Human Leukemia Cell Line Kg-1a, Tumor Stem Cell-like Subpopulations Identification And Enrichment

PART ONE IDENTIFICATION OF CANCER STEM-LIKE SUBPOPULATION CELLS IN HUMAN LEUKEMIA CELL LINES KG-1AObjective: To identify cancer stem-like subpopulation cells in human leukemia cell lines KG-1a.Methods: We observed the morphology and RNA content of KG-1a cells respectively using Wright's staining and acridine orange staining, then detected the cell cycle distribution and the CD34~+CD38~- subpopulation cells of KG-1a cells with flow cytometry, and also determined the expression of CD34 antigen with immunohistochemistry assay and flow cytometry, at last detected the side population-like cells in KG-la cells using fluorescent microscope after Hoechst33342 staining.Results: It was shown that KG-1a cells were primitive and nucleoli were apparent; RNA content in small subpopulation cells was low, and the G0/G1 cells were 15.5% about KG-1a cells. CD34 were generally expressed in cytoplasm and on membrane of KG-1a cells, the percentage of CD34+ cells was 96.3%, and the proportion of CD34~+CD38~- cells was 7.02%; the percentage of side population-like cells in KG-1a cells was 7.60%.Conclusion: Our study suggested that cancer stem-like subpopulation cells existed in human leukemia cell lines KG-1a.PART TWO ENRICHMENT OF CANCER STEM-LIKE SUBPOPULATION CELLS WITH 5-FU IN HUMAN LEUKEMIA CELL LINES KG-1AObjective: To enrich cancer stem cell-like subpopulation in human leukemia cell lines KG-1a with 5-Fluorouracil(5-FU).Methods: Optimal concentration and effect time of 5-FU were determined with in vitro drug sensitivity assay. The CD34~+CD38~- subpopulation in the enriched subpopulation of KG-1a cells was detected with flow cytometry. The RNA content of enriched subpopulation cells was measured by using acridine orange staining, and their expression of ABCG2 mRNA by RT-PCR. The side population-like cells in enriched subpopulation cells were detected using fluorescent microscope after Hoechst33342 staining. The colony-forming ability of this subpopulation cells was evaluated by semi-solid culturing. Results: It was indicated through our research that after treatment of KG-1a cells with 50μg/ml 5-FU for 4 days, the proportion of CD34~+CD38~- cells in enriched subpopulation was elevated by more than 10 folds; The RNA content of enriched subpopulation cells was low, but the ABCG2 mRNA was highly expressed. The percentage of side population-like cells in enriched subpopulation cells was 84.20%. The colony forming ability of enriched subpopulation cells was lower than of non-enriched KG-1a cells.Conclusion: Our study suggested that utilizing 5-FU for killing most proliferating cells, we successfully established the method in which selecting and enriching cancer stem-like subpopulation cells in human leukemia cell lines KG-1a with 5-FU.