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C L Mummery

Publications and source records attributed to C L Mummery.

At least 73 records · Page 4Linked to original sources
PubMed1990

Identification of the type-B receptor for platelet-derived growth factor in human embryonal carcinoma cells.

The human embryonal carcinoma (EC) cell line Tera 2 clone 13 (T2/13) can be induced to differentiate in vitro into neuroectodermal cell types with retinoic acid. Undifferentiated cells are characterized by rapid proliferation, whereas differentiated cells show a prolonged generation time, have a limited life span, and possess new cell-surface markers. In the present study we establish that both differentiated and undifferentiated T2/13 cells express the type-B platelet-derived growth factor (PDGF) receptor mRNA and bind PDGF-BB with high affinity. Differentiation causes a three-fold increase in receptor number per cell and leaves the affinity of the receptors unaffected. These data are the first to describe expression of this receptor in EC cells. The biosynthesis and degradation of this receptor were studied in undifferentiated as well as in differentiated T2/13 cells using an anti-type-B receptor antibody. These experiments revealed that high concentrations of recombinant PDGF-AA did not accelerate receptor metabolism in both cell types. In contrast, human PDGF or recombinant PDGF-BB added to the culture dishes readily increased receptor degradation. These results demonstrate that T2/13 cells express functional type-B PDGF receptors and suggest that cells responsive to PDGF might be present during mammalian development before the onset of mesoderm formation.

Cell Differentiation↗
PubMed1990

Characteristics of embryonic stem cell differentiation: a comparison with two embryonal carcinoma cell lines.

Embryonic stem (ES) cells isolated from late blastocysts can now be maintained in culture in an undifferentiated state provided they are grown in the presence of a specific differentiation inhibitor, known variously as leukaemia inhibiting factor (LIF) or differentiation inhibiting activity (DIA), found at high concentrations in medium conditioned by Buffalo rat liver (BRL) cells. ES cells acquired a differentiated phenotype in monolayer, either when in the absence of LIF/DIA or in the presence of retinoic acid (RA). We have now characterized this bipotential differentiation of ES cells in terms of a series of extracellular matrix and cell surface proteins as well as cytokeratin expression, and compared it with the changes observed during the differentiation of two embryonal carcinoma (EC) cell lines, P19 and F9. ES cells exposed to RA in the presence of LIF/DIA largely resembled F9 EC + RA after 5 days, while ES cells deprived of LIF/DIA formed a culture with mixed phenotype resembling P19 EC + RA. This study therefore establishes the predominantly parietal endoderm-like phenotype of cells derived from ES by RA induction, and suggests that a mixed population of endoderm- and ill-defined mesoderm-like cells are formed after removal of specific inhibitor(s) of differentiation.

Animals↗
PubMed1989

Identification and characterization of polypeptide growth factors secreted by murine embryonal carcinoma cells.

Undifferentiated P19 and PC13 murine embryonal carcinoma (EC) cells have been analyzed for their ability to secrete polypeptide growth factors. This has been carried out by a combination of specific bioassays and the use of biochemical and immunological detection methods. Both P19 and PC13 EC cells secrete a platelet-derived growth factor (PDGF)-like growth factor, a type beta transforming growth factor, and insulin-like growth factors. In addition, PC13 EC cells secrete a heparin-binding growth factor functionally related to fibroblast growth factor, while P19 EC cells secrete transforming growth factor-alpha. This is the first demonstration for secretion of transforming growth factor-alpha by an equivalent of early embryonic cells. The possible paracrine growth stimulating effects of these growth factors have been tested on differentiated derivatives of P19 EC cells, corresponding to all three germ layers. The differences in growth factor production by various embryonal carcinoma cells are discussed in relation to the developmental origin of these cell lines.

Animals↗
PubMed1989

Human teratocarcinoma cells express functional insulin-like growth factor I receptors.

