Susceptibility to Spontaneous Apoptosis of Peripheral Blood Granulocytes from Patients with Myelodysplastic Syndrome and Chronic Myelomonocytic Leukemia.

Introduction: Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemias (CMML) constitute a heterogeneous group of clonal hematopoietic diseases. Cytopenias are induced by an apoptosis-mediated bone marrow suppression mechanism. Survival blood cells may exhibit resistance to bone marrow apoptosis and may constitute useful markers of the disease.

Patients and Methods: Peripheral blood granulocytes were isolated from 14 healthy volunteers, 10 patients with MDS (AR 5, ARSA 1, AREB 4) and 4 patients with CMML using a standard method with dextran sulfate and ficoll-hypaque. Apoptosis was quantified by the percentage of anexina V (ANV) and propidium iodide (PI) cells, changes in mitochondrial membrane potential (JC-1) using flow cytometry and expression of bcl-2, bax, bcl-x, XIAP and mcl-1 by Western blot analysis at 0h. Granulocyte apoptosis was evaluated at 0h and after incubation at 37°C for 24h in RPMI medium enriched with serum from each individual. Statistical significance was determined using the two-tailed Student’s t-test considering p<0.05 significant values.

Results: Elevated percentages of early (ANV+ IP-) and late (ANV+ IP+) apoptotic granulocytes were found after incubation for 24h in patients and volunteers. Differences in the ratio of early and late apoptotic granulocytes between patients and volunteers were not statistically significant. Similar results were obtained when changes in mitochondrial membrane potential were analyzed. Circulating granulocytes from healthy volunteers and from patients contained high levels of proapoptotic Bax protein. No patient expressed antiapoptotic bcl-2, bcl-x, XIAP and mcl-1 proteins.

Conclusion: Granulocytes from patients with MDS and CMML do not exhibit lower susceptibility to spontaneous apoptosis as compared to healthy individuals.