P19 Embryonal Carcinoma Research Articles

Dynamic mitochondrial changes during differentiation of P19 embryonic carcinoma cells into cardiomyocytes.

Murine P19 embryonal carcinoma cells are multipotent cells that can differentiate into cardiomyocytes when treated with dimethyl sulfoxide. This experimental model provides an invaluable tool to study different aspects of cardiac differentiation, such as the function of cardiac‑specific transcription factors and signaling pathways, and the regulation of contractile protein expression. The role of mitochondria during cardiac differentiation is unclear. In this context, we have examined the mitochondrial-related changes in undifferentiated and differentiated P19 cells. We observed that mitochondrial DNA content sharply decreased in P19 cell aggregates compared to undifferentiated cells, accompanied by decreased levels of adenosine triphosphate (ATP) and reactive oxygen species (ROS). Following the aggregation stage, the mitochondrial DNA content reached its highest level on day 7 of the differentiation process, with the intracellular ROS level showing a trend to increase, similar to cellular ATP production. In conclusion, our study on differentiating P19 embryonal carcinoma cells provides new insights into the role of mitochondria in the differentiation of P19 stem cells into beating cardiomyocytes.

Bilobalide induces neuronal differentiation of P19 embryonic carcinoma cells via activating Wnt/β-catenin pathway.

Bilobalide, a natural product extracted from Ginkgo biloba leaf, is known to exhibit a number of pharmacological activities. So far, whether it could affect embryonic stem cell differentiation is still unknown. The main aim of this study was to investigate the effect of bilobalide on P19 embryonic carcinoma cells differentiation and the underlying mechanisms. Our results showed that bilobalide induced P19 cells differentiation into neurons in a concentration- and time-dependent manner. We also found that bilobalide promoted neuronal differentiation through activation of Wnt/β-catenin signaling pathway. Exposure to bilobalide increased inactive GSK-3β phosphorylation, further induced the nuclear accumulation of β-catenin, and also up-regulated the expression of Wnt ligands Wnt1 and Wnt7a. Neuronal differentiation induced by bilobalide was totally abolished by XAV939, an inhibitor of Wnt/β-catenin pathway. These results revealed a novel role of bilobalide in neuronal differentiation from P19 embryonic cells acting through Wnt/β-catenin signaling pathway, which would provide a better insight into the beneficial effects of bilobalide in brain diseases.

Mitochondrial metabolism directs stemness and differentiation in P19 embryonal carcinoma stem cells

The relationship between mitochondrial metabolism and cell viability and differentiation in stem cells (SCs) remains poorly understood. In the present study, we compared mitochondrial physiology and metabolism between P19SCs before/after differentiation and present a unique fingerprint of the association between mitochondrial activity, cell differentiation and stemness. In comparison with their differentiated counterparts, pluripotency of P19SCs was correlated with a strong glycolytic profile and decreased mitochondrial biogenesis and complexity: round, low-polarized and inactive mitochondria with a closed permeability transition pore. This decreased mitochondrial capacity increased their resistance against dichloroacetate. Thus, stimulation of mitochondrial function by growing P19SCs in glutamine/pyruvate-containing medium reduced their glycolytic phenotype, induced loss of pluripotent potential, compromised differentiation and became P19SCs sensitive to dichloroacetate. Because of the central role of this type of SCs in teratocarcinoma development, our findings highlight the importance of mitochondrial metabolism in stemness, proliferation, differentiation and chemoresistance. In addition, the present work suggests the regulation of mitochondrial metabolism as a tool for inducing cell differentiation in stem line therapies.

