New class of polyomavirus mutant that can persist as free copies in F9 embryonal carcinoma cells

1986 ◽  
Vol 6 (11) ◽  
pp. 3920-3927
Author(s):  
K Ariizumi ◽  
H Ariga
Keyword(s):  
Embryonal Carcinoma ◽  
Regulatory Region ◽  
Host Cells ◽  
Circular Dna ◽  
New Class ◽  
Differentiated Cells ◽  
Copy Numbers ◽  
Undifferentiated Cells ◽  
Ec Cells ◽  
F9 Embryonal Carcinoma Cells

A small circular DNA was found extrachromosomally in a clone of F9 embryonal carcinoma (EC) cells at high copy numbers per cell. The DNA was cloned in plasmid pUC19. Restriction endonuclease analyses of the DNA indicated that the DNA (fPyF9) was a mutant of polyomavirus (Py) DNA and had a mutation in a noncoding regulatory region. There have been many reports on the isolation of Py mutants capable of replication in undifferentiated cells. However, fPyF9 was different from other Py mutants in the following aspects: it was harbored stably as a free copy at 1 X 10(4) to 5 X 10(4) copies per cell in EC cells; it replicated in undifferentiated cells better than in differentiated cells; it was extremely rearranged in the sequences of the enhancer B domain; and it carried in the enhancer B domain three copies of an exogenous sequence which does not exist in Py strain A2. From these observations, we propose a new class of Py EC mutant which has an autonomous state similar to that of plasmid and small circular DNA in host cells.

scholarly journals New class of polyomavirus mutant that can persist as free copies in F9 embryonal carcinoma cells.

1986 ◽  
Vol 6 (11) ◽  
pp. 3920-3927 ◽  
Author(s):  
K Ariizumi ◽  
H Ariga
Keyword(s):  
Embryonal Carcinoma ◽  
Regulatory Region ◽  
Host Cells ◽  
Circular Dna ◽  
New Class ◽  
Differentiated Cells ◽  
Copy Numbers ◽  
Undifferentiated Cells ◽  
Ec Cells ◽  
F9 Embryonal Carcinoma Cells

A small circular DNA was found extrachromosomally in a clone of F9 embryonal carcinoma (EC) cells at high copy numbers per cell. The DNA was cloned in plasmid pUC19. Restriction endonuclease analyses of the DNA indicated that the DNA (fPyF9) was a mutant of polyomavirus (Py) DNA and had a mutation in a noncoding regulatory region. There have been many reports on the isolation of Py mutants capable of replication in undifferentiated cells. However, fPyF9 was different from other Py mutants in the following aspects: it was harbored stably as a free copy at 1 X 10(4) to 5 X 10(4) copies per cell in EC cells; it replicated in undifferentiated cells better than in differentiated cells; it was extremely rearranged in the sequences of the enhancer B domain; and it carried in the enhancer B domain three copies of an exogenous sequence which does not exist in Py strain A2. From these observations, we propose a new class of Py EC mutant which has an autonomous state similar to that of plasmid and small circular DNA in host cells.

T-antigen-independent replication of polyomavirus DNA in murine embryonal carcinoma cells

1984 ◽  
Vol 4 (2) ◽  
pp. 317-323
Author(s):  
L Dandolo ◽  
J Aghion ◽  
D Blangy
Keyword(s):  
Embryonal Carcinoma ◽  
T Antigen ◽  
Carcinoma Cells ◽  
Wild Type ◽  
Viral Dna ◽  
Differentiated Cells ◽  
Independent Replication ◽  
Ec Cells ◽  
Early Transcription

Expression of wild-type polyomavirus (Py) is restricted in murine embryonal carcinoma (EC) cells. The block appears to be located at the level of early transcription. Since no T antigen is produced, we investigated the fate of viral DNA upon infection of these cells; we showed that wild-type Py DNA replicates efficiently in all EC cells, probably via a T-antigen-independent mechanism. Furthermore, we studied, at permissive and restrictive temperatures, the replication of tsa (thermosensitive for T antigen) viral DNA of an in vitro-constructed deletion mutant lacking part of the early region coding sequences and of a double mutant carrying both the tsa mutation and the PyEC F9 mutation (allowing expression of early and late viral functions in EC cells). Our results imply that replication of wild-type A2 strain Py DNA can occur in EC cells in the absence of a functional T antigen. However, this protein clearly enhances viral DNA replication and is absolutely required in differentiated cells.

