Cell Death In Lymphocytes Research Articles

The CD16−/CD56bright Subset of NK Cells Is Resistant to Oxidant-Induced Cell Death

Phagocyte-derived reactive oxygen species ("oxygen radicals") have been ascribed a suppressive role in immunoregulation by inducing dysfunction and apoptotic cell death in lymphocytes. Earlier studies show that human NK cells are exceptionally sensitive to oxygen radical-induced apoptosis and functional inhibition. Two subsets of human CD56(+) NK cells have been identified: the highly cytotoxic CD56(dim) cells which constitute >90% of NK cells in peripheral blood, and the less cytotoxic but efficiently cytokine-producing CD56(bright) cells. In this study, we demonstrate that the CD56(bright) subset of NK cells, in contrast to CD56(dim) cells, remains viable and functionally intact after exposure to phagocyte-derived or exogenously added oxygen radicals. The resistance of CD56(bright) cells to oxidative stress was accompanied by a high capacity of neutralizing exogenous hydrogen peroxide, and by a high cell-surface expression of antioxidative thiols. Our results imply that CD56(bright) NK cells are endowed with an efficient antioxidative defense system that protects them from oxygen radical-induced inactivation.

Exogenous hydrogen sulfide induces functional inhibition and cell death of cytotoxic lymphocytes subsets

The toxic effects of exogenous hydrogen sulfide on peripheral blood lymphocytes have been investigated in detail. Hydrogen sulfide is now considered as a gasotransmitter with specific functional roles in different cell types, like neurons and vascular smooth muscle. Here we show that exogenous hydrogen sulfide induces a caspase-independent cell death of peripheral blood lymphocytes that depends on their intracellular glutathione levels, with a physiologically relevant subset specificity for CD8+ T cells and NK cells. Although lymphocyte activation does not modify their sensitivity to HS-, after 24 h exposure to hydrogen sulfide surviving lymphocyte subsets show a dramatically decreased proliferation in response to mitogens and a reduced IL-2 production. Overall, our data demonstrate that HS- reduces the cellular cytotoxic response of peripheral blood lymphocytes as well as their production of IL-2, therefore de-activating the major players of local inflammatory responses, adding new basic knowledge to the clinically well known anti-inflammatory effects of sulfur compounds.

Abnormal activation and cytokine spectra in lymph nodes of people chronically infected with HIV-1

AbstractThere is growing recognition that HIV-1 infection leads to an activation of the immune system that includes perturbations of cytokine expression, redistribution of lymphocyte subpopulations, cell dysfunction, and cell death. Here, we explored the relationships between HIV-1 infection and immune activation in chronically HIV-1–infected human lymph nodes. In addition to CD4 T-cell depletion, we found increased effector T-cell frequencies associated with profound up-regulation of an activation marker CD38 in naive, central memory, and effector CD4+ and CD8+ T cells. Likewise, Fas death receptor (CD95) was more frequently detectable on T cells from HIV-1 nodes. Dendritic cell (DC) depletion was dramatic, with plasmacytoid DCs (PDCs) 40-fold and myeloid DCs (MDCs) 20-fold less frequent in HIV+ nodes than in control nodes. Cytokine dysregulation was evident, with IL-2 and IL-15 as much as 2 or 3 logs greater in infected nodes than in control nodes. Thus, activated effector cells are inappropriately attracted and/or retained in lymphoid tissue in chronic HIV-1 infection. High-level cytokine expression in turn activates and retains more cells at these sites, leading to lymphadenopathy and massive bystander activation that characterizes HIV-1 infection. Strategies targeting these activation pathways may lead to new therapies.

Interleukin-2 Signaling via STAT5 Constrains T Helper 17 Cell Generation

Recent work has identified a new subset of effector T cells that produces interleukin (IL)-17 known as T helper 17 (Th17) cells, which is involved in the pathophysiology of inflammatory diseases and is thought to be developmentally related to regulatory T (Treg) cells. Because of its importance for Treg cells, we examined the role of IL-2 in Th17 generation and demonstrate that a previously unrecognized aspect of IL-2 function is to constrain IL-17 production. Genetic deletion or antibody blockade of IL-2 promoted differentiation of the Th17 cell subset. Whereas STAT3 appeared to be a key positive regulator of RORgammat and IL-17 expression, absence of IL-2 or disruption of its signaling by deletion of the transcription factor STAT5 resulted in enhanced Th17 cell development. We conclude that in addition to the promotion of activation-induced cell death of lymphocytes and the generation of Treg cells, inhibition of Th17 polarization appears to be an important function of IL-2.

