Phosphatidylinositol transfer protein in murine embryonal carcinoma cells during retinoic acid-induced differentiation

Phosphatidylinositol transfer protein (PI-TP) was studied in P19 embryonal carcinoma (EC) cells at different stages of retinoic acid (RA) induced differentiation. Western blot analysis indicated an increased expression of PI-TP (35 kDa) during differentiation. Western blots of isoelectric focusing gels showed that the 35 kDa band consisted of the PI-carrying form of PI-TP (pl 5.5) and of a novel, more acidic form of PI-TP (pl 5.4), levels of both of which increased during differentiation. These increased levels were not reflected in the in vitro PI-transfer activity of the cytosolic fraction nor in the mRNA levels as analyzed by northern blotting. By using indirect immunofluorescence it was shown that PI-TP is localized in the cytoplasm and associated with perinuclear Golgi structures and that this distribution is slightly affected during RA-induced differentiation. Immunoprecipitation of PI-TP from [³²P]P_i labeled cells demonstrated that the level of phosphorylation of PI-TP is high in undifferentiated P19 EC cells and low after 5 days of RA-induced differentiation. These results strongly suggest that changes in the levels of PI-TP are intimately connected with changes in the growth characteristics of P19 EC cells during RA-induced differentiation. It remains to be established to what extent this connection is governed by the recent finding that PI-TP is an essential cytosolic factor in stimulating phospholipase C activity.     

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

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.     

Induction of differentiation of murine embryonal carcinoma cells by ouabain

Murine embryonal carcinoma cells (EC) can be induced to differentiate by a variety of chemical agents, including retinoid acid (RA) and dimethyl acetamide (DMA). However, it is not known how these agents exert their effects. In this study we demonstrate that murine EC cells can also be induced to differentiate by ouabain at concentrations which inhibit Na+, K+-ATPase activity as measured by inhibition of 86Rb+ uptake. Since the pharmacologic action of ouabain is thought to be specific, we investigated the role of Na+, K+-ATPase inhibition and specific metabolic consequences of this inhibition in the induction of EC differentiation, and explored whether this might be a common mode of action for a variety of structurally diverse inducers. Although the Na+, K+-ATPase maintains ion gradients in cells, our studies failed to demonstrate a consistent role for alterations of ion flux or ion concentration on the differentiation process. Ouabain inhibited cell growth, but a direct correlation between the degree of growth inhibition and the extent of differentiation could not be demonstrated. There was also no evidence that RA or DMA induces differentiation by inhibiting the Na+, K+-ATPase. The mechanism of ouabain induction may be mediated by some alternative consequence of Na+, K+-ATPase inhibition, but it appears to be specific for that inducer and cannot be generalized to that of other inducers of EC differentiation.     

The biological role of the Alzheimer amyloid precursor protein in epithelial cells

Proteolytic processing of the Alzheimer amyloid precursor protein (APP) results in the generation of at least two distinct classes of biologically relevant peptides: (1) the amyloid beta peptides which are believed to be involved in the pathogenesis of Alzheimer's disease and (2) the soluble N-terminal ectodomain (sAPP) which exhibits a protective but as yet ill-defined effect on neurons and epithelial cells. In this report we present an overview on the functions of sAPP as an epithelial growth factor. This function involves specific binding of sAPP to membrane rafts and results in signal transduction and various physiological effects in epithelial cells as different as keratinocytes and thyrocytes. At nanomolar concentrations sAPP induces a two to fourfold increase in the rate of cell proliferation and cell migration. Specific inhibition of APP expression by antisense techniques results in decreased sAPP release and in reduced proliferative and motogenic activities. Proliferation and migration are known to be part of complex processes such as wound healing which, therefore, might be facilitated by the growth factor function of sAPP.     

Differential expression of a novel murine non-receptor protein tyrosine phosphatase during differentiation of P19 embryonal carcinoma cells.

