-inducible cDNA clones in F9 cells: One of the early inducible clones encodes a novel protein sharing several highly homologous regions with a Drosophila Polyhomeotic protein

Abstract To elucidate regulatory mechanisms triggering early mammalian differentiation, 17 retinoic acid (RA)-inducible clones were isolated from 1.4·10 5 plaques of cDNA libraries prepared from mouse embryonal carcinoma F9 cells, using the differential plaque hybridization method. Partial nucleotide sequences of these clones demonstrated that ten clones correspond to known genes. Interestingly, only 2 of the 17 clones are among the previously documented up-regulated genes. Therefore, there are many more unidentified genes up-regulated in the course of RA-induced differentiation of F9 cells. As RNAs hybridizable with one of the seven unidentified clones were induced in F9 cells after 3 h of RA treatment, we chose this ‘Rae-28’ clone as being representative of developmentally up-regulated unidentified clones and its properties were characterized. We determined the Rae-28 cDNA sequence and deduced the RAE-28 protein structure. The deduced RAE-28 protein shared several motifs and highly homologous regions with a Drosophila polyhomeotic protein. As the Drosophila polyhomeotic gene is involved in regulating morphogenesis, the rae-28 gene may participate in regulating early mammalian development.

F9 Cells Can be Differentiated toward Two Distinct, Mutually Exclusive Pathways by Retinoic Acid and Sodium Butyrate: (teratocarcinoma/differentiation/retinoic acid/sodium butyrate).

Both retinoic acid (RA) and sodium butyrate (NaB) induce differentiation in embryonal carcinoma F9 cells. Phenotypic changes caused by RA are irreversible, whereas those of NaB are rapid and reversible. In this study, we investigated the effects of combinations of these two agents on F9 cell differentiation and showed that RA had no effect on the cells induced to differentiate with NaB and vice versa. Thus, F9 cells are induced to differentiate along two distinct pathways which are mutually exclusive.

Exemption of Satellite DNA from Demethylation in Immortalized Differentiated Derivatives of F9 Mouse Embryonal Carcinoma Cells

DNA methylation in F9 embryonal carcinoma cells at various stages of retinoic acid-induced differentiation was compared to that in immortalized differentiated derivatives of this cell line. Different repetitive sequences, such as L1 LINE, GAPDH pseudogenes, and major and minor satellite DNA, lost their methylation to similar extents during F9 differentiation into parietal endoderm cells. In the immortalized derivatives 8a and P1, which phenotypically resemble F9 cells at intermediate stages of differentiation, methylation patterns diverged: Methylation of L1 and GAPDH sequences was strongly and moderately diminished, respectively, whereas methylation of satellite DNA was almost as high as that in stem cells. P19 embryonal carcinoma cells and D3 embryonic stem cells possessed methylation patterns similar to those of F9 stem cells. In immortalized cell lines with differentiated phenotypes derived from P19, methylation was diminished uniformly throughout the genome, but the overall level of methylation remained higher than that in terminally differentiated F9 cells. Treatment with the methylation inhibitor deoxyazacytidine halted the proliferation of 8a, P1, and F9 cells and elicited marked changes in morphology. However, markers of differentiation were not induced in F9 cells. The finding that all immortalized differentiated derivatives of embryonal carcinoma cells retained a higher level of DNA methylation—at least in parts of their genomes—than did terminally differentiated cells may indicate a function of demethylation of DNA and of satellite sequences in particular in terminal differentiation of extraembryonic tissues.

A retinoic acid-inducible GATA-binding protein binds to the regulatory region of J6 serpin gene.

