This experimental research was aimed to establish an optimum system of enucleation, purification and identification for preparing the cytoplasts of suspension culture cells in order to undertake cell recombination. Human leukemia HL-60 cells in suspension culture were purified by 42% Percoll density gradient centrifugation and low-speed centrifugation at 1 500r/min, respectively. The purified HL-60 cells were treated with cytochalasin B (CB) alone or combined with colcchicine and enucleated by isopycnic gradient centrifugation on 50% Percoll at 25 degrees C and 34 degrees C, respectively. Cytoplasts made from HL-60 cells were purified through gradient centrifugation by 37%, 38% and 40% Percoll, respectively. The final cytoplasts were identified by Wright-Giemsa staining and 4,6-diamidino-2-phenylidole dihydrochloride (DAPI)/5, 6-carboxyflu-orescein diacetate succinimidyl ester (CFSE) double-staining. The phenotype and mitochondrial membrane potential of HL-60 cytoplasts were analyzed by flow cytometry. The results indicated that the enucleation ratio of HL-60 cells induced by CB combined with colcchicine was up to 91. 98% +/-4. 29%, which was significantly higher than that in CB alone group (74. 95% +/- 3. 02%)(P<0. 01). The rates of enucleation and cytoplast with diameter over 5min in 34 degrees C group were higher than those in 25 degrees C group (all P<0. 01). The cytoplast purities were (95.43 +/- 0. 59)% in 38% Percoll groups,which were higher than those of 40% Percoll (P<0. 05). Nucleus and caryoplasm could be clearly distinguished by DAPI and CFSE double labeling. The results further showed that the phenotype of HL-60 cytoplasts had no significant change, and the activity of the cytoplasts was above 80% within 12h. It is concluded that enucleation throuth density gradient centrifugation on 50% Percoll mediated by CB combined with colcchicine, 38%Percoll of purification followed by DAPI/CFSE double labeling and MMP detection is an optimum scheme for preparation and identification of cytoplast from suspension culture cells.
Cell Compartmentation ; Cell Nucleus ; Cell Separation ; Centrifugation, Density Gradient ; Colchicine ; analogs & derivatives ; pharmacology ; Cytochalasin B ; pharmacology ; Cytoplasm ; HL-60 Cells ; Humans

<b>OBJECTIVEb>To screen differentially expressed genes in cytochalasin B (CB)-induced denucleated K562 cells by restriction display (RD) technique.

<b>METHODSb>The total RNA was isolated and purified from K562 cells before and after CB (10 mug/ml) treatment. The mRNA from both treated and untreated K562 cells were reversely transcribed into cDNA, and the differentially expressed genes were separated using RD technique combined with polyacrylamide gel electrophoresis and sliver staining, followed by cloning, sequencing and homology analysis against GenBank database of these genes.

<b>RESULTSb>Seven differentially expressed genes were identified in CB-treated cells including aquaporin 1 (AQP1) gene, which was verified to be up-regulated after CB treatment by RT-PCR.

<b>CONCLUSIONb>AQP1 gene might be in close association with the regulation of denucleation processes and CB-induced proliferation inhibition of K562 cells.

Aquaporin 1 ; biosynthesis ; genetics ; Cytochalasin B ; pharmacology ; Electrophoresis, Polyacrylamide Gel ; Gene Expression Profiling ; methods ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; K562 Cells ; Oligonucleotide Array Sequence Analysis ; Reverse Transcriptase Polymerase Chain Reaction

Pro-oxidant properties of ascorbate have been studied with uses of brain tissues and neuronal cells. Here we address potential mechanism of ascorbate coupling with glutamate to generate oxidative stress, and the role which oxidized ascorbate (dehydroascorbate) transport plays in oxidative neuronal injury. Ascorbate in neurones can be depleted by adding glutamate in culture medium since endogenous ascorbate can be exchanged with glutamate, which enhances ascorbate/ dehydroascorbate transport by depleting ascorbate in the neurons with the glutamate-heteroexchange. However, ascorbate is known readily being oxidized to dehydroascorbate in the medium. Glutamate enhanced the dehydroascorbate uptake by cells via a glucose transporter (GLUT) from extracellular region, and cytosolic dehydroascorbate enhanced lipid peroxide production and reduced glutathione (GSH) concentrations. Iso-ascorbate, the epimer of ascorbate was ineffective in generating the oxidative stress. These observations support the current concept that the high rates of dehydroascorbate transport via a GLUT after the release of ascorbate by glutamate leads to peroxidation, the role of glutamate on ascorbate/ dehydroascorbate recycling being critical to induce neuronal death via an oxidative stress in the brain injury.
Animals ; Ascorbic Acid/analogs & derivatives/pharmacology ; Biological Transport/drug effects ; Cerebral Cortex/*drug effects/*metabolism ; Cytochalasin B/pharmacology ; Dehydroascorbic Acid/*metabolism ; Glutamic Acid/*pharmacology ; Glutathione/metabolism ; In Vitro ; Lipid Peroxidation/*drug effects ; Male ; Oxidation-Reduction/drug effects ; Oxidative Stress/drug effects ; Rats ; Rats, Sprague-Dawley ; Thiobarbituric Acid Reactive Substances/metabolism