Amplification and chromosomal dispersion of human endogenous retroviral sequences.

Endogenous retroviral sequences in humans have undergone amplification events involving both viral and flanking cellular sequences. We cloned members of an amplified family of full-length endogenous retroviral sequences. Genomic blotting, employing a flanking cellular DNA probe derived from a member of this family, revealed a similar array of reactive bands in both humans and chimpanzees, indicating that an amplification event involving retroviral and associated cellular DNA sequences occurred before the evolutionary separation of these two primates. Southern analyses of restricted somatic cell hybrid DNA preparations suggested that endogenous retroviral segments are widely dispersed in the human genome and that amplification and dispersion events may be linked.     

Activation of endogenous retroviral sequences in human leukemia

Endogenous retroviral sequences have been identified in the genomes of several species including humans. Proteins similar to those of primate endogenous viruses have been found on the surface of various malignant cells in man. To further define this role, we have used a primate retrovirus DNA from the Baboon Endogenous Virus to probe a human genomic library for related sequences. A total of 45 clones homologous to BaEV gag-pol were isolated under low stringency hybridization. Of these, several were found to contain DNA which was expressed as RNA at higher levels in human lymphoid leukemic cells than in normal lymphocytes.     

Cell-cycle characteristics of undifferentiated and differentiating embryonal carcinoma cells

The cell-cycle parameters of undifferentiated and differentiating embryonal carcinoma cells were determined. Undifferentiated cultures of an F9 subclone, OTF9-63, had a 12-hr generation time, 10-hr S, and 2-hr G2 + M. G1 was less than 0.5 hr. In contrast, OTF9-63 cells induced to differentiate by treatment with retinoic acid had a 16.8-hr generation time, 12.5-hr S, 2-hr G2 + M, and 2.3-hr G1. Similar results were obtained with undifferentiated cultures and aggregation-induced differentiating cultures of PSA-1 cells. These data demonstrate that the undifferentiated stem cells have little or no G1, and that both G1 and S lengthen during differentiation.     

Factors regulating endogenous retroviral sequences in human and mouse

Endogenous retroviruses (ERVs) are stably integrated in the genome of vertebrates and inherited as Mendelian genes. The several human ERV (HERV) families and related elements represent up to 5-8% of the DNA of our species. ERVs may be involved in the regulation of adjacent genomic loci, especially promoting the tissue-specific expression of genes; some HERVs may have functional roles, e.g., coding for the placental fusogenic protein, syncytin. This paper reviews the growing evidence about factors that may modulate ERVs, including: cell and tissue types (with special attention to placenta and germ cells), processes related to differentiation and aging, cytokines, agents that disrupt cell functions (e.g., DNA hypomethylating agents) and steroids. Special attention is given to HERVs, due to their possible involvement in autoimmunity and reproduction, as well as altered expression in some cancer types; moreover, different HERV families may deserve specific attention, due to remarkable differences concerning, e.g., expression in tissues. A comparison with factors interacting with murine ERV-related sequences indicates that the mouse may be a useful model for studying some patterns of HERV regulation. Overall, the available evidence identifies the diverse, potential interactions with endogenous or exogenous factors as a promising field for investigating the roles of ERVs in physiology and disease.     

Identification and phylogenetic analysis of novel human endogenous retroviral sequences belonging to the HERV-H family on human X and Y chromosomes

HERV-H, a family of endogenous retroviral elements that has undergone successive expansions in the human genome, includes sequences that are expressed in placenta and T cells. With a PCR approach to the HERV-H using human monochromosomal somatic cell hybrid DNA, we identified 8 new HERV-H sequences on the X chromosome, and one novel HERV-H element, HY-1, the first reported such element on the Y chromosome, and compared these with sequences in the nucleotide sequence database. Phylogenetic analysis indicated that clone HX-1 and BAC clone 523A23 on the X chromosome were found to be in close relationship to the sequences of DJ088A21 on the human chromosome 7q31. This finding allows us to speculate that HERV-H elements may have evolved by intra-chromosomal spread. Our data may relevant to an understanding of human genomic plasticity.     