Spatial and temporal expression of RA70/Scap2 in the developing neural tube

Src kinase-associated phosphoprotein 2 (Ra70/scap2), which was originally isolated as a retinoic acid (RA)-induced gene, associates with molecules that modulate integrin-survival signals. Although RA is essential for vertebrate organogenesis in the posterior region, little is known about the biological role of RA70/Scap2 during development. In the present study, we demonstrate that Ra70/scap2 mRNA is temporally expressed during the RA-induced neuronal differentiation of P19 embryonic carcinoma cells. Homozygous knockout mice in which the Ra70/scap2 gene was replaced with LacZ exhibited embryonic lethality, while heterozygous mice displayed preferential expression of LacZ in posterior neural tissues, including the neural tube and hindbrain during development (E7.5–11.5), but not the forebrain. Ra70/scap2 was expressed in the ependymal layer and ventricular zone in the neural tube, where neuroepithelial cells and neuroblasts with proliferation capacity are localized, respectively. Thus, RA70/Scap2 may be necessary for RA-induced neuronal differentiation from the posterior neuroectoderm.

Foxa1 contributes to the repression of Nanog expression by recruiting Grg3 during the differentiation of pluripotent P19 embryonal carcinoma cells

Transcription factor Foxa1 plays a critical role during neural differentiation and is induced immediately after retinoic acid (RA)-initiated differentiation of pluripotent P19 embryonal carcinoma cells, correlated with the downregulated expression of pluripotency-related genes such as Nanog. To study whether Foxa1 participates in the repression of pluripotency factors, we expressed Foxa1 ectopically in P19 cells and identified that Nanog was repressed directly by Foxa1. We confirmed that Foxa1 was able to interact with Grg3, which is a transcriptional corepressor that expresses in P19 cells as well as during RA-induced P19 cell differentiation. Knockdown of Foxa1 or Grg3 delayed the downregulation of Nanog expression during RA-induced P19 cell differentiation. Furthermore, we found that Foxa1 recruited Grg3 to the Nanog promoter −2kb upstream region and switched the promoter to an inactive chromatin status represented by typical modifications in histone H3. Together, our results suggested a critical involvement of Foxa1 in the negative regulation of Nanog expression during the differentiation of pluripotent stem cells.

The study of P19 stem cell behavior on aligned oriented electrospun poly(lactic‐co‐glycolic acid) nano‐fibers for neural tissue engineering

Recent medical cell therapy using polymeric biomaterial‐loaded stem cells with the capability of differentiation to specific neural population has directed focuses toward the recovery of central nervous system. Definite culture conditions that regulate cell morphogenesis, proliferation, survival, and differentiation to lineage specific cells have a great influence on the cells' life. In this research, random and aligned poly(lactic‐co‐glycolic acid) fibrous scaffolds with nano‐structure were prepared, and the effect of nano‐fiber morphology on the pluripotent P19 embryonic carcinoma cell proliferation and neural differentiation in response to retinoic acid induction was studied. Fiber morphology was assessed by scanning electron microscope (SEM). For in vitro studies, SEM, (4,5‐dimethylazol‐2‐yl)‐2,5‐diphenyl‐2H tetrazolium bromide assay, and immunofluorescent staining were carried out for the assessment of biological properties of the seeded cells. Our results showed that, because of their similarity with natural extracellular matrix, the aligned fibrous substrates provide the suitable surface for cell function as well as the augmentation of cell differentiation. Copyright © 2014 John Wiley & Sons, Ltd.

Efficient and Simple Production of Insulin-Producing Cells from Embryonal Carcinoma Stem Cells Using Mouse Neonate Pancreas Extract, As a Natural Inducer

