Effect of 5-azacytidine treatment on mouse embryonal carcinoma cells

Several properties of embryonal carcinoma (EC) cell lines, such as multipotent PCC4-aza-1 cells and nullipotent F9 cells originating from murine teratocarcinoma cells, were examined after treatment with 5-azacytidine, which produces undermethylated DNA. Drug-treated PCC4-aza-1 cells exhibited morphological changes and differentiated, whereas azacytidine-treated F9 cells displayed no detectable morphological change. After treatment with 5 azacytidine, PCC4-aza-1 cells, whether or not they differentiated, as well as F9 cells, became permissive for polyoma even though both cell types are usually resistant to polyoma. In contrast, only the differentiated azacytidine-treated PCC4-aza-1 cells became sensitive to SV40 infection, i.e., synthesized T antigen, despite the resistance normally shown by such cells to this viral infection. In some PCC4-aza-1 and F9 cells, drug treatment induced expression of H2 antigen but did not derepress plasminogen activator synthesis. These results suggest that undermethylation of certain cellular genes in PCC4-aza-1 and F9 cells is correlated with the establishment of Py permissivity, SV40 sensitivity, H2 antigen expression, and the triggering of a differentiation process. The relationship between the expression of these characters and differentiation is discussed.     

Nuclear activity from F9 embryonal carcinoma cells binding specifically to the enhancers of wild-type polyoma virus and PyEC mutant DNAs.

Although wild-type polyoma virus does not productively infect murine embryonal carcinoma (EC) cells, a number of mutants (PyEC mutants) that do infect undifferentiated EC cells have been isolated. All PyEC mutants have DNA sequence alterations within the enhancer region of the viral genome. This report describes an activity present in nuclear extracts of F9 EC cells which, by "footprint" analyses, binds specifically to a small region of about 20 base pairs (nucleotides 5180-5200) within the subregion of the polyoma enhancer designated as the B or beta element. While no difference in binding of factor was detected between wild-type polyoma enhancer and the enhancers of the PyEC mutants, PyF111 and PyF441, which had been selected for productive infection of F9 cells, definite differences between wild-type and mutants were observed in the digestion patterns of their naked DNAs with either DNAase I or exonuclease III. This difference was restricted to the region around the point mutation (nucleotide 5258) common to these mutant DNAs.     

Production and characterization of monoclonal antibodies to polyomavirus major capsid protein VP1.

Four hybridoma cell lines producing monoclonal antibodies against intact polyoma virions were produced and characterized. These antibodies were selected for their ability to react with polyoma virions in an enzyme-linked immunosorbent assay. The antibodies immunoprecipitated polyoma virions and specifically recognized the major capsid protein VP1 on an immunoblot. Distinct VP1 isoelectric species were immunoprecipitated from dissociated virion capsomere preparations. Two-dimensional gel electrophoresis demonstrated antibody reactivity with specific VP1 species. Monoclonal antibodies E7 and G9 recognized capsomeres containing VP1 species D, E, and F, while monoclonal antibodies C10 and D3 recognized capsomeres containing species B and C. Two of the monoclonal antibodies, E7 and G9, were capable of neutralizing viral infection and inhibiting hemagglutination. The biological activity of the monoclonal antibodies correlated well with the biological function of the species with which they reacted.     

Sequence of EndoA gene encoding mouse cytokeratin and its methylation state in the CpG-rich region.

A genomic clone obtained from mouse liver DNA using a mouse cytokeratin EndoA cDNA probe revealed the complete sequence of the EndoA gene. The gene is divided into nine exons and the exon-intron pattern has been conserved compared to that of other type-II cytokeratin-encoding genes. The 5' upstream, 3' downstream and first and third introns contain potential regulatory sequences, including polyoma virus enhancer motifs (PEA1 and PEA3) and AP-1 elements. The 5' regions upstream of the EndoA, EndoB and Ck8 genes contain homologous sequences surrounding the TATA boxes. In addition, a CpG dinucleotide cluster region was located around the first exon. This CpG cluster region was found to be hypomethylated in endodermal PYS-2 cells, retinoic acid-treated F9 cells, and F9 embryonal carcinoma cells, but hypermethylated in BALB/C 3T3 fibroblast cells that do not express EndoA. These findings may provide a clue to understanding the molecular mechanisms of EndoA gene expression.     

Expression and amplification in transgenic mice of a polyoma virus mutant regulatory region.

