P19 Embryonal Cells Research Articles

Gα12 and Gα13 Mediate Differentiation of P19 Mouse Embryonal Carcinoma Cells in Response to Retinoic Acid

P19 mouse embryonal carcinoma cells can be stimulated to differentiate into endodermal-like, mesodermal-like, and neuronal-like phenotypes in response to specific morphogens. At low concentrations, retinoic acid stimulates P19 embryonal cells to differentiate to cells displaying an endodermal phenotype, whereas at higher concentrations it stimulates differentiation to neuroectoderm. The Galpha12 and Galpha13 subunits of heterotrimeric G-proteins are expressed in the embryonal P19 cells and stimulated in response to retinoic acid as the cells differentiate to endodermal or neuroectodermal phenotypes. Suppression of the expression of either Galpha12 or Galpha13 by antisense RNA is shown to promote cell detachment from substratum and apoptosis. Expression of the constitutively active, mutant form of Galpha12 (Q229L), in contrast, stimulates loss of the embryonal phenotype. Expression of the constitutively active form of Galpha13 (Q226L) stimulates differentiation of the cells from embryonal to endodermal, in the absence of retinoic acid. Thus, both Galpha12 and Galpha13 are essential to stimulation of cell differentiation by retinoic acid. Deficiency of either Galpha12 or Galpha13 increases programmed cell death.

Low mutagenic effects of mitomycin C in undifferentiated embryonic P19 cells are correlated with efficient cell cycle control

Pluripotent undifferentiated embryonic carcinoma cells of line P19 and their differentiated progeny, epithelioid ectoderm-like EPI-7 cells, showed different responses to mitomycin C (MMC) with respect to induction of micronuclei, mutations at the HPRT-locus and cell cycle control. Cytotoxic effects of MMC after a 5-h treatment were lower in undifferentiated P19 cells than in differentiated EPI-7 cells with IC 50 values of 1.3 and 0.25 μM for P19 and EPI-7 cells, respectively. MMC did not induce 6-thioguanine-resistant mutants in P19 cells but significantly increased the mutation frequency in EPI-7 cells with concentrations of 0.25, 0.5 and 1.0 μM MMC. Micronuclei determined by flow-cytometry were induced by MMC in both cell lines at equitoxic concentrations of 4.5 (P19) and 0.75 (EPI-7) μM, reducing the viability in both cell lines to 10%. Whereas the induction of micronuclei in P19 cells was maximal 28 h after treatment and declined thereafter, micronucleus induction peaked 48 h post treatment in EPI-7 cells and remained significantly increased even 67 h after the treatment. Flow-cytometric determination of the distribution of MMC-treated P19 and EPI-7 within the cell cycle revealed a distinct G 2 M -block in P19 cells, whereas EPI-7 cells showed normal progression through S-phase and a negligible G 2 M -block . Therefore, we conclude that the lower effectivity of MMC to induce gene mutations and micronuclei in P19 cells seemed to be correlated with a more efficient cell cycle control in undifferentiated compared to differentiated EPI-7 cells.

High levels of mouse thymosin β4 mRNA in differentiating P19 embryonic cells and during development of cardiovascular tissues

The self assembly of actin and the large number of actin-binding proteins are important in the establishment of cell shape and function during embryogenesis. Thymosin β 4 (T β 4) is a small acidic peptide that participates in the regulation of actin polymerization in mammalian cells. In the present work, we report the presence of the mRNA encoding for T β 4 in mouse embryonic stem cells and its induction in P19 embryonal cells stimulated to differentiate into ectodermal-like (neurons and glia) or mesodermal-like cells (cardiac and skeletal muscle). The induction of T β 4 mRNA in P19 cells was confirmed by in situ hybridization analysis of early mouse postimplantation embryos. Noteworthy, we observed an important hybridization signal in several areas of the embryo specially in blood vessels and in heart tissues, suggesting a role for this peptide in angiogenesis. In conclusion, the results presented here demonstrate the expression of T β 4 gene during early embryogenesis which immediately suggests an important role for this peptide in developmental processes requiring actin-based functions such as the formation of cardiovascular system.

Ras Induces a General DNA Demethylation Activity in Mouse Embryonal P19 Cells

We demonstrate that expression of v-Ha-ras in mouse embryonal P19 cells results in genome-wide demethylation. Analysis of the pattern of methylation of specific genes reveals that different types of genes are demethylated in the ras transfectants: skeletal muscle specific genes, a gene specifically expressed in the adrenal cortex (c21), ubiquitous genes, and exogenously introduced sequences. Transient transfection and in vitro demethylation assays reveal that the ras transfectants express high levels of a general DNA demethylation activity. This demonstrates that the general DNA demethylation activity in mouse embryonal cells is controlled by an important cellular signal transducer and that DNA demethylase is a potential downstream effector of Ras.

Neuronal differentiation of P19 embryonal cells exhibits cell-specific regulation of neurotrophin receptors.

During development, the regulation of expression of the Trk family of tyrosine kinase receptors plays an important role in defining the cellular responses to neurotrophin action. We report here that neurotrophin receptors are differentially expressed in distinct populations of retinoic acid-differentiated P19 cells. TrkB, the tyrosine kinase receptor for brain-derived neurotrophic factor, and LNGFR, the low-affinity receptor for all neutrophins, are preferentially expressed in P19-derived neurones. In contrast, retinoic acid induces the expression of TrkA, the high-affinity receptor for nerve growth factor, and of a non-catalytic form of TrkB, in non-neural subsets of differentiated cells. We propose P19 cells as a model system to study the mechanisms controlling the expression of neurotrophin receptors and the responsiveness of developing neurones to a specific neurotrophin.

Differentiation of a stem cell line toward a neuronal phenotype

This study examined the morphology and the development of inward currents in the course of differentiation of a stem cell toward a neuronal phenotype. Using the P19 embryonal cell line, whole-cell current profiles of P19 cells before, during and after retinoic acid-induced differentiation were matched with their morphology as well as with the expression of neuron-specific enolase-like immunoreactivity. Prior to and during the initial 48 hr of retinoic acid treatment, P19 cells either lacked detectable currents or expressed a voltage-dependent outward potassium current, did not display neuron-like morphology and did not express neuron-specific enolase-like immunoreactivity. Upon completion of retinoic acid treatment, the current profile of fully differentiated P19 cells was hallmarked by a large voltage-dependent inward current which consisted of a sodium current and a smaller cobalt-sensitive calcium component, in addition to the potassium current observed earlier. Such cells invariably emitted neuntes and displayed neuron-specific enolase-like immunoreactivity. Interestingly, coupling was prevalent among P19 cells in the undifferentiated state but was absent in the fully differentiated cultures. In studying cells undergoing neuronal differentiation, these results underscore the importance of taking into account both electrical properties and morphological considerations in determining the degree of differentiation.