Transcription of the J6 gene (a member of the serpin family) is induced in murine F9 teratocarcinoma cells 24-48 h after retinoic acid (RA) treatment. Previously, we have identified a region of the J6 5'-flanking region (-1050 to -738) that is involved in regulating transcription of this gene. In this report, we show that a RA-induced nuclear protein in the F9 cell extract recognizes the GAGATAG sequence which is repeated four times in this regulatory region of the J6 gene. The kinetics of RA induction of the GAGATAG-binding protein correlates with that of J6 mRNA, suggesting that the GAGATAG-binding protein may be involved in the transactivation of the J6 gene in RA-treated F9 cells. Competition experiments demonstrate further that the RA- induced GAGATAG-binding protein is related to the transcription factors GATA-1 and GATA-2. Furthermore, insertion of the RA regulatory region of the J6 gene into a thymidine kinase promoter/chloramphenicol acetyltransferase expression vector causes an increase in chloramphenicol acetyltransferase expression by 5-8-fold in human HeLa cells when co-transfected with human GATA-2 or GATA-3 expression vectors. This suggests that the J6 gene is likely to be transactivated by the GATA-2, GATA-3, or related transcription factor, which is activated by retinoic acid during F9 cell differentiation.

NF-kappa B-independent activation of beta-interferon expression in mouse F9 embryonal carcinoma cells.

We have examined the behaviour of the beta-interferon promoter in mouse F9 embryonal carcinoma cells. In undifferentiated cells, the beta-interferon promoter is not responsive to dsRNA or to Sendai virus. In cells stimulated to differentiate into parietal endoderm by treatment with retinoic acid, the beta-interferon promoter responds to both inducers, but only Sendai virus can activate the transcription factor NF-kappa B previously thought to be essential for beta-interferon induction. Differentiated F9 cells therefore present an unprecedented situation in which induction of the beta-interferon gene does not require NF-kappa B. In addition to these differences, induction by dsRNA, but not by Sendai virus, is significantly enhanced by a pretreatment with interferon (priming). These observations suggest that paramyxo-viruses can participate in beta-interferon induction in a manner that is distinct from a simple generator of dsRNA. Analysis of the promoter requirements for induction in differentiated F9 cells suggests that induction is brought about by a novel mechanism using the currently identified regulatory domains.

The Expression of Retinoid X Receptor Genes Is Regulated by All- trans-and 9- cis-Retinoic Acid in F9 Teratocarcinoma Cells

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

Radiation-induced apoptosis in F9 teratocarcinoma cells.

We have found that F9 murine teratocarcinoma cells undergo morphological changes and internucleosomal DNA fragmentation characteristic of apoptosis after exposure to ionizing radiation. We studied the time course, radiation dose-response, and the effects of protein and RNA synthesis inhibitors on this process. The response is dose dependent in the range 2-12 Gy. Internucleosomal DNA fragmentation can be detected as early as 6 h postirradiation and is maximal by 48 h. Cycloheximide, a protein synthesis inhibitor, and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an RNA synthesis inhibitor, both induced internucleosomal DNA fragmentation in the unirradiated cells and enhanced radiation-induced DNA fragmentation. F9 cells can be induced to differentiate into cells resembling endoderm with retinoic acid. After irradiation, differentiated F9 cells exhibit less DNA fragmentation than stem cells. This indicates that ionizing radiation can induce apoptosis in non-lymphoid tumours. We suggest that embryonic tumour cells may be particularly susceptible to agents that induce apoptosis.

Activin Receptor Type II Gene Expression is Induced During Embryonic Development and Differentiation of Murine F9 Embryonal Carcinoma Cells: (activin receptor/development/endoderm/embryonal carcinoma cell/retinoic acid).

The functional role of activins in mesoderm induction has been shown in early amphibian embryos; however, its role in early mammalian embryogenesis is still unknown. The protein and mRNA for activin subunits are present in mouse preimplantation embryos. In this study we report on the expression of activin receptor type II (ActR-II) gene in pre- and post-implantation mouse embryos and the regulation of ActR-II mRNA in F9 embryonal carcinoma cells that served as a model for murine embryonic development. The results show that ActR-II gene is expressed in ovulated mature oocytes, becomes decreased at 2-celll stage, and is substantially activated in day-6 embryo at the stage when ectoderm and endoderm are formed. When F9 cells are treated with retinoic acid (RA) and undergo endodermal differentiation, ActR-II mRNA is induced. The 6.0 kb transcript is enhanced more than the 3.0 kb transcript. suggesting that these two mRNA species are controlled by different promotors. Addition of cyclic AMP analogue, which further induces differentiation of F9 cells into parietal endoderm, does not further enhance ActR-II gene expression. The effect of RA on ActR-II mRNA levels is direct and does not require ongoing protein synthesis. Furthermore, all-trans-RA is more effective than 9-cis-RA in the induction of ActR-II gene expression. The results of this study demonstrate that the ActR-II gene is activated during endodermal differentiation and suggest that activin action in mammalian embryonic development is mediated through increased activin receptor gene expression.

