P19 Mouse Embryonal Carcinoma Cells Research Articles

Expression of functional CB1 cannabinoid receptors in retinoic acid-differentiated P19 embryonal carcinoma cells

Although primary neuronal cell cultures, usually obtained from embryonic or early postnatal rodents, have been used in vitro to study the neural cannabinoid signalling system, development of cell lines with neural properties exhibiting native expression of cannabinoid receptors is desirable. This study was undertaken to investigate the expression of CB1 and CB2 cannabinoid receptors in neurons that develop from retinoic acid (RA)-primed mouse P19 embryonal carcinoma cells. Both undifferentiated P19 cells and RA-treated P19 neurons were positive, by using reverse transcription-polymerase chain reaction (RT-PCR), for CB1 (but not CB2) mRNA. Neuronal differentiation increased the CB1 mRNA expression, and Western blotting with a CB1 receptor antibody showed a strong immunoreactive band at approximately 62 kDa in membranes from P19-derived neurons. The cannabinoid receptor agonists CP 55,940 and HU-210 produced concentration-dependent inhibition of forskolin-induced (3 microM) cyclic AMP production in the P19-derived neurons (29% at 1 microM CP 55,940 and 34% at 1 microM HU-210), which could be blocked by the CB1-selective receptor antagonist AM251, but not by the CB2-selective antagonist AM630. Furthermore, glutamate (100 microM) induced a sustained increase in [Ca2+]i in P19-derived neurons that could be concentration-dependently blocked by the cannabinoid receptor agonists WIN 55,212-2. Thus, the protocol used provides an in vitro model system expressing CB1 cannabinoid receptors at the level of mRNA, protein, and AM251-sensitive agonist-induced inhibition of intracellular cyclic AMP accumulation, which may be useful to investigate the developmental regulation, expression and function of neuronal cannabinoid receptors.

Micro-RNA Regulation of the Mammalian lin-28 Gene during Neuronal Differentiation of Embryonal Carcinoma Cells

Vertebrate genomes each encode hundreds of micro-RNAs (miRNAs), yet for few of these miRNAs is there empirical evidence as to which mRNA(s) they regulate. Here we report the identification of human lin-28 mRNA as a regulatory target of human miR-125b and its homolog miR-125a. Studies of miR-125b function in mouse P19 embryonal carcinoma cells induced to develop into neurons suggest a role for this regulatory miRNA in mammalian neuronal differentiation, since its increased concentration in these cells contributes to lin-28 downregulation. Within the lin-28 3' untranslated region (UTR) are two conserved miRNA responsive elements (miREs) that mediate repression by miR-125b and miR-125a. Simultaneous deletion of both miREs renders the lin-28 3' UTR almost completely insensitive to these miRNAs, indicating that these two miREs are the principal elements in the lin-28 3' UTR that respond to miR-125. At the 3' end of each element is an adenosine residue that makes a significant contribution to function irrespective of its complementarity to the 5'-terminal nucleotide of miR-125. By contrast to most earlier reports of gene repression by other miRNAs that are imperfectly complementary to their targets, lin-28 downregulation by miR-125 involves reductions in both translational efficiency and mRNA abundance. The decrease in the mRNA concentration is achieved by a posttranscriptional mechanism that is independent of the inhibitory effect on translation.

The role of actin in the apoptotic cell death of P19 embryonal carcinoma cells

The P19 mouse embryonal carcinoma cell line was used as a model for a study of apoptosis accompanying differentiation induced by all-trans retinoic acid (ATRA). Apoptosis was detected both on the basis of morphological features (nuclear fragmentation, blebbing of plasma membrane, and formation of apoptotic bodies), and by using DNA electrophoresis and flow-cytometric measurement of DNA content. Actin cytoskeleton was studied both on morphological and submicroscopic levels. ATRA-treated cells manifested apoptosis-specific changes in the distribution of actin foremost in association with their entry into executive phase of apoptosis, when F-actin cables participated in cell disintegration into apoptotic bodies. Using immunogold labeling, actin was also identified in centers of fragmenting apoptotic nuclei, in the disintegration of which it is likely involved as well. At the same time, a cleavage of actin by active caspase-3 was proved, resulting in the emergence of 32 kDa fragment, termed fractin. Measurement of F-actin and fractin content using flow cytometry showed an unequivocal decrease of F-actin and synchronous increase of fractin in the apoptotic population as compared to non-treated cells. Therefore, our results proved both actin proteolysis and active involvement of specific actin structures in the final cell disintegration during apoptosis in the P19 cells.

