Transient glutathione depletion determines terminal differentiation in HL-60 cells

Abstract

To better define the role of glutathione (GSH) in cell differentiation, the present study measured GSH concentrations during terminal HL-60 cell differentiation, in the presence and absence of differentiation-inducing agents, and in the presence and absence of GSH altering agents. Interestingly, there was a small transient increase in intracellular GSH levels during dimethyl sulfoxide (DMSO) or 1alpha,25-dihydroxyvitamin D3 (VD3) induced differentiation. This increase coincided with an increase in nitroblue tetrazolium (NBT) reduction capacity, a measure of superoxide anion production, but there was no apparent change in the GSH/glutathione disulfide (GSSG) ratio. Surprisingly, treatment of cells with low doses of 1-chloro-2,4-dinitrobenzene (CDNB; 5 microM) or diethylmaleate (DEM; 0.5 mM), which transiently deplete GSH levels to about 40% of control levels, resulted in enhanced differentiation of HL-60 cells exposed to VD3 or all-trans-retinoic acid (ATRA), as well as under un-induced conditions (i.e., spontaneous differentiation). Enhanced differentiation occurred when cells were treated with the GSH-depleting agents 4 hours after treatment with differentiation inducers. These findings indicate that intracellular GSH levels are regulated in a complex fashion during HL-60 cell differentiation, and that transient GSH depletion using low doses of CDNB and DEM enhances the differentiation process.

Figure 1

A transient increase in glutathione occurs during DMSO, and VD3-induced Hl-60 differentiation. Total glutathione content (nanomoles/mg protein) in control cells, and those treated with 1.5% DMSO at 0, 1, 2, 3, 4 and 5 days of culture (A), or with 500 nM VD3 cells at 0, 9, 16, 24 and 48 hours of culture (B). Reduced glutathione (% of total glutathione) was determined for control, DMSO (C), and VD3-treated HL-60 cells (D). Samples were assayed in triplicate, and data are represented as mean ± S.E. of five to six experiments (A and B), and three experiments (C and D). Statistical significance is expressed as *p < 0.05 versus control values.

Figure 2

The transient increase in glutathione precedes changes in S-phase cell cycle distribution and CD11b expression. S-phase cell cycle distribution was assessed after 0, 1, 2, 3, 4 and 5 days of DMSO exposure (A), and 0, 9, 16, 24 and 48 hours of VD3 exposure (B). Total CD11b cell surface marker expression was measured in HL-60 cells after 0, 1, 2, 3, 4 and 5 days of 1.5% DMSO exposure (C), and 0, 9, 24 and 48 hours of 500 nM VD3 treatment (D). Data are represented as mean ± S.E. of three to four experiments. Statistical significance is expressed as *p < 0.05 versus control values.

Figure 3

NBT reduction capacity during DMSO and VD3-induced HL-60 differentiation. NBT reduction capacity in HL-60 cells after 0, 8, 16, 24 and 48 hours of DMSO exposure (A), and after 0, 6, 9 and 24 hours of VD3 treatment (B). Data are represented as mean ± S.E. of three experiments. Statistical significance is expressed as *p < 0.05 versus control values.

Figure 4

Experimental design of GSH depletion experiments during induced HL-60 cell differentiation. HL-60 cells were incubated with GSH depleting agents, 0.5 mM DEM or 0.5 µM CDNB, either with or without 5 mM GSH. Treatment occurred during differentiation, after exposure to maturation agents, but before the transient increase in GSH observed after VD3 treatment. Cells were collected for GSH analysis 3, 6 and 20 hours after the initial incubation with GSH-altering agents. NBT reduction was analyzed at 24 hours, and cell cycle and cell surface marker expression 48 hours after exposure to differentiation inducers, 500 nM VD3 or 500 nM ATRA.

Figure 5

Total glutathione levels in control and VD3-induced HL-60 cells after DEM, and CDNB treatment. Control cells were incubated with either 0.5 mM DEM (A) or 5 µM CDNB (B), in the presence or absence of 5 mM GSH for a 3 hour period. HL-60 cells were treated with 500 nM VD3, and 4 hours later cells were incubated with either 0.5 mM DEM (C) or 5 μM CDNB (D) for 3 hours, in the presence or absence of 5 mM GSH. Cells were collected 3, 6 and 20 hours after treatment with GSH-altering agents for analysis of total intracellular glutathione levels. Samples were analyzed in triplicate for each experiment. Data are represented as mean ± S.E. of three experiments. Statistical significance is expressed as *p < 0.05 versus control values.

Figure 6

DEM and CDNB altered differentiation marker expression in VD3, and ATRA-induced HL-60 cells. HL-60 cells were exposed to 500 nM VD3 or 500 nM ATRA. Approximately 4 hours later cells were treated with either 0.5 mM DEM or 5 µM CDNB for 3 hours, in the presence or absence of 5 mM GSH. At 48 hours after differentiation induction, cells were collected for the analysis of CD11b and CD71 expression (A and B), and S-phase populations (C and D). At 24 hours after VD3 exposure, or ATRA treatment cells were collected to assess NBT reduction capacity (E and F). Data are represented as mean ± S.E. of three to five experiments. Statistical significance is expressed as *p < 0.05 versus control values.

Figure 7

NAC treatment decreases differentiation marker expression in VD3-induced HL-60 cells. HL-60 cells were treated with NAC (5 or 10 mM) 4 hours after treatment with 500 nM VD3. Cell surface marker expression (A), and NBT reduction (B) were assessed 48, and 24 hours after VD3 treatment, respectively (B). Samples were analyzed in triplicate for NBT experiments. Data are represented as mean ± S.E. of three experiments. Statistical significance is expressed as *p < 0.05 versus control values.

Figure 8

DEM, and CDNB enhanced differentiation marker expression in un-induced HL-60 cells. HL-60 cells were treated with either 0.5 mM DEM or 5 µM CDNB for 3 hours, in the presence or absence of 5 mM GSH. Cells were collected at 48 hours for CD11b and CD71 expression (A), and at 24 hours for NBT reduction analysis (B). Samples were analyzed in triplicate for NBT experiments. Data are represented as mean ± S.E. of five to nine experiments. Statistical significance is expressed as *p < 0.05 versus control values.