Apoptosis Machinery Research Articles

Abstract 3328: Caspase activation as an approach to treat multiple myeloma.

Abstract Apoptosis is a cell death program that is essential for normal organ development. Defects in apoptosis lead to uncontrolled cell accumulation resulting in cancer such as multiple myeloma (MM), an incurable plasma cell disorder. MM patients have defective apoptosis machinery largely due to the overexpression of anti-apoptotic proteins such as BCL-2, MCL-1, cIAP1, cIAP2 and XIAP. We are investigating a novel class of procaspase-3 activators as a strategy to activate apoptosis in myeloma cells. The Procaspase Activating Compounds (PAC), PAC-1 and its analog L14R8, were synthesized by Dr. Hergenrother's lab (U. of Illinois, Urbana, IL) and are being developed for clinical application by Vanquish Oncology. PACs convert the inactive zymogen procaspase-3 to caspase-3 which activates apoptosis. Mechanistically, PACs activate procaspase-3 by chelating inhibitory zinc ions thus allowing procaspase-3 to auto-activate to caspase-3. Our hypothesis is that targeting procaspase-3 with PAC agents will bypass inhibitory activity of the anti-apoptotic proteins and induce cell death in myeloma cells. In initial experiments, we examined procaspase-3 expression in a panel of 9 myeloma cell lines and observed that it was expressed in all the cell lines suggesting that PAC agents will be active in myeloma cells. Our investigations with PAC-1 and L14R8 demonstrated that L14R8, but not PAC-1, induced cell death in myeloma cells in a dose- and time-dependent manner. Treatment with 10 μM L14R8 for 24 hours induced >80% apoptosis in U266 and MM.1S cells. Furthermore, L14R8 activity was inhibited by exogenous addition of zinc confirming that the mechanism of L14R8 action is through zinc chelation. The activation of procaspase-3 resulted in the cleavage of its target PARP and decreased the levels of the anti-apoptotic proteins MCL-1 and XIAP. 10 μM L14R8 was also effective in inducing >80% cell death in myeloma cells grown in the presence of protective microenvironment as demonstrated by co-culture experiments with NKtert stromal cells. In addition, when myeloma cells were grown in the presence of exogenous growth factors such as hepatocyte growth factor (50 ng/ml) or interleukin-6 (10 or 50 ng/ml), L14R8 was still able to induce apoptosis (> 60% with 10 μM L14R8 treatment in the presence of either growth factor). Finally, L14R8 was effective in inducing apoptosis in cells resistant to lenalidomide, a standard treatment for myeloma patients. Our future plan is to examine the effectiveness of this drug in primary CD138+ plasma cells (representing malignant myeloma cells). These studies demonstrate that targeting procaspase-3 with L14R8 is an effective strategy to induce myeloma cell death. Citation Format: Shadia Zaman, Rui Wang, Varsha Gandhi. Caspase activation as an approach to treat multiple myeloma. [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 3328. doi:10.1158/1538-7445.AM2013-3328

KLF4 overexpression and apigenin treatment down regulated anti-apoptotic Bcl-2 proteins and matrix metalloproteinases to control growth of human malignant neuroblastoma SK-N-DZ and IMR-32 cells

Neuroblastoma is a childhood tumor that arises from immature neuroblasts of the sympathetic nervous system. Krüpple-like factor 4 (KLF4) is a transcription factor, the precise function of which in neuroblastoma is unclear. We examined the effects of KLF4 overexpression and apigenin (APG) treatment in human malignant neuroblastoma SK-N-DZ and IMR-32 cell lines. KLF4 overexpression in both SK-N-DZ and IMR-32 cell lines was confirmed by laser scanning immunofluorescent confocal microscopy and Western blotting. We found that 100 nM KLF4 plasmid and 25 μM APG synergistically inhibited the growth of SK-N-DZ and IMR-32 cells. We also found increase in KLF4 expression in response to treatment with various concentrations of APG. Combination of KLF4 plasmid and APG treatment significantly increased the amounts of apoptosis in both cell lines when compared with control vector or single treatment. We also noticed that the combination therapy decreased expression of the anti-apoptotic proteins Bcl-2 and Mcl-1, increased expression of the pro-apoptotic proteins Bax, Noxa, and Puma, upregulated p53, and caused activation of caspase-3 for cleavage of the inhibitor of caspase-activated DNase (ICAD) leading to completion of apoptosis machinery. Further, combination of KLF4 overexpression and APG treatment was highly effective in inhibiting migration of both neuroblastoma cell lines and was associated with down regulation of matrix metalloproteinases (MMPs) such as MMP-2 and MMP-9. Collectively, our results from this investigation strongly suggest that KLF4 functions as a tumor suppressor and potentiates the anti-cancer activities of APG in two different human malignant neuroblastoma cell lines.

