Evaluation of Hematopoietic Abnormalities and Apoptosis in Myelodysplastic Syndromes Using Long Term Bone Marrow Culture System and In Situ Nick End Labeling Method

Won, Jong Ho; Hong, Dae Sik; Park, Hee Sook; Lee, Jung Shin

Abstract

OBJECTIVES
The myelodysplastic syndromes (MDS) are a group of acquired clonal hematopoietic disorders characterized by peripheral cytopenias and a hypercellular or normocellular dysplastic bone marrow. The mechanisms responsible for development of MDS are not known. We performed this study to evaluate the hematopoietic abnormalities and apoptosis in MDS.
METHODS
Long-term bone marrow culture (LTBMC) was performed for colony assays, cobblestone area assay, stromal morphologic changes from 7 patients with MDS and 7 normal controls. In situ nick end labeling (ISNEL) method was performed for detection of apoptosis from LTBMC in 7 patients with MDS and 7 normal controls. ISNEL method also performed in bone marrow cell bloc samples in 36 patients with MI3S.
RESULTS
Viability of nonadherent cells from LTBMC of patients with MDS was not decreased compared with normal controls at 1 week, but significantly decreased at 2 and 3 weeks compared with normal controls (P<0.0001). Formation of the cobblestone areas from patients with MDS was slightly decreased compared with normal controls at 1st week, but significantly decreased at 2nd and 3rd weeks compared with normal controls (P<0.0001). Slightly decreased compared with normal controls at 1 week, but significantly decreased at 2 and 3 weeks compared with normal controls (P<0.0001). Stromal layers produced in LTBMC of normal controls and 1 patient with MDS were detected at 1 week and were formed confluent stroma from 3 weeks, but another patients with MDS who did not form a confluent stroma. Patients with MDS had significantly lower colony forming unit granulocyte-macrophage (CFU-GM) compared with normal controls at 1 (P<0.01) and 2 weeks (P<0.001) of LTBMC. Two weeks of LTBMC resulted more profound inhibition of CFU-GM formation than 1 week (P<0.0001). Apoptotic cell death was absent in adherent and non adherent cells from normal controls at 1 and 2 weeks, but massive apoptotic cell death was found in adherent and non adherent cells from patients with MDS at 1 and 2 weeks and the degree of apoptosis was profound at 2 weeks compared with 1 week. Among the 36 patients, fifteen patients demonstrated varying degrees of apoptosis positive cells, 4 having low, 8 intermediates, and 3 high scores. Remaining 21 patients showed absent apoptosis or only occasional positive cells.
CONCLUSION
Hematopoietic abnormalities such as a failure of differentiation are caused by the stromal defects and the biologic basis of the apparent paradox of peripheral cytopenias in the face of hypercellular (or normocellular) marrow is related by intramedullary apoptotic cell death of the stromal and hematopoietic cells.