Date of Award
Doctor of Philosophy (PhD)
Dr. C.B. Harley
Biologically active phorbol esters cause HL-60 promyelocytic leukemia cells, to differentiate into cells resembling normal macrophages. Elevation of intracellular Ca²⁺ by ionophores and activation of protein kinase C (PKC) by phorbol esters often cooperate in a synergistic manner to elicit a full biological response; hence, a similar effect was sought in HL-60 cell differentiation. Assessment by a number of criteria conclusively demonstrated that treatment of HL-60 cells with subthreshold concentrations of phorbol esters in the presence Ca²⁺ ionophores caused macrophage-like differentiation. Synergistic HL-60 cell differentiation was phenotypicaly similar to the TPA-induced response yet was associated with a distinct pattern of gene regulation. In particular, induction of the proto-oncogene c-fos did not correlate with synergistic differentiation. This precluded a number of possible mechanisms which might underlie the observed cooperativity and suggested that Ca²⁺ may independently trigger essential components of the HL-60 differentiation program.
In an immunological survey of various tissues and cell lines, the major PKC substrate of platelets (P47) was detected in HL-60 cells. P47 abundane in HL-60 cells was increased 4.0 ± 0.5 fold upon differentiation with retinoic acid (RA), apparently because of a parallel induction of P47 mRNA. RA-differentiated cells were therefore chosen as an mRNA source from which to clone P47. Immunological screening of a lambda gt11 library yielded positive recombinamts expressing an open reading frame that matched peptide sequences obtained from purified platelet P47. Expression of P47 sequences in vitro and in E. coli showed the the P47 cDNA encoded a protein of Mr 40,087. The P47 sequence was not similar to any other known sequence but did contain a putative Ca²⁺ binding EF-hand and a strong candidate region for phosphorylation by PKC. The 3.0 kb P47 mRNA had some unusual structural features and was restricted to differentiated white blood cells. Analysis of the P47 gene suggested it has been conserved through vertebrate evolution and that in humans it has a complex 5' end. A Msp I polymorphism detected in HL-60 cells by sequencing of cDNA clones was confirmed in the normal human population. Preliminary characterization of human P47 genomic clones confirmed the gene structure deduced from Southern analysis. The P47 sequence refuted two candidate functions for this protein in the platelet.
Tyers, Michael David, "Molecular Aspects of Differentiation in the HL-60 Human Promyelocytic Leukemia Cell Line: Signal Transduction, Gene Regulation and Cloning of the Major Protein Kinase C Substrate of Platelets (P47)" (1988). Open Access Dissertations and Theses. Paper 2013.