Accumulation of microtubule-associated protein 1A (MAP1A) in differentiating P19 embryonal carcinoma cells

1995 ◽  
Vol 73 (9-10) ◽  
pp. 695-702 ◽  
Author(s):  
A. R. Vaillant ◽  
D. L. Brown
Keyword(s):  
Neurite Outgrowth ◽  
Mitotic Spindle ◽  
Embryonal Carcinoma ◽  
Growth Cones ◽  
Carcinoma Cells ◽  
Embryonal Carcinoma Cells ◽  
Protein Levels ◽  
Undifferentiated Cells ◽  
P19 Embryonal Carcinoma Cells ◽  
Ec Cells

We have examined the accumulation of MAP1A in retinoic acid induced P19 embryonal carcinoma (EC) neurons. By immunofluorescent confocal microscopy, MAP1A was detected in the mitotic spindle of undifferentiated cells but was not evident in association with the interphase microtubules in most cells. By day 4 of differentiation, when neurite outgrowth was underway, MAP1A was co-localized with microtubules in all neurites but was absent from growth cones. By day 8, substantial neurite outgrowth had occurred and MAP1A was seen in all processes. At day 12, no further neurite outgrowth was evident and existing neurites were organized into fascicles. Western blotting and ELISA showed that MAP1A protein levels increased during differentiation. Peak accumulation occurred no later than day 8, coinciding with the period of neurite outgrowth, and then decreased after day 8. The results suggest that in differentiating P19 EC cells MAP1A modulates microtubule dynamics during neurite outgrowth.Key words: MAP1A, neuronal cytoskeleton, neurite outgrowth.

Cyclic AMP response element-binding protein and the catalytic subunit of protein kinase A are present in F9 embryonal carcinoma cells but are unable to activate the somatostatin promoter

1992 ◽  
Vol 12 (3) ◽  
pp. 1096-1106
Author(s):  
N Masson ◽  
M Ellis ◽  
S Goodbourn ◽  
K A Lee
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinase A ◽  
Embryonal Carcinoma ◽  
Carcinoma Cells ◽  
Catalytic Subunit ◽  
Embryonal Carcinoma Cells ◽  
Undifferentiated Cells ◽  
F9 Embryonal Carcinoma Cells ◽  
Kinase A

The cyclic AMP (cAMP) response elements (CREs) of the somatostatin and vasoactive intestinal peptide (VIP) promoters contain binding sites for CRE-binding protein (CREB) that are essential for cAMP-regulated transcription. Using F9 embryonal carcinoma cells, we show that the somatostatin and VIP promoters exhibit a differentiation-dependent cAMP response, demonstrating that these promoters are regulated by transcription factors that become active during differentiation. Lack of cAMP responsiveness of the somatostatin promoter in undifferentiated cells is not due to the absence of known positive-acting factors (the catalytic subunit of protein kinase A [cPKA] and CREB) or a general inhibition of protein kinase A activity. Since overexpression of exogenous cPKA and CREB is sufficient to activate the somatostatin promoter in undifferentiated cells, these findings suggest that a negative factor(s) represses endogenous cPKA and CREB. In contrast to their effects on somatostatin, exogenous CREB and cPKA do not activate the VIP promoter. Thus, despite coregulation during differentiation and the ability to bind CREB, the somatostatin and VIP promoters are not coordinately activated by CREB in undifferentiated F9 cells.

scholarly journals T-antigen-independent replication of polyomavirus DNA in murine embryonal carcinoma cells.

1984 ◽  
Vol 4 (2) ◽  
pp. 317-323 ◽  
Author(s):  
L Dandolo ◽  
J Aghion ◽  
D Blangy
Keyword(s):  
Embryonal Carcinoma ◽  
T Antigen ◽  
Carcinoma Cells ◽  
Wild Type ◽  
Viral Dna ◽  
Differentiated Cells ◽  
Independent Replication ◽  
Ec Cells ◽  
Early Transcription

Expression of wild-type polyomavirus (Py) is restricted in murine embryonal carcinoma (EC) cells. The block appears to be located at the level of early transcription. Since no T antigen is produced, we investigated the fate of viral DNA upon infection of these cells; we showed that wild-type Py DNA replicates efficiently in all EC cells, probably via a T-antigen-independent mechanism. Furthermore, we studied, at permissive and restrictive temperatures, the replication of tsa (thermosensitive for T antigen) viral DNA of an in vitro-constructed deletion mutant lacking part of the early region coding sequences and of a double mutant carrying both the tsa mutation and the PyEC F9 mutation (allowing expression of early and late viral functions in EC cells). Our results imply that replication of wild-type A2 strain Py DNA can occur in EC cells in the absence of a functional T antigen. However, this protein clearly enhances viral DNA replication and is absolutely required in differentiated cells.

scholarly journals Cyclic AMP response element-binding protein and the catalytic subunit of protein kinase A are present in F9 embryonal carcinoma cells but are unable to activate the somatostatin promoter.

1992 ◽  
Vol 12 (3) ◽  
pp. 1096-1106 ◽  
Author(s):  
N Masson ◽  
M Ellis ◽  
S Goodbourn ◽  
K A Lee
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinase A ◽  
Embryonal Carcinoma ◽  
Carcinoma Cells ◽  
Catalytic Subunit ◽  
Embryonal Carcinoma Cells ◽  
Undifferentiated Cells ◽  
F9 Embryonal Carcinoma Cells ◽  
Kinase A

