Research On The Effect Of Arsenic Trioxide On CD34~+Cells Derived From Leukemia Cell Line K562

Objective:To explore the effect of arsenic trioxide on CD34+cells derived from leukemia cell line K562.Method:We analyzed the expression of CD34and CD38antigen of K562by flow cytometry isolated CD34-positive and-negative cell subsets from leukemia cell line K562by Magnetic-activated cell sorting method and compared the differences of CD34、 CD38antigen expression between the two cell groups and parents with And the DNA contents, drug resistant proteins (p-gp, ABCG2),methyl cellulose cloned culturing,the gene mRNA expression of BCR-ABL, ABCG2, MDR1, PML, BCL-2, P53and MDM2were measured. The drug sensitivity and the effects of AS2O3with different concentrations and time on proliferation of K562cells, sorted positive cells and K562/A02cells were analyzed by MTT assay. The cell apoptosis were detected with Annexin V-FITC/PI double stained and cell cycle was analyzed with PI single stained in sorted positive cells by flow cytometry. The levels of BCR-ABL, PML, BCL-2, P53, MDM2gene mRNA expression were measured by fluorescence quantitative PCR.Results:The expression of CD34was significantly higher in positive subsets (55.8±20.49%) sorted by Magnetic-activated cell sorting method labeled with CD34MicroBeads from leukemia cell line K562than the sorted negative subsets (0.20±0.17%) and K562cells (1.27±1.17%)(P<0.05).However there was no significant differences in the CD38expression in the three subsets (P>0.05). The scales were23.83±20.22%,1.53±0.72%,1.57±1.40%respectively. The expression of CD34in positive subset was decreased (41.05±0.05%) after cultured for a week, but the was no obvious change in the expression of CD38(1.82±0.11%)(P>0.05). Most of positive populations were in the quiescent period. The percentage of GO/G1phase cells was54.83±0.76%, significantly higher than that of K562cells (36.97±9.05%) and negative cells (39.20±7.80%)(P<0.05). But the ratio of GO/G1phase cells was decreased (45.13±0.78%) after cultured for a week.There was no significant differences in the three subsets (P>0.05). The expression of p-gp and BCRP were significantly higher in positive subsets (p-gp26.23±8.77%, BCRP15.0±1.80%) than K562cells (p-gp3.43±1.44%, BCRP1.97±0.97%) and negative subsets (p-gp3.02±2.94%, BCRP0.63±0.31%)(P<0.05). The positive subsets had stronger colony-forming ability than K562cells and negative subsets in vitro. The number of colony forming was79.50±18.96,13.67±10.97,7.83±5.53respectively (P<0.05). The levels of PML, BCL-2, P53, MDM2gene mRNA expression were significantly higher and BCR-ABL gene expression mRNA was lower in positive subsets than in K562cells and negative subsets (P<0.05). K562cells, sorted positive cells and K562/A02cells were treated with the concentration range of0to8umol/L of AS2O3respectively for24h,48h and72h.The cell proliferation was measured by MTT assay.The proliferation of the three subsets were obviously inhibited by AS2O3and the highest inhibition rate appeared after the cells were treated with8umol/L AS2O3for72h.The effect showed a dose-and time-dependent manner. The inhibitory of sorted positive cells was significant lower than K562and K562/A02cells (P<0.05). AS2O3could induce the apoptosis and necrosis in the three cell populations with the increasing of concentration and extending of action time. The rate of apoptosis and necrosis gradually increased (P<0.05).The effect was significant caused by8umol/L AS2O3for48h.However in the same condition, the apoptosis rate was higher in the positive subsets than in K562and K562/A02cells (P<0.05).The similar effect was seen in the other two subsets. And the maximal apoptosis rate was observed in cells treated with different concentration of AS2O3at24h and48h. AS2O3reduced the ratio of G0/G1phase cells in the positive subsets, and increase the proportion of G2/M phase cells mainly at short acting time (24h).But the percentage of S phase cells increased with action time was prolonging. The mainly effect was seen in24h and48h. The transcription level of BCR-ABL (P210) gene expression gradually increased in sorted positive cells companied with time and dose increasing of As203.The change was obviously in48h,8umol/L, but decreased in K562cells and sorted negative cells with the same condition of AS2O3, the role was similar in the other subsets, and the significant effect was in48h,4umol/L. The effect on K562cells obviously dose and time dependent. The most obvious effect was observed in treatment of4umol/L AS2O3for48h. In separating negative cells,the role of AS2O3presented a dose dependent manner, especially with2,4umol/L. Relative to the negative control, AS2O3could decreased the transcription level of PML, P53, BCL-2, MDM2gene expression both in sorted positive cells and K562cells, with certain concentration dependence.Conclusion:1. The leukemia cell line K562contains a small group of CD34+cells, with low expression of CD38antigen. Compared with K562cells and the sorted negative cells, cd34+K562cells have the characteristics of mostly in the stationary state, higher expression resistant protein p-gp and BCRP, stronger colony-forming ability in vitro, higher expression gene mRNA of MDR1, ABCG2, P53, BCL-2, PML, MDM2, lower expression of BCR-ABL. These suggest that the CD34+cells separated from K562cell line are rich in leukemia stem cells associated with drug resistance and relapse of leukemia.2. AS2O3can inhibit the positive cells proliferation, promote apoptosis and necrosis, exert the function of anti-tumor, possible through decreasing the level of P53, BCL-2, PML, MDM2mRNA expression, reducing the rate of GO/G1phase and partial blocking in S and G2/M phase.