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.

The effect of retinoic acid pretreatment on the ability of murine embryonal carcinoma and inner cell mass cells to participate in chimaera development

Development ◽  
1986 ◽  
Vol 98 (1) ◽  
pp. 99-110
Author(s):  
B. K. Waters ◽  
J. Rossant
Keyword(s):  
Retinoic Acid ◽  
Embryonal Carcinoma ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
P19 Cells ◽  
Acid Pretreatment ◽  
Preimplantation Mouse ◽  
Embryo Cells

Certain embryonal carcinoma (EC) cell lines can colonize the embryo following blastocyst injection or embryo aggregation, giving rise to EC-embryo chimaeras. However, such chimaeras often develop abnormally. For example, diploid P19 cells colonize the embryo readily but resulting chimaeras are usually abnormal, with persistence of tumour cells. Retinoic acid (RA) induces differentiation of EC cells to a variety of cell types in vitro but, in this study, it was shown that pretreatment of P19 cells with RA did not result in more normal development of P19-embryo chimaeras. The only significant effect of RA was to reduce the ability of P19 cells to participate in embryonic development at all after blastocyst injection. RA did not have a direct toxic or teratogenic effect on preimplantation mouse embryos and did not affect the ability of pluripotent embryo cells to colonize chimaeras. Therefore, RA may not be the normal inducer of differentiation in early embryogenesis.

Changes in protein synthesis during differentiation of embryonal carcinoma cells, and a comparison with embryo cells

Development ◽  
1980 ◽  
Vol 59 (1) ◽  
pp. 187-206
Author(s):  
R. H. Lovell-Badge ◽  
M. J Evans
Keyword(s):  
Protein Synthesis ◽  
Embryonal Carcinoma ◽  
Inner Cell Mass ◽  
Embryoid Body ◽  
Cell Mass ◽  
Cell Types ◽  
Embryonic Cell ◽  
Embryoid Bodies ◽  
Ec Cells

Two-dimensional electrophoresis was used to find changes in protein synthesis occurring as pluripotent embryonal carcinoma (EC) cells differentiate to give embryoid bodies in vitro. 2-D patterns from other embryonic cell lines, and from the inner cell mass (ICM) cells of mouse embryos, were also analysed for the expression of those proteins showing some change during embryoid body formation and for overall differences between these and the EC cells. Most changes in protein synthesis occurred before 12 h but endoderm was not discerned morphologically on the outside of EC cell clumps until at least 18 h after their suspension. The number of changes occurring is small compared with the number of polypeptides resolved, but is in line with similar studies. Comparisons with nullipotent EC cells and an endodermal cell line have allowed these changes to be assigned, tentatively, to the different cell types within embryoid bodies, and may allow them to be used as markers of differentiation. Comparisons between the 2-D patterns derived from ICMs and EC cells reveal substantial differences between the two that might not have been expected from their developmental homology. The importance of these differences to their pluripotentiality is 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.

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.

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 Expression of the Metalloproteinase ADAM8 Is Upregulated in Liver Inflammation Models and Enhances Cytokine Release In Vitro

2021 ◽  
Vol 2021 ◽  
pp. 1-17
Author(s):  
Tanzeela Awan ◽  
Aaron Babendreyer ◽  
Justyna Wozniak ◽  
Abid Mahmood Alvi ◽  
Viktor Sterzer ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inflammatory Mediators ◽  
Liver Tissue ◽  
Stellate Cell ◽  
Hepatoma Cell ◽  
Cell Types ◽  
Chronic Liver ◽  
Liver Inflammation ◽  
Tnf Α

Acute and chronic liver inflammation is driven by cytokine and chemokine release from various cell types in the liver. Here, we report that the induction of inflammatory mediators is associated with a yet undescribed upregulation of the metalloproteinase ADAM8 in different murine hepatitis models. We further show the importance of ADAM8 expression for the production of inflammatory mediators in cultured liver cells. As a model of acute inflammation, we investigated liver tissue from lipopolysaccharide- (LPS-) treated mice in which ADAM8 expression was markedly upregulated compared to control mice. In vitro, stimulation with LPS enhanced ADAM8 expression in murine and human endothelial and hepatoma cell lines as well as in primary murine hepatocytes. The enhanced ADAM8 expression was associated with an upregulation of TNF-α and IL-6 expression and release. Inhibition studies indicate that the cytokine response of hepatoma cells to LPS depends on the activity of ADAM8 and that signalling by TNF-α can contribute to these ADAM8-dependent effects. The role of ADAM8 was further confirmed with primary hepatocytes from ADAM8 knockout mice in which TNF-α and IL-6 induction and release were considerably attenuated. As a model of chronic liver injury, we studied liver tissue from mice undergoing high-fat diet-induced steatohepatitis and again observed upregulation of ADAM8 mRNA expression compared to healthy controls. In vitro, ADAM8 expression was upregulated in hepatoma, endothelial, and stellate cell lines by various mediators of steatohepatitis including fatty acid (linoleic-oleic acid), IL-1β, TNF-α, IFN-γ, and TGF-β. Upregulation of ADAM8 was associated with the induction and release of proinflammatory cytokines (TNF-α and IL-6) and chemokines (CX3CL1). Finally, knockdown of ADAM8 expression in all tested cell types attenuated the release of these mediators. Thus, ADAM8 is upregulated in acute and chronic liver inflammation and is able to promote inflammation by enhancing expression and release of inflammatory mediators.

