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T-cell reconstitution after bone marrow transplant (BMT) is characterized, for at least 1 year, by the expansion of populations of T cells with a primed/memory phenotype and by reverse CD4/CD8 proportions. T lymphocytes from 26 BMT patients (mostly adults) were obtained at various times after transplantation (from 45 to ≥730 days) and were tested for susceptibility to spontaneous apoptosis and anti-Fas triggered apoptosis in vitro. Substantial proportions of CD4+ and CD8+ cells generated during the first year after transplantation, but not by day 730, exhibited in these assays decreased mitochondrial membrane potential (▵Ψm) and apoptotic DNA fragmentation. The apoptotic phenotype tended to disappear late in the follow-up period, when substantial absolute numbers of naive (CD45RA+/CD62-L+) T cells had repopulated the peripheral blood compartment of the BMT patients. The rate of spontaneous cell death in vitro was significantly correlated with lower levels of ex vivo Bcl-2 protein, as assessed by cytofluorometry and Western blot analysis. In contrast, the levels of Bax protein remained unchanged, resulting in dysregulated Bcl-2/Bax ratios. Cell death primarily concerned the expanded CD8+/CD45R0+ subpopulation, although CD45R0− subpopulations were also involved, albeit to a lesser extent. These results show that the T-cell regeneration/expansion occurring after BMT is accompanied by decreased levels of Bcl-2 and susceptibility to apoptosis.

Human neutrophils are terminally differentiated cells that spontaneously undergo apoptosis in tissue culture. Apoptosis in these cells can be delayed by culture in the presence of granulocyte colony-stimulating factor or other inflammatory mediators. Neutrophils were found to contain an acid endonuclease that appeared to be responsible for the internucleosomal DNA cleavage that accompanies apoptosis. As measured by a plasmid nicking assay, this endonuclease had a molecular weight (M,) of 35,000, a pH optimum of 5.5, and a threshold for activity of pH 6.6 to 6.8. It was weakly inhibited by divalent cations (Ca’’, Mg2+, and Zn2+) and more strongly inhibited by aurintricarboxylic acid EUTROPHILS ARE terminally differentiated cells with a life span of hours to days. In culture, they undergo programmed cell death or apoptosis, an event characterized among other things by the condensation of chromatin and the digestion of genomic DNA into oligonucleosomal fragments (manifested in apoptotic cells as altered nuclear morphology and the appearance of nucleosomal ladders on gel electrophoresis of DNA), whereas energy stores and membrane integrity remain for the most part intact.’ In the absence of cytokines, apoptosis occurs in 30% to 50% of the cells by 24 hours. Apoptosis in neutrophils can be delayed, but not prevented, by cytokines such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin- 1 (IL- I), or lipopolysaccharide.*.’ Apoptosis-associated DNA degradation in other systems has been attributed to any of several endonucleases, including a calcium- and magnesium-dependent endonuclease: the calcium-dependent enzyme DNase I,’ and an acidic endonuclease similar or identical to DNase IL6 In the present report we characterize an acid endonuclease as the only DNase detectable in mature human neutrophils and show that, when the intracellular pH of neutrophils is lowered into the range in which this nuclease is active, internucleosomal DNA cleavage is seen. N

