Cell Death Phenotype Research Articles

Loss of ceramide kinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts.

Arabidopsis thaliana plants that lack ceramide kinase, encoded by ACCELERATED CELL DEATH5 (ACD5), display spontaneous programmed cell death late in development and accumulate substrates of ACD5. Here, we compared ceramide accumulation kinetics, defense responses, ultrastructural features, and sites of reactive oxygen species (ROS) production in wild-type and acd5 plants during development and/or Botrytis cinerea infection. Quantitative sphingolipid profiling indicated that ceramide accumulation in acd5 paralleled the appearance of spontaneous cell death, and it was accompanied by autophagy and mitochondrial ROS accumulation. Plants lacking ACD5 differed significantly from the wild type in their responses to B. cinerea, showing earlier and higher increases in ceramides, greater disease, smaller cell wall appositions (papillae), reduced callose deposition and apoplastic ROS, and increased mitochondrial ROS. Together, these data show that ceramide kinase greatly affects sphingolipid metabolism and the site of ROS accumulation during development and infection, which likely explains the developmental and infection-related cell death phenotypes. The acd5 plants also showed an early defect in restricting B. cinerea germination and growth, which occurred prior to the onset of cell death. This early defect in B. cinerea restriction in acd5 points to a role for ceramide phosphate and/or the balance of ceramides in mediating early antifungal responses that are independent of cell death.

Identification and characterization of suppressor mutants of spl11- mediated cell death in rice.

Lesion mimic mutants have been used to dissect programmed cell death (PCD) and defense-related pathways in plants. The rice lesion-mimic mutant spl11 exhibits race nonspecific resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae and the fungal pathogen Magnaporthe oryzae. Spl11 encodes an E3 ubiquitin ligase and is a negative regulator of PCD in rice. To study the regulation of Spl11-mediated PCD, we performed a genetic screen and identified three spl11 cell-death suppressor (sds) mutants. These suppressors were characterized for their resistance to X. oryzae pv. oryzae and M. oryzae and for their expression of defense-related genes. The suppression of the cell-death phenotypes was generally correlated with reduced expression of defense-related genes. When rice was challenged with avirulent isolates of M. oryzae, the disease phenotype was unaffected in the sds mutants, indicating that the suppression might be Spl11-mediated pathway specific and may only be involved in basal defense. In addition, we mapped one of the suppressor mutations to a 140-kb interval on the long arm of rice chromosome 1. Identification and characterization of these sds mutants should facilitate our efforts to elucidate the Spl11-mediated PCD pathway.

Differential necrotic lesion formation in soybean cultivars in response to soybean mosaic virus

In this study, we examined the necrosis phenotype on leaves of two cultivars of soybean (ZheA8901 and Nannong1138-2) that show varying level of resistance to soybean mosaic virus (SMV). The necrotic symptoms seen on inoculated and systemic leaves of soybean cultivar ZheA8901 were reminiscent of programmed cell death (PCD). The cell death phenotypes were evaluated using the TUNEL method, quantification of hydrogen peroxide (H2O2) and salicylic acid, callose production, as well as by monitoring expression of defence genes GmPR-1 and GmNPR1. Our results show that SMV inoculation induced PCD on ZheA8901 is associated with rapid increase in H2O2, increased SA and callose accumulation and higher defence gene expression.

GSK3 Inhibitors Regulate MYCN mRNA Levels and Reduce Neuroblastoma Cell Viability through Multiple Mechanisms, Including p53 and Wnt Signaling

Neuroblastoma is an embryonal tumor accounting for approximately 15% of childhood cancer deaths. There exists a clinical need to identify novel therapeutic targets, particularly for treatment-resistant forms of neuroblastoma. Therefore, we investigated the role of the neuronal master regulator GSK3 in controlling neuroblastoma cell fate. We identified novel GSK3-mediated regulation of MYC (c-MYC and MYCN) mRNA levels, which may have implications for numerous MYC-driven cancers. In addition, we showed that certain GSK3 inhibitors induced large-scale cell death in neuroblastoma cells, primarily through activating apoptosis. mRNA-seq of GSK3 inhibitor-treated cells was performed and subsequent pathway analysis revealed that multiple signaling pathways contributed to the loss of neuroblastoma cell viability. The contribution of two of the signaling pathways highlighted by the mRNA-seq analysis was functionally validated. Inhibition of the p53 tumor suppressor partly rescued the cell death phenotype, whereas activation of canonical Wnt signaling contributed to the loss of viability, in a p53-independent manner. Two GSK3 inhibitors (BIO-acetoxime and LiCl) and one small-molecule Wnt agonist (Wnt Agonist 1) demonstrated therapeutic potential for neuroblastoma treatment. These inhibitors reduced the viability of numerous neuroblastoma cell lines, even those derived from high-risk MYCN-amplified metastatic tumors, for which effective therapeutics are currently lacking. Furthermore, although LiCl was lethal to neuroblastoma cells, it did not reduce the viability of differentiated neurons. Taken together our data suggest that these small molecules may hold potential as effective therapeutic agents for the treatment of neuroblastoma and other MYC-driven cancers.