Radioprotection against DNA Damage by an Extract of Indian Green mussel, Perna viridis (L)

This study describes the radioprotective ability of a hydrolysate prepared using an enzyme-acid hydrolysis method from the green mussel Perna viridis in terms of its ability to prevent radiation-induced damage in plasmid DNA, cell death, reactive oxygen species (ROS) formation, and DNA damage in mice lymphocytes. The mussel hydrolysate (MH) present during irradiation showed significant protection from gamma-radiation-induced strand breaks in plasmid DNA as evaluated by gel electrophoresis. Viability studies by trypan blue dye exclusion and MTT assay showed that preincubation of mice splenic lymphocytes with MH protected them from gamma-radiation-mediated killing. Moreover, the presence of MH during irradiation of isolated mice lymphocytes significantly decreased the DNA damage, as measured by comet assay. Measurement of intracellular ROS by dichlorofluorescein fluorescence revealed that the presence of MH effectively reduced the ROS generated in lymphocytes by both chemical method and gamma-irradiation. Prevention of DNA damage both in plasmid and lymphocytes and cell death in lymphocytes appears correlated with reduction of oxidatively generated free radicals. It is concluded that protection against radiation-induced cell death and DNA damage by MH was attributable to reduction of reactive free radical species generated by gamma-radiation.

Osteopontin-induced relapse and progression of autoimmune brain disease through enhanced survival of activated T cells

Relapses and disease exacerbations are vexing features of multiple sclerosis. Osteopontin (Opn), which is expressed in multiple sclerosis lesions, is increased in patients' plasma during relapses. Here, in models of multiple sclerosis including relapsing, progressive and multifocal experimental autoimmune encephalomyelitis (EAE), Opn triggered recurrent relapses, promoted worsening paralysis and induced neurological deficits, including optic neuritis. Increased inflammation followed Opn administration, whereas its absence resulted in more cell death of brain-infiltrating lymphocytes. Opn promoted the survival of activated T cells by inhibiting the transcription factor Foxo3a, by activating the transcription factor NF-kappaB through induction of phosphorylation of the kinase IKKbeta and by altering expression of the proapoptotic proteins Bim, Bak and Bax. Those mechanisms collectively suppressed the death of myelin-reactive T cells, linking Opn to the relapses and insidious progression characterizing multiple sclerosis.

CNTO328 Abrogates Glucocorticoid (GC) Resistance Conferred by Interleukin (IL)-6 and the Bone Marrow Microenvironment in Multiple Myeloma (MM).

Given the critical role that IL-6 plays in MM cell proliferation, survival, and resistance to GCs, we evaluated the ability of CNTO328, a chimeric monoclonal IL-6 neutralizing antibody, to overcome GC resistance in cell line models of human MM. In the presence of IL-6, the MM cell lines ANBL-6 and KAS-6 were resistant to the cytotoxic activity of dexamethasone (Dex) as assessed by cell viability assays both in suspension culture and in the context of patient-derived stromal cells. Resistance to dexamethasone was readily reversed by CNTO328, but not an isotype control antibody, in suspension culture. For example, in the case of the ANBL-6 model, viability was reduced by 12% with CNTO328 alone, 8% with Dex, but 74% with the combination, consistent with a synergistic interaction Given the ability of other growth factors in the bone marrow microenvironment to confer GC resistance in preclinical models of MM, we evaluated the activity of the CNTO328 and Dex combination in ANBL-6 and KAS-6 cells using a physiologically-relevant MM cell/patient-derived bone marrow stromal cell co-culture system. Importantly, bone marrow stromal cells rendered ANBL-6 and KAS-6 cells resistant to Dex in cell viability assays, and CNTO328 was able to reestablish Dex sensitivity, thus confirming a central role of IL-6 in bone marrow stroma-mediated GC resistance. Furthermore, treatment of ANBL-6 and KAS-6 cells with Dex alone did not induce apoptosis in this co-culture system, whereas the combination of CNTO328 and Dex led to a synergistic induction of apoptosis. In KAS-6 cells, IL-6-mediated Dex resistance was not overcome using pharmacologic inhibitors to p38, PI-3 kinase, mTor or MEK, suggesting that other IL-6 signaling pathways are likely involved. In contrast, the mTor inhibitor rapamycin was capable of sensitizing ANBL-6 cells to Dex in the presence of IL-6, suggesting that this pathway may be relevant to IL-6-mediated GC resistance in these cells. Induction of the pro-apoptotic Bcl-2 family member, Bim, has been shown to play an important role in GC-mediated cell death in lymphocytes as well as preclinical lymphoma and acute lymphoblastic leukemia models. Interestingly, although treatment of ANBL-6 cells in the presence of IL-6 with either CNTO328 or dexamethasone did not lead to induction of Bim, the combination led to a 3.3-fold increase in its expression. Taken together, the above data demonstrate that inhibition of IL-6 signaling with CNTO328 can effectively overcome IL-6-mediated GC resistance even in the presence of bone marrow stroma, and provide a compelling rationale for translation of this combination into clinical trials for patients suffering from MM. Furthermore, we show that the ability of CNTO328 to overcome GC resistance may be mediated in part by its ability to reverse IL-6-mediated repression of GC-induced Bim expression. Studies evaluating the relevance of Bim modulation in IL-6-mediated GC resistance and the molecular pathways that mediate this effect are on-going.

