Spontaneous differentiation in the colonies of a nullipotent embryonal carcinoma cell line (F9)

Experiments were undertaken to reveal the spontaneous differentiation capacity of the nullipotent F9 embryonal-carcinoma (EC) cell line in colonies derived from single cells. Culture conditions which allowed the development of neuroblasts in colonies of the multipotent EC cell line (PCC3) were worked out, and comparative studies on neuroblast differentiation in PCC3 and F9 colonies were conducted. Neural-cell-specific silver impregnation, selective staining of cells having electrically excitable membranes with merocyanine 540 and the observation of nerve processes were considered as differentiation markers. The appearance of neuroblasts in F9 and PCC3 colonies could be detected from the 6th day after seeding. The development of neuroblasts was less prevalent in high-density cultures, especially in the case of F9. By the 8th day in differentiating colonies, PCC3 cells lost much of their colony-forming activity, while F9 cells preserved their original high plating efficiency, in spite of advanced differentiation. The determination of growth parameters during differentiation in colonies led to the conclusion that F9 cells had lost certain growth-control mechanisms which normally restrict the clonal growth of EC cells. It is suggested that the phenomenon of nullipotence may be analysed in terms of the coordinated regulation of proliferation and differentiation of EC cells.     

Spontaneous differentiation in the colonies of a nullipotent embryonal carcinoma cell line (F9)

Abstract. Experiments were undertaken to reveal the spontaneous differentiation capacity of the nullipotent F9 embryonal-carcinoma (EC) cell line in colonies derived from single cells. Culture conditions which alllowed the development of neuroblasts in colonies of the multipotent EC cell line (PCC3) were worked out, and comparative studies on neuroblast differentiation in PCC 3 and F 9 colonies were conducted. Neural-cell-specific silver impregnation, selective staining of cells having electrically excitable membranes with merocyanine 540 and the observation of nerve processes were considered as differentiation markers. The appearance of neuroblasts in F9 and PCC3 colonies could be detected from the 6th day after seeding. The development of neuroblasts was less prevalent in high-density cultures, especially in the case of F9. By the 8th day in differentiating colonies, PCC3 cells lost much of their colony-forming activity, while F9 cells preserved their original high plating efficiency, in spite of advanced differentiation. The determination of growth parameters during differentiation in colonies led to the conclusion that F9 cells had lost certain growth-control mechanisms which normally restrict the clonal growth of EC cells. It is suggested that the phenomenon of nullipotence may be analysed in terms of the coordinated regulation of proliferation and differentiation of EC cells.     

Overexpression of uvomorulin in a compaction-negative F9 mutant cell line

The mutant F9 cell line F9att-5.51 synthesizes reduced amounts of uvomorulin (UM) protein and we hypothesized earlier (Adamson, Baribault, and Kemler, Dev. Biol. (1990), 138, 338) that this may account for its inability to compact into tightly aggregated balls of cells. Subsequently, when 5.51 cells are treated with retinoic acid to stimulate their differentiation, they are unable to form embryoid bodies as do wild-type cells which form an outer epithelial layer of visceral endoderm cells. We have now examined the possibility that the UM protein made in the mutant line is defective, but find that it is normal in structure and stability. The gene coding for UM appears to be normal as does the mRNA which is synthesized at a normal rate but is severely reduced in steady-state measurements of mutant cells. A rescue experiment was performed by increasing levels of UM in mutant cells by means of transfection with a UM expression vector. The resulting cells expressed abundant UM mRNA and protein but were still unable to form compacted aggregates and did not differentiate into embryoid bodies. Interestingly, the stability of endogenous UM mRNA was improved in the presence of exogenous UM; therefore, a positive feedback mechanism contributes to low mRNA levels in mutant cells. The accumulated data suggest that UM in 5.51 cells is unable to mount a compaction activity because a distal connecting link in the multicomponent process initiated by UM is missing or or aberrant. The missing component is likely to connect UM to actin and the cytoskeleton of the cell.     

Induction of F9 cell differentiation by transient exposure to retinoic acid.

The F9 cell is a mouse embryonal teratocarcinoma which can be induced to differentiate into visceral endoderm by treatment with retinoic acid (RA). Treatment with RA in conventional studies was carried out in the constant presence of RA. Here we demonstrate that treatment with RA can be as short as 3 hrs to induce differentiation of F9 cells. Morphology, alpha-fetoprotein gene activity, and temporal patterns of F9 cell differentiation are the same with both short- and long-term treatment with RA.     

