Homeostatic proliferation and survival of naïve and memory T cells.

The immune system relies on homeostatic mechanisms in order to adapt to the changing requirements encountered during steady-state existence and activation by antigen. For T cells, this involves maintenance of a diverse repertoire of naïve cells, rapid elimination of effector cells after pathogen clearance, and long-term survival of memory cells. The reduction of T-cell counts by either cytotoxic drugs, irradiation, or certain viruses is known to lead to lymphopenia-induced proliferation and restoration of normal T-cell levels. Such expansion is governed by the interaction of TCR with self-peptide/MHC (p/MHC) molecules plus contact with cytokines, especially IL-7. These same ligands, i.e. p/MHC molecules and IL-7, maintain naïve T lymphocytes as resting cells under steady-state T-cell-sufficient conditions. Unlike naïve cells, typical "central" memory T cells rely on a combination of IL-7 and IL-15 for their survival in interphase and for occasional cell division without requiring signals from p/MHC molecules. Other memory T-cell subsets are less quiescent and include naturally occurring activated memory-phenotype cells, memory cells generated during chronic viral infections, and effector memory cells. These subsets of activated memory cells differ from central memory T cells in their requirements for homeostatic proliferation and survival. Thus, the factors controlling T-cell homeostasis can be seen to vary considerably from one subset to another as described in detail in this review.

Neuronal survival induced by neurotrophins requires calmodulin

It has been reported that phosphoinositide 3-kinase (PI 3-kinase) and its downstream target, protein kinase B (PKB), play a central role in the signaling of cell survival triggered by neurotrophins (NTs). In this report, we have analyzed the involvement of Ca2+ and calmodulin (CaM) in the activation of the PKB induced by NTs. We have found that reduction of intracellular Ca2+ concentration or functional blockade of CaM abolished NGF-induced activation of PKB in PC12 cells. Similar results were obtained in cultures of chicken spinal cord motoneurons treated with brain-derived neurotrophic factor (BDNF). Moreover, CaM inhibition prevented the cell survival triggered by NGF or BDNF. This effect was counteracted by the transient expression of constitutive active forms of the PKB, indicating that CaM regulates NT-induced cell survival through the activation of the PKB. We have investigated the mechanisms whereby CaM regulates the activation of the PKB, and we have found that CaM was necessary for the proper generation and/or accumulation of the products of the PI 3-kinase in intact cells.

GD3 Synthase Overexpression Sensitizes Hepatocarcinoma Cells to Hypoxia and Reduces Tumor Growth by Suppressing the cSrc/NF-κB Survival Pathway

Abstract Background: Hypoxia-mediated HIF-1a stabilization and NF-kB activation play a key role in carcinogenesis by fostering cancer cell survival, angiogenesis and tumor invasion. Gangliosides are integral components of biological membranes with an increasingly recognized role as signaling intermediates. In particular, ganglioside GD3 has been characterized as a proapoptotic lipid effector by promoting cell death signaling and suppression of survival pathways. Thus, our aim was to analyze the role of GD3 in hypoxia susceptibility of hepatocarcinoma cells and in vivo tumor growth. Methodology/Principal Findings: We generated and characterized a human hepatocarcinoma cell line stably expressing GD3 synthase (Hep3B-GD3), which catalyzes the synthesis of GD3 from GM3. Despite increased GD3 levels (2-3 fold), no significant changes in cell morphology or growth were observed in Hep3B-GD3 cells compared to wild type Hep3B cells under normoxia. However, exposure of Hep3B-GD3 cells to hypoxia (2% O2) enhanced reactive oxygen species (ROS) generation, resulting in decreased cell survival, with similar findings observed in Hep3B cells exposed to increasing doses of exogenous GD3. In addition, hypoxia-induced c-Src phosphorylation at tyrosine residues, NF-kB activation and subsequent expression of Mn-SOD were observed in Hep3B cells but not in Hep3B-GD3 cells. Moreover, MnTBAP, an antioxidant with predominant SOD mimetic activity, reduced ROS generation, protecting Hep3B-GD3 cells from hypoxia-induced death. Finally, lower tumor growth, higher cell death and reduced Mn-SOD expression were observed in Hep3B-GD3 compared to Hep3B tumor xenografts. Conclusion: These findings underscore a role for GD3 in hypoxia susceptibility by disabling the c-Src/NF-kB survival pathway resulting in lower Mn-SOD expression, which may be of relevance in hepatocellular carcinoma therapy.

