Date of Award
5-1987
Document Type
Open Access Dissertation
Degree Name
Doctor of Philosophy (PhD)
Abstract
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.)
Recommended Citation
Frank, David Alan, "The regulation of tyrosine phosphorylation during the differentiation of a human leukemia" (1987). Yale Medicine Thesis Digital Library. 2200.
https://elischolar.library.yale.edu/ymtdl/2200
