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1.
Cell ; 181(6): 1329-1345.e24, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32445698

RESUMO

Posterior fossa A (PFA) ependymomas are lethal malignancies of the hindbrain in infants and toddlers. Lacking highly recurrent somatic mutations, PFA ependymomas are proposed to be epigenetically driven tumors for which model systems are lacking. Here we demonstrate that PFA ependymomas are maintained under hypoxia, associated with restricted availability of specific metabolites to diminish histone methylation, and increase histone demethylation and acetylation at histone 3 lysine 27 (H3K27). PFA ependymomas initiate from a cell lineage in the first trimester of human development that resides in restricted oxygen. Unlike other ependymomas, transient exposure of PFA cells to ambient oxygen induces irreversible cellular toxicity. PFA tumors exhibit a low basal level of H3K27me3, and, paradoxically, inhibition of H3K27 methylation specifically disrupts PFA tumor growth. Targeting metabolism and/or the epigenome presents a unique opportunity for rational therapy for infants with PFA ependymoma.


Assuntos
Ependimoma/genética , Ependimoma/metabolismo , Epigenoma/genética , Neoplasias Infratentoriais/genética , Neoplasias Infratentoriais/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular , Proliferação de Células/genética , Metilação de DNA/genética , Epigenômica/métodos , Histonas/genética , Histonas/metabolismo , Humanos , Lactente , Lisina/genética , Lisina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mutação/genética
2.
Mol Cell ; 79(5): 846-856.e8, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32755594

RESUMO

Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.


Assuntos
Proliferação de Células/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Resveratrol/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular , Resistência a Medicamentos/genética , Humanos , Hidroxiureia/farmacologia , Células Jurkat , Nucleotídeos/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Sirtuína 1/metabolismo , Estilbenos/farmacologia
3.
J Biol Chem ; 290(34): 20841-20855, 2015 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-26124276

RESUMO

Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is depicted by a gain in cell volume (oncosis), swelling of organelles, plasma membrane leakage, and subsequent loss of intracellular contents. Although considered as different cell death entities, there is an overlap between apoptosis and necrosis. In this sense, mounting evidence suggests that both processes can be morphological expressions of a common biochemical network known as "apoptosis-necrosis continuum." To gain insight into the events driving the apoptosis-necrosis continuum, apoptotically proficient cells were screened facing several apoptotic inducers for the absence of type II apoptotic nuclear morphologies. Chelerythrine was selected for further studies based on its cytotoxicity and the lack of apoptotic nuclear alterations. Chelerythrine triggered an early plasma membrane leakage without condensed chromatin aggregates. Ultrastructural analysis revealed that chelerythrine-mediated cytotoxicity was compatible with a necrotic-like type of cell death. Biochemically, chelerythrine induced the activation of caspases. Moreover, the inhibition of caspases prevented chelerythrine-triggered necrotic-like cell death. Compared with staurosporine, chelerythrine induced stronger caspase activation detectable at earlier times. After using a battery of chemicals, we found that high concentrations of thiolic antioxidants fully prevented chelerythrine-driven caspase activation and necrotic-like cell death. Lower amounts of thiolic antioxidants partially prevented chelerythrine-mediated cytotoxicity and allowed cells to display type II apoptotic nuclear morphology correlating with a delay in caspase-3 activation. Altogether, these data support that an early and pronounced activation of caspases can drive cells to undergo a form of necrotic-like regulated cell death.


Assuntos
Antineoplásicos/farmacologia , Caspases/metabolismo , Cromatina/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Necrose/enzimologia , Clorometilcetonas de Aminoácidos/farmacologia , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzofenantridinas/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspases/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Cromatina/ultraestrutura , Colchicina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Necrose/induzido quimicamente , Necrose/genética , Neurônios , Nocodazol/farmacologia , Peptidomiméticos/farmacologia , Quinolinas/farmacologia , Rotenona/farmacologia , Transdução de Sinais , Estaurosporina/farmacologia , Tapsigargina/farmacologia
4.
J Biol Chem ; 289(27): 18752-69, 2014 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-24838313

