F9 Teratocarcinoma Cells Research Articles

Secretion of alpha1,3-galactosyltransferase by cultured cells and presence of enzyme in animal sera.

Glycosyltransferases are normally synthesized as membrane-anchored proteins. However, we recently found that the murine enzyme UDP-Gal:Gal beta1 -->4GLcNAc (Gal to Gal) alpha1,3 galactosyltransferase (alpha1,3GT) is secreted in a soluble form into media by mouse teratocarcinoma F9 cells (Cho SK, Yeh J-C, Cho M, Cummings RD (1996) J Biol Chem 271: 3238-46). To study the biosynthesis of this enzyme and whether secretion of the soluble enzyme is a general phenomenon, a solid-phase assay was developed for the alpha1,3GT activity. A recombinant and soluble form of the murine alpha1,3GT was produced in H293 cells (H293-alpha1,3GT) to aid in optimizing the assay. Desialylated orosomucoid was used as an immobilized acceptor in coated microtiter plates. The formation of product was detected by a biotinylated human-derived anti-alpha-Gal IgG and streptavidin conjugated to either alkaline phosphatase or the recombinant bioluminescent protein aequorin. Enzyme activity was dependent on the concentrations of asialoorosomucoid, UDP-Gal, alpha1,3GT and the time of incubation. The assay was also useful in monitoring alpha1,3GT activity during enzyme enrichment procedures. Using this assay, we found that alpha1,3GT activity was present in both cell extracts and culture media of several mammalian cell lines. Enzyme activity was also present in the sera from several mammals, but activity was absent in the sera from either humans or baboons. Our results demonstrate the development of a novel assay for the alpha1,3GT and provide evidence that secretion of the enzyme is a common biological phenomenon.

Analysis of connexin43 gene expression induced by retinoic acid in F9 teratocarcinoma cells

All-trans retinoic acid (10 −7 M) induces cell-cell communication and expression of the gap junction protein connexin43 in mouse F9 teratocarcinoma cells. Northern blot analysis revealed an increase of connexin43 mRNA after treatment with retinoic acid, accompanied by an increase of the mRNA of collagen IV, a differentiation marker. To address the question at what level gene expression is enhanced by retinoic acid, nuclear run-on experiments were carried out. There was no detectable change in the level of newly transcribed connexin43 mRNA. Therefore, we postulate a post-transcriptional mechanism responsible for the regulation of connexin43 mRNA levels by retinoic acid.

DNA methylation patterns in tumors derived from F9 cells resemble methylation at the blastula stage

We show here that the genome of F9 teratocarcinoma cells growing in culture is heavily methylated but undergoes massive demethylation in tumors derived by subcutaneous injection of the cells. This demethylation occurs primarily in single copy gene sequences. As a result imprinted genes acquire their characteristic monoallelic methylation patterns while non-imprinted genes undergo demethylation. The overall methylation pattern in the tumors resembles the pattern observed in the mouse preimplantation embryo. The F9 cells in the tumors apparently recognize imprinted genes, distinguish them from non-imprinted genes and change the methylation of both gene classes to the pattern which characterizes the embryo. These cells therefore have the potential of providing an abundant source of protein factors involved in establishing the methylation pattern during embryo development.

A retinoic acid/cAMP-responsive enhancer containing a cAMP responsive element is required for the activation of the mouse thrombomodulin-encoding gene in differentiating F9 cells

