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Joanna C. Bakowska - One of the best experts on this subject based on the ideXlab platform.
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Oxidative Stress in Caenorhabditis elegans: Protective Effects of Spartin
PloS one, 2015Co-Authors: Timothy Truong, Zachary A. Karlinski, Christopher P. O'hara, Maleen Cabe, Hongkyun Kim, Joanna C. BakowskaAbstract:Troyer syndrome is caused by a mutation in the SPG20 gene, which results in complete loss of expression of the protein spartin. We generated a genetic model of Troyer syndrome in worms to explore the locomotor consequences of a null mutation of the Caenorhabditis elegans SPG20 orthologue, F57B10.9, also known as spg-20. Spg-20 mutants showed decreased length, crawling speed, and thrashing frequency, and had a shorter lifespan than wild-type animals. These results suggest an age-dependent decline in motor function in mutant animals. The drug paraquat was used to induce oxidative stress for 4 days in the animals. We measured survival rate and examined locomotion by measuring crawling speed and thrashing frequency. After 4 days of paraquat exposure, 77% of wild-type animals survived, but only 38% of spg-20 mutant animals survived. Conversely, animals overexpressing spg-20 had a survival rate of 95%. We also tested lifespan after a 1 hour exposure to sodium azide. After a 24 hour recovery period, 87% of wild type animals survived, 57% of spg-20 mutant animals survived, and 82% of animals overexpressing spg-20 survived. In the behavioral assays, spg-20 mutant animals showed a significant decrease in both crawling speed and thrashing frequency compared with wild-type animals. Importantly, the locomotor phenotype for both crawling and thrashing was rescued in animals overexpressing spg-20. The animals overexpressing spg-20 had crawling speeds and thrashing frequencies similar to those of wild-type animals. These data suggest that the protein F57B10.9/SPG-20 might have a protective role against oxidative stress.
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The role of spartin and its novel ubiquitin binding region in DALIS occurrence.
Molecular biology of the cell, 2014Co-Authors: Amelia B. Karlsson, Christopher Hooper, Alexander Shekhtman, Jacqueline Washington, Valentina Dimitrova, Joanna C. BakowskaAbstract:Troyer syndrome is an autosomal recessive hereditary spastic paraplegia (HSP) caused by frameshift mutations in the SPG20 gene that results in a lack of expression of the truncated protein. Spartin is a multifunctional protein, yet only two conserved domains--a microtubule-interacting and trafficking domain and a plant-related senescence domain involved in cytokinesis and mitochondrial physiology, respectively--have been defined. We have shown that overexpressed spartin binds to the Ile44 hydrophobic pocket of ubiquitin, suggesting spartin might contain a ubiquitin-binding domain. In the present study, we demonstrate that spartin contributes to the formation of dendritic aggresome-like induced structures (DALIS) through a unique ubiquitin-binding region (UBR). Using short hairpin RNA, we knocked down spartin in RAW264.7 cells and found that DALIS frequency decreased; conversely, overexpression of spartin increased the percentage of cells containing DALIS. Using nuclear magnetic resonance spectroscopy, we characterized spartin's UBR and defined the UBR's amino acids that are key for ubiquitin binding. We also found that spartin, via the UBR, binds Lys-63-linked ubiquitin chains but does not bind Lys-48-linked ubiquitin chains. Finally, we demonstrate that spartin's role in DALIS formation depends on key residues within its UBR.
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SPG20−/− mice reveal multimodal functions for Troyer syndrome protein spartin in lipid droplet maintenance, cytokinesis and BMP signaling
Human molecular genetics, 2012Co-Authors: Benoît Renvoisé, Julia Stadler, Rajat Singh, Joanna C. Bakowska, Craig BlackstoneAbstract:Hereditary spastic paraplegias (HSPs; SPG1-48) are inherited neurological disorders characterized by lower extremity spasticity and weakness. Loss-of-function mutations in the SPG20 gene encoding spartin cause autosomal recessive Troyer syndrome (SPG20), which has additional features of short stature, cognitive deficits and distal amyotrophy. To identify cellular impairments underlying Troyer syndrome, we generated SPG20-/- mice, which exhibit progressive gait defects. Although gross central nervous system pathology appeared largely normal, cerebral cortical neurons cultured from neonatal SPG20-/- mice exhibited increased axon branching, a phenotype suppressed by reintroducing spartin and which required its interaction with the endosomal sorting complex required for transport (ESCRT)-III protein IST1. Analysis of the bone morphogenetic protein (BMP) signaling pathway in SPG20-/- embryonic fibroblasts indicated that Smad1/5 phosphorylation is modestly elevated, possibly due to alterations in BMP receptor trafficking. Cytokinesis was impaired in embryonic fibroblasts cultured from SPG20-/- mice, and binucleated chondrocytes were prominent in epiphyseal growth plates of bones in SPG20-/- mice, perhaps explaining the short stature of patients. Finally, adipose tissue from SPG20-/- female mice exhibited increased lipid droplet (LD) numbers and alterations in perilipin levels, supporting a role for spartin in LD maintenance. Taken together, our results support multimodal functions for spartin that provide important insights into HSP pathogenesis.
