| K562 cell is a highly malignant human erythroleukemia cell line that can be induced in vitro to differentiate by a number of chemical agents. The induced erythroid differentiations are characterized by decreases of cell proliferation, increases of hemoglobin expression, and the tendency of karyopyknosis and spontaneous denucleation.As spontaneous denucleation is one of the important markers of erythroid differentiation of K562 cell, We design an experimental protocol in which the K562 cells were induced to enucleate, in a hope to find the gene expression profile changes that underlies cellular denucleation. Cytochalasin B (CB) is a chemical agent capable of destroying the assembly of micro filament specifically. The assembly of microfilament was also inhibited by CB. The combined effects weaken the connections between nucleus and cytoplasm, which facilitate the nucleus to be enucleated.In our experiments, it was observed the denucleation efficiency of K562 cell is positively correlated with CB concentration and the duration of CB treatment. CB at low dose can inhibit the cell proliferation obviously and at high dose can cause cell death.To further understand the molecular mechanisms of CB in denucleation of K562 cell, we applied the Restriction Display (RD) technique combined with polyacrylamide gel electrophoresis and sliver staining, to separate the differentially expressed genes. We found 7 genes were differentially expressed in the treated group, which were sequenced and analyzed. Aquaporin 1 gene was confirmed to be associated closely with the regulation of K562 cell erythroid differentiation.We also applied RD technique to construct the restriction cDNA library, from which, cDNA fragments were collected for the microarray preparation. 277 human genes of RD products were spotted on a glass slide in our laboratory by using Cartesian Microarrayer. Total RNA was isolated from K562 cells before and after CB treatment and mRNA were purified. BothmRNA from the treated K562 cells and the untreated control K562 cells were reverse transcribed into cDNA and labeled with two different fluorescence dyes: Cy5 or Cy3, using a labeling method of restriction display technique. The labeled cDNA were hybridized to the K562 microarray at 42℃ for 12-18h. The gene expression microarray were scanned by using a dual-laser scanner (ScanArray Lite) and the data were statistically analyzed with the Quantarray software package. Among the 277 target genes, 18 down-regulated genes (Cy3/Cy5>2.0) were identified after CB treatment. Most down-regulated genes were correlated with cellular proliferation, signal transduction and transcription factors. Cancer metastasis related gene (Laminin receptor, LR) and cell signal transduction gene (G protein) has a significant down-expression (Cy3/Cy5>3.0). The results were further verified by using RT-PCR.In summary, we used the advantages of the high-throughput, efficiency and sensitivity of cDNA microarray to analyze the differentially expressed genes after CB treatment. This study could provide theoretical basis for further elucidation of the molecular mechanism of tumor cells differentiation and can provide reference to the selection of clinical anti-tumor drugs as well as to the therapeutic approaches in genetic level against erythroleukemia.
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