Clonal lines of teratocarcinoma cells in vitro: Differentiation and cytogenetic characteristics

Clonal lines of embryonal carcinoma cells have been established in culture from four independently-derived transplantable teratocarcinomas of mice: three from strain C3H and one from strain 129/Sv. Cells from all lines retain the capacity to differentiate into a variety of tissue types both in tumors formed following the injection of cells into syngeneic animals and in vitro under appropriate culture conditions. Analysis of their G-banded chromosomes indicated that the four lines have near-diploid but not absolutely normal karyo-types. The same chromosomal abnormalities were often present in more than one line. Tetraploid embryonal carcinoma cells made by Colcemid or cytochalasin B treatment were also pluripotential in spite of chromosomal instability. Hybrid cells were readily obtained between diploid or tetraploid embryonal carcinoma cells and mouse 3T3 fibroblasts. Hybrid cells failed to differentiate and were contact inhibited like the 3T3 parent.     

Expression of fragile X in human-mouse somatic cell hybrids

Fragile X expression was studied in human-mouse cell hybrids prepared from lymphocytes and fibroblasts obtained from a mentally retarded male. The patient showed a fragile X in 29-35.5% of his lymphocytes in medium 199 (M199) and in M199 plus fluorodeoxyuridine (FdU). One lymphocyte hybrid clone showed no expression in M199 and low expression in M199 + FdU. The other lymphocyte hybrid clone showed significantly increased expression in both media, comparable to levels in the parental cells. Fibroblast cultures from the patient showed no fragile X expression in M199 and 17% expression in M199 + FdU. Fragile X expression was also found in fibroblast hybrid clones in M199 and was significantly enhanced by the addition of FdU. Fragile X expression in one clone was consistently lower than in the other two clones and in the parental fibroblasts. Our results indicate that the level of fragile X expression varies in the hybrid clones, since frequencies similar to those of parental cells and suppressed frequencies were found. The presence or absence of a specific human chromosome did not correlate with the level of fragile X expression.     

Maintenance of replication patterns in human-mouse hybrids retaining only one human chromosome.

The time of termination of DNA replication of human chromosomes in human-mouse hybrids retaining only one human chromosome was analyzed. Hybrids between SV40-transformed human skin fibroblasts and mouse peritoneal macrophages were used for these studies. Data obtained from hybrids containing only human chromosome 7 or 17 were compared with data from related hybrids containing additional human chromosomes. When either human chromosome 7 or 17 was present alone, it terminated replication at the same stage of the S phase as in hybrids in which other human chromosomes were present (relative to the time of termination of replication of the mouse chromosomes). In comparing the hybrids containing single human chromosomes, it was found that chromosome 17 terminated replication much earlier than chromosome 7. Therefore, the relationship between the replication times of these chromosomes normally observed in human cells was maintained in the hybrids in the absence of all other human chromosomes. The results also indicate that the presence of SV40 gene sequences in chromosomes 7 and 17 did not alter the relative times of termination of replication of those chromosomes.     

Kinetics of human light chain synthesis by human-mouse myeloma hybrids

Human myeloma cells from a patient suffering from a lambda Bence-Jones myeloma were hybridized with a continuous mouse myeloma cell line. Several hybrids were obtained, two of which produced the same lambda chain produced by the patient. They have proved to be stable over long periods of time in continuous cell culture. Kinetics of growth of this line proved to be linear over 72 h. This growth rate is independent of any possible nutrient depletion effect from the media. Rate of production of lambda chain was highest (45 micrograms/10(6) cells/24 h) when cell density was between 3-5 X 10(5)/ml and decreased as cell density increased. Efficiency of human light chain synthesis in these hybrids is similar to murine Ig synthesis by the producer parental mouse cells. Human-mouse myeloma hybrids that produce large quantities of human Igs can be established routinely, are stable over long periods of time, and can be used as a model system for the study of the control of synthesis of human Igs.     

Adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase immunoprecipitation reactions in human-mouse and human-hamster cell hybrids.

Male New Zealand White rabbits were immunized with human adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which were purified about 2000-fold and 800-fold, respectively, from erythrocytes by DEAE-cellulose chromatography, ammonium sulfate precipitation and preparative polyacrylamide gel electrophoresis. Specific immunoprecipitations of APRT and HGPRT were achieved with the antisera that were obtained and by using polyethylene glycol as a substitute for goat anti-(rabbit) gamma globulin. The activities of the human forms of these enzymes, whether from red blood cells or from cultured cells, were almost completely eliminated under the conditions of immunoprecipitation used. Little or no reduction of APRT and HGPRT activities from mouse and Chinese hamster cells was observed. This discriminatory capacity of the antisera was successfully used for the identification of human APRT and HGPRT in human-mouse and human-hamster cell hybrids using the immunoprecipitation reaction.     

