X-chromosome activity in heterokaryons and hybrids between mouse fibroblasts and teratocarcinoma stem cells

To determine whether 2X-active cells contain factors capable of reactivating the inactive mammalian X chromosome, fibroblast lines, having a cytologically or genetically marked inactive X, were fused with 2X-active mouse embryos or ovarian teratocarcinoma stem cells. Fusions with 2–16 cell embryos were uninformative because no mitosis occurred in heterokaryons. Fusions with 2X-active teratocarcinoma cells, and screening for re-expression of alleles on the inactive X showed that reactivation did not occur with detectable frequency in heterokaryon. Hybridization of HPRT^?M. musculus × M. caroli cells with XO HPRT^? teratocarcinoma cells yielded hybrids with a frequency of >10^?6; these hybrids all expressed the Hpt allele on the inactive M. caroli X, but not the M. caroliGpd or Pgk. Late replication-banding studies of hybrids and 6-thioguanine-resistant revertants showed that the reactivated Hp⁺ allele was still located on the late replicating X. Similar results were obtained with hybridization of this line to 1X-active (male-derived) fibroblast lines, indicating that hybridization per se, rather than a specific factor contributed by the teratocarcinoma cell partner, was reponsible for the frequent localized derepression of the Hpt⁺ allele on the inactive X.     

Isolation of human fibroblast heterokaryons with two-colour flow sorting (FACS II)

Red and green fluorescent polystyrene beads were used to label different populations of cultured human skin fibroblasts. After co-cultivation for 24 h no exchange of label could be observed. Twenty-four hours after fusion the population of red-green heterofluorescent cells was sorted out with a FACS II cell sorter. Microscopical examination of the heterofluorescent population 20 h after sorting indicated, that with Sendai-induced fusion, 50–60% of these viable cells contained more than one nucleus. After polyethylene glycol (PEG)-induced fusion, between 70 and 85% bi- or multinucleated cells were present in the sorted and cultured fraction. Autoradiography using [³H]thymidine indicated that the sorted heterofluorescent bikaryons were in fact heterokaryons.     

Low expression of activin a in mouse and human embryonic teratocarcinoma cells

TGFβ family factors play an important role in regulating the balance of self-renewal and differentiation of mouse and human pluripotent stem and embryonic teratocarcinoma cells. The expression patterns of TGFβ family signaling ligands and functional roles of these signaling pathways differ significantly in mouse and human embryonic stem cells, but the activity and functional role of these factors in mouse and human embryonic teratocarcinoma cells were not sufficiently investigated. Comparative quantitative real-time PCR analysis of the expression of TGFβ family factors in mouse embryonic stem, embryonic germ, and embryonic teratocarcinoma cells showed that embryonic teratocarcinoma cells express lower ActivinA than pluripotent stem cells but similar levels of factors Nodal, Lefty1, TGFβ1, BMP4, and GDF3. In human nullipotent embryonic teratocarcinoma PA-1 cells, most factors of the TGFβ family (ACTIVINA, NODAL, LEFTY1, BMP4, and GDF3) are expressed at lower levels than in human embryonic stem cells. Thus, in mouse and human nullipotent teratocarcinoma cells, the expression of ActivinA is significantly reduced compared with embryonic stem cells. Presumably, these differences may be associated with changes in the functional activity of the respective signaling pathways and deregulation of proliferative and antiproliferative mechanisms in embryonic teratocarcinoma cells.     

Uncoupling of muscle-specific protein expression in myocyte X myoblast heterokaryons

The inducibility of several rat skeletal muscle proteins was examined in heterokaryons formed by fusing differentiated chick myocytes to undifferentiated rat myoblasts. Chicken and rat proteins were distinguished using species-specific antibodies or by their different migrations in polyacrylamide or agarose gels. Both rat skeletal myosin light chain 1 and rat α-tropomyosin were induced in the heterokaryons. In contrast, neither rat acetylcholine receptors nor creatine kinase could be detected. These results suggest that chick myocytes may contain quantities of regulatory factors that are sufficient for the activation of some but not all of these rat muscle-specific proteins within the cellular context of the heterokaryon.     

Electrical excitability and chemosensitivity of mouse neuroblastoma X mouse or human fibroblast hybrids

Intracellular microelectrode recording techniques were used to measure passive membrane properties, electrical excitability and chemosensitivity of mouse neuroblastoma cells and somatic cell hybrids formed between these cells and either L cells or human diploid fibroblasts. Different clones of the hybrid cells showed varying degrees of neuronal or fibroblastic membrane-differentiated function; a selection technique involving incubation of the cells with aminopterin gave quite homogeneous non-dividing populations of cells within a given clone of the neuroblastoma x L cell hybrids. Despite relatively uniform chromosomal numbers within a given clone, the neuroblastoma x human fibroblast hybrids were morphologically and electrophysiologically heterogeneous. The possibility is considered that this may represent the effect of variable segregation of the human chromosomal complement.     

