We used flow cytometry to analyze the cell cycle, DNA damage, and apoptosis in hematopoietic subsets in MDS marrow. Subsets were assigned using CD45, side scatter, CD34, and CD71. Cell cycle fractions were analyzed using DRAQ 5 (DNA content) and MPM-2 (mitoses). DNA damage was assessed using p-H2A.X, and apoptosis using Annexin V. Compared to controls, MDS patients demonstrated no increased mitoses in erythroid, myeloid, or CD34+ cells. Myeloid progenitors demonstrated increased G2 cells, which with no increased mitoses suggested delayed passage through G2. Myeloid progenitors demonstrated increased p-H2A.X, consistent with DNA damage causing this delay. Annexin V reactivity was equivalent in MDS and controls. Results for each parameter varied among hematopoietic compartments, demonstrating the need to analyze compartments separately. Our results suggest that peripheral cytopenias in MDS are due to delayed cell cycle passage of marrow progenitors and that this delayed passage and leukemic progression derive from excessive DNA damage. 1. Introduction Myelodysplastic syndrome (MDS) is characterized by life-threatening peripheral blood cytopenias and a propensity to progress to acute myeloid leukemia (AML). Pathogenetic explanations for both characteristics remain elusive. MDS is a serious health problem, especially in the expanding elderly population, where incidence approaches 80 cases per 100,000 population per year [1–5]. There is no effective curative strategy for MDS in elderly patients, and in younger patients curative treatment consists of allogeneic stem cell transplantation, which is expensive with associated morbidity and mortality [6–8]. A current pathogenetic model of MDS is hyperproliferation of marrow progenitors but poor production of circulating cells due to excessive in vivo apoptosis; however, this model is not supported by the absence of hyperuricemia as a defining characteristic of the disease and fails to explain the propensity of MDS to progress to AML. An alternative model is that MDS is inherently a mutator phenotype characterized by increased DNA damage, that causing impaired cell cycling, failure of production of peripheral blood cells, and leukemic transformation. Improved treatment strategies for MDS require clarification of its pathogenesis. To investigate these issues we used multiparametric flow cytometry to analyze the cell cycle, including mitotic events, DNA damage, and apoptosis in individual hematopoietic precursor compartments in marrow samples from patients with MDS. 2. Methods 2.1. Patients MDS patients receiving no
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