Plasma cells (PCs) are the terminally differentiated effector cells of the humoral immune system. The majority of PCs are short-lived and undergo programmed cell death in the form of apoptosis after a few days of intensive immunoglobulin secretion. Despite potentially wide-ranging implications for infection control, auto-immunity, and PC dyscrasias, the mechanisms that govern the initiation and execution of PC apoptosis are poorly understood. We used two well-established murine systems of PC differentiation and immunohistochemistry of human lymphoid tissue sections to study the regulation of PC apoptosis. IgM-secreting post-mitotic CD138+B220− PCs were differentiated in vitro from primary mouse splenic B cells using cytokines and LPS and purified by magnetic selection. Murine I.29mu+ B lymphoma cells were induced to undergo plasmacytic differentiation by stimulation with LPS. In both systems, terminal PC differentiation is followed by spontaneous apoptosis of half of the PCs within 48h, similar to PC apoptosis in vivo. We found that a sharp increase in endoplasmatic reticulum (ER) stress, which is caused by an imbalance between secretory load and capacity in the ER, occurs in PCs that have completed differentiation and begin to undergo apoptosis. In parallel, susceptibility specifically to ER stress-induced apoptosis but not to other apoptotic stimuli increases substantially in differentiated PCs, despite an ongoing ER stress response and expansion of the secretory machinery. Caspase-12, which has been linked specifically to ER stress-induced apoptosis, is activated and processed during programmed PC death. Using the specific inhibitor of caspase-12, zATADfmk, we found that caspase-12 mediates apoptotic DNA fragmentation and chromatin condensation in PCs undergoing apoptosis but not in B cells undergoing tunicamycin-induced apoptosis. In contrast, the major apoptotic effector caspases (caspase-9, caspase-3, caspase-7) downstream of the mitochondria become resistant to activation by apoptotic ER stress during terminal PC differentiation and are not activated during PC apoptosis. We observed that he pan-caspase inhibitor, zVADfmk, completely blocks tunicamycin-induced apoptosis in B cells but does not inhibit PC apoptosis or tunicamycin-induced cell death in PCs. Using the small molecule PAC-1, which specifically activates caspase-3 by targeting a “safety-catch” amino acid sequence that keeps caspase-3 inactive, we found that caspase-3 is stabilized in its inactive form in PCs and human myeloma cell lines, but not in B cells. Immunohistochemistry of human lymphoid tissue sections demonstrated that most primary reactive PCs and extramedullary myeloma cells undergo spontaneous apoptosis in vivo without activation of caspase-3. Thus, ER stress plays a major role in limiting the life span of short-lived PCs and activates caspase-12, which mediates nuclear apoptosis specifically in PCs. The major apoptotic effector caspases, however, become resistant to activation during terminal PC differentiation, and PC apoptosis is largely independent of caspases downstream of the mitochondria. These observations lead us to propose that developmentally regulated inhibition of key apoptotic caspases, which rapidly execute apoptosis in most cells, has evolved in PCs as a means to delay apoptosis under conditions of increasing ER stress linked to immunoglobulin secretion. Overwhelming ER stress ultimately limits the life span of short-lived PCs by inducing apoptosis using alternative mechanism involving caspase-12, which is redundant for the execution of ER stress-induced apoptosis in cells that can activate the classical effector caspases.

Iron overload has been shown to induce DNA damage and may be implicated in leukocyte apoptosis. Previously we have shown that indirect markers of apoptosis such as caspase activity and Bax levels were higher in leukocytes of thalassemia patients. We have now assessed whether nucleosomal DNA fragmentation, a hallmark of apoptosis, is higher in leukocytes of iron-overloaded thalassemia patients compared with control subjects. Methods: Thalassemia Clinical Research Network patients participating in the Novartis CICL670A0107 trial (a randomized comparison of deferasirox, an oral iron chelator, vs. DFO) were eligible and 44 (25 male, mean age: 21.8 ± 11.1 yrs) were enrolled in the study. Fasting blood samples were obtained after a 5-day washout of DFO prior to commencing treatment with study drug, and 24 hours post-chelator at 1, 6, and 12 months on study. Thirty healthy controls matched for age, sex, race and antioxidant usage (15 male, mean age 24.5 ± 9 yrs) also supplied a blood sample. Plasma, serum and cells were separated by centrifugation. The classic marker for apoptosis, nucleosomal DNA fragmentation was determined by a high throughput ELISA assay that used monoclonal antibodies directed against single- and double-stranded DNA bound to histones (H1, H2A, H2B, H3, and H4) and thus specifically detected mono- and oligonucleosomes (measured in ng/μg leukocyte protein). Detection of free mono- or oligonucleosomes is a direct measurement of apoptotic DNA fragmentation. DNA fragmentation results were log-transformed prior to analysis and means are reported. Results: At baseline, thalassemia patients had elevated nucelosomes compared to the controls (351.5 vs. 74.9 ng/μg leukocyte protein, p = 0.001). This high level of leukocyte DNA fragmentation was unchanged throughout the study. Furthermore, nucleosomal DNA fragmentation was positively correlated with CRP (r = 0.3, p = 0.01), WBC (r = 0.2, p = 0.05) and ANC (r = 0.2, p = 0.04), suggesting apoptosis is present in increased inflammatory states. Conclusions: This finding demonstrates that nucleosomal DNA fragmentation is higher in thalassemia compared to control patients and thus provides further evidence that there is increased apoptosis in circulating leukocytes of thalassemia patients.

