The Experts below are selected from a list of 3135 Experts worldwide ranked by ideXlab platform
Alfred H. Merrill - One of the best experts on this subject based on the ideXlab platform.
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Development of pheochromocytoma in Ceramide Synthase 2 null mice
Endocrine-related cancer, 2015Co-Authors: Woo-jae Park, Alfred H. Merrill, Yael Pewzner-jung, Ori Brenner, Aviram Kogot-levin, Ann Saada, Anthony H FutermanAbstract:Pheochromocytoma (PCC) and paraganglioma are rare neuroendocrine tumors of the adrenal medulla and sympathetic and parasympathetic paraganglia, for which mutations in ∼15 disease-associated genes have been identified. We now document the role of an additional gene in mice, the Ceramide Synthase 2 (CerS2) gene. CerS2, one of six mammalian CerS, synthesizes Ceramides with very-long (C22-C24) chains. The CerS2 null mouse has been well characterized and displays lesions in several organs including the liver, lung and the brain. We now demonstrate that changes in the sphingolipid acyl chain profile of the adrenal gland lead to the generation of adrenal medullary tumors. Histological analyses revealed that about half of the CerS2 null mice developed PCC by ∼13 months, and the rest showed signs of medullary hyperplasia. Norepinephrine and normetanephrine levels in the urine were elevated at 7 months of age consistent with the morphological abnormalities found at later ages. Accumulation of ceroid in the X-zone was observed as early as 2 months of age and as a consequence, older mice displayed elevated levels of lysosomal cathepsins, reduced proteasome activity and reduced activity of mitochondrial complex IV by 6 months of age. Together, these findings implicate an additional pathway that can lead to PCC formation, which involves alterations in the sphingolipid acyl chain length. Analysis of the role of sphingolipids in PCC may lead to further understanding of the mechanism by which PCC develops, and might implicate the sphingolipid pathway as a possible novel therapeutic target for this rare tumor.
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ablation of Ceramide Synthase 2 causes chronic oxidative stress due to disruption of the mitochondrial respiratory chain
Journal of Biological Chemistry, 2013Co-Authors: Hila Zigdo, Alfred H. Merrill, Aviram Kogotlevi, Joowo Park, Ruth Goldschmid, Samuel Kelly, Avigdo Scherz, Yael Pewznerjung, A Saada, Anthony H FutermaAbstract:Ceramide is a key intermediate in the pathway of sphingolipid biosynthesis and is an important intracellular messenger. We recently generated a Ceramide Synthase 2 (CerS2) null mouse that cannot synthesize very long acyl chain (C22-C24) Ceramides. This mouse displays severe and progressive hepatopathy. Significant changes were observed in the sphingolipid profile of CerS2 null mouse liver, including elevated C16-Ceramide and sphinganine levels in liver and in isolated mitochondrial fractions. Because Ceramide may be involved in reactive oxygen species (ROS) formation, we examined whether ROS generation was affected in CerS2 null mice. Levels of a number of anti-oxidant enzymes were elevated, as were lipid peroxidation, protein nitrosylation, and ROS. ROS were generated from mitochondria due to impaired complex IV activity. C16-Ceramide, sphingosine, and sphinganine directly inhibited complex IV activity in isolated mitochondria and in mitoplasts, whereas other Ceramide species, sphingomyelin, and diacylglycerol were without effect. A fluorescent analog of sphinganine accumulated in mitochondria. Heart mitochondria did not display a substantial alteration in the sphingolipid profile or in complex IV activity. We suggest that C16-Ceramide and/or sphinganine induce ROS formation through the modulation of mitochondrial complex IV activity, resulting in chronic oxidative stress. These results are of relevance for understanding modulation of ROS signaling by sphingolipids.
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Ablation of Ceramide Synthase 2 strongly affects biophysical properties of membranes
Journal of lipid research, 2012Co-Authors: Liana C. Silva, Alfred H. Merrill, Elad L. Laviad, Yael Pewzner-jung, Oshrit Ben David, Johnny Stiban, Sibali Bandyopadhyay, Manuel Prieto, Anthony H FutermanAbstract:Little is known about the effects of altering sphingolipid (SL) acyl chain structure and composition on the biophysical properties of biological membranes. We explored the biophysical consequences of depleting very long acyl chain (VLC) SLs in membranes prepared from lipid fractions isolated from a Ceramide Synthase 2 (CerS2)-null mouse, which is unable to synthesize C22–C24 Ceramides. We demonstrate that ablation of CerS2 has different effects on liver and brain, causing a significant alteration in the fluidity of the membrane and affecting the type and/or extent of the phases present in the membrane. These changes are a consequence of the depletion of VLC and unsaturated SLs, which occurs to a different extent in liver and brain. In addition, ablation of CerS2 causes changes in intrinsic membrane curvature, leading to strong morphological alterations that promote vesicle adhesion, membrane fusion, and tubule formation. Together, these results show that depletion of VLC-SLs strongly affects membrane biophysical properties, which may compromise cellular processes that critically depend on membrane structure, such as trafficking and sorting.
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Hydrolyzed fumonisins HFB1 and HFB2 are acylated in vitro and in vivo by Ceramide Synthase to form cytotoxic N-acyl-metabolites.