Neurotoxicity of coral snake phospholipases A2 in cultured rat hippocampal neurons

The neurotoxicity of two secreted Phospholipases A2 from Brazilian coral snake venom in rat primary hippocampal cell culture was investigated. Following exposure to Mlx-8 or Mlx-9 toxins, an increase in free cytosolic Ca2+ and a reduction in mitochondrial transmembrane potential (ΔΨm) became evident and occurred prior to the morphological changes and cytotoxicity. Exposure of hippocampal neurons to Mlx-8 or Mlx-9 caused a decrease in the cell viability as assessed by MTT and LDH assays. Inspection using fluorescent images and ultrastructural analysis by scanning and transmission electron microscopy showed that multiphase injury is characterized by overlapping cell death phenotypes. Shrinkage, membrane blebbing, chromatin condensation, nucleosomal DNA fragmentation and the formation of apoptotic bodies were observed. The most striking alteration observed in the electron microscopy was the fragmentation and rarefaction of the neuron processes network. Degenerated terminal synapses, cell debris and apoptotic bodies were observed among the fragmented fibers. Numerous large vacuoles as well as swollen mitochondria and dilated Golgi were noted. Necrotic signs such as a large amount of cellular debris and membrane fragmentation were observed mainly when the cells were exposed to highest concentration of the PLA2-neurotoxins. PLA2s exposed cultures showed cytoplasmic vacuoles filled with cell debris, clusters of mitochondria presented mitophagy-like structures that are in accordance to patterns of programmed cell death by autophagy. Finally, we demonstrated that the sPLA2s, Mlx-8 and Mlx-9, isolated from the Micrurus lemniscatus snake venom induce a hybrid cell death with apoptotic, autophagic and necrotic features. Furthermore, this study suggests that the augment in free cytosolic Ca2+ and mitochondrial dysfunction are involved in the neurotoxicity of Elapid coral snake venom sPLA2s.

JNK signaling is needed to tolerate chromosomal instability

Chromosomal instability (CIN), as a common feature of tumors, represents a potential therapeutic target if ways can be found to specifically cause apoptosis in unstably dividing cells. We have previously shown that if signaling through the JNK pathway is reduced, apoptosis is triggered in models of chromosomal instability induced by loss of the spindle checkpoint. Here we identify components upstream and downstream of JNK that are able to mediate this effect, and test the involvement of p53 and DNA damage in causing apoptosis when JNK signaling is reduced in CIN cells. We show that cell cycle progression timing has a strong effect on the apoptosis seen when JNK signaling is reduced in genetically unstable cells: a shortened G2 phase enhances the apoptosis, while lengthening G2 rescues the JNK-deficient CIN cell death phenotype. Our findings suggest that chromosomal instability represents a significant stress to dividing cells, and that without JNK signaling, cells undergo apoptosis because they lack a timely and effective response to DNA damage.

Turning up the heat: heat stress induces markers of programmed cell death in Plasmodium falciparum in vitro

