Acute lymphoblastic leukemia-derived extracellular vesicles affect quiescence of hematopoietic stem and progenitor cells.

Patient-derived xenografted (PDX) models were generated through the transplantation of primary acute lymphoblastic leukemia (ALL) cells into immunodeficient NSG mice. We observed that ALL cells from mouse bone marrow (BM) produced extracellular vesicles (EVs) with specific expression of inducible heat shock protein HSP70, which is commonly activated in cancer cells. Taking advantage of this specific expression, we designed a strategy to generate fluorescent HSP70-labeled ALL EVs and monitor the impact of these EVs on endogenous murine BM cells ex vivo and in vivo. We discovered that hematopoietic stem and progenitor cells (HSPC) were mainly targeted by ALL EVs, affecting their quiescence and maintenance in the murine BM environment. Investigations revealed that ALL EVs were enriched in cholesterol and other metabolites that contribute to promote the mitochondrial function in targeted HSPC. Furthermore, using CD34+ cells isolated from cord blood, we confirmed that ALL EVs can modify quiescence of human HSPC. In conclusion, we have discovered a new oncogenic mechanism illustrating how EVs produced by proliferative ALL cells can target and compromise a healthy hematopoiesis system during leukemia development.

HS-AFM and SERS Analysis of Murine Norovirus Infection: Involvement of the Lipid Rafts.

Studies on human norovirus are severely hampered by the absence of a cell culture system until the discovery of murine norovirus (MNV). The cell membrane domains called lipid rafts have been defined as a port of entry for viruses. This study is conducted to investigate murine norovirus binding on the mouse leukemic monocyte macrophage cell line. Lipid raft related structures are extracted from cells by detergent treatment resulting detergent-resistant membrane (DRMs) domains. The real-time polymerase chain reaction technique is performed to detect the viral genome, thereby the MNV binding on the DRMs. The interactions between MNV and DRMs are investigated by high-speed atomic force microscopy (HS-AFM) combined with surface-enhanced Raman spectroscopy (SERS). The inoculation of the virus onto cells results in the aggregations of detergent-resistant membrane domains significantly. The characteristic Raman band of MNV is found in inoculated samples. To be sure that these results are originated from specific interactions between DRM and MNV, methyl-ß-cyclo-dextrin (MßCD) is applied to disrupt lipid rafts. The MNV binding on DRMs is precluded by the MßCD treatment. The cholesterols chains are defined as a key factor in the interactions between norovirus and DRMs. The authors conclude that the MNV binding involves the presence of DRMs and cholesterol dependent.

Association between Admission Serum 25-Hydroxyvitamin D Levels and Functional Outcome of Thrombolyzed Stroke Patients.

BACKGROUND: 25-Hydroxyvitamin D (25(OH)D) deficiency is a frequent condition in patients who suffer a stroke, and several studies suggested that it may be associated with a poorer prognosis. The aim of this study was to investigate specifically the association between 25(OH)D levels and functional outcome at 3 months in ischemic stroke patients treated with intravenous thrombolysis. METHODS: Consecutive ischemic stroke patients who received intravenous thrombolysis were enrolled between 2010 and 2013. Baseline characteristics were collected, and serum concentrations of 25(OH)D were measured within the first 24 hours after admission and were analyzed according to the quartiles of their distribution (<25 nmol/L versus ≥ 25 nmol/L). Multivariable ordinal logistic regression was used to evaluate the association between 25(OH)D and 3-month functional outcome assessed by the modified Rankin score. RESULTS: Three hundred fifty-two patients were included (mean age 68.6 ± 15.8, 50.7% women, mean 25(OH)D level 45 ± 25 nmol/L). The characteristics of the patients only differed with regard to higher premorbid functional impairment in patients with low 25(OH)D. In univariate analysis, the risk of functional impairment in patients with low 25(OH)D levels was greater than that in patients with higher 25(OH)D levels (odds ratio [OR] 2.10, 95% confidence interval [CI]: 1.35-3.27, P = .001). This association was still observed after adjustment for confounding variables (OR 1.70, 95% CI: 1.06-2.71, P = .027). CONCLUSION: A low serum 25(OH)D level is associated with worse functional outcome in patients with acute ischemic stroke treated with intravenous thrombolysis. Further investigations are required to understand the underlying mechanisms of this association.