Combined loss of the BH3-only proteins Bim and Bmf restores B-cell development and function in TACI-Ig transgenic mice.

Terminal differentiation of B cells depends on two interconnected survival pathways, elicited by the B-cell receptor (BCR) and the BAFF receptor (BAFF-R), respectively. Loss of either signaling pathway arrests B-cell development. Although BCR-dependent survival depends mainly on the activation of the v-AKT murine thymoma viral oncogene homolog 1 (AKT)/PI3-kinase network, BAFF/BAFF-R-mediated survival engages non-canonical NF-κB signaling as well as MAPK/extracellular-signal regulated kinase and AKT/PI3-kinase modules to allow proper B-cell development. Plasma cell survival, however, is independent of BAFF-R and regulated by APRIL that signals NF-κB activation via alternative receptors, that is, transmembrane activator and CAML interactor (TACI) or B-cell maturation (BCMA). All these complex signaling events are believed to secure survival by increased expression of anti-apoptotic B-cell lymphoma 2 (Bcl2) family proteins in developing and mature B cells. Curiously, how lack of BAFF- or APRIL-mediated signaling triggers B-cell apoptosis remains largely unexplored. Here, we show that two pro-apoptotic members of the 'Bcl2 homology domain 3-only' subgroup of the Bcl2 family, Bcl2 interacting mediator of cell death (Bim) and Bcl2 modifying factor (Bmf), mediate apoptosis in the context of TACI-Ig overexpression that effectively neutralizes BAFF as well as APRIL. Surprisingly, although Bcl2 overexpression triggers B-cell hyperplasia exceeding the one observed in Bim(-/-)Bmf(-/-) mice, Bcl2 transgenic B cells remain susceptible to the effects of TACI-Ig expression in vivo, leading to ameliorated pathology in Vav-Bcl2 transgenic mice. Together, our findings shed new light on the molecular machinery restricting B-cell survival during development, normal homeostasis and under pathological conditions. Our data further suggest that Bcl2 antagonists might improve the potency of BAFF/APRIL-depletion strategies in B-cell-driven pathologies.

JNK Inhibition Reduced Retinal Ganglion Cell Death after Ischemia/Reperfusion In Vivo and after Hypoxia In Vitro.

Mitogen-activated protein kinases (MAPKs) are key regulators that have been linked to cell survival and death. Among the main classes of MAPKs, c-jun N-terminal kinase (JNK) has been shown to mediate cell stress responses associated with apoptosis. In Vitro, hypoxia induced a significant increase in 661W cell death that paralleled increased activity of JNK and c-jun. 661W cells cultured in presence of the inhibitor of JNK (D-JNKi) were less sensitive to hypoxia-induced cell death. In vivo, elevation in intraocular pressure (IOP) in the rat promoted cell death that correlated with modulation of JNK activation. In vivo inhibition of JNK activation with D-JNKi resulted in a significant and sustained decrease in apoptosis in the ganglion cell layer, the inner nuclear layer and the photoreceptor layer. These results highlight the protective effect of D-JNKi in ischemia/reperfusion induced cell death of the retina.

