Expression of the Brachyury gene during mesoderm development in differentiating embryonal carcinoma cell cultures

Development ◽  
1994 ◽  
Vol 120 (1) ◽  
pp. 115-122 ◽  
Author(s):  
G. Vidricaire ◽  
K. Jardine ◽  
M.W. McBurney
Keyword(s):  
Bone Morphogenetic Proteins ◽  
Embryonal Carcinoma ◽  
Carcinoma Cell ◽  
Cell Aggregation ◽  
Cell Types ◽  
Carcinoma Cells ◽  
Embryonal Carcinoma Cell ◽  
P19 Cells ◽  
Mesoderm Development ◽  
P19 Embryonal Carcinoma Cells

When aggregated and treated with dimethyl sulfoxide (DMSO), P19 embryonal carcinoma cells differentiate into cell types normally derived from the mesoderm and endoderm including epithelium and cardiac and skeletal muscle. The Brachyury gene is expressed transiently in these differentiating cultures several days before the appearance of markers of the differentiated cell types. The expression of Brachyury is not affected by DMSO but is induced by cell aggregation, which requires extracellular calcium. Expression of Brachyury is also induced by various members of the TGF beta family such as activin and bone morphogenetic proteins. D3 is a mutant clone of P19 cells selected for its failure to differentiate when aggregated in DMSO. Aggregated D3 cells express Brachyury mRNA suggesting that the mutation(s) responsible for the phenotype of D3 cells is downstream of the chain of events initiated by Brachyury expression.

scholarly journals Lineage-specific transformation after differentiation of multipotential murine stem cells containing a human oncogene.

1986 ◽  
Vol 6 (2) ◽  
pp. 617-625 ◽  
Author(s):  
J C Bell ◽  
K Jardine ◽  
M W McBurney
Keyword(s):  
Embryonal Carcinoma ◽  
Carcinoma Cell ◽  
Cell Lineage ◽  
Cell Types ◽  
Embryonal Carcinoma Cell ◽  
Fibroblast Cells ◽  
P19 Cells ◽  
Differentiated Cells ◽  
Embryonal Carcinoma Cell Line ◽  
Low Levels

We transfected the human EJ bladder carcinoma oncogene (Ha-rasEJ-1) into multipotential embryonal carcinoma cell line P19. The transgenic P19(ras+) cells expressed high levels of both the mRNA and the p21EJ protein derived from the oncogene. When cultured in the presence of retinoic acid, P19(ras+) cells differentiated and developed into the same spectrum of differentiated cell types as the parental P19 cells (namely, neurons, astrocytes, and fibroblast-like cells). Thus, it seems unlikely that the Ha-ras-1 proto-oncogene product plays a role in initiation of differentiation or in the choice of differentiated cell lineage. Most of the P19(ras+)-derived differentiated cells contained relatively low levels of p21EJ and were nontransformed, whereas certain cells with fibroblast-like morphology continued to express the Ha-rasEJ-1 gene at high levels and were transformed (i.e., immortal and anchorage independent). Fibroblasts derived from P19 cells did not become transformed following transfection of the Ha-rasEJ-1 oncogene, suggesting that transformation of the fibroblast cells only occurred if the oncogene was present and expressed during the early stages of the developmental lineage.

Lineage-specific transformation after differentiation of multipotential murine stem cells containing a human oncogene

1986 ◽  
Vol 6 (2) ◽  
pp. 617-625
Author(s):  
J C Bell ◽  
K Jardine ◽  
M W McBurney
Keyword(s):  
Embryonal Carcinoma ◽  
Carcinoma Cell ◽  
Cell Lineage ◽  
Cell Types ◽  
Embryonal Carcinoma Cell ◽  
Fibroblast Cells ◽  
P19 Cells ◽  
Differentiated Cells ◽  
Embryonal Carcinoma Cell Line ◽  
Low Levels