Plasma phospholipid transfer protein (PLTP) modulates adaptive immune functions through alternation of T helper cell polarization.

OBJECTIVE: Plasma phospholipid transfer protein (PLTP) is a key determinant of lipoprotein metabolism, and both animal and human studies converge to indicate that PLTP promotes atherogenesis and its thromboembolic complications. Moreover, it has recently been reported that PLTP modulates inflammation and immune responses. Although earlier studies from our group demonstrated that PLTP can modify macrophage activation, the implication of PLTP in the modulation of T-cell-mediated immune responses has never been investigated and was therefore addressed in the present study. Approach and results: In the present study, we demonstrated that PLTP deficiency in mice has a profound effect on CD4+ Th0 cell polarization, with a shift towards the anti-inflammatory Th2 phenotype under both normal and pathological conditions. In a model of contact hypersensitivity, a significantly impaired response to skin sensitization with the hapten-2,4-dinitrofluorobenzene (DNFB) was observed in PLTP-deficient mice compared to wild-type (WT) mice. Interestingly, PLTP deficiency in mice exerted no effect on the counts of total white blood cells, lymphocytes, granulocytes, or monocytes in the peripheral blood. Moreover, PLTP deficiency did not modify the amounts of CD4+ and CD8+ T lymphocyte subsets. However, PLTP-deficiency, associated with upregulation of the Th2 phenotype, was accompanied by a significant decrease in the production of the pro-Th1 cytokine interleukin 18 by accessory cells. CONCLUSIONS: For the first time, this work reports a physiological role for PLTP in the polarization of CD4+ T cells toward the pro-inflammatory Th1 phenotype.

T Lymphocyte Inhibition by Tumor-Infiltrating Dendritic Cells Involves Ectonucleotidase CD39 but Not Arginase-1.

T lymphocytes activated by dendritic cells (DC) which present tumor antigens play a key role in the antitumor immune response. However, in patients suffering from active cancer, DC are not efficient at initiating and supporting immune responses as they participate to T lymphocyte inhibition. DC in the tumor environment are functionally defective and exhibit a characteristic of immature phenotype, different to that of DC present in nonpathological conditions. The mechanistic bases underlying DC dysfunction in cancer responsible for the modulation of T-cell responses and tumor immune escape are still being investigated. Using two different mouse tumor models, we showed that tumor-infiltrating DC (TIDC) are constitutively immunosuppressive, exhibit a semimature phenotype, and impair responder T lymphocyte proliferation and activation by a mechanism involving CD39 ectoenzyme.

[Mass spectrometry and inherited metabolic diseases diagnosis]. / Spectrométrie de masse et diagnostic des maladies héréditaires du métabolisme.

Tandem mass spectrometry is used for the diagnosis and following of metabolic disorders for acylcarnitine profiling and with liquide chromatography to explore creatin metabolism and amino and bile acids disorders. The analysis of organic acids by GC-MS is still the reference method for the diagnosis of inherited disorders of organiques acides and sterols. These techniques are also used to perform "in vitro" functional tests.

Human monocyte-derived suppressor cells control graft-versus-host disease by inducing regulatory forkhead box protein 3-positive CD8+ T lymphocytes.