Activation of thrombomodulin-encoding gene ( TM) transcription in murine teratocarcinoma F9 cells occurs during differentiation in response to retinoic acid (RA) and dibutyryl cAMP (dbcAMP). To define regulatory elements involved in TM activation, reporter plasmids containing various lengths of the 5' flanking region of the mouse TM promoter fused to the cat gene were examined in transfected F9 cells. Stable transfectants showed no CAT activity in undifferentiated or RA-treated cells, and only those plasmids containing a 431-bp region approximately 700 bp upstream of the transcription start point showed significant CAT activity on subsequent treatment with RA/dbcAMP. Transfer of this region to an enhancerless SV40 promoter conferred position-independent responsiveness to RA/dbcAMP. Within this region is a 46-bp domain that contains potential transcription factor binding sites, including a cAMP responsive element (CRE) and a direct repeat sequence (DR). CAT assays using reporters lacking these sites showed that both contribute to TM expression in differentiating F9 cells. Gel retardation experiments demonstrated protein binding to both the CRE and DR sequences, and suggested that the factor interacting with the DR influences binding of a factor to the CRE site. Binding of both factors disappeared in differentiated cells, suggesting they are required for induction but not continued expression of TM. The CpG methylation pattern of the TM promoter was identical in undifferentiated and differentiated F9 cells. DNase I hypersensitivity studies showed increased DNase I resistance and the loss of a hypersensitive site in differentiated F9 cells. We conclude that an enhancer-like region containing a DR sequence and CRE is necessary for the induction of TM expression in differentiating F9 cells, that methylation does not play a role in the regulation of TM, and that the RA/cAMP response is associated with specific alterations to chromatin structure.

DNA Demethylation In Vitro: Involvement of RNA

An in vitro system for studying DNA demethylation has been established using extracts from tissue culture cells. This reaction, which is unusually resistant to proteinase K, takes place through the removal of a 5-methylcytosine nucleotide unit from the DNA substrate and its conversion to an RNase-sensitive form. It is likely that this represents the in vivo mechanism, as well, since extracts from L8 mybolasts specifically demethylate an α-actin gene, while extracts from F9 teratocarcinoma cells specifically demodify the Aprt CpG island. After pretreatment with proteinase K, these extracts demethylate both genes equally, suggesting that gene specificity may be controlled by protein factors.

Murine endodermal F9E cells, derived from the teratocarcinoma line F9, contain high basal levels of retinoic acid receptors (RARs and RXRs) but are not sensitive to the actions of retinoic acid

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

In vitro induction of differentiation by ginsenoides in F9 teratocarcinoma cells.

The aim of this study was to determine the ability of the ginsenosides, extracts of Panax ginseng C.A. Meyer, to cause differentiation of F9 teratocarcinoma stem cells as a model system. F9 stem cells cultured in the presence of the ginsenosides together with dibutyryl cyclic AMP (dbcAMP) became parietal endoderm-like cells. Moreover, the expression of differentiation marker genes, such as laminin B1 and type IV collagen, was increased after treatment with the ginsenosides. Among the various purified ginsenosides, Rh1 and Rh2 were the most effective at causing differentiation of F9 cells. Since ginsenosides and glucocorticoid hormone have similar chemical structures, we examined the possibility of the involvement of a glucocorticoid receptor (GR) in the differentiation process induced by the ginsenosides. According to Southwestern blot analysis, a 94 kDa protein regarded as a GR was detected in F9 cells cultured in the medium containing the ginsenosides Rh1 or Rh2. In addition, F9 stem cells treated with the ginsenosides Rh1 or Rh2 and with RU486, a glucocorticoid antagonist with a high affinity for the GR, did not differentiate into endoderm cells morphologically, and the expression of laminin B1 gene was not induced in these cells. In a gel mobility shift assay, protein factors capable of binding to the glucocorticoid responsive element (GRE) specifically were detected in nuclear extracts of the ginsenoside-treated F9 cells. Moreover, overexpression of GR by cotransfection of GR expression vector and GRE-luciferase vector enhanced the transactivation activity of GRE promoter in the presence of ginsenosides Rh1 or Rh2 and was further augmented by dbcAMP. In addition, ginsenosides Rh1 and Rh2 bound to a GR assessed by whole-cell binding assay, even though the specific binding affinity was weaker compared to dexamethasone. Based on these data, we suggest that the ginsenosides Rh1 and Rh2 cause the differentiation of F9 cells and the effects of ginsenosides might be exerted via binding with a GR or its analogous nuclear receptor.