Malaria is characterised by cyclical febrile episodes that result from the rupture of mature schizont-infected erythrocytes releasing merozoites. In patients infected with Plasmodium falciparum, fever may reach peak temperatures as high as 41 °C. Febrile episodes typically have a deleterious effect on parasites and probably benefit the host by aiding parasite clearance; however, the parasite may also gain advantage from limiting its burden on the host and prolonging infection to ensure development and transmission of slow-maturing gametocytes. Programmed cell death (PCD) may provide the parasite with a mechanism of self-limitation, although the occurrence and phenotype of PCD in the erythrocytic stages remain controversial due to conflicting data. This study aimed to characterise the cell death phenotype of P. falciparum in response to in vitro heat stress. A variety of biochemical markers of PCD, including DNA fragmentation, mitochondrial dysregulation and phosphatidylserine externalisation, as well as morphological studies of Giemsa-stained thin smears and real-time microscopy were utilised to characterise the phenotype. Heat stress decreased P. falciparum growth and development in vitro. Late-stage parasites were more susceptible, although early stages were more affected than expected. Early-stage parasites exposed to 41 °C exhibited markers of an apoptosis-like PCD phenotype, including DNA fragmentation and mitochondrial depolarisation. Heat-stressed late-stage parasites showed no significant DNA fragmentation or mitochondrial dysregulation; however, cytoplasmic vacuolisation was suggestive of an autophagy-like form of PCD. Our results therefore showed that biochemical and morphological markers of PCD varied with intra-erythrocytic parasite development and that P. falciparum exhibited facets of both apoptosis- and autophagy-like phenotypes after exposure to febrile temperatures, which may reflect a unique PCD phenotype.

To Each His Own: Fever Induces Different Suicide Mechanisms in Early and Late Stage Plasmodium falciparum Malaria Parasites in vitro

Malaria is characterised by cyclical febrile episodes that result from the rupture of mature schizont-infected erythrocytes releasing merozoites. In patients infected with Plasmodium falciparum, fever may reach peak temperatures as high as 41°C, last for 2-6 hours and recur every 48 hours, in accordance with the parasite life cycle. Febrile episodes typically have a deleterious effect on parasites, and probably benefit the host by aiding parasite clearance; however, a reduction in parasitemia may also be an advantage for the parasite by limiting the burden of infection on the host and prolonging infection to ensure development and transmission of slow-maturing gametocytes. Although programmed cell death (PCD) has been identified in the mosquito stages of the parasite, data regarding the erythrocytic stages are limited and often conflicting and thus the occurrence of PCD and the phenotype remain controversial. This study aimed to characterise the cell death phenotype of P. falciparum in response to in vitro heat stress similar to fever periods experienced during malaria paroxysms.Synchronised early and late stage P. falciparum 3D7 cultures were exposed to 41°C for 2 hours or maintained as controls at 37°C. Parasitemia was monitored with thiazole orange flow cytometry. Numerous biochemical markers of PCD were assessed by flow cytometric assays immediately after heat stress and 24 hours later. DNA fragmentation was measured with the Terminal deoxynucleotidyl transferase-mediated Nick End Labelling (TUNEL) assay; mitochondrial membrane polarisation was measured with 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)]; and phosphatidylserine (PS) externalisation of infected erythrocytes was quantified by Annexin V binding. Morphological studies of Giemsa-stained thin smears and real-time microscopy were also utilised to characterise the parasite response to heat stress.Exposure to 41°C decreased the parasitemia of both early and late stage P. falciparum in vitro. Ring stage parasites were more affected than expected, although some ring stage parasites developed into trophozoites similar to control parasites maintained at 37°C. Biochemical markers showed evidence of DNA fragmentation and mitochondrial depolarisation suggesting that ring stage parasites exhibited an apoptosis-like phenotype of PCD, although infected erythrocytes showed no significant increase in PS externalisation.Late stage control parasites maintained at 37°C replicated within 24 hours to form newly-invaded rings with increased parasitemia. In contrast, heat stress caused a reduction in the number of late stage parasites and the remaining parasites failed to invade new erythrocytes. Although infected erythrocytes exhibited increased PS externalisation, the parasites exhibited very little DNA fragmentation and no mitochondrial dysregulation. Giemsa-stained thin smears showed some late stage parasites with cytoplasmic vacuolisation, which was suggestive of an autophagy-like form of PCD. Real-time microscopy revealed the movement of many late stage parasites inside host erythrocytes after heat stress, indicating that these parasites were alive, consistent with a recovery in parasitemia after two cycles of continued culture at 37°C.Data regarding the possible induction of PCD by heat stress in P. falciparum are scarce and conflicting. Our results showed biochemical and morphological markers of PCD that varied with intra-erythrocytic parasite development and suggested that P. falciparum exhibited both apoptosis- and autophagy-like phenotypes of PCD after exposure to febrile temperatures. However, these cell death markers may also represent different facets of a PCD pathway in P. falciparum that is distinct from that in metazoans. The identification and exploitation of an intrinsic cell death pathway unique to P. falciparum may provide novel targets for eliminating the parasite in malaria patients. Disclosures:No relevant conflicts of interest to declare.

