Dimethyl sulfoxide enhances polyethylene glycol-mediated somatic cell fusion

The efficiency of fusion of human diploid cells by polyethylene glycol was greatly enhanced by addition of dimethyl sulfoxide. The extent of fusion was directly proportional to the concentrations of both of these compounds. At all except the highest concentrations, cell loss was moderate to minimal and perturbation of cell cycle function as measured by [^3H]thymidine labeling indices and mitotic indices was minimal in the surviving cells. This technique is potentially useful for heterokaryon studies as well as for the isolation of hybrids of mammalian somatic cells.     

Rapid kinetics of polyethylene glycol-mediated fusion

Experiments are described in which the kinetics of fusion and subsequent cytoplasmic mixing are examined in human diploid fibroblastlike cells following exposure to polyethylene glycol-dimethyl sulfoxide. Analyses were performed on autoradiographic preparations in which one parental cell strain was prelabeled with tritiated amino acids. The results indicate that these events occur rapidly after exposure to the fusogen.     

High-efficiency polyethylene glycol-mediated transformation of mammalian cells

A new, high-efficiency method for transformation of mammalian cells with nucleic acids is described which yields 10⁵–10⁶ plaques/g poliovirus infectious RNA (iRNA). The optimized procedure consists of two steps: (1) exposure of cells to iRNA in a high ionic-strength buffer followed by (2) a brief exposure to a 35% polyethylene glycol (PEG) solution. Optimized conditions for each variable in the procedure are described. Under optimized conditions for PEG-mediated transformation with RNA, large numbers of transformants are recovered with plasmid DNA as well. The procedure presented is similar to other high-efficiency PEG-mediated methods previously described for the genetic transformation of both nonprotoplasted Escherichia coliand yeast.     

Increased polyethylene glycol-mediated fusion competence in mitotic cells of a mouse lymphoid cell line

Spontaneous mitotic cells of the mouse leukemic cell line GF7 are preferentially included in cell fusion products after treatment with polyethylene glycol (PEG). This implies a unique configuration of the natural mitotic membrane which is particularly vulnerable to induction of fusion by PEG. Colcemidarrested GF7 mitotic cells, however, are excluded from PEG-induced cell fusion products, suggesting that colcemid reverses the membrane configuration which is susceptible to the action of PEG. When Sendai virus is used as the fusogenic agent, bothcolcemidarrested and spontaneous mitotic cells are selectively fused. There must, therefore, be an essential membranefusogen reaction which is characteristically different for each of these agents.     

Improved techniques for the induction of mammalian cell hybridization by polyethylene glycol

Modifications in the techniques for the induction of mammalian somatic cell hybridization by polyethylene glycol (PEG) have led to procedures that are rapid, simple, and effective. The basic improvements, for both monolayer and suspension fusions, are a short exposure to PEG and a rapid dilution of PEG following treatment. There is a marked effect of PEG concentration on cell hybridization, and there seem to be inherent differences between cells in terms of the extent of cell fusion induced by PEG.     

Interferon production by human lymphoblastoid cells is stimulated by inducers of Friend cell differentiation.

A variety of structurally unrelated substances stimulated production of human lymphoblastoid interferon in Namalwa cells. Active substances include short-chain fatty acids, dimethyl sulfoxide, and hexamethylene bisacetamide. These substances are established inducers of erythropoietic differentiation in mouse erythroleukemic spleen cells (Friend cells). When Namalwa cells were cultured for 24 hr or more in the presence of 1–2.5 mM n-butyric acid and then induced with Sendai virus, a 30-fold increase in interferon yields over untreated Namalwa cells was observed. Propionic acid and n-valeric acid at a concentration of 5 mM caused a similar increase in interferon production and caproic acid at 5 mM stimulated it 8-fold. Substituted or unsaturated fatty acids were inert. Dimethyl sulfoxide increased interferon production at 280 mM and hexamethylene bisacetamide at 10 mM. All substances which enhanced interferon production blocked thymidine incorporation into Namalwa cell DNA at concentrations equal to those effective in interferon stimulation.     

AEV-transformed erythroleukemia cell induced differentiation: Expression of specific cell membrane antigenic molecules

Summary A simultaneous decay of the expression of Im 140 kDa, Im 150 kDa and Im 160 kDa high MW membrane antigens, concomitant with the cell proliferation arrest, was observed during erythropoietin induced differentiation ofts 34 AEV-transformed erythroid cells cultivated at the restrictive temperature. Expression of embryo-immature antigens was maintained during induced differentiation of erythroleukemia cells, but their MW shifted from 50 to 48 kDa, which corresponds to the MW of embryo-immature antigens detected on normal erythroid cells. In the absence of erythropoietin at the restrictive temperature, conditions under which thets 34 AEV-transformed erythroid cells fail to differentiate and maintain their capacity to proliferate, the expression of high MW antigens as well as the expression of embryoimmature antigens remained unaffected. Therefore, it is shown that the expression of specific membrane antigens is modulated under conditions rendering the erythroleukemia cell differentiation process possible.With 3 Figures     

Parameters of polyethylene glycol-induced cell fusion and hybridization in lymphoid cell lines

Methods employing polyethylene glycol (PEG) as a cell-fusion agent for mouse and human lymphoid cell lines were devised. Variables in these procedures were systematically explored. Under optimal conditions, PEG will induce 56% of the cells in the mouse cultures and 18% of the cells in the human cultures tested to undergo fusions resulting in multinucleated products. Intraspecific hybrids between mouse leukemic cells occur at a frequency of 26 × 10^–5, and interspecific hybrids between mouse leukemic cells and Chinese hamster fibroblasts occur at 4 × 10^–5. Differences in the requirements for maximum Sendai virus activity and PEG fusogenic activity are discussed.     

