Preparation and characterization of chick erythrocyte nuclei from heterokaryons

A method for the isolation of reactivated chick erythrocyte nuclei from heterokaryons was developed. The heterokaryons were produced by fusing chick erythrocytes with HeLa or L cells in the presence of inactivated Sendai virus. At various time intervals after fusion nuclei were isolated directly from the monolayer by treatment with an acidic detergent solution. Chick erythrocyte nuclei were then separated from other nuclei (HeLa or L cell) by centrifugation on sucrose gradients. The purified preparation of reactivated chick erythrocyte nuclei was shown to be free from other nuclei and cytoplasmic contamination. By using L cells which had been labelled with ³H-leucine before fusion or heterokaryons labelled after fusion it was demonstrated that labelled mouse proteins migrate from the cytoplasm of the heterokaryons into the reactivating chick erythrocyte nuclei. ³H-uridine labelling of heterokaryons made by fusing UV-irradiated chick erythrocytes with L cells failed to reveal any significant migration of mouse RNA into the chick erythrocyte nuclei.     

Interaction of human and chick DNA repair functions in UV-irradiated xeroderma pigmentosum-chick erythrocyte heterokaryons

Fusion of chick erythrocytes with human primary fibroblasts results in the formation of heterokaryons in which the inactive chick nuclei become reactivated. The expression of chick DNA repair functions was investigated by the analysis of the DNA repair capacity after exposure to ultraviolet (UV) irradiation of such heterokaryons obtained after fusion of chick erythrocytes with normal human or xeroderma pigmentosum (XP) cells of complementation groups A, B, C and D. Unscheduled DNA synthesis (UDS) in normal human nuclei in these heterokaryons is suppressed during the first 2–4 days after fusion. The extent and duration of this suppression is positively correlated with the number of chick nuclei in the heterokaryons. Suppression is absent in heterokaryons obtained after fusion of chicken embryonic fibroblasts with XP cells (complementation group A and C).Restoration of DNA repair synthesis is found after fusion in XP nuclei of all complementation groups studied. It occurs rapidly in XP group A nuclei, starting one day after fusion and reaching near normal human levels after 5–8 days. In nuclei of the B, C and D group increased levels of UDS are found 5 days after fusion. At 8 days after fusion the UDS level is about 50% of that found in normal human nuclei. The pattern of UDS observed in the chick nuclei parallels that of the human counterpart in the fusion. A fast complementation pattern is also observed in chick fibroblast-XP group A heterokaryons resulting within 24 h in a UDS level comparable with that in chick fibroblast-normal human heterokaryons. In heterokaryons obtained after fusion of chick fibroblasts with XP group C cells UDS remains at the level of chick cells. These data suggest that reactivation of chick erythrocyte nuclei results in expression of repair functions which are able to complement the defects in the XP complementation groups A, B, C and D.     

Synthesis of rat myosin light chains in heterokaryons formed between undifferentiated rat myoblasts and chick skeletal myocytes

The control of gene expression during terminal myogenesis was explored in heterokaryons between differentiated and undifferentiated myogenic cells by analyzing the formation of species specific myosin light chains of chick and rat skeletal muscle. Dividing L6 rat myoblasts served as the biochemically undifferentiated parent. The differentiated parental cells were mononucleated muscle cells (myocytes) that were obtained from primary cultures of embryonic chick thigh muscle by blocking myotube formation with EGTA and later incubating the postimitotic cells in cytochalasin B. Heterokaryons were isolated by the selective rescue of fusion products between cells previously treated with lethal doses of different cell poisons. 95-99% pure populations of heterokaryons formed between undifferentiated rat myoblasts and differentiated chick myocytes were obtained. The cells were labeled with [35S]methionine, and whole cell extracts were analyzed on two-dimensional polyacrylamide gels. These heterokaryons synthesize the light chain of chick myosin and both embryonic and adult light chains of rat skeletal myosin. Control homokaryons formed by fusing undifferentiated cells to themselves did not synthesize skeletal myosin light chains. Control heterokaryons formed between undifferentiated rat myoblasts and chick fibroblasts also failed to synthesize myosin light chains. These results indicate that differentiated chick muscle cells provide some factor that induces L6 myoblasts to synthesize rat myosin light chains. This system provides a model for investigating the processes by which differentiated cell functions are induced.     