JAK/STAT autocontrol of ligand-producing cell number through apoptosis

During development, specific cells are eliminated by apoptosis to ensure that the correct number of cells is integrated in a given tissue or structure. How the apoptosis machinery is activated selectively in vivo in the context of a developing tissue is still poorly understood. In the Drosophila ovary, specialised follicle cells [polar cells (PCs)] are produced in excess during early oogenesis and reduced by apoptosis to exactly two cells per follicle extremity. PCs act as an organising centre during follicle maturation as they are the only source of the JAK/STAT pathway ligand Unpaired (Upd), the morphogen activity of which instructs distinct follicle cell fates. Here we show that reduction of Upd levels leads to prolonged survival of supernumerary PCs, downregulation of the pro-apoptotic factor Hid, upregulation of the anti-apoptotic factor Diap1 and inhibition of caspase activity. Upd-mediated activation of the JAK/STAT pathway occurs in PCs themselves, as well as in adjacent terminal follicle and interfollicular stalk cells, and inhibition of JAK/STAT signalling in any one of these cell populations protects PCs from apoptosis. Thus, a Stat-dependent unidentified relay signal is necessary for inducing supernumerary PC death. Finally, blocking apoptosis of PCs leads to specification of excess adjacent border cells via excessive Upd signalling. Our results therefore show that Upd and JAK/STAT signalling induce apoptosis of supernumerary PCs to control the size of the PC organising centre and thereby produce appropriate levels of Upd. This is the first example linking this highly conserved signalling pathway with developmental apoptosis in Drosophila.

<i>In Vitro</i> Biological Evaluation of Anti-Tumor Effect of the Chelate-Setting Hydroxyapatite Cement

Cancers frequently metastasize to bone, where it leads to secondary tumor growth, and osteolytic bone degradation. Bone metastases are often associated with fractures and severe pain resulting in decreased quality of life. Accordingly, effective therapies to inhibit the development or progression of bone metastases will have important clinical benefits. Bone cement, one of the powerful tools as bone substitutes, is used to fill the resection voids. The aim of this study was to develop a local drug delivery system using HAp cement as a carrier of chemotherapeutic agents. In the present study, we have fabricated chelate-setting apatite cements (IP6-HAp cements) using HAp particles surface-modified with inositol hexaphosphate (IP6) and evaluated their anti-tumor effect. Human osteosarcoma (HOS) cultured on IP6-HAp cements (over 3000 ppm IP6) resulted in inhibition of cell growth. DNA microarray analysis indicated changes in the expression of apoptosis-related genes on IP6-HAp cement surface-modified with 5000 ppm IP6 compared with HAp cement, suggesting activation of apoptosis machinery by IP6-HAp cement. To clarify the mechanism of anti-tumor effect of IP6-HAp cement, the properties of cement were investigated. The release kinetics of IP6 from IP6-HAp cement showed that the level of released IP6 was insufficient to induce anti-tumor activity. These results led us to consider that locally high concentration of IP6 which was released from cement acts on the cells directly as anti-tumor agent and induces the apoptosis. Consequently, IP6-HAp cement might gain the anti-tumor effect and act as a carrier for local drug delivery system.

ASSESSING THE ROLE OF CASPASE ACTIVITY AND METACASPASE EXPRESSION ON VIRAL SUSCEPTIBILITY OF THE COCCOLITHOPHORE, EMILIANIA HUXLEYI (HAPTOPHYTA).