Induction of apoptosis in Jurkat cells by photoexcited psoralen derivatives: Implication of mitochondrial dysfunctions and caspases activation

The prevailing form of cell death in lymphocytes exposed to psoralen plus UVA light (PUVA), was investigated. We studied the well known drug 8-methoxypsoralen (8-MOP) and an angular derivatives: angelicin (ANG). We evaluated the induction of apoptosis in a human tumor T-cell line (Jurkat). Both compounds provoke a significant induction of apoptosis at 24 h from irradiation as demonstrated by a remarkable percentage of cells Annexin-V positive. We investigated the effects of the psoralen derivatives upon UVA irradiation on the cell cycle. The flow cytometric analysis of propidium labeled cells indicates that treatment induces, in a dose dependent manner, a massive accumulation of cells, for both compounds, in G2–S phase after 24 h from the irradiation. We have focused our attention on the mitochondrial functionality after irradiation in the presence of psoralen derivatives. We evaluated, by flow cytometry, (i) the mitochondrial potential (Δ ψ mt), (ii) the production of reactive oxygen species (ROS) and (iii) the oxidation of cardiolipin, a phospholipid restricted to the inner mitochondrial membrane. Furthermore the activation of caspases -3, -8 and -9 was also investigated. The obtained data indicated that, upon UVA irradiation, the two compounds induce a strong decrease in mitochondrial functions and activate caspase-3, -8 and -9.

Molecular mechanisms of lymphocyte apoptosis injury in chronic viral infection

Programmed cell death of lymphocytes in patients with chronic infections induced by tick-borne encephalitis, hepatites B and C was investigated. The injury character in realization of lymphocyte apoptosis is shown to depend on molecular features of the infectious agent. Apoptotic death of lymphocytes was elevated after their incubation in vitro with dexamethazone, etopozide and in the environment without serum. Receptor-dependent and mitochondrial paths of apoptotic signal conduction ways are preferably modulated in chronic virus persistence.

Curcumin induces cell death without oligonucleosomal DNA fragmentation in quiescent and proliferating human CD8+ cells.

Cytotoxic CD8+ cells play an important role in determining host response to tumor, thus chemotherapy is potentially dangerous as it may lead to T cells depletion. The purpose of this study was to elucidate the propensity of quiescent and proliferating human CD8+ cells to undergo cell death upon treatment with curcumin, a natural dye in Phase I of clinical trials as a prospective chemopreventive agent. We treated human quiescent or proliferating CD8+ cells with 50 microM curcumin or irradiated them with UVC. Cell death symptoms such as decreased cell viability, chromatin condensation, activation of caspase-3 and specific DFF40/CAD endonuclease and oligonucleosomal DNA fragmentation were analyzed using MTT test, microscopic observation, Western blotting and flow cytometry. Curcumin decreased cell viability, activated caspase-3 and decreased the level of DFF45/ICAD, the inhibitor of the DFF40/CAD endonuclease. However, this did not lead to oligonucleosomal DNA degradation. In contrast, UVC-irradiated proliferating, but not quiescent CD8+ cells revealed molecular and morphological changes characteristic for apoptosis, including oligonucleosomal DNA fragmentation. Curcumin can induce cell death in normal human lymphocytes both quiescent and proliferating, without oligonucleosomal DNA degradation which is considered as a main hallmark of apoptotic cell death. Taking into account the role of CD8+ cells in tumor response, their depletion during chemotherapy could be particularly undesirable.