Two hybrid gene constructs consisting of wild-type and mutant polyoma regulatory regions fused to a bacterial reporter gene were inserted in the mouse germline. Both transgenes were expressed in a large number of different organs. However, marker gene expression controlled by the polyoma wild-type regulatory region was not detectable in the early embryo and remained low throughout the life of the animal while expression controlled by the polyoma F9-1 mutation was detectable in blastocysts and was significantly higher at later stages of development. The F9-1 hybrid gene was also amplifiable when large T-antigen was supplied in trans to mice or to kidney cells derived from these transgenic mice. Amplification resulted in the appearance of several hundred copies of episomal transgenes and a marked increase of marker gene RNA and protein. Our results suggest that the F9-1 mutation does not alter the target spectrum of gene expression in vivo but does create a more efficient enhancer element in the polyoma early control region. Transgene amplification based upon use of the polyoma regulatory elements may be a means of increasing expression of genes in transgenic mice.     

Isolation and characterization of polyoma virus mutants which grow in murine embryonal carcinoma and trophoblast cells.

Mouse trophoblast cell lines established from cultured midterm placenta and a cell line obtained from cultured blastocyst resemble trophectoderm cells. These cells are resistant to infection by wild-type polyoma virus. We have isolated six polyoma virus mutants capable of growing in trophoblast cell lines. Restriction enzyme analyses and marker rescue experiments revealed that the genetic changes necessary for the growth of these mutants ( PyTr mutants) in trophoblast cells were located in a regulatory region of the genome between the origin of viral DNA replication and the region encoding the viral structural proteins. PyTr mutants are, therefore, similar to PyEC mutants, described by others, which are able to grow in embryonal carcinoma cell lines such as F9 or PCC4. The nucleotide sequence of two independently obtained PyTr mutants has an identical 26-bp deletion from nucleotide 5131 to 5156. This deleted region is replaced by either the sequence GGGA or by viral DNA sequences that flank this deletion. PyECF9 mutants grow well in trophoblast and trophectoderm cells, but PyTr mutants do not grow in F9 or PCC4 cells.     

Retinoic acid selectively activates the ERK2 but not JNK/SAPK or P38 map kinases when inducing myeloid differentiation

Summary Among the three major mitogen-activated protein kinase (MAPK) cascades—the extracellular signal regulated kinase (ERK) pathway, the c-JUN N-terminal/stress-activated protein kinase (JNK/SAPK) pathway, and the reactivating kinase (p38) pathway—retinoic acid selectively utilizes ERK but not JNK/SAPK or p38 when inducing myeloid differentiation of HL-60 human myeloblastic leukemia cells. Retinoic acid is known to active ERK2. The present data show that the activation is selective for this MAPK pathway. JNK/SAPK or p38 are not activated by retinoic acid. Presumably because it activates relevant signaling pathways including MAPK, the polyoma middle T antigen, as well as certain transformation defective mutants thereof, is known to promote retinoic acid-induced differentiation, although the mechanism of action is not well understood. The present results show that consistent with the selective involvement of ERK2, ectopic expression of either the polyoma middle T antigen or its dl23 mutant, which is defective for PLCγ and PI-3 kinase activation, or the Δ205 mutant, which in addition is also weakened for activation of src-like kinases, caused no enhanced JNK/SAPK or p38 kinase activity that promoted the effects of retinoic acid. However, all three of these polyoma antigens are known to enhance ERK2 activation and promote differentiation induced by retinoic acid. Polyoma-activated MAPK signaling relevant to retinoic acid-induced differentiation is thus restricted to ERK2 and does not involve JNK/SAPK or p38. Taken together, the data indicate that among the three parallel MAPK pathways, retinoic acid-induced HL-60 myeloid differentiation selectively depends on activating ERK but not the other two MAPK pathways, JNK/SAPK or p38, with no apparent cross talk between pathways. Furthermore, the striking ability of polyoma middle T antigens to promote retinoic acid-induced differentiation appears to utilize ERK, but not JNK/SPK or p38 signaling.     

Vitamin A inhibition of polyoma virus replication

Vitamin A (retinoic acid) inhibited polyoma virus replication in confluent mouse embryo cells. A significant, dose dependent inhibition was observed when cell monolayers were pretreated with concentrations of vitamin A (10(-8) to 10(-6) M) thought to approximate those found in vivo. This inhibitory effect could be reduced by increasing the input multiplicity of infection. Growth curves of polyoma virus in the presence and absence of vitamin A suggested that vitamin A actually inhibited, and did not simply delay, virus replication. The cell density dependence of this inhibitory effect suggested its association with the prevailing level of cellular DNA synthesis. Vitamin A caused a significant decrease in overall (viral plus cellular) DNA synthesis. Other viruses which do not require induction of host cell DNA synthesis for their replication in confluent, non-dividing cells were not inhibited by vitamin A. These results are consistent with the known inhibitory effects of vitamin A on papovavirus infection in vivo and suggest a mechanism of vitamin A action at the level of the infected cell.     

