Pattern of chick gene activation in chick erythrocyte heterokaryons

The reactivation of chicken erythrocyte nuclei in chick-mammalian heterokaryons resulted in the activation of chick globin gene expression. However, the level of chick globin synthesis was dependent on the mammalian parental cell type. The level of globin synthesis was high in chick erythrocyte-rat L6 myoblast heterokaryons but was 10-fold lower in chick erythrocyte-mouse A9 cell heterokaryons. Heterokaryons between chick erythrocytes and a hybrid cell line between L6 and A9 expressed chick globin at a level similar to that of A9 heterokaryons. Erythrocyte nuclei reactivated in murine NA neuroblastoma, 3T3, BHK and NRK cells, or in chicken fibroblasts expressed less than 5% chick globin compared with the chick erythrocyte-L6 myoblast heterokaryons. The amount of globin expressed in heterokaryons correlated with globin mRNA levels. Hemin increased beta globin synthesis two- to threefold in chick erythrocyte-NA neuroblastoma heterokaryons; however, total globin synthesis was still less than 10% that of L6 heterokaryons. Distinct from the variability in globin expression, chick erythrocyte heterokaryons synthesized chick constitutive polypeptides in similar amounts independent of the mammalian parental cell type. Approximately 40 constitutive chick polypeptides were detected in heterokaryons after immunopurification and two-dimensional gel electrophoresis. The pattern of synthesis of these polypeptides was similar in heterokaryons formed by fusing chicken erythrocytes with rat L6 myoblasts, hamster BHK cells, or mouse neuroblastoma cells. Three polypeptides synthesized by non-erythroid chicken cells but less so by embryonic erythrocytes were conspicuous in heterokaryons. Two abundant erythrocyte polypeptides were insignificant in non-erythroid chicken cells and in heterokaryons.     

Phenotypic expression in chick erythrocyte X rat myoblast hybrids and in chick myoblast X rat myoblast hybrids

Attempts were made to reprogram chick erythrocyte nuclei to specify the synthesis of chick myosin. Chick erythrocytes were fused with rat myogenic cells with the aid of UV-inactivated Sendai virus. In the heterokaryons and hybrid myotubes which resulted from this fusion, the erythrocyte nuclei resumed RNA synthesis and formed nucleoli. Although some new chick antigens developed in those myotubes which contained fully reactivated chick erythrocyte nuclei, accumulation of chick myosin could not be detected by immunological methods. Neither small heterokaryons nor large hybrid myotubes which were actively synthesizing rat myosin reacted with antibodies directed against chick myosin. A small number of mononucleated cells, believed to be synkaryons formed by mitotic division of heterokaryons, did, however, react strongly with antibodies directed against chick myosin and showed a cross striation typical of skeletal muscle. The frequency of such cells was too low, however, to permit karyological analysis or further characterization of the antigen. Hybrids between chick myoblasts and rat myoblasts produced both chick and rat myosin thus indicating that simultaneous translation of chick and rat mRNA for myosin in a common cytoplasm was possible. In summary the evidence obtained suggested that reprogramming of chick erythrocyte nuclei, if it did occur in the present system, was a rare phenomenon.The possibility that hybrids between chick erythrocytes and rat myoblasts expressed markers typical of an erythroid phenotype was examined by immune staining with antibodies directed against chick haemoglobin. The results suggested that haemoglobin was introduced into hybrid cells by erythrocytes which failed to lyse before fusion. The intensity of this immune fluorescence decreased with increasing time after fusion. The rate at which this decrease occurred was not affected by inhibition of RNA synthesis. Thus, there was no evidence for the accumulation of haemoglobin in the hybrid cells.     

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.     

Reactivation of chick erythrocyte nuclei in heterokaryons with temperature-sensitive Chinese hamster cells.

Chinese hamster cell line K12 is temperature-sensitive for the initiation of DNA synthesis. K12 cells synchronized by serum deprivation were collected in early G1(G0). Heterokaryons were formed by fusing chick erythrocytes with serum-starved K12 cells through the use of UV-irradiated Sendai virus. At the permissive temperature (36.5 degrees C), erythrocyte nuclei in heterokaryons enlarged, the chromatin dispersed, and erythrocyte nuclei synthesized DNA at about the same time as the K12 nuclei. At the restrictive temperature (41 degrees C), erythrocyte nuclei enlarged, but neither erythrocyte nor K12 nuclei initiated DNA synthesis. When erythrocyte nuclei were fused with Wg-1A cells, the wild-type parent for ts K12 cells, both kinds of nuclei synthesized DNA at 36.5 degrees C and 41 degrees C. Activation of erythrocyte nuclei was inefficient in heterokaryons incubated in low-serum medium. The results indicate that serum factors and a cellular function defined by the K12 mutation are required for activation of chick erythrocyte nuclear DNA synthesis.     

