The regulation of tyrosine phosphorylation during the differentiation of a human leukemia

David Alan Frank, Yale University.

This is an Open Access Thesis


HL-60 promyelocytic leukemia cells are an excellent model system for studying hematopoietic differentiation. This study sought to determine how tyrosine phosphorylation and the enzymes which regulate it are controlled during HL-60 differentiation.HL-60 cells normally contain about 1.5% of their phosphoaminoacids as phosphotyrosine. Upon granulocytic differentiation, phosphotyrosine content decreases to 0.1 to 0.3% of phosphoaminoacids. HL-60 cells possess a tyrosine kinase activity located in the particulate fraction which can phosphorylate a number of tyrosine-containing substrates in a time- and temperature-dependent fashion. It has a k$\sb{\rm m}$ for ATP of about 20 uM, a pH optimum of 6.4, and a preference for Mn$\sp{2+}$ (12 mM) as a co-factor; it is further stimulated by Zn$\sp{2+}$, does not respond to insulin or epidermal growth factor, is activated by detergents, and is inactivated by N-ethyl-maleimide.During the granulocytic differentiation of HL-60, there is a 2.5- to 3.5-fold increase in tyrosine kinase activity, and a 5- to 8-fold increase in phosphotyrosine phosphatase activity. Two cell lines resistant to dimethyl sulfoxide-induced differentiation were derived and employed to show that these changes were differentiation-specific and not non-specific drug effects. Several anthracycline antibiotics and a hypoxanthine-guanine phosphoribosyl transferase-deficient subline sensitive to induction of differentiation by 6-thioguanine were used to demonstrate that these changes were not secondary to growth inhibition, but were due to differentiation per se. A murine myelomonocytic leukemia, WEHI-3B, was also studied and found to display similar changes in phosphotyrosine and tyrosine kinase and phosphotyrosine phosphatese activities with differentiation.Monocytic differentiation of HL-60 cells was found to be accompanied by an order of magnitude decrease in phosphotyrosine residues, a 2-fold increase in tyrosine kinase activity, and a 10-fold increase in phosphotyrosine phosphatase activity.Thus, both granulocytic and monocytic differentiation of HL-60 cells are accompanied by a large fall in total phosphotyrosine, an increase in tyrosine kinase activity, and a still larger increase in phosphotyrosine phosphatase activity. This system represents an excellent model for dissecting the importance of phosphotyrosine regulation. (Abstract shortened with permission of author.)