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SPG20 protein spartin associates with cardiolipin via its plant-related senescence domain and regulates mitochondrial Ca2+ homeostasis.
PloS one, 2011Co-Authors: Dinesh C. Joshi, Joanna C. BakowskaAbstract:Hereditary spastic paraplegias (HSPs) are a group of neurological disorders characterized clinically by spasticity of lower limbs and pathologically by degeneration of the corticospinal tract. Troyer syndrome is an autosomal recessive HSP caused by a frameshift mutation in the spartin (SPG20) gene. Previously, we established that this mutation results in a lack of expression of the truncated mutant spartin protein. Spartin is involved in many cellular processes and associates with several intracellular organelles, including mitochondria. Spartin contains a conserved plant-related senescence domain at its C-terminus. However, neither the function of this domain nor the roles of spartin in mitochondrial physiology are currently known. In this study, we determined that the plant-related senescence domain of spartin interacts with cardiolipin but not with two other major mitochondrial phospholipids, phosphatidylcholine and phosphatidylethanolamine. We also found that knockdown of spartin by small interfering RNA in a human neuroblastoma cell line resulted in depolarization of the mitochondrial membrane. In addition, depletion of spartin resulted in a significant decrease in both mitochondrial calcium uptake and mitochondrial membrane potential in cells treated with thapsigargin. Our results suggest that impairment of mitochondrial calcium uptake might contribute to the neurodegeneration of long corticospinal axons and the pathophysiology of Troyer syndrome.
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SPG20 protein spartin is recruited to midbodies by ESCRT-III protein Ist1 and participates in cytokinesis
Molecular biology of the cell, 2010Co-Authors: Benoît Renvoisé, Joanna C. Bakowska, Rell L. Parker, Dong Yang, James H. Hurley, Craig BlackstoneAbstract:Hereditary spastic paraplegias (HSPs, SPG1-46) are inherited neurological disorders characterized by lower extremity spastic weakness. Loss-of-function SPG20 gene mutations cause an autosomal recessive HSP known as Troyer syndrome. The SPG20 protein spartin localizes to lipid droplets and endosomes, and it interacts with tail interacting protein 47 (TIP47) as well as the ubiquitin E3 ligases atrophin-1-interacting protein (AIP)4 and AIP5. Spartin harbors a domain contained within microtubule-interacting and trafficking molecules (MIT) at its N-terminus, and most proteins with MIT domains interact with specific ESCRT-III proteins. Using yeast two-hybrid and in vitro surface plasmon resonance assays, we demonstrate that the spartin MIT domain binds with micromolar affinity to the endosomal sorting complex required for transport (ESCRT)-III protein increased sodium tolerance (Ist)1 but not to ESCRT-III proteins charged multivesicular body proteins 1-7. Spartin colocalizes with Ist1 at the midbody, and depletion of Ist1 in cells by small interfering RNA significantly decreases the number of cells where spartin is present at midbodies. Depletion of spartin does not affect Ist1 localization to midbodies but markedly impairs cytokinesis. A structure-based amino acid substitution in the spartin MIT domain (F24D) blocks the spartin-Ist1 interaction. Spartin F24D does not localize to the midbody and acts in a dominant-negative manner to impair cytokinesis. These data suggest that Ist1 interaction is important for spartin recruitment to the midbody and that spartin participates in cytokinesis.
Craig Blackstone - One of the best experts on this subject based on the ideXlab platform.