Molecular nutrition & food research, 2007Co-Authors: Michaela Seiferlein, Elaine Wang, M. Cameron Sullards, Jeremy C. Allegood, Hans-ulrich Humpf, Kenneth A. Voss, Alfred H. MerrillAbstract:Fumonisins B 1 and B 2 (FB 1 and FB 2 ) are the most abundant members of the fumonisins - mycotoxins that are produced by Fusarium verticillioides and are natural inhibitors of Ceramide Synthase. Their hydrolyzed forms, HFB 1 and HFB 2 (also called AP 1 and AP 2 ) are found in some foods, and they are not only inhibitors of Ceramide Synthase but also undergo acylation by this enzyme. This study characterized the conversion of HFB 1 and HFB 2 by Ceramide Synthase to their respective N-acylated metabolites using rat liver microsomes and palmitoyl-CoA or nervonoyl-CoA as cosubstrates, and examined animals that had been dosed with hydrolyzed fumonisins to ascertain if acylation occurs in vivo. Using an HPLC-MS/MS method that allowed the sensitive and selective detection of the acylation products, both HFB 1 and HFB 2 were found to be metabolized in vitro to nervonoyl- or palmitoyl-HFB 1 and -HFB 2 (i. e. C 24:1 -HFB 1/2 and C 16 -HFB 1/2 , respectively). The apparent v max was considerably higher for formation of C 24:1 HFB 1 (157 pmol/min/mg protein) than for formation of C 16 HFB 1 (8.7 pmol/min/mg protein). The acylation products also inhibited Ceramide Synthase and significantly reduced the number of viable cells in an in vitro [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] assay using a human colonic cell line (HT29). Furthermore, HPLC-MS/MS analysis of tissues from rats given intraperitoneal doses of HFB 1 confirmed that formation of N-acylHFB 1 occurs in vivo to produce metabolites with fatty acids of various chain lengths. The contribution of acylated HFB 1 and HFB 2 metabolites to fumonisin toxicity in vivo warrants further investigation.
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[3] Ceramide Synthase
Methods in Enzymology, 2004Co-Authors: Elaine Wang, Alfred H. MerrillAbstract:Publisher Summary Ceramide Synthase catalyzes the acylation of sphinganine, sphingosine, and other long chain sphingoid bases to form their N-acyl derivatives, which are usually referred to as “dihydroCeramides” (N-acylsphinganines) and “Ceramides” (N-acylsphingosines). Ceramide Synthase is, therefore, involved in both the early steps of de novo sphingolipid biosynthesis (the acylation of sphinganine) and the recycling of free sphingoid bases generated in cells—for example, from Ceramide hydrolysis—or taken up from exogenous sources. Ceramide Synthase activity is conventionally regarded as the acylation of sphingoid bases using fatty acyl-CoA of varying fatty acid chain length, although there have been occasional reports of Ceramide synthesis via the condensation of sphingosine and a free fatty acid, perhaps by the reversal of ceramidase. The significance of this latter pathway for sphingolipid metabolism is unclear; therefore, this article deals only with the fatty acyl-CoA-dependent reaction. Ceramide Synthase is of interest not only because it forms the lipid backbone for complex sphingolipid biosynthesis, but also as the target of a number of microbial toxins and as a possible participant in the induction of apoptosis by various stimuli and toxins.
Anthony H Futerman - One of the best experts on this subject based on the ideXlab platform.
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LPS-mediated septic shock is augmented in Ceramide Synthase 2 null mice due to elevated activity of TNFα-converting enzyme.
FEBS Letters, 2015Co-Authors: Ashish Saroha, Yael Pewzner-jung, Anthony H FutermanAbstract:Abstract Tumor necrosis factor α (TNFα) is an inflammatory cytokine that plays an intimate role in septic shock. Injection of high levels of lipopolysaccharide induces septic shock and death in mice within 30 h, whereas Ceramide Synthase 2 (CerS2) null mice, defective in the synthesis of very-long acyl chain Ceramides, die within ∼10 h. The augmented rate of death of CerS2 null mice is due to elevated levels of TNFα secretion as a result of enhanced activity of TNFα-converting enzyme (TACE). We discuss the relationship between the sphingolipid acyl chain length and TACE activity and the relevance of this data to septic shock.
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Development of pheochromocytoma in Ceramide Synthase 2 null mice
Endocrine-related cancer, 2015Co-Authors: Woo-jae Park, Alfred H. Merrill, Yael Pewzner-jung, Ori Brenner, Aviram Kogot-levin, Ann Saada, Anthony H FutermanAbstract:Pheochromocytoma (PCC) and paraganglioma are rare neuroendocrine tumors of the adrenal medulla and sympathetic and parasympathetic paraganglia, for which mutations in ∼15 disease-associated genes have been identified. We now document the role of an additional gene in mice, the Ceramide Synthase 2 (CerS2) gene. CerS2, one of six mammalian CerS, synthesizes Ceramides with very-long (C22-C24) chains. The CerS2 null mouse has been well characterized and displays lesions in several organs including the liver, lung and the brain. We now demonstrate that changes in the sphingolipid acyl chain profile of the adrenal gland lead to the generation of adrenal medullary tumors. Histological analyses revealed that about half of the CerS2 null mice developed PCC by ∼13 months, and the rest showed signs of medullary hyperplasia. Norepinephrine and normetanephrine levels in the urine were elevated at 7 months of age consistent with the morphological abnormalities found at later ages. Accumulation of ceroid in the X-zone was observed as early as 2 months of age and as a consequence, older mice displayed elevated levels of lysosomal cathepsins, reduced proteasome activity and reduced activity of mitochondrial complex IV by 6 months of age. Together, these findings implicate an additional pathway that can lead to PCC formation, which involves alterations in the sphingolipid acyl chain length. Analysis of the role of sphingolipids in PCC may lead to further understanding of the mechanism by which PCC develops, and might implicate the sphingolipid pathway as a possible novel therapeutic target for this rare tumor.