Using iodinated insulin-like growth factors (IGFs) we have detected receptors for IGF-I at the cell surface of the clonally derived human embryonal carcinoma cell line Tera 2 clone 13. Affinity crosslinking of IGFs to Tera 2 clone 13-derived membrane preparations revealed the presence of proteins with features of both type-I and type-II IGF receptors. Treatment of Tera 2 clone 13 cells with retinoic acid to induce differentiation results in an increased number of cell surface receptors, apparently without altering the ratio of type-I and type-II receptors. In addition, Tera 2 clone 13 IGF-I receptors catalyze (auto)phosphorylation at tyrosine upon IGF-I and insulin binding. These findings suggest that type-I IGF receptors might be involved in mediating the effects of IGFs and insulin upon the proliferation of Tera 2 clone 13 cells.

Cell Line↗
PubMed1989

Transforming growth factor-beta and its receptor are differentially regulated in human embryonal carcinoma cells.

The human embryonal carcinoma cell lines Tera-2 clone 13 and NTera-2 clone D1 can be induced by retinoic acid to differentiate in vitro into neuroectodermal derivatives. The undifferentiated cells are rapidly proliferating and tumorigenic, whereas retinoic-acid-treated cells possess a decreased growth rate, lose their transformed phenotype and show a finite lifespan. Differentiation is accompanied by a marked increase in the levels of mRNA for TGF-beta 1 and TGF-beta 2 and the production of TGF-beta activity. Just like murine embryonal carcinoma cells the growth of Tera-2 clone 13 cells is not affected by the addition of either TGF-beta 1 or TGF-beta 2 to the culture medium. In contrast to published data on murine embryonal carcinoma cells, Tera-2 clone 13 and NTera-2 clone D1 cells bind TGF-beta 1 with high affinity, which is due to the presence of type-III TGF-beta receptors. Furthermore, and again in contrast to murine embryonal carcinoma cells, treatment of the human embryonal carcinoma cells with retinoic acid causes a nearly complete loss of TGF-beta 1 binding sites. These results are discussed in the light of similarities and differences in the regulation of growth and differentiation of human and murine embryonal carcinoma cell lines.

Carcinoma↗
PubMed1989

Production of insulin-like growth factors, platelet-derived growth factor, and transforming growth factors and their role in the density-dependent growth regulation of a differentiated embryonal carcinoma cell line.

P19 EPI-7, a differentiated murine embryonal carcinoma cell line with an epithelioid morphology, does not require external growth factors for proliferation under clonal and subconfluent conditions. At saturation density, however, cells become quiescent in the G1/G0 phase of the cell cycle from which they can be restimulated, particularly upon addition of epidermal growth factor. Medium conditioned by confluent P19 EPI-7 cultures is able to enhance clonal outgrowth of this cell line, suggesting that autocrine growth factor loops may be acting in these cells. Analysis of conditioned serum-free medium shows that this cell line produces a platelet-derived growth factor-like growth factor, next to a type beta transforming growth factor and large amounts of insulin-like growth factor II (IGF-II) and an IGF-binding protein with high specificity for IGF-II. This latter observation has been confirmed by the use of a specific bioassay for IGFs, based on their ability to specifically stimulate proliferation of MCF-7 human breast cancer cells. The amount of IGF-II produced (0.5 mg/liter conditioned medium) makes P19 EPI-7 one of the best producing cell lines for this factor described so far. Receptor cross-linking analysis shows that this cell line contains IGF-I receptors, but no specific receptors for IGF-II. Depending on the conditions tested, transforming growth factor-beta 1 either act as a growth-stimulating factor or as a strong growth inhibitory factor. These data demonstrate that upon cellular differentiation, embryonal carcinoma cells can be formed which produce polypeptide growth factors and are also able to respond to such factors. These observations are discussed in the light of the role of autocrine and paracrine growth stimulation processes during early murine development.

Animals↗
PubMed1988

Regulation of intracellular pH during the G1/S-phase transition of the neuroblastoma cell cycle.