Two distinct sequence elements mediate retroviral gene expression in embryonal carcinoma cells.

Moloney murine leukemia virus (M-MuLV) and M-MuLV-derived retroviral vectors are not expressed in early mouse embryos or in embryonal carcinoma cells. M-MuLV-derived mutants or M-MuLV-related variants which transduce the neomycin phosphotransferase gene can, however, induce drug resistance in embryonal carcinoma cells with high efficiency. In this study we investigated the sequences critical for retroviral gene expression in two different embryonal carcinoma cell lines, F9 and PCC4. We show that two synergistically acting sequence elements mediate expression in embryonal carcinoma cells. One of these is located within the U3 region of the viral long terminal repeat, and the second one is in the 5' untranslated region of the retrovirus. The latter element, characterized by a single point mutation, affects the level of stable RNA in infected cells, suggesting a regulatory mechanism similar to that of human immunodeficiency virus in human T cells.     

Hypermethylation of human DNA sequences in embryonal carcinoma cells and somatic tissues but not in sperm.

Certain human DNA sequences are much less methylated at CpG sites in sperm than in various adult somatic tissues. The DNA of term placenta displays intermediate levels of methylation at these sequences (Sp-0.3 sequences). We report here that pluripotent embryonal carcinoma (EC) cells derived from testicular germ cell tumors are hypermethylated at the three previously cloned Sp-0.3 sequences and seven newly isolated sequences that exhibit sperm-specific hypomethylation. In contrast to their hypermethylation in EC cells, the Sp-0.3 sequences are hypomethylated in a line of yolk sac carcinoma cells, which like placenta, represent an extraembryonic lineage. These DNA sequences, therefore, appear to be subject to coordinate changes in their methylation during differentiation, probably early in embryogenesis, despite their diversity in copy number (1 to 10(4] and primary structure. Two of these Sp-0.3 sequences are highly homologous to DNA sequences in human chromosomal regions that might be recombination hotspots, namely, a cryptic satellite DNA sequence at a fragile site and the downstream region of the beta-globin gene cluster.     

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.     

Isolation and characterization of a multipotent clone of human embryonal carcinoma cells

Histopathological studies suggest that the stem cells of human teratomas may be classified into two major categories: nullipotent stem cells, and multipotent stem cells, capable both of self-renewal and differentiation into a wide range of somatic and extraembryonic cell types. We have isolated a multipotent stem cell clone from the human teratoma cell line GCT 27, and compared its properties to a nullipotent clone derived from the same strain. The multipotent clone GCT 27 X-1 gave rise to colonies of mixed cell morphology in vitro. Analysis of cell surface, cytostructural and extracellular matrix markers in GCT 27 X-1 cells showed that the stem cells of this line were very similar in phenotype to nullipotent cells. The two cell clones were predominantly hypotriploid, and contained several marker chromosomes in common. GCT 27 X-1 was feeder-cell-dependent for continuous growth in vitro; removal of the feeder layer resulted in differentiation of the stem cells into a variety of cell types, some with characteristics of extraembryonic endoderm, others showing neuronal properties. When transplanted into nude mice, GCT 27 X-1 cells gave rise to teratocarcinomas containing embryonal carcinoma stem cells, and many other cell types: yolk sac carcinoma cells; cells producing alphafetoprotein or human chorionic gonadotrophin; glandular, columnar, cuboidal, and squamous epithelium; primitive mesenchyme and cartilage; neuroectodermal cells. Nullipotent GCT 27 C-1 cells could form colonies in the absence of feeder layers, but multipotent GCT 27 X-1 cells could not. While a range of known growth factors and related substances failed to substitute for feeder layers in supporting the growth of GCT 27 X-1 stem cells, supernatants from yolk sac carcinoma cell line GCT 44 could partially replace the feeder cell requirement. Thus, the results revealed a basic difference in growth control between these multipotent and nullipotent human embryonal carcinoma cells, and suggested a possible paracrine regulatory pathway between multipotent stem cells and yolk sac carcinoma cells.     

Characterization of a tyrosine kinase signaling pathway in undifferentiated P19 embryonal carcinoma cells.