Aging and cell death in the other yeasts,Schizosaccharomyces pombeandCandida albicans

How do cells age and die? For the past 20years, the budding yeast, Saccharomyces cerevisiae, has been used as a model organism to uncover the genes that regulate lifespan and cell death. More recently, investigators have begun to interrogate the other yeasts, the fission yeast, Schizosaccharomyces pombe, and the human fungal pathogen, Candida albicans, to determine if similar longevity and cell death pathways exist in these organisms. After summarizing the longevity and cell death phenotypes in S.cerevisiae, this mini-review surveys the progress made in the study of both aging and programed cell death (PCD) in the yeast models, with a focus on the biology of S.pombe and C.albicans. Particular emphasis is placed on the similarities and differences between the two types of aging, replicative aging, and chronological aging, and between the three types of cell death, intrinsic apoptosis, autophagic cell death, and regulated necrosis, found in these yeasts. The development of the additional microbial models for aging and PCD in the other yeasts may help further elucidate the mechanisms of longevity and cell death regulation in eukaryotes.

Bendless modulates JNK-mediated cell death and migration in Drosophila

The TNF-JNK pathway is a highly conserved signaling pathway that regulates a wide spectrum of biological processes including cell death and migration. To further delineate this pathway, we carried out a genetic screen for dominant modifiers of the cell death phenotype triggered by ectopic expression of Eiger (Egr), the Drosophila TNF ortholog. Here we show that Bendless (Ben), an E2 ubiquitin-conjugating enzyme, modulates Egr-induced JNK activation and cell death through dTRAF2. Furthermore, Ben physically interacts with dTRAF2 and regulates Egr-induced dTRAF2 polyubiquitination. Finally, Ben is required for JNK-dependent tumor progression, cell migration, oxidative stress resistance and longevity. Our results indicate that Ben constitutes an essential component of the evolutionarily conserved TNF-JNK pathway that modulates cell death and invasion, tumor progression, stress response and lifespan in metazoans.

Involvement of poly(ADP-ribose) polymerase in paraptotic cell death of D. discoideum

Paraptosis is mediated by several proteins, poly(ADP-ribose) polymerase being one of them. D. discoideum lacks caspases thus providing a better system to dissect out the role of PARP in paraptosis. The cell death phenotype in unicellular eukaryote, D. discoideum is similar to the programmed cell death phenotype of multicellular animals. However, the events downstream to the death signal of PCD in D. discoideum are yet to be understood. Our results emphasize that oxidative stress in D. discoideum lacking caspases leads to PARP activation, mitochondrial membrane potential changes, followed by the release of apoptosis inducing factor from mitochondria. AIF causes large scale DNA fragmentation, a hallmark feature of paraptosis. The role of PARP in paraptosis is reiterated via PARP inhibition by benzamide, PARG inhibition by gallotannin and PARP down-regulation, which delays paraptosis. PARP, PARG and AIF interplay is quintessential in paraptosis of D. discoideum. This is the first report to establish the involvement of PARP in the absence of caspase activity in D. discoideum which could be of evolutionary significance and gives a lead to understand the caspase independent paraptotic mechanism in higher organisms.

The Components of Drosophila Histone Chaperone dCAF-1 Are Required for the Cell Death Phenotype Associated with rbf1 Mutation

A Polycomb group protein, Posterior sex combs (Psc), was identified in a genetic screen designed to find factors that can specifically induce morphological defects in rbf1 mutant eyes. We discovered that rbf1 mutations enhance developmental phenotypes caused by Psc overexpression such as ectopic cell death and disorganized ommatidia. Our genetic analysis revealed that Psc-induced developmental defects are strongly influenced by CAF1p55, which is a shared component of several chromatin-associated complexes including a histone chaperone complex, chromatin assembly factor-1 (dCAF-1). Interestingly, the expression levels of dCAF-1 components, CAF1p105 and CAF1p180, are increased in rbf1 mutants, whereas the expression level of CAF1p55 itself remains relatively unchanged. We demonstrated that the increased levels of CAF1p105 and CAF1p180 are required for the hypersensitivity of rbf1 mutant cells to Psc-induced cell death and for the developmentally regulated cell death normally observed in rbf1 mutant eyes. We propose that Caf1p105 and Caf1p180 are important determinants of cell death sensitivity in rbf1 mutant cells and contribute to the genetic interaction between Psc and rbf1.