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SPG20−/− mice reveal multimodal functions for Troyer syndrome protein spartin in lipid droplet maintenance, cytokinesis and BMP signaling
Human molecular genetics, 2012Co-Authors: Benoît Renvoisé, Julia Stadler, Rajat Singh, Joanna C. Bakowska, Craig BlackstoneAbstract:Hereditary spastic paraplegias (HSPs; SPG1-48) are inherited neurological disorders characterized by lower extremity spasticity and weakness. Loss-of-function mutations in the SPG20 gene encoding spartin cause autosomal recessive Troyer syndrome (SPG20), which has additional features of short stature, cognitive deficits and distal amyotrophy. To identify cellular impairments underlying Troyer syndrome, we generated SPG20-/- mice, which exhibit progressive gait defects. Although gross central nervous system pathology appeared largely normal, cerebral cortical neurons cultured from neonatal SPG20-/- mice exhibited increased axon branching, a phenotype suppressed by reintroducing spartin and which required its interaction with the endosomal sorting complex required for transport (ESCRT)-III protein IST1. Analysis of the bone morphogenetic protein (BMP) signaling pathway in SPG20-/- embryonic fibroblasts indicated that Smad1/5 phosphorylation is modestly elevated, possibly due to alterations in BMP receptor trafficking. Cytokinesis was impaired in embryonic fibroblasts cultured from SPG20-/- mice, and binucleated chondrocytes were prominent in epiphyseal growth plates of bones in SPG20-/- mice, perhaps explaining the short stature of patients. Finally, adipose tissue from SPG20-/- female mice exhibited increased lipid droplet (LD) numbers and alterations in perilipin levels, supporting a role for spartin in LD maintenance. Taken together, our results support multimodal functions for spartin that provide important insights into HSP pathogenesis.
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Targeted disruption of the Mast syndrome gene SPG21 in mice impairs hind limb function and alters axon branching in cultured cortical neurons
neurogenetics, 2010Co-Authors: Cynthia Soderblom, Julia Stadler, Craig Blackstone, Henri Jupille, Oleg Shupliakov, Michael C. HannaAbstract:Mast syndrome (SPG21) is a childhood-onset, autosomal recessive, complicated form of hereditary spastic paraplegia (HSP) characterized by dementia, thin corpus callosum, white matter abnormalities, and cerebellar and extrapyramidal signs in addition to spastic paraparesis. A nucleotide insertion resulting in premature truncation of the SPG21 gene product maspardin underlies this disorder, likely leading to loss of protein function. In this study, we generated SPG21−/− knockout mice by homologous recombination as a possible animal model for SPG21. Though SPG21−/− mice appeared normal at birth, within several months they developed gradually progressive hind limb dysfunction. Cerebral cortical neurons cultured from SPG21−/− mice exhibited significantly more axonal branching than neurons from wild-type animals, while comprehensive neuropathological analysis of SPG21−/− mice did not reveal definitive abnormalities. Since alterations in axon branching have been seen in neurons derived from animal models of other forms of HSP as well as motor neuron diseases, this may represent a common cellular pathogenic theme.
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SPG20 protein spartin is recruited to midbodies by ESCRT-III protein Ist1 and participates in cytokinesis
Molecular biology of the cell, 2010Co-Authors: Benoît Renvoisé, Joanna C. Bakowska, Rell L. Parker, Dong Yang, James H. Hurley, Craig BlackstoneAbstract:Hereditary spastic paraplegias (HSPs, SPG1-46) are inherited neurological disorders characterized by lower extremity spastic weakness. Loss-of-function SPG20 gene mutations cause an autosomal recessive HSP known as Troyer syndrome. The SPG20 protein spartin localizes to lipid droplets and endosomes, and it interacts with tail interacting protein 47 (TIP47) as well as the ubiquitin E3 ligases atrophin-1-interacting protein (AIP)4 and AIP5. Spartin harbors a domain contained within microtubule-interacting and trafficking molecules (MIT) at its N-terminus, and most proteins with MIT domains interact with specific ESCRT-III proteins. Using yeast two-hybrid and in vitro surface plasmon resonance assays, we demonstrate that the spartin MIT domain binds with micromolar affinity to the endosomal sorting complex required for transport (ESCRT)-III protein increased sodium tolerance (Ist)1 but not to ESCRT-III proteins charged multivesicular body proteins 1-7. Spartin colocalizes with Ist1 at the midbody, and depletion of Ist1 in cells by small interfering RNA significantly decreases the number of cells where spartin is present at midbodies. Depletion of spartin does not affect Ist1 localization to midbodies but markedly impairs cytokinesis. A structure-based amino acid substitution in the spartin MIT domain (F24D) blocks the spartin-Ist1 interaction. Spartin F24D does not localize to the midbody and acts in a dominant-negative manner to impair cytokinesis. These data suggest that Ist1 interaction is important for spartin recruitment to the midbody and that spartin participates in cytokinesis.
