Evidence for involvement of tumor necrosis factor-alpha in apoptotic deathof bone marrow cells in myelodysplastic syndromes

We previously reported excessive apoptosis and high levels of tumor necrosi s factor-alpha (TNF-alpha) in the bone marrows of patients with myelodyspla stic syndromes (MDS), using histochemical techniques. The present studies p rovide further circumstantial evidence for the involvement of TNF-alpha in apoptotic death of the marrow cells in MDS. Using our newly developed in si tu double-labeling technique that sequentially employs DNA polymerase (DNA Pol) followed by terminal deoxynucleotidyl transferase (TdT) to label cells undergoing apoptosis, we have characterized DNA fragmentation patterns dur ing spontaneous apoptosis in MDS bone marrow and in HL60 cells treated with TNF-alpha or etoposide (Vp16). Clear DNA laddering detected by gel electro phoresis in MDS samples confirmed the unique length of apoptotic DNA fragme nts (180-200 bp). Surprisingly, however, phenotypically heterogeneous popul ation of MDS cells as well as the homogenous population of HL60 cells showe d three distinct labeling patterns after double labeling-only DNA-Pol react ion, only TdT reaction, and a combined DNA Pol + TdT reaction, albeit in di fferent cohorts of cells. Each labeling pattern was found at all morphologi cal stages of apoptosis. MDS mononuclear cells, during spontaneous apoptosi s in 4 hr cultures, showed highest increase in double-labeled cells (DNA Po l + TdT reaction). Interestingly, this was paralleled by TNF-alpha-induced apoptosis in HL60 cells. In contrast, VP16 treatment of HL60 cells led to i ncreased apoptosis in cells showing only TdT reaction. The double-labeling technique was applied to normal bone marrow and peripheral blood mononuclea r cells after treatment with known endonucleases that specifically cause 3' recessed (BamHI), 5' recessed (PstI), or blunt ended (DraI) double-strande d DNA breaks. It was found that the DNA-Pol reaction in MDS and HL60 cells corresponds to 3' recessed DNA fragments, the TdT reaction to 5' recessed a nd/or blunt ended fragments, and a combined "DNA Pol + TdT reaction" corres ponds to a copresence of 3' recessed with 5' recessed and/or blunt ended fr agments. Clearly, therefore, apoptotic DNA fragments, in spite of a unique length, may have differently staggered ends that could be cell (or tissue) specific and be selectively triggered by different inducers of apoptosis. T he presence of TNF-alpha-inducible apoptotic DNA fragmentation pattern in M DS supports its involvement in these disorders and suggests that anti-TNF-a lpha (or anticytokine) therapy may be of special benefit to MDS patients, w here no definitive treatment is yet available. (C) 1999 Wiley-Liss, Inc.