Induced muscle differentiation in an embryonal carcinoma cell line.

Cells of the teratocarcinoma-derived line P19S1801A1 (01A1) are pluripotent embryonal carcinoma cells and can be induced to differentiate when aggregated and exposed to dimethyl sulfoxide. Many nonneural cell types appear in dimethyl sulfoxide-treated cultures, cardiac and skeletal muscle being the most easily identified. We have used immunofluorescence procedures with monoclonal antibodies directed against muscle myosin to confirm and quantitate the number of muscle cells formed. A monoclonal antibody reactive with an embryonal carcinoma-specific surface antigen was used to confirm the disappearance of undifferentiated cells after dimethyl sulfoxide treatment. Cardiac muscle cells developed within 4 to 5 days of drug exposure, but skeletal muscle cells did not become evident until 7 to 8 days. We have isolated a mutant cell line (D3) which appears to be incapable of muscle development but which does form neurons and glial cells when exposed to high retinoic acid concentrations. We propose that this system will be useful for investigation of the means by which pluripotent cells become committed to development along the striated muscle lineages.     

Analysis of a nontumorigenic embryonal carcinoma cell line

Embryonal carcinoma (EC) cells have proven to be of particular value in studies of both oncogenesis and mammalian development as well as in evaluating the relationship between these two phenomena. We have infected EC cells with a retrovirus in an effort to obtain by insertional mutagenesis cell lines defective in either differentiative or oncogenic potentials. One such cell line, identified originally by its unique morphological phenotype, is abnormal with respect to both parameters. These cells do not differentiate along typical EC cell lineages, possibly having lost their ability to elaborate endodermal derivatives. They do, however, retain certain cell surface markers characteristic of EC cells and lose these markers after exposure to retinoic acid. Most significantly, they also fail to form tumors in vivo in syngeneic mice, although they grow as well as the parental cells in vitro. Southern blot analysis indicates that this variant cell line has a single viral insert and the original cell was probably hemizygous for the insertion site, suggesting that a single gene may regulate both the tumorigenic and differentiative capacities of the cell.     

A new yeast artificial chromosome vector designed for gene transfer into mammalian cells

This report describes the construction of a new yeast artificial chromosome (YAC) vector designed for gene transfer into mammalian cells. For ease of use, the two arms of the vector were cloned separately. The vector harbours the Neo and Hyg genes for dominant selection in mammalian cells, a putative human origin of replication, a synthetic matrix attachment region and two loxP sites (one on each arm). The cloning ability of the vector was demonstrated by successful propagation of the cDNA of the cystic fibrosis gene, CFTR, as a YAC in Saccharomyces cerevisiae. A YAC containing the entire CFTR gene was also constructed by retrofitting the two arms of a pre-existing clone (37AB12) with the two arms of the novel vector. Both the cDNA and entire gene containing YACs were circularized in yeast by inducible expression of the Cre recombinase. Recombination occurred very specifically at the loxP sequences present on the two arms of the YAC. Applications of the vector to gene transfer are discussed.     

An origin-deficient yeast artificial chromosome triggers a cell cycle checkpoint

Checkpoint controls coordinate entry into mitosis with the completion of DNA replication. Depletion of nucleotide precursors by treatment with the drug hydroxyurea triggers such a checkpoint response. However, it is not clear whether the signal for this hydroxyurea-induced checkpoint pathway is the presence of unreplicated DNA, or rather the persistence of single-stranded or damaged DNA. In a yeast artificial chromosome (YAC) we have engineered an approximately 170 kb region lacking efficient replication origins that allows us to explore the specific effects of unreplicated DNA on cell cycle progression. Replication of this YAC extends the length of S phase and causes cells to engage an S/M checkpoint. In the absence of Rad9 the YAC becomes unstable, undergoing deletions within the origin-free region.     