As part of their strategy to infect the globally important coccolithophore, Emiliania huxleyi (Lohmann) W.W. Hay & H.P. Mohler, Coccolithoviruses trigger and regulate the host's programmed cell death (PCD) machinery during lytic infection. The induction and recruitment of host metacaspases, specialized, ancestral death proteases that facilitate viral lysis, suggests they may be important subcellular determinants to infection. We examined the "basal" levels and patterns of caspase activity and metacaspase expression in exponentially growing resistant and sensitive E.huxleyi strains and linked them with susceptibility to E.huxleyi virus 1 (EhV1). Resistant E.huxleyi strains were consistently characterized by low caspase specific activity and a relatively simple metacaspase expression profile. In contrast, sensitive E.huxleyi strains had markedly elevated caspase specific activity and consistently expressed more diverse metacaspase proteins. Using pooled data sets from triplicate experiments, we observed statistically significant linear correlations between infectivity, caspase activity, and metacaspase expression, with each strain forming distinct clusters, within a gradient in viral susceptibility. At the same time, we observed positive correlations between the expression of a subset of metacaspase proteins and lower susceptibility, suggestive of potential protective roles. Our findings implicate the importance of subtle differences in the basal physiological regulation of the PCD machinery to viral resistance or sensitivity and cell fate.

CDKN1C/P57 Is Regulated by the Notch Target Gene Hes1 and Induces Senescence in Human Hepatocellular Carcinoma

CDKN1C/P57 is a cyclin-dependent kinase inhibitor implicated in different human cancers, including hepatocellular carcinoma (HCC); however, little is known regarding the role of CDKN1C/P57 and its regulation in HCC. In this study, we show that the down-regulation of Notch1 and Notch3 in two HCC cell lines resulted in Hes1 down-regulation, CDKN1C/P57 up-regulation, and reduced cell growth. In line with these data, we report that CDKN1C/P57 is a target of transcriptional repression by the Notch effector, Hes1. We found that the up-regulation of CDKN1C/P57 by cDNA transfection decreased tumor growth, as determined by growth curve, flow cytometry analysis, and cyclin D1 down-regulation, without affecting the apoptosis machinery. Indeed, the expression of Bax, Noxa, PUMA, BNIP(3), and cleaved caspase-3 was not affected by CDKN1C/P57 induction. Morphologically CDKN1C/p57-induced HCC cells became flat and lengthened in shape, accumulated the senescence-associated β-galactosidase marker, and increased P16 protein expression. Evaluation of senescence in cells depleted both for Hes1 and CDKN1C/P57 revealed that the senescent state really depends on the accumulation of CDKN1C/p57. Finally, we validated our in vitro results in primary HCCs, showing that Hes1 protein expression inversely correlates with CDKN1C/P57 mRNA levels. In addition, reduced Hes1 protein expression is accompanied by a shorter time to recurrence after curative resection, suggesting that Hes1 may represent a biomarker for prediction of patients with poor prognosis.

LDOC1 Gene Expression in Two Patients with Head and Neck Squamous Cell Carcinomas and Parkinson's Disease

Introduction Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers in the world. Risk factors for this cancer include tobacco and alcohol use, ultraviolet light exposure, and viral infection. Parkinson's disease is one of the most common neurodegenerative disorders, with a prevalence of 3% in persons over the age of 65 years. Apoptosis is a programmed cell death machinery pivotal for normal development, the establishment of highly organized neuronal circuitry, and the elimination of cancer cells. It has been suggested that increased expression of proapoptotic genes is associated with head tumors. One of these genes is the leucine zipper, down-regulated in cancer 1 (LDOC1) gene. Case report We report two interesting cases of a 79-year-old man and a 98-year-old woman, both with Parkinson's disease and well-differentiated multiple HNSCC, in whom we evaluated the possible differential expression of LDOC1. Results We found that LDOC1 gene expression was increased in both patients compared with three male and three female controls. Conclusions These findings suggest that apoptosis may play a pathogenetic role in HNSCC.