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a rapid Ceramide Synthase activity using nbd sphinganine and solid phase extraction
Journal of Lipid Research, 2015Co-Authors: Rotem Tidhar, Kacee H Sims, Eden Rosenfeldgur, Walter Shaw, Anthony H FutermanAbstract:Ceramides are synthesized by six mammalian ce- ramide Synthases (CerSs), each of which uses fatty acyl- CoAs of different chain lengths for N -acylation of the sphingoid long-chain base. We now describe a rapid and reli- able CerS assay that uses a fl uorescent N-(6-((7-nitrobenzo- 2-oxa-1,3-diazol-4-yl) (NBD ) sphinganine substrate followed by separation of the NBD-lipid substrate and products using solid phase extraction (SPE) C18 chromatography. SPE chromatography is a quick and reliable alternative to TLC, and moreover, there is no degradation of either NBD-sphinganine or NBD-Ceramide. We have optimized the assay for use with minimal amounts of protein in a minimal volume. This assay will prove useful for the analysis of CerS activity, which is of particular importance in light of the growing involvement of CerS in cell regulation and in the pathology of human diseases. —Tidhar, R., K. Sims, E. Rosenfeld-Gur, W. Shaw, and A. H. Futerman. A rapid Ceramide Synthase activity using NBD-sphinganine and solid phase extraction. J. Lipid Res . 2015. 56: 193-199.
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Ceramide Synthases expression and role of Ceramide Synthase-2 in the lung: insight from human lung cells and mouse models.
PloS one, 2013Co-Authors: Irina Petrache, Elad L. Laviad, Yael Pewzner-jung, Krzysztof Kamocki, Christophe Poirier, Kelly S. Schweitzer, Mary Van Demark, Matthew J. Justice, Walter C. Hubbard, Anthony H FutermanAbstract:Increases in Ceramide levels have been implicated in the pathogenesis of both acute or chronic lung injury models. However, the role of individual Ceramide species, or of the enzymes that are responsible for their synthesis, in lung health and disease has not been clarified. We now show that C24- and C16-Ceramides are the most abundant lung Ceramide species, paralleled by high expression of their synthetic enzymes, Ceramide Synthase 2 (CerS2) and CerS5, respectively. Furthermore, the Ceramide species synthesis in the lung is homeostatically regulated, since mice lacking very long acyl chain C24-Ceramides due to genetic deficiency of CerS2 displayed a ten-fold increase in C16-Ceramides and C16-dihydroCeramides along with elevation of acid sphingomyelinase and CerS5 activities. Despite relatively preserved total lung Ceramide levels, inhibition of de novo sphingolipid synthesis at the level of CerS2 was associated with significant airflow obstruction, airway inflammation, and increased lung volumes. Our results suggest that Ceramide species homeostasis is crucial for lung health and that CerS2 dysfunction may predispose to inflammatory airway and airspace diseases.
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Ablation of Ceramide Synthase 2 strongly affects biophysical properties of membranes
Journal of lipid research, 2012Co-Authors: Liana C. Silva, Alfred H. Merrill, Elad L. Laviad, Yael Pewzner-jung, Oshrit Ben David, Johnny Stiban, Sibali Bandyopadhyay, Manuel Prieto, Anthony H FutermanAbstract:Little is known about the effects of altering sphingolipid (SL) acyl chain structure and composition on the biophysical properties of biological membranes. We explored the biophysical consequences of depleting very long acyl chain (VLC) SLs in membranes prepared from lipid fractions isolated from a Ceramide Synthase 2 (CerS2)-null mouse, which is unable to synthesize C22–C24 Ceramides. We demonstrate that ablation of CerS2 has different effects on liver and brain, causing a significant alteration in the fluidity of the membrane and affecting the type and/or extent of the phases present in the membrane. These changes are a consequence of the depletion of VLC and unsaturated SLs, which occurs to a different extent in liver and brain. In addition, ablation of CerS2 causes changes in intrinsic membrane curvature, leading to strong morphological alterations that promote vesicle adhesion, membrane fusion, and tubule formation. Together, these results show that depletion of VLC-SLs strongly affects membrane biophysical properties, which may compromise cellular processes that critically depend on membrane structure, such as trafficking and sorting.
Zvi Fuks - One of the best experts on this subject based on the ideXlab platform.