A differentiation-defective concanavalin-A-resistant variant of a pluripotent embryonal carcinoma cell line

A concanavalin-A(Con A)-resistant variant of the pluripotent mouse embryonal carcinoma cell line, PSA1-NG2, was isolated. This variant, designated NG2-2.16, fails to exhibit the extensive spontaneous differentiation displayed by PSA1-NG2 in colonies in vitro and in tumours in vivo. The molecular nature of the defect in NG2-2.16 cells was not revealed by quantitative studies of the binding, uptake and metabolism of tritiated Con A, or by Western blotting of membrane and whole cell homogenates, thus indicating the defect to be the result of a more subtle molecular alteration. Statistical evidence suggests that the same mutation is responsible for both the Con A resistance and the lack of spontaneous differentiation. NG2-2.16 cells were induced to differentiate by exposure to retinoic acid, suggesting that the mutation affects the regulation of differentiation rather than the potential for differentiation.     

alpha-Difluoromethylornithine induces differentiation of a human embryonal carcinoma cell line in vitro

Human embryonal carcinoma cells could serve as a useful model system for analysis of early human development. A limited number of human embryonal carcinoma cell lines have been generated from in vivo tumors. We report here that alpha-difluoromethylornithine, a specific enzyme-activated inhibitor of ornithine decarboxylase activity, can induce differentiation in human embryonal carcinoma cells. The differentiated phenotype could be distinguished from undifferentiated cells by altered cellular morphology, biochemical and cell surface antigenic properties. These results suggest that alterations in the intracellular levels of polyamines may play a role in human embryonal carcinoma cell differentiation, and possibly human embryogenesis.     

Characterization of a new human cell line derived from a xenografted embryonal carcinoma

Summary A human cell line has been established from a transplantable xenografted human testicular tumor, which, both in the original tumor and in the xenograft, exhibited the histological characteristics of an undifferentiated malignant teratoma (embryonal cell carcinoma). The cells in culture were undifferentiated by biochemical, morphological, and ultrastructural criteria, growing as small islands of cells that tended to form aggregates at high density. The cells showed some variation in chromosome number with 30 to 40% of the cells having a normal human karyotype. The cells expressed high levels of alkaline phosphatase, which by heat inactivation and inhibition studies was 40 to 50% placental type alkaline phosphatase. None of the cultures produced human chorionic gonadotrophin, alphafetoprotein, carcinoembryonic antigen, or fibronectin, although at high cell densities plasminogen activator could be detected at low levels. Cell surface studies showed that the cells shared antigens with the murine embryonal carcinoma cell line F9, expressedβ ₂-microglobulin at very low and variable levels, and bound the lectin peanut agglutinin. These studies suggest that this cell line has some of the characteristics described for murine embryonal carcinoma cell lines.     

Evidence for an intraclonal random shift between two types of cell cycle times in an embryonal carcinoma cell line

In a recent paper we reported the discovery of an intraclonal bimodal-like cell cycle time variation within the multipotent embryonal carcinoma (EC) PCC3 N/1 line growing in the exponential phase in the undifferentiated state. The variability was found to be localized in the G1 period. Furthermore, an inverse relation between cell size and cell generation time was found in the cell system analysed. It was suggested that the bimodal-like intraclonal time variability previously reported was attributable to an intraclonal shift between two types of cell-growth-rate cycles and that the cell-growth cycle has a supramitotic character, being dissociated from the DNA-division cycle. The growth rate heterogeneity in the cell population was found to need three cell cycles to reach full dispersion in time. This was assumed to be due to a decreased inheritance from sister cell pairs to second cousin cell pairs. Thus, the interesting feature is that in one and the same multipotent cell line there was evidence for an intraclonal instability with a random shift between two types of cell cycle differing in the duration of their G1 period.     

Purification of a tumor-specific PNA-binding glycoprotein, gp200, from a human embryonal carcinoma cell line.

A 200-kDa peanut agglutinin (PNA)-binding glycoprotein, gp200, has been purified and partially characterized from the human embryonal carcinoma cell line, HT-E (833k). Tissue distribution analysis of this molecule by lectin blotting with PNA of detergent-extracted proteins from human cell lines and tissues demonstrated expression limited to nonseminomatous germ cell tumors. The 200-kDa protein was purified with lectin affinity and gel filtration chromatography. Purification to apparent homogeneity was demonstrated by one- and two-dimensional gel electrophoresis. Characterization of gp200 revealed it to be a surface integral membrane glycoprotein; however, gp200 could also be purified from the culture media of EC cells, suggesting gp200 has an extracellular role. The carbohydrate groups of gp200 are N-linked and partially sialylated and contain terminal galactose residues. These initial studies suggest that the PNA-defined glycoprotein, gp200, is a candidate for a nonseminomatous germ cell tumor marker.     