Gene expression in flow sorted mouse teratocarcinoma X human fibroblast heterokaryons

Abstract. Mouse teratocarcinoma cells and primary human fibroblasts were fluorescently labelled with fluorescein isothiocyanate (F1TC)- and trimethylrhodamine isothiocyanate (TR1TC)-stearylamine respectively. After fusion populations highly enriched for red-green heterokaryons (around 80%) were isolated from the fusion mixture using a FACS II cell sorter.
To study gene expression in the early hybrids [³⁵S] methionine-labelled proteins synthesized by the sorted cells at two and three days after fusion were analysed by two-dimensional gel electrophoresis. Three spots were denser in gels of the fused cells than in those of 1:1 mixtures of parental cells. For one of these proteins it could be demonstrated that this reflects the enhanced synthesis of a mouse-specific protein present only in small amounts in teratocarcinoma cells. All three proteins were synthesized in relatively large amounts by differentiated mouse cells.
Collagen (type I) synthesis by the sorted hybrid cells was studied by analysing the [³H] proline-labelled material secreted into the medium. Analysis by sodium dodecyl sulphate (SDS)-gel electrophoresis and two-dimensional non-equilibrium pH gradient electrophoresis showed that the material secreted by the fused cells five days after fusion was the same as that secreted by the human fibroblasts. No evidence was obtained for synthesis of mouse α2(I) collagen. The amount of collagen produced by the sorted cells five days after fusion was about half the amount produced by the human fibroblasts. Immunofluorescence studies also showed that collagen synthesis was not suppressed after fusion both in heterokaryons and synkaryons.
In conclusion, we did not find evidence for activation of a previously completely silent mouse gene in the fused cells. The results show, however, that the fused cells do resemble the differentiated fibroblasts rather than the undifferentiated teratocarcinoma cells.     

Expression of lysosomal enzymes in human mutant fibroblast-chick erythrocyte heterokaryons

The generation of enzymes located in lysosomes, in cytosol or in endoplasmatic reticulum/Golgi complex is studied in heterokaryons in which chick erythrocyte nuclei are reactivated. The lysosomal enzymes, alpha-glucosidase (alpha-glu) and beta-galactosidase (beta-gal), are synthesized in heterokaryons obtained after fusion of chick erythrocytes with human fibroblasts of patients with Pompe's disease (alpha-glu-deficient) and GM1-gangliosidosis (beta-gal-deficient), respectively. The enzymes appear to be of chick origin and their activities can be detected at first around 4 days after fusion, i.e., at a time when the nucleoli in the erythrocyte nuclei have been reactivated. Maximal activities are reached around 15 days after fusion. No generation of the lysosomal enzyme beta-hexosaminidase is detected in the heterokaryons up to 23 days after fusion of chick erythrocyte with either beta-hexosaminidase A- and B-deficient fibroblasts (Sandhoff's disease) or beta-hexosaminidase A-deficient fibroblasts (Tay-Sachs disease). Similarly no expression of the cytosol enzyme glucose-6-phosphate dehydrogenase (G6PD) is fond up to 30 days after fusion, when chick erythrocytes are fused with fibroblasts from two different G6PD-deficient cell strains (residual activities of 4 and 20% respectively). Indirectly we examined N-acetyl-glucosamine-1-phosphate transferase activity, an enzyme located in the endoplasmic reticulum/Golgi region. This enzyme is needed for the phosphorylation of the lysosomal hydrolases and absence of its activity is the cause of the multiple lysosomal enzyme deficiencies in patients with I-cell disease. The retention of both, chick and human beta-galactosidase in the experiments in which I-cell fibroblasts were fused with chick erythrocytes indicates a reactivation of the gene coding for this phosphorylating enzyme. It also implies that this step in the processing of human lysosomal enzymes is not species-specific.     

Complementation analysis of xeroderma pigmentosum variants

DNA repair after UV exposure was studied in multinucleate cells, obtained after fusion of excision-defective and variant xeroderma pigmentosum fibroblasts. Optimal fusion conditions were determined, facilitating the measurement of DNA replication in heterokaryons. In unirradiated multikaryons, entry into the S phase was depressed, when compared with unfused cells. The extent of the depression of S phase entry was dependent on the fusion conditions. In heterokaryons obtained after fusion of XP variant (6 different strains) with excision-defective XP (three cell strains from complementation groups A, C and D) both unscheduled DNA synthesis and postreplication repair after UV irradiation were restored to normal levels. In contrast, complementation was not observed after pairwise fusion of the XP variant cell strains. These results suggest that the XP variants comprise a single complementation group, different from complementation groups A, C and D.     