Brain‐derived neurotrophic factor induces a rapid dephosphorylation of tau protein through a PI‐3Kinase signalling mechanism

The microtubule-associated protein tau is essential for microtubule stabilization in neuronal axons. Hyperphosphorylation and intracellular fibrillar formation of tau protein is a pathology found in Alzheimer's disease (AD) brains, and in a variety of neurodegenerative disorders referred to as 'taupathies'. In the present study, we investigated how brain-derived neurotrophic factor (BDNF), an extracellular factor that is down-regulated in AD brains, affects tau phosphorylation. BDNF stimulation of neuronally differentiated P19 mouse embryonic carcinoma cells resulted in a rapid decrease in tau phosphorylation, at phosphorylation sites recognized by Tau 1, AT 8, AT 180 and p 262-Tau antibodies. K 252 a, a tyrosine receptor kinase (Trk) inhibitor, attenuated this dephosphorylation event, suggesting that BNDF activation of TrkB is responsible for the tau dephosphorylation. In addition, BDNF had no affect on tau phosphorylation in the presence of wortmannin, a PI-3 Kinase inhibitor, or lithium, a GSK 3 beta inhibitor, suggesting that these two kinases are part of the signaling transduction cascade leading from TrkB receptor activation to tau dephosphorylation. These results suggest a link between a correlate of AD, decrease in BDNF levels and an AD pathology, tau hyperphosphorylation.

Using the Chorions of Fertilized Zebrafish Eggs as a Biomaterial for the Attachment and Differentiation of Mouse Stem Cells

The development of proper biomaterials is critical for the success of cell therapy and modern tissue engineering. Here, we extruded the yolk and remaining inner mass from fertilized zebrafish eggs and used the resulting chorions as a biomaterial for the differentiation and attachment of mouse P19 embryonic carcinoma (EC) cells. Cells inserted into the chorion showed the spontaneous formation of embryoid body due to the repulsive cell adhesion of the chorion and differentiated specifically into neural cells and cardiomyocytes. In contrast, dissolved chorion extracellular matrix (ECM) conferred enhanced cell attachment on it, suggesting that a unique property of the zebrafish chorion with nanoporous structure appears to be responsible for the simple and controllable embryoid formation for stem cell differentiation. These results indicate that chorions from fertilized zebrafish eggs may be used as an extracellular matrix alternative and applied for stem cell differentiation to specific cell lineages.

Induction of Ectopic Olfactory Structures and Bone Morphogenetic Protein Inhibition by Rossy, a Group XII Secreted Phospholipase A2

The secreted phospholipases A(2) (sPLA(2)s) comprise a family of small secreted proteins with the ability to catalyze the generation of bioactive lipids through glycophospholipid hydrolysis. Recently, a large number of receptor proteins and extracellular binding partners for the sPLA(2)s have been identified, suggesting that these secreted factors might exert a subset of their broad spectrum of biological activities independently of their enzymatic activity. Here, we describe an activity for the sPLA(2) group XII (sPLA(2)-gXII) gene during Xenopus laevis early development. In the ectoderm, sPLA(2)-gXII acts as a neural inducer by blocking bone morphogenetic protein (BMP) signaling. Gain of function in embryos leads to ectopic neurogenesis and to the specification of ectopic olfactory sensory structures, including olfactory bulb and sensory epithelia. This activity is conserved in the Drosophila melanogaster, Xenopus, and mammalian orthologs and appears to be independent of the lipid hydrolytic activity. Because of its effect on olfactory neurogenesis, we have renamed this gene Rossy, in homage to the Spanish actress Rossy de Palma. We present evidence that Rossy/sPLA(2)-gXII can inhibit the transcriptional activation of BMP direct-target gene reporters in Xenopus and mouse P19 embryonic carcinoma cells through the loss of DNA-binding activity of activated Smad1/4 complexes. Collectively, these data represent the first evidence for signaling cross talk between a secreted phospholipase A(2) and the BMP/transforming growth factor beta pathways and identify Rossy/sPLA(2)-gXII as the only factor thus far described which is sufficient to induce anterior sensory neural structures during vertebrate development.