The role of raptor in cell death

Selon les statistiques, les maladies cancéreuses sont en augmentation dans les pays en développement ainsi que dans les pays industrialisés. Ceci peut s'expliquer largement par les habitudes alimentaires, le tabagisme, les infections, le manque d'activité physique, la pollution et le stress, entre autres. Ainsi, l'Organisation Mondiale de la Santé (OMS) prévoit une augmentation de la fréquence des cancers avec 15 millions de nouveaux cas par an en 2020. La transformation d'une cellule normale en une cellule cancéreuse se déroule en plusieurs étapes avec, au niveau moléculaire, différentes mutations ciblant des protéines régulant la croissance cellulaire. Un des exemples de protéines qui participent au contrôle des voies cellulaires impliquées lors de la prolifération des cellules sont les complexes de protéines mTORCl et mTORC2 (« mammalian target of rapamycin complex 1 and 2 »). Ces complexes mTORCl et mTORC2 activent des processus anaboliques (la synthèse de protéines et de lipides, le métabolisme énergétique, entre autres) et inhibent en même temps des voies de catabolismes cellulaires (autophagie et synthèse de lysosomes). Ils sont souvent mutés dans de nombreux cas de cancers, c'est pourquoi ils sont la cible de nombreux traitements anti-cancéreux. Pour ces raisons, nous nous sommes intéressés aux mécanismes d'actions moléculaires des drogues qui ciblent les complexes mTORCl et mTORC2. Nous avons ainsi découvert qu'une molécule présente uniquement dans le complexe mTORCl, raptor, était clivée en un fragment plus petit lors du traitement de cellules cancéreuses avec des drogues. Des molécules activées durant la mort cellulaire programmée par apoptose, les caspases, se sont révélées responsables du clivage de raptor. Nous avons ensuite décrit de façon précise les sites de clivage de raptor par les caspases durant la mort cellulaire. Il s'est avéré que le clivage de raptor affaiblissait son interaction avec mTOR au sein du complexe mTORCl, ce qui participe à l'inactivation de mTORCl lors de traitements avec des molécules anti-cancéreuses. Ces résultats nous ont permis de mieux comprendre les mécanismes d'actions de différentes drogues anti-cancéreuses au niveau du complexe mTORCl, ce qui peut être utile pour la synthèse de nouvelles molécules ciblant mTORCl ainsi que pour lutter contre les mécanismes de résistance chimiothérapeutiques. -- La protéine « mammalian target of rapamycin » (mTOR) est une sérine/thréonine kinase qui est hautement conservée des protistes à l'être humain. Deux complexes mTOR existent : le complexe 1 mTOR (mTORCl) et le complexe 2 mTOR (mTORC2). Ils régulent positivement des processus anaboliques (synthèse de protéines et de lipides, le métabolisme énergétique, l'organisation du cytosquelette, la survie cellulaire) et négativement des voies cataboliques (autophagic, biogenèse de lysosomes). Les complexes mTORCl et mTORC2 sont sensibles aux signaux mitogéniques tels que les acides aminés, le glucose, les facteurs de croissance, l'état énergétique (ATP) et les niveaux d'oxygène et induisent des voies de croissance cellulaire essentielles. La voie cellulaire regulée par mTORCl peut être hyperactivée dans de nombreux cancers humains. Puisque plusieurs voies cellulaires convergent et régulent les complexes mTORCl et mTORC2, des mutations dans les kinases en amont peuvent mener à une dérégulation de l'activation de mTOR. Des stratégies thérapeutiques ont été développées pour cibler les complexes mTORCl et mTORC2, ainsi que les kinases en amont qui régulent mTOR. Plusieurs drogues ciblant mTORCl, telles que la rapamycine et la curcumine, affectent l'interaction entre mTOR et un composant spécifique de mTORCl, raptor. Dans cette étude, nous nous sommes intéressés aux mécanismes moléculaires des drogues qui ciblent mTORCl, ainsi que leur effet déstabilisant sur l'interaction entre mTOR et raptor dans des lignées cellulaires de lymphomes. Nous avons démontré que raptor était clivé en un fragment de lOOkDa après traitement avec la rapamycine, la curcumine, l'étoposide, la cisplatine, la staurosporine et le ligand Fas (FasL). Etant donné