Immortalized phenotype and the presence of active oncogenes correlate with the capacity of culture cells to induce reactivation of DNA synthesis in macrophage nuclei in heterokaryons

Several types of culture cells with limited life span (rat embryo fibroblasts, rat chondrocytes and mouse premacrophages) were found to be unable to induce the reactivation of DNA synthesis in the nuclei of non-dividing differentiated cells (mouse peritoneal resident macrophages) in heterokaryons. By contrast, malignant HeLa cells have this ability. In heterokaryons formed by fusion of mouse macrophages with HE239 cells (Syrian hamster fibroblasts transformed with a ts mutant of the SV40 virus), DNA synthesis in macrophage nuclei is reactivated only at the permissive temperature (33° C), at which viral T antigen is stable. Immortalization of rat chondrocytes by transfection with p53 gene enables to induce DNA synthesis in macrophage nuclei upon fusion. All the evidence indicates that the function of immortalizing oncogenes is necessary for the resumption of the DNA synthesis in macrophage nuclei in heterokaryons.     

Fusion and hybridization of marsupial and eutherian cells. Heterokaryon formation

Marsupial x eutherian cell hybrids would be very useful for studies of mammalian genetics and cell biology. A critical step in the formation of such hybrids is the fusion of cells to form heterokaryons. We have examined many different combinations of marsupial and eutherian cells for their ability to fuse, and we have found that all combinations yielded heterokaryons, but with different frequencies, depending on the cell types used. Ranked in order of decreasing ability to fuse with eutherian cells, the marsupial cell types were; established lines, primary diploid fibroblasts and lymphocytes. In all fusion experiments there was a marked preference for the formation of homokaryons compared with heterokaryons. It was possible to control the numbers and types of heterokaryons formed by varying the input ratio of parental cells.     

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.     

Evidence for endogenous polypeptide-mediated inhibition of cell-cycle transit in human diploid cells

Previous studies have shown that the senescent phenotype is dominant with respect to DNA synthesis in fusions between late passage and actively replicating human diploid fibroblasts. Brief postfusion treatments with the protein synthesis inhibitor cycloheximide (CHX) or puromycin have been found to significantly delay (by 24-48 h) the inhibition of entry into DNA synthesis of young nuclei in heterokaryons after fusion with senescent cells. A significant fraction of the senescent nuclei incorporated tritiated thymidine in CHX-treated heterokaryons. The optimal duration of exposure to CHX was 1-3 h immediately after fusion, although treatments beginning as late as 9 h after fusion elevated the heterokaryon labeling index. Prefusion treatments with CHX were without a significant effect. These results are consistent with the interpretation that regulatory cell cycle inhibitor(s) which are dependent upon protein synthesis may be present in heterokaryons between senescent and actively replicating cells.     

Bidirectional reprogramming of mouse embryonic stem cell/fibroblast hybrid cells is initiated at the heterokaryon stage

Immunofluorescent analysis of markers specific for parental genomes was used to study heterokaryons and hybrid cells soon after the fusion of diploid embryonic stem (ES) cells marked with green fluorescent protein and diploid fibroblasts labeled by blue fluorescent beads. Heterokaryons were identified by an analysis of parental mitochondrial DNAs. Within 20 h after fusion, most heterokaryons (up to 80%) had a fibroblast-like phenotype, being positive for typical fibroblast markers (collagen type I, fibronectin, lamin A/C) and for the modification me3H3K27 chromatin marking the inactive X chromosome but being negative for Oct4 and Nanog. Approximately 20% of heterokaryons had an alternative ES-like phenotype being positive for Oct4 and Nanog, with signs of reactivation of the previously inactive X-chromosome but negative for fibroblast markers. Hybrid cells having alternative phenotypes were easily identified from 24-48 h. The level of DNA methylation at the promoter of the fibroblast Oct4 allele in the ES-like hybrid cells at day 4 was similar to that of ES cells but at the same time, both parental Oct4 alleles were heavily methylated in fibroblast-like hybrid cells. Thus, bidirectional reprogramming initiated at the heterokaryon stage seems to lead to the formation of two types of hybrid cells with alternative dominance of the parental genomes. However, the further fates of two types of hybrid cells are different: ES-like hybrid cells form colonies at 4-6 days but no colonies are derived from the fibroblast-like hybrid cells. The latter grow as disconnected single cells and are unable to form colonies, like mouse embryonic fibroblasts.     

