Mitotic segregation of cytoplasmic determinants for chloramphenicol resistance in mammalian cells. I: Fusion with mouse cell lines

The segregation of cytoplasmically inherited chloramphenicol (CAP) resistance in mouse cells was investigated in fusions between CAP-resistant cells or cytoplasts (enucleated cells) and CAP-sensitive cells of varying tissue origin. All hybrids formed in cell-cell fusions were initially CAP-resistant, indicating that CAP resistance is dominant. Hybrids from fusions of cells of the same tissue origin (homologous) were stably CAP-resistant, whereas the hybrid population from fusions of different origins (heterologous) showed a rapid diminution of average CAP resistance. Individual hybrid clones from these heterologous fusions also showed an overall loss of CAP resistance, and a wide variation in CAP resistance which is consistent with a large number of genetic determinants (possibly mitochondrial DNA molecules) contributing to the CAP phenotype. Similar results were obtained from cytoplast-cell fusions, so the observed CAP segregation is not the result of nuclear-nuclear interactions. This segregation of CAP resistance constitutes a second criterion of cytoplasmic inheritance in mammalian cells.     

Polyethylene-glycol-mediated cybrid formation: high-efficiency techniques and cybrid formation without enucleation.

Polyethylene glycol (PEG) can be used to promote the fusion of enucleated cytoplasts from chloramphenicol (CAP)-resistant mouse cells with intact cells, resulting in the formation of viable cybrids. The techniques are simple and highly efficient, yielding up to one viable cybrid per 20 intact cells fused. It also seems that PEG can be used to induce cybrid formation without the necessity of prior enucleation of the CAP-resistant cells.     

Cytoplasmic transfer of chloramphenicol resistance in human tissue culture cells

The cytoplasmic inheritance of human chloramphenicol (cap) resistance has been demonstrated by removing the nuclei of cells of the CAP-resistant HeLa strain 296-1 (enucleation) and fusing them to a CAP-sensitive HeLa strain lacking nuclear thymidine kinase. Plating the fusion products in bromodeoxyuridine and CAP resulted in the growth of about 150 colonies/10(6) parent cells plated. Permanent cell lines (cybrids) grown from such fusions have been designated HEB. A recloned HEB cybrid (HEB7A) has also been enucleated and fused to hypoxanthine phosphoribosyl transferase (HPRT)-deficient HeLa cells (S3AG1) and HPRT-deficient lymphocytes (WAL-2A). Cybrids were selected in thioguanine and CAP. In the fusion of enucleated (en) HEB7A to S3AG1, 1,200 colonies/10(6) parents were observed. Fusion of enHEB7A to WAL-2A was done in mass culture and cybrids were obtained on three separate occasions. In every case the parental controls were negative. All isolates tested from the above fusions have the CAP-resistant characteristics, in vivo and in vitro, of the enucleated parent and the nuclear characteristics of the CAP-sensitive parent, such as chromosome number, morphology, and specific isozyme and chromosome markers. Therefore, it can be concluded that CAP resistance is coded in the cytoplasm and not in the nucleus of 296-1 cells. Furthermore, this resistance can be transferred to cells of widely different origin and differentiated state. These studies represent the first genetic evidence of cytoplasmic inheritance in human cells.     

Cytoplasmic transfer of microtubule organizing centers in mouse tissue culture cells

A chloramphenicol-resistant, aminopterin-sensitive cell line (AMT) derived from a mouse mammary tumor MT-29240 was enucleated, and the cytoplasts were fused with nucleated chloramphenicol-sensitive, HAT-resistant SV40 3T3 mouse cells. The resulting cytoplasmic hybrids (cybrids) were selected for their resistance to chloramphenicol and the chromosome complement of the SV40 3T3 cells. In addition to transfer of chloramphenicol resistance, these cybrid clones, as studied in the electron microscope, contained the intracisternal A particle phenotype characteristic of only the AMT cells. The cytoplasmic microtubule complex (CMTC) in these cybrids was also studied and appears to resemble the elaborate CMTC of the AMT cells more closely than the more reduced CMTC of the SV40 3T3. When treated with a colcemid block and then a brief reverse, the microtubule organizing centers (MTOC) appear as bright fluorescent foci when tubulin antibody and indirect immunofluorescence techniques are used. When AMT or SV40 3T3 cells are treated in this manner, only one MTOC is present in interphase cells. One clone of these cybrids, however, contained two MTOCs in interphase cells. This CMTC and MTOC phenotype has persisted in this cybrid clone for over 3 months of continuous culture.     

