Simultaneous assessment of cell kinetics and programmed cell death in bone marrow biopsies of myelodysplastics reveals extensive apoptosis as the probable basis for ineffective hematopoiesis.

Despite hypercellular bone marrows (BM), peripheral cytopenias are the rule in patients with myelodysplastic syndromes (MDS). This study examined the roles played by cell birth and cell death rates in generating this paradox. Cell kinetics from BM biopsies of 35 MDS patients were measured using intravenous infusions of either iododeoxyuridine or bromodeoxyuridine, or both. Degree of apoptosis or programmed cell death (PCD) was estimated using in situ end-labeling of DNA directly from BM biopsies, which were simultaneously double-labeled from proliferation/PCD. MDS were found to be highly proliferative disorders with large numbers of myeloid, erythroid, and megakaryocytic cells synthesizing DNA. Median cycling time (Tc) of myeloblasts was more rapid than that of patients with acute myeloid leukemia (44.1 hr vs. 56.0 hr). Interestingly, most marrow cells of all three lineages in 32 of 34 evaluable cases were undergoing PCD. In 19 of 32 patients, greater than 75% cells were apoptotic. Surprisingly, large numbers of S-phase cells were found to be simultaneously undergoing PCD, as were stromal cells of the BM microenvironment. We conclude that the extensive apoptosis in hematopoietic cells effectively cancels the high birth rate resulting in ineffective hematopoiesis and accounting for deficient bone marrow function.

Novel in situ double labeling for simultaneous detection of proliferation and apoptosis.

We describe a novel double-labeling method to simultaneously investigate proliferation and apoptosis from plastic-embedded biopsy specimens (PEBs). Infusions of bromo- and/or iododeoxyuridine (BrdU/IudR) were given to 10 patients, five with acute myeloid leukemia (AML) and five with myelodysplastic syndromes (MDS), and S-phase cells were measured in PEBs using a monoclonal anti-IudR/BrdU antibody. Apoptosis was measured by in situ end-labeling (ISEL) of DNA. The results demonstrate that both AML and MDS are highly proliferative disorders but that there is almost no apoptosis in the former, whereas extensive apoptosis was observed in the latter. Double labeling revealed that large numbers of S-phase cells in MDS were simultaneously undergoing apoptosis. We conclude that the high cell death in MDS cancels the high cell birth, resulting in a functionally aplastic marrow and thus accounting for the observed ineffective hematopoiesis. On the other hand, AML is rapidly fatal, probably owing to high cell birth with no or minimal cell death. Therapeutic strategies to prevent intramedullary programmed cell death of hematopoietic precursors should be evaluated in MDS, and efficacy of chemotherapy in AML can be assessed by measuring the induction of apoptosis in post-treatment biopsy specimens.

In situ end labelling of DNA to detect apoptotic cell death in a variety of human tumours.

The present studies illustrate clinical applications of in situ end labelling (ISEL) of DNA to detect apoptosis in a variety of human malignancies including myelodysplastic syndromes (MDS, n=10), non-Hodgkin's lymphoma (NHL, n=10), head and neck cancer (n = 3), breast cancer (n = 1) and cervical cancer (n = 1). These studies also describe a new in situ double labelling technique to detect apoptosis and proliferation (S-phase cells) simultaneously in the same section of plastic embedded tissue. In vivo intravenous infusions of thymidine analogues (i.e. bromodeoxyuridine (BrdU) and/or iododeoxyuridine (IUdR)) followed by their detection with a specific monoclonal antibody in a plastic embedded biopsy, combined with ISEL in the same section, facilitated simultaneous estimations of apoptosis and proliferation. The most salient finding of these studies was excessive apoptosis in MDS including the cells in S-phase as indicated by uniquely double labelled cells in their bone marrow biopsies. On the other hand, a very low degree of apoptosis was observed in NHL and other solid tumours. Moreover, the solid tumours exhibited definite compartments of apoptosis and proliferation. Further experiments are underway to confirm these findings in a larger study in order to design appropriate therapeutic modalities for these disorders.