Oxygen Radicals Induce Poly(ADP-Ribose) Polymerase-Dependent Cell Death in Cytotoxic Lymphocytes

Cytotoxic T cells and NK cells will acquire features of apoptosis when exposed to oxygen radicals, but the molecular mechanisms underlying this phenomenon are incompletely understood. We have investigated the role of two enzyme systems responsible for execution of cell death, caspases and the poly(ADP-ribose) polymerase (PARP). We report that although human cytotoxic lymphocytes were only marginally protected by caspase inhibitors, PARP inhibitors completely protected lymphocytes from radical-induced apoptosis and restored their cytotoxic function. The radical-induced, PARP-dependent cell death was accompanied by nuclear accumulation of apoptosis-inducing factor and a characteristic pattern of large-fragment DNA degradation. It is concluded that the PARP/apoptosis-inducing factor axis is critically involved in oxygen radical-induced apoptosis in cytotoxic lymphocytes.

DX5+NKT cells induce the death of colitis‐associated cells: involvement of programmed death ligand‐1

NKT cells are activated by CD1d and show an immune regulating function. Here, we investigated whether DX5+ NKT cells could be used to reduce colitis in a chronic colitis mouse model and studied the potential immunological mechanisms involved. Chronic colitis was induced either by transfer of enriched CD62L+ CD4+ T cells to severe-combined-immunodeficient mice or by feeding dextran sodium sulfate to immune competent mice. DX5+ NKT cells were transferred to mice with chronic colitis. Co-transfer of DX5+ NKT cells, but not CD8+ control cells, prevented the onset of colitis, and the immune regulatory effect of DX5+ NKT cells was completely abrogated by injecting CD1d blocking antibody. Moreover, DX5+ NKT cells reduced established colitis in both chronic colitis models. In vitro, DX5+ NKT cells induced cell death of colon-infiltrating lymphocytes isolated from diseased mice. This effect was inhibited in the presence of either anti-CD1d or anti-programmed death ligand-1 (PD-L1) blocking antibodies. The specific potency of DX5+ NKT cells in regulating chronic colitis in two mouse models is demonstrated. In vitro testing suggests that DX5+ NKT cells activated by CD1d induce cell death of colitis-inducing lymphocytes, which is mediated through PD-L1. Therefore, DX5+ NKT cells could be important in the regulation of immune responses associated with chronic colitis.

GENETIC DISORDERS OF PROGRAMMED CELL DEATH IN THE IMMUNE SYSTEM

Human genetics offers new possibilities for understanding physiological regulatory mechanisms and disorders of the immune system. Genetic abnormalities of lymphocyte cell death programs have provided insights into mechanisms of receptor biology and principles of immune homeostasis and tolerance. Thus far, there are two major diseases of programmed cell death associated with inherited human mutations: the autoimmune lymphoproliferative syndrome and the caspase-eight deficiency state. We describe the details of their molecular pathogenesis and discuss how these diseases illustrate important concepts in immune regulation and genetics.