SPATIAL EXPRESSION PATTERNS OF EXTRACELLULAR GLUTATHIONE PEROXIDASE (EGPx) DURING MURINE POST-IMPLANTATION DEVELOPMENT SUGGEST A PROTECTIVE ROLE FOR THE EMBRYO. † 356

Extracellular glutathione peroxidase (EGPx) is a member of a selenium-dependent antioxidant enzyme family. To better understand its potential functions in vivo, we examined the postimplantation expression of EGPx mRNA from E7.5 to E16.5 of mouse development. During this period, the message for EGPx shifted major sites of accumulation, but maintained high concentration at borders between the developing embryo and its environment. Thus, at E7.5 (neural plate stage) the highest EGPx expression was found in the maternally-derived deciduum, with minor accumulation in the visceral yolk sac endoderm. At E9.5, the major sites of expression were the yolk sac endoderm and developing heart musculuture. By E16.5, EGPx mRNA expression persisted in the yolk sac endoderm, but also accumulated significantly in a ventral to dorsal gradient in the developing skin. Expression in the heart and lungs was primarily restricted to atrially-derived myocytes. At E16.5, we also detected significant EGPx mRNA accumulation in the intestinal epithelium and minor accumulation in developing kidney, the principle site of EGPx synthesis in the adult. Since EGPx is expressed in the yolk sac endoderm, we explored its in vitro expression in F9 teratocarcinoma cells. EGPx mRNA is induced along with α-fetoprotein when F9 cells are differentiated toward visceral, but not parietal endoderm. Further studies of EGPx function and gene regulation can now be performed in this in vitro system.

Transcriptional Regulation of α1,3-Galactosyltransferase in Embryonal Carcinoma Cells by Retinoic Acid: MASKING OF LEWIS X ANTIGENS BY α-GALACTOSYLATION

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

Correlation of ploidy and cyclin B1, P34cdc2 and PCNA expression in F9 teratocarcinoma cells.

Correlation of ploidy and cyclin B1, P34cdc2 and PCNA expression in F9 teratocarcinoma cells.

P450-dependent enzymes as targets for prostate cancer therapy

Metastatic prostate adenocarcinoma is a leading cause of cancer-related deaths among men. First line treatment is primarily aimed at blocking the synthesis and action of androgens. As primary endocrine treatment, androgen deprivation is usually achieved by orchidectomy or LHRH analogues, frequently combined with androgen receptor antagonists in order to block the residual adrenal androgens. However, nearly all the patients will eventually relapse. Available or potential second line therapies include, among others, alternative endocrine manipulations and chemotherapy. Cytochrome P450-dependent enzymes are involved in the synthesis and/or degradation of many endogenous compounds, such as steroids and retinoic acid. Some of these enzymes represent suitable targets for the treatment of prostate cancer. In first line therapy, inhibitors of the P450-dependent 17,20-lyase may achieve a maximal androgen ablation with a single drug treatment. Ketoconazole at high dose blocks both testicular and adrenal androgen biosynthesis but its side-effects, mainly gastric discomfort, limit its widespread use. A series of newly synthesized, more selective, steroidal 17,20-lyase inhibitors related to 17-(3-pyridyl)androsta-5,16-dien-3β-ol, may open new perspectives in this field. In prostate cancer patients who relapse after surgical or medical castration, therapies aiming at suppressing the remaining adrenal androgen biosynthesis (ketoconazole) or producing a medical adrenalectomy (aminoglutethimide + hydrocortisone) have been used, but are becoming obsolete with the generalization of maximal androgen blockade in first line treatment. The role of inhibition of aromatase in prostate cancer therapy, which was postulated for aminoglutethimide, could not be confirmed by the use of more selective aromatase inhibitors, such as formestane. An alternative approach is represented by liarozole fumarate (LIA), a compound that blocks the P450-dependent catabolism of retinoic acid (RA). In vitro, it enhances the antiproliferative and differentiation effects of RA in cell lines that express RA metabolism, such as F9 teratocarcinoma and MCF-7 breast carcinoma cells. In vivo, monotherapy with LIA increases RA plasma levels and, to a greater extent, endogenous tissue RA levels leading to retinoid-mimetic effects. In the rat Dunning prostate cancer models, it inhibits the growth of androgen-independent as well as androgen-dependent carcinomas relapsing after castration. Concurrently, changes in the pattern of cytokeratins characteristic of increased differentiation were observed. Early clinical trials show that LIA, in second or third line therapy in metastatic prostate cancer, induces PSA responses in about 30% of unselected patients. In some patients regression of soft tissue metastasis has been observed. In a subgroup of patients, an important relief of metastatic bone pain was also noted.

