Chapter 18 Induction of Mammalian Somatic Cell Hybridization by Polyethylene Glycol

Davidson, Richard L.; Gerald, Park S.

doi:10.1016/s0091-679x(08)60223-x

Cited by 100 publications

(51 citation statements)

References 11 publications

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“…PEG induces cell fusion (1) and is widely used in somatic cell hybridization studies. This study was initiated AND TRON to examine whether the toxic effects of PEG treatment, which are frequently encountered in cell fusion experiments and noticed by dye exclusion tests, may be utilized as alternative to other methods of cell permeabilization (9,22).…”

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confidence: 99%

Permeabilization of lymphocytes with polyethylene glycol 1000. Discrimination of permeabilized cells by flow cytometry

1982

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The toxicity of polyethylene glycol 1000 (PEG) used similarly as in cell hybridization experiments, has been studied by flow cytometry, measuring the light scattering and fluorescence distributions of PEG‐treated human lymphocytes stained with propidium iodide, fluorescein diacetate and acridine orange (AO). The sensitivity of these tests to detect permeabilized, or potentially dead cells, was equal. In addition, PEG proved to interfere with AO staining most likely through the inhibition of its binding to nucleic acids. The decrease of AO fluorescence in cells killed by PEG was unexpected since intercalation of propidium iodide was the same as in alcohol fixed cells. Permeabilization of cells by PEG appears to be an all‐or‐none phenomenon, accompanied by entrance of PEG into the cells. The findings are described in the context of a review of the currently used flow cytometric techniques to discriminate viable and lethally affected cells; also, the problems of interpretation are discussed.

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confidence: 99%

Permeabilization of lymphocytes with polyethylene glycol 1000. Discrimination of permeabilized cells by flow cytometry

1982

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“…Treatment in suspension can result in the formation of fused cells (unpublished data). Prolonged exposure to PEG can be toxic to cells (11). We observed a breakdown of DNA at 24 h and a loss of colony-forming ability.…”

Section: Discussionmentioning

confidence: 74%

“…This report describes the use of polyethylene glycol (PEG) to permeabilize Chinese hamster V-79 cells. PEG, like inactivated Sendai virus (12,16) and lysolecithin (10), can produce fusion of mammalian cells (11).…”

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Permeabilization of ultraviolet-irradiated Chinese hamster cells with polyethylene glycol and introduction of ultraviolet endonuclease from Micrococcus luteus.

Yarosh

Setlow

1981

Mol. Cell. Biol.

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Chinese hamster V-79 cells were made permeable by treatment with polyethylene glycol and then incubated with a Micrococcus luteus extract containing ultraviolet-specific endonuclease activity. This treatment introduced nicks in irradiated, but not in unirradiated, deoxyribonucleic acid. The nicks remained open for at least 3 h; there was no loss of endonuclease-sensitive sites, and no excision of dimers as measured by chromatography was detected. In addition, there was no increase in ultraviolet resistance in treated cells. This suggests that the absence of a significant amount of excision repair in rodent cells is due to the lack of both incision and excision capacity.

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“…The molecular mass and concentration of PEG was typical of that used in prior studies where injured brain tissue, and spinal cord in vivo and in situ, was clearly protected by polymer administration (Koob et al, 2005;Borgens and Shi, 2000;Laverty et al, 2004). The range of molecular mass used in these studies was based on the literature of PEG-mediated cell fusion and an evolution of practical concerns about administering the polymer in laboratory animal and canine clinical studies (~1000-3000·Da) Davidson et al, 1976;Laverty et al, 2004;Borgens, 2001).…”

Section: Discussionmentioning

confidence: 99%

Neuroprotection from secondary injury by polyethylene glycol requires its internalization

Liu-Snyder

Logan

Shi

et al. 2007

Journal of Experimental Biology

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Recently this laboratory, in collaboration with the Veterinary Clinical Sciences Department of Purdue University and the College of Veterinary Medicine at Texas A&M University, has shown that intravenous injections of an ~30% solution of polyethylene glycol (PEG; 3500·Da) in sterile saline can produce unexpected recovery of functions in naturally produced, severe, canine spinal cord injury. This occurred in neurologically complete, paraplegic dogs treated in a hospital setting (Laverty et al., 2004). Supportive laboratory animal data for initiating this preliminary clinical trial will be described below; however, the long record of safe usage of PEG in human medicine (Working et al., 1997) has pressed these experiments to the brink of human clinical trials in spinal cord injury (SCI) and traumatic brain injury (TBI) (Sofamor Danek/Medtronics Corporation).Though the behavioral responses to injected and topically applied PEG in various models of neurotrauma are consistently positive Here we provide further evidence that the ability of PEG to reduce or limit secondary injury and/or lipid peroxidation (LPO) of membranes requires entry of PEG into the cytosol, further suggesting a physical interaction with the membranes of organelles such as mitochondria as the initial event leading to neurorepair/neuroprotection.We have evaluated this relationship in vitro using acrolein, a potent endogenous toxin that is a product of LPO. Acrolein can pass through cell membranes with ease, inducing progressive LPO in 'bystander' cells, and the production of even more acrolein by inducing its own production. Immediate application of PEG (10·mmol·l -1 , 2000·Da) to poisoned neurons in vitro was unable to rescue them from necrosis and death. Furthermore, threedimensional confocal microscopy of fluorescently decorated PEG shows that it does not enter these cells for up to 2·h after application. By this time the mechanisms of necrosis are likely irreversible. Additionally, severe oxygen and or glucose deprivation of spinal cord white matter in vitro also initiates LPO. Addition of potent free radical scavengers such as ascorbic acid or superoxide dismutase (SOD) is able to interfere with this process, but PEG is not. Taken together, these data are consistent with the hypothesis that PEG is able to rescue mechanically damaged cells, based on a restructuring of the damaged plasmalemma. Furthermore, in compromised cells with an intact cell membrane, PEG must first gain access to the cytosol where this same capability may be useful in restoring the integrity of cellular organelles such as mitochondria, though the intracellular concentration of the polymer must be significant relative to the concentration of toxins produced by LPO in order to rescue the cell.

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Chapter 18 Induction of Mammalian Somatic Cell Hybridization by Polyethylene Glycol

Cited by 100 publications

References 11 publications

Permeabilization of lymphocytes with polyethylene glycol 1000. Discrimination of permeabilized cells by flow cytometry

Permeabilization of lymphocytes with polyethylene glycol 1000. Discrimination of permeabilized cells by flow cytometry

Permeabilization of ultraviolet-irradiated Chinese hamster cells with polyethylene glycol and introduction of ultraviolet endonuclease from Micrococcus luteus.

Neuroprotection from secondary injury by polyethylene glycol requires its internalization

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