Cytoplasmic suppression of malignancy

Summary Using both normal and transformed rat liver epithelial cells to prepare cytoplasmic hybrids (cybrids) we have found evidence to support the theory that the cytoplasm from a normal cell can suppress tumorigenicity. A unique aspect of this study is that all of the cells utilized, both normal and malignantly transformed, were derived from an original cloned cell. We found that fusing cytoplasts from normal cells to malignantly transformed whole cells resulted in cybrid clones which, when injected into newborn rat pups, isogenic with those from which the cell culture was initiated, yielted tumors in 51% of the animals injected compared to 92% of the animals injected with the tumorigenic parent. Those animals that did develop tumors from the cybrid cells survived longer than those injected with cells from the tumorigenic parent. Thus, the cybrid, formed of cytoplasm from both parents, was less tumorigenic than the malignantly transformed parent cell. When reconstituted cells were prepared by fusing cytoplasts from normal cells with karyoplasts from malignantly transformed cells, a situation in which essentially all of the cytoplasm of the reconstituted cell is derived from normal cells, the tumorigenic phenotype was extinguished. This work was supported in part by United States Public Health Service grant CA12056, and grant CA09100 from the National Cancer Institute, Bethesda, MD. This work is partial fulfillment for the degree of Doctor of Philosophy for B.A.I.     

The preparation and properties of cytoplasts from Ehrlich ascites tumor cells

A method is described in which cytochalasin B is used to fractionate Ehrlich ascites tumor cells into cytoplasts and (nucleated) karyoplasts. The plasma membrane and cytoplasm are selectively removed from these cells by this method such that the cytoplasts rarely contain membranous organelles (e.g., mitochondria) which are retained in the karyoplast during fractionation. ATP concentrations similar to those found in whole cells and glycolytic activity were measured in cytoplasts in the presence but not the absence of glycose. Cytoplasts also actively transport Na⁺, K⁺, and α-aminoisobutyric acid to steadystate distribution ratios similar to those found in whole cells. It was concluded that these cytoplasts are a simplified model system for the study of active transport in Ehrlich cells.     

Isolation and characterization of intraspecific cybrids : Effect of mitochondrial DNA on their cellular properties

Cybrid clones were obtained by fusing whole cells of rat glioma C6BU-1, resistant to 5-bromodeoxyuridine (BrdU), with cytoplasts of embryonic rat 3Y1CAP cells, resistant to chloramphenicol (CAP), in selective medium with BrdU and CAP. The clones resistant to BrdU and CAP were confirmed to be cybrids by chromosome and mtDNA analyses. More than half the mtDNA of all the cybrid clones was from the 3Y1CAP cells. After cultivation of a cybrid clone Y22 for 3 months in the absence of CAP, subclones were isolated. One subclone Y22-22 contained predominantly mitochondrial DNA (mtDNA) from the 3Y1CAP cells. Using this subclone, the effects of the mitochondrial genome on cellular properties were examined. The growth patterns, expression of glioma-specific beta-adrenergic receptor, and composition of the major proteins of C6BU-1 cells were not affected by transmitted mtDNA from the 3Y1CAP cells. This procedure for isolating cells containing predominantly foreign mtDNA will be useful in studies on the interaction between genomes of the mitochondria and nucleus.     

New Djungarian hamster cell lines with selective cytoplasmic and nuclear genetic markers