BAD-Dependent Regulation of Fuel Metabolism and KATP Channel Activity Confers Resistance to Epileptic Seizures

Neuronal excitation can be substantially modulated by alterations in metabolism, as evident from the anticonvulsant effect of diets that reduce glucose utilization and promote ketone body metabolism. We provide genetic evidence that BAD, a protein with dual functions in apoptosis and glucose metabolism, imparts reciprocal effects on metabolism of glucose and ketone bodies in brain cells. These effects involve phosphoregulation of BAD and are independent of its apoptotic function. BAD modifications that reduce glucose metabolism produce a marked increase in the activity of metabolically sensitive K(ATP) channels in neurons, as well as resistance to behavioral and electrographic seizures in vivo. Seizure resistance is reversed by genetic ablation of the K(ATP) channel, implicating the BAD-K(ATP) axis in metabolic control of neuronal excitation and seizure responses.

Abstract 2194: KLF4 overexpression and apigenin treatment inhibited cell migration and induced apoptosis in human malignant neuroblastoma SK-N-DZ and IMR32 cells

Abstract Neuroblastoma is a childhood tumor that arises from neuroblasts of the sympathetic nervous system and mostly occurs in adrenal gland. Despite major advances in our understanding of its biology and treatment strategies, malignant neuroblastoma still remains mostly incurable. So, identification of novel molecular targets and exploration of new therapeutic agents are urgently needed. Krüpple-like factor 4 (KLF4) is a transcription factor that shows divergent functions in different malignancies; however, the precise function of KLF4 in malignant neuroblastoma is unclear. In this study, we examined the effects of KLF4 overexpression and apigenin (APG) treatment in human malignant neuroblastoma SK-N-DZ and IMR32 cell lines. We transfected malignant neuroblastoma cells with the plasmid vector overexpressing KLF4 or the control vector and monitored the KLF4 overexpression by staining the cells with FITC conjugated KLF4 antibody and DAPI followed by laser scanning confocal microscopy. Further, KLF4 overexpression in both SK-N-DZ and IMR32 cell lines was confirmed by Western blotting. Then, we determined the reduction in cell viability after transfecting the cells with various concentrations of KLF4 expression vector and treating the cells with APG alone and in combinations. We found that 100 nM KLF4 expression vector and 25 µM APG acted synergistically for most effectively inhibiting the growth of SK-N-DZ and IMR32 cells. We also performed in vitro cell migration assay to monitor inhibition of cell migration and found that combination of KLF4 and APG was highly effective in inhibiting the migration of both neuroblastoma cell lines. Further, we examined the efficacy of combination of KLF4 and APG in induction of apoptosis in SK-N-DZ and IMR32 cells by in situ Wright staining and Annexin V-FITC/PI staining. Combination of KLF4 and APG significantly increased the amounts of apoptosis in both cell lines when compared with control vector or single treatment. We performed more Western blotting to determine the molecular mechanisms for inhibition of cell migration and induction of apoptosis. Matrix metalloproteinases (MMPs), especially MMP2 and MMP9, are responsible for proteolytic processing of extracellular matrix structural proteins and highly implicated in cancer cell migration. We found down regulation of both MMP2 and MMP9 after treatment with combination KLF4 and APG. We also noticed that combination therapy decreased expression of the anti-apoptotic protein Mcl-1, increased expression of the pro-apoptotic proteins Noxa and Puma, and caused activation of caspase-3 for cleavage of ICAD (inhibitor of caspase-activated DNase) leading to completion of apoptosis machinery. Collectively, our results strongly suggest that KLF4 functions as a tumor suppressor and potentiates anti-cancer activities of APG in different human malignant neuroblastoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2194. doi:1538-7445.AM2012-2194

NFκB and ubiquitination: partners in disarming RIPK1-mediated cell death

The mechanisms regulating cell survival and thus its corollary, cell death, have been intensively studied over the last two decades. Recent studies have shed new light into how non-degradative ubiquitination of the kinase RIPK1 is critical in determining this cell fate. In this review, we summarize recent findings on how ubiquitination of RIPK1 constitutes a survival signal through both NFκB-independent and NFκB-dependent mechanisms. However, in the absence of ubiquitination, RIPK1 becomes a death-signaling molecule capable of engaging both the caspase-dependent apoptosis machinery and the recently described RIPK3-dependent necroptosis machinery. Another layer of complexity is now emerging in that components of the ubiquitin-modifying machinery are themselves regulated by proteolytic processing. This survival/death regulatory mechanism has been best analyzed in the context of TNF receptor signaling, but it is likely that principles learned from TNFR may be applicable to other immune receptors including the antigen and Toll-like receptors.