Protein phosphorylation on tyrosine residues is one of the major mechanisms of cell signal transduction and is regulated by protein tyrosine kinases and protein tyrosine phosphatases. Here we report the molecular cloning of an additional member of the protein tyrosine phosphatase-family from differentiated murine P19 embryonal carcinoma cells. This non-receptor protein tyrosine phosphatase, P19-PTP, does not contain regulatory sequences, homologous to the ones found in other non-receptor PTPases. P19-PTP is differentially expressed during in vitro differentiation of P19 EC cells, in that P19-PTP mRNA could only be detected in embryoid bodies, derived from P19 cells.     

Cell-cell interaction can influence drug-induced differentiation of murine embryonal carcinoma cells

When cultured in the presence of either retinoic acid (RA) or dimethyl sulfoxide (DMSO), aggregates of the P19 line of mouse embryonal carcinoma (EC) cells differentiate and the spectrum of cell types formed depends on the drug dose. It is shown here the EC cells rapidly lose their colony-forming ability when cultured as aggregates in the presence of DMSO. This loss of plating efficiency (PE) also occurs rapidly following RA treatment. Loss of PE has been used as a quantitative procedure for assessing the rate of drug-induced differentiation. The relationship between drug dose and loss of PE is much steeper for DMSO than for RA, suggesting that these two drugs affect different stages of the differentiation decision-making apparatus. Mutant EC cell lines (D3 and RAC65) do not differentiate in the presence of drug-inducers (DMSO and RA, respectively). Neither differentiation-deficient mutant has an altered ability to form gap junctions. When D3 and P19 cells were mixed within the same DMSO-treated aggregates, the D3 cells remained undifferentiated and the P19 cells differentiated much less efficiently than if they were cultured in the absence of the D3 cells. When RAC65 and P19 cells were mixed in RA-treated aggregates, each cell responded to the drug as though the other were absent. Thus RA behaves as a cell-autonomous inducer of differentiation, whereas DMSO-induced differentiation seems to be mediated by interactions between neighboring cells.     

Cytoplasmic and nuclear protein synthesis during in vitro differentiation of murine ICM and embryonal carcinoma cells

Protein syntheses during in vitro differentiation of inner cell masses (ICM) isolated from mouse blastocysts and of pluripotent embryonal carcinoma cells (ECC) were compared by two-dimensional electrophoretic analysis of [³⁵S]methionine-labeled cells. While most of the polypeptides found in ICM, ECC, and embryoid bodies (EB) derived from them were common to all four preparations, some distinct differences were noted. More polypeptides changed in intensity during the differentiation of ICM than during the differentiation of ECC. Analysis of ECC prior to differentiation revealed that only some of the polypeptides abundant in ICM were present, while at the same time, some of the polypeptides abundant in ICM-EB were being synthesized. These data indicate that ECC represent cells further advanced in development than the cells of ICM isolated from 4-day-old blastocysts. The EB derived from ECC also differ from those from ICM. Comparison of EB derived from ICM and ECC with cells of the parietal yolk sac line, PYS, indicates that all three synthesize two polypeptides abundant in EB. These two polypeptides can, therefore, be used as biochemical markers of parietal entoderm differentiation. Pluripotent ECC synthesize small amounts of characteristic EB proteins and the 10-nm filament protein (also found in PYS cells but not in EB). This indicates that small numbers of differentiated or differentiating cells are present in pluripotent ECC cultures.     

Overexpression of amyloid precursor protein increases copper content in HEK293 cells

Amyloid precursor protein (APP) is a transmembrane glycoprotein widely expressed in mammalian tissues and plays a central role in Alzheimer’s disease. However, its physiological function remains elusive. Cu²⁺ binding and reduction activities have been described in the extracellular APP135-156 region, which might be relevant for cellular copper uptake and homeostasis. Here, we assessed Cu²⁺ reduction and ⁶⁴Cu uptake in two human HEK293 cell lines overexpressing APP. Our results indicate that Cu²⁺ reduction increased and cells accumulated larger levels of copper, maintaining cell viability at supra-physiological levels of Cu²⁺ ions. Moreover, wild-type cells exposed to both Cu²⁺ ions and APP135-155 synthetic peptides increased copper reduction and uptake. Complementation of function studies in human APP751 transformed Fre1 defective Saccharomyces cerevisiae cells rescued low Cu²⁺ reductase activity and increased ⁶⁴Cu uptake. We conclude that Cu²⁺ reduction activity of APP facilitates copper uptake and may represent an early step in cellular copper homeostasis.     