Differential display of proteins involved in the neural differentiation of mouse embryonic carcinoma P19 cells by comparative proteomic analysis

Mouse embryonic carcinoma P19 cell has been used extensively as a model to study molecular mechanisms of neural differentiation in vitro. After retinoic acid (RA) treatment and aggregation, P19 cells can differentiate into neural cells including neurons and glial cells. In this study, comparative proteomic analysis is utilized to approach the protein profiles associated with the RA-induced neural differentiation of P19 cells. Image analysis of silver stained two-dimensional gels indicated that 28 protein spots had significantly differential expression patterns in both quantity and quality. With mass spectrometry analysis and protein functional exploration, many proteins demonstrated an association with distinct aspects of neural differentiation. These proteins were gag polyprotein, rod cGMP-specific 3',5'-cyclic phosphodiesterase, 53 kDa BRG1-associated factor A, N-myc downstream regulated 1, Vitamin D receptor associated factor 1, stromal cell derived factor receptor 1, phosphoglycerate mutase, Ran-specific GTPase-activating protein, and retinoic acid (RA)-binding protein. While some cytoskeleton-related proteins such as beta cytoskeletal actin, gamma-actin, actin-related protein 1, tropomyosin 1, and cofilin 1 are related to cell migration and aggregation, other proteins have shown a relationship with distinct aspects of neural differentiation including energy production and utilization, protein synthesis and folding, cell signaling transduction, and self-protection. The differential expression patterns of these 28 proteins indicate their different roles during the neural differentiation of P19 cells. As an initial step toward unveiling the regulations involved in the commitment of pluripotent cells to a neural fate, information from this study may be helpful to uncover the molecular mechanisms of neural differentiation.

An efficient cell separation system using 3D-asymmetric microelectrodes

An efficient 3D-asymmetric microelectrode system for high-throughput was designed and fabricated to enhance sorting sensitivities to the dielectric properties-size, morphology, conductivity, and permittivity-of living cells. The principle of the present system is based on the use of the relative strengths of negative dielectrophoretic and drag forces, as in a conventional 3D-microelectrode system. Whereas the typical 3D-microelectrode system has a constant electric field magnitude due to the constant width of the microelectrodes and a fixed gap between face-to-face microelectrodes, the present 3D-asymmetric microelectrode system has electric fields of continuously varying magnitudes along the transverse direction of a channel owing to the changing widths of the electrodes in the half-circular shaped cross section of the microchannel. Thus, varying dielectric forces are generated, leading to increased sorting sensitivity through differentially induced forces to definitely distinct cell types. Numerical analysis verified the improved sensitivity of the present system for sorting living cells. The feasibility of using the newly fabricated system under experimental conditions was tested by demonstrating that a mixed population of mouse P19 embryonic carcinoma (EC) and red blood cells (RBCs) was effectively sorted to different wells depending on their respective relative physical properties.

DNA rearrangement activity during retinoic acid-induced neural differentiation of P19 mouse embryonal carcinoma cells.

Because of the many superficial similarities between the immune system and the central nervous system, it has long been speculated that somatic DNA recombination is, like the immune system, involved in brain development and function. To examine whether or not the V(D)J recombination signals of the immune system work in an in vitro neural differentiation model, the P19 mouse embryonal carcinoma cell line was transfected with a reporter gene that is designed, when rearranged, to express bacterial beta-galactosidase, which was previously reported to exhibit somatic DNA recombination in the transgenic mouse brain. The cloned cells were then induced into neural cells by retinoic acid treatment. This neural induction treatment resulted in the cloning of a P19 cell line that showed a high incidence of beta-galactosidase-positive cells. Most of these beta-galactosidase-positive cells were immunocytochemically identified as either neurons, neuroepithelial cells, or astrocytes. The 5'-end sequences of the beta-galactosidase transcripts expressed in the induced cells were analyzed, and sequences were found that seemed to reflect DNA rearrangement through re-integration of the reporter gene into the host genome. However, the V(D)J recombination signals did not work in the in vitro model. These results suggested that DNA rearrangement activity though integration increased during neural differentiation of P19 cells.