Glyceryl monooleate as a fusogen for the formation of heterokaryons and interspecific hybrid cells

Glyceryl monooleate was used to induce heterokaryon formation between mouse LS fibroblasts and hen erythrocytes during 15 min incubation at 37 °C. Many of the heterokaryons that were formed contained haemoglobin since cell fusion occurred without complete haemolysis of the hen erythrocytes. Following fusion, the plasma membranes of the heterokaryons and their subcellular organelles were apparently intact and relatively undamaged. Although the results of individual experiments were variable, clones of viable hybrid cells were obtained on treatment of mouse 3T3 TK- fibroblasts and chinese hamster wg 3 IMP- fibroblasts with glyceryl monooleate up to 32 times more frequently than in untreated, control cultures. Karyogram analyses confirmed the presence of both sets of parental chromosomes in all hybrid cells studied. Clones of hybrid cells were isolated and subcultured successfully for several months in HAT medium.     

Extinction of muscle-specific properties in somatic cell heterokaryons

In studies of gene regulation using somatic cell fusion techniques, the analysis of heterokaryons circumvents several problematic aspects of the more traditional approach utilizing proliferating hybrid cells. We have analyzed the expression of muscle specific properties in heterokaryons between muscle and nonmuscle cells in order to investigate whether differentiating cells contain regulatory factors that repress the expression of alternative developmental pathways. Heterokaryons and cybrids were derived from polyethylene glycol-mediated fusion of differentiated mononucleate chicken myocytes with mouse melanoma cells, mouse melanoma cytoplasts, chicken fibroblasts, or other chicken myocytes. Our results demonstrate that fusion of a myocyte with a nonmyogenic cell generally results in extinction of muscle-specific properties in the immediate fusion product. Myocyte X melanoma heterokaryons ceased to express the skeletal muscle forms of myosin, desmin and creatine kinase, reinitiated DNA synthesis, and showed a loss of spontaneous fusion competence within 96 hr after their formation. Although chicken myocyte X mouse melanoma heterokaryons showed extinction of muscle specific properties, they continued to synthesize protein and to incorporate [3H]hypoxanthine, presumably due to the continued production of constitutive chicken HPRT. That presence of the melanoma nucleus was required for extinction to be observed was demonstrated by the continued expression of muscle proteins in cybrids between chicken myocytes and melanoma cytoplasts. Significantly, heterokaryons between chicken myocytes and chicken fibroblasts also exhibited extinction of muscle proteins, demonstrating for the first time that extinction is not restricted to fusions in which at least one parental cell type was derived from an established cell line. Our results strongly support the notion that extinction reflects cell-type specific gene regulatory mechanisms operative during development.     

5-Azacytidine permits gene activation in a previously noninducible cell type

We previously reported that silent muscle genes in fibroblasts could be activated following fusion with muscle cells to form heterokaryons. This activation did not require changes in chromatin structure involving significant DNA synthesis. We report here that muscle gene activation was never observed when HeLa cells were used as the nonmuscle fusion partner. However, if HeLa cells were treated with 5-azacytidine (5-aza-CR) prior to fusion, muscle gene expression was induced in the heterokaryons. The genes for both an early (5.1H11 cell surface antigen) and a late (MM-creatine kinase) muscle function were activated, but were frequently not coordinately expressed. These results suggest that the expression of two muscle genes, which is usually sequential, is not interdependent. Furthermore, changes induced by 5-aza-CR, presumably in the level of DNA methylation, are required for muscle genes in HeLa cells to be expressed in response to putative trans-acting regulatory factor(s) present in muscle cells.     

DNA synthesis in the heterokaryons of non-dividing differentiated cells and culture cells with various proliferative potentials

Resident peritoneal mouse macrophages (non-dividing differentiated cells) were fused with mouse embryo fibroblasts (cells with a limited lifespan), NIH 3T3 and C3H 10T 1/2 cells ('immortal' cell lines) and SV 3T3 cells (a malignant cell line). DNA synthesis was investigated in the resultant heterokaryons. No inhibitory effect upon the transition of NIH 3T3 and mouse embryo fibroblasts nuclei to the S-phase was observed. C3H 10T 1/2, NIH 3T3 and SV 3T3 cells induced the reactivation of DNA synthesis in the macrophage nuclei in the heterokaryons. At the same time, no replication was detected in the macrophage nuclei after fusion with mouse embryo fibroblasts.     