Human glycoprotein hormone production in human-human and human-mouse somatic cell hybrids

The production of both protein and steroid hormones was studied utilizing somatic cell hybrids formed with human choriocarcinoma cells. The human JEG-3 cell line produced the species and organ-specific hormone human chorionic gonadotropin (hCG), the steroid hormone progesterone, and converted 19-carbon steroids to estrogens. Hybrids formed with human VA-2 cells, mouse Cl 1D cells and mouse 3T3-4EF cells had detectable hCG synthesis in 20 of 41 total clones. There was no detectable progesterone or 19-carbon aromatization to estrogens in any hybrids. These data demonstrate that the differentiated function of human protein hormone production can be retained in inter- and intra-specific somatic cell hybrids. These results also suggest that protein hormone production can occur independently of steroid production in these cells of placental origin.     

Identification of human and mouse chromosomes in human-mouse hybrids by centromere fluorescence

Differences between the centromeric regions of mouse and human chromosomes have been revealed with a staining technique which is based on the quenching by 5-bromodeoxyuridine of the fluorescence of the dye 33258 Hoechst. The differences can be used to distinguish between human and mouse chromosomes in human-mouse hybrids.     

Somatic cell hybrids between totipotent mouse teratocarcinoma and rat hepatoma cells.

We have produced somatic cell hybrids between totipotent mouse teratocarcinoma cells and rat hepatoma cells. These hybrids were tested for the expression of liver specific functions expressed in the hepatoma cell parent and for their ability to differentiate when injected into nude mice. The results of this study indicate that hybrid cell clones do not resemble either of the parental cells, since they do not produce albumin and tyrosine aminotransferase that are expressed in the rat hepatoma parent, and are incapable of forming either teratocarcinomas or hepatomas when injected in experimental animals.     

Gene expression of differentiated parent in teratocarcinoma cell hybrids. Repression or reprogramming?

The hybrid cell line H422 was constructed by fusing embryonal carcinoma (EC) cells of the PCC4AzaRCapR cell line with lymphocytes from a 129-tw32 mouse inbred strain female. An apparently complete extinction of stage-specific gene products of the lymphocyte parent was inferred from comparison of protein maps in two-dimensional PAGE of parental and hybrid cells, and from the reactivity patterns of monoclonal antibodies in radioimmunobinding assays. Furthermore, the hybrid cells display a true EC phenotype and EC functions, tumorigenicity and pluripotency. Taken together, the available evidence suggests either that the lymphocyte genome is a 'silent passenger' as far as the differentiated functions of hybrid cells are concerned, or that the developmentally restricted lymphocyte genome is reprogrammed in the hybrid cells back to a non-determined stage and both genomes thus act in a concerted manner.     

Immunochemical analysis of the N-acetyl hexosaminidases in human-mouse hybrids made using a double selective system.

A human-mouse hybrid, DUR 4 (Solomon et al., 1976), containing a human X/15 translocation chromosome and also chromosome 5, among other human chromosomes, was used in a double selection system to obtain hybrids of four different types: X/15+ 5+, X/15- 5+, X/15+, 5-. Standard positive and negative selection systems were used for the X chromosome, and negative selection for chromosome 5 was done with diphtheria toxin. The assignment of HEXB and presently only when both the X/15 chromosome and chromosome 5 were present. A "HEXA-like" band segregated with chromosome 15 (or X/15) but independently of chromosome 5. This component, unlike HEXA, does not contain human HEXbeta antigen.     

Supermelanotic hybrids between mouse teratocarcinoma and mouse melanoma cells

Each of four kinds of teratocarcinoma cells, OTT6050P, PCC4, PSA1 and LT, derived from 129 or LT mouse strain, was fused with B16-CAPr melanoma cells derived from C57BL/6J by using Sendai virus. The resultant hybrids were morphologically melanotic melanoma cells which were larger and more heavily pigmented than the parental B16-CAPr melanoma cells. The chromosome analysis and GPI electrophoresis demonstrated that all hybrids were products of fusion between a single teratocarcinoma cell and a single melanoma cell. The pigmentation in the hybrids between a 129 teratocarcinoma cell and a melanoma cell was much stronger than that in hybrids between an LT teratocarcinoma cell and a melanoma cell. This phenomenon was consistent with the difference of coat color between 129 and LT mouse strain. From these results, it was suggested that the genes of teratocarcinoma cells involved in the pigmentation are activated in the hybrids with B16-CAPr melanoma cells.     

