RESUMO
Calcium (Ca2+) is a versatile intracellular second messenger that regulates several signaling pathways involved in growth, development, stress tolerance, and immune response in plants. Autoinhibited Ca2+-ATPases (ACAs) play an important role in the regulation of cellular Ca2+ homeostasis. Here, we systematically analyzed the putative OsACA family members in rice, and according to the phylogenetic tree of OsACAs, OsACA9 was clustered into a separated branch in which its homologous gene in Arabidopsis thaliana was reported to be involved in defense response. When the OsACA9 gene was knocked out by CRISPR/Cas9, significant accumulation of reactive oxygen species (ROS) was detected in the mutant lines. Meanwhile, the OsACA9 knock out lines showed enhanced disease resistance to both rice bacterial blight (BB) and bacterial leaf streak (BLS). In addition, compared to the wild-type (WT), the mutant lines displayed an early leaf senescence phenotype, and the agronomy traits of their plant height, panicle length, and grain yield were significantly decreased. Transcriptome analysis by RNA-Seq showed that the differentially expressed genes (DEGs) between WT and the Osaca9 mutant were mainly enriched in basal immune pathways and antibacterial metabolite synthesis pathways. Among them, multiple genes related to rice disease resistance, receptor-like cytoplasmic kinases (RLCKs) and cell wall-associated kinases (WAKs) genes were upregulated. Our results suggest that the Ca2+-ATPase OsACA9 may trigger oxidative burst in response to various pathogens and synergically regulate disease resistance and leaf senescence in rice.
Assuntos
Resistência à Doença , Oryza , Resistência à Doença/genética , Adenosina Trifosfatases/metabolismo , Oryza/metabolismo , Senescência Vegetal , Filogenia , Regulação da Expressão Gênica de Plantas , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismoRESUMO
Mutations in IDH genes occur frequently in acute myeloid leukemia (AML) and other human cancers to generate the oncometabolite R-2HG. Allosteric inhibition of mutant IDH suppresses R-2HG production in a subset of patients with AML; however, acquired resistance emerges as a new challenge, and the underlying mechanisms remain incompletely understood. Here we establish isogenic leukemia cells containing common IDH oncogenic mutations by CRISPR base editing. By mutational scanning of IDH single amino acid variants in base-edited cells, we describe a repertoire of IDH second-site mutations responsible for therapy resistance through disabling uncompetitive enzyme inhibition. Recurrent mutations at NADPH binding sites within IDH heterodimers act in cis or trans to prevent the formation of stable enzyme-inhibitor complexes, restore R-2HG production in the presence of inhibitors, and drive therapy resistance in IDH-mutant AML cells and patients. We therefore uncover a new class of pathogenic mutations and mechanisms for acquired resistance to targeted cancer therapies. SIGNIFICANCE: Comprehensive scanning of IDH single amino acid variants in base-edited leukemia cells uncovers recurrent mutations conferring resistance to IDH inhibition through disabling NADPH-dependent uncompetitive inhibition. Together with targeted sequencing, structural, and functional studies, we identify a new class of pathogenic mutations and mechanisms for acquired resistance to IDH-targeting cancer therapies. This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Leucemia Mieloide Aguda , Humanos , NADP , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutação , Aminoácidos/genética , Isocitrato DesidrogenaseRESUMO
Fish meal is increasingly being replaced by plant protein raw materials, meanwhile, it brings phytic acid, which combines with phosphorus to form phytate phosphorus and leads to a low utilization rate of phosphorus in shrimp. To solve this problem, this study investigated the effects of phytase supplementation on growth performance, phosphorus utilization, antioxidants, and digestion in red swamp crayfish (Procambarus clarkii). Crayfish (initial mean weight: 8.69 ± 0.15 g, N = 324) were randomly divided into six groups each with three replicates of 18 individuals each, and hand-fed for 8 weeks with one of six experimental diets (50 and 490 g kg-1 animal and plant protein raw material, respectively): negative control (NC; 11.0 g kg-1 phosphorus), positive control (PC; 15 g kg-1 NaH2PO4 added to NC; 14.7 g kg-1 phosphorus), and phytase supplementation diets (P1-P4: 0.1, 0.2, 0.4, and 0.6 g kg-1 phytase added to NC, respectively). The feeding trial was performed in a micro-flow water culture system. P2 showed a significantly higher weight gain rate (WGR), specific growth rate, protein efficiency ratio, and protein retention efficiency (PRE) but showed the lowest feed conversion ratio (FCR) than other groups. Broken-line regression analyses using WGR, FCR, and PRE as evaluation indices showed that the optimal dietary phytase supplementation level was 0.233, 0.244, and 0.303 g kg-1, respectively. P2 showed the highest crude protein content of whole crayfish and abdominal muscle, and phosphorus deposition rate, which was significantly higher than that in NC and PC. P3 showed the highest calcium and phosphorus contents in whole crayfish and phosphorus content in abdominal muscle, and calcium and inorganic phosphorus content in serum, which were significantly higher than those in NC. P3 showed significantly lowest serum alkaline phosphatase, alanine aminotransferase, aspartate transaminase activities, malondialdehyde content in hepatopancreas, and highest catalase activity, which were significantly lower and higher, respectively, than those in NC and PC. In summary, the addition of 0.2-0.4 g kg-1 phytase significantly improves the growth performance, feed utilization, digestive enzyme activity, and antioxidant of P. clarkii, which has a similar effect to the direct addition of NaH2PO4 at 15 g kg-1 to the feed.