Changes in Expression of Fibroblast Surface Antigen (SFA) during Cytodifferentiation and Heterokaryon Formation

Fibroblast surface antigen (SF antigen, SFA) is a major glycoprotein antigen detected in connective tissue cells (primitive mesenchymal cells, fibroblasts, and astroglial cells). In this study the expression of SFA was followed during differentiation of the mesenchymal cells of the mouse metanephros and during heterokaryon formation produced by Sendai-virus induced fusion of human fibroblasts and chick red blood cells. It was demonstrated by immunofluorescence that SFA was lost from the kidney mesenchymal cells when they differentiate into epithelial cells of the secretory tubuli. During this process SFA became detectable in the basement membrane formed around the tubuli. In cell fusion experiments human SFA which was present as fibrillar network on the surface of cultured fibroblasts, was gradually lost from the heterokaryons when the incorporated chick nuclei became activated. These two sets of experiments indicate that SFA can be used as a phenotypic marker of Cytodifferentiation.     

DNA Repair in Human/Embryonic Chick Heterokaryons: Ability of Each Species to Aid the Other in the Removal of Ultraviolet-Induced Damage

Cultured human and embryonic chick fibroblasts possess different enzyme-mediated processes to repair cyclobutyl pyrimidine dimers induced in their deoxyribonucleic acid (DNA) by ultraviolet (UV) radiation. While dimers are corrected in human cells by excision repair, a photoenzymatic repair process exists in embryonic chick cells for the removal of these potentially deleterious UV photoproducts. We have utilized a sensitive enzymatic assay to monitor the disappearance, i.e. repair, of dimer-containing sites in fused populations of human and chick cells primarily consisting of multinucleate human/chick heterokaryons. Fused cultures were constructed such that UV photoproducts were present only in chick DNA when evaluating excision repair and only in human DNA when evaluating photoenzymatic repair. Based on the kinetics of site removal observed in these cultures we are led to conclude the following: Within heterokaryons per se the photoreactivating enzyme derived from chick nuclei and at least one excision-repair enzyme (presumably a UV endonuclease) derived from human nuclei act on UV-damaged DNA in foreign nuclei with an efficiency equal to that displayed toward their own nuclear DNA. Hence, after cell fusion these chick and human repair enzymes are apparently able to diffuse into foreign nuclei and once therein competently attack UV-irradiated DNA independently of its origin. In harmony with the situation in nonfused parental cultures, in heterokaryons the chick photoenzymatic repair process rapidly removed all dimer-containing sites from human DNA including the residual fraction normally acted upon slowly by the human excision-repair process.     

Complementation of genetic disease: a velocity sedimentation procedure for the enrichment of heterokaryons

Methodology is described to enrich for heterokaryons after mammalian cell fusion. A heterogeneous cell mixture can be separated on a Sta-Put apparatus into fractions of uniform size cells by sedimentation through a 1% bovine serum albumin-5% Ficoll gradient. Unfused RAG and LM/TK- cells, differing by 10% in diameter, have been sorted by size; following fusion, larger and faster sedimenting cells were shown to be hybrids. This methodology can be utilized in genetic complementation studies of human genetic diseases where selection procedures for proliferating hybrids do not exist. When fibroblasts from individuals with Tay-Sachs disease [deficient in hexosaminidase A (HEX A-)] and Sandhoff-Jatzkewitz disease (HEX A- and HEX B-) are fused, HEX A is generated, demonstrating complementation of two different mutations. After Sta-Put fractionation, the HEX A complementation product was associated with the faster sedimenting multinuclear cells and not with the mononuclear parental cells. This methodology will facilitate detection of genetic differences in fibroblasts from related inherited disorders.     

Inhibition of DNA synthesis in proliferating human diploid fibroblasts by fusion with senescent cytoplasts

Cytoplasts were prepared from senescent human diploid fibroblasts. The cytoplasts were fused to young human diploid fibroblasts and DNA synthesis was analyzed in the fusion products. DNA synthesis was inhibited (greater than or equal to 40%) in the senescent cytoplast fusion products when compared to unfused young cells or young cytoplasts fused with young cells. These results are consistent with previous experiments that have shown the blockage of DNA synthesis in both nuclei of heterokaryons from fusions of senescent and young human diploid fibroblast cells. Furthermore, these results support the postulate that senescent cells synthesize a specific substance(s), which is present in the cytoplasm of the senescent cell that inhibits DNA synthesis.