Regulated expression of a transfected human cardiac actin gene during differentiation of multipotential murine embryonal carcinoma cells

1988 ◽  
Vol 8 (1) ◽  
pp. 406-417
Author(s):  
M A Rudnicki ◽  
M Ruben ◽  
M W McBurney
Keyword(s):  
Cardiac Muscle ◽  
Thymidine Kinase ◽  
Embryonal Carcinoma ◽  
Actin Gene ◽  
P19 Cells ◽  
Cardiac Muscle Cells ◽  
Cardiac Actin ◽  
Ec Cells ◽  
Actin Promoter ◽  
Actin Mrna

P19 embryonal carcinoma (EC) cells are multipotential stem cells which can be induced to differentiate in vitro into a variety of cell types, including cardiac muscle cells. A cloned human cardiac actin (CH-actin) gene was transfected into P19 cells, and stable transformants were isolated. Low levels of CH-actin mRNA were present in transformed EC cells, but a marked increase in the level of CH-actin mRNA was found as these cells differentiated into cardiac muscle. The accumulation of CH-actin mRNA paralleled that of the endogenous mouse cardiac actin mRNA. A chimeric gene, which consisted of the CH-actin promoter linked to the herpes simplex virus thymidine kinase coding region, was constructed and transfected into P19 cells. In these transformants, the thymidine kinase protein was located almost exclusively in cardiac muscle cells and was generally not detectable in EC or other nonmuscle cells. These results suggest that the transfected CH-actin promoter functions in the appropriate developmental and tissue-specific manner during the differentiation of multipotential EC cells in culture.

Expression of the K-fgf proto-oncogene is controlled by 3' regulatory elements which are specific for embryonal carcinoma cells

1990 ◽  
Vol 10 (6) ◽  
pp. 2475-2484
Author(s):  
A M Curatola ◽  
C Basilico
Keyword(s):  
Dna Sequences ◽  
Embryonal Carcinoma ◽  
Protein S ◽  
Cell Types ◽  
Regulatory Elements ◽  
Developmentally Regulated ◽  
Cis Acting ◽  
Dna Elements ◽  
Ec Cells ◽  
Cat Expression

Expression of the K-fgf/hst proto-oncogene appears to be restricted to cells in the early stages of development, such as embryonal carcinoma (EC) cells. When EC cells are induced to differentiate, K-fgf expression is drastically repressed. To identify cis-acting DNA elements responsible for this type of regulation, we constructed a plasmid in which cat gene expression was driven by about 1 kilobase of upstream K-fgf human DNA sequences, including the putative promoter, and transfected it into undifferentiated F9 EC cells or HeLa cells as prototypes of cells which express or do not express, respectively, the K-fgf proto-oncogene. This plasmid was essentially inactive in both cell types, and the addition of more than 8 kilobases of DNA sequences upstream of the K-fgf promoter did not lead to any increase in chloramphenicol acetyltransferase (CAT) expression. On the other hand, when we inserted in this plasmid DNA sequences which are 3' of the human K-fgf coding sequences, we could detect a significant stimulation of CAT activity. Analysis of these sequences led to the identification of enhancerlike DNA elements which are part of the 3' noncoding region of K-fgf exon 3 and promote CAT expression only in undifferentiated mouse F9 or human NT2/D1 EC cells, but not in HeLa, 3T3, or differentiated F9 cells, therefore mimicking the physiological expression of the K-fgf proto-oncogene. Similar elements are also present in the 3' region of the murine K-fgf proto-oncogene, in a region showing high homology to the human K-fgf sequences. These regulatory elements can promote CAT expression from heterologous promoters in an EC-specific manner, suggesting that they interact with a specific cellular transacting protein(s) whose expression is developmentally regulated.

Targeting RNF8 Effectively Reverse Cisplatin and Doxorubicin Resistance in Endometrial Cancer

2020 ◽  
Author(s):  
Ben Yang ◽  
Wang Ke ◽  
Yingchun Wan ◽  
Tao Li
Keyword(s):  
Endometrial Cancer ◽  
Cell Lines ◽  
Quantitative Polymerase Chain Reaction ◽  
Mrna Levels ◽  
Mice Model ◽  
Expression Levels ◽  
Gynecological Malignancy ◽  
Ec Cells

Abstract Background Endometrial cancer (EC) is one of the most frequent gynecological malignancy worldwide. However, resistance to chemotherapy remains one of the major difficulties in the treatment of EC. Thus, there is an urgent requirement to understand mechanisms of chemoresistance and identify novel regimens for patients with EC. Methods Cisplatin and doxorubicin resistant cell lines were acquired by continuous exposing parental EC cells to cisplatin or doxorubicin for 3 months. Cell viability was determined by using MTT assay. Protein Expression levels of protein were examined by western blotting assay. mRNA levels were measured by quantitative polymerase chain reaction (qPCR) assay. Ring finger protein 8 (RNF8) knockout cell lines were generated by clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 gene editing assay. Nonhomologous end joining (NHEJ) efficiency were quantified by plasmid based NHEJ assay. DNA double strand breaks (DSB) were generated using laser micro-irradiation. Protein recruitment to DSB was analyzed by immunofluorescent assay. Tumor growth was examined by AN3CA xenograft mice model. Results We found that protein and mRNA expression levels of RNF8 were significantly increased in both cisplatin and doxorubicin resistant EC cells. Cell survival assay showed that RNF deficiency significantly enhanced the sensitivity of resistant EC cells to cisplatin and doxorubicin (P < 0.01). In addition, chemoresistant EC cells exhibited increased NHEJ efficiency. Knockout of RNF8 in chemoresistant EC cells significantly reduced NHEJ efficiency and prolonged Ku80 retention on DSB. Moreover, cisplatin resistant AN3CA xenograft showed that RNF8 deficiency overcame cisplatin resistance. Conclusions Our in vitro and in vivo assays provide evidence for RNF8, which is a NHEJ factor, serving as a promising, novel target in EC chemotherapy.

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