Microarray‐assisted fine mapping of quantitative trait loci on chromosome 15 for susceptibility to seizure‐induced cell death in mice

Prior studies with crosses of the FVB/NJ (FVB; seizure-induced cell death-susceptible) mouse and the C57BL/6J (B6; seizure-induced cell death-resistant) mouse revealed the presence of a quantitative trait locus (QTL) on chromosome 15 that influenced susceptibility to kainic acid-induced cell death (Sicd2). In an earlier study, we confirmed that the Sicd2 interval harbors gene(s) conferring strong protection against seizure-induced cell death through the creation of the FVB.B6-Sicd2 congenic strain, and created three interval-specific congenic lines (ISCLs) that encompass Sicd2 on chromosome 15 to fine-map this locus. To further localise this Sicd2 QTL, an additional congenic line carrying overlapping intervals of the B6 segment was created (ISCL-4), and compared with the previously created ISCL-1-ISCL-3 and assessed for seizure-induced cell death phenotype. Whereas all of the ISCLs showed reduced cell death associated with the B6 phenotype, ISCL-4, showed the most extensive reduction in seizure-induced cell death throughout all hippocampal subfields. In order to characterise the susceptibility loci on Sicd2 by use of this ISCL and identify compelling candidate genes, we undertook an integrative genomic strategy of comparing exon transcript abundance in the hippocampus of this newly developed chromosome 15 subcongenic line (ISCL-4) and FVB-like littermates. We identified 10 putative candidate genes that are alternatively spliced between the strains and may govern strain-dependent differences in susceptibility to seizure-induced excitotoxic cell death. These results illustrate the importance of identifying transcriptomics variants in expression studies, and implicate novel candidate genes conferring susceptibility to seizure-induced cell death.

Bax phosphorylation association with nucleus and oligomerization after neonatal Hypoxia-ischemia

Neonatal hypoxia-ischemia (HI) is a common occurrence in preterm and low-birth-weight infants, and the incidence of low-birth-weight and preterm births is increasing. Characterization of brain injury after HI is of critical importance in developing new treatments that more accurately target the injury. After severe HI, neuronal cells undergo necrosis and secondary apoptosis of the surrounding cells as a result of neuroinflammation. We sought to characterize the biochemical pathways associated with cell death after HI. Bax, a cell death signaling protein, is activated after HI and translocates to the nucleus, endoplasmic reticulum, and mitochondria. The translocation patterns of Bax affect the resultant cell death phenotype (necrotic or apoptotic) observed. Although Bax is known to oligomerize once it is activated, less is known about the factors that control its translocation and oligomerization. We hypothesize that Bax kinase-specific phosphorylation determines its oligomerization and intracellular localization. Using well-established in vivo and in vitro models of neonatal HI, we characterized Bax oligomerization and multiorganelle translocation. We found that HI-dependent phosphorylation of Bax determines its oligomerization status and multiorganelle localization, and, ultimately, the cell death phenotype observed. Understanding the mechanisms of Bax translocation will aid in the rational design of therapeutic strategies that decrease the trauma resulting from HI-associated inflammation.

Disruption of Fumarylacetoacetate Hydrolase Causes Spontaneous Cell Death under Short-Day Conditions in Arabidopsis

Fumarylacetoacetate hydrolase (FAH) hydrolyzes fumarylacetoacetate to fumarate and acetoacetate, the final step in the tyrosine (Tyr) degradation pathway that is essential to animals. Deficiency of FAH in animals results in an inborn lethal disorder. However, the role for the Tyr degradation pathway in plants remains to be elucidated. In this study, we isolated an Arabidopsis (Arabidopsis thaliana) short-day sensitive cell death1 (sscd1) mutant that displays a spontaneous cell death phenotype under short-day conditions. The SSCD1 gene was cloned via a map-based cloning approach and found to encode an Arabidopsis putative FAH. The spontaneous cell death phenotype of the sscd1 mutant was completely eliminated by further knockout of the gene encoding the putative homogentisate dioxygenase, which catalyzes homogentisate into maleylacetoacetate (the antepenultimate step) in the Tyr degradation pathway. Furthermore, treatment of Arabidopsis wild-type seedlings with succinylacetone, an abnormal metabolite caused by loss of FAH in the Tyr degradation pathway, mimicked the sscd1 cell death phenotype. These results demonstrate that disruption of FAH leads to cell death in Arabidopsis and suggest that the Tyr degradation pathway is essential for plant survival under short-day conditions.