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regulation of Ceramide Synthase mediated crypt epithelium apoptosis by dna damage repair enzymes
Cancer Research, 2010Co-Authors: Jimmy A Rotolo, Richard Kolesnick, Adriana Haimovitzfriedman, Judith Mesicek, Jerzy G Maj, Jeanphilip Truman, Zvi FuksAbstract:Acute endothelial cell apoptosis and microvascular compromise couple gastrointestinal tract irradiation to reproductive death of intestinal crypt stem cell clonogens (SCCs) following high-dose radiation. Genetic or pharmacologic inhibition of endothelial apoptosis prevents intestinal damage, but as the radiation dose is escalated, SCCs become directly susceptible to an alternate cell death mechanism, mediated via Ceramide Synthase (CS)-stimulated de novo synthesis of the proapoptotic sphingolipid Ceramide, and p53-independent apoptosis of crypt SCCs. We previously reported that ataxia-telangiectasia mutated deficiency resets the primary radiation lethal pathway, allowing CS-mediated apoptosis at the low-dose range of radiation. The mechanism for this event, termed target reordering, remains unknown. Here, we show that inactivation of DNA damage repair pathways signals CS-mediated apoptosis in crypt SCCs, presumably via persistent unrepaired DNA double-strand breaks (DSBs). Genetic loss of function of sensors and transducers of DNA DSB repair confers the CS-mediated lethal pathway in intestines of sv129/B6Mre11(ATLD1/ATLD1) and C57BL/6(Prkdc/SCID) (severe combined immunodeficient) mice exposed to low-dose radiation. In contrast, CS-mediated SCC lethality was mitigated in irradiated gain-of-function Rad50(s/s) mice, and epistasis studies order Rad50 upstream of Mre11. These studies suggest unrepaired DNA DSBs as causative in target reordering in intestinal SCCs. As such, we provide an in vivo model of DNA damage repair that is standardized, can be exploited to understand allele-specific regulation in intact tissue, and is pharmacologically tractable.
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Reversal of Radiation Resistance in LNCaP Cells by Targeting Apoptosis through Ceramide Synthase
Cancer Research, 1999Co-Authors: Mark Garzotto, Adriana Haimovitz-friedman, Margaret White-jones, Robert Huryk, Carlos Cardon-cardo, Wen-chieh Liao, Warren D. W. Heston, Richard Kolesnick, Zvi FuksAbstract:Cell lines derived from human prostate cancer are regarded as relatively resistant to both radiation-induced clonogenic death and apoptosis. Here we attempted to modulate the response of LNCaP prostate cancer cells to radiation therapy (XRT) by pretreatment with 12- O -tetradecanoylphorbol acetate (TPA), a known apoptogenic agent in LNCaP cells. Using plateau-phase cultures, we investigated the response of these cells to XRT, TPA, and a combination of XRT and TPA. LNCaP irradiation did not result in Ceramide generation or apoptosis. However, pretreatment with TPA enabled XRT to generate Ceramide via activation of the enzyme Ceramide Synthase and signal apoptosis. Apoptosis was abrogated by the competitive inhibitor of Ceramide Synthase, fumonisin B 1 . Furthermore, when transplanted orthotopically into the prostate of nude mice, LNCaP cells produced tumors that recapitulated the responses of LNCaP cells in vitro . XRT or TPA failed to signal apoptosis in LNCaP tumors, whereas a combination of the two resulted in substantial (20–25%) apoptosis within 24 h. There was an additional benefit associated with this regimen because TPA pretreatment protected the adjacent rectum from radiation-induced apoptosis. This represents the first description of signaling-based therapy designed to overcome one form of radiation resistance expressed preferentially in LNCaP human prostate cancer cells.
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ataxia telangiectasia mutated gene product inhibits dna damage induced apoptosis via Ceramide Synthase
Journal of Biological Chemistry, 1999Co-Authors: Wen-chieh Liao, Roger S Persaud, Maureen Mcloughlin, Desiree Ehleiter, Magtouf Gatei, Richard Kolesnick, Adriana Haimovitzfriedman, Martin F. Lavin, Ning Zhang, Zvi FuksAbstract:Abstract DNA double-stranded breaks (dsb) activate surveillance systems that identify DNA damage and either initiate repair or signal cell death. Failure of cells to undergo appropriate death in response to DNA damage leads to misrepair, mutations, and neoplastic transformation. Pathways linking DNA dsb to reproductive or apoptotic death are virtually unknown. Here we report that metabolic incorporation of 125I-labeled 5-iodo-2′deoxyuridine, which produces DNA dsb, signaled de novo Ceramide synthesis by post-translational activation of Ceramide Synthase (CS) and apoptosis. CS activation was obligatory, since fumonisin B1, a fungal pathogen that acts as a specific CS inhibitor, abrogated DNA damage-induced death. X-irradiation yielded similar results. Furthermore, inhibition of apoptosis using the peptide caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone did not affect CS activation, indicating this event is not a consequence of induction of apoptosis. ATM, the gene mutated in ataxia telangiectasia, is a member of the phosphatidylinositol 3-kinase family that constitutes the DNA damage surveillance/repair system. Epstein-Barr virus-immortalized B cell lines from six ataxia telangiectasia patients with different mutations exhibited radiation-induced CS activation, Ceramide generation, and apoptosis, whereas three lines from normal patients failed to manifest these responses. Stable transfection of wild type ATM cDNA reversed these events, whereas antisense inactivation of ataxia telangiectasia-mutated gene product in normal B cells conferred the ataxia telangiectasia phenotype. We propose that one of the functions of ataxia telangiectasia-mutated gene product is to constrain activation of CS, thereby regulating DNA damage-induced apoptosis.