CML is a clonal myeloproliferative disease in which Bcr-Abl deregulated tyrosine kinase (TK) activity impairs blood cells homeostasis leading to altered growth, homing and apoptosis. Imatinib mesylate (Glivec, Novartis), an ATP competitor molecule, is the gold standard therapy for Bcr-Abl positive diseases, but resistance is increasingly encountered, mainly due to point mutations in the abl kinase portion of the molecule. Second generation TK inhibitors (Dasatinib, Brystol-Myers Squibb and Nilotinib, Novartis) can bind mutated forms of the protein, but T315I mutation appear unresponsive to TK treatments. Apoptosis is a cell suicide mechanism activated to remove redundant, damaged, or infected cells. An essential component of the apoptotic machinery, the caspase family, consists of a group of intracellular cysteine proteases that can be divided into initiator/upstream caspases (2, 8, 9, and 10 caspases), which activate the downstream/effector caspases (3, 6 and 7 caspases). Cleavage of a selected group of substrates by effector caspases is responsible for dismantling of essential cell components, which results in morphological and biochemical changes that characterise apoptotic cell death: cytoskeletal rearrangement, cell membrane blebbing, nuclear condensation and DNA fragmentation. Based on cell line experiments we focused our attention on some members of the pyrrolo[1,2-b][1,2,5]benzothiadiazepines (PBTDs) family to test their potential apoptotic activity in CML. Important apoptotic activity was demonstrated on K562, K562 R-IM, HL -60 ,U937 and Jurkatt cell lines, as evidenced with the concentration and the percentage of the cell death quantified by measuring PI-uptake by flow cytometry, DNA fragmentation, and analyzed by agarose gel electrophoresis generating a characteristic ladder pattern of discontinuous DNA fragments. Three PBTDs coumpounds (RS 678, RS779, RS 2630 RS) were then incubated with peripheral blood and/or bone marrow cells isolated from 30 CML untreated, 25 imatinib-resistant, 10 Dasatinib resistant, and 1 Nilotinib resistant patients. Cells were incubated at the concentration 10μM for,16,24, and 48 hours. Apoptotic DNA fragmentation was tested by agarose gels electrophoresis and was present in all but one patients in a percentage varying from 60 to 85%, compared to 0 to 5% of internal controls. Among the 25 imatinib-resistant patient 23 were tested for abl mutation using D-HPLC. Five patients showed T315I, one F317L, one each M351V, D276G, Y253H, and 2 patients showed 2 mutations (F317l and M315T; and G250G, and F359V). Only one patient Dasatinib reistant carrying M351V mutation showed very low apoptosis (5–10%), while the 5 patients with T315I mutation underwent high apoptotic DNA fragmentation. Apoptosis seem to be triggered by PBTDs through activation of initiator caspases 8 and 9 and effector caspases 3 as eveidenced by western blotting. Although more experiments are needed, these compounds appear promising for a preclinical approach in CML.

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disease driven by the Bcr-Abl hybrid protein expressed in the hemopoietic stem cell compartment. Bcr-Abl deregulated tyrosine kinase (TK) activity, impairs blood cells homeostasis leading to altered growth, homing and apoptosis. Imatinib mesylate (Glivec), an ATP competitor molecule active on Abl and Bcr-Abl TKs, is the gold standard therapy for Bcr-Abl positive CML, however long-term efficacy of the drug is unknown, and resistance has been described. In the present study we focused our attention on some members of the pyrrolo [1,2-b][1,2,5] benzothiadiazepines (PBTDs) family for their potential apoptotic activity. Coumpounds were tested on K562, NPE and ARO cell lines. After showing important apoptotic activity, the compounds were tested on CML cells from 10 patients at onset and 2 patients in blast crisis Imatinib-resistant. Our data evidenced strong activity in apoptosis induction in all patients, including patients that have developed blast crisis under Imatinib treatment. Three compounds (RS779, RS735 and RS678) were selected among the PBTDs. At first compounds were incubated with CML K562 cell line showing DNA fragmentation and the characteristic morphological features of apoptosis such as shrinkage, chromatin condensation, membrane blebbing, evaluated by electronic microscopy analysis. NPE and ARO (neoplastic thyroid cells) cell lines were also tested showing similar results. At this point we decided to test compounds on cells from CML patients. We harvested peripheral blood cells from 10 consecutive CML patients at onset, and also peripheral blood cells from 2 blast crisis evolved under Imatinib treatment. Peripheral blood samples were incubated with PBTDs for 8,16, and 24 hours at 10μM concentration in order to make a direct comparison of apoptotic potencies. Apoptotic DNA fragmentation was tested by agarose gels electrophoresis and was present in all patients generating a characteristic ladder pattern of discontinuous DNA fragments. The characteristic morphological features such as shrinkage, chromatin condensation and membrane blebbing were also evidenced. RS-678 and RS-779 caused strong dose- and time-dependent induction of apoptosis. The DNA fragmentation became apparent at 8 h and increased up to 16h. We also examined the expression of apoptosis-related genes in 6/10 patients at onset. In cells treated with (RS-678) and (RS-779) compounds, expression of pro-apoptotic bax and procaspase-3 genes were increased. The b-actin DNA band used as control, was distributed at similar levels in all samples, while expression of the anti apoptotic bcl-XL and bcl-2 genes were decreased. These results suggest that the apoptotic effect of these compounds is likely mediated through regulation of genes involved in apoptosis, and/or cell survival at the transcriptional level. Among the three compounds tested RS-678 and RS-779 were found to be the most active both in cell lines and in patient samples, whereas RS-735 had little or no effect. These findings suggest that PBTDs could be promising candidates as a novel therapeutic agent for Bcr-Abl-positive CML.