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Interaction of the SPG21 protein ACP33/maspardin with the aldehyde dehydrogenase ALDH16A1
neurogenetics, 2009Co-Authors: Michael C. Hanna, Craig BlackstoneAbstract:Mast syndrome (SPG21) is an autosomal-recessive complicated form of hereditary spastic paraplegia characterized by dementia, thin corpus callosum, white matter abnormalities, and cerebellar and extrapyramidal signs in addition to spastic paraparesis. A nucleotide insertion resulting in premature truncation of the SPG21 gene product acidic cluster protein 33 (ACP33)/maspardin underlies this disorder, likely causing loss of protein function. However, little is known about the function of maspardin. Here, we report that maspardin localizes prominently to cytoplasm as well as to membranes, possibly at trans -Golgi network/late endosomal compartments. Immunoprecipitation of maspardin with identification of coprecipitating proteins by mass spectrometry revealed the aldehyde dehydrogenase ALDH16A1 as an interacting protein. This interaction was confirmed using overexpressed proteins as well as by fusion protein pull down experiments, and these proteins colocalized in cells. Further studies of the function of ALDH16A1 and the role of the maspardin–ALDH16A1 interaction in neuronal cells may clarify the cellular pathogenesis of Mast syndrome.
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Lack of spartin protein in Troyer syndrome: a loss-of-function disease mechanism?
Archives of neurology, 2008Co-Authors: Joanna C. Bakowska, Heng Wang, Baozhong Xin, Charlotte J. Sumner, Craig BlackstoneAbstract:Background Hereditary spastic paraplegias (SPG1-SPG33) are characterized by progressive spastic weakness of the lower limbs. A nucleotide deletion (1110delA) in the ( SPG20 ; OMIM275900) spartin gene is the origin of autosomal recessive Troyer syndrome. This mutation is predicted to cause premature termination of the spartin protein. However, it remains unknown whether this truncated spartin protein is absent or is present and partially functional in patients. Objective To determine whether the truncated spartin protein is present or absent in cells derived from patients with Troyer syndrome. Design Case report. Setting Academic research. Patients We describe a new family with Troyer syndrome due to the 1110delA mutation. Main Outcome Measures We cultured primary fibroblasts and generated lymphoblasts from affected individuals, carriers, and control subjects and subjected these cells to immunoblot analyses. Results Spartin protein is undetectable in several cell lines derived from patients with Troyer syndrome. Conclusions Our data suggest that Troyer syndrome results from complete loss of spartin protein rather than from the predicted partly functional fragment. This may reflect increased protein degradation or impaired translation.
Andrew H Crosby - One of the best experts on this subject based on the ideXlab platform.
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Endogenous spartin, mutated in hereditary spastic paraplegia, has a complex subcellular localization suggesting diverse roles in neurons
Experimental cell research, 2006Co-Authors: Dimitri Robay, Heema Patel, Michael A Simpson, Nigel A. Brown, Andrew H CrosbyAbstract:Mutation of spartin (SPG20) underlies a complicated form of hereditary spastic paraplegia, a disorder principally defined by the degeneration of upper motor neurons. Using a polyclonal antibody against spartin to gain insight into the function of the endogenous molecule, we show that the endogenous molecule is present in two main isoforms of 85 kDa and 100 kDa, and 75 kDa and 85 kDa in human and murine, respectively, with restricted subcellular localization. Immunohistochemical studies on human and mouse embryo sections and in vitro cell studies indicate that spartin is likely to possess both nuclear and cytoplasmic functions. The nuclear expression of spartin closely mirrors that of the snRNP (small nuclear ribonucleoprotein) marker alpha-Sm, a component of the spliceosome. Spartin is also enriched at the centrosome within mitotic structures. Notably we show that spartin protein undergoes dynamic positional changes in differentiating human SH-SY5Y cells. In undifferentiated non-neuronal cells, spartin displays a nuclear and diffuse cytosolic profile, whereas spartin transiently accumulates in the trans-Golgi network and subsequently decorates discrete puncta along neurites in terminally differentiated neuroblastic cells. Investigation of these spartin-positive vesicles reveals that a large proportion colocalizes with the synaptic vesicle marker synaptotagmin. Spartin is also enriched in synaptic-like structures and in synaptic vesicle-enriched fraction.
