Abstract

The myelodysplastic syndromes (MDS) are a diverse group of primary bone marrow disorders characterized by persistent cytopenias and a variable rate of progression to acute myelogenous leukemia (AML). A consistent finding in the bone marrow of patients with MDS is increased apoptosis and a defect in terminal differentiation of maturing cells. We hypothesized that MDS cells are unable to tolerate the increasing oxygen tension experienced during hematopoiesis as cells move out of the hypoxic stem cell niche and this intolerance leads to increased apoptosis. In order to test this hypothesis, we have studied the growth in methylcellulose colonies of bone marrow progenitors in eight samples from patients with MDS at normoxia (21% O2) and at hypoxia (3% O2). In 5/8 samples there is a greater than 8-fold (range 8.5–15.9) increase in colony number under hypoxic conditions. The remaining three MDS samples, while only demonstrating an average increase in colony number of 2.4-fold, demonstrated significantly larger colonies of various lineages. Comparatively, in six normal patient samples the augmentation averaged 1.5-fold and never exceeded 2-fold, with a statistically significant (p=0.01) difference in the mean fold-change between normal and MDS marrows. These results suggest that MDS cells have an impaired ability to tolerate increasing levels of oxygen and also suggests a methodology for expansion of MDS cells for further biochemical analysis of this phenotype. The cellular response to hypoxia is modulated in part through the mitochondrion and significant genetic data suggests that a mitochondrial abnormality is associated with MDS. In order to examine mitochondrial potential in MDS progenitor cells, we have adapted a multiparameter flow cytometric strategy for the simultaneous analysis of mitochondrial potential (ψm) in defined hematopoietic progenitors using the potentiometric dye tetramethyl rhodamine ethyl ester (TMRE). These studies confirm that in normal bone marrow samples, ψm increases with development from the hematopoietic stem cell (HSC) to differentiated progenitor cells. Analysis of marrow from a patient with early MDS (refractory cytopenia with multilineage dysplasia, IPSS Low) and a 10-fold increase in colony number under hypoxic conditions demonstrates the absence of this progressive increase in ψm through myeloid development when compared to normal controls. Additionally, two samples from patients with progression of their MDS to frank AML demonstrated a markedly elevated ψm in the blast cells, consistent with the elimination of the mitochondrial pathology during the genesis of the leukemic clone. Analysis of mitochondrial potential in additional MDS samples is ongoing and will be presented. These data suggest that a defect in the mitochondrial adaptation to increasing oxygen tension experienced during hematopoietic differentiation may explain the impaired growth of MDS progenitor cells.

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