Gα13 Signals via p115RhoGEF Cascades Regulating JNK1 and Primitive Endoderm Formation

The heterotrimeric G-protein G(13) mediates the formation of primitive endoderm from mouse P19 embryonal carcinoma cells in response to retinoic acid, signaling to the level of activation of c-Jun N-terminal kinase. The signal linkage map from MEKK1/MEKK4 to MEK1/MKK4 to JNK is obligate in this G alpha(13)-mediated pathway, whereas that between G alpha(13) and MEKKs is not known. The overall pathway to primitive endoderm formation was shown to be inhibited by treatment with Clostridium botulinum C3 exotoxin, a specific inactivator of RhoA family members. Constitutively active G alpha(13) was found to activate RhoA as well as Cdc42 and Rac1 in these cells. Although constitutively active Cdc42, Rac1, and RhoA all can activate JNK1, only the RhoA mutant was able to promote formation of primitive endoderm, mimicking expression of the constitutively activated G alpha(13). Expression of the constitutively active mutant form of p115RhoGEF (guanine nucleotide exchange factor) was found to activate RhoA and JNK1 activities. Expression of the dominant negative p115RhoGEF was able to inhibit activation of both RhoA and JNK1 in response to either retinoic acid or the expression of a constitutively activated mutant of G alpha(13). Expression of the dominant negative mutants of RhoA as well as those of either Cdc42 or Rac1, but not Ras, attenuated G alpha(13)-stimulated as well as retinoic acid-stimulated activation of all three of these small molecular weight GTPases, suggesting complex interrelationships among the three GTPases in this pathway. The formation of primitive endoderm in response to retinoic acid also could be blocked by expression of dominant negative mutants of RhoA, Cdc42, or Rac1. Thus, the signal propagated from G alpha(13) to JNK requires activation of p115RhoGEF cascades, including p115RhoGEF itself, RhoA, Cdc42, and Rac1. In a concerted effort, RhoA in tandem with Cdc42 and Rac1 activates the MEKK1/4, MEK1/MKK4, and JNK cascade, thereby stimulating formation of primitive endoderm.

Directing pluripotent cell differentiation using “diced RNA” in transient transfection

Embryonic stem (ES) and embryonic carcinoma (EC) cells are pluripotent and have the capacity to differentiate into many cell types. The ability to direct their differentiation should have considerable practical applications. Here, we first report the use of diced short interfering RNAi against Oct4 in a transient approach, to direct differentiation of ES towards the trophectoderm lineage. We then apply this approach to downregulate Smad4 in mouse P19 EC cells. We have found that this leads to an increase in the levels of Pax6 (a neuroectoderm marker), reduction in the levels of Brachyury (a mesoderm marker), and a 3-fold increase in the number of betaIII tubulin-positive colonies when these cells were allowed to differentiate. This indicates a redirection of cell fate towards the neuroectoderm lineage. Thus, transient RNAi could provide a valuable tool to direct pluripotent cells along specific pathways of differentiation while circumventing permanent genetic changes.

Retinoic acid regulates the expression of PBX1, PBX2, and PBX3 in P19 cells both transcriptionally and post-translationally.

Pre-B cell leukemia transcription factors (PBXs) are important co-factors for the transcriptional regulation mediated by a number of Hox proteins during embryonic development. It was previously shown that the expression of several Pbx genes is elevated in mouse embryo limb buds and embryonal carcinoma P19 cells upon retinoic acid (RA) treatment although the mechanism of this induction is not well understood. In this report, we demonstrate that PBX1a, PBX1b, PBX2, and PBX3 mRNAs and PBX1/2/3 proteins are induced during endodermal and neuronal differentiation of P19 cells in a RAR-dependent subtype-unspecific manner following RA treatment. The increases in both PBX1 mRNA and PBX3 mRNA levels are secondary responses to RA treatment requiring new proteins synthesis while the increase in PBX2 mRNA is a primary response. The RA-dependent increases in PBX1 mRNA, PBX2 mRNA, and PBX3 mRNA levels are likely to be transcriptionally regulated since the stability of these mRNAs does not change. In addition, the half-lives of PBX1/2/3 proteins are significantly extended by RA treatment. Two possible mechanisms could contribute to the stabilization of PBX proteins: PBX proteins associate with RA-dependent increased levels of MEIS proteins, and RA may decrease the proteasome dependent degradation of PBX proteins.