Inflammatory Biomarkers in Acute Coronary Syndromes

The inflammatory etiology of atherosclerosis has prompted a search for biomarkers of inflammation that predict risk for coronary artery disease and its sequelae. Within the acute coronary syndromes (ACS), inflammatory biomarkers may provide independent information regarding pathophysiology, prognosis, and optimal therapeutic strategies. On the basis of the hypothesis that different pathophysiological processes provide nonoverlapping information regarding risk stratification and disease management, this review series addresses biomarkers for each step in the inflammatory process that leads to ACS. Part I reviewed cytokines; this part reviews acute-phase reactants and biomarkers of endothelial cell activation; subsequent parts will address biomarkers of oxidative stress, angiogenesis, extracellular matrix degradation, and platelet activation. In the acute-phase response, cytokines drive production of acute-phase reactants, defined by >25% change in circulating concentration during an inflammatory response. These markers may also remain elevated chronically because of continuing inflammatory stimuli (Table). View this table: Inflammatory Biomarkers in ACS ### C-Reactive Protein C-reactive protein (CRP) is a pentraxin acute-phase protein, members of which are evolutionarily conserved in most vertebrates.1 Hepatocytes and possibly smooth muscle cells and macrophages transcriptionally activate production of CRP in response to inflammatory cytokines, including interleukin-1 and interleukin-6.2 CRP is a robust clinical marker because of its stability, reproducible results, and ease of assay. Although it was originally proposed as a nonspecific marker of inflammation, several reports suggest that CRP may play a direct pathophysiological role in the development and progression of atherosclerosis. Proposed mechanisms include induction of endothelial dysfunction,3 promotion of foam cell formation,4 inhibition of endothelial progenitor cell survival and differentiation,5 and activation of complement in atherosclerotic plaque intima6 and ischemic myocardium.7 Patients with ACS have elevations in CRP in association with their presenting symptoms. There appears to be a bimodal CRP response among patients with ACS. In some patients, CRP may remain elevated for …

Resveratrol Induces Apoptosis in an Activated B-Cell Lymphoma Line OCI-LY3

RATIONALE: Keeping with data showing the polyphenolic compound resveratrol has proven anti-tumor properties in several distinct cell lines, we examined efficacy in a subset of diffuse large B cell lymphoma (DLBCL), activated B-cell (ABC) lymphoma.METHODS: OCI-LY3 cells were cultured and prepared for flow cytometry, western blot analysis, electrophoretic mobility shift assay (EMSA) analysis and siRNA experiments.RESULTS: Resveratrol, which has been previously shown to induce 5% cell death in normal human lymphocytes at a concentration of 100μM, was shown to induce substantial (>70%) cell death in OCI-LY3 cells. We have found that the mechanism underlying cell death involves the downregulation of the AKT/MEK/ERK pathway, as well as the anti-apoptotic protein Bcl-xL. The downregulation of Bcl-xL occurs despite the upregulation of NF-κB, an anti-apoptotic transcription factor which (i) is a major part of the unique ABC gene signature and (ii) binds specifically to κB elements in the Bcl-xL promoter and is thought to cause upregulation of the Bcl-xL gene.CONCLUSIONS: Resveratrol induces apoptosis in the ABC DLBCL cell line OCI-LY3 in a dose-dependent manner involving the inactivation of the AKT/MEK/ERK pathway and reduction in Bcl-xL protein, despite the resveratrol-induced upregulation of NF-κB. Thus, we show a mechanism in which resveratrol plays an anti-cancerous role and present data that resveratrol is a potent inducer of cell death in a subset of lymphoma in which traditional chemotherapy remains fairly ineffective. RATIONALE: Keeping with data showing the polyphenolic compound resveratrol has proven anti-tumor properties in several distinct cell lines, we examined efficacy in a subset of diffuse large B cell lymphoma (DLBCL), activated B-cell (ABC) lymphoma. METHODS: OCI-LY3 cells were cultured and prepared for flow cytometry, western blot analysis, electrophoretic mobility shift assay (EMSA) analysis and siRNA experiments. RESULTS: Resveratrol, which has been previously shown to induce 5% cell death in normal human lymphocytes at a concentration of 100μM, was shown to induce substantial (>70%) cell death in OCI-LY3 cells. We have found that the mechanism underlying cell death involves the downregulation of the AKT/MEK/ERK pathway, as well as the anti-apoptotic protein Bcl-xL. The downregulation of Bcl-xL occurs despite the upregulation of NF-κB, an anti-apoptotic transcription factor which (i) is a major part of the unique ABC gene signature and (ii) binds specifically to κB elements in the Bcl-xL promoter and is thought to cause upregulation of the Bcl-xL gene. CONCLUSIONS: Resveratrol induces apoptosis in the ABC DLBCL cell line OCI-LY3 in a dose-dependent manner involving the inactivation of the AKT/MEK/ERK pathway and reduction in Bcl-xL protein, despite the resveratrol-induced upregulation of NF-κB. Thus, we show a mechanism in which resveratrol plays an anti-cancerous role and present data that resveratrol is a potent inducer of cell death in a subset of lymphoma in which traditional chemotherapy remains fairly ineffective.