Two new Djungarian hamster cell lines which are resistant to chloramphenicol (CAP) are described. The clonal DMCAP subline was obtained by incubation of HPRT-deficient DM-15 cells for 6 months in the medium containing 50 micron/ml of CAP. Resistance to CAP is determined in DMCAP cells by the cytoplasm: cytoplasts from these cells could transmit resistance to CAP into sensitive cells, such as L or DMCH-2/1 cells by hybridization. However, after transplantation of DMCAP nuclei into L cytoplasts, the resulting hybrid cells lost resistance to CAP to a great extent. Using the capacity of DMCAP cytoplasts to transfer CAP-resistance, we obtained a line of hybrids (cyt. DMCAP X DMCH-2/1) which was resistant to 8-azaguanine, CAP and colchicine. As in the original DMCH-2/1 cell line, colchicine-resistance in the cybrid line appeared to be associated with gene amplification. Thus, chromosomal analysis showed that the karyotype of the hybrids was identical to that of DMCH-2/1 cells. Both contained marker chromosomes with homogeneously staining regions (HSRs) and, during incubation in the colchicine-free medium, lost resistance to colchicine. The loss of resistance was accompanied by a decrease in the number of cells containing chromosomes with HSRs and an increase in the number with double minutes (DMs). Many cells containing small chromatin bodies in their cytoplasm also appeared. These chromatin bodies may be DMs lost from the nucleus during mitosis. These new sublines with cytoplasmic and nuclear genetic markers may be useful in the further study of cytoplasmic-nuclear interactions, particularly, in the analysis of possible activities of the DNA fragments which appear in the cytoplasm during reversion to colchicine sensitivity.     

Non-selective isolation of fibroblast-cybrids by flow sorting

Red- and green-fluorescing polystyrene beads were used to label different populations of cultured human fibroblasts. After enucleation of the green-fluorescing population, the cytoplasts were fused with red-fluorescing cells. Twenty-four hours after cell fusion the population of red-green heterofluorescent cells was isolated with a FACS II cell sorter. When Lesch-Nyhan fibroblasts (HPRT⁻) were fused with cytoplasts from control fibroblasts (HPRT⁺) more than 95% of the sorted cells were heterofluorescent and 90% of the sorted cells showed HPRT⁺ activity. Therefore almost all sorted heterofluorescent cells are true cybrids. With this procedure for cybrid isolation, earlier complementation studies using cybrids from different variants of β-galactosidase deficiency could be confirmed.     

Expression of monoamine oxidase A and B activities in mouse cybrids and hybrids

The inheritance of monoamine oxidase (MAO; EC1.4.3.4) was studied in cultured cells using techniques of somatic cell genetics. Cells of a mouse neuroblastoma variant line lacking MAO activity were fused to cytoplasts prepared from a mouse L cell line which expresses MAO activity and is resistant to chloramphenicol (a cytoplasmically inherited trait). The resulting cybrids were resistant to chloramphenicol, but did not recover MAO activity, indicating that the loss of activity in the neuroblastoma parent was not the result of an inherited lesion in a cytoplasmically transmitted gene. This cybrid cells were then fused to rat hepatoma cells expressing both A and B types of MAO activity. A resulting hybrid line, grown in medium containing hypoxanthine, aminopterin and thymidine (HAT) to select cells that had retained HPRT activity and hence the rat X chromosome, expressed both types of activity, but at a reduced level compared to the hepatoma parent. This finding indicates that the genetic lesion in the neuroblastoma cells resulting in loss of MAO activity is not phenotypically dominant, and that both A and B types of activity can be conferred together to neuroblastoma cells which normally express only the A type of MAO activity.     

Cellular aging studied by the reconstruction of replicating cells from nuclei and cytoplasms isolated from normal human diploid cells

Cytochalasin B (CB) was used to enucleate cells (cytoplasts) and to obtain karyoplasts (nuclei) from the human diploid fetal lung fibroblast strain WI-38. Fusion of cytoplasts and nuclei from young and old cells was accomplished with the aid of inactivated Sendai virus. Viable nuclei may be obtained from the karyoplast pellet after passage through a layer of bovine albumin which retains any contamination cytoplasts. The majority of successful fusions forming “whole cells” occurred when cytoplast from “old” cultures (PDL 40–51) and karyoplasts from “young” cultures were used (PDL 12–22), but almost always resulted in limited division of the viable reconstructed cells. When successful fusion occurred between “young” cytoplasts and “young” karyoplasts the number of cell divisions obtained was comparable to control cells kept under similar conditions.     

Induction of erythroid differentiation by cytoplast fusion in mouse erythroleukemia (Friend) cells

An intracellular activity, which is induced by dimethyl sulfoxide (DMSO) or hexamethylenebisacetamide (HMBA) and leads to erythroid differentiation in mouse Friend cells, was characterized by cell fusion between genetically marked intact cells and cytoplasts. For this, a procedure for rapid selection of cybrids was devised by sensitizing non-fused cells with oligomycin. We were able to demonstrate that cytoplasts derived from DMSO- (or HMBA)-treated cells trigger erythroid differentiation upon fusion with UV-irradiated cells. The activity in the cytoplasts remained only transiently and its induction was inhibited by biologically active phorbol esters or cycloheximide. The activity, however, was not induced in cytoplasts by directly treating them with DMSO (or HMBA). These results indicate that (1) the intracellular erythroid-inducing activity is located in cytoplasts, (2) it acts in trans and induces erythroid differentiation as a dominant factor and (3) its production requires de novo nuclear protein synthesis. The mechanisms of the induction of the intracellular activity and of how it triggers erythroid differentiation are discussed.     