BACKGROUND: Adoptive transfer of immunosuppressive cells has emerged as a promising strategy for the treatment of immune-mediated disorders. However, only a limited number of such cells can be isolated from in vivo specimens. Therefore efficient ex vivo differentiation and expansion procedures are critically needed to produce a clinically relevant amount of these suppressive cells. OBJECTIVE: We sought to develop a novel, clinically relevant, and feasible approach to generate ex vivo a subpopulation of human suppressor cells of monocytic origin, referred to as human monocyte-derived suppressive cells (HuMoSCs), which can be used as an efficient therapeutic tool to treat inflammatory disorders. METHODS: HuMoSCs were generated from human monocytes cultured for 7 days with GM-CSF and IL-6. The immune-regulatory properties of HuMoSCs were investigated in vitro and in vivo. The therapeutic efficacy of HuMoSCs was evaluated by using a graft-versus-host disease (GvHD) model of humanized mice (NOD/SCID/IL-2Rγc(-/-) [NSG] mice). RESULTS: CD33+ HuMoSCs are highly potent at inhibiting the proliferation and activation of autologous and allogeneic effector T lymphocytes in vitro and in vivo. The suppressive activity of these cells depends on signal transducer and activator of transcription 3 activation. Of therapeutic relevance, HuMoSCs induce long-lasting memory forkhead box protein 3-positive CD8+ regulatory T lymphocytes and significantly reduce GvHD induced with human PBMCs in NSG mice. CONCLUSION: Ex vivo-generated HuMoSCs inhibit effector T lymphocytes, promote the expansion of immunosuppressive forkhead box protein 3-positive CD8+ regulatory T cells, and can be used as an efficient therapeutic tool to prevent GvHD.

Agreement of seven 25-hydroxy vitamin D3 immunoassays and three high performance liquid chromatography methods with liquid chromatography tandem mass spectrometry.

BACKGROUND: Several recent studies have shown some discrepancies between 25-hydroxyvitamin D [25(OH)D] assay methods, despite some improvement in the past few years. The accuracy of 25(OH)D assay methods is still a real challenge for clinical laboratories. The aim of this study was to assess the agreement between a large panel of routine assays and a two-dimensional liquid chromatography/tandem mass spectrometry (2D LC-MS/MS) method, selected as the reference method. METHODS: Forty-nine human plasma samples with only endogenous 25(OH)D3 were analyzed with 11 different methods, especially with three LC-UV methods that differed in the extraction step. Seven routine immunoassays were also tested: two manual (RIA and EIA from IDS) and five fully-automated methods. The results of the 25(OH)D3 assays were compared with those of the 2D LC-MS/MS method using weighted Deming regression analysis, Bland-Altman plots and concordance correlation coefficient (CCC). The ability of these methods to properly classify patients was evaluated by sorting results depending on vitamin D status. RESULTS: The CCC was >0.90 for the three LC-UV methods and for most of the automated IA, meaning substantial agreement with 2D LC-MS/MS results. The ability to properly classify patients according to their vitamin D status was overall satisfactory for most of the methods tested (concordance >90%). CONCLUSIONS: The immunoassays available on Liaison, Isys, Architect and Elecsys, together with our in-house LC-UV method preceded by an SLE step met the minimum requirements for the assessment of vitamin D status in clinical laboratories.

Development of abdominal aortic aneurysm is decreased in mice with plasma phospholipid transfer protein deficiency.

Plasma phospholipid transfer protein (PLTP) increases the circulating levels of proatherogenic lipoproteins, accelerates blood coagulation, and modulates inflammation. The role of PLTP in the development of abdominal aortic aneurysm (AAA) was investigated by using either a combination of mechanical and elastase injury at one site of mouse aorta (elastase model) or continuous infusion of angiotensin II in hyperlipidemic ApoE-knockout mice (Ang II model). With the elastase model, complete PLTP deficiency was associated with a significantly lower incidence and a lesser degree of AAA expansion. With the Ang II model, findings were consistent with those in the elastase model, with a lower severity grade in PLTP-deficient mice, an intermediate phenotype in PLTP-deficient heterozygotes, and a blunted effect of the PLTP-deficient trait when restricted to bone marrow-derived immune cells. The protective effect of whole-body PLTP deficiency in AAA was illustrated further by a lesser degree of adventitia expansion, reduced elastin degradation, fewer recruited macrophages, and less smooth muscle cell depletion in PLTP-deficient than in wild-type mice, as evident from comparative microscopic analysis of aorta sections. Finally, cumulative evidence supports the association of PLTP deficiency with reduced expression and activity levels of matrix metalloproteinases, known to degrade elastin and collagen. We conclude that PLTP can play a significant role in the pathophysiology of AAA.