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.     

Regional localization on the human X of DNA segments cloned from flow sorted chromosomes.

Fluorescence activated sorting of chromosomes from 49,XXXXY human lymphoblasts has been used to obtain DNA enriched for the human X. This DNA was cloned in lambda phage Charon 21A to obtain a library of approximately 60,000 pfu. Phage inserts free of human highly repeated DNA sequences are localized to different regions of the human X by two independent hybridization analyses. The first utilized comparative hybridization to rodent-human hybrid cell DNA samples containing all or known portions of the human X, while the second was based on hybridization dosage to DNA samples from human cell lines differing in the number of X chromosomes or X chromosome segments. Of five unique sequence inserts tested, three were X chromosome specific and were localized to regions Xpter leads to Xcen, Xql leads to Xq22 and Xq24 leads to Xqter, respectively. The library presented here represents a highly enriched source of human X chromosome-specific DNA sequences.     

Antigen expression of mouse spleen × thymocyte heterokaryons

The expression of Thy 1.2 and thymic leukemia (TL) antigens by heterokaryons of spleen cells of strain A mice (A-S) and AKR thymocytes (AKR-T) was determined. The A-S parental cells do not express TL antigens, although strain A thymocytes are TL-positive. Approximately 25% of A-S cells express Thy 1.2 antigens; however, AKR-T cells express a different Thy 1 antigen (Thy 1.1) and are phenotypically negative for TL expression. AKR-T × A-S heterokaryons were prepared with the aid of inactivated Sendai virus. Identification of heterokaryons was facilitated by prior isotopic labeling of AKR-T but not A-S cells, and the finding by autoradiography of binucleated cells with one radioactively labeled and one non-labeled nucleus. Antigenic expression of these fused cells was determined by exposure of the cells to specific antiserum and complement prior to autoradiography. 24 hr after fusion, fused cells were resistant to the cytotoxic effects of TL antiserum and fresh complement. However, a large proportion of these cells was lysed by treatment with antiserum directed against the Thy 1.2 antigen.     

Selection of proliferating cybrid cells by dual laser flow sorting : Isolation of teratocarcinoma X neuroblastoma and teratocarcinoma X endoderm cybrids

An alternative method for the isolation of proliferating cybrid cells was developed, and was used to obtain teratocarcinoma X neuroblastoma and teratocarcinoma X endoderm cybrids. Enucleated neuroblastoma (or endoderm) cells labelled with fluorescent microspheres were fused with (HPRT-deficient) unlabelled teratocarcinoma cells. The cells in the fusion mixture were stained with the vital DNA stain Hoechst 33342 and the cybrids, containing both a fluorescent nucleus and fluorescent beads, were isolated by dual laser flow sorting. The purity of the sorted fraction, as determined by the percentage of cells showing HPRT activity, was 86 and 57% for the neuroblastoma and endoderm cybrids, respectively. After single cell sorting in wells of Terasaki microtest plates, clones of proliferating cybrids were obtained with cloning efficiencies of 33% (neuroblastoma and endoderm cybrids respectively. After single cell sorting in wells of Terasaki microtest parental cell lines were analysed by two-dimensional gel electrophoresis. A number of differences were found between the parental cell lines but all isolated colonies (sixteen neuroblastoma cybrids and eight endoderm cybrids) resembled the teratocarcinoma parent. These results therefore give no evidence for the existence of cytoplasmic factors in neuroblastoma or endoderm cells capable of inducing permanent differentiation of teratocarcinoma cells.     

Study of X chromosome abnormality in XX males using bivariate flow karyotype analysis and flow sorted dot blots

We have used bivariate flow karyotype analysis to quantify aberrant X chromosome size in 11 XX males. With one exception, the patients could be grouped into those with an X homologue difference greater than normal (Group A, n = 3) and into those whose X homologue difference could not be distinguished from female controls (Group B, n = 7). The range of sizes of the aberrant X chromosome in Y-sequence positive patients agrees with the variable nature of the X-Y interchange in these individuals as determined by the use of Y-specific DNA probes and Southern blotting analysis. In one patient it was possible to sort separately the normal and the X-Y interchanged homologues for dot blot analysis. The presence of Y sequences and an increased dose of the zinc finger gene, ZFY, were detected in the X-Y interchanged homologue. In preliminary studies of 5 male and 6 female controls, it was noted that a consistent difference between the two X homologues in females was found which could not be totally explained by errors of the fitting procedure. We suggest that this difference could be due to X inactivation and that the two X homologues in females might be distinguishable.     