Simplified construction and characterization of yeast artificial chromosome libraries.

Three yeast artificial chromosome (YAC) libraries were constructed using two human cell lines and the pYAC-RC vector. The main differences from the previously described methods were: i) genomic DNA was digested in low melting point (LMP) agarose blocks with the rare cutting enzyme ClaI; ii) DNA was ligated in melted LMP agarose after agarase treatment; iii) spheroplast regeneration plating was done in calcium alginate thin layer. In addition, a panel of PCR primers was used to identify quickly the presence in the libraries of repetitive and single copy human DNA sequences.     

Analysis of chromosome 21 yeast artificial chromosome (YAC) clones.

Chromosome 21 contains genes relevant to several important diseases. Yeast artificial chromosome (YAC) clones, because they span > 100 kbp, will provide attractive material for initiating searches for such genes. Twenty-two YAC clones, each of which maps to a region of potential relevance either to aspects of the Down syndrome phenotype or to one of the other chromosome 21-associated genetic diseases, have been analyzed in detail. Clones total approximately 6,000 kb and derive from all parts of the long arm. Rare restriction-site maps have been constructed for each clone and have been used to determine regional variations in clonability, methylation frequency, CpG island density, and CpG island frequency versus gene density. This information will be useful for the isolation and mapping of new genes to chromosome 21 and for walking in YAC libraries.     

Commitment in a murine embryonal carcinoma cell line during differentiation induced by retinoic acid

Murine embryonal carcinoma (EC) cells are induced to differentiate when cultured in the presence of retinoic acid (RA). Whereas the EC cells have a high plating efficiency, the differentiated cells have little or no colony-forming ability under the same conditions. We have assumed that the loss of colony-forming ability following exposure of EC cells to RA corresponds to the irreversible commitment of EC cells to differentiate. We found that uncommitted EC cells persist in RA-treated aggregates of EC cells and that the proportion of EC cells stabilizes at a level inversely related to the RA concentration. Both experimental evidence and mathematical modelling results are consistent with the interpretation that there is a dynamic equilibrium achieved by a balance between the processes of EC cell proliferation and differentiation. Since different cell types are induced by different RA concentrations, our results suggest that the commitment to differentiate is not related in any simple way to the developmental program which ensues.     

States of developmental commitment of a mouse embryonal carcinoma cell line differentiating along a neural pathway

The embryonal carcinoma cell line PCC7-S-AzaR1 (clone 1009) has been shown to differentiate in the presence of all-trans retinoic acid and dibutyryl cAMP into cells of predominantly neural properties (Paulin, D., H. Jakob, F. Jacob, K. Weber, and M. Osborn. 1982. Differentiation. 22:90-99). By analyzing the marker expression of derivatives in further detail, we characterized the two major cell phenotypes as neuron- and fibroblast-like and the two minor ones as astroglia- and endothelial-like. The stability of developmental commitment of clone 1009 was tested by recloning. The isolated subclones exhibited different patterns of chemically induced derivatives, with some of them (denoted N-clones) producing only a single (neuronal) cell type. As shown by long-term cultures in the absence of retinoic acid, the properties of isolated subclones remained essentially stable. In contrast to the clones producing neuron-like and other derivatives upon induced differentiation, the (exclusively neuronal) derivatives of N-clones detached and died within a few days in culture. If maintained in the presence of other neural cell types, however, their survival was dramatically extended indicating a requirement for specific interactions with other cells of the same tissue. The patterns of derivatives obtained from N-clones depended on the chemical nature of the substrate on which they were grown. Thus, when seeded on laminin-coated surfaces before induced differentiation, N-clones developed not only to neuron-like derivatives but rather to the same four derivatives observed with the original cell pool. These and further results suggest a common cell lineage of the identified phenotypes. The isolated subclones of uninduced cells probably represent different states of commitment within the same developmental pathway. Their stability offers the opportunity to analyze the nature of cellular commitment on the cellular, molecular, and genetic levels. This makes the family of clones derived from PCC7-S-AzaR1 (clone 1009) cells an advantageous in vitro model of mammalian brain early ontogenesis.     