[Evaluation of the new Hevylite™ IgA assay for the diagnosis and follow-up of monoclonal gammopathies]. / Évaluation du nouveau test Hevylite™ IgA dans le diagnostic et le suivi des gammapathies monoclonales.

Multiple myeloma diagnosis and follow-up are based on monoclonal protein measurement. The estimation of monoclonal immunoglobulin production requires serum protein electrophoresis, immunoelectrophoresis and free light chain assay. However these classical assays have some limitations. Hevylite™ IgA (Binding Site) is a new nephelometric/turbidimetric assay allowing the IgA κ and IgA λ measurement. The aim of this study was to determine the performance of this assay, for the diagnosis and follow-up of myeloma patients at different stages. Sixty seven frozen sera from 26 patients were assayed. Total IgA, IgA κ, IgA λ concentrations, serum protein electrophoresis and serum immunofixation were performed at diagnosis and during follow-up. All myeloma patients had an abnormal IgA κ/IgA λ ratio at diagnosis. During disease monitoring, the IgA κ or IgA λ concentrations correlated well with the electrophoretic estimation of the monoclonal spike and the values of total IgA. Hevylite™ test was more sensitive than serum protein electrophoresis and provided numerical and reproductible assessment of the monoclonal and non-monoclonal isotype. The IgA κ/IgA λ ratio allowed early prediction of disease relapse. Hevylite™ is an interesting assay especially when the monoclonal IgA comigrates on electrophoresis with normal proteins making impossible a reliable densitometric estimation. Hevylite™ might become an important assay in the biological exploration of gammopathies.

Lipid rafts: a signalling platform linking lipoprotein metabolism to atherogenesis.

Lipid rafts are microdomains of the plasma membrane which are enriched in cholesterol and sphingolipids. They serve as a platform for signal transduction, in particular during immune and inflammatory responses. As hypercholesterolemia and inflammation are two key elements of atherogenesis, it is conceivable that the cholesterol and cholesterol oxide content of lipid rafts might influence the inflammatory signalling pathways, thus modulating the development of atherosclerosis. In support of this emerging view, lipid rafts have been shown to be involved in several key steps of atherogenesis, such as the oxysterol-mediated apoptosis of vascular cells, the blunted ability of high density lipoproteins (HDL) to exert anti-inflammatory effects, and the exacerbated secretion of pro-inflammatory cytokines by immune cells. Additional studies are now required to address the relative contribution of lipid raft abnormalities to the pathophysiology of atherosclerosis and cardiovascular disease.

Mitochondrion-dependent N-terminal processing of outer membrane Mcl-1 protein removes an essential Mule/Lasu1 protein-binding site.