RESUMO

Caspase-dependent apoptosis is a controlled type of cell death characterized by oligonucleosomal DNA breakdown and major nuclear morphological alterations. Other kinds of cell death do not share these highly distinctive traits because caspase-activated DNase (DFF40/CAD) remains inactive. Here, we report that human glioblastoma multiforme-derived LN-18 cells do not hydrolyze DNA into oligonucleosomal fragments after apoptotic insult. Furthermore, their chromatin remains packaged into a single mass, with no signs of nuclear fragmentation. However, ultrastructural analysis reveals that nuclear disassembly occurs, although compacted chromatin does not localize into apoptotic nuclear bodies. Caspases become properly activated, and ICAD, the inhibitor of DFF40/CAD, is correctly processed. Using cell-free in vitro assays, we show that chromatin from isolated nuclei of LN-18 cells is suitable for hydrolysis into oligonuclesomal fragments by staurosporine-pretreated SH-SY5Y cytoplasms. However, staurosporine-pretreated LN-18 cytoplasms do not induce DNA laddering in isolated nuclei from either LN-18 or SH-SY5Y cells because LN-18 cells express lower amounts of DFF40/CAD. DFF40/CAD overexpression makes LN-18 cells fully competent to degrade their DNA into oligonucleosome-sized fragments, and yet they remain unable to arrange their chromatin into nuclear clumps after apoptotic insult. Indeed, isolated nuclei from LN-18 cells were resistant to undergoing apoptotic nuclear morphology in vitro. The use of LN-18 cells has uncovered a previously unsuspected cellular model, whereby a caspase-dependent chromatin package is DFF40/CAD-independent, and DFF40/CAD-mediated double-strand DNA fragmentation does not warrant the distribution of the chromatin into apoptotic nuclear bodies. The studies highlight a not-yet reported DFF40/CAD-independent mechanism driving conformational nuclear changes during caspase-dependent cell death.


Assuntos
Apoptose , Caspases/metabolismo , Montagem e Desmontagem da Cromatina , Fragmentação do DNA , Desoxirribonucleases/metabolismo , Glioblastoma/patologia , Nucleossomos/genética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , DNA/química , DNA/genética , DNA/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Glioblastoma/genética , Humanos , Peso Molecular , Nucleossomos/efeitos dos fármacos , Estaurosporina/farmacologia
5.
J Biol Chem ; 288(13): 9200-15, 2013 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-23430749

RESUMO

Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD(-/-) cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3'-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3'-OH ends in single-strand rather than double-strand DNA nicks/breaks.


Assuntos
Apoptose , Caspases/metabolismo , Cromatina/química , Quebras de DNA de Cadeia Simples , Fragmentação do DNA , Desoxirribonucleases/metabolismo , Animais , Bisbenzimidazol/farmacologia , Morte Celular , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Clonagem Molecular , DNA/metabolismo , Dano ao DNA , Endonucleases/metabolismo , Citometria de Fluxo/métodos , Humanos , Camundongos , Modelos Biológicos , Mutação , Neuroblastoma/metabolismo , Nucleossomos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Azul Tripano/farmacologia
6.
Biochim Biophys Acta ; 1833(5): 1085-95, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23369735

