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 (> 60 ng/ml) of pure recombinant CD, no more than a 70%
inhibition of myeloid colony formation was seen. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/6244 |
| Date | 11 1900 |
| Creators | Abdel-Kader, A. Karim |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Format | 10855869 bytes, application/pdf |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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