Mcl-1, a pro-survival member of the Bcl-2 family located at the mitochondrial outer membrane, is subject to constitutive ubiquitylation by the Bcl-2 homology 3-only E3 ligase, Mule/Lasu1, resulting in rapid steady-state degradation via the proteasome. Insertion of newly synthesized Mcl-1 into the mitochondrial outer membrane is dependent on its C-terminal transmembrane segment, but once inserted, the N terminus of a portion of the Mcl-1 molecules can be subject to proteolytic processing. Remarkably, this processing requires an intact electrochemical potential across the inner membrane. Three lines of evidence directed at the endogenous protein, however, indicate that the resulting Mcl-1ΔN isoform resides in the outer membrane: (i) full-length Mcl-1 and Mcl-1ΔN resist extraction by alkali but are accessible to exogenous protease; (ii) almost the entire populations of Mcl-1 and Mcl-1ΔN are accessible to the membrane-impermeant Cys-reactive agent 4-acetamido-4'-[(iodoacetyl)amino]stilbene-2,2'-disulfonic acid; and (iii) Mcl-1 and Mcl-1ΔN exhibit equivalent chemical cross-linking to Bak in intact mitochondria, an Mcl-1 binding partner located in the outer membrane. In addition to the Mule Bcl-2 homology 3 domain, we show that interaction between Mcl-1 and Mule also requires the extreme N terminus of Mcl-1, which is lacking in Mcl-1ΔN. Thus, Mcl-1ΔN does not interact with Mule, exhibits reduced steady-state ubiquitylation, evades the hyper-rapid steady-state degradation that is observed for full-length Mcl-1 in response to treatments that limit global protein synthesis, and confers resistance to UV stress-induced cell death.

Plasma phospholipid transfer protein deficiency in mice is associated with a reduced thrombotic response to acute intravascular oxidative stress.

OBJECTIVE: Earlier in vitro studies suggested a putative role for the plasma phospholipid transfer protein (PLTP) in the modulation of blood coagulation. The effect of PLTP expression on blood coagulation under both basal and oxidative stress conditions was compared here in wild-type and PLTP-deficient (PLTP-/-) mice. METHODS AND RESULTS: Under basal conditions, PLTP deficiency was associated with an extended tail bleeding time despite a significant depletion of vascular α-tocopherol content and an impairment of endothelial function. When acute oxidative stress was generated in vivo in the brain vasculature, the steady state levels of oxidized lipid derivatives, the extent of blood vessel occlusion, and the volume of ischemic lesions were more severe in wild-type than in PLTP-/- mice. CONCLUSIONS: In addition to its recognized hyperlipidemic, proinflammatory, and proatherogenic properties, PLTP increases blood coagulation and worsens the extent of ischemic lesions in response to acute oxidative stress. Thus, PLTP arises here as a cardiovascular risk factor for the late thrombotic events occurring in the acute phase of atherosclerosis.

Vitamin E transport, membrane incorporation and cell metabolism: Is alpha-tocopherol in lipid rafts an oar in the lifeboat?

Vitamin E is composed of closely related compounds, including tocopherols and tocotrienols. Studies of the last decade provide strong support for a specific role of alpha-tocopherol in cell signalling and the regulation of gene expression. It produces significant effects on inflammation, cell proliferation and apoptosis that are not shared by other vitamin E isomers with similar antioxidant properties. The different behaviours of vitamin E isomers might relate, at least in part, to the specific effects they exert at the plasma membrane. alpha-Tocopherol is not randomly distributed throughout the phospholipid bilayer of biological membranes, and as compared with other isomers, it shows a propensity to associate with lipid rafts. Distinct aspects of vitamin E transport and metabolism is discussed with emphasis on the interaction between alpha-tocopherol and lipid rafts and the consequences of these interactions on cell metabolism.

7-ketocholesterol incorporation into sphingolipid/cholesterol-enriched (lipid raft) domains is impaired by vitamin E: a specific role for alpha-tocopherol with consequences on cell death.