que ces drogues ont été décrites comme induisant I'apoptose, l'utilisation d'un inhibiteur de caspases (z- VAD-fmk) a révélé que le clivage de raptor, lors de la mort cellulaire, était dépendant des caspases. Des essais caspases in vitro ont permis d'identifier la caspase-6 (ainsi que probablement d'autres caspases) comme étant une protéase impliquée dans le clivage de raptor. La séquence protéique de raptor a montré potentiellement plusieurs sites de clivage de caspases aux extrémités amino-terminale et carboxy-terminale. La mutagénèse a permis d'identifier les sites de clivages de raptor par les caspases comme étant DEAD LTD (acides aminés 17-23) et DDADD (acides aminés 939¬943). De plus, le clivage de raptor corrèle avec l'inhibition de l'activité de mTORCl envers ces substrats (S6K et 4E-BP1). Nous avons aussi observé que le clivage de raptor affaiblissait l'interaction entre mTOR et raptor, ce qui indique que ce clivage est une étape critique dans l'inhibition de mTORCl durant I'apoptose. Pour terminer, la mutagénèse du site de clivage de raptor DDADD a montré une résistance à la mort cellulaire de cellules cancéreuses. Notre travail de recherche a révélé un nouveau mécanisme moléculaire qui module l'organisation et l'activité de mTORCl, ce qui peut être d'un grand intérêt pour les recherches dans le domaine de mTOR ainsi que pour la découverte de molécules ciblant mTORCl. -- The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase, which is highly conserved from yeast to humans. Two different mTOR complexes exist: the mTOR complex 1 (mTORCl) and the mTOR complex 2 (mTORC2). They positively regulate anabolic processes (protein and lipid synthesis, energy metabolism, cytoskeleton organization, cell survival) and negatively regulate catabolic pathways (autophagy, lysosome biogenesis). The mTORCl and mTORC2 respond to mitogenic stimuli such as amino acids, glucose, growth factors, energy levels (ATP) and oxygen levels and drive essential cellular growth pathways. The mTORCl pathway can be found hyperactivated in numerous human cancers. As various cellular pathways converge and regulate mTORCl and mTORC2, mutations in upstream protein kinases can lead to a deregulated mTOR activation. Different therapeutic strategies have been developped to target mTORCl, mTORC2, as well as upstream protein kinases regulating mTOR pathways. Various drugs targeting mTORCl, such as rapamycin and curcumin, affect the interaction between mTOR and a specific mTORCl component, raptor. In this study, we investigated the molecular mechanisms of drugs targeting mTORCl, as well as their destabilizing effect on the mTOR-raptor interaction in lymphoma cell lines. We demonstrated that raptor was processed into a lOOkDa fragment after treatment with rapamycin, curcumin, etoposide, cisplatin, staurosporine and FasL. As these drugs were reported to induce apoptosis, the use of a pan-caspase inhibitor (z-VAD-fmk) revealed that the cleavage of raptor under cell death was caspase-dependent. In vitro caspase assays were performed to identify caspases-6 (and probably other caspases) as an important cysteine protease implicated in the cleavage of raptor. Analysis of raptor protein sequence showed several putative caspase-specific cleavage sites at the N-terminal and the C-terminal ends. Mutagenesis studies allowed us to identify the DEADLTD (amino acids 17-23) and the DDADD (amino acids 939-943) as the caspase-dependent cleavage residues of raptor. Furthermore, the cleavage of raptor correlated with inhibition of mTORCl activity towards its specific targets (4E-BP1 and S6K). We also highlighted that raptor processing weakened the interaction between mTOR and raptor, indicating that raptor cleavage is a critical step in the mTORCl inhibition process during apoptosis. Finally, mutagenesis of raptor C-terminal cleavage site (DDADD) conferred resistance to the chemotherapeutic-mediated cell death cascade of cancer cell. Our research work highlighted a new molecular mechanism modulating mTORCl organization and activity, which can be of great interest in the mTOR field research and for designing drugs trageting mTORCl.