An attractive approach to replace the destroyed insulin-producing cells (IPCs) is the generation of functional β cells from stem cells. Embryonal carcinoma (EC) stem cells are pluripotent cells which can differentiate into all cell types. The present study was carried out to establish a simple nonselective inductive culture system for generation of IPCs from P19 EC cells by 1–2 weeks old mouse pancreas extract (MPE). Since, mouse pancreatic islets undergo further remodeling and maturation for 2–3 weeks after birth, we hypothesized that the mouse neonatal MPE contains essential factors to induce in vitro differentiation of pancreatic lineages. Pluripotency of P19 cells were first confirmed by expression analysis of stem cell markers, Oct3/4, Sox-2 and Nanog. In order to induce differentiation, the cells were cultured in a medium supplemented by different concentrations of MPE (50, 100, 200 and 300 µg/ml). The results showed that P19 cells could differentiate into IPCs and form dithizone-positive cell clusters. The generated P19-derived IPCs were immunoreactive to proinsulin, insulin and insulin receptor beta. The expression of pancreatic β cell genes including, PDX-1, INS1 and INS2 were also confirmed. The peak response at the 100 µg/ml MPE used for investigation of EP300 and CREB1 gene expression. When stimulated with glucose, these cells synthesized and secreted insulin. Network analysis of the key transcription factors (PDX-1, EP300, CREB1) during the generation of IPCs resulted in introduction of novel regulatory candidates such as MIR17, and VEZF1 transcription factors, as well as MORN1, DKFZp761P0212, and WAC proteins. Altogether, we demonstrated the possibility of generating IPCs from undifferentiated EC cells, with the characteristics of pancreatic β cells. The derivation of pancreatic cells from EC cells which are ES cell siblings would provide a valuable experimental tool in study of pancreatic development and function as well as rapid production of IPCs for transplantation.

A rapid and efficient method for neuronal induction of the P19 embryonic carcinoma cell line

P19 mouse embryonic carcinoma cells are conventionally induced to differentiate into neural cells by suspension culture in the presence of retinoic acid to form cell aggregates, followed by adhesion culture in a poly-l-lysine-coated dish. Drawbacks of this procedure include it taking more than 10 days to obtain mature neurons, and non-neuronal proliferating cells occupying the majority of the cell population with time. Here, we show a novel method for the rapid and efficient neurogenesis of P19 cells, without aggregate formation in a suspension culture. The new approach is based on an adherent serum-free culture in a laminin-coated dish in the presence of FGF8, a γ-secretase inhibitor, and cytosine arabinoside. The new method efficiently induced P19 cells to differentiate into neurons within 4 days, and subsequently into mature neurons that were responsive to several neurotransmitters, giving spontaneous neuronal network activity within 6 days. The novel method accelerated neuritogenesis and enhanced population of neuron selectively compared to the conventional method. Proliferating non-neuronal cells were eliminated by adding cytosine arabinoside during neuronal maturation. The method is useful for studying neuronal differentiation or activities.

Synergistic action in P19 pluripotential cells of retinoic acid and Wnt3a on Cdx1 enhancer elements

Cdx1 encodes a homeodomain protein that regulates expression of some Hox genes. Cdx1 itself is known to be regulated in the primitive streak/tailbud by both retinoic acid (RA) and Wnt3a. Cdx1 in eutherian mammals has two retinoic acid response elements (RAREs), located upstream and in the first intron, and each is adjacent to structural Lef/Tcf motifs. Upstream Lef/Tcf motifs respond to canonical Wnt signalling to activate Cdx1 synergistically with RA. By combined use of reporter assays, immunofluorescence and flow cytometry in mouse P19 embryonal carcinoma cells we show that the Cdx1 intron Lef/Tcf motif also responds to Wnt3a signalling. Synergy between individual Cdx1 RARE and Lef/Tcf motifs can occur whether they are adjacent or distant in the gene. Part, though not all, of the Cdx1 stimulation by RA (in absence of added Wnt3a) likely depends upon Wnt protein produced by the cells themselves, since it is inhibited by mutation of Lef/Tcf motifs, or by IWP-2, an inhibitor of Wnt production. RA and Wnt3a stimulate Cdx1 by increasing both the proportion of P19 cells that are expressing and also their mean level of expression. The expressing/non-expressing sub-populations do not simply correspond with those that express a marker of pluripotentiality, Nanog. We conclude that RA and Wnt3a activate Cdx1 synergistically by overlapping use of both upstream and intron enhancers, and that mouse embryonal carcinoma cell populations display heterogeneity in their response to these activators.