Enhancement of mast cell differentiation in vitro by T cell factor(s)

Mast cell colonies were obtained when lymph node cells of horse serum-immunized Balb/c mice were cultured in a horse serum-containing medium on embryonic fibroblast monolayer. In order to characterized precursors of mast cells, mesenteric lymph node cells from the immunized mice were fractionated to obtain nonadherent cells, a B cell-depleted fraction and a T cell-depleted fraction; and each fraction was cultured on fibroblast monolayer. Mast cell colonies developed from nonadherent cells and from the B cell-depleted fraction but not from the T cell-depleted fraction. However, cultures of the same T cell-depleted fraction developed mast cell colonies if cell-free supernatant obtained from culture of horse serum-primed T cells was added. Soluble factors promoting mast cell growth were not obtained when the same T cells were incubated in horse serum-free medium. It appears that the majority of mast cell precursors in the lymph nodes are nonadherent cells and bear neither immunoglobulin nor Thy 1 antigen. The results also suggested that soluble factor(s) released from antigen-stimulated T cells enhanced the differentiation of the precursors to mature mast cells.     

Cell differentiation rates of Friend murine erythroleukemia variants isolated by sib selection

Variants of Friend erythroleukemia cells were isolated which produced a high frequency (98%) or low frequency (2%) of hemoglobinized cells after induction with dimethylsulfoxide. Repeated subcloning and sib selection allowed enrichment of different cell lines without the use of mutagens or drug selection. The cell lines do not differ in growth rates, plating efficiencies, or chromosome numbers. The differences in inducibility phenotype were stable for more than 260 cell generations. In addition to differences in induction by dimethylsulfoxide and butyric acid, the cell lines also differ in spontaneous rates of cell differentiation. These results suggest that differences in differentiation rates are an inherited property of cells which is amplified in the presence of nonphysiological inducing agents.     

Heterogeneity of the response to inducers of differentiation and to cytostatics of tumor cell populations

The purpose of the experiments was to establish whether individual cells of a tumor cell population, or clonal lines derived from its express the differentiated phenotype, or respond heterogeneously following treatment with inducers of differentiation or with cytostatic drugs. The human cell lines used in this study were: HL-60 promyelocytic leukemia, K562 erythroleukemia, BHM-97 and A2058 melanoma, and A-1, A-2, A-4 and A-6 clones of A2058 line. Inducers of differentiation were phorbol myristate acetate (PMA), dimethylsulfoxide (DMSO) and retinoic acid (RA); cytostatics: adriamycin (ADM), 5-fluorouracil (5-FU), dacarbazine (DTIC), cis-platin (platidiam, PD) and arabinosyl cytosine (ara-C). Expression of the differentiated phenotype was shown by cell attachment (HL-60), hemoglobin production (K562), dendrit formation (A2058, BHM-97). Individual cells expressed the differentiated phenotype heterogeneously in all types of cell populations. Clone A-4 was the most, and clone A-6 the least sensitive to PMA. The drug sensitivity of the clones was different and drug-dependent. It is concluded that induction of differentiation as another approach to therapy of cancer, similar to anticancer drug therapy, also implies disadvantages due to population heterogeneity. Combinations of cytostatics with differentiation inducers might result in improved therapeutic effects.     

Sex chromosome differentiation revealed by genomic in-situ hybridization

In this work, genomic in-situ hybridization (GISH) was used to study the sex chromosome molecular differentiation on chromosomes of male and female individuals of the isopod crustacean Asellus aquaticus. As a composite hybridization probe, we contemporaneously used male and female whole genomic DNA differently labelled in the presence of an excess of unlabelled DNA of the female homogametic sex. The karyotype of A. aquaticus normally displays eight homomorphic chromosome pairs, but a heteromorphic sex chromosome pair is present in about a quarter of the males of a natural population previously identified by us. GISH did not reveal any sex chromosome molecular differentiation on the male and female homomorphic sex chromosome pair, and the karyotypes of these individuals were equally labelled by the male- and female-derived probe, while the heteromorphic Y chromosome showed a differentially labelled region only with the male-derived probe. This region evidently contains male-specific sequences but, because no similar hybridized region is observed on the male homomorphic chromosome pair, they are probably not important for sex determination but represent a molecular differentiation acquired from the Y chromosome. This revised version was published online in July 2006 with corrections to the Cover Date.     