Dopa oxidase expression in fibroblast x melanoma fusions. Extinction in heterokaryons and maintenance in non-dividing cybrids

Pigmented B-16 mouse melanoma cells were fused with chick embryo fibroblasts or fibroblast cytoplasts and maintained as heterokaryons or non-dividing cybrids, respectively. These single cells were examined ultrastructurally for evidence or pigment gene expression using a cytochemical test for dopa oxidase, the initial enzyme in the conversion of dopa to melanin. Heterokaryons showed significantly less enzyme activity than control cells, whereas non-dividing cybrids showed no significant difference. Therefore, the presence of the intact nuclear membranes in the heterokaryons did not serve as a barrier to the interactions resulting in extinction of differentiated function(s). However, the presence of the fibroblast nucleus was necessary to elicit continued response.     

H-2 class I antigen expression on mouse teratocarcinoma cell lines

Immunity against PCC3 teratocarcinoma cells (129, H-2 ^b) was induced in allogeneic (C3H, H-2 ^k) mice by preimmunization with L cells (C3H, H-2 ^k) expressing cosmid-introduced K ^b or D ^b genes, but not with nontransfected L cells. In addition, the growth of PCC3 cells in sublethally irradiated (C3H × B6-H-2 ^bm1)F₁ and (C3H × B6-H-2 ^bm13 )F₁ mice bearing the K ^bm1 and D ^bm13 mutations, respectively, was either prevented, stopped, or delayed in comparison with the (C3H × B6)F₁ (k × b) mice, which failed to reject the PCC3 cells. The teratocarcinoma line OC15S was exceptional because it reacted specifically with K^b- and D^b-specific (but not I^b-specific) alloantisera, and because K^b- and D^b-specific antibodies could be absorbed by OC15S cells. The subpopulation of OC15S cells bearing the ECMA-7 antigen characteristic for embryonic carcinoma (EC) cells was isolated by the fluorescence-activated cell sorter and was shown to react specifically with K^b- and D^b-specific antisera. These experiments show that teratocarcinoma cells express antigens similar or identical to the K-and D-region products of differentiated cells. The lack of expression of class I antigens is thus neither a condition nor a consequence of the pluripotentiality of the EC cells. The exact nature of the major histocompatibility complex antigens on EC cells has yet to be established using the methods of molecular biology and biochemistry.     

Extinction of globin gene expression in human fibroblast x mouse erythroleukemia cell hybrids.

We have chosen human fibroblast x mouse erythroleukemia hybrid cells as a model system to examine regulation of unique genes. The globin genes were studied as a marker of erythroid differentiation. Three separate hybrid cell lines were incubated in 2% dimethylsulfoxide, an agent which induces erythroid differentiation of the parental erythroleukemia cells. Neither human nor mouse globin mRNA sequences could be detected by a sensitive molecular hybridization assay which utilized globin complementary D N A. However, td n a from one of the cell lines was shown to contain both the mouse and humand globin genes. Thus, loss of the genes by chromosomal segregation did not account for their failure to be expressed. Cocultivation of the mouse erythroleukemia cells with excess human fibroblasts did not prevent erythroid differentiation of the erythroleukemia cells in the presence of dimethylsulfoxide. Similarly globin gene expression was preserved in tetraploid cells generated by fusion of two erythroleukemia lines. Thus, extinction of globin geneated by fusion of two erythroleukemia lines. Thus, extinction of blobin gene expression in the human fibroblast x erythroleukemia hybrids occurred at the level of mRNA production and appeared to be due to the presence of the fibroblast genome within the hybrial cell.     

In vitro differentiation of mouse teratocarcinoma cells monitored by intermediate filament expression

Teratocarcinoma differentiation has been studied using sera specific for each of the five intermediate filament (IF) classes. These antibodies distinguish cells of epithelial, muscle, neural, astrocytic, and mesenchymal origin. In embryoid bodies, derived from embryo transplants and obtained in the ascitic fluid by transplantation of teratocarcinoma, the cells of the inner cellular mass did not express any of these intermediate filament types while the outer cells expressed cytokeratin. Intermediate filament expression in the embryoid body thus appears analogous to that in the blastocyst and differs from that in embryonal carcinoma (EC) lines. Twelve EC lines have now been shown to express vimentin although in some EC lines not all cells express vimentin. Other established permanent differentiated cell lines, derived from EC lines in vitro or from tumors in vivo, have been characterized with respect to the type of IF they contain. The distribution of different IF types has been examined in EC cells induced to differentiate by addition of retinoic acid. The proportion of cells expressing each type of intermediate filament appears to depend on the EC cell line used, on the inducing agent, and on the length of treatment. Thus, for instance, F9 cells express cytokeratin, PCC3 derivatives express vimentin, many 1009 derivatives express either glial fibrillar acidic protein (GFA) or neurofilament proteins. Overall the results obtained are in excellent agreement with emerging principles of intermediate filament expression during embryonic differentiation, thus emphasizing the potential use of the various EC lines to study differentiation in culture.