Correlation of tumor necrosis factor alpha (TNF alpha) with high Caspase 3-like activity in myelodysplastic syndromes

Increased intramedullary apoptotic death of hematopoietic cells is thought to contribute to the ineffective hematopoiesis in myelodysplastic syndromes (MDS). Furthermore, high amounts of tumor necrosis factor alpha (TNF alpha ) have previously been correlated with apoptosis in MDS marrows. The presen t studies were undertaken to examine the status of two key downstream effec ters of TNF alpha signaling, i.e. Caspase 1 and Caspase 3 enzymes, using a fluorometric assay in the bone marrow aspirate mononuclear cells (BMMNC) in relation to apoptotic DNA fragmentation detected by in situ end-labeling ( ISEL) of DNA and with localization of TNF alpha in the corresponding biopsi es from 14 MDS patients. Both Caspase 1 and 3 were detectable in freshly ha rvested BMMNC, albeit median Caspase 3 levels (47.5 units/mg protein) being almost 10 times higher than Caspase 1 (4.0 units/mg protein). Upon short-t erm culture for 4 h in a serum-supplemented medium in vitro a significant i ncrease was seen in Caspase 3 activity (58.8 +/- 13.9 at 0 h vs. 177.8 +/- 55.2 units/mg protein at 4 h, n = 14, P = 0.017) and in percent cells label ed by ISEL (apoptotic index or AI%: 0.76% +/- 0.25% vs. 3.99% +/- 1.1%, n = 14, P = 0.004, respectively). Caspase I activity increased after 15 min in culture. Interestingly, TNF alpha levels measured by immunohistochemistry correlated with the net increase in Caspase 3 activity after 4 h (rho = 0.5 17, n = 13, P = 0.07) and the starting levels of Caspase 1 at 0 h correlate d with the Caspase 3 levels attained at 4 h (rho = 0,593, n = 13, P = 0.033 ). Additionally when TNF alpha-positive bone marrows (8/ 14) were compared with the negative marrows (6/14) the Caspase 3 levels were significantly hi gher in the TNF alpha-positive marrows (189.6 +/- 66.2 vs. 25.0 +/- 14.6 un its/mg protein, respectively, P = 0.043). The increase in AI%, though not s tatistically significant, was also higher in the TNF alpha-positive marrows . Finally in HL60 cells the effects of different Caspase inhibitors and pen toxifylline (PTX) (interferes with lipid signaling of cytokines) on TNF alp ha-induced apoptosis were evaluated. TNF alpha treatment significantly incr eased AI% (P < 0.003) as compared to the untreated controls. A co-treatment with three Caspase inhibitors, zVAD.FMK (inhibitor of Caspases 1 and 3, 10 mu M/l), Ac.YVAD.FMK (Caspase 1 inhibitor, 1 mu M/l), Ac.DEVD.FMK (Caspase 3 inhibitor, 10 mu M/l) as well as PTX (250 mu M/l) significantly curtaile d the AI% induced by TNF alpha The present studies thus identify the downst ream effecters of TNF alpha-inducible apoptosis in MDS and so also the supp ressors of TNFa apoptotic signaling. These results may have significant cli nical implications in the therapy of MDS in the future. (C) 1999 Published by Elsevier Science Ireland Ltd. All rights reserved.