Implication of different domains of the Leishmania major metacaspase in cell death and autophagy.

Metacaspases (MCAs) are cysteine peptidases expressed in plants, fungi and protozoa, with a caspase-like histidine-cysteine catalytic dyad, but differing from caspases, for example, in their substrate specificity. The role of MCAs is subject to debate: roles in cell cycle control, in cell death or even in cell survival have been suggested. In this study, using a Leishmania major MCA-deficient strain, we showed that L. major MCA (LmjMCA) not only had a role similar to caspases in cell death but also in autophagy and this through different domains. Upon cell death induction by miltefosine or H2O2, LmjMCA is processed, releasing the catalytic domain, which activated substrates via its catalytic dyad His/Cys and a proline-rich C-terminal domain. The C-terminal domain interacted with proteins, notably proteins involved in stress regulation, such as the MAP kinase LmaMPK7 or programmed cell death like the calpain-like cysteine peptidase. We also showed a new role of LmjMCA in autophagy, acting on or upstream of ATG8, involving Lmjmca gene overexpression and interaction of the C-terminal domain of LmjMCA with itself and other proteins. These results allowed us to propose two models, showing the role of LmjMCA in the cell death and also in the autophagy pathway, implicating different protein domains.

Regulation of B Cell Gene Expression and Function by Ikaros, Helios and Bcl6

B lymphocytes constitute a key branch of adaptive immunity by providing specificity to recognize a vast variety of antigens by B cell antigen receptors (BCR) and secreted antibodies. Antigen recognition activates the cells and can produce antibody secreting plasma cells via germinal center reaction that leads to the maturation of antigen recognition affinity and switching of antibody effector class. The specificity of antigen recognition is achieved through a multistep developmental pathway that is organized by interplay of transcription factors and signals through BCR. Lymphoid malignancies arise from different stages of development in abnormal function of transcriptional regulation. To understand the B cell development and the function of B cells, a thorough understanding of the regulation of gene expression is important. The transcription factors of the Ikaros family and Bcl6 are frequently associated with lymphoma generation. The aim of this study was to reveal the targets of Ikaros, Helios and Bcl6 mediated gene regulation and to find out the function of Ikaros and Helios in B cells. This study uses gene targeted DT40 B cell lines and establishes a role for Ikaros family factors Ikaros and Helios in the regulation of BCR signaling that is important at developmental checkpoints, for cell survival and in activation. Ikaros and Helios had opposing roles in the regulation of BCR signals. Ikaros was found to directly repress the SHIP gene that encodes a signaling lipid-metabolizing enzyme, whereas Helios had activating effect on SHIP expression. The findings demonstrate a balancing function for these two Ikaros family transcription factors in the regulation of BCR signaling as well as in the regulation of gene expression. Bcl6 was found to repress plasma cell gene expression program while maintaining gene expression profile of B cells. Analysis of direct Bcl6 target genes suggested novel mechanisms for Bcl6-mediated suppression of plasma cell differentiation and promoting germinal center phenotype.

Lethal weapons : novel approaches for receptor-targeted cancer cell elimination

The currently used forms of cancer therapy are associated with drug resistance and toxicity to healthy tissues. Thus, more efficient methods are needed for cancer-specific induction of growth arrest and programmed cell death, also known as apoptosis. Therapeutic forms of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are investigated in clinical trials due to the capability of TRAIL to trigger apoptosis specifically in cancer cells by activation of cell surface death receptors. Many tumors, however, have acquired resistance to TRAIL-induced apoptosis and sensitizing drugs for combinatorial treatments are, therefore, in high demand. This study demonstrates that lignans, natural polyphenols enriched in seeds and cereal, have a remarkable sensitizing effect on TRAIL-induced cell death at non-toxic lignan concentrations. In TRAIL-resistant and androgen-dependent prostate cancer cells we observe that lignans repress receptor tyrosine kinase (RTK) activity and downregulate cell survival signaling via the Akt pathway, which leads to increased TRAIL sensitivity. A structure-activity relationship analysis reveals that the γ-butyrolactone ring of the dibenzylbutyrolactone lignans is essential for the rapidly reversible TRAIL-sensitizing activity of these compounds. Furthermore, the lignan nortrachelogenin (NTG) is identified as the most efficient of the 27 tested lignans and norlignans in sensitization of androgen-deprived prostate cancer cells to TRAIL-induced apoptosis. While this combinatorial anticancer approach may leave normal cells unharmed, several efficient cancer drugs are too toxic, insoluble or unstable to be used in systemic therapy. To enable use of such drugs and to protect normal cells from cytotoxic effects, cancer-targeted drug delivery vehicles of nanometer scale have recently been generated. The newly developed nanoparticle system that we tested in vitro for cancer cell targeting combines the efficient drug-loading capacity of mesoporous silica to the versatile particle surface functionalization of hyperbranched poly(ethylene imine), PEI. The mesoporous hybrid silica nanoparticles (MSNs) were functionalized with folic acid to promote targeted internalization by folate receptor overexpressing cancer cells. The presented results demonstrate that the developed carrier system can be employed in vitro for cancer selective delivery of adsorbed or covalently conjugated molecules and furthermore, for selective induction of apoptotic cell death in folate receptor expressing cancer cells. The tested carrier system displays potential for simultaneous delivery of several anticancer agents specifically to cancer cells also in vivo.