Lamp-1 does not acquire the large polylactosaminoglycans characteristic of F9 cells

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.

The Induction of jun Genes during the Reversible Changes Induced with Sodium Butyrate on the Differentiation of F9 Cells

Sodium butyrate (NaB) induces the phenotypic changes and differentiation markers in embryonal carcinoma F9 cells (Kosaka et al., Exp. Cell Res. 192, 46, 1991). However, the effects are rapid and reversible and different from those induced with retinoic acid (RA). We examined the expression of jun genes during differentiation induced with NaB and compared it with that of RA induction. Although c-jun expression was induced with RA, rapid and predominant induction of junB expression was specifically observed with NaB, which is regulated by both transcriptional and post-transcriptional mechanisms. These results suggest that the mechanism of differentiation induced with NaB differs from that of RA; the former may be mediated by the junB gene and the latter by c-jun.

Cyclic AMP-Mediated Augmentation of Thrombomodulin Gene Expression: Cell Type-Dependent Usage of Control Regions

We reported that a cell surface thrombin receptor, thrombomodulin (TM), was regulated by cyclic AMP in fibroblasts and in parietal endoderm-like cells derived from F9 embryonal carcinoma cells. In this paper, the genetic basis for augmentation of TM expression by cyclic AMP was studied in F9 and BALB/3T3 cells. Transient expression assays were performed with plasmid constructs containing various 5′ flanking sequences of the TM gene and a reporter gene, chloramphenicol acetyltransferase (CAT). Two regulatory DNA regions, the proximal (-411 to -50) and the distal (-1026 to -850), were located. Interplay of the two regions was suggested using a heterologous thymidine kinase promoter in differentiated F9 cells. Both proximal and distal regions contributed to cyclic AMP-dependent augmentation of CAT expression in differentiated F9 cells, whereas only the proximal region was functional in BALB/3T3 cells. The two cell types responded differently also to a protein synthesis inhibitor, cycloheximide, with respect to TM message accumulation. In BALB/3T3 cells TM message accumulation was refractory to the inhibitor in contrast to that of differentiated F9 cells, which was only partially so. We propose that there are at least two separate genomic DNA regions that regulate cyclic AMP-dependent TM gene expression and that their functions are cell type dependent.

Uvomorulin, LAMP-1, and Laminin Are Substrates for Cell Surface β-1,4-Galactosyltransferase on F9 Embryonal Carcinoma Cells: Comparisons between Wild-Type and Mutant 5.51 att- Cells