Expression of syndecan-1 changes during the differentiation of visceral and parietal endoderm from murine F9 teratocar Cinoma cells

F9 teratocarcinoma stem cells treated with retinoic acid differentiate in suspension into embryoid bodies with an outer layer of visceral endoderm surrounding a core of largely undifferentiated cells. The visceral endoderm-containing embryoid bodies, when plated onto an extracellular matrix coating, give rise to parietal endoderm outgrowth. These in vitro cell cultures mimic both geometrically and biochemically the differentiation of visceral and parietal endoderm in the early mouse embryo and, thus, were used as a model system for the study of molecular and cellular mechanisms underlying the differentiation of the extraembryonic endoderm lineages. We have investigated the expression of syndecan-1, an integral membrane proteoglycan that binds to multiple components of the extracellular matrix and basic FGF, during visceral endoderm differentiation and parietal endoderm outgrowth. Syndecan-1 immunostaining is detected on all cell surfaces in the undifferentiated embryoid bodies and in the differentiating embryoid bodies prior to the formation of the visceral endoderm. Following the differentiation of visceral endoderm, syndecan-1 localizes predominantly to the basal surface of this epithelial layer, while syndecan-1 staining in the core of differentiated embryoid bodies is faint. Quantitation of cell associated syndecan-1 indicates that syndecan-1 is down-regulated during embryoid body differentiation. However, northern analysis shows that the amounts of steady-state syndecan-1 mRNA are the same in undifferentiated versus differentiated embryoid bodies, suggesting post-transcriptional regulation of syndecan-1 expression in the differentiating embryoid body. Analysis of syndecan-1 distribution in the outgrowth culture by immunofluorescence demonstrates that syndecan-1 is absent from the cell surface of parietal endoderm. However, a substantial amount of syndecan-1 is detected inside parietal endoderm cells. While all three cell types release syndecan-1 ectodomain into the culture medium, the parietal endoderm outgrowth releases more syndecan-1 ectodomain than the differentiated embryoid body. These data suggest that the post-transcriptional control and post-translational shedding of syndecan-1 from the cell surface are developmentally regulated during the differentiation of visceral to parietal endoderm and the migration of parietal endoderm.

Effect of Cell Cycle Stage, Dose Rate and Repair of Sublethal Damage on Radiation-Induced Apoptosis in F9 Teratocarcinoma Cells

