Edelfosine-induced metabolic changes in cancer cells that precede the overproduction of reactive oxygen species and apoptosis

Background: Metabolic flux profiling based on the analysis of distribution of stable isotope tracer in metabolites is an important method widely used in cancer research to understand the regulation of cell metabolism and elaborate new therapeutic strategies. Recently, we developed software Isodyn, which extends the methodology of kinetic modeling to the analysis of isotopic isomer distribution for the evaluation of cellular metabolic flux profile under relevant conditions. This tool can be applied to reveal the metabolic effect of proapoptotic drug edelfosine in leukemia Jurkat cell line, uncovering the mechanisms of induction of apoptosis in cancer cells. Results: The study of 13C distribution of Jukat cells exposed to low edelfosine concentration, which induces apoptosis in ¿5% of cells, revealed metabolic changes previous to the development of apoptotic program. Specifically, it was found that low dose of edelfosine stimulates the TCA cycle. These metabolic perturbations were coupled with an increase of nucleic acid synthesis de novo, which indicates acceleration of biosynthetic and reparative processes. The further increase of the TCA cycle fluxes, when higher doses of drug applied, eventually enhance reactive oxygen species (ROS) production and trigger apoptotic program. Conclusion: The application of Isodyn to the analysis of mechanism of edelfosine-induced apoptosis revealed primary drug-induced metabolic changes, which are important for the subsequent initiation of apoptotic program. Initiation of such metabolic changes could be exploited in anticancer therapy.

Mechanisms of apoptosis in retinitis pigmentosa.

Mutations in humans are associated with several forms of inherited retinal dystrophies, such as Retinitis Pigmentosa which lead to retinal cell death and irreversible loss of vision. Genes involved in affected patients mainly encode proteins related to vision physiology including visual cycle and light-dependent phototransduction cascade. As reported in spontaneous and genetically engineered mouse models, apoptosis is a common fate in retinal degeneration, although the triggered signals to retinal apoptosis remain largely unraveled. Several studies highlighted that many of the molecular pathways involved in ocular diseases rely on caspase-dependent or -independent apoptotic mitochondrial pathway involving the Bcl-2 family of proteins. Anti- and pro-apoptotic Bcl-2 members are present in retinal tissues and are thought to play a role in the pathogenesis of several retinal disorders. Since almost no efficient treatments are available so far, it remains a great challenge to decipher the molecular pathways involved in retinal dystrophies and to develop alternative therapies to prevent or inhibit eye defect. Toward this goal, mutation-independent strategies such as molecular therapy provides promising and exciting approaches to deliver anti-apoptotic molecules targeting the Bcl-2 pathway through the use of cell permeable transport peptides. Modulation of common apoptotic signaling pathways may be of outstanding potential to target multiple retinal dystrophies regardless of the primary genetic defect.

Autophagy induction does not protect retina against apoptosis in ischemia/reperfusion model.

The role played by autophagy after ischemia/reperfusion (I/R) in the retina remains unknown. Our study investigated whether ischemic injury in the retina, which causes an energy crisis, would induce autophagy. Retinal ischemia was induced by elevation of the intraocular pressure and modulation of autophagic markers was analyzed at the protein levels in an early and late phase of recovery. Following retinal ischemia an increase in LC3BII was first observed in the early phase of recovery but did not stay until the late phase of recovery. Post-ischemic induction of autophagy by intravitreal rapamycin administration did not provide protection against the lesion induced by the ischemic stress. On the contrary, an increase in the number of apoptotic cells was observed following I/R in the rapamycin treated retinas.

Lentivirus-mediated Bcl-2 expression in betaTC-tet cells improves resistance to hypoxia and cytokine-induced apoptosis while preserving in vitro and in vivo control of insulin secretion.

betaTC-tet cells are conditionally immortalized pancreatic beta cells which can confer long-term correction of hyperglycemia when transplanted in syngeneic streptozocin diabetic mice. The use of these cells for control of type I diabetes in humans will require their encapsulation and transplantation in non-native sites where relative hypoxia and cytokines may threaten their survival. In this study we genetically engineered betaTC-tet cells with the anti-apoptotic gene Bcl-2 using new lentiviral vectors and showed that it protected this cell line against apoptosis induced by hypoxia, staurosporine and a mixture of cytokines (IL-1beta, IFN-gamma and TNF-alpha). We further demonstrated that Bcl-2 expression permitted growth at higher cell density and with shorter doubling time. Expression of Bcl-2, however, did not inter- fere either with the intrinsic mechanism of growth arrest present in the betaTC-tet cells or with their normal glucose dose-dependent insulin secretory activity. Furthermore, Bcl-2 expressing betaTC-tet cells retained their capacity to secrete insulin under mild hypoxia. Finally, transplantation of these cells under the kidney capsule of streptozocin diabetic C3H mice corrected hyperglycemia for several months. These results demonstrate that the murine betaTC-tet cell line can be genetically modified to improve its resistance against different stress-induced apoptosis while preserving its normal physiological function. These modified cells represent an improved source for cell transplantation therapy of type I diabetes.

