The relationship between induction of embryogenesis and chromosome doubling in microspore cultures

Summary. The objective of this paper is to review the relationship between induction of microspore embryogenesis and chromosome doubling. It has been augmented with relative data on chromosome doubling by nuclear fusion. Some of the treatments used for induction of embryogenesis may also lead to doubling of the chromosome number, either through nuclear fusion or endomitosis. High frequencies of spontaneous chromosome doubling in cereal species appear to be induced by treatments that block cell wall formation during the first cell divisions, resulting in coenocytic cells in which the nuclei are able to fuse. The use of mannitol as a pretreatment for induction of embryogenesis in barley, wheat, and maize microspore cultures provides examples of nuclear fusion. The use of antimicrotubule agents for embryo induction via treatments during the first few hours of microspore culture has also resulted in high frequencies of chromosome doubling. Factors such as the doubling agent concentration, temperature during treatment, and duration of treatment may be critical for individual species. Actin filament as well as microtubule assembly studies related to new cell wall formation provide further evidence at the molecular level for the relationship between microspore embryogenesis and chromosome doubling. Correspondence and reprints: Department of Plant Agriculture, University of Guelph, Crop Science Building, Guelph, ON N1G 2W1, Canada.     

Hybridization frequencies of different mammalian cell types by electrofusion

The efficiency of electrofusion of four types of cells: CHO, HeLa, mouse melanoma cells and human skin fibroblasts has been studied. The frequencies of fusion products were determined 1) directly in a closed flow-through fusion chamber after dielectrophoresis and pulsation; 2) after short-term postfusion cultivation period of 5 to 10 minutes; and 3) in various intervals up to 30 hours after fusion induction. No substantial differences were found in the rates of formation of heterokaryons and synkaryons between the individual cell types, and this confirmed the uniformity of the effects of electric fields on diverse cell membranes. After 5 hours of culture the yield of fusion products reached 15 to 35% in various cell combinations and the frequencies of synkaryons reached up to 7% in almost all the combinations studied 24 to 30 hours after fusion.     

Analysis of mutagenesis and sister-chromatid exchanges induced by 5-bromo-2'-deoxyuridine in somatic hybrids derived from Syrian hamster melanoma cells and Chinese hamster ovary cells

Somatic cell hybrids were derived from the fusion of Chinese hamster ovary (CHO) cells and Syrian hamster melanoma cells (2E). These two cell lines had previously been shown to differ in their response to the induction of mutations and sister-chromatid exchanges (SCEs) by 5-bromo-2'-deoxyuridine (BrdUrd) (Kaufman, 1987). The parental cells and a number of representative, independent hybrid clones were tested for their response to both the INC and REP mutagenesis protocols. INC mutagenesis involves the incorporation of BrdUrd into DNA under conditions of deoxyribonucleoside triphosphate (dNTP) pool imbalance, while REP mutagenesis involves the replication of 5-bromouracil-substituted DNA in the presence of dNTP pool imbalance. When tested for the toxic effects of high concentrations of BrdUrd and for the induction of mutations by the INC protocol, the hybrid clones all expressed the 2E phenotype, i.e., sensitivity to relatively low concentrations of BrdUrd and thymidine for the induction of mutations, dNTP pool perturbation, and the toxic effects of BrdUrd. When the hybrid clones were tested for the induction of mutations and SCEs by the REP protocol, it was found that they expressed the 2E phenotype for the induction of mutations and the CHO phenotype for the induction of SCEs. Thus, various aspects of the 2E phenotype, such as high mutation frequencies associated with large dNTP pool perturbations, appeared to be dominantly expressed in the cell hybrids, while the lack of induction of SCEs by these mutagenic conditions in 2E cells was found to be a recessive characteristic.     