Rapid reprogramming of globin gene expression in transient heterokaryons

Interspecific heterokaryons were formed by fusing adult mouse erythroleukemia (MEL) cells and human embryonic/fetal erythroid (K562) cells with each other, or with a variety of mouse and human nonerythroid cell types. Analysis of total cellular RNA isolated 24 hr after fusion revealed that normally inactive globin genes can be activated in these "transient" heterokaryons, in which the nuclei do not fuse. In general, the types of globin genes expressed in the donor erythroid cell are activated in the nucleus of the recipient cell. Therefore, erythroid cells contain transacting regulatory factors that are capable of activating the expression of globin genes in a stage- and tissue-specific manner. These observations also indicate that globin genes are not irreversibly repressed in differentiated cells and that their expression can be rapidly reprogrammed in the presence of the appropriate regulatory factors.     

Erythroid differentiation in a friend erythroleukemic cell x lymphoma hybrid cell line is limited, possibly due to reduced hem levels

Induction of erythroid differentiation has been investigated in a cell hybrid formed between an inducible Friend cell and a lymphoma line (L5178Y) derived from the same strain of mouse (DBA/2). Although globin messenger RNA (mRNA) is induced by DMSO to a level similar to that in the inducible Friend cell parent (about 9 000 molecules/cell) haemoglobin does not accumulate in detectable amounts, nor do morphological changes characteristic of terminal differentiation occur. This failure to accumulate haemoglobin in response to DMSO is due to a reduced rate of globin chain synthesis (6% of total protein synthesis, compared to 25% for the parental Friend cell), and partly to inability of the globin chains synthesized to form tetrameric haemoglobin molecules. Globin chain instability is not the reason why haemoglobin does not accumulate. In comparison, treatment of the hybrid cells with haemin induces about 14% globin synthesis and about 13 000 globin mRNA molecules. These values are somewhat higher than with DMSO. Treatment of hybrid cells with haemin plus DMSO is even more effective; it induces 25% globin synthesis and about 30 000 globin mRNA molecules and terminal differentiation also occurs normally. Whether treated with DMSO or haemin or both, virtually all the globin mRNA molecules seem to be present in polysomes and are therefore presumably in the process of being translated. These results suggest that failure of differentiation in these hybrid cells is due to haem limitation which also prevents the expression of other co-ordinated erythroid functions.     

Expression of differentiated functions in heterokaryons between skeletal myocytes,adrenal cells,fibroblasts and glial cells

The regulation of both muscle and adrenal functions was examined in heterokaryons formed by fusing differentiated chick skeletal myocytes to Y1 mouse adrenal cells. Mouse fast skeletal myosin light chain one (LC1) synthesis was induced and acetylcholine receptor expression was maintained at muscle control levels. Steroid secretion, although reduced compared with Y1 × Y1 adrenal homokaryon control fusions, was nonetheless maintained at relatively high levels. Steroid secretion in the myocyte × adrenal heterokaryons was constitutively expressed and was not increased by exposure to either adrenocorticotrophic hormone or db-cAMP. The population of heterokaryons was thus simultaneously expressing both muscle and adrenal functions. The steroid secretion in these heterokaryons was compared to that in heterokaryons formed by fusing Y1 adrenal cells to either chick skin fibroblasts or rat C6 glial cells. Both of these sets of heterokaryons exhibited low baseline levels of steroid secretion that were inducible to control values by ACTH. These results extend previous observations showing that heterokaryons are functionally very different than cell hybrids, and exhibit a variety of phenotypic interactions. Although fibroblasts suppress muscle functions in heterokaryons, they are permissive for adrenal functions. C6 glial cells are permissive for both adrenal and muscle functions, and along with several other neurectodermal derivatives contain an inducible skeletal myosin light chain gene. Finally, myocytes and Y1 adrenal cells are mutually permissive for their differentiated functions, and Y1 adrenal cells contain an inducible myosin light chain gene.     