Impact of retinoic acid exposure on midfacial shape variation and manifestation of holoprosencephaly in Twsg1 mutant mice.

Holoprosencephaly (HPE) is a developmental anomaly characterized by inadequate or absent midline division of the embryonic forebrain and midline facial defects. It is believed that interactions between genes and the environment play a role in the widely variable penetrance and expressivity of HPE, although direct investigation of such effects has been limited. The goal of this study was to examine whether mice carrying a mutation in a gene encoding the bone morphogenetic protein (BMP) antagonist twisted gastrulation (Twsg1), which is associated with a low penetrance of HPE, are sensitized to retinoic acid (RA) teratogenesis. Pregnant Twsg1+/− dams were treated by gavage with a low dose of all-trans RA (3.75 mg/kg of body weight). Embryos were analyzed between embryonic day (E)9.5 and E11.5 by microscopy and geometric morphometric analysis by micro-computed tomography. P19 embryonal carcinoma cells were used to examine potential mechanisms mediating the combined effects of increased BMP and retinoid signaling. Although only 7% of wild-type embryos exposed to RA showed overt HPE or neural tube defects (NTDs), 100% of Twsg1−/− mutants exposed to RA manifested severe HPE compared to 17% without RA. Remarkably, up to 30% of Twsg1+/− mutants also showed HPE (23%) or NTDs (7%). The majority of shape variation among Twsg1+/− mutants was associated with narrowing of the midface. In P19 cells, RA induced the expression of Bmp2, acted in concert with BMP2 to increase p53 expression, caspase activation and oxidative stress. This study provides direct evidence for modifying effects of the environment in a genetic mouse model carrying a predisposing mutation for HPE in the Twsg1 gene. Further study of the mechanisms underlying these gene-environment interactions in vivo will contribute to better understanding of the pathogenesis of birth defects and present an opportunity to explore potential preventive interventions.

AP-1-mediated expression of brain-specific class IVa β-tubulin in P19 embryonal carcinoma cells.

Expression of brain-specific phenotypes increased in all trans retinoic acid (ATRA)-induced neural differentiation of mouse P19 embryonal carcinoma cells. Among these phenotypes, expression of class IVa β-tubulin isotype (TUBB4a) was particularly enhanced in neural differentiation. Transient transfection assays employing a reporter construct found that ATRA-mediated regulatory region of the TUBB4a gene lay in the region from −83 nt to +137 nt relative to the +1 transcription start site. Site-directed mutagenesis in the AP-1 binding site at −29/−17 suggested that the AP-1 binding site was a critical region for ATRA-mediated TUBB4a expression. Chromatin immunoprecipitation experiments suggested participation of JunD and activating transcription factor-2 (ATF2) in TUBB4a expression. Additionally, exogenous induction of the dominant-negative (dn) type of JunD canceled ATRA-induced upregulation of TUBB4a, and the dn type of ATF2 suppressed even the basal activity. Further immunoblot study revealed an ATRA-mediated increase in JunD protein, while a significant amount of ATF2 protein was constantly produced. These results suggest that differentiation-mediated activation of JunD results in enhanced TUBB4a expression.

A fluorescence microplate screen assay for the detection of neurite outgrowth and neurotoxicity using an antibody against βIII-tubulin