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A new locus for autosomal recessive complicated hereditary spastic paraplegia (SPG26) maps to chromosome 12p11.1–12q14
Journal of Medical Genetics, 2005Co-Authors: P A Wilkinson, Johanna A. Reed, Kamini Kalidas, L. Bastaki, Elena Samilchuk, Michael A Simpson, R. Khan, Thomas T Warner, H Patel, Andrew H CrosbyAbstract:The term hereditary spastic paraplegia (HSP) is used to describe a group of clinically and genetically heterogeneous disorders in which the defining clinical feature is progressive spasticity and weakness of the lower limbs. The phenotype is traditionally classified as “pure” when symptoms and signs are generally confined to those of a progressive spastic paraparesis, or “complicated” when associated with additional neurological or other clinical features.1 Inheritance may be autosomal dominant, autosomal recessive, or rarely X linked. Overall autosomal dominant inheritance is most commonly associated with pure forms of the disease, whereas autosomal recessive HSP shows greater phenotypic variability, including several well defined syndromes.2,3 To date nine autosomal recessive HSP loci have been identified and causative mutations found in three genes: SPG7 (paraplegin), SPG20 (spartin), and SPG21 (maspardin). SPG7 encodes paraplegin, a mitochondrial protein, which is a member of the AAA protein superfamily (ATPase associated with diverse cellular activities) and is homologous to a number of yeast mitochondrial metalloproteases.4 SPG7 mutations may result in either pure or complicated HSP phenotypes.4,5 Muscle biopsy analysis of patients with SPG7 mutations may show histological evidence of mitochondrial dysfunction4,6 and recently biochemical studies have shown specific defects in mitochondrial respiratory chain function.5,7 Mutations …
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Troyer syndrome revisited
Journal of Neurology, 2004Co-Authors: Christos Proukakis, Michael A. Patton, Heema Patel, Harold Cross, Alan Valentine, Andrew H CrosbyAbstract:Troyer syndrome, originally described in 1967 in an Old Order Amish population, is a complicated form of hereditary spastic paraplegia (HSP) inherited in an autosomal recessive fashion and slowly progressive. The cardinal features are spastic paraparesis, pseudobulbar palsy and distal amyotrophy, together with mild developmental delay and subtle skeletal abnormalities. We report a detailed evaluation of 21 cases of Troyer syndrome in the same Amish population, including three from the original study. Imaging of the brain revealed white matter abnormalities, particularly in the temporoparietal periventricular area. This study, coupled with the recent identification of the gene responsible ( SPG20 , encoding spartin), increases our understanding of this form of HSP.
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Troyer syndrome revisited. A clinical and radiological study of a complicated hereditary spastic paraplegia.
Journal of neurology, 2004Co-Authors: Christos Proukakis, Michael A. Patton, Heema Patel, Harold E. Cross, A. R. Valentine, Andrew H CrosbyAbstract:Troyer syndrome, originally described in 1967 in an Old Order Amish population, is a complicated form of hereditary spastic paraplegia (HSP) inherited in an autosomal recessive fashion and slowly progressive. The cardinal features are spastic paraparesis, pseudobulbar palsy and distal amyotrophy, together with mild developmental delay and subtle skeletal abnormalities. We report a detailed evaluation of 21 cases of Troyer syndrome in the same Amish population, including three from the original study. Imaging of the brain revealed white matter abnormalities, particularly in the temporoparietal periventricular area. This study, coupled with the recent identification of the gene responsible (SPG20, encoding spartin), increases our understanding of this form of HSP.