Analysis of mutagenesis and sister-chromatid exchanges induced by 5-bromo-2′-deoxyuridine in somatic hybrids derived from Syrian hamster melanoma cells and Chinese hamster ovary cells

Somatic cell hybrids were derived from the fusion of Chinese hamster ovary (CHO) cells and Syrian hamster melanoma cells (2E). These two cell lines had previously been shown to differ in their response to the induction of mutations and sister-chromatid exchanges (SCEs) by 5-bromo-2′-deoxyuridine (BrdUrd) (Kaufman, 1987). The parental cells and a number of representative, independent hybrid clones were tested for their response to both the INC and REP mutagenesis protocols. INC mutagenesis involves the incorporation of BrdUrd into DNA under conditions of deoxyribonucleoside triphosphate (dNTP) pool imbalance, while REP mutagenesis involves the replication of 5-bromouracil-substituted DNA in the presence of dNTP pool imbalance. When tested for the toxic effects of high concentrations of BrdUrd and for the induction of mutations by the INC protocol, the hybrid clones all expressed the 2E phenotype, i.e., sensitivity to relatively low concentrations of BrdUrd and thymidine for the induction of mutations, dNTP pool perturbation, and the toxic effects of BrdUrd. When the hybrid clones were tested for the induction of mutations and SCEs by the REP protocol, it was found that they expressed the 2E phenotype for the induction of mutations and the CHO phenotype for the induction of SCEs. Thus, various aspects of the 2E phenotype, such as high mutation frequencies associated with large dNTP pool perturbations, appeared to be dominantly expressed in the cell hybrids, while the lack of induction of SCEs by these mutagenic conditions in 2E cells was found to be a recessive characteristic.     

Prematurely condensed chromosomes of dividing and non-dividing cells in aging human cell cultures.

Chromosomes of dividing and non-dividing aging cells were examined by fusing senescent WI38 cells with mitotic HeLa cells to induce premature chromosome condensation (PCC). Exposure of the WI38 cells to ³H-thymidine 48 h prior to fusion allowed autoradiographic identification of cells that did not synthesize DNA (non-dividing cells). Ninety-six percent of the non-dividing cells, diploid or tetraploid, induced into PCC had single chromatids and were therefore blocked in the G1 phase of the cell cycle. Anomalous centromeric pairing of chromatids was noted in the remaining 4% of the non-dividing cells. Typical G2 configurations (double chromatids) were observed only among labeled (dividing) cells. The efficiency of PCC induction was independent of culture age. In addition, the efficiency of PCC induction was independent of phase in the cell cycle, as shown by comparison of observed frequencies with expected frequencies.     

Production of interspecies somatic/pluripotent heterokaryons using polyethylene glycol (PEG) and selection by imaging flow cytometry for the study of nuclear reprogramming

Fusion of somatic cells to embryonic stem cells induces reprogramming of the somatic nucleus and can be used to study the effect of trans-acting factors from the pluripotent cell over the differentiated nucleus. However, fusion only occurs in a small fraction of the cells exposed to fusogenic conditions, hence the need for a protocol that produces high fusion rate with minimal cell damage, coupled with a method capable of identifying and selecting these rare events. Here, we describe a protocol to induce formation of bi-species mouse pluripotent/bovine somatic heterokaryons, as well as same-species homokaryons, using polyethylene glycol (PEG). To identify bi-species fusion products, heterokaryons were labeled using cell type-specific fluorescent antibodies and selected using imaging (Amnis ImageStream Mark II) and traditional (BD FACSAria I) flow cytometry. Heterokaryons selected with this method produced ES cell-like colonies in vitro. This procedure can be combined with downstream applications such as nucleic acid isolation for RT-PCR and RNA-Seq, and used as a tool to study somatic cell nuclear reprogramming.     

Streptococcal preparation OK-432 promotes fusion efficiency and enhances induction of antigen-specific CTL by fusions of dendritic cells and colorectal cancer cells