ACQUISITION OF CHICK CYTOSOL THYMIDINE KINASE ACTIVITY BY THYMIDINE KINASE-DEFICIENT MOUSE FIBROBLAST CELLS AFTER FUSION WITH CHICK ERYTHROCYTES

Chick-mouse heterokaryons were obtained by UV-Sendai virus-induced fusion of chick erythrocytes with thymidine (dT) kinase-deficient mouse fibroblast [LM(TK^-)] cells. Autoradiographic studies demonstrated that 1 day after fusion, [³H]dT was incorporated into both red blood cell and LM(TK^-) nuclei of 23% of the heterokaryons. Self-fused LM(TK^-) cells failed to incorporate [³H]dT into nuclear DNA. 15 clonal lines of chick-mouse somatic cell hybrids [LM(TK^-)/CRB] were isolated from the heterokaryons by cultivating them in selective hypoxanthine-aminopterin-thymidine-glycine medium. LM(TK^-) and chick erythrocytes exhibited little, if any, cytosol dT kinase activity. In contrast, all 15 LM(TK^-)/CRB lines contained levels of cytosol dT kinase activity comparable to that found in chick embryo cells. Disk polyacrylamide gel electrophoresis and isoelectric focusing analyses demonstrated that the LM(TK^-)/CRB cells contained chick cytosol, but not mouse cytosol dT kinase. The LM(TK^-)/CRB cells also contained mouse mitochondrial, but not chick mitochondrial dT kinase. Hence, the clonal lines were somatic cell hybrids and not LM(TK^-) cell revertants. The experiments demonstrate that chick erythrocyte cytosol dT kinase can be activated in heterokaryons and in hybrid cells, most likely as a result of functions supplied by mouse fibroblast cells.     

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.     

Intracellular signals for developmental hemoglobin switching

We have detected trans-acting factors that regulate developmental hemoglobin switching by fusing erythroid cells of different developmental programs. Adult erythroid cells of one anuran species, Xenopus laevis, were fused with tadpole erythroid cells of another frog, Rana catesbeiana. In a second set of experiments, dimethyl sulfoxide-induced murine erythroleukemia cells, which express only adult mouse globins, were fused with Rana tadpole erythroid cells, which express only embryonic and fetal-like globins. Adult Rana globin gene expression was detected in both sets of transient heterokaryons at 6 hr after fusion. Dot blots and Northern blots of total RNA from the heterokaryons contained material that reacted with an adult Rana alpha-globin probe; newly synthesized adult Rana hemoglobin tetramers were detected with native polyacrylamide gel electrophoresis. These results show that developmental stage-specific transacting factors for globin genes can function across vertebrate classes (mammalia to amphibia) and suggest that the mechanisms that regulate developmental hemoglobin switching are highly conserved.     

Restoration of metabolic cooperation in heterokaryons between HGPRT-deficient mouse A9 fibroblasts and chick embryo erythrocytes

Genetic determinants of metabolic cooperation were studied by fusing chick erythrocytes to HGPRT- mammalian cells. Heterokaryons were then tested for their ability to incorporate [3H]hypoxanthine and to transfer radioactive material to HGPRT- recipient cells. Chick erythrocytes (CE) have nuclei which are inactive but contain the HGPRT gene and some cytoplasmic HGPRT enzyme activity. They are unable, however, to cooperate with HGPRT- cells. Of the two mammalian cell lines used, the human GM29 line is HGPRT- and capable of functioning as a receptor cell in cooperation experiments with HGPRT+ cells. The HGPRT- mouse A9 line on the other hand is unable to cooperate. Immediately after fusion, both types of heterokaryons incorporated [3H]hypoxanthine, indicating the presence of some chick HGPRT enzyme contributed by the erythrocyte partner at the time of fusion. While the CE-GM29 heterokaryons participated in metabolic cooperation shortly after fusion, the CE-A9 heterokaryons did not. However, four days after fusion, i.e., at a time when the erythrocyte nucleus had been reactivated, the CE-A9 heterokaryons did cooperate. This suggests that in CE-A9 heterokaryons the genes required for metabolic cooperation are expressed by the previously dormant chick erythrocyte nucleus.     

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.     

Hepatocytes in heterokaryons do not retard the nuclei of stimulated fibroblasts entering the S period]

Serum-deprived (0.2%) resting and serum-stimulated (10%) proliferating NIH 3T3 mouse fibroblasts were fused with hepatocytes from intact, regenerating and embryonic mouse livers to elucidate mechanisms of liver cell proliferation, DNA synthesis being investigated in nuclei of heterokaryons and non-fused cells using radioautography. Hepatocytes in heterokaryons were found to have no inhibitory effect on the entry of stimulated fibroblast nuclei into the S-period, but on the contrary they were involved in DNA synthesis. In addition, the nuclei in heterokaryons mutually stimulated each other to enter the S-period. In their turn, the resting fibroblasts did not prevent the proliferating hepatocytes from the regenerating and embryonic livers to enter the S-period. Possible reasons of the absence of inhibitory effect of differentiated cells in heterokaryons are discussed. The data obtained enable us to conclude that the mechanism of proliferative process control in resting immortalized cells differs from that in differentiated cells where proliferation seems to be stopped without affecting the endogenous inhibitor postulated for the resting and ageing fibroblasts.     