Autophagy defect is associated with low glucose-induced apoptosis in 661W photoreceptor cells.

Glucose is an important metabolic substrate of the retina and diabetic patients have to maintain a strict normoglycemia to avoid diabetes secondary effects, including cardiovascular disease, nephropathy, neuropathy and retinopathy. Others and we recently demonstrated the potential role of hypoglycemia in diabetic retinopathy. We showed acute hypoglycemia to induce retinal cell death both in vivo during an hyperinsulinemic/hypoglycemic clamp and in vitro in 661W photoreceptor cells cultured at low glucose concentration. In the present study, we showed low glucose to induce a decrease of BCL2 and BCL-XL anti-apoptotic proteins expression, leading to an increase of free pro-apoptotic BAX. In parallel, we showed that, in retinal cells, low glucose-induced apoptosis is involved in the process of autophagosomes formation through the AMPK/RAPTOR/mTOR pathway. Moreover, the decrease of LAMP2a expression led to a defect in the autophagosome/lysosome fusion process. Specific inhibition of autophagy, either by 3-methyladenine or by down-regulation of ATG5 or ATG7 proteins expression, increased caspase 3 activation and 661W cell death. We show that low glucose modifies the delicate equilibrium between apoptosis and autophagy. Cells struggled against low nutrient condition-induced apoptosis by starting an autophagic process, which led to cell death when inhibited. We conclude that autophagy defect is associated with low glucose-induced 661W cells death that could play a role in diabetic retinopathy. These results could modify the way of addressing negative effects of hypoglycemia. Short-term modulation of autophagy could be envisioned to treat diabetic patients in order to avoid secondary complications of the disease.

Expression of B220 on activated T cell blasts precedes apoptosis.

Superantigens are bacterial or viral products that polyclonally activate T cells bearing certain TCR beta chain variable elements. For instance, Vbeta8+ T cells proliferate in response to staphylococcal enterotoxin B (SEB) in vivo and then undergo Fas- and/or TNF-mediated apoptosis. We have recently shown that apoptotic SEB-reactive T cells express the B cell marker B220. Here we report the identification of a novel subset of CD4+ B220+ T cell blasts that are the precursors of these apoptotic cells in SEB-immunized mice. Moreover, we show that the CD4- CD8- B220+ T cells that accumulate in the lymphoid organs of Fas ligand-defective gld mice stably express a form of the B220 molecule which exhibits biochemical similarities to that expressed by activated wild-type T cells, but is distinct from that displayed on the surface of B cells. Surprisingly, we also find a population of CD4+ B220+ pre-apoptotic T cells in FasL-defective gld mice, arguing that these cells can be generated in a Fas-independent fashion. Collectively, our data support a general model whereby upon activation, T cells up-regulate B220 before undergoing apoptosis. When the apoptotic mechanisms are defective, T cells presumably down-regulate their coreceptor molecules but retain expression of B220 as they accumulate in lymphoid organs.

Viral inhibitor of apoptosis vFLIP/K13 protects endothelial cells against superoxide-induced cell death.

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma (KS). HHV-8 encodes an antiapoptotic viral Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (vFLIP/K13). The antiapoptotic activity of vFLIP/K13 has been attributed to an inhibition of caspase 8 activation and more recently to its capability to induce the expression of antiapoptotic proteins via activation of NF-kappaB. Our study provides the first proteome-wide analysis of the effect of vFLIP/K13 on cellular-protein expression. Using comparative proteome analysis, we identified manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant and an important antiapoptotic enzyme, as the protein most strongly upregulated by vFLIP/K13 in endothelial cells. MnSOD expression was also upregulated in endothelial cells upon infection with HHV-8. Microarray analysis confirmed that MnSOD is also upregulated at the RNA level, though the differential expression at the RNA level was much lower (5.6-fold) than at the protein level (25.1-fold). The induction of MnSOD expression was dependent on vFLIP/K13-mediated activation of NF-kappaB, occurred in a cell-intrinsic manner, and was correlated with decreased intracellular superoxide accumulation and increased resistance of endothelial cells to superoxide-induced death. The upregulation of MnSOD expression by vFLIP/K13 may support the survival of HHV-8-infected cells in the inflammatory microenvironment in KS.

TRAMP, a novel apoptosis-mediating receptor with sequence homology to tumor necrosis factor receptor 1 and Fas(Apo-1/CD95).