Dendritic/tumor fusion cell (FC) vaccine is an effective approach for various types of cancer but has not yet been standardized. Antitumor activity can be modulated by different mechanisms such as dendritic cell (DC) maturation state. This study addressed optimal strategies for FC preparations to enhance Ag-specific CTL activity. We have created three types of FC preparations by alternating fusion cell partners: 1) immature DCs fused with autologous colorectal carcinoma cells (Imm-FCs); 2) Imm-FCs followed by stimulation with penicillin-inactivated Streptococcus pyogenes (OK-432) (Imm-FCs/OK); and 3) OK-432-stimulated DCs directly fused to autologous colorectal carcinoma cells (OK-FCs). Both OK-FCs and Imm-FCs/OK coexpressed the CEA, MUC1, and significantly higher levels of CD86, CD83, and IL-12 than those obtained with Imm-FCs. Short-term culture of fusion cell preparations promoted the fusion efficiency. Interestingly, OK-FCs were more efficient in stimulating CD4(+) and CD8(+) T cells capable of high levels of IFN-gamma production and cytolysis of autologous tumor or semiallogeneic targets. Moreover, OK-FCs are more effective inducer of CTL activation compared with Imm-FCs/OK on a per fusion cell basis. The pentameric assay confirmed that CEA- and MUC1-specific CTL was induced simultaneously by OK-FCs at high frequency. Furthermore, the cryopreserved OK-FCs retained stimulatory capacity for inducing antitumor immunity. These results suggest that OK-432 promotes fusion efficiency and induction of Ag-specific CTL by fusion cells. We conclude that DCs fused after stimulation by OK-432 may have the potential applicability to the field of antitumor immunotherapy and may provide a platform for adoptive immunotherapy in the clinical setting.     

Activation of the human beta-interferon gene requires an interferon-inducible factor.

beta-Interferon (beta-IFN) gene expression can be induced by poly(I)-poly(C) or virus, but there is considerable variation in the extent of induction between different cell lines. We characterized two poorly inducible human cell lines, HeLa and 143 thymidine kinase negative (143 tk-), to define cellular factors involved in the activation of the beta-IFN gene. We show that the deficiency in beta-IFN induction in these cells can be complemented by fusion to highly inducible mouse cells. We conclude that the human cells are deficient in a trans-acting factor required for B-IFN gene activation. The level of induction of the beta-IFN gene in HeLa and 143 tk- cells can also be increased by priming with IFN before induction. If IFN priming is carried out in the presence of cycloheximide, a approximately 200-fold increase in induction is observed. We conclude that activation of the beta-IFN gene requires an IFN-inducible factor that is only expressed at low levels in unprimed HeLa and 143 tk- cells.     

Induction of cell fusion by a factor released by the cellular slime mold Polysphondylium pallidum

Heterokaryons and hybrid cells, which are extremely useful for research in cell biology, can be produced artificially by treating cells with either polyethylene glycol or certain inactivated viruses that alter the plasma membrane. We report here a novel cell-fusion inducing factor secreted by CK-8 strain cells of cellular slime mold Polysphondylium pallidum. Treatment of other strains or other species of cellular slime molds, such as NC-4 of Dictyostelium discoideum with the diluted fraction, containing molecules larger than 50 kDa, of the conditioned medium of CK-8 cell culture induces cell fusion at high frequency and produces multinucleated large cells. This cell fusion is inducible between cells of either a single strain or of two different strains of cellular slime molds.Abbreviations BSS Bonner's salt solution - CM conditioned medium - EDTA ethylenediaminetetraacetic acid - F2 fraction containing cell-fusion induction factor - Mr molecular mass     

Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions

A substantial range of protein-protein interactions can be readily monitored in real time using bioluminescence resonance energy transfer (BRET). The procedure involves heterologous coexpression of fusion proteins, which link proteins of interest to a bioluminescent donor enzyme or acceptor fluorophore. Energy transfer between these proteins is then detected. This protocol encompasses BRET1, BRET2 and the recently described eBRET, including selection of the donor, acceptor and substrate combination, fusion construct generation and validation, cell culture, fluorescence and luminescence detection, BRET detection and data analysis. The protocol is particularly suited to studying protein-protein interactions in live cells (adherent or in suspension), but cell extracts and purified proteins can also be used. Furthermore, although the procedure is illustrated with references to mammalian cell culture conditions, this protocol can be readily used for bacterial or plant studies. Once fusion proteins are generated and validated, the procedure typically takes 48-72 h depending on cell culture requirements.     