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ataxia telangiectasia mutated gene product inhibits dna damage induced apoptosis via Ceramide Synthase
Journal of Biological Chemistry, 1999Co-Authors: Wen-chieh Liao, Roger S Persaud, Maureen Mcloughlin, Desiree Ehleiter, Magtouf Gatei, Richard Kolesnick, Adriana Haimovitzfriedman, Martin F. Lavin, Ning Zhang, Zvi FuksAbstract:Abstract DNA double-stranded breaks (dsb) activate surveillance systems that identify DNA damage and either initiate repair or signal cell death. Failure of cells to undergo appropriate death in response to DNA damage leads to misrepair, mutations, and neoplastic transformation. Pathways linking DNA dsb to reproductive or apoptotic death are virtually unknown. Here we report that metabolic incorporation of 125I-labeled 5-iodo-2′deoxyuridine, which produces DNA dsb, signaled de novo Ceramide synthesis by post-translational activation of Ceramide Synthase (CS) and apoptosis. CS activation was obligatory, since fumonisin B1, a fungal pathogen that acts as a specific CS inhibitor, abrogated DNA damage-induced death. X-irradiation yielded similar results. Furthermore, inhibition of apoptosis using the peptide caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone did not affect CS activation, indicating this event is not a consequence of induction of apoptosis. ATM, the gene mutated in ataxia telangiectasia, is a member of the phosphatidylinositol 3-kinase family that constitutes the DNA damage surveillance/repair system. Epstein-Barr virus-immortalized B cell lines from six ataxia telangiectasia patients with different mutations exhibited radiation-induced CS activation, Ceramide generation, and apoptosis, whereas three lines from normal patients failed to manifest these responses. Stable transfection of wild type ATM cDNA reversed these events, whereas antisense inactivation of ataxia telangiectasia-mutated gene product in normal B cells conferred the ataxia telangiectasia phenotype. We propose that one of the functions of ataxia telangiectasia-mutated gene product is to constrain activation of CS, thereby regulating DNA damage-induced apoptosis.
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12 o tetradecanoylphorbol 13 acetate induced apoptosis in lncap cells is mediated through Ceramide Synthase
Cancer Research, 1998Co-Authors: Mark Garzotto, Desiree Ehleiter, Wen-chieh Liao, Adriana Haimovitzfriedman, Zvi Fuks, Margaret Whitejones, Youwei Jiang, Richard KolesnickAbstract:Protein kinase C (PKC) activation is often antiapoptotic, although in a few cell types PKC initiates apoptosis by an unknown mechanism. Recent investigations showed that activation of PKCα by 12- O -tetradecanoylphorbol 13-acetate (TPA) induced apoptosis in LNCaP prostate cancer cells. The present studies examine the mechanism of this effect and show that de novo Ceramide generation through the enzyme Ceramide Synthase is required. TPA induced rapid Ceramide generation, which was detectable by 1 h and increased linearly for 12 h. TPA-induced apoptosis was measurable by 12 h and was progressive for 48 h. Investigations into the mechanism of TPA-induced Ceramide generation revealed that acid and neutral sphingomyelinase activities were not enhanced. However, TPA induced an increase in Ceramide Synthase activity that persisted for at least 16 h. Treatment with fumonisin B1, a specific natural inhibitor of Ceramide Synthase, abrogated both Ceramide production and TPA-induced apoptosis. Ceramide analogues bypassed fumonisin B1 inhibition to initiate apoptosis directly. Thus, Ceramide appears to be a necessary signal for TPA-induced apoptosis in LNCaP cells. This represents the first description of a pathway by which PKC may signal apoptosis.
Richard Kolesnick - One of the best experts on this subject based on the ideXlab platform.
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regulation of Ceramide Synthase mediated crypt epithelium apoptosis by dna damage repair enzymes
Cancer Research, 2010Co-Authors: Jimmy A Rotolo, Richard Kolesnick, Adriana Haimovitzfriedman, Judith Mesicek, Jerzy G Maj, Jeanphilip Truman, Zvi FuksAbstract:Acute endothelial cell apoptosis and microvascular compromise couple gastrointestinal tract irradiation to reproductive death of intestinal crypt stem cell clonogens (SCCs) following high-dose radiation. Genetic or pharmacologic inhibition of endothelial apoptosis prevents intestinal damage, but as the radiation dose is escalated, SCCs become directly susceptible to an alternate cell death mechanism, mediated via Ceramide Synthase (CS)-stimulated de novo synthesis of the proapoptotic sphingolipid Ceramide, and p53-independent apoptosis of crypt SCCs. We previously reported that ataxia-telangiectasia mutated deficiency resets the primary radiation lethal pathway, allowing CS-mediated apoptosis at the low-dose range of radiation. The mechanism for this event, termed target reordering, remains unknown. Here, we show that inactivation of DNA damage repair pathways signals CS-mediated apoptosis in crypt SCCs, presumably via persistent unrepaired DNA double-strand breaks (DSBs). Genetic loss of function of sensors and transducers of DNA DSB repair confers the CS-mediated lethal pathway in intestines of sv129/B6Mre11(ATLD1/ATLD1) and C57BL/6(Prkdc/SCID) (severe combined immunodeficient) mice exposed to low-dose radiation. In contrast, CS-mediated SCC lethality was mitigated in irradiated gain-of-function Rad50(s/s) mice, and epistasis studies order Rad50 upstream of Mre11. These studies suggest unrepaired DNA DSBs as causative in target reordering in intestinal SCCs. As such, we provide an in vivo model of DNA damage repair that is standardized, can be exploited to understand allele-specific regulation in intact tissue, and is pharmacologically tractable.