Reactivation of chick erythrocyte nuclei in young and senescent WI38 cells

The chromatin of the dormant chick nucleus is dispersed in the heterokaryons made by Sendai virus fusion of phase II WI38 cells with chick erythrocyte nuclei. The erythrocyte nucleus resumes RNA synthesis and enters into DNA synthesis with the host nucleus. In the heterokaryons of phase III WI38 cells and chick erythrocytes, the nuclear chromatin is not dispersed and RNA synthesis occurs at a reduced rate. The differences in the physiological state of the young and senescent cells measured by [³H]uridine incorporation into nuclear RNA is reflected in the extent of reactivation of the chick erythrocyte nuclei in the cytoplasm of these cells. The reactivation of the chick nucleus in enucleated fibroblasts parallels the nucleated cells. The results of these studies are interpreted as evidence that there is a specific loss of nuclear function in the senescent cells.     

Repair DNA synthesis in heterokaryons during reactivation of chick erythrocytes fused with human diploid fibroblasts or HeLa cells

Suppression of unscheduled DNA synthesis (UDS) after exposure to ultraviolet (UV) light in the human nuclei results when diploid human fibroblasts are fused with chick erythrocytes. The suppression is positively correlated with the number of erythrocyte nuclei in the heterokaryons, with a maximal effect at 36 h after fusion. Evidence is presented that this suppression is due to lowered levels of the enzymes involved in UDS as a result of inhibition of the RNA synthesis by chick components. No suppression of UDS is detected in the human nuclei of the HeLa-chick erythrocyte heterokaryons. In HeLa cells the rate of RNA synthesis is about 10 times higher than the rate in the normal diploid fibroblasts, and the relatively small inhibitory influence of the chick components will therefore not lead to a limitation of the enzymes involved in UDS in the HeLa-chick erythrocyte heterokaryons.     

DNA synthesis in heterokaryons obtained by the fusion of polymorphonuclear leukocytes and cultured cells possessing different proliferative potentials

Heterokaryons between terminally differentiated polymorphonuclear leukocytes (PL) and culture cells of different proliferative potentials: mouse and rat embryo fibroblasts (EFM, EFR); immortal cells NIH 3T3 and E2; malignant cells NCC2, L929, He239 and SV 3T3,--were obtained by means of electrofusion. Radioautographic study of 3H-thymidine incorporation in the nuclei of heterokaryons showed that all the cells taken for fusion were able to induce reactivation of DNA synthesis in PL nuclei, however, with different rates: 7-37% for EFM and NIH 3T3 and 20-40% for malignant cells. The presence of oncogenes Elan in E2 cells and ras in NCC2 cells increased the rate of PL reactivation approximately twice as compared with the cells of original lines (EFR and NIH 3T3, correspondingly). In parallel to reactivation of DNA synthesis in PL nuclei inhibition of the synthesis in culture cell nuclei in the same heterokaryons was found. The rate of inhibition was about 70% for non-malignant and 23, 40 and 18% for NCC2, L and SV 3T3 cells, respectively. He239 cells, transformed by a temperature-dependent mutant of virus SV40 showed at permissive temperature the increased capacity of inducing reactivation of PL nuclei, though He239 cells susceptibility to inhibitory action of PL nuclei did not change with temperature. According to the behaviour in heterokaryons PL were found to be similar to chick erythrocytes, but differing from them by a pronounced inhibiting effect upon DNA synthesis in the nuclei of malignant cells.     

Viable hybrids between lethally X-irradiated hamster cells and unirradiated mouse cells.

Senda?-virus-induced fusion between heavily X-irradiated hamster cells, BHK21 or RS2-3 (BHK21 cells transformed by Rous Sarcoma Virus), and unirradiated mouse cells, A9 or c11D, give rise to hybrids. These hybrids possess mouse and hamster surface antigens. However, RS2-3 x mouse hybrids do not form heterokaryons with chick-embryo fibroblasts producing infectious Rous sarcoma virus.