Negative regulators of hematopoiesis from normal and leukemic granulocytes
Creators: name: Abdel-Kader, A. Karim
Publisher: name: University of British Columbia
Year: 2009

description: Hematopoiesis is controlled by a dynamic equilibrium between positive and negative growth regulatory signals. Initially, much investigation focused on the positive regulatory signals. The importance of the negative regulators in maintaining the tightly controlled limits on cell numbers seen in vivo is now being appreciated. This thesis describes research into the role of negative regulation in normal and leukemic hematopoiesis. The proteins believed to be responsible for the inhibitory activity of two crude neutrophil preparations, one from patients suffering from chronic myeloid leukemia (CML) and one from normal donors, have been identified. Limited characterization of these activities on normal and leukemic hematopoiesis has also been performed. Previous work in our laboratory described an activity derived from immunoaffinity enriched cell lysates of patients with chronic or acute myeloid leukemia (CML or AML). This material demonstrated significant inhibition of growth of committed progenitor cells (especially CFU-GM) from normal individuals (both human and murine) but did not inhibit equivalent colonies from samples taken from patients with CML. Preliminary attempts to characterize this material (referred to previously as CAMAL) isolated the active constituent to within a 30 to 35 kDa molecular size fraction. Further purification of this activity was undertaken. Using reverse phase high pressure liquid chromatography (rpHPLC) to fractionate the immunoaffinity enriched material, the inhibitory activity was found to elute exclusively in a single rpHPLC fraction corresponding to the leading portion of the peak corresponding to the 29 to 37 kDa serine protease homologue azurocidin/CAP37. The main portion of the azurocidin peak was found to have no inhibitory activity. Two-dimensional gel analysis of the active part of the peak and a reference of azurocidin (isolated from normal azurophilic granules) showed no entity distinct from the higher molecular weight glycoforms of azurocidin. As azurocidin is found within normal neutrophils, we purified this molecule from this source. Only one of six azurocidin preparations from normal donors was found to contain inhibitory activity on normal CFU-GM. Recently, we have devoted our efforts to extend the identification and characterization of a myelopoietic inhibitory activity originally described by Boyum and colleagues from normal neutrophils (147). Using density gradient fractionation of neutrophil lysates, we localized the activity to the cytosol and the specific granules fraction. Using sub-fractionation of granulocytes, ammonium sulfate and heat precipitation coupled with size exclusion and anion exchange chromatography, we have purified the molecule responsible for the inhibitory activity on myeloid progenitors to a single, silver stained band of approximately 15 kDa. Identification of this material as cytidine deaminase (CD) was established by Western blot analysis, the use of recombinant cytidine deaminase and blockage of the inhibitory activity by the known inhibitor of CD activity tetrahydrouridine (THU). Normal human, murine and CML progenitors were equally susceptible to the inhibitory activity of this molecule. In addition the proliferation and clonogenicity of various leukemic cell lines was also inhibited. Interestingly, even at high concentrations (&gt; 60 ng/ml) of pure recombinant CD, no more than a 70% inhibition of myeloid colony formation was seen.