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Reversal of Radiation Resistance in LNCaP Cells by Targeting Apoptosis through Ceramide Synthase
Cancer Research, 1999Co-Authors: Mark Garzotto, Adriana Haimovitz-friedman, Margaret White-jones, Robert Huryk, Carlos Cardon-cardo, Wen-chieh Liao, Warren D. W. Heston, Richard Kolesnick, Zvi FuksAbstract:Cell lines derived from human prostate cancer are regarded as relatively resistant to both radiation-induced clonogenic death and apoptosis. Here we attempted to modulate the response of LNCaP prostate cancer cells to radiation therapy (XRT) by pretreatment with 12- O -tetradecanoylphorbol acetate (TPA), a known apoptogenic agent in LNCaP cells. Using plateau-phase cultures, we investigated the response of these cells to XRT, TPA, and a combination of XRT and TPA. LNCaP irradiation did not result in Ceramide generation or apoptosis. However, pretreatment with TPA enabled XRT to generate Ceramide via activation of the enzyme Ceramide Synthase and signal apoptosis. Apoptosis was abrogated by the competitive inhibitor of Ceramide Synthase, fumonisin B 1 . Furthermore, when transplanted orthotopically into the prostate of nude mice, LNCaP cells produced tumors that recapitulated the responses of LNCaP cells in vitro . XRT or TPA failed to signal apoptosis in LNCaP tumors, whereas a combination of the two resulted in substantial (20–25%) apoptosis within 24 h. There was an additional benefit associated with this regimen because TPA pretreatment protected the adjacent rectum from radiation-induced apoptosis. This represents the first description of signaling-based therapy designed to overcome one form of radiation resistance expressed preferentially in LNCaP human prostate cancer cells.
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ataxia telangiectasia mutated gene product inhibits dna damage induced apoptosis via Ceramide Synthase
Journal of Biological Chemistry, 1999Co-Authors: Wen-chieh Liao, Roger S Persaud, Maureen Mcloughlin, Desiree Ehleiter, Magtouf Gatei, Richard Kolesnick, Adriana Haimovitzfriedman, Martin F. Lavin, Ning Zhang, Zvi FuksAbstract:Abstract DNA double-stranded breaks (dsb) activate surveillance systems that identify DNA damage and either initiate repair or signal cell death. Failure of cells to undergo appropriate death in response to DNA damage leads to misrepair, mutations, and neoplastic transformation. Pathways linking DNA dsb to reproductive or apoptotic death are virtually unknown. Here we report that metabolic incorporation of 125I-labeled 5-iodo-2′deoxyuridine, which produces DNA dsb, signaled de novo Ceramide synthesis by post-translational activation of Ceramide Synthase (CS) and apoptosis. CS activation was obligatory, since fumonisin B1, a fungal pathogen that acts as a specific CS inhibitor, abrogated DNA damage-induced death. X-irradiation yielded similar results. Furthermore, inhibition of apoptosis using the peptide caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone did not affect CS activation, indicating this event is not a consequence of induction of apoptosis. ATM, the gene mutated in ataxia telangiectasia, is a member of the phosphatidylinositol 3-kinase family that constitutes the DNA damage surveillance/repair system. Epstein-Barr virus-immortalized B cell lines from six ataxia telangiectasia patients with different mutations exhibited radiation-induced CS activation, Ceramide generation, and apoptosis, whereas three lines from normal patients failed to manifest these responses. Stable transfection of wild type ATM cDNA reversed these events, whereas antisense inactivation of ataxia telangiectasia-mutated gene product in normal B cells conferred the ataxia telangiectasia phenotype. We propose that one of the functions of ataxia telangiectasia-mutated gene product is to constrain activation of CS, thereby regulating DNA damage-induced apoptosis.
