Glutathione homeostasis during terminal human leukemia-60 (HL-60) cell differentiation

Thesis (Ph. D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Environmental Medicine, 2008.The tripeptide glutathione (L-γ-glutamyl-L-cysteinylglycine; GSH), is the most abundant intracellular thiol and functions as a major regulator of the intracellular redox state. Imbalances in GSH homeostasis are associated with a wide array of disease states, and are implicated in the aging process. For years, research on GSH has focused on its role in detoxification, however a renewed interest in the molecule has focused on its role in major cell processes, including cell differentiation. A number of studies have described GSH status in differentiated cells, however limited information is available concerning GSH status during the process. In addition, many seemingly contradictory reports have been made concerning the effects of altering GSH levels on differentiation outcomes. Studies in this thesis were designed to elucidate GSH status during terminal differentiation, and to determine how altering GSH during the process affects differentiation. It was hypothesized that GSH status is dynamic during terminal differentiation, and that depleting levels of this tripeptide modulates differentiation outcomes. In order to test this hypothesis, HL-60 cells were induced to differentiate into either neutrophil or monocyte-like cells, and GSH levels as well as differentiation marker expression were assessed during the process. Methods used to assess differentiation include flow cytometric analysis of CD11b and CD71 cell surface marker expression and cell cycle distribution, and the NBT reduction assay, used to measure superoxide anion production capability. These studies demonstrated that a transient increase in glutathione is a feature of HL-60 differentiation, and that this phenomenon coincides with increased NBT reduction capacity, but precedes alterations in cell surface marker expression and proliferation. When GSH was depleted during induced HL-60 maturation, differentiation marker expression was enhanced. In contrast, thiol supplementation diminished differentiation marker expression. GSH depletion also increased spontaneous maturation in uninduced HL-60 cells. In summary, these data demonstrate that glutathione levels are dynamic during HL-60 differentiation, and that altering intracellular glutathione content during the process modulates differentiation outcomes. Taken together, these findings suggest that glutathione status plays an important role in modulating differentiation outcomes, and thus may contribute to conditions, and disease states characterized by abnormal differentiation