RESUMO

TNFα can promote either cell survival or cell death. The activation of NF-κB plays a central role in cell survival while its inhibition makes TNFα-triggered cytotoxicity possible. Here, we report that the overexpression of a non-degradable mutant of the inhibitor of NF-κB (super-repressor (SR)-IκBα) sensitizes HeLa cells towards TNFα-induced apoptosis, involving caspases activation and cytocrome C release from the mitochondria. Interestingly, we describe that the specific knockdown of Bcl-xL, but not that of Bcl-2, Bcl-w or Mcl-1, renders cells sensitive to TNFα-induced apoptosis. This cytotoxic effect occurs without altering the activation of NF-κB. Then, the activation of the NF-κB pathway is not sufficient to protect Bcl-xL-downregulated cells from TNFα-induced cell death, meaning that TNFα is not able to promote cell survival in the absence of Bcl-xL. In addition, Bcl-xL silencing does not potentiate the cytotoxicity afforded by the cytokine in SR-IκBα-overexpressing cells. This indicates that TNFα-induced apoptosis in SR-IκBα-overexpressing cells relies on the protein levels of Bcl-xL. We have corroborated these findings using RD and DU-145 cells, which also become sensitive to TNFα-induced apoptosis after Bcl-xL knockdown despite that NF-κB remains activated. Altogether, our results point out that the impairment of the anti-apoptotic function of Bcl-xL should make cells sensitive towards external insults circumventing the TNFα-triggered NF-κB-mediated cytoprotective effect. Hence, the specific inhibition of Bcl-xL could be envisaged as a promising alternative strategy against NF-κB-dependent highly chemoresistant proliferative malignancies.


Assuntos
Proteínas Reguladoras de Apoptose , Proteínas Proto-Oncogênicas c-bcl-2 , Fator de Necrose Tumoral alfa/metabolismo , Proteína bcl-X , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Células HeLa , Humanos , Proteínas I-kappa B/farmacologia , Mitocôndrias , Proteína de Sequência 1 de Leucemia de Células Mieloides , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
7.
J Biol Chem ; 287(10): 7766-79, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22253444

RESUMO

Apoptotic cell death is characterized by nuclear fragmentation and oligonucleosomal DNA degradation, mediated by the caspase-dependent specific activation of DFF40/CAD endonuclease. Here, we describe how, upon apoptotic stimuli, SK-N-AS human neuroblastoma-derived cells show apoptotic nuclear morphology without displaying concomitant internucleosomal DNA fragmentation. Cytotoxicity afforded after staurosporine treatment is comparable with that obtained in SH-SY5Y cells, which exhibit a complete apoptotic phenotype. SK-N-AS cell death is a caspase-dependent process that can be impaired by the pan-caspase inhibitor q-VD-OPh. The endogenous inhibitor of DFF40/CAD, ICAD, is correctly processed, and dff40/cad cDNA sequence does not reveal mutations altering its amino acid composition. Biochemical approaches show that both SH-SY5Y and SK-N-AS resting cells express comparable levels of DFF40/CAD. However, the endonuclease is poorly expressed in the cytosolic fraction of healthy SK-N-AS cells. Despite this differential subcellular distribution of DFF40/CAD, we find no differences in the subcellular localization of both pro-caspase-3 and ICAD between the analyzed cell lines. After staurosporine treatment, the preferential processing of ICAD in the cytosolic fraction allows the translocation of DFF40/CAD from this fraction to a chromatin-enriched one. Therefore, the low levels of cytosolic DFF40/CAD detected in SK-N-AS cells determine the absence of DNA laddering after staurosporine treatment. In these cells DFF40/CAD cytosolic levels can be restored by the overexpression of their own endonuclease, which is sufficient to make them proficient at degrading their chromatin into oligonucleosome-size fragments after staurosporine treatment. Altogether, the cytosolic levels of DFF40/CAD are determinants in achieving a complete apoptotic phenotype, including oligonucleosomal DNA degradation.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Fragmentação do DNA , Desoxirribonucleases/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Desoxirribonucleases/genética , Inibidores Enzimáticos/farmacologia , Humanos , Mutação , Proteínas de Ligação a Poli-ADP-Ribose , Estaurosporina/farmacologia
8.
Aging Cell ; 20(4): e13331, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33660365

