Fluorouracil selectively enriches stem-like leukemic cells in a leukemic cell line

Abstract

Recent studies have reported that cancer stem cells (CSCs) could be isolated from solid cancer cell lines, in which the purity of CSCs was higher than that from tumor tissues. Separation of CSCs from leukemic cell lines was rarely reported. In this study, CD34(+)CD38(-)stem-like cell subsets in human KG-1a leukemic cell line were enriched by cytotoxic agent 5-fluorouracil (5-FU). After 4 days incubation of KG-1a cell line with 5-FU (50 microg/ml), the CD34(+)CD38(-) subpopulation of cell lines was enriched more than 10 times. The enriched cells had proliferate potential in vitro, low level of RNA transcription and Hoechst 33342 dye efflux ability, accompanied by high expression of ATP-binding cassette transporter protein ABCG2. Our findings suggest that treatment with 5-FU offers an easy method to isolate leukemic stem-like subpopulation. It can facilitate studies of leukemic stem cell biology and the development of new therapeutic strategies.

Keywords: 5-fluorouracil; KG-1a; cell line; leukemia; stem cell.

Figure 1

Dose-and time-dependent curves of 5-FU incubation with KG-1a cells. a, KG-1a cells were incubated with different concentrations of 5-FU for 24 h, and the killing effect of 5-FU was in a dose-dependent manner, with 50 μg / ml as the optimal concentration. b, KG-1a cells were incubated with 50 μg / ml 5-FU for different durations, and the killing effect of 5-FU was in a time-dependent manner, with 4 d as the optimal duration.

Figure 2

Detection of CD34⁺CD38^- subpopulation in KG-1a cells. It shows the flow cytometric analysis of CD34 and CD38 expression. FITC denotes fluorescein isothiocyanate, and PE phycoerythrin. a, KG-1a cells incubated at 50 μg/mL of 5-FU for 4 d. The enriched cells had a high percentage of CD34^-CD38^- subpopulation. b, KG-1a cells.

Figure 3

Colony formation assay in vitro semi-solid culture (Inverted microscope×400). a, (KG-1a +5-FU) group: KG-1a cells incubated at 50 μg/mL of 5-FU for 4 d. The enriched cells cultured in semisolid medium on 14 d formed clones and the number of colony forming cells was more than 50. b, KG-1a group: Majority of cells in this group only formed clusters. c, comparison on the colony-forming rate between two groups,*, p < 0.05. Error bars correspond to mean ± SD.

Figure 3

Colony formation assay in vitro semi-solid culture (Inverted microscope×400). a, (KG-1a +5-FU) group: KG-1a cells incubated at 50 μg/mL of 5-FU for 4 d. The enriched cells cultured in semisolid medium on 14 d formed clones and the number of colony forming cells was more than 50. b, KG-1a group: Majority of cells in this group only formed clusters. c, comparison on the colony-forming rate between two groups,*, p < 0.05. Error bars correspond to mean ± SD.

Figure 4

Acridine orange staining of nucleic acid in KG-1a cells (Fluorescence microscope ×200). a, (KG-1a +5-FU) group: KG-1a cells incubated at 50 μg/mL of 5-FU for 4 d. The enriched cells had low level of RNA content; b, KG-1a group. c, comparison on the percentages of the cells with orange fluorescence between the two groups,*, p < 0.05. Error bars correspond to mean ± SD. The green fluorescence represents DNA content and the orange fluorescence represents RNA content. Three independent experiments were performed. A representative of the experimental results was shown.

Figure 4

Acridine orange staining of nucleic acid in KG-1a cells (Fluorescence microscope ×200). a, (KG-1a +5-FU) group: KG-1a cells incubated at 50 μg/mL of 5-FU for 4 d. The enriched cells had low level of RNA content; b, KG-1a group. c, comparison on the percentages of the cells with orange fluorescence between the two groups,*, p < 0.05. Error bars correspond to mean ± SD. The green fluorescence represents DNA content and the orange fluorescence represents RNA content. Three independent experiments were performed. A representative of the experimental results was shown.

Figure 5

The Hoechst 33342 negative/low staining cells rate in KG-1a cells. KG-1a cells were stained by Hoechst 33342 and the cells were observed under inverted fluorescence microscope. Three independent experiments were performed. (KG-1a +5-FU) group: KG-1a cells incubated at 50 μg/mL of 5-FU for 4 d. The enriched cells had a high percentage of negative/low staining cells *, p < 0.05. Error bars correspond to mean ± SD.

Figure 6

Expression of ABCG2 mRNA and protein in KG-1a cells. The enriched cells had high expression of ABCG2 mRNA and protein. a, analysis by RT-PCR of expression of ABCG2 mRNA. b, analysis by Western blotting of the expression of ABCG2 protein. +5-FU: KG-1a cells incubated with 50 μg/mL of 5-FU for 4 d; -5-FU: KG-1a cells without 5-FU.