Unequal homologous recombination of human DNA on a yeast artificial chromosome.

We examined unequal homologous DNA recombination between human repetitive DNA elements located on a yeast artificial chromosome (YAC) and transforming plasmid molecules. A plasmid vector containing an Alu element, as well as a sequence identical to a unique site on a YAC, was introduced into yeast and double recombinant clones analyzed. Recombination occurs between vector and YAC Alu elements sharing as little as 74% identity. The physical proximity of an Alu element to the unique DNA segment appears to play a significant role in determining the frequency with which that element serves as a recombination substrate. In addition, cross-over points of the recombination reaction are largely confined to the ends of the repetitive element. Since a similar distribution of crossover sites occurs during unequal homologous recombination in human germ and somatic tissue, we propose that similar enzymatic processes may be responsible for the events observed in our system and in human cells. This suggests that further examination of the enzymology of unequal homologous recombination of human DNA within yeast may yield a greater understanding of the molecular events which control this process in higher eukaryotes.     

Transgenic mice generated by pronuclear injection of a yeast artificial chromosome.

Transgenic mice have become invaluable for analysing gene function and regulation in vivo. However, the size of constructs injected has been limited by the cloning capacity of conventional vectors, a constraint that could be overcome with yeast artificial chromosomes (YACs). We investigated the feasibility of making transgenic mice with YACs by pronuclear injection of a small YAC carrying a gene encoding tyrosinase. Use of a vector with a conditional centromere allowed fifteenfold amplification of the YAC in yeast and its recovery in high yield. The albino phenotype of the recipient mice was rescued demonstrating the correct expression of the tyrosine gene from the construct. Furthermore, the telomeric sequences added by the yeast integrated into the mouse genome and did not reduce efficiency of integration. Using this technique future experiments with longer YACs will allow the expression of gene complexes such as Hox and the globin gene clusters to be analysed in transgenic animals.     

Construction and characterization of a yeast artificial chromosome library containing 1.5 equivalents of human chromosome 21.

A library of yeast artificial chromosomes (YACs) was constructed from a human/hamster somatic cell hybrid containing human chromosome 21 (q11-qter). Cells were embedded in agarose, and the DNA was partially digested with EcoRI, released into solution by agarase treatment of the agarose plugs, ligated into pYAC4, and transferred into yeast. Double screening of the yeast transformants with human and hamster genomic DNA allowed the selection of clones hybridizing only with human DNA. The library consists of 321 clones, amounting to 1.5 equivalents (61 Mb) of chromosome 21. The mean YAC size calculated from 178 clones is 190 +/- 100 kb. Screening of the library with eight sequence-tagged sites gave six positives. Among 21 YACs tested by in situ hybridization, 17 mapped to chromosome 21.     

Establishment of a pluripotent embryonal carcinoma cell line not expressing SSEA-1 and ECMA-7 phenotypes

A murine embryonal carcinoma (EC) cell line heterozygous for t0 recessive lethal mutation has been established from an embryo-derived transplantable teratocarcinoma TC1Ph of the genotype (129-T/t0 X C3H/Di)t0/+. The EC cell line, designated EC1Ph, and two cloned sublines, EC1Ph/a and EC1Ph/b, maintain the diploid karyotype (40, XY) and give rise to teratocarcinomas with differentiated derivatives of EC cells after inoculation into syngeneic recipients. The cloned sublines express low or zero amounts of SSEA-1 and ECMA-7 stage-specific antigens. At some passages, the EC1Ph line and the cloned subline EC1Ph/b express a significant quantity of class I H-2 antigens. This unusual EC phenotype resembles that of human teratocarcinoma cell lines.