Ectopic expression of an A-type lamin does not interfere with differentiation of lamin A-negative embryonal carcinoma cells

1991 ◽  
Vol 100 (3) ◽  
pp. 589-598 ◽  
Author(s):  
M. Peter ◽  
E.A. Nigg
Keyword(s):  
Cell Differentiation ◽  
Cell Lines ◽  
Embryonal Carcinoma ◽  
Ectopic Expression ◽  
Cell Types ◽  
Biochemical Properties ◽  
Lamin A ◽  
Embryonal Carcinoma Cell ◽  
P19 Cells ◽  
Developmental Potential

The nuclear lamina is an intermediate filament-type network underlying the inner nuclear membrane. It is believed to be important for nuclear envelope integrity and the organization of interphase chromatin. On the basis of biochemical properties and sequence criteria, vertebrate lamin proteins are classified as either A- or B-type. While B-type lamins are expressed in almost all cell types, no A-type lamins are present in early vertebrate embryos or undifferentiated embryonal carcinoma cell lines. Intriguingly, expression of A-type lamins occurs concomitant with cell differentiation and embryonic development. These findings have led to the hypothesis that A-type lamins might play a role in establishing or stabilizing cell-type specific differences in nuclear organization, which in turn might relate to the developmental potential of a cell. To test this hypothesis, we have stably expressed chicken lamin A in undifferentiated murine embryonal carcinoma (P19) cells, and examined the consequences of ectopic lamin A expression for the differentiation state and potential of these cells. Our results demonstrate that the P19 cells, although normally devoid of lamin A, properly incorporate and process chicken lamin A. Moreover, the stably transfected cell lines maintain the properties of undifferentiated cells, demonstrating that expression of lamin A does not directly induce differentiation. Conversely, when exposed to retinoic acid, an inducer of differentiation, lamin A-expressing P19 cells are able to differentiate normally. Taken together, our results suggest that unscheduled expression of A-type lamins is not sufficient to deregulate cell differentiation programs. The implications of these findings for the possible role for lamin A expression during development are discussed.

Cell lines with developmental potential restricted to mesodermal lineages isolated from differentiating cultures of pluripotential P19 embryonal carcinoma cells

Development ◽  
1989 ◽  
Vol 107 (2) ◽  
pp. 361-372
Author(s):  
M.A. Rudnicki ◽  
K.R. Reuhl ◽  
M.W. McBurney
Keyword(s):  
Connective Tissue ◽  
Cell Lines ◽  
Embryonal Carcinoma ◽  
Type I Collagen ◽  
Cell Types ◽  
Type I ◽  
P19 Cells ◽  
Developmental Potential ◽  
Differentiated Cells ◽  
P19 Embryonal Carcinoma Cells

Embryonal carcinoma (EC) cells are developmentally pluripotential cells which can be induced to differentiate in cell culture to form a wide variety of cell types. To investigate the lineage relationships between cells of different types, we set out to isolate cell lines with multiple but restricted developmental potentials from differentiating cultures of P19 cells, a line of EC. By selecting for differentiated cells capable of anchorage-independent growth, we isolated cell lines which differentiated in high density cultures to form at least two cell types; myocytes that resembled fetal skeletal muscle cells and loose connective tissue cells that secreted large amounts of type I collagen. These results suggest that skeletal myocytes and connective tissue share a common precursor and that stem cells with limited but multiple developmental potentials can be isolated from differentiating cultures of P19 cells.

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.

scholarly journals Functional cloning of mouse chromosomal loci specifically active in embryonal carcinoma stem cells.

1988 ◽  
Vol 8 (8) ◽  
pp. 3251-3259 ◽  
Author(s):  
K Bhat ◽  
M W McBurney ◽  
H Hamada
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Dna Sequences ◽  
Embryonal Carcinoma ◽  
Regulatory Elements ◽  
Embryonal Carcinoma Cell ◽  
P19 Cells ◽  
Embryonal Carcinoma Cell Line ◽  
Cellular Dna ◽  
Chromosomal Loci