Changes in active K+ and Na+ influx during the cell cycle of neuroblastoma (clone Neuro-2A) have suggested activation of an Na+, H+ exchange system during the G1/S-phase transition. Here we report that pHi, measured by the digitonin null-point method, is constant during G1-phase and the G1/S-phase transition and decreases in early S-phase. In addition pHi is shown to be most sensitive to the diuretic amiloride in the G1/S-phase transition, in agreement with the ion influx data. It is concluded from these data, that pHi is tightly regulated during the early cell cycle phases by the Na+, H+ exchange system, in particular during the G1/S-phase transition.

Amiloride↗
PubMed1988

Differentially regulated production of platelet-derived growth factor and of transforming growth factor beta by a human teratocarcinoma cell line.

The human teratocarcinoma stem cell line Tera-2 clone 13 is induced by retinoic acid to differentiate in vitro into endodermal or neuroectodermal cell types. In the absence of externally added growth factors, Tera-2 clone 13 cells proliferated at the same rate as in the presence of serum growth factors. Analysis of serum-free medium conditioned by Tera-2 clone 13 cells showed the presence of a polypeptide immunologically and biochemically related to platelet-derived growth factor (PDGF). In addition transforming growth factor beta (TGF-beta), but no TGF-alpha production could be detected. Tera-2 clone 13 cells specifically expressed high levels of the A-chain mRNA, but not the B-chain mRNA of PDGF. During retinoic acid induced differentiation the level of A-chain mRNA became markedly reduced. In contrast the TGF-beta mRNA levels increased significantly upon differentiation. The implications of these findings are discussed in terms of regulation of growth and differentiation in early embryos as well as in (human) teratocarcinomas.

Cell Differentiation↗
PubMed1987

Epidermal growth factor receptor expression during morphological differentiation of pheochromocytoma cells, induced by nerve growth factor or dibutyryl cyclic AMP.

Rat pheochromocytoma cells (clone PC12) possess functional surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells respond to NGF as well as to dibutyryl cyclic AMP (dbcAMP) by arrest of cell proliferation and initiation of morphological differentiation, while EGF acts as a mitogen. Exposure of PC12 cells to NGF for several days resulted in a complete loss of rapid EGF responses, such as membrane ruffling and activation of active K+ transport. EGF binding studies revealed that this loss of EGF responses was due to an almost complete reduction of the number of EGF binding sites. In contrast, exposure of PC12 cells to dbcAMP for 2 days did not affect the rapid EGF responses, despite the morphological differentiation. Moreover, EGF binding studies demonstrated a twofold increase in the number of high-affinity binding sites and a small increase in the number of low-affinity sites. In addition, exposure of the cells to dbcAMP caused a twofold increase of EGF-receptor phosphotyrosine kinase activity. These results indicate that neither EGF-binding or the presence of EGF receptors nor the rapid EGF responses are sufficient for persistent proliferation, on one hand, or sufficient to avoid morphological differentiation, on the other.

Animals↗
PubMed1987

Commitment to differentiation induced by retinoic acid in P19 embryonal carcinoma cells is cell cycle dependent.

The rate at which P19 embryonal carcinoma cells in monolayer culture become anchorage dependent during differentiation induced by retinoic acid (RA) was investigated. In both nonsynchronized cultures and cultures synchronized by mitotic selection, the ability to grow in semisolid medium, characteristic of the malignant stem cell, decreased after a lag period of about 12 hr in the continuous presence of RA, prior to an increase in cell generation time. However, striking differences between synchronized and nonsynchronized cultures were observed in their commitment to differentiation following RA removal. After only 2 hr of exposure to RA, synchronized cells continued a program of differentiation in which they became anchorage dependent, while at least 24 hr of exposure was required for exponentially growing cells to become similarly committed. Induction of anchorage dependence by RA was also strikingly cell cycle dependent; 2 or 4 hr of exposure of synchronized cells to RA in G1 phase, when the intrinsic capacity for soft agar growth is low, was sufficient to commit cells to anchorage dependence, but a similar exposure in S phase was not. Together, these results suggested that interactions between cells in different cell cycle phases in asynchronous cultures influenced commitment since exposure to RA for more than one cycle (13 hr) was required for all cells to become anchorage dependent. Increased plasminogen activator secretion and epidermal growth factor binding, markers of certain differentiated cell types, increased only 3 and 5 days after RA addition, respectively, and were not induced by pulsed exposure to RA of less than 24 hr, even in synchronized cells.