There are at least two different modes of cell death after treatment with ionizing radiation. The first is a failure to undergo sustained cell division despite metabolic survival, and we refer to this end point as "classical reproductive cell death." The second is a process that results in loss of cell integrity. This second category includes cellular necrosis as well as apoptosis. Earlier studies in our laboratory showed that the predominant mechanism of cell death for irradiated F9 cells is apoptosis, and there is no indication that these cells die by necrosis. We have therefore used cells of this cell line to reassess basic radiobiological principles with respect to apoptosis. Classical reproductive cell death was determined by staining colonies derived from irradiated cells and scoring colonies of less than 50 cells as reproductively dead and colonies of more than 50 cells as survivors. Cells that failed to produce either type of colony (detached from the plate or disintegrated) were scored as having undergone apoptosis. Using these criteria we found that the fraction of the radiation-killed F9 cells that died by apoptosis did not vary when cells were irradiated at different stages of the cell cycle despite large variations in overall survival. This suggests that the factors that influence radiation sensitivity throughout the cell cycle have an equal impact on apoptosis and classical reproductive cell death. There was no difference in cell survival between split doses and single doses of X rays, suggesting that sublethal damage repair is not a factor in radiation-induced apoptosis of F9 cells. Apoptosis was not affected by changes in dose rate in the range of 0.038-4.96 Gy/min.

Novel flow-cytometric method for separating cell types in differentiated F9 embryoid bodies.

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

Role of LFB3 in Cell-specific cAMP Induction of the Urokinase-type Plasminogen Activator Gene

In previous work we suggested that a kidney-specific transcription factor LFB3 cooperates with cAMP-response element (CRE)-binding proteins within a cAMP regulatory unit comprised of three protein-binding domains and located 3.4 kilobase pairs upstream of the urokinase-type plasminogen activator (uPA) gene in LLC-PK1 cells (Menoud, P.-A., Matthies, R., Hofsteenge, J., and Nagamine, Y. (1993) Nucleic Acids Res. 21, 1845-1852). The two domains contain a CRE-like sequence, and the third domain is recognized by LFB3. The absolute requirement of LFB3 as well as the cooperation among the three domains for cAMP regulation were confirmed by transient transfection assays in F9 teratocarcinoma cells, in which the level of LFB3 was negligible. Suspecting a possible feedback regulation of LFB3 mRNA expression during cAMP-dependent uPA gene induction in LLC-PK1 cells, we measured LFB3 mRNA levels after cAMP treatment and found a strong reduction. This reduction was not due to a change in template activity of the LFB3 gene because run-on transcription showed no significant change in LFB3 gene transcription. RNA synthesis inhibitor-chase experiments indicated that the down-regulation was post-transcriptional. Interestingly, when the inhibitor was added at the same time as cAMP, the cAMP-induced decrease in LFB3 mRNA levels was abrogated, suggesting that ongoing RNA synthesis is required for the decrease. Similar effects on LFB3 mRNA metabolism were observed with all agents that induce uPA mRNA in LLC-PK1 cells, including 12-O-tetradecanoylphorbol-13-acetate, okadaic acid, colchicine, and cytochalasin. We discuss the significance of this regulation in uPA gene expression.

Different Response to Retinoic Acid of Two Teratocarcinoma Cell Lines

Retinoic acid (RA), a well-known inducer of differentiation, has been shown to regulate its own receptor gene expression in F9 teratocarcinoma cells. The homologous regulation of receptors by RA might be critical for RA-induced F9 cell differentiation. F9 cell lines from two different laboratories, named F9-1 and F9-2, were compared for retinoic acid receptor (RAR) and retinoid x receptor (RXR) gene expression in response to RA. The data show that both F9-1 and F9-2 cell lines are embryonal carcinoma cells, but of different phenotypes and different sensitivity to RA. In F9-1 cells, RA regulates all three RARs (α, β, and γ), two RXRs (α and γ), two activin receptors (ActR II and IIB), and tissue-specific plasminogen activator (t-PA) gene expression. In F9-2 cells RA regulates only the RARβ, RXRα, and t-PA genes. The induction of mRNA levels was much higher in F9-1 than in F9-2 cells. Different basal RARγ and RXRγ mRNA levels were also noted. In these two cell lines F9-2 cells expressed greater amounts of RARγ1, γ2, and γ3 mRNA isoforms, but lacked RXRγ mRNA compared with F9-1 cells. Since RARγ1 has been shown to exert an antagonistic effect on other types of RA receptors, the decreased sensitivity of F9-2 cells to RA might be due to its high level of RARγ1 and/or low level of RXRγ. This notion was in part supported by gel shift assay which demonstrated constitutive binding of RARγ to a RA responsive element (RARβE) in F9-2 cells. Further, the binding of nuclear protein to RARβE was increased upon RA treatment in F9-1 cells, but not in F9-2 cells. These differences in the regulation of RA receptors might determine the sensitivity of the two substrains of F9 cells to RA.