Systems biology of yeast cell death

Programmed cell death (PCD) (including apoptosis) is an essential process, and many human diseases of high prevalence such as neurodegenerative diseases and cancer are associated with deregulations in the cell death pathways. Yeast Saccharomyces cerevisiae, a unicellular eukaryotic organism, shares with multicellular organisms (including humans) key components and regulators of the PCD machinery. In this article, we review the current state of knowledge about cell death networks, including the modeling approaches and experimental strategies commonly used to study yeast cell death. We argue that the systems biology approach will bring valuable contributions to our understanding of regulations and mechanisms of the complex cell death pathways.

On Programmed Cell Death inPlasmodium falciparum: Status Quo

Conflicting arguments and results exist regarding the occurrence and phenotype of programmed cell death (PCD) in the malaria parasite Plasmodium falciparum. Inconsistencies relate mainly to the number and type of PCD markers assessed and the different methodologies used in the studies. In this paper, we provide a comprehensive overview of the current state of knowledge and empirical evidence for PCD in the intraerythrocytic stages of P. falciparum. We consider possible reasons for discrepancies in the data and offer suggestions towards more standardised investigation methods in this field. Furthermore, we present genomic evidence for PCD machinery in P. falciparum. We discuss the potential adaptive or nonadaptive role of PCD in the parasite life cycle and its possible exploitation in the development of novel drug targets. Lastly, we pose pertinent unanswered questions concerning the PCD phenomenon in P. falciparum to provide future direction.

Understanding the Molecular Machinery of Apoptosis

Understanding the Molecular Machinery of Apoptosis

Regulation of anti‐apoptotic BCL2‐proteins by non‐canonical interactions: The next step forward or two steps back?

All aspects of cellular biology affect the process of regulated cell death, or apoptosis, and disruption of this process is a causative event in many diseases. Therefore, a comprehensive understanding of all pathways that regulate apoptosis would increase our knowledge of basic cellular functions, as well as the etiologies of many diseases. In turn, we may be able to use this knowledge to better treat patients with diseases, including cancer. Although the basic signaling pathway that regulates apoptosis has been known for over 10 years, we still have much to learn about the upstream signaling components that can directly regulate the core apoptosis machinery. The focus of this review will be to direct attention to non-canonical regulators of the BCL2-family of proteins, especially our void of understanding of such interactions, and the controversy that surrounds some such interactions.

C-Jun Regulates Glucocorticoid-Mediated BIM Induction and Apoptosis in Leukemia Cells: An Alternative Target to Overcome Glucocorticoid-Resistance,

