Apparent species restriction in the isolation of proliferating embryonal carcinoma cell hybrids

Proliferating somatic cell hybrids are readily obtained from the fusion of a metabolic co-operation-defective mouse embryonal carcinoma cell variant (R5/3) to LMTK- (1 fibroblastic mouse line derived from L-M) and to derivatives of the R5/3 parental cell line PC13. However it has not yet proved possible to produce growing hybrids from fusions between R5/3 and any non-mouse cell line. It appears, from both this work and that of others that although polyethylene glycol-induced fusion occurs between embryonal carcinoma cells and other cell types viable hybrids cannot always be isolated.     

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.     

Enhanced expression of alkaline phosphatase in hybrids between neuroblastoma and embryonal carcinoma

Three independent hybrid cell lines were isolated from the fusion of clonal lines of embryonal carcinoma and neuroblastoma. A series of subclones was subsequently derived from the original hybrid clones. In early hybrid generations all hybrid lines showed enhancement of alkaline phosphatase activity, expressing 2--8 times the activity of the teratoma parental line. The overexpression of APase appears to take place in the stationary phase of the growth cycle. Segregation for very high levels of APase activity was observed among subclones of one hybrid line. Specific activities of the segregants ranged from 0.1 to 133. Results of heat denaturation studies are consistent with the hypothesis that it is the embryonal carcinoma APase that is being expressed in the hybrids.     

Reversal of X-inactivation in female mouse somatic cells hybridized with murine teratocarcinoma stem cells in vitro

A series of near-diploid embryonal carcinoma-like hybrid cells were obtained from polyethylene glycol mediated cell fusion between murine embryonal carcinoma cells (PSA-6TG1 or OTF9-63) having one X chromosome and thymocytes or bone marrow cells from female mice carrying Cattanach's or Searle's translocation. Prior to fusion with EC cells the somatic cells are presumed to contain only one active X chromosome. Following hybrid formation, the chronology of X chromosome replication and the expression of X-linked gene Pgk-1 indicated that all X chromosomes contributed by both parents were active in these hybrids. Experiments were performed to rule out the possibility that the hybrids were formed by fusion of EC cells with rare somatic cells in which both X chromosomes were active. Taken together the data indicate that within four days of fusion there is reactivation of the entire inactive X chromosome.     

Serum-free culture of PC13 murine embryonal carcinoma cells

The requirements for the serum-free culture of PC13 murine embryonal carcinoma cells were determined. Supplementation of a 50:50 mixture of Dulbecco's modified Eagles medium and MCDB104 with transferrin (5 μg/ml), human high-density lipoprotein (HDL) (100 μg/ml), and human low-density lipoprotein (LDL) (50 μg/ml) supported growth comparable to that observed with 5% foetal calf serum. Media supplementation with lipoproteins apparently substitutes for the effects of insulin, desoctapeptide insulin (DOP), or multiplication-stimulating activity (MSA) on EC cell multiplication. Clonal growth of PC13 EC cells in this serum-free medium could only be achieved in the presence of suitable feeder cell monolayers. These observations demonstrate that PC13 EC cells do not have an absolute requirement for exogenous mitogens to support multiplication.     

Injection of murine embryonal carcinoma cells and embryo-derived pluripotential cells into mouse blastocysts

Two pluripotential mouse cell lines, the OTT 6050-derived cell line TCE and the embryo-derived stem cell line BLC-1, were injected into blastocysts to analyze their developmental potential. The contribution of TCE cells to the embryo was found to be limited and sporadic. There was no indication of a preferential colonization of extraembryonal membranes or developmentally related tissues in adult chimeras. BLC-1 cells failed to colonize the embryo. This indicates that a normal karyotype, pluripotency, and cell surface markers which are shared by cells of early embryos are not necessarily sufficient markers for their ability to participate in embryogenesis.     

The effects of methylmercury on the cytoskeleton of murine embryonal carcinoma cells