Modulation of thrombospondin expression during differentiation of embryonal carcinoma cells

The thrombospondins (TSPs) are a family of extracellular glycoproteins that display distinct patterns of temporal and spatial expression during development. In this study, we investigated the expression of two of the TSPs–TPS1 and TSP2– during the course of differentiation of embryonal carcinoma cells in vitro. We report that both TSP1 and TSP2 mRNA and protein synthesis are induced during the differentiation of P19EC cells into neurons, glial cells, and fibroblasts. Immunofluorescence studies indicate that TSP1 displays a fibrillar pattern of staining, characteristic of an extracellular matrix protein, in differentiated P19EC cells. In contrast, TSP2 is cell-associated and is present on differentiated P19EC cells and on primary neurons and glial cells obtained from a 17-day embyronic mouse cerebral cortex. Interestingly, although both TSP1 and TSP2 are more prevalent in areas of differentiated cells, they display distinct patterns of deposition. These observations suggest that TSP1 and TSP2 may function differently during neurogenesis. The response of TSP1 and TSP2 to differentiation of P19EC cells indicates that this cell system will serve as a valuable model for the study of TSP expression and function during neurogenesis. © 1994 Wiley-Liss, Inc.     

A heparin binding protein whose expression increases during differentiation of embryonal carcinoma cells to parietal endoderm cells: cDNA cloning and sequence analysis

A cDNA clone isolated from a lambda gt11 expression library of teratocarcinoma OTT6050 specifies for a glycoprotein with a molecular weight of about 44,000. The new glycoprotein was termed heparin binding protein-44 (HBP-44), since it was absorbed to a heparin-agarose column and was eluted from it by a buffer containing 1.5 M NaCl. HBP-44 mRNA was intensely expressed in PYS-2 parietal endoderm cells and in the kidney, and the RNA level increased about 10-fold during differentiation of F9 embryonal carcinoma cells to parietal endoderm cells. From the cDNA sequence, HBP-44 was concluded to be rich in charged amino acids, and large segments of the protein appeared to form alpha-helixes. The protein was considered to be anchored to the membrane by a cluster of hydrophobic amino acids present in the N-terminal region. Indeed, the N-terminal sequence of HBP-44 was homologous to asialoglycoprotein receptor, which is anchored to the membrane by the N-terminal region. Furthermore, a portion of the N-terminal region of HBP-44 was homologous to the leucine zipper domain. Except for the N-terminal region, HBP-44 had over-all homology with structural proteins such as myosin heavy chain. We propose that HBP-44 is extruded from plasma membranes and interacts with heparin and related molecules and that it is involved in the interactions of plasma membranes with basement membranes.     

Sodium butyrate induces histone hyperacetylation and differentiation of murine embryonal carcinoma cells

Cells from embryonal carcinoma (EC) lines 6050AJ and PCC4.aza 1R differentiate in response to treatment with sodium butyrate as well as retinoic acid (RA) or hexamethylenebisacetamide (HMBA). Murine 6050AJ EC cells exposed to sodium butyrate possess hyperacetylated forms of histones H4 and altered forms of histones H2a and H2b, whereas histones from cells treated with other inducers appear to be unaffected. These results might indicate that the mechanism by which sodium butyrate promotes differentiation of EC cells is different from the ways in which RA and HMBA act. Differentiation-defective PCC4(RA)-1 EC cells fail to respond to RA, presumably because they possess minimal amounts of active binding protein for RA (cRABP). Sodium butyrate treatment of these cells results in only a modest level of differentiation. On the other hand, exposure to sodium butyrate plus RA leads to extensive differentiation. As is the case with 6050AJ cells, PCC4(RA)-1 cells treated with sodium butyrate also contain hyperacetylated histones. Furthermore, these cells now possess high levels of cRABP. The latter observations suggest that sodium butyrate has the ability to reactivate a silent cRABP gene in PCC4(RA)-1 cells and thereby lead to extensive differentiation via the retinoid pathway when RA is added.     