The majority of environmental and commercial chemicals have not been evaluated for their potential to cause neurotoxicity. We have investigated if neuron specific anti-βIII-tubulin antibodies are useful in a microplate assay of neurite outgrowth of retinoic acid-induced neurons from mouse P19 embryonal carcinoma cells. By incubating the P19-derived neurons with the primary anti-βIII-tubulin antibody and a secondary Alexa Fluor 488-conjugated antibody, followed by measuring the fluorescence in a microplate reader, a time-dependent increase in anti-βIII-tubulin immunofluorescence was observed. The relative fluorescence units increased by 4.3-fold from 2 to 10days in culture. The results corresponded well with those obtained by semi-automatic tracing of neurites in fluorescence microscopy images of βIII-tubulin-labeled neurons. The sensitivity of the neurite outgrowth assay using a microplate reader to detect neurotoxicity produced by nocodazole, methyl mercury chloride and okadaic acid was significantly higher than for a cell viability assay measuring intracellular fluorescence of calcein-AM. The microplate-based method to measure toxicity targeting neurites using anti-βIII-tubulin antibodies is however less sensitive than the extracellular lactate dehydrogenase activity assay to detect general cytotoxicity produced by high concentrations of clomipramine, or glutamate-induced excitotoxicity.In conclusion, the fluorescence microplate assay for the detection of neurite outgrowth by measuring changes in βIII-tubulin immunoreactivity is a rapid and sensitive method to assess chemical- or toxin-induced neurite toxicity.

Nspc1 regulates the key pluripotent Oct4–Nanog–Sox2 axis in P19 embryonal carcinoma cells via directly activating Oct4

Nspc1 is an identified transcription repressor. However, transiently up-regulated or down-regulated Nspc1 in P19 embryonal carcinoma cells affects expression levels of Oct4, Sox2 and Nanog in a positive correlation. Luciferase activity assays verified that Nspc1 regulates the Oct4 promoter in a dose dependent manner. ChIP assay shows that Nspc1 activates Oct4 by directly binding to the (−1021 to −784) region of Oct4 promoter. Dominant negative analysis indicated the activation is dependent on the retinoid acid response element (RARE). We demonstrated Nspc1 has a positive role in maintaining the pluripotency of P19 cells by directly regulating Oct4.

RING finger protein 10 regulates retinoic acid‐induced neuronal differentiation and the cell cycle exit of P19 embryonic carcinoma cells

Rnf10 is a member of the RING finger protein family. Recently, a number of RING finger proteins were reported to be involved in neuronal differentiation, development, and proliferation. In this study, we observed that the mRNA levels and protein expression of Rnf10 increase significantly upon the retinoic acid-induced neuronal differentiation of P19 cells. Knockdown of Rnf10 by RNA interference significantly impaired neuronal differentiation of P19 cells by attenuating the expression of neuronal markers. Cell cycle profiling revealed that Rnf10-depleted cells were unable to establish cell cycle arrest after RA treatment. In agreement with flow cytometry analysis, increased cell proliferation was observed after RA induction in Rnf10 knockdown cells as determined by a BrdU incorporation assay. Moreover, like Rnf10, the mRNA levels and protein expression of p21 and p27 also increased upon RA induction. Rnf10 knockdown only resulted in a reduction of p21 expression, while p27 and p57 expression remained unchanged, indicating that Rnf10 may regulate cell cycle exit through the p21 pathway. Ectopic p21 expression partially rescued the effect of Rnf10 depletion on the neuronal differentiation of P19 cells. Collectively, these results showed that increase in Rnf10 expression upon RA induction is necessary for the positive regulation of cyclin kinase inhibitor p21 expression, which leads to cell cycle arrest and is critical for neuronal differentiation.

Sox2 Level Is a Determinant of Cellular Reprogramming Potential

Epiblast stem cells (EpiSCs) and embryonic stem cells (ESCs) differ in their in vivo differentiation potential. While ESCs form teratomas and efficiently contribute to the development of chimeras, EpiSCs form teratomas but very rarely chimeras. In contrast to their differentiation potential, the reprogramming potential of EpiSCs has not yet been investigated. Here we demonstrate that the epiblast-derived pluripotent stem cells EpiSCs and P19 embryonal carcinoma cells (ECCs) exhibit a lower reprogramming potential than ESCs and F9 ECCs. In addition, we show that the low reprogramming ability is due to the lower levels of Sox2 in epiblast-derived stem cells. Consistent with this observation, overexpression of Sox2 enhances reprogramming efficiency. In summary, these findings suggest that a low reprogramming potential is a general feature of epiblast-derived stem cells and that the Sox2 level is a determinant of the cellular reprogramming potential.