Animals↗
PubMed1987

Cell cycle analysis during retinoic acid induced differentiation of a human embryonal carcinoma-derived cell line.

Several subclones of the human embryonal carcinoma (EC) cell line Tera-2 can be induced to differentiate in monolayer culture by retinoic acid (RA) to a flattened cell type with reduced growth rate. Using a method based on the transition probability model, we have analysed changes in cell cycle kinetics of Tera-2 cells during the differentiation process. Growth inhibition was shown to occur without a lag period and to be partly due to an increase in the duration of the S-phase, but with a relatively greater contribution from an increase in the duration of G1-phase. Since the fraction of the cell population in the G1-phase then doubled, cells accumulated in this part of the cycle. In contrast, the reduced proliferation rate of two murine EC cell lines, PC13 and P19, treated with RA occurs after a lag period of about two cell cycles and is mainly attributable to an increase in the duration of the S-phase. The results illustrate a differential response of human and murine EC cells to growth regulation by RA and again emphasize that although the stem cells of murine teratocarcinomas may provide a useful model, they are not identical to their human counterparts.

Cell Cycle↗
PubMed1986

Protein kinase C and phorbol ester receptor expression related to growth and differentiation of nullipotent and pluripotent embryonal carcinoma cells.

We have characterized effects of phorbol, 12-myristate 13 acetate (PMA) on growth and differentiation in a nullipotent embryonal carcinoma (EC) cell line, F9, in a pluripotent EC line, P19, and in the differentiated derivatives of these cells, In P19EC and F9EC PMA addition resulted in inhibition of growth, while in the differentiated derivates PMA was mitogenic. PMA did not induce differentiation in EC cells but potentiated the retinoic acid (RA) induced differentiation in P19EC, although, not in F9EC. Rapid morphological changes by PMA were seen in P19EC and two differentiated derivatives which represent different stages of differentiation. In F9 no rapid morphological changes were induced by PMA. Using [3H]phorbol dibutyrate as a ligand we showed that during differentiation into endoderm-like cells the number of phorbol ester receptors increases, while in epithelial-like derivatives no increase is found. In differentiated cells with an increased number of phorbol ester receptors, the cytoplasmic Ca2+- and phospholipid-dependent protein kinase (the putative receptor for phorbol esters) activity was also increased. Only in those derivatives where the number of phorbol ester receptors is increased, is the binding of epidermal growth factor (EGF) inhibited by PMA. These results suggest a relationship between levels of expression of phorbol ester receptors, cytoplasmic protein kinase C and biological effects, namely rapid morphological changes, altered growth, potentiation of RA induced differentiation, and inhibition of EGF binding.

Animals↗
PubMed1986

Establishment of a differentiated mesodermal line from P19 EC cells expressing functional PDGF and EGF receptors.