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ataxia telangiectasia mutated gene product inhibits dna damage induced apoptosis via Ceramide Synthase
Journal of Biological Chemistry, 1999Co-Authors: Wen-chieh Liao, Roger S Persaud, Maureen Mcloughlin, Desiree Ehleiter, Magtouf Gatei, Richard Kolesnick, Adriana Haimovitzfriedman, Martin F. Lavin, Ning Zhang, Zvi FuksAbstract:Abstract DNA double-stranded breaks (dsb) activate surveillance systems that identify DNA damage and either initiate repair or signal cell death. Failure of cells to undergo appropriate death in response to DNA damage leads to misrepair, mutations, and neoplastic transformation. Pathways linking DNA dsb to reproductive or apoptotic death are virtually unknown. Here we report that metabolic incorporation of 125I-labeled 5-iodo-2′deoxyuridine, which produces DNA dsb, signaled de novo Ceramide synthesis by post-translational activation of Ceramide Synthase (CS) and apoptosis. CS activation was obligatory, since fumonisin B1, a fungal pathogen that acts as a specific CS inhibitor, abrogated DNA damage-induced death. X-irradiation yielded similar results. Furthermore, inhibition of apoptosis using the peptide caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone did not affect CS activation, indicating this event is not a consequence of induction of apoptosis. ATM, the gene mutated in ataxia telangiectasia, is a member of the phosphatidylinositol 3-kinase family that constitutes the DNA damage surveillance/repair system. Epstein-Barr virus-immortalized B cell lines from six ataxia telangiectasia patients with different mutations exhibited radiation-induced CS activation, Ceramide generation, and apoptosis, whereas three lines from normal patients failed to manifest these responses. Stable transfection of wild type ATM cDNA reversed these events, whereas antisense inactivation of ataxia telangiectasia-mutated gene product in normal B cells conferred the ataxia telangiectasia phenotype. We propose that one of the functions of ataxia telangiectasia-mutated gene product is to constrain activation of CS, thereby regulating DNA damage-induced apoptosis.
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12 o tetradecanoylphorbol 13 acetate induced apoptosis in lncap cells is mediated through Ceramide Synthase
Cancer Research, 1998Co-Authors: Mark Garzotto, Desiree Ehleiter, Wen-chieh Liao, Adriana Haimovitzfriedman, Zvi Fuks, Margaret Whitejones, Youwei Jiang, Richard KolesnickAbstract:Protein kinase C (PKC) activation is often antiapoptotic, although in a few cell types PKC initiates apoptosis by an unknown mechanism. Recent investigations showed that activation of PKCα by 12- O -tetradecanoylphorbol 13-acetate (TPA) induced apoptosis in LNCaP prostate cancer cells. The present studies examine the mechanism of this effect and show that de novo Ceramide generation through the enzyme Ceramide Synthase is required. TPA induced rapid Ceramide generation, which was detectable by 1 h and increased linearly for 12 h. TPA-induced apoptosis was measurable by 12 h and was progressive for 48 h. Investigations into the mechanism of TPA-induced Ceramide generation revealed that acid and neutral sphingomyelinase activities were not enhanced. However, TPA induced an increase in Ceramide Synthase activity that persisted for at least 16 h. Treatment with fumonisin B1, a specific natural inhibitor of Ceramide Synthase, abrogated both Ceramide production and TPA-induced apoptosis. Ceramide analogues bypassed fumonisin B1 inhibition to initiate apoptosis directly. Thus, Ceramide appears to be a necessary signal for TPA-induced apoptosis in LNCaP cells. This represents the first description of a pathway by which PKC may signal apoptosis.
Wen-chieh Liao - One of the best experts on this subject based on the ideXlab platform.
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Reversal of Radiation Resistance in LNCaP Cells by Targeting Apoptosis through Ceramide Synthase
Cancer Research, 1999Co-Authors: Mark Garzotto, Adriana Haimovitz-friedman, Margaret White-jones, Robert Huryk, Carlos Cardon-cardo, Wen-chieh Liao, Warren D. W. Heston, Richard Kolesnick, Zvi FuksAbstract:Cell lines derived from human prostate cancer are regarded as relatively resistant to both radiation-induced clonogenic death and apoptosis. Here we attempted to modulate the response of LNCaP prostate cancer cells to radiation therapy (XRT) by pretreatment with 12- O -tetradecanoylphorbol acetate (TPA), a known apoptogenic agent in LNCaP cells. Using plateau-phase cultures, we investigated the response of these cells to XRT, TPA, and a combination of XRT and TPA. LNCaP irradiation did not result in Ceramide generation or apoptosis. However, pretreatment with TPA enabled XRT to generate Ceramide via activation of the enzyme Ceramide Synthase and signal apoptosis. Apoptosis was abrogated by the competitive inhibitor of Ceramide Synthase, fumonisin B 1 . Furthermore, when transplanted orthotopically into the prostate of nude mice, LNCaP cells produced tumors that recapitulated the responses of LNCaP cells in vitro . XRT or TPA failed to signal apoptosis in LNCaP tumors, whereas a combination of the two resulted in substantial (20–25%) apoptosis within 24 h. There was an additional benefit associated with this regimen because TPA pretreatment protected the adjacent rectum from radiation-induced apoptosis. This represents the first description of signaling-based therapy designed to overcome one form of radiation resistance expressed preferentially in LNCaP human prostate cancer cells.