Meiosis reinitiation as a model system for the study of cell division and cell differentiation

In this paper, we review our findings concerning the control of meiosis reinitiation in starfish oocytes and discuss recent advances that lead to characterization of the maturation promoting factor (MPF) responsible for G2-M transition. It is now agreed that appearance of this factor, which triggers nuclear envelope breakdown, chromosome condensation and metaphase spindle formation, corresponds to the activation of a M-phase specific H1-kinase. MPF has been shown to be constituted of equimolar amounts of a 34 kDa catalytic subunit protein homologous to the yeast cdc2/CDC28 gene product and a cyclin protein homologous to the yeast cdc13 gene product. "In vivo" and "in vitro" studies based on the use of inhibitors of protein synthesis, protein kinases, phosphoprotein phosphatases and proteases lead to a better understanding of the complex series of events which regulate activation and inactivation of MPF. In the unfertilized metaphase 2-arrested vertebrate oocyte, it has also been shown that stabilization of MPF depends on the kinase activity of the c-mos protooncogene. This review attempts to illustrate how the significant progress made in the understanding of the regulation of cell cycle transverse directly resulted from the convergence of observations in multidisciplinary studies in yeast genetics, development and oncogenesis. It also offers a model for considering the highly integrated events which, starting at the level of the plasma membrane, may eventually result in early cell differentiation.     

A conditionally immortalized cell line model for the study of human prostatic epithelial cell differentiation

In the normal human prostate, undifferentiated proliferative cells reside in the basal layer and give rise to luminal secretory cells. There are, however, few epithelial cell lines that have a basal cell phenotype and are able to differentiate. We set out to develop a cell line with these characteristics that would be suitable for the study of the early stages of prostate epithelial cell differentiation. We produced a matched pair of conditionally immortalized prostate epithelial and stromal cell lines derived from the same patient. The growth of these cells is temperature dependent and differentiation can be induced following a rise in culture temperature. Three-dimensional co-cultures of these cell lines elicited gland-like structures reminiscent of prostatic acini. cDNA microarray analysis of the epithelial line demonstrated changes in gene expression consistent with epithelial differentiation. These genes may prove useful as markers for different prostate cell types. The cell lines provide a model system with which to study the process of prostatic epithelial differentiation and stromal-epithelial interactions. This may prove to be useful in the development of differentiation-targeted prostate cancer therapies.     

The adenovirus Ela gene induces differentiation of F9 teratocarcinoma cells.

Undifferentiated F9 cells transfected with plasmids encoding adenovirus E1a gene products underwent radical morphological changes. They ceased to express the SSEA-1 stem cell marker antigen and started to express a number of the characteristics of the differentiated state that is induced in F9 cells by treatment with retinoic acid. In particular, they expressed keratin intermediate filaments and acquired the ability to synthesise simian virus 40 tumor antigens after virus infection. The transfected cells expressed the E1a proteins, and this expression was necessary to induce the phenotypic changes, since a coisogenic plasmid encoding only a truncated 70-amino-acid E1a polypeptide and the transfection procedure itself did not detectably after the morphology or marker expression of the F9 stem cells. The phenotypic change was induced by both 13S and 12S cDNA plasmids. We discuss these results in the context of known E1a functions and with reference to the other oncogenes and external factors that can cause F9 cell differentiation.     

Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation

Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60. The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and poly-morphonuclear leukocytes (PMN) over 9 d of culture in 1.3 percent dimethylsulfoxide (DMSO). As the HL-60 cells mature, the rate of O(2-) production increase 18-fold, with a progressive shortening of the lag time required for activation. Hexosemonophosphate shunt activity rises concomitantly. Ingestin of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities. Degranulation, as measured by release of β-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective. However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of O(2-) generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100 percent that of normal PMN. DMSO- induced differentiation of HL-60 cells is a promising model for myeloid development.     