A novel member of the tumor necrosis factor (TNF) receptor family, designated TRAMP, has been identified. The structural organization of the 393 amino acid long human TRAMP is most homologous to TNF receptor 1. TRAMP is abundantly expressed on thymocytes and lymphocytes. Its extracellular domain is composed of four cysteine-rich domains, and the cytoplasmic region contains a death domain known to signal apoptosis. Overexpression of TRAMP leads to two major responses, NF-kappaB activation and apoptosis. TRAMP-induced cell death is inhibited by an inhibitor of ICE-like proteases, but not by Bcl-2. In addition, TRAMP does not appear to interact with any of the known apoptosis-inducing ligands of the TNF family.

Bax-induced apoptosis in Leber's congenital amaurosis : a dual role in rod and cone degeneration

Purpose:We previously observed that anti- and pro-apoptotic genes of the Bcl-2 family were differentially expressed during the development of LCA in the Rpe65-/- mouse model (Cottet et al. 2006). Moreover, we reported that activation and translocation of pro-apoptotic Bax to the mitochondria was associated with apoptosis of rod photoreceptors as the disease progressed (Cottet et al. 2008). In this study we challenged whether disruption of the pro-apoptotic pro-apoptotic Bax protein is sufficient to protect photoreceptor cells against apoptosis. Methods:Apoptosis of photoreceptor cells was addressed by TUNEL assay on flatmounted retinas. Counting of the rod nuclei within the ONL was performed following hematoxylin/eosin histological staining of retina sections. Expression level and localization of photoreceptor gene markers were assessed by quantitative PCR and immunohistological analyses. Results:While expression of rod photoreceptor genes was decreased in Rpe65-deficient retina, expression level remained unchanged in Rpe65-/- / Bax-/- mice. Moreover, OS dysorganization and shortening as well as decrease in ONL thickness observed in diseased retina were prevented in mice lacking functional Bax protein. TUNEL assay confirmed that Bax-dependent rod photoreceptor apoptosis was abolished in Rpe65-/- / Bax-/- mice. However, early and fast degeneration of cone cells was not prevented in Rpe65-/- / Bax-/- mice, indicating that Bax-induced apoptotic pathway was not involved in the degenerating process of cones in Rpe65-deficient retina. Conclusions:Altogether, these data show for the first time that a single genetic mutation can trigger two independent apoptotic pathways in rod and cone photoreceptors in LCA disease. While pro-apoptotic Bax is essential to trigger rod photoreceptor apoptosis, early degeneration of cones is not dependent on Bax-mediated apoptotic pathway in Rpe65-deficientmice.

The fight of viruses against apoptosis.

The induction of apoptosis of virus-infected cells is an important host defense mechanism against invading pathogens. Some viruses express anti-apoptotic proteins that efficiently block apoptosis induced by death receptors or in response to stress signaled through mitochondria. Viral interference with host cell apoptosis leads to enhanced viral replication and may promote cancer.

Early apoptosis of rod photoreceptors in Rpe65(-/-) mice is associated with the upregulated expression of lysosomal-mediated autophagic genes.

RPE65-related Leber's congenital amaurosis (LCA) is a rod-cone dystrophy whose clinical outcome is mainly attributed to the loss of rod photoreceptors followed by cone degeneration. Pathogenesis in Rpe65(-/-) mice is characterized by a slow and progressive degeneration of rods dependent on the constitutive activation of unliganded opsin. We previously reported that this opsin-mediated apoptosis of rods was dependent on Bcl-2-apoptotic pathway and Bax-induced pro-death activity. In this study, we report early initial apoptosis in the newly differentiated retina of Rpe65(-/-) mice. Apoptotic photoreceptors were identified as rods and resulted from pathological phototransduction signaling. This wave of early apoptosis triggered Bcl-2-related pathway and Bax apoptotic activity, while activation of the caspases was not induced. Following cellular stress, multiple signaling pathways are initiated which either commit cells to death or trigger pro-survival responses including autophagy. We report that Bcl-2-related early rod apoptosis was associated with the upregulation of autophagy markers including chaperone-mediated autophagy (CMA) substrate receptor LAMP-2 and lysosomal hydrolases Cathepsin S and Lysozyme. This suggests that lysosomal-mediated autophagy may be triggered in response to early rod apoptosis in Rpe65-LCA disease. These results highlight that Rpe65-related primary stress induces early signaling events, which trigger Bax-induced-apoptotic pathway and autophagy-mediated cellular response. These events may determine retinal cell fate, progression and severity of the disease.

Functional glycosylation of dystroglycan is crucial for thymocyte development in the mouse.