Pre-B Cell Leukemia Transcription Factor (PBX) Proteins Are Important Mediators for Retinoic Acid-dependent Endodermal and Neuronal Differentiation of Mouse Embryonal Carcinoma P19 Cells

Pre-B cell leukemia transcription factors (PBXs) act as cofactors in the transcriptional regulation mediated by Homeobox proteins during embryonic development and cellular differentition. PBX1 protein is expressed throughout murine embryonic development, and its deletion in mice disrupts chondrogenesis. PBX protein levels are also increased in mouse embryonal carcinoma P19 cells during retinoic acid (RA)-induced differentiation. To elucidate the role of PBX proteins in this process, we stably overexpressed PBX1b antisense mRNA in P19 cells (PBX1b-AS cells). PBX1b-AS cells did not differentiate to neuronal or endodermal cells following treatment with RA suggesting PBX proteins are required for both processes. Furthermore we demonstrated that PBX proteins regulate the RA-dependent induction in the mRNA levels of bone morphogenetic protein 4 (BMP4) and Decorin (DCN) in P19 cells using both PBX1b-AS cells and PBX1 small interfering RNA. Chromatin immunoprecipitation assays further demonstrated that PBX proteins directly bind to the promoter of Bmp4 and Dcn in vivo in a RA-dependent fashion. In addition, type I and type II BMP receptor mRNA levels were also increased in P19 cells following RA treatment; however, this was PBX-independent. Taken together these data demonstrate that PBX proteins are required for RA-induced differentiation of P19 cells and that PBX proteins regulate the expression of BMP4 and DCN during this differentiation process.

Long-lasting RNAi activity in mammalian neurons

The effect of RNA interference (RNAi) induced by synthetic small interfering RNAs (siRNAs) on proliferating mammalian cells appears to last for approximately 3–7 days after its induction. Here we show that the RNAi activity induced by a synthetic 21-nucleotide siRNA duplex in postmitotic neurons, mouse primary hippocampal neurons and neurons that differentiated from mouse embryonal carcinoma P19 cells persists for at least 3 weeks, suggesting long-lasting RNAi activity in mammalian neurons. In addition, we also show that an apoptotic (or antiviral) pathway triggered by long dsRNAs is generated during neuronal differentiation of P19 cells, by which the sequence-specific RNAi activity involving long dsRNA appears to be masked.

213.Culture of mouse male germ cells for genetic manipulations

The ability to maintain germ cells in culture offers the opportunity to manipulate their differentiation and to deliver new genes through the germline. However the study of male germ cell biology is hindered by the lack of an in vitro culture system that supports germ cell maintenance and differentiation, and the lack of efficient gene transfer technologies. To address this, we developed a short term (1-6 day) in vitro germ cell-Sertoli cell co-culture system using testes of 6 day old mice, in which the only germ cell type present is the spermatogonium. To establish culture conditions favourable for maintaining germ cells in culture, we compared different substrates (plastic, laminin, poly-L-lysine, Matrigel) and cell culture additives (insulin/transferrin, retinoic acid, lactic acid, pyruvic acid). Using immunocytochemistry, we observed that germ cell numbers varied depending on the substrate, and was greatly improved on laminin or Matrigel. The culture additives tested had no effect. We noted a striking difference in germ cell growth depending on the mouse strain used, with C57BlXCBA F1 cross > Swiss > BalbC >> C57Bl/6J. To investigate gene transfer into germ cells, constructs were generated using promoters identified as exhibiting germ cell-specific expression in transgenic animals (EF1-α, Oct4, Stra8) with EGFP and lacz reporter genes. From this baseline, we have started gene transfer experiments. Initial transfection experiments were performed in mouse testis cell lines (GC1, GC2, TM3, TM4) and the P19 mouse embryonal carcinoma cell line. Several commercial transfection agents and a non-commercial product (1) were used, as was electroporation, however these did not yield reporter expression in germ cells. As these techniques require proliferating cells, we are now undertaking a study with the addition of growth factors in culture (BMP4, activin, GDNF, FSH) to enhance germ cell proliferation and therefore improve transfection efficiencies. We will also employ lentiviral transduction, as this is reportedly 100% efficient and independent of proliferation. (1) Celebi et al. (2002) Mol. Reprod. Dev. 62,477–482.