Disabled-2 mediates c-Fos suppression and the cell growth regulatory activity of retinoic acid in embryonic carcinoma cells

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

Developmental control of a promoter-specific factor that is also regulated by the E1A gene product

We have detected a cellular factor in F9 teratocarcinoma cells that recognizes an adenovirus E1A inducible promoter. This factor, termed E2F, was previously identified in HeLa cells and was found at increased levels as a function of the E1A gene product. Upon differentiation of F9 cells with retinoic acid and cAMP, the factor declines to near undetectable levels, consistent with the control of this factor by E1A and the presence of a cellular E1A-like activity in F9 cells but not in differentiated F9 cells. Finally, if the E1A gene is introduced into differentiated cells by an adenovirus infection, there is a large increase in the level of the factor. We suggest that the control of E2F during F9 differentiation is indeed due to an E1A-like activity.     

Characterization of cloned sequences complementary to F9 cell double-stranded RNA and their expression during differentiation

In a cDNA library prepared from the RNA of cultured murine F9 teratocarcinoma cells, we identified sequences exhibiting strong hybridization to double-stranded RNA (dsRNA) of F9 cells but weak hybridization to mouse liver dsRNA. Northern-blot hybridization of RNA extracted from F9 cells with or without treatment with retinoic acid revealed differences in the expression of some of these sequences in undifferentiated and differentiated cells. As shown by Southern-blot hybridization experiments, these differences of expression were not related to a gross rearrangement of the corresponding genomic DNA sequences of the differentiated cells. When RNA from F9 cells was used, one of the cloned dsRNA-related sequences selected mRNA which was translated in vitro to a polypeptide with an Mr of 24,000; the level of this mRNA was reduced in F9 cells that had been treated with retinoic acid. Our results show that the differentiation of F9 cells induced by retinoic acid results in the differential expression of some middle-repetitive sequences.     

The effect of dibutyryl cyclic AMP and butyrate on F9 teratocarcinoma cellular retinoic acid-binding protein activity

F9 teratocarcinoma cells contain a cellular retinoic acid-binding protein (CRABP) that may mediate the retinoic acid-induced differentiation of this cell line. Specific [3H]retinoic acid binding to CRABP in F9 stem cell cytosol is protein-dependent, reaches equilibrium within 4 h at 4 degrees C, and yields 643 +/- 105 fmol of [3H]retinoic acid per mg of protein with an apparent dissociation constant of 9.2 +/- 1.1 nM. When F9 stem cells are grown in the presence of either dibutyryl cyclic AMP or sodium butyrate, CRABP activity is stimulated 2-4-fold. The effect of these drugs on CRABP activity is both time and concentration-dependent, resulting in an increase in the number of binding sites for [3H]retinoic acid with no change in their affinity. The new [3H]retinoic acid-binding sites have a sedimentation coefficient of 2 S and are not displaced by excess retinol. When F9 stem cells are grown in the presence of cyclic 8-bromo-AMP or cholera toxin, no increase in CRABP activity is observed. We conclude that the stimulation of CRABP activity by dibutyryl cyclic AMP may result from the action of butyrate. In addition, the stimulation of retinoic acid-induced F9 cell differentiation by cyclic AMP analogs (Strickland, S., Smith, K.K., and Marotti, K.R. (1980) Cell 21, 347-355) and the inhibition of this differentiation by butyrate (Levine R. A., Campisi, J., Wang, S.-Y., and Gudas, L. J. (1984) Dev. Biol. 105, 443-450) are not correlated with increases or decreases, respectively, in the level of CRABP activity.     

The level of CRABP-I expression influences the amounts and types of all-trans-retinoic acid metabolites in F9 teratocarcinoma stem cells.

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.     

Stimulation of pp60c-src tyrosyl kinase activity in polyoma virus-infected mouse cells is closely associated with polyoma middle tumor antigen synthesis

We have examined the effect of polyoma virus infection of primary mouse embryo cells on the tyrosyl kinase activity associated with the cellular src gene product, pp60c-src. The results of our studies demonstrate that infection of mouse cells with wild-type polyoma virus or viral mutants capable of transforming rodent cells in culture and inducing tumors in animals results in the stimulation of pp60c-src tyrosyl kinase activity. The level of pp60c-src kinase stimulation in infected cells was found to be proportional to both the oncogenic potential of the virus strain used for infection and the characteristic phenotype of rodent cells transformed by the various strains of polyoma virus. Stimulation of pp60c-src kinase activity was not observed in mouse cells infected with transformation-defective strains of polyoma virus. In examining the kinetics of pp60c-src kinase stimulation in mouse cells at various times following wild-type polyoma virus infection, we found that the level of pp60c-src kinase activity correlated directly with the synthesis of polyoma virus-encoded tumor antigens. By comparing wild-type polyoma virus with other viral mutants in these experiments, we conclude that the stimulation of pp60c-src kinase activity in mouse cells following polyoma virus infection is associated with the synthesis of middle tumor antigen.