RESUMO

Telomere erosion in cells with insufficient levels of the telomerase reverse transcriptase (TERT), contributes to age-associated tissue dysfunction and senescence, and p53 plays a crucial role in this response. We undertook a genome-wide CRISPR screen to identify gene deletions that sensitized p53-positive human cells to telomerase inhibition. We uncovered a previously unannotated gene, C16ORF72, which we term Telomere Attrition and p53 Response 1 (TAPR1), that exhibited a synthetic-sick relationship with TERT loss. A subsequent genome-wide CRISPR screen in TAPR1-disrupted cells reciprocally identified TERT as a sensitizing gene deletion. Cells lacking TAPR1 or TERT possessed elevated p53 levels and transcriptional signatures consistent with p53 upregulation. The elevated p53 response in TERT- or TAPR1-deficient cells was exacerbated by treatment with the MDM2 inhibitor and p53 stabilizer nutlin-3a and coincided with a further reduction in cell fitness. Importantly, the sensitivity to treatment with nutlin-3a in TERT- or TAPR1-deficient cells was rescued by loss of p53. These data suggest that TAPR1 buffers against the deleterious consequences of telomere erosion or DNA damage by constraining p53. These findings identify C16ORF72/TAPR1 as new regulator at the nexus of telomere integrity and p53 regulation.


Assuntos
Aminobenzoatos , Peptídeos e Proteínas de Sinalização Intercelular , Naftalenos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Transdução de Sinais , Telomerase , Proteína Supressora de Tumor p53 , Humanos , Aminobenzoatos/farmacologia , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Técnicas de Inativação de Genes , Imidazóis/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Naftalenos/farmacologia , Piperazinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Telomerase/antagonistas & inibidores , Telomerase/genética , Telomerase/metabolismo , Telômero/metabolismo , Transdução Genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/genética
9.
Cancers (Basel) ; 13(21)2021 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-34771741

RESUMO

Glioblastoma (GBM) is a highly aggressive brain tumor and almost all patients die because of relapses. GBM-derived cells undergo cell death without nuclear fragmentation upon treatment with different apoptotic agents. Nuclear dismantling determines the point-of-no-return in the apoptotic process. DFF40/CAD is the main endonuclease implicated in apoptotic nuclear disassembly. To be properly activated, DFF40/CAD should reside in the cytosol. However, the endonuclease is poorly expressed in the cytosol and remains cumulated in the nucleus of GBM cells. Here, by employing commercial and non-commercial patient-derived GBM cells, we demonstrate that the natural terpenoid aldehyde gossypol prompts DFF40/CAD-dependent nuclear fragmentation. A comparative analysis between gossypol- and staurosporine-treated cells evidenced that levels of neither caspase activation nor DNA damage were correlated with the ability of each compound to induce nuclear fragmentation. Deconvoluted confocal images revealed that DFF40/CAD was almost completely excluded from the nucleus early after the staurosporine challenge. However, gossypol-treated cells maintained DFF40/CAD in the nucleus for longer times, shaping a ribbon-like structure piercing the nuclear fragments and building a network of bridged masses of compacted chromatin. Therefore, GBM cells can fragment their nuclei if treated with the adequate insult, making the cell death process irreversible.

10.
J Mol Biol ; 433(23): 167294, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34662547

RESUMO

Activating mutations in the epidermal growth factor receptor (EGFR) are common driver mutations in non-small cell lung cancer (NSCLC). First, second and third generation EGFR tyrosine kinase inhibitors (TKIs) are effective at inhibiting mutant EGFR NSCLC, however, acquired resistance is a major issue, leading to disease relapse. Here, we characterize a small molecule, EMI66, an analog of a small molecule which we previously identified to inhibit mutant EGFR signalling via a novel mechanism of action. We show that EMI66 attenuates receptor tyrosine kinase (RTK) expression and signalling and alters the electrophoretic mobility of Coatomer Protein Complex Beta 2 (COPB2) protein in mutant EGFR NSCLC cells. Moreover, we demonstrate that EMI66 can alter the subcellular localization of EGFR and COPB2 within the early secretory pathway. Furthermore, we find that COPB2 knockdown reduces the growth of mutant EGFR lung cancer cells, alters the post-translational processing of RTKs, and alters the endoplasmic reticulum (ER) stress response pathway. Lastly, we show that EMI66 treatment also alters the ER stress response pathway and inhibits the growth of mutant EGFR lung cancer cells and organoids. Our results demonstrate that targeting of COPB2 with EMI66 presents a viable approach to attenuate mutant EGFR signalling and growth in NSCLC.


