There is increasing evidence that the balance between the production of intracellular reactive oxygen species and the levels of GSH and other antioxidants modulates redox sensitive signaling pathways that regulate many cellular processes, including proliferation and differentiation; however, the precise mechanisms responsible for these complex responses and interactions remain largely unknown.9
The present study examined intracellular GSH levels during HL-60 cell differentiation, and the effects of two GSH depletors, CDNB and DEM, on the differentiation process. The results demonstrate that there is a small transient increase in total intracellular glutathione, but no apparent change in GSH/GSSG ratio, during both granulocytic and monocytic HL-60 maturation. This increase in intracellular glutathione precedes the changes in proliferation and CD11b cell surface marker expression, but coincides with an increase in NBT reduction capacity, a measure of superoxide anion production. In addition, treatment with low doses of CDNB and DEM to transiently decrease GSH levels augmented both induced and spontaneous differentiation, whereas NAC supplementation diminished it. Taken together with previous findings in other cell systems, the present data are consistent with a model in which reactive oxygen species act as signal mediators of cell differentiation. Thus, stimulation with a differentiation inducer leads to reactive oxygen species production, which in turn activate cell differentiation by an as yet undefined mechanism, as well as compensatory responses to deal with these reactive moieties, including transiently elevated GSH levels ( and
). When GSH levels are transiently depleted, reactive oxygen species availability is temporarily enhanced, leading to the activation of the cell differentiation program. Conversely, when thiol levels are augmented via the addition of NAC, reactive oxygen species are quenched, and as a result less oxidative signals are available to activate redox-sensitive differentiation pathways, and the cell differentiation program is impeded. On the other hand, under conditions of severe and sustained GSH depletion, excessive accumulation of reactive intermediates may cause inappropriate activation of signaling cascades or other cell injury, and thus thwart the normal cell differentiation program. Additional studies are needed to test this model, and to define the signaling pathways involved in these responses.
Although some previous studies have examined the role of GSH concentrations on cell differentiation, these studies have examined the effects of relatively drastic and sustained GSH depletion on this process, and most have depleted cellular GSH levels prior to or simultaneously with the induction of differentiation.23
Not surprisingly, differentiation is impaired under these severe conditions. In contrast, the present findings demonstrate that a more moderate and transient GSH depletion that is applied during the maturation process leads to enhanced HL-60 cell differentiation. In particular, the results show that when HL-60 cells were treated with the GSH-depleting agents at a time point that coincided with the differentiation-associated increase in intracellular GSH, differentiation marker expression was enhanced.
Consistent with this model, NAC treatment diminished differentiation marker expression, the opposite effect of GSH depletion treatments; however, it did so without increasing GSH levels. Note that NAC itself is a potent antioxidant, and has been shown to inhibit transcription factors involved in GSH synthesis. NAC inhibits NF-E2-related factor-2 (Nrf2), and thus downregulates the transcription of γ-glutamylcysteine ligase, the rate-limiting enzyme in GSH synthesis.62
These observations suggest that the thiol groups of GSH or NAC are likely critical for altering differentiation outcomes.
In addition to enhancing differentiation marker expression in induced HL-60 cells, CDNB and DEM also augmented spontaneous cell differentiation. Thus, altering thiol abundance not only affects monocytic and granulocytic pathways upregulated by VD3 and ATRA, respectively, but also stimulates the mechanisms responsible for spontaneous maturation. These findings further support the hypothesis that GSH levels are important determinants of differentiation events.
The present results also reveal that glutathione levels are regulated in a complex fashion during maturation. A small transient increase in glutathione levels was observed during both granulocytic and monocytic differentiation induction, suggesting that this may be a general feature of the differentiation process. Of significance, the measured increase in NBT reduction capacity coincided with the transient increase in GSH at 9 hours after VD3 exposure, and at 1 day after DMSO treatment, indicating a simultaneous increase in GSH and superoxide anion production capacity. Because oxidative stimuli are known to increase the expression of the rate limiting enzyme in GSH synthesis and hence elevate GSH levels,43
the initial increase in superoxide anion may cause GSH levels to become transiently higher. Although this association provides a possible explanation for the transient increase, no causal relationship has yet been established. Despite the fact that the increase in superoxide anion production may be a contributing factor to the transient increase in GSH, another possibility exists. HL-60 cells do not differentiate as a synchronous population, and thus there will always be some cells that are further along the differentiation pathway. As a result, some cells will attain the ability to produce superoxide anion earlier than others, and these data may be a reflection of this phenomenon.
Although these findings reveal a novel feature of HL-60 differentiation, many details concerning the nature of this transient increase still remain unclear. No changes in the GSH/GSSG ratio were detected in the current studies, and thus the transient increase in GSH appears to be explained by an increase in the absolute levels of glutathione (proportional increases in both GSH and GSSG levels). An increase in the absolute level of glutathione will affect any cell process that is dependent on this tripeptide’s availability, including enzyme activity, transport activity, glutathionylation, and cysteine availability. It is possible that higher glutathione levels may be required for some differentiation event, although additional studies are needed to test this possibility.
While these data do not support the hypothesis that a shift in intracellular thiol-redox status (a change in GSH/GSSG) is responsible for the transient increase in glutathione, this notion cannot be discounted. It is clear that thiol-redox status is not uniform throughout intracellular organelles, and differs within specific cellular compartments.63
If variations in GSH/GSSG do exist within the cell, then localized shifts in the GSH/GSSG ratio may be playing a critical role in redox signaling events, and hence cell processes such as differentiation. Additional studies are needed to identify possible changes within specific cellular compartments.
If GSH levels are an important factor in general differentiation events, then agents that alter its concentrations will affect differentiation outcomes. As mentioned earlier, diseases of differentiation (e.g., cancer, autoimmune disease and aging) are also associated with abnormal GSH levels. Although at this point the relationship is only a correlative one, understanding how GSH affects cellular differentiation may provide important insight into the etiology or treatment of these conditions. These findings may also have implications for in utero development. If the developing conceptus is exposed to environmental or infectious agents that deplete GSH levels, then orchestrated differentiation events may be disrupted, possibly leading to or contributing to birth defects, low birth weight, or increased propensity for disease later in life.