The amyloid precursor protein locus and very-late-onset Alzheimer disease

Although mutations in the amyloid-beta precursor protein (APP) gene are known to confer high risk of Alzheimer disease (AD) to a small percentage of families in which it has early onset, convincing evidence of a major role for the APP locus in late-onset AD has not been forthcoming. In this report, we have used a covariate-based affected-sib-pair linkage method to analyze the chromosome 21 clinical and genetic data obtained on affected sibships by the National Institute of Mental Health Alzheimer Disease Genetics Initiative. The baseline model (without covariates) gave a LOD score of 0.02, which increases to 1.43 when covariates representing the additive effects of E2 and E4 are added. Larger increases in LOD scores were found when age at last examination/death (LOD score 5.54; P=.000002) or age at onset plus disease duration (LOD score 5.63; P=.000006) were included in the linkage model. We conclude that the APP locus may predispose to AD in the very elderly.     

Visceral-endoderm-like cell lines induce differentiation of murine P19 embryonal carcinoma cells

When P19 embryonal carcinoma (EC) cells were cocultured with cells from one of several established visceral-endoderm-like cell lines, the EC cells were rapidly induced to aggregate and differentiate, into cell types including mesoderm-derived cardiac and skeletal muscle. Neither parietal-endoderm- nor mesoderm-like cell lines induced aggregation or differentiation of EC cells in coculture, although a cell line with both parietal and visceral endoderm characteristics induced aggregation but not differentiation. Also, without the feeder cells aggregates of P19 failed to differentiate, provided that serum in the culture medium had been previously passed over dextran-coated charcoal to remove lipophilic substances, which may include endogenous retinoids. All experiments were carried out using serum treated in this way. Taken together, the results demonstrated that aggregation was necessary, but not sufficient, to make P19 EC cells differentiate. Direct contact between the two cell types was not necessary, since even when separated by an agar layer in cocultures, aggregates of P19 still differentiated. Medium conditioned by cells of the END-2 line, a visceral-endoderm-like derivative of P19, was particularly potent in inducing endodermal and mesodermal differentiation of single P19 aggregates, confirming the involvement of a diffusible factor secreted specifically by visceral-endoderm-like cells in this process.     

Determinants of retrovirus gene expression in embryonal carcinoma cells.

The expression of Moloney murine leukemia virus is restricted in embryonal carcinoma (EC) cells. To characterize specific mutations necessary for expression of retroviruses in EC cells, we analyzed the expression of retrovirus mutants and recombinants thereof in EC cell lines F9 and PCC4. DNA sequence comparison and functional studies allowed us to define three point mutations in the enhancer region of the viral mutants at positions -345, -326, and -166 and two point mutations within the 5'-untranslated region of the viral genome at positions +164 and +165 that were essential for retrovirus expression in EC cells. DNA fragments derived from either the wild type or mutant viruses were used to search for sequence-specific DNA-binding factors in nuclear extracts from undifferentiated PCC4 cells. A cellular factor was found to bind strongly to sequences within the enhancer region (-354 to -306) of wild-type viruses but only weakly to sequences derived from mutant viruses. This factor was named ECF-I (for EC cell factor I). Retroviral expression in EC cells correlates with decreased binding affinity for ECF-I.     

Neural differentiation following culture of embryonal carcinoma cells in a serum-free defined medium

The embryonal carcinoma line C17-S₁ clone 1003 is multipotential in vivo. When the cells are grown in vitro in serum-containing medium most of them remain undifferentiated, while a few differentiate into a unique morphologic type of epithelioid cell. If 1003 cells are passaged into a defined medium containing insulin, transferrin, selenium, and fibronectin they grow for six to eight generations at the same rate as in serum-containing medium. During this time, all the cells of the culture differentiate into a limited number of phenotypes with neuroepithelial and neuronal cells predominating. Differentiation could be obtained in the defined medium at relatively low cell densities. Exogenous fibronectin is required for cell attachment to the substratum, and when absent the cells form aggregates in which differentiation still occurs. Low amounts of serum added to the defined medium allow multiplication and maintenance of cells of undifferentiated phenotype and prevent differentiation into neuronal cells.