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Analysis of hematopoietic progenitor cell cycle control in the myeloproliferative disorders

University of British Columbia

The myeloproliferative disorders (MPD) comprise an interesting group of hematological neoplasms in which clonal expansion is initiated at the level of the pluripotent stem cell compartment but differentiation proceeds essentially normally. Although evidence for the involvement of specific genetic changes exist in one of these diseases (chronic myeloid leukemia, CML), the nature of the lesion that permits the progeny of a single stem cell to dominate the mature cell compartment has not been elucidated. Application of clonal assay systems to the study of the MPD has provided information about the numbers, proliferative capacity, physical properties and the responsiveness to regulatory factors of hemopoietic progenitors from all cell lineages. However, clonal assays can offer only limited information about the processes that underly stem cell regulation. Some of the limitations imposed by these assays may be overcome by the use of long-term cultures in which primitive and pluripotent progenitors may be maintained for at least 2 months. The purpose of this thesis was to determine if consistent alterations in cell cycle activity were characteristic of progenitors in MPD patients, and to evaluate the potential of the long-term culture system for further investigations of any changes observed. The proliferative behaviour of clonogenic progenitors from the blood and bone marrow of a large number of MPD patients was compared with that of normal individuals using the ³H-thymidine cell suicide technique. These experiments showed that all progenitor classes in the blood and marrow of patients with CML and polycythemia vera (PV), which in normal individuals are quiescent, had a significant component of cycling cells. In addition, a consistent association of this abnormality in cycling control with expression of erythropoietin (EP)-independence in patients with essential thrombocytosis (ET) was revealed. Further studies were then undertaken to determine if these abnormalites could be reproduced in vitro. Experiments with normal marrow showed that the most primitive progenitor classes located in the adherent fraction of standard long-term cultures undergo cyclic changes of proliferative activity with each weekly addition of new growth medium. These studies suggested that the proliferative activity of normal primitive hemopoietic cells may be both positively and negatively regulated by close range interactions with marrow stromal elements. In contrast, in similar experiments with long-term cultures established with PV marrow, where maintenance of neoplastic cells could be documented, analogous primitive progenitor cells in the adherent layer failed to return to a quiescent state and remained continuously in cycle. From previous experiments with CML patients, it was already known that Ph¹ -positive progenitors usually disappeared rapidly in long-term cultures established with CML marrow. Therefore, as an alternative approach CML peripheral blood cells were seeded onto preestablished normal marrow adherent layers, since preliminary studies had suggested that this would allow sufficient numbers of primitive Ph¹-positive progenitors to be maintained for cycling studies. Analysis of such cultures together with studies of appropriate normal controls, revealed the same lack of cycling regulation in CML as previously shown for PV. In addition, studies of control cultures showed that in the absence of an adherent layer, normal peripheral blood progenitors cycle continuously, again suggesting that one regulatory function of the adherent layer is to maintain normal progenitors in a quiescent state. These studies demonstrate that consistent abnormalities of cell cycle control characterize the primitive progenitor compartments in MPD patients, and that these abnormalities can be reproduced in vitro. Initial findings with the long-term marrow system suggest that these abnormalities may be due to the in-sensitivity of primitive neoplastic cell types to respond to factors that arrest their normal counterparts from further progression through the cell cycle.

Text

2010-07-30

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http://hdl.handle.net/2429/27036

Pathology

University of British Columbia

Graduate