Studies on the behavior of meiotic protoplasts II. Induction of a high fusion frequency in protoplasts from liliaceous plants

A method for the induction of high fusion frequencies in meioticprotoplasts of a lily and Trillium is described. Fusion wasinduced during the rapid isolation of protoplasts by quicklybringing about physical contact between protoplasts. Fused protoplastswere obtained in 50 to 90% yields from prophase cells and in30 to 60% yields from cells in later phases. Large protoplastswith more than 10 nuclei were frequently produced. During culture,meiotic development was not synchronous in these protoplasts;each multinudeate maintaining a high degree of synchrony. (Received February 28, 1973; )     

Multi-patterned dynamics of mitochondrial fission and fusion in a living cell

Mitochondria are highly-dynamic organelles, but it is challenging to monitor quantitatively their dynamics in a living cell. Here we developed a novel approach to determine the global occurrence of mitochondrial fission and fusion events in living human epithelial cells (Hela) and mouse embryonic fibroblast cells (MEF). Distinct patterns of sequential events including fusion followed by fission (Fu-Fi), the so-called "kiss and run" model previously described, fission followed by fusion (Fi-Fu), fusion followed by fusion (Fu-Fu), and fission followed by fission (Fi-Fi) were observed concurrently. The paired events appeared in high frequencies with short lifetimes and large sizes of individual mitochondria, as compared to those for unpaired events. The high frequencies of paired events were found to be biologically significant. The presence of membrane uncoupler CCCP enhanced the frequency of paired events (from both Fu-Fi and Fi-Fu patterns) with a reduced mitochondrial size. Knock-out of mitofusin protein Mfn1 increased the frequency of fission with increased lifetime of unpaired events whereas deletion of both Mfn1 and Mfn2 resulted in an instable dynamics. These results indicated that the paired events were dominant but unpaired events were not negligible, which provided a new insight into mitochondrial dynamics. In addition to kiss and run model of action, our data suggest that, from a global visualization over an entire cell, multiple patterns of action appeared in mitochondrial fusion and fission.     

Nuclear membrane fusion in electrofused mammalian cells

Fusion of nuclei was studied in electrofused cells using staining procedures and DNA flow cytometry. Homogeneous and heterogeneous electrofusion of Ehrlich ascites tumor cells. Muntjac cells and V79-S181 cells were performed in balanced-salt solutions at low temperature. Incubation of the cells subjected to electrofusion in fusion media for about 2 h was required to complete cell fusion and, in particular, nuclear membrane fusion. Under optimum electrofusion conditions it was found that fusion of nuclei is a very frequent event. Half of the fused cells (about 30 to 50% of the field-exposed cells) underwent nuclear membrane fusion. It is shown that the high frequency of nuclear membrane fusion in electrofused, unsynchronised cells resulted from intracellular dielectrophoresis occurring during cell alignment. In accordance with theory, maximum nuclear membrane fusion was observed using alignment fields of between 1 and 4 MHz (depending on the cell species), that is above the frequencies at which the plasmalemma capacity no longer shielded the cell interior from participation in the conduction process. In this frequency range a potential difference can be built up across the nuclear membrane leading to repositioning of the nuclei into the contact zone of the plasmalemmas of two attached cells. This intracellular dielectrophoresis apparently facilitated fusion of nuclei once intermingling of the plasma membranes had occurred. It was further demonstrated that exponentially growing cells showed higher cell fusion rates than cells taken from the unfed plateau phase. One, but not the only reason, might be the higher ATP content of exponentially growing cells compared to cells of the plateau phase. Addition of external ATP to plateau phase cells during electrofusion resulted, in accordance with this assumption, in an increase of fusion frequency, whereas ATP had apparently no effect on the fusion yield of exponentially growing cells. G1 cells obtained by mitotic selection after nocodazole-induced blockage in metaphase also showed higher cellular and nuclear membrane fusion yields than exponentially growing cells. Most importantly, it could be demonstrated both experimentally and theoretically that electrofusion of cells in a dielectrophoretically aligned chain is controlled by a simple law of probability resulting predominantly in fusion of two cells independent of the number of cells in the chain. The likelihood of fusion of various numbers of cells in a chain is given by the appropriate power of the probability of two-cell fusion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Use of gene knockouts in cultured cells to study apoptosis