We transfected the human EJ bladder carcinoma oncogene (Ha-rasEJ-1) into multipotential embryonal carcinoma cell line P19. The transgenic P19(ras+) cells expressed high levels of both the mRNA and the p21EJ protein derived from the oncogene. When cultured in the presence of retinoic acid, P19(ras+) cells differentiated and developed into the same spectrum of differentiated cell types as the parental P19 cells (namely, neurons, astrocytes, and fibroblast-like cells). Thus, it seems unlikely that the Ha-ras-1 proto-oncogene product plays a role in initiation of differentiation or in the choice of differentiated cell lineage. Most of the P19(ras+)-derived differentiated cells contained relatively low levels of p21EJ and were nontransformed, whereas certain cells with fibroblast-like morphology continued to express the Ha-rasEJ-1 gene at high levels and were transformed (i.e., immortal and anchorage independent). Fibroblasts derived from P19 cells did not become transformed following transfection of the Ha-rasEJ-1 oncogene, suggesting that transformation of the fibroblast cells only occurred if the oncogene was present and expressed during the early stages of the developmental lineage.

scholarly journals Synthesis and processing of small B2 transcripts in mouse embryonal carcinoma cells.

1990 ◽  
Vol 10 (8) ◽  
pp. 4058-4067 ◽  
Author(s):  
T S Bladon ◽  
C J Frégeau ◽  
M W McBurney
Keyword(s):  
Embryonal Carcinoma ◽  
Consensus Sequence ◽  
Rna Polymerase Iii ◽  
Cell Types ◽  
Nucleocytoplasmic Transport ◽  
Carcinoma Cells ◽  
Embryonal Carcinoma Cells ◽  
B2 Rna ◽  
P19 Embryonal Carcinoma Cells ◽  
Rna Levels

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

scholarly journals Expression of simian virus 40 large T antigen in embryonal carcinoma cell hybrids.

1984 ◽  
Vol 4 (8) ◽  
pp. 1657-1660 ◽  
Author(s):  
A Tunnacliffe ◽  
L V Crawford ◽  
P Goodfellow
Keyword(s):  
Embryonal Carcinoma ◽  
Carcinoma Cell ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Carcinoma Cells ◽  
Embryonal Carcinoma Cell ◽  
Embryonal Carcinoma Cells ◽  
Large T Antigen ◽  
Early Region

Previous work has shown that murine embryonal carcinoma cells are refractory to infection with various viruses, including simian virus 40. Thus, large T and small t antigens, the products of the simian virus 40 early region, are not produced when the virus infects embryonal carcinoma cells, in contrast to other cell types. We show, by qualitative and quantitative analyses, that embryonal carcinoma cell hybrids, containing a simian virus 40 early region integrated into human DNA, are capable of producing viral large T antigen.

scholarly journals Induction and repression of mammalian achaete-scute homologue (MASH) gene expression during neuronal differentiation of P19 embryonal carcinoma cells

Development ◽  
1992 ◽  
Vol 114 (1) ◽  
pp. 75-87 ◽  
Author(s):  
J.E. Johnson ◽  
K. Zimmerman ◽  
T. Saito ◽  
D.J. Anderson
Keyword(s):  
Retinoic Acid ◽  
Neuronal Differentiation ◽  
Embryonal Carcinoma ◽  
Model System ◽  
Neuronal Morphology ◽  
Carcinoma Cells ◽  
P19 Cells ◽  
Embryonal Carcinoma Cells ◽  
Bhlh Genes ◽  
P19 Embryonal Carcinoma Cells

MASH1 and MASH2, mammalian homologues of the Drosophila neural determination genes achaete-scute, are members of the basic helix-loop-helix (bHLH) family of transcription factors. We show here that murine P19 embryonal carcinoma cells can be used as a model system to study the regulation and function of these genes. MASH1 and MASH2 display complementary patterns of expression during the retinoic-acid-induced neuronal differentiation of P19 cells. MASH1 mRNA is undetectable in undifferentiated P19 cells but is induced to high levels by retinoic acid coincident with neuronal differentiation. In contrast, MASH2 mRNA is expressed in undifferentiated P19 cells and is repressed by retinoic acid treatment. These complementary expression patterns suggest distinct functions for MASH1 and MASH2 in development, despite their sequence homology. In retinoic-acid-treated P19 cells, MASH1 protein expression precedes and then overlaps expression of neuronal markers. However, MASH1 is expressed by a smaller proportion of cells than expresses such markers. MASH1 immunoreactivity is not detected in differentiated cells displaying a neuronal morphology, suggesting that its expression is transient. These features of MASH1 expression are similar to those observed in vivo, and suggest that P19 cells represent a good model system in which to study the regulation of this gene. Forced expression of MASH1 was achieved in undifferentiated P19 cells by transfection of a cDNA expression construct. The transfected cells expressing exogenous MASH1 protein contained E-box-binding activity that could be super-shifted by an anti-MASH1 antibody, but exhibited no detectable phenotypic changes. Thus, unlike myogenic bHLH genes, such as MyoD, which are sufficient to induce muscle differentiation, expression of MASH1 appears insufficient to promote neurogenesis.