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SPG20 is mutated in troyer syndrome an hereditary spastic paraplegia
Nature Genetics, 2002Co-Authors: Heema Patel, Michael A. Patton, Christos Proukakis, Ruth Hershberger, Francesca D. Ciccarelli, H Cross, Victor A Mckusick, Peer Bork, Andrew H CrosbyAbstract:Troyer syndrome (TRS) is an autosomal recessive complicated hereditary spastic paraplegia (HSP) that occurs with high frequency in the Old Order Amish. We report mapping of the TRS locus to chromosome 13q12.3 and identify a frameshift mutation in SPG20, encoding spartin. Comparative sequence analysis indicates that spartin shares similarity with molecules involved in endosomal trafficking and with spastin, a molecule implicated in microtubule interaction that is commonly mutated in HSP.
Hao Zhang - One of the best experts on this subject based on the ideXlab platform.
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methylation induced silencing of SPG20 facilitates gastric cancer cell proliferation by activating the egfr mapk pathway
Biochemical and Biophysical Research Communications, 2018Co-Authors: Zhangjian Zhou, Wei Wang, Xin Xie, Yongchun Song, Chengxue Dang, Hao ZhangAbstract:Abstract Spastic paraplegia 20 methylation was characterized in gastric cancer in our previous study. However, its mechanism remains unknown. Cell proliferation, colony formation, flow cytometry, wound healing, in vitro Transwell assays and in vivo xenografts were performed. A nomogram model was established to make a more accurate prognostic prediction for gastric cancer patients. Knockout of Spastic paraplegia 20 promoted gastric cancer cell proliferation, G2/M arrest in vitro and tumor growth in vivo. The EGFR/MAPK pathway was activated as a consequence of Spastic paraplegia 20 deletion. EGFR kinase or ERK1/2 inhibitors impaired Spastic paraplegia 20 knockout-induced cancer cell growth. Gastric cancer patients with poor spartin expression (72/161, 44.7%) exhibited a worse prognosis compared with the high expression group with median survival times of 16 and 54 months, respectively. The nomogram model stratified gastric cancer patients into 3 distinct prognostic groups with 3-year survival rates of 100%, 77%, and 35%. Furthermore, it had a better discrimination than the TNM staging system (C index: 0.785, AIC: 752.8708 VS. C index: 0.712; AIC: 775.1223). Methylation-induced Spastic paraplegia 20 silencing facilitates gastric cancer cell proliferation by activating the EGFR/MAPK signaling pathway. The nomogram based on spartin expression provided significantly better discrimination compared with the traditional AJCC TNM staging system and provided an individualized prediction of the survival for gastric cancer patient survival.
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Methylation-induced silencing of SPG20 facilitates gastric cancer cell proliferation by activating the EGFR/MAPK pathway.
Biochemical and biophysical research communications, 2018Co-Authors: Zhangjian Zhou, Wei Wang, Xin Xie, Yongchun Song, Chengxue Dang, Hao ZhangAbstract:Abstract Spastic paraplegia 20 methylation was characterized in gastric cancer in our previous study. However, its mechanism remains unknown. Cell proliferation, colony formation, flow cytometry, wound healing, in vitro Transwell assays and in vivo xenografts were performed. A nomogram model was established to make a more accurate prognostic prediction for gastric cancer patients. Knockout of Spastic paraplegia 20 promoted gastric cancer cell proliferation, G2/M arrest in vitro and tumor growth in vivo. The EGFR/MAPK pathway was activated as a consequence of Spastic paraplegia 20 deletion. EGFR kinase or ERK1/2 inhibitors impaired Spastic paraplegia 20 knockout-induced cancer cell growth. Gastric cancer patients with poor spartin expression (72/161, 44.7%) exhibited a worse prognosis compared with the high expression group with median survival times of 16 and 54 months, respectively. The nomogram model stratified gastric cancer patients into 3 distinct prognostic groups with 3-year survival rates of 100%, 77%, and 35%. Furthermore, it had a better discrimination than the TNM staging system (C index: 0.785, AIC: 752.8708 VS. C index: 0.712; AIC: 775.1223). Methylation-induced Spastic paraplegia 20 silencing facilitates gastric cancer cell proliferation by activating the EGFR/MAPK signaling pathway. The nomogram based on spartin expression provided significantly better discrimination compared with the traditional AJCC TNM staging system and provided an individualized prediction of the survival for gastric cancer patient survival.
Francesco Merli - One of the best experts on this subject based on the ideXlab platform.