Abstract 3486Glucocorticoids (GC) are common components in many chemotherapeutic protocols for lymphoid/myeloid malignancies, including acute lymphoblastic leukemia (ALL). However, patients often develop resistance to GC on relapse. Resistance to GC in ALL can be associated with defects in apoptosis machinery not in the GC receptor. Thus, targeting downstream molecules may lead to the development of new therapeutic strategies.GC-induced apoptosis is through the intrinsic mitochondria-dependent pathway. The BCL-2 family proteins are central regulatory proteins in this pathway. We and others have previously shown that BIM (BCL-2 Interacting Mediator of cell death), a pro-apoptotic BCL-2 family protein, is up-regulated by dexamethasone (Dex) treatment in ALL cells and plays an essential role in Dex-induced apoptosis (Lu et al., 2006). Furthermore, BIM is inactivated by extracellular signal-regulated kinase (ERK)-mediated phosphorylation by survival/growth factors (Harada et al., 2004). We therefore hypothesized that co-treatment with Dex and MEK/ERK inhibitors will promote apoptosis in ALL cells through BIM up-regulation and activation, resulting in cell death. Significantly, we have demonstrated that MEK inhibitors synergistically promote Dex lethality in a variety of ALL cell lines, in which BIM is a critical molecule in cell death (Rambal et al. 2009).Since BIM is capable to interact with all anti-apoptotic BCL-2 family proteins (i.e. BCL-2, BCL-XL, MCL-1), we hypothesized that down-regulation/inactivation of the anti-apoptotic BCL-2 family proteins is also effective to enhance the activity of BIM. We found that down-regulation of anti-apoptotic BCL-2 family proteins, especially MCL-1, enhanced GC-induced cell death. We then targeted MCL-1 in ALL cells by using GX15-070 (obatoclax), a small molecule inhibitor for anti-apoptotic BCL-2 family proteins. Treatment with GX15-070 in both Dex-sensitive and -resistant ALL cells showed effective growth inhibition and cell death (Heidari et al. 2010). GX15-070 treatment in ALL could be an alternative regimen to overcome GC-resistance.To further explore new treatments to overcome resistance to GC, it is important to elucidate entire signaling pathways for the activation of BIM in GC treatment. It has been indicated that Dex-induced BIM is regulated mainly by transcription, however the molecular mechanisms, including responsible transcription factors, of how Bim is induced are unclear. In this study, we examined the role of c-Jun in Dex-induced Bim transcriptional regulation in ALL cells. Dex treatment induced c-Jun mRNA and protein in parallel with BIM induction. BIM induction decreased in cells harboring dominant-negative c-Jun protein and increased in cells with overexpression of c-Jun. On the Bim promoter, c-Jun bound to and activated the AP-1 binding site at about −2.7kb from the transcription start site. Furthermore, pre-treatment with SB203580, a p38-MAPK inhibitor, decreased the amount of Dex-induced mRNAs and proteins of c-Jun and BIM, suggesting that p38-MAPK activation is upstream of c-Jun and BIM induction. Treatment with RU486, a GC receptor (GR) antagonist, blocked Dex-induced p38-MAPK phosphorylation, c-Jun and BIM induction, and apoptosis. In conclusion, we identified the critical signaling pathway for GC-induced apoptosis, i.e. GC–>GR–>p38-MAPK–>c-Jun–>BIM–>Apoptosis. Targeting this pathway may be an alternative approach to overcome GC-resistance in leukemia treatment. Disclosures:No relevant conflicts of interest to declare.

Análise interactômica da VDAC (voltage-dependent anion selective channel) nos cérebros aviar, bovino e murino

A VDAC é a proteína mais abundante na membrana mitocondrial externa. Exerce o controle da atividade desta organela através da regulação da troca de metabólitos e tem função crucial no mecanismo de apoptose. Em nosso caso, os estudos dos complexos protéicos, das interações entre a VDAC e outras proteínas presentes no interior do neurônio que auxiliam na manutenção das funções das organelas e da célula, fazem parte da chamada interactômica. O presente estudo determinou o interactoma do complexo protéico Hexoquinase-VDAC-ANT presente em cérebros murino, bovino e aviar. Nosso objetivo foi identificar se as expressões diferenciadas da VDAC1 e VDAC2 verificadas nos cérebros murino, aviar e bovino, estão associadas a diferenças nos interactomas dessas proteínas. Este estudo revelou que as espécies aviar e bovina apresentaram o maior número de complexos protéicos contendo VDACs (5) quando comparadas com os neurônios de rato (1), o que é indicativo de uma cinética diferencial de montagem ou desmontagem do complexo. Além disso, a VDAC mitocondrial neuronal aviar também interage com mais proteínas em relação à VDAC mitocondrial neuronal bovina, o que é resultado de uma composição de subunidades diferenciada. Tais resultados indicam diferenças significativas quanto ao metabolismo energético e apoptótico no cérebro aviar, bovino e murino, existindo interações diferenciais da VDAC no cérebro aviar.