Cellular and biochemical parameters of exercise-induced oxidative stress: Relationship with training levels

To better clarify the relationship between physical activity and oxidative stress, we determined the effects of a maximal test in 18 young subjects with different training levels (six professional Athletes and 12 non-agonists (NA)). Redox homeostasis (total antioxidant activity (TAS), vitamin C and glutathione (GSH)), oxidative damage (diene conjugation and hemolysis), lymphocyte cell death and repair systems (apoptosis, micronuclei and Hsp70 expression) were evaluated. We found that agonistic training led to a chronic oxidative insult (high baseline values of oxidized glutathione (GSSG), micronuclei and hemolysis). On the contrary, NA with the lowest level of training frequency showed a well balanced profile at rest, but they were more susceptible to exercise-induced variations (GSSG/GSH and diene increased values), respect to the NA with an higher level of training. As almost all the parameters employed in this study showed inter-individual variations, the GSSG/GSH ratio remains the most sensitive and reliable marker of oxidative stress, accordingly with other data just reported in the literature.

The Role of Apoptotic Inducing Factor (AIF) in Acute Myeloid Leukemia (AML).

Apoptosis is a “physiological cell suicide program” and plays an integral role in a variety of biological events. Numerous genes are involved: Apoptosis inducing factor (AIF) is a newly discovered apoptogenic flavoprotein with NADH oxidase activity that is located in the intermembrane space of mitochondria. The mechanism of induction of cell death by AIF remains unclear. Some data suggest that AIF may play a central role in the regulation of caspase-independent cell death and participate in multiple cell death paradigms. When cells are entering apoptosis, AIF will translocate from the mitochondria to the nuclei causing DNA fragmentation and chromatin condensation. AIF also induces cell death in normal lymphocytes and leukemia cells, but the data is very limited. In this study, we investigate 1) whether AIF inducing cell death is caspase-independent and related to MMP, 2) whether AIF expression levels can be prognostic in primary AML. To determine AIF nuclear translocation in cells undergoing apoptosis is caspase-dependent or caspase-independent, OCI/AML3 cells were treated with several chemotherapeutic agents (Paclitaxol, Vincristine, Ara-C, and Doxorubicin with/without pan-caspase inhibitor IDN-1529). Cells were then fixed, immuno-stained, and analyzed. Results demonstrate that AIF translocated from mitochondria to the nucleus when cell undergo apoptosis, and that it is largely caspase-independent than caspase-dependent. To examine how the mitochondrial membrane potential (MMP) is involved in AIF's nuclear translocation, OCI/AML3 cells was stained with CMXRos and MitoTrack-green after drug treatment with/without caspase inhibitors and then analyzed by flow cytometry. 90% of untreated cells retained normal MMP. Vincristine and paclitaxol treated cells had a more complete depolarization of mitochondria (75.6% ± 4.3% and 79.3% ± 5.4%, respectively), even when caspase inhibitor was added (51.4% ± 3%; p=0.025; and 52.7% ± 5.1%; p=0.006, respectively). These results suggest that the higher ratio of AIF nuclear-translocation observed in cells undergoing apoptosis in Vincristine and paclitaxel treated cells is due to changes in MMP. To further test the function of AIF in AML, small interference RNA (siRNA) was employed to knockdown AIF gene expression in U937 cells and treated with the triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO). AIF siRNA was delivered by electroporation. Results suggest that nuclear translocation of AIF is prevented by silencing AIF expression. Down-regulation of AIF prior to CDDO exposure significantly reduced CDDO-induced growth inhibition (p<0.002). CDDO-induced apoptosis was decreased by approximately 50% in TUNEL assay. To test AIF expression in AML patients, we use immunobloting of bone marrow (BM) and peripheral blood (PB) cells. Twenty-five samples from AML patients were analyzed and the results demonstrated the correlation between blast count and AIF levels (p=0.04). Comparing PB from leukemia patients with normal PB, the p volume is 0.02. Taken together, these results suggest that 1) AIF is inducing cell death through a caspase-independent and caspase-dependent pathway related to MMP, 2) The difference of AIF expression levels in AML suggest a role in the regulation of apoptosis. It may be used as a prognosis marker.

Prostate-Apoptosis-Response-Gene-4 Increases Sensitivity to TRAIL-Induced Apoptosis.