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Abstract 5198: 5AZA and decitabine exhibit different molecular effects on non-Hodgkin lymphoma cells: Involvement of DNMT3a
Molecular and Cellular Biology Genetics, 2019Co-Authors: Raffaele Frazzi, Tonia De Simone, Olga Serra, Annamaria Buschini, Laura Canovi, Tiziana De Luca, Francesco MerliAbstract:Introductory sentence. Demethylating drugs represent a powerful tool for epigenetic modulation of chromatin structure in cancer cells. 5-azacytidine (5-AZA) and 5-aza-2’-deoxycytidine (decitabine) are promising therapeutic solutions also in mature B-cell neoplasms, although the molecular mechanisms of action have yet to be elucidated. Here we describe their effects on non-Hodgkin lymphoma cells and demonstrate how hypomethylation is not homogeneous across the genome. Experimental procedures. Toledo (GC-derived) and NU-DUL-1 (ABC-like) diffuse large-B cell lymphoma (DLBCL; non-Hodgkin) cell lines. Peripheral blood mononuclear cells (PBMCs) isolation. Epitect methyl II methylation qPCR and global chromatin methylation quantitative assays. Immunoblottings. Proliferation assays. CpG islands bioinformatics analysis. Comet assay and phosphorylation of gamma-H2AX histone assay. Gene silencing through siRNAs. New data. A panel of 9 CpG islands within a set of genes that we previously identified has been investigated as a potential target for hypomethylating activity of 5AZA and decitabine. KLF4, DAPK1 and SPG20 promoters (located on chr. n°9 the first two and n°3 the latter) are not affected by a single dose of 5AZA after 48h or 7 days of incubation (the CpG islands within these promoters are fully unmethylated in healthy PBMCs). However, global chromatin demethylation is clearly visible either after 48h or 7 days in both cell lines. By comparison, decitabine demethylating effects return to the initial values after 7 days. MZB1 promoter results demethylated by both treatments, while MGMT is demethylated only in NU-DUL-1 by decitabine but not 5AZA. These data suggest that hypomethylating agents act selectively on discrete regions of the genome. 5AZA and decitabine markedly down-regulate DNMT1 in a dose-dependent fashion and activate PARP. Despite DNMT1 downregulation, KLF4, DAPK1 and SPG20 promoters result unaffected. DNMT1 silencing in our cells do not cause any change in the promoter methylation of the selected targets. The de novo methyltransferase DNMT3a is expressed only by NU-DUL-1 and its silencing leads to a partial MZB1 demethylation and to the significant decrease in global chromatin methylation whereas DNMT1 silencing does not. In order to assess an involvement of the genotoxic damage, Comet assays were performed up to 24 hours of incubation with the drugs. We demonstrate that 5AZA does not induce any significant genotoxic damage while decitabine causes a tail formation starting at 6h after drug administration. Conclusions. Our data show that 5AZA and decitabine exhibit different mechanisms of action on lymphoma cells. Different DLBCL-derived cell lines display different changes when exposed to single doses of hypomethylating drugs, underlying the fact that cell of origin may play a role during the response. DNMT3a contributes to chromatin methylation in lymphoma cells. Citation Format: Raffaele Frazzi, Tonia De Simone, Olga Serra, Annamaria Buschini, Laura Canovi, Tiziana De Luca, Francesco Merli. 5AZA and decitabine exhibit different molecular effects on non-Hodgkin lymphoma cells: Involvement of DNMT3a [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5198.
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Methylation changes of SIRT1, KLF4, DAPK1 and SPG20 in B-lymphocytes derived from follicular and diffuse large B-cell lymphoma
Leukemia research, 2017Co-Authors: Raffaele Frazzi, Eleonora Zanetti, Mariaelena Pistoni, Ione Tamagnini, Riccardo Valli, Luca Braglia, Francesco MerliAbstract:Diffuse large-B cell lymphomas (DLBCL) and follicular lymphomas (FL) are the most represented subtypes among mature B-cell neoplasms and originate from malignant B lymphocytes. Methylation represents one of the major epigenetic mechanisms of gene regulation. Silent information regulator 1 (SIRT1) is a class III lysine-deacetylase playing several functions and considered to be a context-dependent tumor promoter. We present the quantitative methylation, gene expression and tissue distribution of SIRT1 and some key mediators related to lymphoma pathogenesis in B lymphocytes purified from biopsies of follicular hyperplasias, FL and DLBCL. SIRT1 mRNA levels are higher in FL than follicular hyperplasias and DLBCL. B cell lymphoma 6 (BCL6) positively correlates with SIRT1. SIRT1 promoter shows a methylation decrease in the order: follicular hyperplasia - FL - DLBCL. Kruppel-like factor 4 (KLF4), Death-associated protein kinase 1 (DAPK1) and Spastic Paraplegia 20 (SPG20) methylation increase significantly in FL and DLBCL compared to follicular hyperplasias. Gene expression of DAPK1 and SPG20 inversely correlates with their degree of methylation. Our findings evidence a positive correlation between SIRT1 and BCL6 expression increase in FL. SIRT1 methylation decreases in FL and DLBCL accordingly and this parallels the increase of KLF4, DAPK1 and SPG20 methylation.