The avian DT40 cell system represents a novel method to generate loss of function mutations in vertebrate cells. These chicken B lymphoma cells undergo homologous recombination at very high frequencies and can thus be used to "knock out" genes believed to function in apoptotic processes. The knockout cells can then be used to determine how the cell death process is modulated after induction of apoptosis and to order components in cell death pathways. The system can be further modified, using tetracycline-responsive promoters, to allow expression of wild-type cDNAs to rescue "knockout cells" if the gene of interest is essential. Alternatively, cDNA expression constructs containing mutations or deletions in the cDNA encoding the absent protein can be used to delineate functional domains. cDNA expression libraries or known proteins believed to function downstream of the target in a signal transduction pathway could also be transfected into the knockout cell line, and the resultant cells could be assayed for complementation and/or rescue of the apoptotic alteration/defect. Finally, the system has recently been adapted to allow disruption of human genes in DT40/human hybrid cell lines thereby potentially extending this system for use in studying human genes.     

Clastogenic effects of bleomycin, cyclophosphamide, and ethyl methanesulfonate on resting and proliferating human B- and T-lymphocytes

The effects of bleomycin (BM), cyclophosphamide (CP), and ethyl methanesulfonate (EMS) on the frequencies of chromosomal aberrations were tested in mitogen-stimulated highly purified human B- and T-lymphocytes. In unstimulated G0/G1 B- and T-lymphocytes the clastogen induction of chromosome fragments was investigated in prematurely condensed chromosomes (PCC) induced by cell fusion with xenogenic mitotic cells. BM, CP (with metabolic activation), and EMS induced a significant increase in chromosome aberrations in proliferating human B- and T-lymphocytes. There were no significant differences in the BM-induced aberration rates between the cell populations. CP and EMS induced more aberrations in T- than in B-lymphocytes. In the PCC tests, BM-exposed G0/G1 lymphocytes showed dose-dependent high yields of chromosome fragments. No significant differences between B- and T-lymphocytes were observed. CP and EMS induced no clear increase in fragments in either cell population.     

Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.     

Quantitative monitoring for secreted production of human interleukin-2 in stable insect Drosophila S2 cells using a green fluorescent protein fusion partner

Human interleukin-2 (hIL-2) production in Escherichia coli and insect cell/baculovirus expression systems can be inefficient. Here we investigated secreted production of hIL-2 fused with green fluorescent protein (GFP) as a versatile fusion partner in optimized stably transfected insect Drosophila melanogaster S2 cells. This nonlytic S2 insect cell expression system employs a plasmid vector and allows for secretion of functional human proteins. We report that, following stable transfection and induction, S2 cells secreted hIL-2 as a fusion protein (approximately 2.3 microg/mL yield), with a secretion efficiency of approximately 90%. Regression analysis indicated a single linear relationship existed between GFP fluorescence and hIL-2 mass in both whole cell and secreted medium samples, indicating that in vivo monitoring and quantification of target foreign protein expression and even secretion is possible using this system. The simple comparative measurement of GFP fluorescence also allowed monitoring of secretion efficiency during periods of high GFP/hIL-2 expression.     

SCEs are induced by replication of BrdU-substituted DNA templates, but not by incorporation of BrdU into nascent DNA

After 3 rounds of DNA replication in the presence of BrdU, third-division metaphase cells can be scored for the frequencies of SCEs that occurred during cycles 1 and 2, and also for the frequency of SCE during cycle 3. This procedure was used to resolve the issue of SCE induction by replication of BrdU-substituted DNA templates versus induction by BrdU incorporation into nascent DNA. It was observed that third-cycle SCE frequencies in CHO are dependent upon the amount of BrdU that was present during cycles 1 and 2 and are independent of the BrdU concentration during the third cycle. It is therefore BrdU serving as a template, rather than BrdU being incorporated, that initiates the SCE event. A model is proposed that produces reasonable fits to the observed data. It also predicts a true background or spontaneous SCE frequency of 3 per cell per cycle as previously reported by Heartlein et al. (Mutation Res., 107 (1983) (103-109). The predicted single twin ratio is higher than that reported by Wolff and Perry (Exp. Cell Res., 93 (1975) 23-30), and possible explanations for this discrepancy are discussed.