| Arsenic is a natural substance that has been used medicinally for over 2,400 years. In 1970s physicians in China began using arsenic trioxide to treat acute promyelocytic leukemia (APL) and achieved promising results,including clinical remission of several cases. After that,the studies aimed at the functions and mechanisms of arsenic trioxide carried out extensively. It showed that arsenic trioxide could be used not only in the treatment of APL,but also in other malignant tumors,such as gastric cancer,lung cancer,carcinoma of prostate,ect. The mechanisms of such treatment have been proposed as inhibition of proliferation and angiogenesis,as well as induction of differentiation and apoptosis,as has been tested by various in vivo and in vitro experiments.In our experiments,it has also been demonstrated that after the treatment of arsenic trioxide,the K562 cells has undergone major morphological changes,which included nuclear shrinkage,membrane bleb and scattered apoptotic bodies. DNA gel electrophoresis also discovered that the typical "DNA ladder" phenomena in the treatment group,while the control group showed the regular genomic banding. All of these indicated that arsenic trioxide was a powerful chemotherapeutic agent and the cells in the treatment group were induced to apoptosis.To further understand the molecular mechanisms of arsenic trioxide treatment in the induced cellular apoptosis,we applied the Restriction Display-PCR (RD-PCR) technique combined with polyacrylamide gel electrophoresis and sliver staining techniques to separate the differentially expressed genes. We found 11 genes were differentially expressed in the treatment group,which the genes were sequenced and analyzed,showing that the isolated gene fragments were closely associated with the inhibition of cellular proliferation and the induction of apoptosis.Then,we applied the RD-PCR technique to isolated gene expression sequence tags (EST) from treated and controlled K562 cells. 852 ESTs wereisolated totally. Using these ESTs,a cDNA microarray was prepared,so as to study the gene expression profiles of the K562 cells,prior or after arsenic trioxide treatment. Total cellular RNA were extracted,40 micrograms of the total RNA were used in the reverse transcription reaction,using Superscript II reverse transcriptase,Oligo (dT) i8 primers,and Cy3-dCTP or Cy5-dCTP for the experimental and control group respectively. The labeled cDNAs were hybridized to microarrays at 42C for 12 h-18 h. The microarrays were scanned by using a dual-laser scanner (ScanArray Lite) and the data were statistically analyzed with the Quantarray software package. 25 genes were found to be significantly down-regulated,including genes encoding the transcription factors,metabolite enzymes,signal transduction protein,kinases,etc. Further analysis revealed 6 genes were involved in the apoptosis pathway. We also found in our study that IGFs may also play a role in the arsenic trioxide induced apoptosis.In summary,we used the cDNA microarrays to analyze the differentially expressed genes in the arsenic trioxide induced K562 cells. Our results further confirmed in gene expression level that growth arrest and apoptosis could be induced by arsenic trioxide. Such induced apoptosis may also involve the functional inhibition of IGF.Our results also demonstrated that microarray technology was a powerful tool in analyzing the gene expression profile of induced gene expression. Our in-house made cDNA microarray could be successfully applied to the analysis of high throughput and parallel analysis of hundreds of genes.
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