Phosphoproteomic analysis of AML14.3D10 cell line as a model system of eosinophilia

Eosinophils act as effectors in the inflammatory reactions of allergic diseases including atopic dermatitis. Atopic dermatitis patients and others with allergic disorders suffer from eosinophilia, an accumulation of eosinophils due to increased survival or decreased apoptosis of eosinophils. In this study, a differential phosphoproteome analysis of AML14.3D10 eosinophil cell line after treatment with IL-5 or dexamethasone was conducted in an effort to identify the phosphoproteins involved in the proliferation or apoptosis of eosinophils. Proteins were separated by 2-DE and alterations in phosphoproteins were then detected by Pro-Q Diamond staining. The significant quantitative changes were shown in nineteen phosphoproteins including retinoblastoma binding protein 7, MTHSP75, and lymphocyte cytosolic protein 1. In addition, seven phosphoproteins including galactokinase I, and proapolipoprotein, were appeared after treatment with IL-5 or dexamethasone. Especially, the phospho-APOE protein was down-regulated in IL-5 treated AML14.3D10, while the more heavily phosphorylated APOE form was induced after dexamethasone treatment. These phosphoproteome data for the AML14.3D10 cell line may provide clues to understand the mechanism of eosinophilia as well as allergic disorders including atopic dermatitis.     

Family tree analysis of a transformed cell line and the transition probability model for the cell cycle

Variation in intermitotic time between individual cells in culture can be ascribed to the occurrence of random transitions in the cell cycle. We have analysed a family tree of mouse neuroblastoma cells, and observed that variation in difference in intermitotic time between sister cells is smaller than between cousin cells, and this difference is again smaller than between second-cousin and unrelated cells. This observation is incompatible with all transition probability models presented so far. We propose a model for the cell cycle with a single random transition, but with the additional assumption that the (two) system parameters may show variability within the population such that the closer cells are in their relation to each other, the closer their values of the system parameters will be. This model describes correctly the behaviour of the family tree of the cell line and in addition is able to explain why differences in intermitotic time between sister cells are exponentially distributed, while intermitotic times themselves are more or less normally distributed. Methods have been described to quantify the various system parameters.     

The NCI-N87 cell line as a gastric epithelial barrier model for drug permeability assay

The objective of this study was to evaluate the human NCI-N87 cell line as a model for gastric permeability drug studies under pH conditions of the stomach. The optimal conditions that led NCI-N87 cells to form a typical differentiated gastric epithelial barrier were a seeding density of 2.5 × 10⁵ cells/cm² on porous inserts and growth in serum-complemented RPMI-1640 medium until 18–27 days post-confluency. The resulting cell monolayers showed moderately high transepithelial electrical resistance (TEER) values of about 500 Ω cm², cells of polygonal morphology expressing E-cadherin and ZO-1 proteins at their contact surfaces, and production of mucus clusters. The monolayers withstood apical pH of 7.4 down to 3.0 with the basal pH fixed at 7.4. The apparent permeability coefficients (P_app) of model compounds were evaluated in the apical-to-basolateral and basolateral-to-apical directions under different pH gradients. The monolayers were impermeable to the integrity marker Lucifer Yellow (low P_app of 0.3–1.1 × 10⁻⁶ cm/s). The furosemide P_app (0.4–1.5 × 10⁻⁵ cm/s) were slightly dependent on pH but remained moderate. The caffeine P_app (4.2–5.0 × 10⁻⁵ cm/s) were higher and insensitive to pH changes. The NCI-N87 cell line provides a useful in vitro tool to assess gastric drug permeability and absorption under physiologic conditions prevailing in the human stomach.     

The human promyelocytic HL60 cell line: a model of myeloid cell differentiation using dimethylsulphoxide, phorbol ester and butyrate.

The release of the reactive oxygen species that accompanies the oxidative burst was studied in HL60 cells differentiated with either dimethylsulphoxide, butyrate or phorbol myristate acetate in order to establish the extent to which differentiated cells are phenotypically similar to human neutrophils, monocytes and macrophages. When phorbol myristate acetate was used as a stimulus, the rates of superoxide production by dimethylsulphoxide and butyrate differentiated HL60 cells was not significantly different from those observed in neutrophils and monocytes isolated from normal peripheral blood. Similar results were obtained when luminol-dependent chemiluminescence was measured in the presence of horseradish peroxidase using phorbol myristate acetate as the stimulus. However, in the absence of horseradish peroxidase, the luminol-dependent chemiluminescence in the dimethylsulphoxide and butyrate-differentiated HL60 cells was significantly lower than that of the control cells isolated from human blood, reflecting the absence of myeloperoxidase in the differentiated cells. In contrast, HL60 cells differentiated by phorbol myristate acetate failed to show any increased generation of superoxide or luminol-dependent chemiluminescence upon stimulation. Impaired release of lysosomal enzymes by the chemically differentiated cells suggests impairments in the extent of differentiation resulting in cells with defective azurophilic degranulation processes. It is concluded that HL60 cells differentiated by the above agents are somewhat controversial models of promyelocyte differentiation into typical neutrophilic, monocytic and macrophage-like cells.     