Immunofuorescence staining with antibodies to tubulin and vimentin and staining with phalloidin have been used to examine the effects of methylmercury on the cytoskeleton of embryonal carcinoma cells in culture. Exposure of embryonal carcinoma cells to methylmercury (0.01 to 10 m) resulted in concentration- and time-dependent disassembly of microtubules in interphase and mitotic cells. These effects were reversible when cultures were washed free of methylmercury. Spindle microtubules were more sensitive than those of interphase cells. Spindle damage resulted in an accumulation of cells in prometaphase/metaphase, which; correlated with a temporary delay in the resumption of normal proliferation rate upon removal of methylmercury. Of the interphase cytoskeletal components, microtubules were the first affected by methylmercury. Vimentin intermediate filaments appeared relatively insensitive to methylmercury, but showed a reorganization secondary to the microtubule disassembly. Actin microfilaments appeared unchanged in cells showing complete absence of microtubules. Our results 1) support previous reports suggesting that microtubules are a primary target of methylmercury, 2) document a differential sensitivity of mitotic and interphase microtubule systems and 3) demonstrate the relative insensitivities of other cytoskeletal components.Abbreviations -MEM alpha minimal essential medium - EC embryonal carcinoma cells - McHg methylmercury - PBS phosphate buffered saline - SB microtubule stabilizing buffer     

Interactions between diploid embryonal carcinoma cells and early embryonic cells

Two diploid embryonal carcinoma (EC) cell lines, P10 and P19, differ in their response to the embryonic environment. P10 produces mostly normal chimeras following injection into blastocysts, whereas P19 produces mostly abnormal chimeras. In this study, P10 cells were aggregated with morulae, and all resulting fetuses were chimeric with very large contributions from the EC cells. However, all embryos were abnormal. Following aggregation of P19 cells with morulae, very few embryos were recovered and they were all non-chimeric. Both P10 and P19 were capable of forming functional gap junctions with morula cells and with the ICM of the blastocyst but not with trophoblast, showing that differences in the ability to make junctional contact with the embryo cannot explain the differences between the two cell lines.     

Coordinate expression of parietal endodermal functions in hybrids of embryonal carcinoma and endodermal cells.

A derivative, FOT5, of the F9 murine embryonal carcinoma cell line which is resistant to ouabain and thioguanine was fused with a near diploid parietal endodermal cell line, PFHR9, Hybrid clones (ENEC1 to ENEC5) were isolated in HAT Medium containing ouabain at a frequency of approximately 2 x 10(-4). The DNA contents and chromosome number of the ENEC hybrids were approximately the sum of those of the parents. Five hybrid cell lines examined in detail expressed the following parietal endodermal functions: plasminogen activator activity, basement membrane proteins, and endodermal cytoskeletal proteins. Embryonal carcinoma characteristic functions (tumorigenicity, a stage specific embryonic antigen, and high alkaline phosphatase activity) were extinguished in the hybrids. No hybrid clones with embryonal carcinoma morphology were observed among 1,358 hybrid clones examined. Hybrids, propagated for over 100 generations, continued to express endodermal functions and not embryonal carcinoma functions. The coordinate expression of endodermal functions and the extinction of embryonal carcinoma functions in the ENEC hybrids suggest that the parietal endodermal cells contain diffusible activities which extinguish embryonal carcinoma functions and possibly cause the embryonal carcinoma genome to express parietal endodermal characteristics.     

Epstein-Barr virus in somatic cell hybrids between mouse cells and human nasopharyngeal carcinoma cells.

Somatic cell hybrids between mouse cells and cells derived directly from NPC biopsies were produced in order to study the association of the Epstein-Barr virus (EBV) genome and the expression of Epstein-Barr nuclear antigen (EBNA) with the human chromosome(s). All attempts to correlate the presence of EBV-DNA and the expression of EBNA with the presence of a particular human chromosome(s) showed that the segregation of EBV-DNA or of EBNA and human chromosomes was dysconcordant. The data, therefore, suggest that in the hybrids studied the presence of EBA-DNA is not determined by the presence of a specific human chromosome.     

Expression of simian virus 40 large T antigen in embryonal carcinoma cell hybrids.

Previous work has shown that murine embryonal carcinoma cells are refractory to infection with various viruses, including simian virus 40. Thus, large T and small t antigens, the products of the simian virus 40 early region, are not produced when the virus infects embryonal carcinoma cells, in contrast to other cell types. We show, by qualitative and quantitative analyses, that embryonal carcinoma cell hybrids, containing a simian virus 40 early region integrated into human DNA, are capable of producing viral large T antigen.     

Male murine embryonal carcinoma cell line selectively metastatic to the ovaries and adrenals

Developmentally pluripotent embryonal carcinoma cells were isolated from chromosomally male embryo-derived teratocarcinoma and adapted to in vitro growth without a feeder layer. The uncloned original cell line as well as clones derived from it have a tendency to selectively localize to the ovaries and adrenals upon intravenous injection into adult female mice, but only to the adrenals when injected into male mice. The overall take of injected tumor cells was lower in males and the tumors formed slower in males than in females. These findings suggest that the growth of this karyotypically male embryonal carcinoma could be under hormonal regulation.     

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.