Synthesis and processing of small B2 transcripts in mouse embryonal carcinoma cells

1990 ◽  
Vol 10 (8) ◽  
pp. 4058-4067
Author(s):  
T S Bladon ◽  
C J Frégeau ◽  
M W McBurney
Keyword(s):  
Embryonal Carcinoma ◽  
Consensus Sequence ◽  
Rna Polymerase Iii ◽  
Cell Types ◽  
Nucleocytoplasmic Transport ◽  
Carcinoma Cells ◽  
Embryonal Carcinoma Cells ◽  
B2 Rna ◽  
P19 Embryonal Carcinoma Cells ◽  
Rna Levels

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

Transient expression of the proto-oncogene int-1 during differentiation of P19 embryonal carcinoma cells

1989 ◽  
Vol 9 (3) ◽  
pp. 1357-1361
Author(s):  
E Schuuring ◽  
L van Deemter ◽  
H Roelink ◽  
R Nusse
Keyword(s):  
Retinoic Acid ◽  
Neural Development ◽  
Time Course ◽  
Embryonal Carcinoma ◽  
Dose Dependence ◽  
Carcinoma Cells ◽  
P19 Cells ◽  
Embryonal Carcinoma Cells ◽  
P19 Embryonal Carcinoma Cells

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

Effects of taxol on the polymerization and posttranslational modification of class III β-tubulin in P19 embryonal carcinoma cells

1995 ◽  
Vol 73 (9-10) ◽  
pp. 687-694 ◽  
Author(s):  
Nicole B. Laferrière ◽  
D. L. Brown
Keyword(s):  
Neurite Outgrowth ◽  
Posttranslational Modification ◽  
Embryonal Carcinoma ◽  
Carcinoma Cells ◽  
Specific Class ◽  
P19 Cells ◽  
Embryonal Carcinoma Cells ◽  
Class Iii ◽  
P19 Embryonal Carcinoma Cells ◽  
Β Tubulin

Undifferentiated P19 embryonal carcinoma cells and P19 cells induced to differentiate along a neuronal pathway by 10−6 M retinoic acid were treated with taxol to examine the effects of this microtubule-stabilizing drug on the subcellular sorting of class III β-tubulin and on neurite outgrowth. P19 cells were grown on cover slips and then treated with taxol at concentrations of 10−6 to 10−9 M for 24 h. The microtubule cytoskeleton was examined after double-immunofluorescence labelling with a monoclonal antibody to α-tubulin (YOL 1/34) and a monoclonal neuron-specific class III β-tubulin antibody (TuJ1). Treatment of undifferentiated P19 cells with concentrations of taxol greater than 4 × 10−8 M caused microtubule bundling and multiple aster formation and promoted polymerization of the low levels of class III β-tubulin found in these cells. In neurons, at 2 × 10−8 M taxol, bundling of microtubules at the base of the neurite was apparent. At taxol concentrations greater than 1 × 10−7 M, enhanced assembly of class III β-tubulin was apparent, although long neurites were not observed. Using isoelectric focusing followed by western blotting, we detected an additional isoform of class III β-tubulin after treatment with 10−6 M taxol. These results indicate taxol treatment alters the normal subcellular sorting of tubulin isotypes, promotes the polymerization and posttranslational modification of class III β-tubulin, and interferes with neurite outgrowth.Key words: tubulin, taxol, microtubule, posttranslational modification, neurite outgrowth.

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