Preliminary Studying Of The Relationship Between Multidrug Resistances And Side Population Cells In Leukemia K562, K562/A02 Cell Line And The Study Of Genes

Objective:By comparing the percentage of SP cells in K562 parental cells with K562/A02 drug-resistant cells and analyzing DNA conten,resistance protein,in vitro clonal culturing and related gene expression in K562,K562/A02 cell line and sorting the SP,NSP subsets in K562 cell line to further study the biological characteristics of SP cells in K562,K562/A02 cell line and the relationship among SP cell,cancer stem cells and multidrug resistance.Methods:Drug sensitivity was measured by MTT assay of K562/A02 drug-resistant cell line and its parent cells to different drugs.With the characteristics of stem cells being capable of efflux fluorescent dye Hoechst33342,Fluorescence activated cell analysis(FACS) was employed for detecting the percentage of SP cells in K562 and K562/A02 cell line and analyzing the characteristics of DNA content,multidrug resistance proteins(P-gp,ABCG2),methyl cellulose clonal culturing and the gene mRNA expression of ABCG2,MDR1,SMO,BCR/ABL^P210 was measured by RT-PCR in K562,K562/A02 cell line and sorting the SP,NSP subpopulation in K562 cell line.Results:It was shown that the resistance indexes of K562/A02 to ADM,EPI,VP16 and vineristine(VCR) were 303.03±12.40,48.08±2.11,45.25±2.63 and 40.10±3.26 respectively,indicating its multi-drug resistant property.After Hoechst33342 staining,SP cells detected in K562 and K562/A02 cell line by flow cytometrye, which accounted for 2.7±0.5%,96.1±3.2%.multidrug resistance protein P-gp expression in K562/A02 cell were significantly higher than K562 cell(P＜0.05), Although there were no differences between these two cell lines in P-gp expression, most of the two cell lines in S/G2/M phase and in vitro clonal culturing.The sorting SP,NSP subsets in K562 cell line,ABCG2 expression in K562 SP subsets was significant higher than NSP subsets(P＜0.05),P-gp expression in K562 SP and NSP subpopulation did not have a significant deviation(P＞0.05).Most of K562 SP subpopulation were quiescent,the G0/G1 phase cells accounted for about 82.71±3.12%was higher than NSP subpopulation(P＜0.05).The colony-forming ability of K562 SP subpopulation was significantly stronger than K562 NSP.The mRNA expression of the MDR1 gene in the K562/A02 cell line was significant higer than that in the K562 cell line by RT-PCR.ABCG2,SMO,BCR/ABL^P210 gene expression in the two cell lines did not have a significant deviation(P＞0.05),comparing K562SP with K562NSP population,ABCG2,SMO gene in K562SP was significantly expressed,but BCR/ABL^P210 fusion gene expression was negative.Conclusions:The K562 cell line contains a small group of SP cells,According to the significant differences between the SP and the NSP subpopulation,MDR proteins,the majority in a quiescent period,stronger ability of colony formation in vitro,the positive SMO gene and negative Bcr/abl fusion gene,it is possible that leukemia stem cells are enriched in SP cells.The majority of side cluster cells in enriched K562/A02 cell line.P-gp was highly expressed,most of the cells were in S/G2/M phase, colony-forming ability in vitro is weak.We speculated that SP cells phenotypes in the K562/A02 not by stem cells determine the characteristics of dye efflux,it is more associated with acquired multidrug resistance,not rich in cancer stem cells.Multidrug resistance protein ABCG2 may play a decisive role in the SP phenotype of K562 cells, P-gp may play a major role in SP phenotype of K562/A02 cell line.