The cyclic AMP (cAMP) response elements (CREs) of the somatostatin and vasoactive intestinal peptide (VIP) promoters contain binding sites for CRE-binding protein (CREB) that are essential for cAMP-regulated transcription. Using F9 embryonal carcinoma cells, we show that the somatostatin and VIP promoters exhibit a differentiation-dependent cAMP response, demonstrating that these promoters are regulated by transcription factors that become active during differentiation. Lack of cAMP responsiveness of the somatostatin promoter in undifferentiated cells is not due to the absence of known positive-acting factors (the catalytic subunit of protein kinase A [cPKA] and CREB) or a general inhibition of protein kinase A activity. Since overexpression of exogenous cPKA and CREB is sufficient to activate the somatostatin promoter in undifferentiated cells, these findings suggest that a negative factor(s) represses endogenous cPKA and CREB. In contrast to their effects on somatostatin, exogenous CREB and cPKA do not activate the VIP promoter. Thus, despite coregulation during differentiation and the ability to bind CREB, the somatostatin and VIP promoters are not coordinately activated by CREB in undifferentiated F9 cells.

Distribution of desmosomal proteins in F9 embryonal carcinoma cells and epithelial cell derivatives

1992 ◽  
Vol 103 (1) ◽  
pp. 69-80
Author(s):  
K.T. Trevor ◽  
L.S. Steben
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Embryonal Carcinoma ◽  
Cell Types ◽  
Insoluble Fraction ◽  
Epithelial Surface ◽  
Ec Cells ◽  
Steady State Levels ◽  
F9 Embryonal Carcinoma Cells ◽  
Triton X

In diverse epithelia, cytoskeletal keratin intermediate filaments (IFs) associated with the cytoplasmic face of intercellular junctional desmosomes. The processes underlying desmosome formation and keratin IF interactions remain unclear. We have examined F9 embryonal carcinoma (EC) cell differentiation as a model for embryonic development of epithelial surface desmosomes. As determined by immunofluorescence microscopy and biochemical protein techniques, F9 EC cells, which lack surface desmosomes and keratin IFs, express the desmosomal proteins desmoplakins I and II (DP I/II), desmoglein I (DG I) and plakoglobin (PK). DP I/II are present at low level and are relatively soluble in buffer containing Triton X-100. Immunofluorescence localizes DP I/II to the juxtanuclear, centrosomal region. Species of DG I and PK are detected in both the Triton X-100-soluble and -insoluble protein fractions. DG I appears dispersed throughout the cell while PK resides at cell-cell boundaries. In epithelial cell cultures induced by retinoic acid (RA) treatment, each of the desmosomal proteins is organized into punctate desmosome-like structures with the appearance of simple epithelial K8/K18 IFs. The steady-state levels of DP I/II and PK increase with a partitioning of the majority of the desmosomal components into the insoluble fraction. In epithelial cells which lack distinct surface desmosomes, an intracellular association of keratin bundles with DP I/II is observed, suggesting that keratin filaments may facilitate the translocation of these desmosomal components to the cell surface. Parietal endoderm-like cells, derived by treatment with RA and dibutyryl cAMP, are analogous to F9 EC cells in that the cells express desmosomal components and do not display surface desmosomes. Moreover, K8 and K18 do not form distinct filaments, and the protein and RNA levels of K8 are low relative to epithelial cells induced by RA alone. The F9 system appears to be a relevant model for studies of desmosome assembly and the potential interactions of desmosomal proteins and keratin IFs in embryonic epithelial cell types.

scholarly journals PC12 and THP-1 Cell Lines as Neuronal and Microglia Model in Neurobiological Research

2021 ◽  
Vol 11 (9) ◽  
pp. 3729
Author(s):  
Katarzyna Balon ◽  
Benita Wiatrak
Keyword(s):  
Cell Culture ◽  
Dna Damage ◽  
Cell Lines ◽  
Inflammatory Factor ◽  
Research Model ◽  
Differentiated Cells ◽  
Undifferentiated Cells ◽  
Primary Cell Lines ◽  
Neurobiological Research ◽  
The Impact

Models based on cell cultures have become a useful tool in modern scientific research. Since primary cell lines are difficult to obtain and handle, neoplasm-derived lines like PC12 and THP-1 offer a cheap and flexible solution for neurobiological studies but require prior differentiation to serve as a neuronal or microglia model. PC12 cells constitute a suitable research model only after differentiation by incubation with nerve growth factor (NGF) and THP-1 cells after administering a differentiation factor such as phorbol 12-myristate-13-acetate (PMA). Still, quite often, studies are performed on these cancer cells without differentiation. The study aimed to assess the impact of PC12 or THP-1 cell differentiation on sensitivity to harmful factors such as Aβ25-35 (0.001–5 µM) (considered as one of the major detrimental factors in the pathophysiology of Alzheimer’s disease) or lipopolysaccharide (1–100 µM) (LPS; a pro-inflammatory factor of bacterial origin). Results showed that in most of the tests performed, the response of PC12 and THP-1 cells induced to differentiation varied significantly from the effect in undifferentiated cells. In general, differentiated cells showed greater sensitivity to harmful factors in terms of metabolic activity and DNA damage, while in the case of the free radicals, the results were heterogeneous. Obtained data emphasize the importance of proper differentiation of cell lines of neoplastic origin in neurobiological research and standardization of cell culture handling protocols to ensure reliable results.

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