Chromosomal loci that are specifically active in embryonal carcinoma stem cells were cloned from the mouse genome by functional selection. P19 cells, a pluripotent embryonal carcinoma cell line, were transfected with an enhancer trap (a plasmid containing an enhancerless inactive neo gene), and NEO+ transformants were isolated. All of the NEO+ cell lines retained pluripotency and expressed the neo gene. When the cells were induced to differentiate, most of the cell lines continued to express the neo gene, while the neo gene in some cell lines became repressed. From the latter group of cell lines, we have cloned the integrated neo gene plus the flanking cellular DNA sequences. Three of the six cloned DNAs possessed a high NEO+-transforming activity in undifferentiated P19 cells. Among these three, two (015 and 052) were inactive in differentiated P19 cells and NIH 3T3 cells, while the remaining one was active in these differentiated cells. Deletion analysis suggested that both 015 and 052 contain two regulatory elements (promoter and enhancer) of cellular DNA origin. The putative enhancer and promoter are separated by at least 6 kilobases in 015 and 1 kilobase in 052. Therefore, 015 and 052 cloned fragments contain regulatory DNA elements that are specifically active in the embryonal carcinoma stem cells.

A transfected H-ras oncogene does not inhibit differentiation of cardiac and skeletal muscle from embryonal carcinoma cells

1989 ◽  
Vol 67 (9) ◽  
pp. 590-596 ◽  
Author(s):  
Michael A. Rudnicki ◽  
Kenneth R. Reuhl ◽  
Michael W. McBurney
Keyword(s):  
Skeletal Muscle ◽  
Cell Lines ◽  
Embryonal Carcinoma ◽  
Cell Types ◽  
P19 Cells ◽  
Ras Oncogene ◽  
Specific Expression ◽  
Ec Cells ◽  
Myoblast Cell

P19 embryonal carcinoma (EC) cells can be induced to differentiate in vitro into a variety of cell types, including cardiac and skeletal myocytes. We have isolated P19 cells stably transformed with either the activated human H-ras oncogene or with a chimeric gene in which the H-ras oncogene was controlled by a muscle-specific promoter. These P19 lines exhibited ubiquitous and muscle-specific expression of the activated H-ras protein, respectively. In both lines of P19 cells, normal cardiac and skeletal muscle differentiation was observed. Since the activated H-ras prevents differentiation of myoblast cell lines, our results suggest that the EC-derived muscle progenitor cell differs from continuous myoblast cell lines, perhaps by lacking a complementing oncogene responsible for myoblast immortalization.Key words: embryonal carcinoma, oncogene, ras, differentiation, myogenesis.

Functional cloning of mouse chromosomal loci specifically active in embryonal carcinoma stem cells

1988 ◽  
Vol 8 (8) ◽  
pp. 3251-3259
Author(s):  
K Bhat ◽  
M W McBurney ◽  
H Hamada
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Dna Sequences ◽  
Embryonal Carcinoma ◽  
Regulatory Elements ◽  
Embryonal Carcinoma Cell ◽  
P19 Cells ◽  
Embryonal Carcinoma Cell Line ◽  
Cellular Dna ◽  
Chromosomal Loci

Chromosomal loci that are specifically active in embryonal carcinoma stem cells were cloned from the mouse genome by functional selection. P19 cells, a pluripotent embryonal carcinoma cell line, were transfected with an enhancer trap (a plasmid containing an enhancerless inactive neo gene), and NEO+ transformants were isolated. All of the NEO+ cell lines retained pluripotency and expressed the neo gene. When the cells were induced to differentiate, most of the cell lines continued to express the neo gene, while the neo gene in some cell lines became repressed. From the latter group of cell lines, we have cloned the integrated neo gene plus the flanking cellular DNA sequences. Three of the six cloned DNAs possessed a high NEO+-transforming activity in undifferentiated P19 cells. Among these three, two (015 and 052) were inactive in differentiated P19 cells and NIH 3T3 cells, while the remaining one was active in these differentiated cells. Deletion analysis suggested that both 015 and 052 contain two regulatory elements (promoter and enhancer) of cellular DNA origin. The putative enhancer and promoter are separated by at least 6 kilobases in 015 and 1 kilobase in 052. Therefore, 015 and 052 cloned fragments contain regulatory DNA elements that are specifically active in the embryonal carcinoma stem cells.

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.

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