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

We previously reported excessive apoptosis and high levels of tumor necrosis factor-alpha (TNF-alpha) in the bone marrows of patients with myelodysplastic syndromes (MDS), using histochemical techniques. The present studies provide further circumstantial evidence for the involvement of TNF-alpha in apoptotic death of the marrow cells in MDS. Using our newly developed in situ 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 during spontaneous apoptosis in MDS bone marrow and in HL60 cells treated with TNF-alpha or etoposide (VP16). Clear DNA laddering detected by gel electrophoresis in MDS samples confirmed the unique length of apoptotic DNA fragments (180-200 bp). Surprisingly, however, phenotypically heterogeneous population of MDS cells as well as the homogenous population of HL60 cells showed three distinct labeling patterns after double labeling--only DNA-Pol reaction, only TdT reaction, and a combined DNA Pol + TdT reaction, albeit in different cohorts of cells. Each labeling pattern was found at all morphological stages of apoptosis. MDS mononuclear cells, during spontaneous apoptosis in 4 hr cultures, showed highest increase in double-labeled cells (DNA Pol + TdT reaction). Interestingly, this was paralleled by TNF-alpha-induced apoptosis in HL60 cells. In contrast, VP16 treatment of HL60 cells led to increased apoptosis in cells showing only TdT reaction. The double-labeling technique was applied to normal bone marrow and peripheral blood mononuclear cells after treatment with known endonucleases that specifically cause 3' recessed (BamHI), 5' recessed (PstI), or blunt ended (DraI) double-stranded 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 and/or blunt ended fragments, and a combined "DNA Pol + TdT reaction" corresponds to a copresence of 3' recessed with 5' recessed and/or blunt ended fragments. 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. The presence of TNF-alpha-inducible apoptotic DNA fragmentation pattern in MDS supports its involvement in these disorders and suggests that anti-TNF-alpha (or anticytokine) therapy may be of special benefit to MDS patients, where no definitive treatment is yet available.