Dual Face Apoptotic Machinery: From Initiator of Apoptosis to Guardian of Necroptosis

In this issue of Immunity, Bonnet etal. (2011) show that skin-specific ablation of the adaptor protein FADD sensitizes keratinocytes to RIPK3-dependent necrotic cell death, which leads to severe skin inflammation.

The yolk cell of the zebrafish blastula harbors functional apoptosis machinery.

We recently described the implication of the Bcl-2 related antiapoptotic Nrz protein during early zebrafish development. Nrz knock-down induces calcium-dependent cytoskeleton remodeling leading to margin constriction and premature embryo lethality. In the YSL, nrz knock-down embryos exhibit some typical features of apoptosis such as mitochondrial transmembrane potential loss and cytochrome c release. However, downstream caspase-3 activation has not been detected so far. Here, we report that the YSL contains fully functional apoptotic machinery that can activate caspase-3 following zBax ectopic expression. Furthermore, we present evidence that caspase-3 activation is actually detectable in nrz knock-down embryos when premature margin constriction is prevented.

Anti-Apoptotic Machinery Protects the Necrotrophic Fungus Botrytis cinerea from Host-Induced Apoptotic-Like Cell Death during Plant Infection

Necrotrophic fungi are unable to occupy living plant cells. How such pathogens survive first contact with living host tissue and initiate infection is therefore unclear. Here, we show that the necrotrophic grey mold fungus Botrytis cinerea undergoes massive apoptotic-like programmed cell death (PCD) following germination on the host plant. Manipulation of an anti-apoptotic gene BcBIR1 modified fungal response to PCD-inducing conditions. As a consequence, strains with reduced sensitivity to PCD were hyper virulent, while strains in which PCD was over-stimulated showed reduced pathogenicity. Similarly, reduced levels of PCD in the fungus were recorded following infection of Arabidopsis mutants that show enhanced susceptibility to B. cinerea. When considered together, these results suggest that Botrytis PCD machinery is targeted by plant defense molecules, and that the fungal anti-apoptotic machinery is essential for overcoming this host-induced PCD and hence, for establishment of infection. As such, fungal PCD machinery represents a novel target for fungicides and antifungal drugs.

Melanocortin 4 Receptor Activation Protects Against Testicular Ischemia-Reperfusion Injury by Triggering the Cholinergic Antiinflammatory Pathway

Melanocortins (MC) trigger a vagus nerve-mediated cholinergic-antiinflammatory pathway projecting to the testis. We tested whether pharmacological activation of brain MC receptors might protect the testis from the damage induced by ischemia-reperfusion. Adult male rats were subjected to 1-h testicular ischemia, followed by 24-h reperfusion [testicular ischemia-reperfusion (TI/R)]. Before TI/R, groups of animals were subjected to bilateral cervical vagotomy, or pretreated with the nicotinic acetylcholine receptor antagonist chlorisondamine or the selective MC(4) receptor antagonist HS024. Immediately after reperfusion, rats were ip treated with saline or the MC analog [Nle(4),D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH) (340 μg/kg). We evaluated testicular IL-6 and TNF-α by Western blot analysis and organ damage by light microscopy. Some experimental groups were prepared for neural efferent activity recording along the vagus nerve starting 30 min after treatment with NDP-α-MSH or saline, and for a 30-min period. Additional groups of TI/R rats were treated for 30 d with saline, NDP-α-MSH, chlorisondamine plus NDP-α-MSH, or HS024 plus NDP-α-MSH to evaluate spermatogenesis, organ damage, and the apoptosis machinery. After a 24-h reperfusion, in TI/R saline-treated rats, there was an increase in IL-6 and TNF-α expression and a marked damage in both testes. NDP-α-MSH inhibited IL-6 and TNF-α expression, decreased histological damage, and increased neural efferent activity. Furthermore, NDP-α-MSH administration for 30 d greatly improved spermatogenesis, reduced organ damage, and inhibited apoptosis. All positive NDP-α-MSH effects were abrogated by vagotomy, chlorisondamine, or HS024. Our data suggest that selective MC(4) receptor agonists might be therapeutic candidates for the management of testicular torsion.