Glycoprotein 330/Low Density Lipoprotein Receptor-related Protein-2 Mediates Endocytosis of Low Density Lipoproteins via Interaction with Apolipoprotein B100

The ability of glycoprotein 330/low density lipoprotein receptor-related protein-2 (LRP-2) to function as a lipoprotein receptor was investigated using cultured mouse F9 teratocarcinoma cells. Treatment with retinoic acid and dibutyryl cyclic AMP, which induces F9 cells to differentiate into endoderm-like cells, produced a 50-fold increase in the expression of LRP-2. Levels of the other members of the low density lipoprotein (LDL) receptor (LDLR) family, including LDLR, the very low density lipoprotein receptor, and LRP-1, were reduced. When LDL catabolism was examined in these cells, it was found that the treated cells endocytosed and degraded at 10-fold higher levels than untreated cells. The increased LDL uptake coincided with increased LRP-2 activity of the treated cells, as measured by uptake of both 125I-labeled monoclonal LRP-2 antibody and the LRP-2 ligand prourokinase. The ability of LDL to bind to LRP-2 was demonstrated by solid-phase binding assays. This binding was inhibitable by LRP-2 antibodies, receptor-associated protein (the antagonist of ligand binding for all members of the LDLR family), or antibodies to apoB100, the major apolipoprotein component of LDL. In cell assays, LRP-2 antibodies blocked the elevated 125I-LDL internalization and degradation observed in the retinoic acid/dibutyryl cyclic AMP-treated F9 cells. A low level of LDL endocytosis existed that was likely mediated by LDLR since it could not be inhibited by LRP-2 antibodies, but was inhibited by excess LDL, receptor-associated protein, or apoB100 antibody. The results indicate that LRP-2 can function to mediate cellular endocytosis of LDL, leading to its degradation. LRP-2 represents the second member of the LDLR family identified as functioning in the catabolism of LDL.

The kinetics of induction of Hox1.6 and C-jun mRNA during three different ways of inducing differentiation in teratocarcinoma F9 cells.

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

Regulation of gene transcription during retinoic acid induced differentiation of F9 teratocarcinoma cells

Regulation of gene transcription during retinoic acid induced differentiation of F9 teratocarcinoma cells

Induction by adenovirus-5 E1A of the differentiation phenotype of F9 teratocarcinoma cells involves a conserved region (CR1) of E1A

The effects of the E1A protein of adenovirus-5 on the differentiation program of F9 teratocarcinoma cells were examined by the stable introduction of plasmids that expressed wild-type or mutated forms of E1A. Constitutive expression of plasmids for most of the mutant E1As induced loss of expression of the cell-surface antigen SSEA-1 and the enhanced expression of genes specific for the differentiated phenotype of F9 cells, such as genes for laminin B1, tissue-type plasminogen activator (tPA) and type IV collagen, as well as the altered cell morphology that is associated with the differentiated state. However, such changes were not observed in the case of genes for mutant proteins from which a conserved region (CR1) of E1A had been deleted. Furthermore, no significant induction of expression of the c- jun gene or transactivation of the c- jun-CAT reporter gene were observed when the sequence that encodes CR1 of E1A had been deleted. A palindromic sequence element (DRE) of the c- jun promoter was essential for the E1A-mediated up-regulation of the c- jun gene. These results imply that CR1 is required for activation of the c- jun gene and that it is implicated in the growth arrest, expression of parietal endoderm-specific functions and the orderly differentiation of F9 cells.