Cholesterol oxides, in particular 7-ketocholesterol, are proatherogenic compounds that induce cell death in the vascular wall when localized in lipid raft domains of the cell membrane. Deleterious effects of 7-ketocholesterol can be prevented by vitamin E, but the molecular mechanism involved is unclear. In this study, unlike gamma-tocopherol, the alpha-tocopherol vitamin E form was found to prevent 7-ketocholesterol-mediated apoptosis of A7R5 smooth muscle cells. To be operative, alpha-tocopherol needed to be added to the cells before 7-ketocholesterol, and its anti-apoptotic effect was reduced and even suppressed when added together or after 7-ketocholesterol, respectively. Both pre- and co-treatment of the cells with alpha-tocopherol resulted in the redistribution of 7-ketocholesterol out of the sphingolipid/cholesterol-enriched (lipid raft) domains. In turn, fewer amounts of alpha-tocopherol associated with lipid rafts on 7-ketocholesterol-pretreated cells compared with untreated cells, with no prevention of cell death in this case. In further support of the implication of lipid raft domains, the dephosphorylation/inactivation of Akt-PKB was involved in the 7-ketocholesterol-induced apoptosis. Akt-PKB dephosphorylation was prevented by alpha-tocopherol, but not gamma-tocopherol pretreatment.

7beta-Hydroxycholesterol and 25-hydroxycholesterol-induced interleukin-8 secretion involves a calcium-dependent activation of c-fos via the ERK1/2 signaling pathway in THP-1 cells: oxysterols-induced IL-8 secretion is calcium-dependent.

Oxysterols found in oxidized low-density lipoproteins are probably involved in the appearance of atheroma; some are cytotoxic and some able to induce cytokine secretion. An oxysterol-induced interleukin-8 (IL-8) secretion in human monocytes/macrophages has been previously noticed, but the mechanisms remained unclear. In this paper, we investigated the signaling pathways leading to the induction of IL-8 secretion in monocytic THP-1 cells treated with 7beta-hydroxycholesterol, a cytototoxic oxysterol, or with 25-hydroxycholesterol, an oxysterol non-cytotoxic toward this cell line. The oxysterol-induced IL-8 secretion appears to be a calcium-dependent phenomenon as shown by the use of calcium channel blockers, which strongly decreased IL-8 secretion and IL-8 messenger RNA (mRNA) levels. Fluo-3 staining used in flow cytometry and video microscopy revealed an oxysterol-induced Ca(2+) influx, varying according to the oxysterol studied, leading to the activation of the MEK/ERK1/2 pathway as demonstrated by Western blot analysis. ERK activation led to an increase of c-fos mRNA and/or an activation of c-fos. Luciferase reporter gene assay using constructs of the human IL-8 gene promoter and Transam assay revealed the involvement of the AP-1 transcription factor in oxysterol-dependent IL-8 secretion. These results demonstrate that oxysterol-induced IL-8 secretion is a calcium-dependent phenomenon involving the MEK/ERK1/2 pathway leading to the activation of IL-8 gene via AP-1 (c-fos).

In high-risk patients, combination of antiinflammatory procedures during cardiopulmonary bypass can reduce incidences of inflammation and oxidative stress.

Previous studies showed that cardiopulmonary bypass (CPB) was directly associated with a global activation of the inflammatory response, production of oxygen free radicals, and signs of myocardial injury. We therefore evaluated, in the weakest patients, the biological and clinical benefits of a therapeutic optimization of CPB through the combination of several antiinflammatory procedures. High-risk patients undergoing cardiac surgery under CPB were included in this prospective randomized study. Control patients (n = 14) underwent conventional CPB, and treated patients (n = 13) underwent a CPB with Baxter Duraflo II heparin-coated circuits, high doses of aprotinin, and pre-CPB hemofiltration. Usual clinical hemodynamic and biological criteria, inflammation, and oxidative stress markers were measured before, during, and to the second postoperative day. Free radicals were quantified using electronic spin resonance spectroscopy with a spin trap. Significantly lower concentrations of C-reactive protein, interleukin-6, creatine kinase-MB, I-troponin, lactic acid, and systemic free radicals were observed in the plasma of treated patients. These patients had a reduction of postoperative complications and of the length of stay in the intensive care unit. Therefore, pre-CPB therapeutic optimization can reduce the inflammatory response, lower the level of oxidative stress, and help to ameliorate clinical outcome in high-risk patients.