An Evolutionarily Conserved N-terminal Acetyltransferase Complex Associated with Neuronal Development

We previously identified mNAT1 (murine N-terminal acetyltransferase 1) as an embryonic gene that is expressed in the developing brain and subsequently down-regulated, in part, by the onset of N-methyl-d-aspartate (NMDA) receptor function. By searching the data base we discovered a second closely related gene, mNAT2. mNAT1 and mNAT2 are highly homologous to yeast NAT1, a gene known to regulate entry into the G0 phase of the cell cycle. However, in the absence of further characterization, including evidence that mammalian homologues of NAT1 encode functional acetyltransferases, the significance of this relationship has been unclear. Here we focus on mNAT1. Biochemical analysis demonstrated that mNAT1 and its evolutionarily conserved co-subunit, mARD1, assemble to form a functional acetyltransferase. Transfection of mammalian cells with mNAT1 and mARD1 followed by immunofluorescent staining revealed that these proteins localize to the cytoplasm in both overlapping and separate compartments. In situ hybridization demonstrated that throughout brain development mNAT1 and mARD1 are highly expressed in areas of cell division and migration and are down-regulated as neurons differentiate. Finally, mNAT1 and mARD1 are expressed in proliferating mouse P19 embryonic carcinoma cells; treatment of these cells with retinoic acid initiates exit from the cell cycle, neuronal differentiation, and down-regulation of mNAT1 and mARD1 as the NMOA receptor 1 gene is induced. The results provide the first direct evidence that vertebrate homologues of NAT1 and ARD1 form an evolutionarily conserved N-terminal acetyltransferase and suggest that expression and down-regulation of this enzyme complex plays an important role in the generation and differentiation of neurons.

Lagepläne

Pluripotent mouse P19 embryonal carcinoma (EC) cells have been extensively used as a developmental model system because they can differentiate in the presence of retinoic acid (RA) into derivatives of all three germ layers depending on RA dosage and culture conditions. The expression of several genes has been shown to be induced in RA-treated P19 EC cells and, interestingly, some of these genes may play important roles during mouse embryogenesis. In view of the increasing evidence that RA is a crucial signaling molecule during vertebrate development, we have initiated a study aimed at the systematic isolation of genes whose expression is induced in P19 cells at various times after exposure to RA. We describe here an efficient differential subtractive hybridization cloning strategy which was used to identify additional RA-responsive genes in P19 cells. Fifty different cDNA fragments corresponding to RA-induced genes were isolated. Ten cDNAs represent known genes, 4 of which have already been described as RA-inducible, while the remaining 40 correspond to novel genes. Many of these cDNA sequences represent low-abundance mRNAs. Kinetic analysis of mRNA accumulation following RA treatment allowed us to characterize four classes of RA-responsive genes. We also report the sequence and expression pattern in mouse embryos and adult tissues of one of these novel RA-inducible genes, Stral, and show that it corresponds to the mouse ligand for the Cek5 receptor protein-tyrosine kinase.

The blue copper ceruloplasmin induces aggregation of newly differentiated neurons: a potential modulator of nervous system organization

Ceruloplasmin (CP) is a copper-dependent ferroxidase. It regulates iron metabolism and is involved in inflammation, angiogenesis, and protection against oxidative stress. CP also modulates K + channel activity in neuroblastoma cells and affects cardiodynamics of isolated hearts. Considering the presence of CP in the nervous system and the importance of iron ions and K + channels in neuronal activity, we postulated a role of CP in neuronal development. This hypothesis was tested using the P19 mouse embryonal carcinoma cell line, a model of neuronal differentiation. Addition of CP to the culture medium of newly differentiated P19 neurons induced cell aggregation within 24 h. This effect was concentration-dependent half-maximal at 50 nM, and not associated with necrosis, apoptosis or changes in secretory function. Deglycosylated CP was aggregative but not denatured CP, copper salts, His 2Cu complex, or other copper enzymes or serum proteins. CP-induced aggregation was less pronounced with aging neurons and seemed not to involve K + channels. Immunocytofluorescence analysis demonstrated that digoxigenin-labeled CP bound to P19 neurons and the proportion of responding neurons decreased with aging. The interaction of digoxigenin-labeled CP with neurons was half-maximal at 120 nM by enzyme-linked immunosorbent assay and displaced by unlabeled CP. Our data indicate a specific aggregative action of CP on young neurons in vitro, possibly involving CP receptors. A potential developmental role of CP in nervous system organization is thus demonstrated.