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.     

Characterization of mitochondrial DNA in chloramphenicol-resistant interspecific hybrids and a cybrid

We have examined the restriction endonuclease cleavage patterns exhibited by the mitochondrial DNAs (mtDNA) of four chloramphenicol-resistant (CAPR) human x mouse hybrids and one CAPR cybrid derived from CAPR HeLa cells and CAPS mouse RAG cells. Restriction fragments of mtDNAs were separated by electrophoresis and transferred by the Southern technique to diazobenzyloxymethyl paper. The covalently bound DNA fragments were hybridized initially with 32P-labeled complementary RNA (cRNA) prepared from human mtDNA and, after removal of the human probe, hybridized with mouse [32P]cRNA prepared from mouse mtDNA. Three hybrids which preferentially segregated human chromosomes and the cybrid exhibited mtDNA fragments indistinguishable from mouse cells. One hybrid, ROH8A, which exhibited "reverse" chromosome segregation, contained only human mtDNA. The pattern of chromosome and mtDNA segregation observed in these hybrids and the cybrid support the hypothesis that a complete set of human chromosomes must be retained if a human-mouse hybrid is to retain human mitochondrial DNA.     

Replicative potentials of various fusion products between WI-38 and SV40 transformed WI-38 cells and their components

Hybrid cells derived from whole-cell fusions of replicating phase-II normal fibroblast cells (WI-38s) with SV40 transformed WI-38 fibroblast cells (CL-1s) demonstrated that the majority of the hybrid experimental cells still maintained a finite life-span. Approximately 2% demonstrated sustained and possibly indefinite replication. Experimental binucleate cells and subsequent hybrid synkaryons were also formed by fusing CL-1 karyoplasts into phase-II WI-38 replicating normal fibroblasts. In addition, viable cells were constructed from WI-38 fibroblast cytoplasts with CL-1 karyoplasts. Sustained replication was not observed in these crosses.     

Functional activity of enucleated human polymorphonuclear leukocytes

Enucleated human polymorphonuclear leukocytes (PMN) were prepared by centrifuging isolated, intact PMN over a discontinuous Ficoll gradient that contained 20 microM cytochalasin B. The enucleated cells (PMN cytoplasts) contained about one-third of the plasma membrane and about one-half of the cytoplasm present in intact PMN. The PMN cytoplasts contained no nucleus and hardly any granules. The volume of the PMN cytoplasts was about one-fourth of that of the original PMN. Greater than 90% of the PMN cytoplasts had an "outside-out" topography of the plasma membrane. Cytoplasts prepared from resting PMN did not generate superoxide radicals (O2-) or hydrogen peroxide. PMN cytoplasts incubated with opsonized zymosan particles or phorbol-myristate acetate induced a respiratory burst that was qualitatively (O2 consumption, O2- and H2O2 generation) and quantitatively (per unit area of plasma membrane) comparable with that of intact, stimulated PMN. Moreover, at low ratios of bacteria/cells, PMN cytoplasts ingested opsonized Staphylococcus aureus bacteria as well as did intact PMN. At higher ratios, the cytoplasts phagocytosed less well. The killing of these bacteria by PMN cytoplasts was slower than by intact cells. The chemotactic activity of PMN cytoplasts was very low. These results indicate that the PMN apparatus for phagocytosis, generation of bactericidal oxygen compounds, and killing of bacteria, as well as the mechanism for recognizing opsonins and activating PMN functions, are present in the plasma membrane and cytosol of these cells.     