Identification and characterization of new genes and pathways regulating spermatogonial cell fate

Spermatogenesis, i.e sperm production in the seminiferous tubules of the testis, is a complex process that takes over one month to complete. Life-long ability of sperm production ultimately lies in a small population of undifferentiated cells, called spermatogonial stem cells (SSCs). These cells give rise to differentiating spermatogonia, which are committed to mature into spermatozoa. SSCs represent a heterogeneous population of cells and many aspects of their basic biology are still unknown. Understanding the mechanisms behind the cell fate decision of these cells is important to gain more insights into the causes of infertility and testis cancer. In addition, an interesting new aspect is the use of testis-derived stem cells in regenerative medicine. Our data demonstrated that adult mouse testis houses a population of Nanog-expressing spermatogonia. Based on mRNA and protein analysis these cells are enriched in stage XII of the mouse seminiferous epithelial cycle. The cells derived from this stage have the highest capacity to give rise to ES cell-like cells which express Oct4 and Nanog. These cells are under tight non- GDNF regulation but their fate can be dictated by activating p21 signalling. Comparative studies suggested that these cells are regulated like ES cells. Taken together these data imply that pluripotent cells are present in the adult mammalian testis. CIP2A (cancerous inhibitor of PP2A) has been associated with tumour aggressiveness and poor prognosis. In the testis it is expressed by the descendants of stem cells, i.e. the spermatogonial progenitor cells. Our data suggest that CIP2A acts upstream of PLZF and is needed for quantitatively normal spermatogenesis. Classification of CIP2A as a cancer/testis gene makes it an attractive target for cancer therapy. Study on the CIP2A deficient mouse model demonstrates that systemic inhibition of CIP2A does not severely interfere with growth and development or tissue or organ function, except for the spermatogenic output. These data demonstrate that CIP2A is required for quantitatively normal spermatogenesis. Hedgehog (Hh) signalling is involved in the development and maintenance of many different tissues and organs. According to our data, Hh signalling is active at many different levels during rat spermatogenesis: in spermatogonia, spermatocytes and late elongating spermatids. Localization of Suppressor of Fused (SuFu), the negative regulator of the pathway, specifically in early elongating spermatids suggests that Hh signalling needs to be shut down in these cells. Introduction of Hh signalling inhibitor resulted in an increase in germ cell apoptosis. Follicle-stimulating hormone (FSH) and inhibition of receptor tyrosine kinases resulted in down-regulation of Hh signalling. These data show that Hh signalling is under endocrine and paracrine control and it promotes germ cell survival.

Survival following orbital exenteration at a tertiary brazilian hospital

Objective: to analyze the epidemiology, clinical features and survival rate of patients undergoing orbital exenteration (OE) in a tertiary referral hospital. Methods : we conducted a retrospective study of all patients undergoing OE at the Hospital das Clínicas, FMUSP between January 2007 and December 2012. We collected data records related to gender, age, origin, length of stay, duration of the disease, other treatments related to the disease, number of procedures outside of the face related to the disease, follow-up and histological diagnosis. Results : we treated 37 patients in the study period. The average survival in one year was 70%, in two years, 66.1%, and 58.3% in three years. There was no significant difference in the one-year survival related to histological diagnosis (p=0.15), days of hospitalization (p=0.17), gender (p=0.43), origin (p=0.78), disease duration (p=0.27) or the number of operations for the tumor (p=0.31). Mortality was higher in elderly patients (p=0.02). The average years of life lost was 33.9 in patients under 60 years, 14.7 in patients in the 61-80 years range and 11.3 in patients over 80 years. Conclusion : the present series of cases is significant in terms of prevalence of orbital exenteration; on the other hand, it shows one of the lowest survival rates in the literature. This suggests an urgent need for improved health care conditions to prevent deforming, radical resections.