SLC30A3促进人髓性白血病细胞K562向早期红系分化

 目的 研究SLC30A3在红系分化中的作用。 方法 在人髓性白血病K562细胞系中用质粒载体过表达SLC30A3后,检测细胞红系分化指标CD235、ε-、γ-和β-珠蛋白的表达量及细胞增殖速度。流式细胞术检测CD235的表达,荧光实时定量PCR检测珠蛋白mRNA水平,Western blot法检测转录因子GATA-2蛋白水平,MTS法检测细胞增殖速度。结果 过表达SLC30A3使K562细胞的红系分化特异标志CD235的表达升高,CD235阳性细胞率由对照组的34.25%±16.89%增加至95.7%±0.14%,ε-、γ-和β-珠蛋白的表达明显升高,红系分化相关转录因子GATA-2的表达降低,细胞增殖速度加快。结论 SLC30A3促进人髓性白血病K562细胞系向早期红系分化。     

Polyethylene glycol-induced mammalian cell hybridization: Effect of polyethylene glycol molecular weight and concentration

The effects of polyethylene glycol (PEG) molecular weight and concentration on mammalian cell hybridization were studied. The peak hybridization-inducing activity with all grades of PEG from 400–6000 was found to occur in the concentration range of 50–55%. However, changes in concentration were seen to have different quantitative effects with different grades of PEG. For monolayer fusions, PEG 1000 at 50% seems to be the optimal combination of PEG molecular weight and concentration, in terms of both efficiency of hybridization and relative insensitivity to dilution effects.     

A simple method for decreasing the toxicity of polyethylene glycol in mammalian cell hybridization

The yield of hybrid colonies after fusion of mammalian cells with polyethylene glycol (PEG) is increased if the cells are fused in Ca²⁺-free medium, and kept in Ca²⁺-free medium for at least 15 min after fusion. The protective effect of Ca²⁺-free medium is much more obvious when Baker PEG is used than when fusion is carried out with Koch- Light PEG. The increased yield of hybrid colonies is shown to be due to a reduced toxicity rather than to an increased efficiency of cell fusion. These improvements have been found to apply to a variety of cell lines, and also when cell fusion is carried out in suspension. This technique should be particularly useful in studies on mammalian cell hybridization using cell lines that are particularly sensitive to the toxic effect of PEG.     

Lipid changes associated with erythroid differentiation of Friend erythroleukemia cells

Friend erythroleukemia cells were induced to differentiate by dimethyl sulfoxide (DMSO) and hexamethylene-bis-acetamide (HBMA) in order to investigate whether their lipid characteristics, common to other systems of transformed cells, revert to a normal differentiation pattern. DBA/2 mouse erythrocytes were examined as a model of terminal differentiation in erythroid lineage. Variants of erythroleukemia cells not inducible to erythroid differentiation by DMSO and HMBA were also used in this study, in order to test whether lipid modifications occurring in differentiated erythroleukemia cells were related to the differentiation process or caused by specific effects of the inducers. Friend erythroleukemia cells showed the same lipid characteristics as those found in other transformed cell types. That is, a high level of ether-linked lipids and low percentages of long chain polyunsaturated fatty acids along with an accumulation of monoenoic fatty acids in phospholipids. These lipid characteristics remained unchanged when erythroleukemia cells were induced to differentiation by either DMSO or HMBA. However, other lipid components of erythroleukemia cells, e.g., phosphatidylethanolamine and triglycerides, were affected by erythroid differentiation. There were also changes of some lipid components of erythroleukemia cells, such as cholesteryl esters, which were related to specific effects of the inducers. Both DMSO- and HMBA-resistant variants differed from the inducible erythroleukemia cells, mainly in their ether-linked phospholipid pattern.     

人参皂甙Rg3在体外对白血病K562细胞增殖和诱导分化的影响**

背景：文献报道人参皂甙Rg3具有抗肿瘤作用,但对白血病作用研究很少。 目的：观察人参皂甙Rg3对白血病K562细胞的作用,并探讨其相关机制。 方法：以白血病K562细胞为靶细胞,实验分为对照组和人参皂甙Rg3组,人参皂甙Rg3组分别添加10,20,40,80,       100 mg/L Rg3。 结果与结论：MTT检测结果显示,不同浓度人参皂甙Rg3组K562细胞生长抑制率均显著高于对照组(P < 0.05~0.01)。NBT结果显示,人参皂甙Rg3组培养1,2,3 d K562细胞还原率均高于对照组(P < 0.01);人参皂甙Rg3组部分K562细胞体积变小,核仁消失,同时阳性细胞增多,细胞向成熟分化;流式细胞仪检测显示人参皂甙Rg3组培养2 d细胞周期G2期增高了3.84倍。提示人参皂甙Rg3在体外可抑制K562细胞增殖活性,其机制可能与人参皂甙Rg3将K562细胞周期阻滞在G2期,使细胞不能进行正常的有丝分裂,从而抑制细胞增殖有关。