PEG-mediated Cell Fusion Research Articles

Cell fusion in tumor progression: the isolation of cell fusion products by physical methods.

BackgroundCell fusion induced by polyethylene glycol (PEG) is an efficient but poorly controlled procedure for obtaining somatic cell hybrids used in gene mapping, monoclonal antibody production, and tumour immunotherapy. Genetic selection techniques and fluorescent cell sorting are usually employed to isolate cell fusion products, but both procedures have several drawbacks.ResultsHere we describe a simple improvement in PEG-mediated cell fusion that was obtained by modifying the standard single-step procedure. We found that the use of two PEG undertreatments obtains a better yield of cell fusion products than the standard method, and most of these products are bi- or trinucleated polykaryocytes. Fusion rate was quantified using fluorescent cell staining microscopy. We used this improved cell fusion and cell isolation method to compare giant cells obtained in vitro and giant cells obtained in vivo from patients with Hodgkin's disease and erythroleukemia.ConclusionsIn the present study we show how to improve PEG-mediated cell fusion and that cell separation by velocity sedimentation offers a simple alternative for the efficient purification of cell fusion products and to investigate giant cell formation in tumor development.

Effects of chromosomal polyploidy on survival of colon cancer cells

Tetraploid cells are frequently observed in the inflamed mucosal epithelial cells of the patients with Barrett's esophagus or chronic ulcerative colitis. Polyploidy often occurs during cell fusion, abortive cell cycle, and endoreplication. Most tetraploid cells are engaged to apoptotic pathway, but some remaining stable tetraploid cells consequently cause aneuploidization and chromosomal instability. We investigated whether tetraploid cells could acquire survival advantage and hold a dominant position for natural selection. We established tetraploid cell line (HCT116GH) from parental diploid colorectal cancer cell line (HCT116) via PEG-mediated cell fusion and compared its cell viability, cell cycle response and apoptotic fractions responded to H₂O₂) with diploid HCT116 and p53 suppressed HCT116/H6 cell lines. Using MTT assay, plating efficiency and clonogenicity, we evaluated the survival of each cell line. Tetraploid cell line HCT116GH demonstrated an 83 fold greater resistance to 100 microM H₂O₂ than the parental diploid HCT116, and 6 fold greater than even the p53 negative diploid HCT116/E6. Cellular sensitivity, G2/M arrests, and apoptotic proportion were observed less in response to H₂O₂ in HCT116GH compared with HCT116 and HCT116/E6. HCT116GH expressed lower level of p53 and p21 than diploid HCT116. Stable tetraploid cell lines showed enhanced viability in comparison to parental diploid cell lines. The enhanced viability observed in tetraploidization surpassed that from downregulation of p53. Frequent appearance of tetraploid cells in stressful condition can be caused by natural selection owing to their enhanced viability and may consequently contribute to cancer cell transformation.

ESCs Require PRC2 to Direct the Successful Reprogramming of Differentiated Cells toward Pluripotency

Embryonic stem cells (ESCs) are pluripotent, self-renewing, and have the ability to reprogram differentiated cell types to pluripotency upon cellular fusion. Polycomb-group (PcG) proteins are important for restraining the inappropriate expression of lineage-specifying factors in ESCs. To investigate whether PcG proteins are required for establishing, rather than maintaining, the pluripotent state, we compared the ability of wild-type, PRC1-, and PRC2-depleted ESCs to reprogram human lymphocytes. We show that ESCs lacking either PRC1 or PRC2 are unable to successfully reprogram B cells toward pluripotency. This defect is a direct consequence of the lack of PcG activity because it could be efficiently rescued by reconstituting PRC2 activity in PRC2-deficient ESCs. Surprisingly, the failure of PRC2-deficient ESCs to reprogram somatic cells is functionally dominant, demonstrating a critical requirement for PcG proteins in the chromatin-remodeling events required for the direct conversion of differentiated cells toward pluripotency.

Expression of the Human Dp 71 (Apo-Dystrophin-1 ) Gene from a 760-kb DMD-YAC Transferred to Mouse Cells

A 760-kb YAC was constructed by homologous recombination in yeast, containing the genes located in the distal portion of the DMD gene. The YAC was introduced in mouse LA-9 cells by PEG-mediated cell fusion. One transformant accommodated an intact DMD-YAC, i.e. a full copy of the DMD internal Dp 116, Dp 71 and Dp 40 genes (apo-dystrophin-2, -1 and -3, respectively). We have studied the expression of the various gene products derived from the introduced DMD-YAC. RT-PCR revealed expression of human Dp 71 but not of Dp 116 or Dp 40. Remarkably, differences were observed in processing of the 3' region of the endogenous mouse and the human transcripts, due to different splicing of exons 71 (absent in human and present in mouse transcript) and 78 (present in human and absent in mouse transcript). The splicing pattern of the human transcript is the same as that of the major Dp 71 (apo-dystrophin-1) product in human blood. The observed splicing differences may be caused by either species-specific exon use and/or by cis-acting factors, e.g. the upstream transcript composition, because we have no evidence for endogenous Dp 71 expression.

The use of insulin-enriched culture medium and human-human hybridoma formation: a preliminary study.

Human lymphoblastoid cell line WI-L2-729-HFZ was fused with human lymph-node lymphocytes in one fusion and with human spleen cells in another fusion to generate human-human hybridomas. In both, increasing doses of insulin were added to the HAT medium immediately after the PEG-mediated cell fusion (10(-1)-10(-5) IU/ml) and the number of clones formed was determined 3 weeks later. 10(-3) IU/ml of insulin resulted in a 2- to 5-fold increase in the number of clones generated compared to the control plates. In view of the known difficulties in generating high-yield human-human hybridoma fusions, it is suggested that the use of insulin-supplemented HAT medium may provide a more efficient way in obtaining such clones.

Enhancement of polyethylene glycol-mediated cell hybridization by inducers of erythroleukemia cell differentiation

Dimethyl sulfoxide (DMSO) has many biological effects, which include enhancement of polyethylene glycol (PEG) -mediated cell fusion, induction of cell differentiation in erythroleukemia and other cell systems, and cryoprotection of cells from freezing damage. In this study, compounds which induce erythroleukemia cell differentiation were tested for their ability to enhance PEG-mediated cell fusion. It was found that many compounds which induce erythroleukemia cell differentiation also promote cell membrane fusion as well as protect cells against freezing damage. Hence, many inducers of erythroleukemia cell differentiation have direct and similar effects on cell membranes. This study also demonstrates previously unrecognized effects of cryoprotective agents and cell fusogens on the differentiated state of cultured cells.

Isolation and characterization of CHO cell mutants with altered asparagine synthetase.

Two asparagine auxotrophic mutants (N3, N4) were isolated from the Gat- strain of Chinese hamster ovary cells, using a selection procedure modified from that of Goldfarb et al. (1). The defect in these mutants is due to a deficiency in asparagine synthetase activity. N3, in particular, had no measurable enzyme activity. Complementation analysis by PEG-mediated cell fusion showed that the auxotrophic phenotype behaved as a recessive trait; complementation was obtained between N3 or N4 and the pseudoauxotroph, Asn3, which has a temperature-sensitive asparagyl-tRNA synthetase activity. Revertants obtained by plating N3 or N4 in asparagine-free medium had about normal levels of asparagine synthetase activity and were produced with a probability of about 10(-6) per cell per generation. Three particular revertants of N3 and one revertant of N4 were shown to have asparagine synthetase activities that were different in thermolability from that of the wild type. This observation is consistent with the suggestion that N3 and N4 have defective structural genes rather than defective regulatory genes for asparagine synthetase.