| Objective: In the present study, apoptosis arrest of Ph(+) cells play a very important role in the pathogenesis of chronic myeloid leukemia. In the process of drugs investigation, how to induce apoptosis of Ph+ cells is the major target. In the study, apoptosis phases, the earlier stages of apoptosis and the later stages of apoptosis, were elucidated with the using of the apoptotic model of K562 cells treated with harringtonine. Furthermore, apoptosis-associated proteins were screened and identified with the technique of proteome and the possible apoptosis-inducing mechanisms were discussed.Methods: Flow cytometry was used to distinguish K562 cells that were in the earlier stages of apoptosis from those that were in the later stages of apoptosis by annexinVand PI staining. Moreover, fluorescence staining, DNA electrophoresis and fluorescence activated cell sorting(FACS) were used to characterize the process of apoptosis of K-562 cells treated with HT and the phase of cell cycle arrested. The technique of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) coupled with computer image analysis was used to explore the changes in protein expression during the different stages of apoptosis. In combination with database searching, proteins were identified by peptide mass fingerprint using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).Results:1 When treated the K-562 cells with 10ug/ml harringtonine for 5,12,24 hours,the percentage of the early apoptotic cells were 28.3%, 20.1%, 18.1% respectively. In contrast, the percentage of the late apoptotic cells were 9.1%,15.3%, 20.2% respectively. The similar tendency was observed by the PI staining solution, fluorescence staining and DNA electrophoresis. With a prolonged time period, flow cytometric analysis demonstrated that the HT-induced K-562 cells were arrested in Go/Gi phase.The results suggest us that K-562 cells treated with HT for 5 hours are in the early stage of apoptosis. In contrast, HT-induced K562 cells for 24 hours have already undergone an apoptosis death.2 The 2-D PAGE was used to detected proteins from control cells, cells induced by HT for 5 and 24 hours respectively. Proteins resolved on the wet gels were visualized using ImageScanner densitometer and the digitized images subjected to rigorous analysis using the Imagemaster 2D Elite v3.01 software system. There were evident relativity and comparability among proteins maps of the three groups. Further, Statistics analysis showed 3 spots disappeared, 7 spots with evidently decreasing intensity, 10 spots with strongly increased intensity in the apoptotic cells induced by HT for 24 hours.3 Ten spots were selected on the basis of the intensity and the significant difference in abundance of changes. The MALDI-TOF was used for the direct identification of these proteins. In combination with database searching, eight proteins were successfully identified by MALDI-TOF and two proteins can't be identified definitely. Five proteins involved in the apoptosis were keratin 9, basic transcription factor 3(BTF3), tryptophanyl-tRNAsynthetase, DJ-1 and prohibitin.Conclusions: The up-regulation of tryptophanyl-tRNA synthetase, DJ-1, prohibitin and the down-regulation of BTF3 were connected with inhibition of transcription and protein synthesis in the apoptotic K562 cells induced by HT, whereas the up-regulation of keratin9 was related with resistance-apoptosis. It is contribute to understand the mechanisms of apoptosis of K562 cells treated with HT that studying further the functions of these apoptosis-associated proteins. |
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