Genomic Analysis of effect of bufalin on HL60 Cells

Bufalin, a natural toxin isolated from the Chinese toad venom preparation Chan ’su, has been used in leukemia therapy in the mainland China by inducing human leukemia cells to differentiation and apoptosis. In the present study, oligonucleotide microarrays were used to assess profiles of target gene regulation at several points at a 48h period by bufalin at an appropriate concentration in the human promyelocytic leukemia cell line HL60. Data from the gene expression profiles indicated that bufalin altered the expression of 1206 genes which are distributed in multiple profiles of regulation. Results are consistent with bufalin regulating HL60 cells proliferation and differentiation as well as apoptosis. Keywords: time course For microarray experiment, the cells in late log phase of growth were seeded in the 100mm flask in fresh medium at a concentration of 1×106/ml and bufalin was added to 12.5 nM concentration with 10 µM stock solution in PBS. Control cultures were maintained in medium supplemented with vehicle (DMSO) at a 1:10,000 dilution. No growth and differentiation effects of DMSO were observed under these culture conditions in HL-60 cells. All cells were grown in the dark. RNA prepared from bufalin treated cells was used to synthesize fluorescent cDNA probe, followed by hybridization to the human oligonucleotide microarray. We have done double hybridization experiments with fluorescence switch with the same RNA preparation. The fidelity of microarray we used was evaluated with untreated HL60 cells by conducting the self-to-self gene expression profiling with dyeswap strategy. The experimental results indicated that only four genes were found to be altered in their expression level by ≥ 1.5 fold, and no gene was found to be altered in their expression level by ≥ 2 fold. Considering the very low false-positive ratio (4/21,329 for 1.5 fold or 0 for 2 fold), the ratio cut-off value for significant differential expression was chosen at 1.5 fold for 6h, and 2 fold for 12h, 24h, and 48h. The self-to-self gene expression profiling of untreated HL60 cells with dyeswap strategy also serves as the 0h time point experiment. For microarray analysis with dyeswap strategy experiment, if at least one intensity value of Cy5 or Cy3 on two slides was more than 800, then that gene was selected as an expressed gene. According to this standard, there were 4820, 6318, 6325 and 6710 genes expressed at 6h, 12h, 24h and 48h respectively and total 8050 genes expressed in control and bufalin treated HL60 cells. For identification of differentially expressed genes, if Cy5 and Cy3 intensity value on one slide were both under 800, the Cy5 or Cy3 intensity value on other slide must be more than 3000. There were 21, 272, 461, 659 genes were found to be altered in their expression level by ≥ 2 fold at 6h, 12h, 24h and 48h respectively. Since the cut off 1.5 fold for changed gene expression was often selected and so the changed genes were selected for 6h at such cut off of 1.5 fold. Then there were 102 genes for 6h and was used for analysis below. Then total 1206 genes expression were changed at least one time point.