Aza-induced cardiomyocyte differentiation of P19 EC-cells by epigenetic co-regulation and ERK signaling

Stem cells in cell based therapy for cardiac injury is being potentially considered. However, genetic regulatory networks involved in cardiac differentiation are not clearly understood. Among stem cell differentiation models, mouse P19 embryonic carcinoma (EC) cells, are employed for studying (epi)genetic regulation of cardiomyocyte differentiation. Here, we comprehensively assessed cardiogenic differentiation potential of 5-azacytidine (Aza) on P19 EC-cells, associated gene expression profiles and the changes in DNA methylation, histone acetylation and activated-ERK signaling status during differentiation. Initial exposure of Aza to cultured EC-cells leads to an efficient (55%) differentiation to cardiomyocyte-rich embryoid bodies with a threefold (16.8%) increase in the cTnI+ cardiomyocytes. Expression levels of cardiac-specific gene markers i.e., Isl-1, BMP-2, GATA-4, and α-MHC were up-regulated following Aza induction, accompanied by differential changes in their methylation status particularly that of BMP-2 and α-MHC. Additionally, increases in the levels of acetylated-H3 and pERK were observed during Aza-induced cardiac differentiation. These studies demonstrate that Aza is a potent cardiac inducer when treated during the initial phase of differentiation of mouse P19 EC-cells and its effect is brought about epigenetically and co-ordinatedly by hypo-methylation and histone acetylation-mediated hyper-expression of cardiogenesis-associated genes and involving activation of ERK signaling.

Csn3 Gene Is Regulated by All-Trans Retinoic Acid during Neural Differentiation in Mouse P19 Cells

κ-Casein (CSN3) is known to play an essential role in controlling the stability of the milk micelles. We found that the expression of Csn3 was induced by all-trans retinoic acid (ATRA) during neural differentiation in P19 embryonal carcinoma cells from our study using DNA microarray. In this paper, we describe the detailed time course of Csn3 expression and the induction mechanism of Csn3 transcription activation in this process. The Csn3 expression was induced rapidly and transiently within 24 h of ATRA treatment. Retinoic acid receptor (RAR)-specific agonists were used in expression analysis to identify the RAR subtype involved upregulation of Csn3; a RARα-specific agonist mimicked the effects of ATRA on induction of Csn3 expression. Therefore, RARα may be the RAR subtype mediating the effects of ATRA on the induction of Csn3 gene transcription in this differentiation-promoting process of P19 cells. We found that the promoter region of Csn3 contained a typical consensus retinoic acid response element (RARE), and this RARE was necessary for ATRA-dependent transcriptional regulation. We confirmed that RARα bound to this RARE sequence in P19 cells. These findings indicated that the Csn3 expression is upregulated via ATRA-bound RARα and binding of this receptor to the RARE in the Csn3 promoter region. This will certainly serve as a first step forward unraveling the mysteries of induction of Csn3 in the process of neural differentiation.

Reciprocal regulation of the basic helix–loop–helix/Per–Arnt–Sim partner proteins, Arnt and Arnt2, during neuronal differentiation