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ataxia telangiectasia mutated gene product inhibits dna damage induced apoptosis via Ceramide Synthase
Journal of Biological Chemistry, 1999Co-Authors: Wen-chieh Liao, Roger S Persaud, Maureen Mcloughlin, Desiree Ehleiter, Magtouf Gatei, Richard Kolesnick, Adriana Haimovitzfriedman, Martin F. Lavin, Ning Zhang, Zvi FuksAbstract:Abstract DNA double-stranded breaks (dsb) activate surveillance systems that identify DNA damage and either initiate repair or signal cell death. Failure of cells to undergo appropriate death in response to DNA damage leads to misrepair, mutations, and neoplastic transformation. Pathways linking DNA dsb to reproductive or apoptotic death are virtually unknown. Here we report that metabolic incorporation of 125I-labeled 5-iodo-2′deoxyuridine, which produces DNA dsb, signaled de novo Ceramide synthesis by post-translational activation of Ceramide Synthase (CS) and apoptosis. CS activation was obligatory, since fumonisin B1, a fungal pathogen that acts as a specific CS inhibitor, abrogated DNA damage-induced death. X-irradiation yielded similar results. Furthermore, inhibition of apoptosis using the peptide caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone did not affect CS activation, indicating this event is not a consequence of induction of apoptosis. ATM, the gene mutated in ataxia telangiectasia, is a member of the phosphatidylinositol 3-kinase family that constitutes the DNA damage surveillance/repair system. Epstein-Barr virus-immortalized B cell lines from six ataxia telangiectasia patients with different mutations exhibited radiation-induced CS activation, Ceramide generation, and apoptosis, whereas three lines from normal patients failed to manifest these responses. Stable transfection of wild type ATM cDNA reversed these events, whereas antisense inactivation of ataxia telangiectasia-mutated gene product in normal B cells conferred the ataxia telangiectasia phenotype. We propose that one of the functions of ataxia telangiectasia-mutated gene product is to constrain activation of CS, thereby regulating DNA damage-induced apoptosis.
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ataxia telangiectasia mutated gene product inhibits dna damage induced apoptosis via Ceramide Synthase
Journal of Biological Chemistry, 1999Co-Authors: Wen-chieh Liao, Roger S Persaud, Maureen Mcloughlin, Desiree Ehleiter, Magtouf Gatei, Richard Kolesnick, Adriana Haimovitzfriedman, Martin F. Lavin, Ning Zhang, Zvi FuksAbstract:Abstract DNA double-stranded breaks (dsb) activate surveillance systems that identify DNA damage and either initiate repair or signal cell death. Failure of cells to undergo appropriate death in response to DNA damage leads to misrepair, mutations, and neoplastic transformation. Pathways linking DNA dsb to reproductive or apoptotic death are virtually unknown. Here we report that metabolic incorporation of 125I-labeled 5-iodo-2′deoxyuridine, which produces DNA dsb, signaled de novo Ceramide synthesis by post-translational activation of Ceramide Synthase (CS) and apoptosis. CS activation was obligatory, since fumonisin B1, a fungal pathogen that acts as a specific CS inhibitor, abrogated DNA damage-induced death. X-irradiation yielded similar results. Furthermore, inhibition of apoptosis using the peptide caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone did not affect CS activation, indicating this event is not a consequence of induction of apoptosis. ATM, the gene mutated in ataxia telangiectasia, is a member of the phosphatidylinositol 3-kinase family that constitutes the DNA damage surveillance/repair system. Epstein-Barr virus-immortalized B cell lines from six ataxia telangiectasia patients with different mutations exhibited radiation-induced CS activation, Ceramide generation, and apoptosis, whereas three lines from normal patients failed to manifest these responses. Stable transfection of wild type ATM cDNA reversed these events, whereas antisense inactivation of ataxia telangiectasia-mutated gene product in normal B cells conferred the ataxia telangiectasia phenotype. We propose that one of the functions of ataxia telangiectasia-mutated gene product is to constrain activation of CS, thereby regulating DNA damage-induced apoptosis.
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12 o tetradecanoylphorbol 13 acetate induced apoptosis in lncap cells is mediated through Ceramide Synthase
Cancer Research, 1998Co-Authors: Mark Garzotto, Desiree Ehleiter, Wen-chieh Liao, Adriana Haimovitzfriedman, Zvi Fuks, Margaret Whitejones, Youwei Jiang, Richard KolesnickAbstract:Protein kinase C (PKC) activation is often antiapoptotic, although in a few cell types PKC initiates apoptosis by an unknown mechanism. Recent investigations showed that activation of PKCα by 12- O -tetradecanoylphorbol 13-acetate (TPA) induced apoptosis in LNCaP prostate cancer cells. The present studies examine the mechanism of this effect and show that de novo Ceramide generation through the enzyme Ceramide Synthase is required. TPA induced rapid Ceramide generation, which was detectable by 1 h and increased linearly for 12 h. TPA-induced apoptosis was measurable by 12 h and was progressive for 48 h. Investigations into the mechanism of TPA-induced Ceramide generation revealed that acid and neutral sphingomyelinase activities were not enhanced. However, TPA induced an increase in Ceramide Synthase activity that persisted for at least 16 h. Treatment with fumonisin B1, a specific natural inhibitor of Ceramide Synthase, abrogated both Ceramide production and TPA-induced apoptosis. Ceramide analogues bypassed fumonisin B1 inhibition to initiate apoptosis directly. Thus, Ceramide appears to be a necessary signal for TPA-induced apoptosis in LNCaP cells. This represents the first description of a pathway by which PKC may signal apoptosis.