CONTROL OF DIFFERENTIATION OF HUMAN PROMYELOCYTES

This proposal is directed at elucidation of the biochemical mechanisms
involved in differentiation of human promyelocytic leukemia cells (HL-60).
Treatment of HL-60 cells with phorbol esters result in differentiation to a
macrophage-like cell. The biochemical mechanisms by which phorbol esters
induce differentiation in this leukemic cell line remains unclear. It has
been hypothesized that the phorbol ester induced differentiation is
dependent on the activation of a calcium/phospholipid-dependent protein
kinase (C-kinase). Phorbol esters stimulate a number of early biochemical
changes which have been felt to be a result of activation of C-kinase. We
have recently described a compound, Bryostatin, which activates HL-60
C-kinase both in vitro and in whole cells, but when applied to HL-60 cells
in culture does not induce differentiation. In addition, Bryostatin when
added concomitantly with phorbol esters prevents the differentiation
event. This data suggests that activation of C-kinase is not totally
sufficient for HL-60 differentiation. This proposal will focus on the role of C-kinase in phorbol ester induced
differentiation of HL-60 cells. Bryostatin will be an important tool to
aid in this analysis. We shall first examine whether the early biochemical
changes induced after phorbol ester treatment, phosphorylation of specific
proteins, phospholipid synthesis and sodium/hydrogen ion flux, occur as a
result of C-kinase activation. By examining the effects of Bryostatin, we
will establish whether or not these events occur as part of a chain of
events initiated by activation of C-kinase or are independent events.
Using Bryostatin, will next examine if changes in the nucleus occurring
with differentiation are a direct result of C-kinase binding and activation
of phosphorylation of specific nuclear substrates. Finally using
counterflow centrifugal elutriation to synchronize HL-60 cells, we will
examine whether phorbol ester effects on biochemical changes and
differentiation are limited to a specific phase in the cell cycle. Studies described in this proposal will aid in the understanding of the
role of phorbol esters in HL-60 differentiation. This information could
then be used to help understand the block in differentiation in human
promyelocytic leukemia.