Embryonal carcinoma (EC) cells are the malignant stem cells of teratocarcinoma and have the capacity to proliferate in the absence of serum growth factors. As yet no receptor protein tyrosine kinases have been identified on undifferentiated EC cells and as a consequence tyrosine kinase signaling pathways could not be studied in these cells. We have used stably transfected P19 embryonal carcinoma cells expressing a well-characterized receptor protein tyrosine kinase, the human epidermal growth factor receptor (hEGF-R) to study protein tyrosine kinase signaling mechanisms in undifferentiated EC cells. Here we report that the ectopically expressed hEGF-R contains EGF-inducible autophosphorylation activity and is rapidly internalized and degraded upon ligand binding. In addition, the exogenous hEGF-R confers EGF-responsiveness to these cells in that inositol phosphate formation and cytoplasmic-free Ca2+ concentration are enhanced in response to EGF. Furthermore, the Na+/H+ exchanger is activated in response to EGF, leading to a sustained rise in intracellular pH. Our results show that undifferentiated P19 EC cells contain the necessary components of protein tyrosine kinase signal transduction machinery.     

Cytoskeleton of human embryonal carcinoma cells

Monoclonal antibodies to cytoskeletal proteins were used to study the intermediate filament proteins of human embryonal carcinoma (EC) cell lines, tumors produced in nude mice from these cell lines, and surgically removed testicular germ cell tumors. It was found that all cells of tumor lines 2102Ep, 1156 and Tera 1 react with antibodies to low molecular weight keratin proteins. By immunoblotting of SDS gels it was found that these lines expressed three keratin polypeptides (40K, 45K and 52K). Clonal line NTera-2 derived from Tera-2 differed from the above listed cell lines in that only 10% of the cells expressed the 40K keratin polypeptide. Upon treatment with retinoic acid 70% of NTera-2 cells became reactive with the antibody to the 40K keratin polypeptide. All cell lines contained a small population of vimentin-positive cells. The number of vimentin-positive cells could be increased by retinoic acid treatment of NTera-2 cells or by seeding the 2102Ep cells at low cell density. Neurofilament-positive cells could be induced in the cell line NTera-2 by retinoic acid treatment. Tumors produced from NTera-2 cells injected into nude mice contained cells reacting with antibodies to keratin, vimentin, neurofilament proteins and desmin. Keratin polypeptides were immunohistochemically demonstrated in embryonal carcinoma, yolk sac carcinoma and trophoblastic components of solid human germ cell tumors. Atypical intratubular cells ('carcinoma in situ') also reacted with antibodies to keratin.     

Molecular genetic characterization of the RD-114 gene family of endogenous feline retroviral sequences.

RD-114 is a replication-competent, xenotropic retrovirus which is homologous to a family of moderately repetitive DNA sequences present at ca. 20 copies in the normal cellular genome of domestic cats. To examine the extent and character of genomic divergence of the RD-114 gene family as well as to assess their positional association within the cat genome, we have prepared a series of molecular clones of endogenous RD-114 DNA segments from a genomic library of cat cellular DNA. Their restriction endonuclease maps were compared with each other as well as to that of the prototype-inducible RD-114 which was molecularly cloned from a chronically infected human cell line. The endogenous sequences analyzed were similar to each other in that they were colinear with RD-114 proviral DNA, were bounded by long terminal redundancies, and conserved many restriction sites in the gag and pol regions. However, the env regions of many of the sequences examined were substantially deleted. Several of the endogenous RD-114 genomes contained a novel envelope sequence which was unrelated to the env gene of the prototype RD-114 env gene but which, like RD-114 and endogenous feline leukemia virus provirus, was found only in species of the genus Felis, and not in other closely related Felidae genera. The endogenous RD-114 sequences each had a distinct cellular flank which indicates that these sequences are not tandem but dispersed nonspecifically throughout the genome. Southern analysis of cat cellular DNA confirmed the conclusions about conserved restriction sites in endogenous sequences and indicated that a single locus may be responsible for the production of the major inducible form of RD-114.