Regulation of c-myc Gene by Nitric Oxide via Inactivating NF-κB Complex in P19 Mouse Embryonal Carcinoma Cells

Nitric oxide (NO) may regulate gene expression by directly modifying redox state-sensitive residues of transcription factors. Here we show that the NO donor, sodium nitroprusside (SNP), rapidly represses c-myc gene transcription in a protein synthesis-independent manner in P19 embryonal carcinoma cells by inactivation of NF-kappa B. SNP treatment reduces the DNA binding ability of the constitutively active NF-kappa B heterodimer, p65/p50, and its consequent transactivation of the c-myc promoter. Repression can be blocked by the peroxynitrite scavenger, deferoxamine, but not by dithiothreitol, which triggers reduction of S-nitrosylated residues. In HEK293 cells, where tumor necrosis factor-alpha can activate NF-kappa B, SNP likewise suppresses the binding of the active NF-kappa B complex, restoring the binding of the repressive p50/p50 homodimer complex. This effect of SNP in HEK293 cells is also blocked by deferoxamine. Chromatin immunoprecipitation analysis of SNP-treated P19 cells reveals reduced association of p65, but not of p50, with the promoter region of the endogenous c-myc gene. SNP-induced p65 dissociation was associated with the recruitment of histone deacetylase 1 and 2 to the endogenous c-myc gene promoter and the subsequent deacetylation of its chromatin histone. This study is the first to demonstrate that NO modulates the transcriptional activity of the c-myc gene promoter by dissociating the active form of NF-kappa B and replacing it with a repressive NF-kappa B complex, correlated with the recruitment of gene-silencing histone deacetylases. In light of findings that NF-kappa B stimulates Myc oncoprotein expression in cancers, our findings suggest that NO should be investigated as a prospective therapeutic cancer agent.

The Adhesion and Differentiation-inhibitory Activities of the Immunoglobulin Superfamily Member, Carcinoembryonic Antigen, Can Be Independently Blocked

The external domains of Ig superfamily members are involved in multiple binding interactions, both homophilic and heterophilic, that initiate molecular events leading to the execution of diverse cell functions. Human carcinoembryonic antigen (CEA), an Ig superfamily cell surface glycoprotein used widely as a clinical tumor marker, undergoes homophilic interactions that mediate intercellular adhesion. Recent evidence supports the view that deregulated overexpression of CEA has an instrumental role in tumorigenesis through the inhibition of cell differentiation and the disruption of tissue architecture. The CEA-mediated block of the myogenic differentiation of rat L6 myoblasts depends on homophilic binding of its external domains. We show here that L6 transfectant cells expressing CEA can "trans-block" the myogenesis of juxtaposed differentiation-competent L6 transfectant cells expressing a deletion mutant of CEA (DeltaNCEA). This result implies the efficacy of antiparallel CEA-CEA interactions between cells in the differentiation block. In addition, DeltaNCEA can acquire differentiation blocking activity by cross-linking with specific anti-CEA antibodies, thus implying the efficacy of parallel CEA-CEA interactions on the same cell surface. The myogenic differentiation blocking activity of CEA was demonstrated by site-directed mutations to involve three subdomains of the amino-terminal domain, shown previously to be critical for its intercellular adhesion function. Monovalent Fab fragments of monoclonal antibodies binding to the region bridging subdomains 1 and 2 could both inhibit intercellular adhesion and release the myogenic differentiation block. Amino acid substitutions Q80A, Q80R, and D82N in subdomain 3, QNDTG, however, were found to completely ablate the differentiation blocking activity of CEA but had no effect on intercellular adhesion activity. A cyclized peptide representing this subdomain was the most effective at releasing the differentiation block.