Aggregation of pluripotent P19 embryonal carcinoma (EC) cells in the presence of DMSO induces differentiation to various mesodermal cell types, including spontaneously contracting muscle. We have established clonal cell lines from these cultures and characterized one (MES-1) in particular for its response to growth factors. In contrast to the undifferentiated stem cells, but as a number of myoblast and muscle cell lines, MES-1 cells respond to both carbachol and bradykinin by the rapid release of Ca2+ from intracellular stores. In addition, MES-1 express receptors for and respond mitogenically to epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Isolated membranes from these cells retain the capacity to bind both ligands; addition of EGF to membranes induces endogenous phosphorylation of several proteins, including the EGF receptor itself and a 38 kD protein, while addition of PDGF specifically induces phosphorylation of the PDGF receptor. By contrast, other derivatives of P19, isolated from retinoic acid (RA)-treated aggregates and resembling neuroectodermal or endodermal cell types respond only to EGF; PDGF neither binds nor induces phosphorylation and a mitogenic response in these cells. During differentiation from EC cells therefore MES-1 cells developed a combination of growth factor receptor characteristics typical of somatic mesodermal cells and indicate that such receptors on EC-derived mesodermal cells are also functional.

Animals↗
PubMed1985

Two receptor classes for epidermal growth factor on pheochromocytoma cells, distinguishable by temperature, lectins, and tumor promoters.

Rat pheochromocytoma cells (clone PC12) display cell surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF) and therefore provide a useful model system with which to study the role of these receptors in the regulation of proliferation and differentiation. In this paper PC12 cells are demonstrated to possess two classes of EGF receptors, a high-affinity class with 7,600 sites per cell and an apparent dissociation constant (Kd) of 0.05 nM, and a low-affinity class with 62,000 sites per cell and a Kd of 14.1 nM. These findings are contrary to literature data (Huff et al., 1981; Vale and Shooter, 1983) but can be explained in part by differences in experimental conditions. Binding studies at 37 degrees C compared with room temperature demonstrated similar affinities of both classes, but during prolonged incubation at 37 degrees C, the binding capacities of both classes decreased. Furthermore the high-affinity class was sensitive to lectins, such as concanavalin A (Con A), and to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Both compounds caused a decrease of the affinity of the high-affinity class without affecting the low-affinity class. At high concentrations of Con A or TPA, a decrease of the apparent number of binding sites of the low-affinity class was also observed. The similarities between the characteristics of EGF binding and NGF binding in PC12 cells are striking and will be discussed.

Animals↗
PubMed1985

Clonal variants of differentiated P19 embryonal carcinoma cells exhibit epidermal growth factor receptor kinase activity.

Differentiated clonal cell lines were isolated from pluripotent P19 embryonal carcinoma (EC) cells treated as aggregates with retinoic acid. Two were characterized in detail. The lines differ in morphology, proliferation rate, the production of plasminogen activator, and in their mitogenic response to insulin but both produce extracellular matrix proteins and can be serially passaged over extended periods, in contrast to differentiated derivatives of many other EC lines. Further, both lines have receptors for and respond mitogenically to epidermal growth factor (EGF). Endogenous phosphorylation of several proteins, including the EGF receptor (150 kDa) and a 38-kDa protein, is induced by EGF in membranes isolated from these cells. Preincubation of membranes with EGF renders them able to catalyze phosphorylation of tyrosine residues in exogenously added peptide substrates. High voltage electrophoresis confirmed the tyrosine specificity of the phosphorylation on the 150- and 38-kDa bands. By contrast, similar experiments in undifferentiated cells showed that intact P19 EC neither bind nor respond to EGF mitogenically and EGF induces no changes in phosphorylation in isolated membranes.

Animals↗
PubMed1984

A short-term screening test for teratogens using differentiating neuroblastoma cells in vitro.

Thirty-nine teratogenic and 18 nonteratogenic compounds were tested using an assay based on the ability of agents to interfere with normal growth and differentiation of murine neuroblastoma cells (clone N1E-115) in culture. Induction of differentiation in cells under growth-promoting conditions and inhibition of differentiating cells over a period of 7 days was dose-dependent, with the lowest effective dose not being highly toxic. Eighty-six percent of the compounds were correctly identified by the assay. The proportions of both false negatives (10%) and false positives (22%) were of the same order or better than in current, comparable tests. The possibilities offered by the system in rapid screening for teratogenic potential of environmental agents are discussed.

Animals↗