γ-Secretase Inhibitors Induce Erythroid Differentiation in Erythroid Leukemia Cell Lines

Materials and Methods

Cells and GSIs. Two erythroid leukemia cell lines were used. The AA cell line was established by Dr. A. Arai from blast cells from a 72-year-old male with acute pure erythroid leukemia. AA cells were cultured in 10% fetal calf serum (FCS)-supplemented RPMI-1640 medium without erythropoietin. Flow cytometric analysis showed that AA cells strongly express glycophorin A. AA cells express Notch1 and Jagged1 proteins (data not shown) according to immunoblotting studies (11). HEL cells were supplied by the Japanese Collection of Research Bioresources (Ibaraki, Japan). Three kinds of GSIs, GSI-IX (DAPT), GSI-XII (Z-IL-CHO) and GSI-XXI (Compound E) were purchased from Calbiochem (USA) and dissolved in dimethyl sulphoxide (DMSO).

Cell growth assay. The effects of GSIs on cell growth were examined using a colorimetric WST-1 assay. Briefly, 1×10³ cells were cultured in 96-well culture plates in 0.1 ml of 10% FCS-supplemented RPMI-1640 medium in the presence of increasing concentrations of GSIs. WST-1 and 1-methoxy-5-methylphenazinium methylsulfate (Dojindo Laboratories, Japan) were added after 7 days, at the manufacturer's recommended concentrations. The optical density (OD) was measured using an ELISA plate reader. Cell growth is shown as a percentage of the mean OD value of control cells cultured without GSIs.

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Figure 1.

Dose–response curves for the growth of AA and HEL cells treated with γ-secretase inhibitors (GSIs). Cells were cultured with increasing concentrations of GSIs. Growth was examined after 7 days using a colorimetric assay. Growth is shown as a percentage of the mean optical density value of control cells. *p<0.05, Significantly different from the control.

Colony assay. The effects of the GSIs on colony formation were examined in methylcellulose cultures. Using 96-well culture plates, 2×10² cells/well were plated in RPMI-1640 medium containing 0.8% methylcellulose and 10% FCS, with or without GSIs. Colonies containing more than 20 cells were counted under an inverted microscope after 7 days in culture.

Cell differentiation. Cytospin preparations of cells from 5-day suspension cultures with GSIs were stained with Wright solution and observed under a light microscope to examine cell morphology. Hemoglobin production was evaluated by diaminofluorene (DAF) staining (12). The DAF stock solution contained 1% 2,7-diaminofluorene in 90% acetic acid. The working solution was prepared by mixing 0.1 ml of DAF stock solution with 0.1 ml of hydrogen peroxide and 10 ml of 0.2 M Tris-HCl buffer (pH 7.0). DAF working solution was added to the cell suspension or was layered onto the methylcellulose cultures. Cells or colonies stained blue after 10 minutes were considered to be hemoglobin positive.

Quantitative reverse transcription-polymerase chain reaction (RT-PCR). The effects of GSIs on gene expression were examined using quantitative RT-PCR. First-strand cDNAs were synthesized from RNA extracted from cells treated with GSIs for 24 hours. Quantitative PCR was performed using a FastStart DNA Master SYBR Green I kit, a LightCycler (Roche Diagnostics, Germany) and LightCycler primer sets for β-ACTIN and HES1. The expression level of mRNA was normalized to the corresponding β-ACTIN mRNA level, which was measured concurrently.