The capacity of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) to preferentially induce apoptosis in malignant cells while sparing normal tissues renders it an attractive therapeutic agent. Nevertheless, the molecular determinants governing sensitivity towards TRAIL remain to be defined. Acknowledging the previously demonstrated deregulation of prostate-apoptosis-response-gene-4 (par-4) in ex vivo cells of patients suffering from acute and chronic lymphatic leukemia, we here tested the hypothesis that expression of par-4 influences sensitivity to TRAIL. We here show, that expression of par-4 in T-lymphoblastic Jurkat cells i) considerably increases TRAIL-induced apoptosis; ii) enforces cleavage of c-FlipL and the subsequent activation of the initiator caspases-8 and -10; iii) does not alter expression of the Bcl-2 family members Bax and Bak; iv) does not enhance the disruption of mitochondrial membrane potential; v) promotes down-regulation the inhibitor-of-apoptosis proteins cIAP-1, cIAP-2, XIAP and survivin; vi) concomitantly augments activation of the executioner caspases-6 and -7. Moreover we prove the crucial role of caspase-8 in par-4-promoted apoptosis by demonstrating that inhibition of caspase-8 considerably reduces TRAIL-induced apoptosis in mock- as well as par-4-transfected Jurkat clones and reverses the described molecular changes. In conclusion, we here provide first evidence that expression of par-4 determines sensitivity to TRAIL-induced lymphocytic cell death and outline the responsible molecular determinants.

Apoptosis of allospecifically activated human helper T cells is blocked by calcineurin inhibition

Background Classical transplantation immunosuppression relies heavily upon the interruption of interleukin-2 (IL-2) signaling by calcineurin inhibition. However, recent evidence in murine models suggests that IL-2 is necessary for activation-induced cell death (AICD) of allograft-specific lymphocytes. Methods We examined the apoptotic effects of the calcineurin inhibitor cyclosporine A and mTOR inhibitor rapamycin on the apoptotic alterations that occur in allospecifically activated human lymphocytes in a one-way mixed lymphocyte culture (MLC). Results Cyclosporine increased caspase-3 activation in MLC, which corresponded with a decrease in lymphocyte apoptosis in MLC. Cyclosporine also reduced apoptosis in the CD4+ helper T cell subset, while CD8+ cells had similar or increased apoptosis when compared to controls. In contrast, rapamycin-treated cultures had normal levels of CD4+ T cell apoptosis when compared to control MLC, with decreases seen in CD8+ T lymphocytes. Conclusions In humans, blockade of IL-2 receptor signal with rapamycin allows apoptosis of allospecifically activated CD4+ lymphocytes to occur, while blockade of IL-2 production with cyclosporine results in decreased apoptosis in this T cell subset. As helper T cells are integral to the immune response, these results may explain the tolerogenic effects of rapamycin.

Transcription inhibition by flavopiridol: mechanism of chronic lymphocytic leukemia cell death

AbstractFlavopiridol is active against chronic lymphocytic leukemia (CLL) cells in vitro and in the treatment of advanced stage disease, but the mechanisms of these actions remain unclear. Originally developed as a general cyclin-dependent kinase inhibitor, flavopiridol is a potent transcriptional suppressor through the inhibition of positive transcription elongation factor b (P-TEFb; CDK9/cyclin T). P-TEFb phosphorylates the C-terminal domain (CTD) of RNA polymerase II to promote transcriptional elongation. Because most CLL cells are not actively cycling, and their viability is dependent upon the continuous expression of antiapoptotic proteins, we hypothesized that flavopiridol induces apoptosis in CLL cells through the transcriptional down-regulation of such proteins. This study demonstrated that flavopiridol inhibited the phosphorylation of the CTD of RNA polymerase II in primary CLL cells and reduced RNA synthesis. This was associated with a decline of the transcripts and the levels of short-lived antiapoptotic proteins such as myeloid cell leukemia 1 (Mcl-1), and resulted in the induction of apoptosis. The B-cell lymphoma 2 (Bcl-2) protein level remained stable, although its mRNA was consistently reduced, suggesting that the outcome of transcriptional inhibition by flavopiridol is governed by the intrinsic stability of the individual transcripts and proteins. The dependence of CLL-cell survival on short-lived oncoproteins may provide the biochemical basis for the therapeutic index in response to flavopiridol. (Blood. 2005;106:2513-2519)