Regulation of cell fate by c-FLIP phosphorylation

Programmed cell death is an important physiological cellular process that maintains homeostasis and protects multicellular organisms from diseases. Apoptosis is the principal mode of cell death, which eliminates unwanted cells and an enormous effort has been made to understand the molecular mechanisms of the signaling pathway and its regulatory systems. Irregular apoptosis often has life-threatening consequences to humans, including cancer, autoimmune diseases and degenerative diseases. In cancer for example, cell death is an attractive target to eradicate uncontrollably proliferating cells that have disregard pro-apoptotic signaling. Targeted therapeutic approaches are not as effective as once expected, since now we know that the cell death pathways are not sole entities in cells, but are highly associated with various cellular processes. Proteins that regulate apoptosis can also control non-apoptotic signaling pathways. For example, c-FLIP is a protein that can either inhibit or promote caspase-8 activation, which is required to induce apoptosis. Not only has c-FLIP opposing effects on initiating apoptosis, but it also regulates various pro-survival signaling pathways in the cell. It is well known that protein expression level is a determinant of how c-FLIP can regulate different signaling pathways, but other regulatory mechanisms potentially affecting the role of c-FLIP are less well understood. This work addresses novel insights into the mechanisms of c-FLIP post-translational modifications and their functional consequences. We have identified that phosphorylation is an important inception for subcellular localization of c-FLIP, thereby dictating which apoptotic and non-apoptotic signaling pathways c-FLIP could regulate to promote cell survival. Furthermore, we have constructed mathematical models to unite independent studies to establish more systematic c-FLIP signaling pathways to understand the dynamics of extrinsically-induced apoptosis.

Veratridine increases the survival of retinal ganglion cells in vitro

Neuronal cell death is an important phenomenon involving many biochemical pathways. This degenerative event has been studied to understand how the cells activate the mechanisms that lead to self-destruction. Target cells and afferent cells play a relevant role in the regulation of natural cell death. We studied the effect of veratridine (1.5, 3.0, 4.5 and 6.0 µM) on the survival of neonatal rat retinal ganglion cells in vitro. Veratridine (3.0 µM), a well-known depolarizing agent that opens the Na+ channel, promoted a two-fold increase in the survival of retinal ganglion cells kept in culture for 48 h. This effect was dose-dependent and was blocked by 1.0 µM tetrodotoxin (a classical voltage-dependent Na+ channel blocker) and 30.0 µM flunarizine (a Na+ and Ca2+ channel blocker). These results indicate that electrical activity is also important for the maintenance of retinal ganglion cell survival in vitro

The effect of porphyrins on normal and transformed mouse cell lines in the presence of visible light

Photodynamic therapy consists of the uptake of a photosensitizing dye, often a porphyrin, by tumor tissue and subsequent irradiation of the tumor with visible light of an appropriate wavelength matched to the absorption spectrum of the photosensitizing dye. This class of molecules produces reactive oxygen species when activated by light, resulting in a direct or indirect cytotoxic effect on the target cells. Photodynamic therapy has been used in the treatment of cancer but the technology has a potential for the treatment of several disease conditions mainly because of its selectivity. However, it is not clear why the porphyrins are retained preferentially by abnormal tissue. This paper describes a study of the effect of the association of porphyrin and visible light on two mouse fibroblast cell lines: A31, normal cells and B61, an EJ-ras transformed variant of A31. Two water-soluble porphyrins were used, a positively charged one, tetra(N-methyl-4-pyridyl)porphyrin chloride, and a negatively charged one, tetra(4-sulfonatophenyl)porphyrin-Na salt (TPPS4) in order to assess the effect on cell survival. The results suggest that the B61 cell line is more sensitive to incubation with the anionic porphyrin (TPPS4) followed by light irradiation and that the anionic porphyrin is more efficient in killing the cells than the cationic porphyrin.

Multicellular spheroids of bone marrow stromal cells: a three-dimensional in vitro culture system for the study of hematopoietic cell migration

Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.