MYCN and HDAC2 cooperate to repress miR-183 signaling in neuroblastoma

MYCN is a master regulator controlling many processes necessary for tumor cell survival. Here, we unravel a microRNA network that causes tumor suppressive effects in MYCN-amplified neuroblastoma cells. In profiling studies, histone deacetylase (HDAC) inhibitor treatment most strongly induced miR-183. Enforced miR-183 expression triggered apoptosis, and inhibited anchorage-independent colony formation in vitro and xenograft growth in mice. Furthermore, the mechanism of miR-183 induction was found to contribute to the cell death phenotype induced by HDAC inhibitors. Experiments to identify the HDAC(s) involved in miR-183 transcriptional regulation showed that HDAC2 depletion induced miR-183. HDAC2 overexpression reduced miR-183 levels and counteracted the induction caused by HDAC2 depletion or HDAC inhibitor treatment. MYCN was found to recruit HDAC2 in the same complexes to the miR-183 promoter, and HDAC2 depletion enhanced promoter-associated histone H4 pan-acetylation, suggesting epigenetic changes preceded transcriptional activation. These data reveal miR-183 tumor suppressive properties in neuroblastoma that are jointly repressed by MYCN and HDAC2, and suggest a novel way to bypass MYCN function.

Abstract 2103: XB05: a promising small molecule for cancer therapy.

Abstract XB05 is synthetic small molecule which was serendipitously discovered to have potent anticancer properties. XB05 displays selectivity for malignant cells over non-malignant cells. Human myeloid leukemia cells (U937) and human lung cancer cells (A549) treated with XB05 show an increase in oxidative stress, DNA damage and a mixed apoptotic and necrotic mechanism of cell death. We conducted a COMPARE analysis to search for correlations between XB05 response and gene expression using publicly available microarray studies of the National Cancer Institute 60 cell line panel. This analysis uncovered a correlation between cell death in response to XB05 and expression of the transcription factor, SOX9. We find that SOX9 is differentially expressed across a panel of human lung cancer cell lines. MTT proliferation and clonogenic cell survival assays suggest that SOX9 expression positively correlates with the sensitivity of lung cancer cells to XB05. Furthermore, we show that siRNA knockdown of SOX9 recapitulates the phenotype of XB05-induced cell death, and that SOX9 may be downregulated at the protein level by XB05 treatment. Overall, these data demonstrate potent activity of XB05 against human cancer cells via a novel mechanism of action, and a potential role for SOX9 as a biomarker for selection and monitoring of therapy with XB05. Citation Format: Francesca R. Salipur, Elsa M. Reyes Reyes, Bo Xu, Ned Smith, Jian Cai, Gerald B. Hammond, Paula J. Bates. XB05: a promising small molecule for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2103. doi:10.1158/1538-7445.AM2013-2103

Abstract 3041: A tumor-suppressive role of the IL-21R/Jak/STAT3 pathway in the germinal center B cell subtype of diffuse large B cell lymphomas.