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Abstract 4451: Different patterns of SIRT1, KLF4, DAPK1 and SPG20 methylation in B lymphocytes correlate with the clinical parameters of non-Hodgkin lymphoma subtypes
Molecular and Cellular Biology Genetics, 2016Co-Authors: Raffaele Frazzi, Eleonora Zanetti, Mariaelena Pistoni, Ione Tamagnini, Riccardo Valli, Francesco MerliAbstract:Background. DNA methylation is one of the best studied epigenetic modifications and one major constituent of the epigenome of a cell. It contributes to normal development as well to carcinogenesis. Nowadays, many efforts are being made in order to use DNA methylation as a biomarker. The aim of our work is to characterize the expression and methylation of SIRT1, HIC1, BCL6, KLF4 and other genes relevant for Non-Hodgkin lymphomas (NHL) pathogenesis. Methods. Immunohistochemistry (IHC) on 72 formalin-fixed paraffin embedded tissue sections (FFPE). B-lymphocytes were purified from 36 biopsies of follicular hyperplasias (non-malignant B-lymphocytes), follicular lymphomas (FL) and diffuse large B-cell lymphomas (DLBCL). Gene expression were analysed by quantitative retrotranscribedPCR (qRTPCR). Quantitative CpG promoter methylation analysis was performed by pyrosequencing after bisulfite conversion or by Methyl II array qPCR on genomic DNA. Results. In a total of 72 FFPE samples of follicular hyperplasias (n = 17), FL (n = 36) and DLBCL (n = 19), SIRT1 staining is localized in the germinal center of the majority of follicular hyperplasias and FL samples. SIRT1 localizes preferentially in the centroblasts of the GC of the follicles where it correlates with Ki67. BCL6 is uniformly positive in follicular hyperplasias and FL, but heterogeneously distributed in DLBCL. Interestingly, SIRT1 and BCL6 expression correlate in FL. By quantitative pyrosequencing we analyzed 3 CpG sites for the SIRT1 promoter (corresponding to the binding sites for CREB, ARID and PPARG transcription factors). Follicular hyperplasias display higher methylation levels (52.88%) than FL (38.36%) and DLBCL (32.65%) on SIRT1 promoter suggesting a possible inverse correlation between tumor aggressiveness and SIRT1 methylation. Next, we selected a panel of genes whose expression is linked to lymphoma pathogenesis. By Methyl II array qPCR, we show that BCL6 methylation does not vary among samples. KLF4, DAPK1 and SPG20, show statistically significant methylation increases in FL and DLBCL compared to follicular hyperplasias, indicating a possible role of these proteins in lymphoma pathogenesis. On the contrary, no significant differences are observed for the other markers MZB1, MGMT, LMO2 and ASXL1. Notably, KLF4, DAPK1 and SPG20 mRNA expression levels anti-correlate with their promoter methylation in FL. Conclusions. Epigenetic changes in SIRT1 methylation inversely correlate with NHL aggressiveness (decreasing in the order: follicular hyperplasias - FL - DLBCL), while KLF4, DAPK1 and SPG20 show a methylation increase that correlates with tumor aggressiveness. These data suggest that different patterns of methylation correlate with the clinical and prognostic parameters of these NHL subtypes. Citation Format: Raffaele Frazzi, Eleonora Zanetti, Mariaelena Pistoni, Ione Tamagnini, Riccardo Valli, Francesco Merli. Different patterns of SIRT1, KLF4, DAPK1 and SPG20 methylation in B lymphocytes correlate with the clinical parameters of non-Hodgkin lymphoma subtypes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4451.