Analyses of deoxycytidylate deaminase molecular forms in human-mouse and monkey-mouse somatic cell hybrids

Disc polyacrylamide gel electrophoresis (disc PAGE) analyses have revealed that mouse, human, and monkey cytosol deoxycytidylate (dCMP) deaminases differ in electrophoretic mobility, so that mixtures of mouse and human, mouse and monkey, and human and monkey enzymes can be separated. To learn whether the genes for dCMP deaminase and thymidine (dT) kinase are genetically linked, disc PAGE analyses of cytosol fractions from human-mouse and monkey-mouse somatic cell hybrids were carried out. The interspecific somatic cell hybrids were derived from the fusion of cytosol dT kinase deficient mouse cells with cytosol dT kinase-positive human and monkey cells: they contained mostly mouse chromosomes and a few primate chromosomes, including the determinant for primate cytosol dT kinase. The disc PAGE analyses demonstrated that the human-mouse and monkey-mouse somatic cell hybrids contained a dCMP deaminase activity with an electrophoretic mobility characteristic of mouse dCMP deaminase. Enzymes with electrophoretic mobilities characteristic of human and monkey dCMP deaminases were not demonstrable. These findings suggest that primate cytosol dT kinase and dCMP deaminase are coded on different chromosomes, or that the formation in hybrid cells of an active primate dCMP deaminase is suppressed. Chick-mouse somatic cell hybrids containing chick but not mouse cytosol dT kinase were also analyzed. The chick-mouse hybrid cells contained cytosol dCMP deaminase activity, but it was not possible to establish whether the enzyme was of murine or avian origin because of the similarity in electrophoretic mobility between the chick and mouse enzymes. Human and mouse cells contained low levels of mitochondrial dCMP deaminase activity. In contrast to dT kinase isozymes, however, mitochondrial and cytosol dCMP deaminases were electrophoretically indistinguishable.This investigation was aided by Grant Q-163 from the Robert A. Welch Foundation and by USPHS Grants CA-06656-12 and 1-K6-AI 2352 from the National Cancer Institute and the National Institute of Allergy and Infectious Diseases.     

Necessity for two human chromosomes for human chorionic gonadotropin production in human-mouse hybrids

Through a series of human-mouse hybrids we have identified that two human chromosomes, 10 and 18, must be present for production of the pregnancy protein hormone human chorionic gonadotropin (hCG). Human choriocarcinoma cells producing hCG were hybridized to mouse cells. From 49 independent clones three hybrid clones continued to produce whole hCG. Chromosomal analysis was done on the 3 producer clones and 5 nonproducer clones. The additional 41 nonproducer clones were genetically characterized by isozymes. Only when chromosomes 10 and 18 were present in a clone would the whole hCG molecule be produced. Clones with only 10 or only 18 did not produce hormone. Nine subclones of a producer clone confirmed this observation. Three subclones retaining both 10 and 18 continued to produce hCG. This study demonstrated the need to use cellular chromosome data and population enzyme data to identify two chromosomes necessary for hCG production in heterogeneous human-mouse hybrids.     

Phenotype and surface antigens of mouse teratocarcinoma x fibroblast cell hybrids

Numerous colonies of hybrids between PCC4-aza 1 teratocarcinoma cells and fibroblasts of the heteroploid Cl.1D cell line were examined. All of the hybrids were fibroblasts showing extinction of the multiple developmental potentialities of the teratocarcinoma cell parent, irrespective of whether the teratocarcinoma parent was diploid or tetraploid. The hybrids did not show loss of any specific chromosome contributed by the PCC4-aza 1 cell parent. In contrast with the PCC4 parental cells which carry F9 antigens and do not express H-2b, the hybrids do not express F9 antigens and carry H-2 alloantigens of both parental specificities. These results suggest that in hybrids whose phenotype is that of the Cl.1D parent, a change may occur in the genetic program of the teratocarcinoma cells.     

Hemoglobin synthesis in cell hybrids formed between teratocarcinoma and friend erythroleukemia cells

Viable hybrid cells have been isolated following fusion of Friend erythroleukemia cells and undifferentiated teratocarcinoma cells. The hybrids formed between near-diploid parental cells resembled Friend cells in their ability to grow in suspension and to synthesize hemoglobin in the presence of the chemical inducers dimethyl sulfoxide (DMSO) and ouabain. Erythropoietin (EPO) was effective in inducing hemoglobin synthesis in some of the hybrid cell lines. The hemoglobins synthesized by the hybrids were of the adult forms, but were quantitatively different from those hemoglobins synthesized by the parental Friend cells, suggesting that the fusion event modulated the expression of the hemoglobin chain genes.