BACKGROUND: Alpha-dystroglycan (alpha-DG) is a cell surface receptor providing a molecular link between the extracellular matrix (ECM) and the actin-based cytoskeleton. During its biosynthesis, alpha-DG undergoes specific and unusual O-glycosylation crucial for its function as a high-affinity cellular receptor for ECM proteins. METHODOLOGY/PRINCIPAL FINDINGS: We report that expression of functionally glycosylated alpha-DG during thymic development is tightly regulated in developing T cells and largely confined to CD4(-)CD8(-) double negative (DN) thymocytes. Ablation of DG in T cells had no effect on proliferation, migration or effector function but did reduce the size of the thymus due to a significant loss in absolute numbers of thymocytes. While numbers of DN thymocytes appeared normal, a marked reduction in CD4(+)CD8(+) double positive (DP) thymocytes occurred. In the periphery mature naïve T cells deficient in DG showed both normal proliferation in response to allogeneic cells and normal migration, effector and memory T cell function when tested in acute infection of mice with either lymphocytic choriomeningitis virus (LCMV) or influenza virus. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that DG function is modulated by glycosylation during T cell development in vivo and that DG is essential for normal development and differentiation of T cells.

Individual caspase-10 isoforms play distinct and opposing roles in the initiation of death receptor-mediated tumour cell apoptosis.

The cysteine protease caspase-8 is an essential executioner of the death receptor (DR) apoptotic pathway. The physiological function of its homologue caspase-10 remains poorly understood, and the ability of caspase-10 to substitute for caspase-8 in the DR apoptotic pathway is still controversial. Here, we analysed the particular contribution of caspase-10 isoforms to DR-mediated apoptosis in neuroblastoma (NB) cells characterised by their resistance to DR signalling. Silencing of caspase-8 in tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive NB cells resulted in complete resistance to TRAIL, which could be reverted by overexpression of caspase-10A or -10D. Overexpression experiments in various caspase-8-expressing tumour cells also demonstrated that caspase-10A and -10D isoforms strongly increased TRAIL and FasL sensitivity, whereas caspase-10B or -10G had no effect or were weakly anti-apoptotic. Further investigations revealed that the unique C-terminal end of caspase-10B was responsible for its degradation by the ubiquitin-proteasome pathway and for its lack of pro-apoptotic activity compared with caspase-10A and -10D. These data highlight in several tumour cell types, a differential pro- or anti-apoptotic role for the distinct caspase-10 isoforms in DR signalling, which may be relevant for fine tuning of apoptosis initiation.

The TRAF3-binding site of human molluscipox virus FLIP molecule MC159 is critical for its capacity to inhibit Fas-induced apoptosis.

Members of the viral Flice/caspase-8 inhibitory protein (v-FLIP) family prevent induction of apoptosis by death receptors through inhibition of the processing and activation of procaspase-8 and -10 at the level of the receptor-associated death-inducing signaling complex (DISC). Here, we have addressed the molecular function of the v-FLIP member MC159 of the human molluscum contagiosum virus. MC159 FLIP powerfully inhibited both caspase-dependent and caspase-independent cell death induced by Fas. The C-terminal region of MC159 bound TNF receptor-associated factor (TRAF)3, was necessary for optimal TRAF2 binding, and mediated the recruitment of both TRAFs into the Fas DISC. TRAF-binding-deficient mutants of MC159 showed impaired inhibition of FasL-induced caspase-8 processing and Fas internalization, and had reduced antiapoptotic activity. Our findings provide evidence that a MC159/TRAF2/TRAF3 complex regulates a new aspect of Fas signaling, and identify MC159 FLIP as a molecule that targets multiple features of Fas-induced cell death.

Differential involvement of MEK kinase 1 (MEKK1) in the induction of apoptosis in response to microtubule-targeted drugs versus DNA damaging agents.

MEK kinase 1 (MEKK1) is a 196-kDa enzyme that is involved in the regulation of the c-Jun N-terminal kinase (JNK) pathway and apoptosis. In cells exposed to genotoxic agents including etoposide and cytosine arabinoside, MEKK1 is cleaved at Asp874 by caspases. The cleaved kinase domain of MEKK1, itself, stimulates caspase activity leading to apoptosis. Kinase-inactive MEKK1 expressed in HEK293 cells effectively blocks genotoxin-induced apoptosis. Treatment of cells with taxol, a microtubule stabilizing agent, did not induce MEKK1 cleavage in cells, and kinase-inactive MEKK1 expression failed to block taxol-induced apoptosis. MEKK1 became activated in HEK293 cells exposed to taxol, but in contrast to etoposide-treatment, taxol failed to increase JNK activity. Taxol treatment of cells, therefore, dissociates MEKK1 activation from the regulation of the JNK pathway. Overexpression of anti-apoptotic Bcl2 blocked MEKK1 and taxol-induced apoptosis but did not block the caspase-dependent cleavage of MEKK1 in response to etoposide. This indicates Bcl2 inhibition of apoptosis is, therefore, downstream of caspase-dependent MEKK1 cleavage. The results define the involvement of MEKK1 in the induction of apoptosis by genotoxins but not microtubule altering drugs.