Multiplexed flow cytometric analyses of pro- and anti-inflammatory cytokines in the culture media of oxysterol-treated human monocytic cells and in the sera of atherosclerotic patients.

BACKGROUND: Some oxysterols are identified in atheromatous plaques and in plasma of atherosclerotic patients. We asked whether they might modulate cytokine secretion on human monocytic cells. In healthy and atherosclerotic subjects, we also investigated the relationships between circulating levels of C-reactive protein (CRP), conventional markers of hyperlipidemia, some oxysterols (7beta-hydroxycholesterol, 7-ketocholesterol, and 25-hydroxycholesterol), and various cytokines. METHODS: Different flow cytometric bead-based assays were used to quantify some cytokines (IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, G-CSF, GM-CSF, IFN-gamma, MCP-1, MIP-1beta, or TNF-alpha) in the culture media of oxysterol-treated U937 and THP-1 cells, and in the sera of healthy and atherosclerotic subjects. CRP and markers of hyperlipidemia were determined with routine analytical methods. Oxysterols were quantified by gas chromatography/mass spectrometry. Flow cytometric and biochemical methods were used to measure IL-8 mRNA levels, intracellular IL-8 content, and protein phosphorylation in the mitogenic extracellular kinase/extracellular signal-regulated kinase1/2 (MEK/ERK1/2) signaling pathway. RESULTS: All oxysterols investigated are potent in vitro inducers of MCP-1, MIP-1beta, TNF-alpha, and/or IL-8 secretion, the latter involving the MEK/ERK1/2 cell signaling pathway. In healthy and atherosclerotic subjects, no relationships were found between cytokines (IL-8, IL-1beta, IL-6, IL-10, TNF-alpha, IL-12, and MCP-1), CRP, conventional markers of hyperlipidemia, and oxysterols. However, in patients with arterial disorders of the lower limbs, small but statistically significant differences in the circulating levels of CRP, TNF-alpha, and IL-10 were observed comparatively to healthy subjects and according to the atherosclerotic stage considered. CONCLUSIONS: Flow cytometric bead-based assays are well adapted to measure variations of cytokine secretion in the culture media of oxysterol-treated cells and in the sera of healthy and atherosclerotic subjects. They underline the in vitro proinflammatory properties of oxysterols and may permit to distinguish healthy and atherosclerotic subjects, as well as various atherosclerotic stages.

Activation of caspase-3-dependent and -independent pathways during 7-ketocholesterol- and 7beta-hydroxycholesterol-induced cell death: a morphological and biochemical study.

On treatment with 7-ketocholesterol (7-keto) or 7beta-hydroxycholesterol (7beta-OH), which are major oxysterols in atherosclerotic plaques, the simultaneous identification of oncotic and apoptotic cells suggests that these compounds activate different metabolic pathways leading to various modes of cell death. With U937, MCF-7 (caspase-3 deficient), MCF-7/c3 cells (stably transfected with caspase-3), we demonstrate that caspase-3 is essential for caspase-9, -7, -8 activation, for Bid degradation mediating mitochondrial cytochrome c release, for cleavage of poly(ADP-ribose) polymerase and inhibitor of the caspase-activated deoxyribonuclease, and, at least in part, for internucleosomal DNA fragmentation. The crucial role of caspase-3 was supported by the use of z-VAD-fmk and z-DEVD-fmk, which abolished apoptosis and the associated events. However, inactivation or lack of caspase-3 did not inhibit 7-keto- and 7beta-OH-induced cell death characterized by staining with propidium iodide, loss of mitochondrial potential. The mitochondrial release of apoptosis-inducing factor and endonuclease G was independent of the caspase-3 status, which conversely played major roles in the morphological aspects of dead cells. We conclude that caspase-3 is essential to trigger 7-keto- and 7beta-OH-induced apoptosis, that these oxysterols simultaneously activate caspase-3-dependent and/or -independent modes of cell death.