Studies on the Cloudman melanoma cell line as a model for the action of MSH

A review of the studies done at Yale on the role of MSH in regulating pigmentation and growth of Cloudman (S91) melanoma cells is presented. The areas covered include the isolation and analyses of mutant cell lines unresponsive to MSH; the role of cyclic AMP, cyclic AMP-dependent protein kinases, and protein phosphorylation reactions in the response of MSH; new regulators of the melanogenesis pathway; the cytotoxicity of melanin precursors; the development of methodology for synthesizing 125I-beta-MSH; the use of this ligand to study receptors for MSH; and the chemical and biological properties of phosphorylated isomers of L-dopa, a new class of compounds exhibiting potent bio-activity toward melanocytes. All of the experiments described were carried out in the Department of Dermatology at the Yale University School of Medicine during the tenure of Dr. Aaron B. Lerner as chairman.     

The induction of antigenic changes in a teratocarcinoma stem cell line (F9) by retinoic acid

Treatment of embryonal carcinoma cells F9 with retinoic acid results in the appearance of epithelioid cells resembling endoderm which synthesize basement membrane protein and plasminogen activator. Concomitant with the appearance of these properties of differentiated cells, the epithelial cells cease to express SSEA-1, an antigenic determinant characteristic of teratocarcinoma stem cells and early mouse embryos. Our evidence indicates that the phenotypic changes that accompany retinoic acid treatment of embryonal carcinoma cells are irreversible and a consequence of the differentiation of the cells into endoderm.     

Expression of fibroblast growth factor by F9 teratocarcinoma cells as a function of differentiation

F9 teratocarcinoma stem cells treated with retinoic acid (RA) and dibutyryl cAMP (but2 cAMP) differentiate into embryonic parietal endoderm. Using heparin-affinity chromatography, endothelial cell proliferation assays, immunoprecipitation, and Western analysis with antibodies specific for acidic and basic fibroblast growth factors (FGFs), we detected biologically active FGF in F9 cells only after differentiation. A bovine basic FGF cDNA probe hybridized to 2.2-kb mRNAs in both F9 stem and parietal endoderm cells and to a 3.8-kb mRNA in F9 stem cells. A genomic DNA probe for acidic FGF hybridized to a 5.8-6.0-kb mRNA in both F9 stem and parietal endoderm cells, and to a 6.0-6.3-kb mRNA only in parietal endoderm cells. Although these FGF mRNAs were present in the stem cells, we could find no evidence that F9 stem cells synthesized FGFs, whereas differentiated F9 cells synthesized both acidic and basic FGF-like proteins. We conclude that biologically active factors with properties characteristic of acidic and basic FGF are expressed by F9 parietal endoderm cells after differentiation. Differentiating embryonic parietal endoderm thus may serve as a source of FGF molecules in the developing blastocyst, where these factors appear to play a central role in subsequent embryogenesis.     

Multidrug-resistant phenotype influences the differentiation of a human colon carcinoma cell line.

The human colon carcinoma cell line HT29-D4, which constitutively expresses a very low level of the MDR1 gene product, was made multidrug resistant by transfection with a human MDR1 cDNA from the pHaMDR1/A expression vector and selection by colchicine. Resistant clones were 3- to 15-fold resistant to colchicine and were cross-resistant to doxorubicin (3- to 4-fold). MDR1 gene expression was associated with the expression of functional P-glycoprotein (gp-170); the function was reversed by verapamil and cyclosporin A. HT29-D4 cells are able to differentiate in vitro by replacement of glucose by galactose in the culture medium and also to release the carcinoembryonic antigen (CEA). Under these culture conditions, MDR1 mRNA and gp-170 were always expressed and the protein remained functional. Upon galactose treatment, resistant clones were less differentiated since they showed a heterogeneous monolayer organization accompanied by heterogeneous staining of cell-surface CEA and a high decrease (60-90%) of CEA release.