Assuntos
Proteína Coatomer/genética , Proteína Coatomer/metabolismo , Descoberta de Drogas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/genética , Descoberta de Drogas/métodos , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos
11.
Nat Commun ; 12(1): 979, 2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33579912

RESUMO

Glioblastoma (GBM) is a deadly cancer in which cancer stem cells (CSCs) sustain tumor growth and contribute to therapeutic resistance. Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising target in GBM. Using two orthogonal-acting inhibitors of PRMT5 (GSK591 or LLY-283), we show that pharmacological inhibition of PRMT5 suppresses the growth of a cohort of 46 patient-derived GBM stem cell cultures, with the proneural subtype showing greater sensitivity. We show that PRMT5 inhibition causes widespread disruption of splicing across the transcriptome, particularly affecting cell cycle gene products. We identify a GBM splicing signature that correlates with the degree of response to PRMT5 inhibition. Importantly, we demonstrate that LLY-283 is brain-penetrant and significantly prolongs the survival of mice with orthotopic patient-derived xenografts. Collectively, our findings provide a rationale for the clinical development of brain penetrant PRMT5 inhibitors as treatment for GBM.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Apoptose , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Epigenômica , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Proteína-Arginina N-Metiltransferases/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/genética , Splicing de RNA , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Nat Commun ; 11(1): 2396, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32409666

RESUMO

Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective, and cell-active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 results in drastically reduced levels of arginine monomethylated HSP70 family stress-associated proteins. Structural and biochemical analyses reveal that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibits methylation of both constitutive and inducible forms of HSP70, and leads to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.


Assuntos
Arginina/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Estresse Fisiológico , Animais , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Metilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9
14.
Neuro Oncol ; 18(7): 950-61, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26755073

RESUMO

BACKGROUND: Glioblastoma (GBM) or grade IV astrocytoma is one of the most devastating human cancers. The loss of DFF40/CAD, the key endonuclease that triggers oligonucleosomal DNA fragmentation during apoptosis, has been linked to genomic instability and cell survival after radiation. Despite the near inevitability of GBM tumor recurrence after treatment, the relationship between DFF40/CAD and GBM remains unexplored. METHODS: We studied the apoptotic behavior of human GBM-derived cells after apoptotic insult. We analyzed caspase activation and the protein levels and subcellular localization of DFF40/CAD apoptotic endonuclease. DFF40/CAD was also evaluated in histological sections from astrocytic tumors and nontumoral human brain. RESULTS: We showed that GBM cells undergo incomplete apoptosis without generating oligonucleosomal DNA degradation despite the correct activation of executioner caspases. The major defect of GBM cells relied on the improper accumulation of DFF40/CAD at the nucleoplasmic subcellular compartment. Supporting this finding, DFF40/CAD overexpression allowed GBM cells to display oligonucleosomal DNA degradation after apoptotic challenge. Moreover, the analysis of histological slices from astrocytic tumors showed that DFF40/CAD immunoreactivity in tumoral GFAP-positive cells was markedly reduced when compared with nontumoral samples. CONCLUSIONS: Our data highlight the low expression levels of DFF40/CAD and the absence of DNA laddering as common molecular traits in GBM. These findings could be of major importance for understanding the malignant behavior of remaining tumor cells after radiochemotherapy.


Assuntos
Apoptose/genética , Caspases/metabolismo , DNA/metabolismo , Desoxirribonucleases/deficiência , Exorribonucleases/genética , Glioblastoma/enzimologia , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , DNA/genética , Humanos , Proteínas de Ligação a Poli-ADP-Ribose
15.
Cell Res ; 25(6): 653-4, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25930710

RESUMO

DNA hydrolysis is a biochemical process often associated with different forms of cell death, including apoptosis. In a recent paper published in Cell Discovery, Du et al. report that synaptic acetylcholinesterase (AChE-S) shows an unexpected enzymatic activity as DNase switched on after cytotoxic insults.

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