Basic helix–loop–helix/Per–Arnt–Sim (bHLH/PAS) transcription factors function broadly in development, homeostasis and stress response. Active bHLH/PAS heterodimers consist of a ubiquitous signal-regulated subunit (e.g., hypoxia-inducible factors, HIF-1α/2α/3α; the aryl hydrocarbon receptor, AhR) or tissue-restricted subunit (e.g., NPAS1/3/4, Single Minded 1/2), paired with a general partner protein, aryl hydrocarbon receptor nuclear translocator (Arnt or Arnt2). We have investigated regulation of the neuron-enriched Arnt paralogue, Arnt2. We find high Arnt/Arnt2 ratios in P19 embryonic carcinoma cells and ES cells are dramatically reversed to high Arnt2/Arnt on neuronal differentiation. mRNA half-lives of Arnt and Arnt2 remain similar in both parent and neuronal differentiated cells. The GC-rich Arnt2 promoter, while heavily methylated in Arnt only expressing hepatoma cells, is methylation free in P19 and ES cells, where it is bivalent with respect to active H3K4me3 and repressive H3K27me3 histone marks. Typical of a ‘transcription poised’ developmental gene, H3K27me3 repressive marks are removed from Arnt2 during neuronal differentiation. Our data are consistent with a switch to predominant Arnt2 expression in neurons to allow specific functions of neuronal bHLH/PAS factors and/or to avoid neuronal bHLH/PAS factors from interfering with AhR/Arnt signalling.

Testosterone enhances cardiomyogenesis in stem cells and recruits the androgen receptor to the MEF2C and HCN4 genes

Since a previous study (Goldman-Johnson et al., 2008 [4]) has shown that androgens can stimulate increased differentiation of mouse embryonic stem (mES) cells into cardiomyocytes using a genomic pathway, the aim of our study is to elucidate the molecular mechanisms regulating testosterone-enhanced cardiomyogenesis. Testosterone upregulated cardiomyogenic transcription factors, including GATA4, MEF2C, and Nkx2.5, muscle structural proteins, and the pacemaker ion channel HCN4 in a dose-dependent manner, in mES cells and P19 embryonal carcinoma cells. Knock-down of the androgen receptor (AR) or treatment with anti-androgenic compounds inhibited cardiomyogenesis, supporting the requirement of the genomic pathway. Chromatin immunoprecipitation (ChIP) studies showed that testosterone enhanced recruitment of AR to the regulatory regions of MEF2C and HCN4 genes, which was associated with increased histone acetylation. In summary, testosterone upregulated cardiomyogenic transcription factor and HCN4 expression in stem cells. Further, testosterone induced cardiomyogenesis, at least in part, by recruiting the AR receptor to the regulatory regions of the MEF2C and HCN4 genes. These results provide a detailed molecular analysis of the function of testosterone in stem cells and may offer molecular insight into the role of steroids in the heart.

Effects of cannabinoids and related fatty acids upon the viability of P19 embryonal carcinoma cells

Compounds acting on the cannabinoid (CB) receptors are involved in the control of cell fate, and there is an emerging consensus that CBs have anticancer effects. However, the CB-mediated effects are contradictory since some studies suggest stimulatory effects on cancer cell proliferation, and CBs have been shown to stimulate both proliferation and differentiation of other mitotic cells such as stem and progenitor cells. In this study, the concentration-dependent effects of synthetic and endogenous CBs on the viability of mouse P19 embryonal carcinoma (EC) cells have been examined by using fluorescence assays of cell membrane integrity, cell proliferation, oxidative stress, and detection of apoptosis and necrosis. All compounds examined produced a concentration-dependent decrease in cell viability in the micromolar range, with the potent CB receptor agonist HU 210 and the enantiomer HU 211 (with no CB receptor activity) being the most potent compounds examined with apparent IC50 values of 1 and 0.6 μM, respectively. The endogenous CB anandamide showed similar potency and efficacy as structurally related polyunsaturated fatty acids with no reported activity at the CB receptors. The rapid (within hours) decrease in cell viability induced by the examined CBs suggests cytocidal rather than antiproliferative effects and is dependent on the plating cell population density with the highest toxicity around 100 cells/mm(2). The CB-induced cytotoxicity, which appears to involve CB receptors and the sphingomyelin-ceramide pathway, is a mixture of both apoptosis and necrosis that can be blocked by the antioxidants α-tocopherol and N-acetylcysteine. In conclusion, both synthetic and endogenous CBs produce seemingly unspecific cytotoxic effects in the P19 EC cells.