P. Venugopal

Correlation of tumor necrosis factor α (TNFα) 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 α (TNFα) have previously been correlated with apoptosis in MDS marrows. The present studies were undertaken to examine the status of two key downstream effectors of TNFα 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 TNFα in the corresponding biopsies from 14 MDS patients. Both Caspase 1 and 3 were detectable in freshly harvested 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-term culture for 4 h in a serum-supplemented medium in vitro a significant increase 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 labeled by ISEL (apoptotic index or AI%: 0.76%±0.25% vs. 3.99%±1.1%, n=14, P=0.004, respectively). Caspase 1 activity increased after 15 min in culture. Interestingly, TNFα levels measured by immunohistochemistry correlated with the net increase in Caspase 3 activity after 4 h (ρ=0.517, n=13, P=0.07) and the starting levels of Caspase 1 at 0 h correlated with the Caspase 3 levels attained at 4 h (ρ=0.593, n=13, P=0.033). Additionally when TNFα-positive bone marrows (8/14) were compared with the negative marrows (6/14) the Caspase 3 levels were significantly higher in the TNFα-positive marrows (189.6±66.2 vs. 25.0±14.6 units/mg protein, respectively, P=0.043). The increase in AI%, though not statistically significant, was also higher in the TNFα-positive marrows. Finally in HL60 cells the effects of different Caspase inhibitors and pentoxifylline (PTX) (interferes with lipid signaling of cytokines) on TNFα-induced apoptosis were evaluated. TNFα treatment significantly increased 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 μM/l), Ac.YVAD.FMK (Caspase 1 inhibitor, 1 μM/l), Ac.DEVD.FMK (Caspase 3 inhibitor, 10 μM/l) as well as PTX (250 μM/l) significantly curtailed the AI% induced by TNFα The present studies thus identify the downstream effectors of TNFα-inducible apoptosis in MDS and so also the suppressors of TNFα apoptotic signaling. These results may have significant clinical implications in the therapy of MDS in the future.

P15ink4b gene methylation and expression in normal, myelodysplastic, and acute myelogenous leukemia cells and in the marrow cells of cured lymphoma patients

P15INK4B methylation and expression was studied in bone marrow cells obtained from normal individuals, from patients who had been cured of lymphoma, and from patients with either MDS or AML. The level of p15 methylation was very low in normal BM cells and in CD34+ and CD34− subpopulations (0–6.5%; med, = 2.5%). P15INK4B transcripts were present in each of these cell populations. In contrast, methylation was the usual situation in MDS and AML marrows. The presence of methylation of the p15INK4B gene did not always indicate an absence of expression nor was expression always present if methylation was absent. P15INK4B methylation was studied in the marrows of nine patients (one studied twice) who had been cured of lymphoma and in whom hemopoiesis was believed to be normal. Increased methylaton was present in all 10 marrows. These data indicate that p15INK4B methylation is likely to be a very early event in the development of the secondary hematologic disorders.

Effect of interferon-α on CD20 antigen expression of B–cell chronic lymphocytic leukemia

Chimeric CD20 monoclonal antibody as alternative therapy in relapsed low–grade non–Hodgkin’s lymphoma (NHL) has produced responses in nearly 50% of patients. Augmenting CD20 expression on tumor cells and/or inducing its expression may increase the cell kill and effectiveness of antibody therapy. Peripheral blood lymphocytes from 19 patients with B–cell chronic lymphocytic leukemia (B–CLL) were incubated in vitro in the presence of interferon– f (IFN– f) (500 U/ml and 1000 U/ml) for 24 and 72 hours. The effect on CD20 expression was studied by flow cytometry. The differences in the percentage positivity, the mean fluorescence intensity (MFI), and the product of percentage positivity and MFI were used to assess upregulation. There was a significant upregulation of CD20 expression on B cells seen at both concentrations after 24–hour priming (p < 0.01). B–CLL cells cultured for 72 hours in the presence of IFN– f also showed upregulation of CD20 expression; however, the degree of upregulation was m...