Cloned mice derived from embryonic stem cell karyoplasts and activated cytoplasts prepared by induced enucleation

Our objective was to induce enucleation (IE) of activated mouse oocytes to yield cytoplasts capable of supporting development following nuclear transfer. Fluorescence microscopy for microtubules, microfilaments, and DNA was used to evaluate meiotic resumption after ethanol activation and the effect of subsequent transient treatments with 0.4 micro g/ml of demecolcine. Using oocytes from B6D2F1 (C57BL/6 x DBA/2) donors, the success of IE of chromatin into polar bodies (PBs) was dependent on the duration of demecolcine treatment and the time that such treatment was initiated after activation. Similarly, variations in demecolcine treatment altered the proportions of oocytes exhibiting a reversible compartmentalization of chromatin into PBs. Treatment for 15 min begun immediately after activation yielded an optimized IE rate of 21% (n = 80) when oocytes were evaluated after overnight recovery in culture. With this protocol, 30-50% of oocytes were routinely scored as compartmentalized when assessed 90 min postactivation. No oocytes could be scored as such following overnight recovery, with 66% of treated oocytes cleaving to the 2-cell stage (n = 80). Activated cytoplasts were prepared by mechanical removal of PBs from oocytes whose chromatin had undergone IE or compartmentalization. These cytoplasts were compared with mechanically enucleated, metaphase (M) II cytoplasts whose activation was delayed in nuclear transfer experiments using HM-1 embryonic stem cells. Using oocytes from either B6D2F1 or B6CBAF1 (C57BL/6 x CBA) donors, the in vitro development of cloned embryos using activated cytoplasts was consistently inferior to that observed using MII cytoplasts. Live offspring were derived from both oocyte strains using the latter, whereas a single living mouse was cloned from activated B6CBAF1 cytoplasts.     

Nuclear control of tumorigenicity in cells reconstructed by PEG-induced fusion of cell fragments

The techniques of somatic cell hybridization have provided a valuable means of studying mechanisms of regulation of mammalian cell differentiation and transformation. Most previous studies have indicated that fusions between tumorigenic and nontumorigenic cells result in hybrid cells that are usually tumorigenic. In recent years it has been demonstrated that the phenotypic expression of tumorigenicity is at least partially due to the extensive chromosome loss that occurs in most interspecific and some intraspecific hybrid cells. In the present study we have utilized enucleation techniques that permit cells to be divided into nuclear (karyoplast) and cytoplasmic (cytoplast) cell fragments. Even though these nuclear and cytoplasmic fragments are metabolically stable for short periods of time, in our hands they ultimately degenerate. Viable cells can be reconstructed by PEG-induced fusion of karyoplasts to cytoplasts. Since reconstructed cells apparently do not segregate chromosomes, they may provide a clearer understanding of the interactions between the nucleus and the cytoplasm in the control of the expression of tumorigenicity. We have reconstructed cells using karyoplasts from the tumorigenic Y-1 cell line and cytoplasts from a nontumorigenic cell line, A-MT-BU-A1. In addition we have reconstructed cells containing Y-1 cytoplasts and A-MT-BU-A1 karyoplasts. The reconstructed cells porduced were assayed for tumorigenicity by their ability to grow in soft agar and in nude mice. The results of these experiments indicate that the reconstructed cells containing a tumorigenic nucleus and a nontumorigenic cytoplasm ultimately are tumorigenic and conversely the reconstructed cells containing a nontumorigenic nucleus and a tumorigenic cytoplasm are nontumorigenic. These experiments support the concept that with these cell lines the nucleus (karyoplast) is sufficient to control the phenotypic expression of tumorigenicity.     

Cytoplasmically inherited mutations of a human cell line resulting in deficient mitochondrial protein synthesis.

A large number of mutants deficient in mitochondrial protein synthesis (mtPS-) have been isolated from the human cell line VA2-B by subjecting cells partially depleted of their mtDNA to mutagenic treatments thought to be specific for mtDNA. Each of these mtPS- mutants has less than 10% of the wild-type rate of mitochondrial protein synthesis, exhibits reduced cytochrome oxidase and rutamycin sensitive ATPase activities, requires high concentrations of glucose, and grows indefinitely in the presence of 100 micrograms/ml of chloramphenicol (CAP). Fusion of cytoplasts from seven mtPS- mutants to the nucleated thioguanine-resistant VA2-B derivative TG-6 has yielded numerous cybrid clones which grow in CAP plus thioguanine, whereas almost no clones have resulted from the fusion of nucleated mtPS- cells to TG-6 cells: these results suggest that the gene(s) coding for the phenotype of mtPS- cells is localized in the cytoplasm (mtDNA?).     

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.