Abstract Diffuse large B cell lymphoma (DLBCL) is the most common type of B cell malignancy in the adult population. Based on their patterns of gene expression, DLBCLs can be divided into two major subgroups: the germinal center (GC) B cell like (GCB-DLBCL) and the activated B cell like (ABC-DLBCL) cases. Current studies suggest that the cell-of-origin for both subgroups is a normal GC B cell. The difference lies in the degree of maturation. While the GCB variant corresponds to the GC centroblasts that abundantly express BCL6 with no NF-kappaB activation, the ABC variant phenotypically resembles activated GC centrocytes and plasma cell precursors that have received activating signals (e.g. IL-21 and CD40L) from follicular T helper (Tfh) cells and thus contain activated STAT3 and NF-kappaB due to mutations in a high percentage of cases. Although it is commonly accepted that pathogenesis of B cell lymphomas such as DLBCL is intimately linked to impaired B cell differentiation, a direct examination of this concept has not been made for GCB-DLBCL. In a previous study, we showed that the IL-21R/Jak/Stat3 signaling pathway plays a critical role in post-GC plasma cell differentiation, while concurrent signals transduced through the CD40/NF-kappaB axis potentiate the activity of Jak/Stat3 through two distinct mechanisms. In the current study, we report that IL-21-triggered Stat3 activation is often defective in GCB-DLBCL cell lines. Impaired STAT3 activation results in failure to commit to the plasma cell fate as evidenced by lack of Blimp-1 induction. The IL-21 insensitive cell lines either have very low levels of IL-21R or lack the Jak3 protein. Aberrant expression of the components of the IL-21R/Jak/STAT3 signaling pathway can also be detected in subsets of primary GCB-DLBCL samples. Lentivirus mediated reconstitution of IL-21R and Jak3 in the defective cell lines restored IL-21 responsiveness and induced a delayed cell death phenotype, indicating that low level expression of a single signaling molecule is responsible for the IL-21R signaling defect in a given cell line. We have sequenced the coding regions of the IL21R, Jak3 and STAT3 genes in 3 cell lines, but did not find any mutations. Subsequent analyses showed that the IL-21R abnormality is a transcriptional defect associated with Sp1 expression changes, while silencing of the Jak3 gene is caused by promoter methylation. Collectively, our results suggest that in GCB-DLBCL, the IL-21R/Jak/STAT3 pathway plays a novel, tumor suppressive role, and its epigenetic inactivation may promote lymphoma development by blocking terminal B cell differentiation. Citation Format: B. Hilda Ye, Enguang Bi, J. Jessica Yu, Yun Mai, B. Belinda Ding, Yiting Yu, Amit Verma, Rita Shaknovich, Wing C. Chan. A tumor-suppressive role of the IL-21R/Jak/STAT3 pathway in the germinal center B cell subtype of diffuse large B cell lymphomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3041. doi:10.1158/1538-7445.AM2013-3041

Cell Death Control: The Interplay of Apoptosis and Autophagy in the Pathogenicity of Sclerotinia sclerotiorum

Programmed cell death is characterized by a cascade of tightly controlled events that culminate in the orchestrated death of the cell. In multicellular organisms autophagy and apoptosis are recognized as two principal means by which these genetically determined cell deaths occur. During plant-microbe interactions cell death programs can mediate both resistant and susceptible events. Via oxalic acid (OA), the necrotrophic phytopathogen Sclerotinia sclerotiorum hijacks host pathways and induces cell death in host plant tissue resulting in hallmark apoptotic features in a time and dose dependent manner. OA-deficient mutants are non-pathogenic and trigger a restricted cell death phenotype in the host that unexpectedly exhibits markers associated with the plant hypersensitive response including callose deposition and a pronounced oxidative burst, suggesting the plant can recognize and in this case respond, defensively. The details of this plant directed restrictive cell death associated with OA deficient mutants is the focus of this work. Using a combination of electron and fluorescence microscopy, chemical effectors and reverse genetics, we show that this restricted cell death is autophagic. Inhibition of autophagy rescued the non-pathogenic mutant phenotype. These findings indicate that autophagy is a defense response in this necrotrophic fungus/plant interaction and suggest a novel function associated with OA; namely, the suppression of autophagy. These data suggest that not all cell deaths are equivalent, and though programmed cell death occurs in both situations, the outcome is predicated on who is in control of the cell death machinery. Based on our data, we suggest that it is not cell death per se that dictates the outcome of certain plant-microbe interactions, but the manner by which cell death occurs that is crucial.

Melittin peptide kills Trypanosoma cruzi parasites by inducing different cell death pathways

Antimicrobial peptides (AMPs) are components of the innate immune response that represent desirable alternatives to conventional pharmaceuticals, as they have a fast mode of action, a low likelihood of resistance development and can act in conjunction with existing drug regimens. AMPs exhibit strong inhibitory activity against both Gram-positive and Gram-negative bacteria, fungi, viruses, metazoans and other parasites, such as the protozoan Leishmania. Melittin is a naturally occurring AMP, which comprises 40–50% of the dry weight of Apis mellifera venom. Our group has recently shown that crude A. mellifera venom is lethal to Trypanosoma cruzi, the Chagas disease etiologic agent, and generates a variety of cell death phenotypes among treated parasites. Here, we demonstrate that the melittin affected all of T. cruzi developmental forms, including the intracellular amastigotes. The ultrastructural changes induced by melittin suggested the occurrence of different programmed cell death pathways, as was observed in A. mellifera-treated parasites. Autophagic cell death appeared to be the main death mechanism in epimastigotes. In contrast, melittin-treated trypomastigotes appeared to be dying via an apoptotic mechanism. Our findings confirm the great potential of AMPs, including melittin, as a potential source of new drugs for the treatment of neglected diseases, such as Chagas disease.