Intercellular adhesions in F9 embryonal carcinoma cells are primarily dependent upon two types of cell adhesion molecules: uvomorulin (Um or E-cadherin), which facilitates intercellular adhesion by homophilic binding, and cell surface β-1,4-galactosyltransferase (GalTase), which binds terminal N-acetylglucosamine residues on consociate glycoprotein substrates on adjacent cell surfaces. The variant F9 cell line, 5.51 att-, undergoes initial cell aggregation, but fails to form the mature intercellular adhesions characteristic of wild-type F9 cells. The 5.51 att- cells show reduced Um expression; however, previous studies have shown that reduced levels of Um are not responsible for the att- mutant phenotype. Therefore, in this study we determined whether the att- mutant phenotype was the result of altered expression of surface GalTase or of its consociate glycoprotein substrates. Surface GalTase was found to be expressed normally on 5.51 att- cells. Furthermore, the initial intercellular adhesions characteristic of reaggregating 5.51 att- cells were dependent upon surface GalTase, since anti-GalTase antibodies inhibited 5.51 intercellular adhesion. Preliminary studies showed that the interaction of surface GalTase with its consociate glycoprotein substrates was abnormal on 5.51 att- cells. To define the biochemical basis for this observation, specific glycoprotein substrates for cell surface GalTase were identified and their expression and utilization were compared between wild-type and 5.51 att- cells. In wild-type cells, uvomorulin (Um), lysosome-associated membrane protein-1 (LAMP-1), and laminin (Lm) were shown to be substrates for cell surface GalTase, suggesting that they may participate in GalTase-specific adhesions. Moreover, the interaction between GalTase and these glycoproteins exhibited characteristic changes during retinoic acid-induced F9 cell differentiation. In contrast to that seen on wild-type cells, surface GalTase interaction with Um, LAMP-1, and Lm was atypical on 5.51 att- cells, resulting, in part, from increased synthesis of oligosaccharide substrates for surface GalTase, as determined by size exclusion and lectin affinity chromatography. N -acetylglucosaminyltransferase activity was elevated in 5.51 att- cells and likely responsible for the increased expression of GalTase oligosaccharide substrates. These results suggest that mutant 5.51 att- cells are able to maintain residual intercellular adhesions because of increased expression of oligosaccharide substrates for surface GalTase. The impact of aberrant GalTase-specific adhesions on the ability of 5.51 att- cells to form mature intercellular adhesions and to differentiate in response to retinoic acid is discussed.

Perlecan gene expression precedes laminin gene expression during differentiation of F9 embryonal carcinoma cells.

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

Suppression of tumorigenicity in temperature-sensitive mutants of embryonal carcinoma cells by induction of cell differentiation at non-permissive temperature.

The temperature-sensitive (ts) mutants of cell differentiation derived from the mouse teratocarcinoma cell line F9, when exposed to non-permissive temperature, undergo stem-cell differentiation concomitant with a transient retardation of cell-cycle progression. By incubating these mutant strains at non-permissive temperature, we were able to study the relationship between cell differentiation and tumorigenicity. Upon exposure to non-permissive temperature, the mutant cells undergo extensive differentiation and lose their ability to initiate tumors in vivo, but retain their in vitro proliferative potential. Our data suggest that the loss of tumorigenicity is not caused by altered proliferative potential, but rather by cell differentiation. We therefore suggest that the loss of proliferative potential and the onset of cell differentiation in teratocarcinoma F9 cells are 2 independent events which can be genetically dissected, and that there is (a) crucial step(s) in the differentiation pathway at which these ts mutant cells lose their tumorigenicity.

Characterization of the mouse thrombomodulin gene and functional analysis of the 5′ flanking region in F9 teratocarcinoma cells

The mouse thrombomodulin (TM) gene was examined and shown to be a single copy gene lacking introns. Two different clones each containing the entire mouse TM gene were isolated and the nucleotide sequence of a 1.4 kb fragment comprising the 5′ untranslated region and 1.2 kb of flanking sequences was determined. The transcriptional initiation site was located 30 bp downstream from a classical TATA motif within this fragment. This site was used in BALB/c 3T3 cells constitutively expressing TM, and when TM expression was induced in F9 teratocarcinoma cells in response to retinoic acid (RA) and dibutyryl cAMP (dbcAMP). A reporter construct consisting of the 1.4 kb fragment fused to the chloramphenicol acetyl transferase (CAT) gene was used to examine promoter function in F9 cells. CAT activity was induced on exposure to RA and dbcAMP and mimicked the pattern of expression of the endogenous TM gene. Induction of CAT activity did not depend on a sequence resembling a palindromic retinoic acid/thyroid hormone response element. We conclude that the 1.4 kb fragment contains the mouse TM promoter together with elements that control the induction of TM expression in differentiating F9 cells.

Differentiation-dependent activation of interferon-stimulated gene factors and transcription factor NF-kappa B in mouse embryonal carcinoma cells.

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.