Assuntos
6-Fitase , Fósforo na Dieta , 6-Fitase/farmacologia , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Antioxidantes/farmacologia , Astacoidea/metabolismo , Cálcio/metabolismo , Dieta/veterinária , Suplementos Nutricionais/análise , Digestão , Fósforo , Fósforo na Dieta/farmacologia , Ácido Fítico/metabolismo , Proteínas de PlantasRESUMO
As a new species in aquaculture, the lipid nutrition requirement for the juvenile redclaw crayfish Cherax quadricarinatus on a dietary basis on a practical formula needs to be evaluated accurately. In this study, the optimum dietary lipid level was explained by analyzing the growth performance, antioxidant state, lipid metabolism, and gut microbiota of C. quadricarinatus after an eight-week cultivation trial. Six diets with different soybean oil levels (named L0, L2, L4, L6, L8, and L10) were fed to C. quadricarinatus (11.39 ± 0.28 g). The results indicated that the specific growth rate and weight gain of crayfish fed the L4 and L6 diets were significantly higher than those of the other groups (P < 0.05). By the analysis of a second-order polynomial regression model according to growth performance (weight gain rate), the optimum lipid level in a practical diet for juvenile C. quadricarinatus was 9.67%. The survival, condition factor, and hepatosomatic index of crayfish were not significantly affected by dietary oil levels (P > 0.05). As the level of dietary lipids increased, the total antioxidant capacity and glutathione peroxidase activity in serum showed a tendency to rise and then fall and the enzyme activity was highest in crayfish fed the L6 diet. Gut lipase and pepsin activities showed the highest value in crayfish fed the L6 diet. There was no significant difference in acetyl-CoA carboxylase and carnitine palmitoyltransferase-1 contents in crayfish among all the groups (P > 0.05). The relative abundance of Proteobacteria in the phylum and Citrobacter in the genus showed a significant decrease in crayfish of the L10 diet, while the relative abundance of Firmicutes in the phylum markedly increased compared to that of the other groups (P < 0.05). In summary, the results indicated that the 10.39% (L6 diet) dietary lipid level could induce better growth performance, antioxidant ability, and digestive enzyme activity. Most of the fatty acid composition of muscle is not closely related to the fatty acid composition of the diet. Moreover, the composition and diversity of the gut microbiota of C. quadricarinatus were changed by high dietary lipid levels.
RESUMO
Cancers develop from the accumulation of somatic mutations, yet it remains unclear how oncogenic lesions cooperate to drive cancer progression. Using a mouse model harboring NRasG12D and EZH2 mutations that recapitulates leukemic progression, we employ single-cell transcriptomic profiling to map cellular composition and gene expression alterations in healthy or diseased bone marrows during leukemogenesis. At cellular level, NRasG12D induces myeloid lineage-biased differentiation and EZH2-deficiency impairs myeloid cell maturation, whereas they cooperate to promote myeloid neoplasms with dysregulated transcriptional programs. At gene level, NRasG12D and EZH2-deficiency independently and synergistically deregulate gene expression. We integrate results from histopathology, leukemia repopulation, and leukemia-initiating cell assays to validate transcriptome-based cellular profiles. We use this resource to relate developmental hierarchies to leukemia phenotypes, evaluate oncogenic cooperation at single-cell and single-gene levels, and identify GEM as a regulator of leukemia-initiating cells. Our studies establish an integrative approach to deconvolute cancer evolution at single-cell resolution in vivo.
Assuntos
Carcinogênese/genética , Carcinogênese/metabolismo , Leucemia/genética , Leucemia/metabolismo , Análise de Célula Única , Animais , Apoptose , Ciclo Celular , Proteína Potenciadora do Homólogo 2 de Zeste/deficiência , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Epigenômica , GTP Fosfo-Hidrolases , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Humanos , Leucemia Mieloide Aguda , Proteínas de Membrana , Camundongos Knockout , Mutação , Células Mieloides , Oncogenes , Fenótipo , TranscriptomaRESUMO
The DNA-sensing enzyme cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) regulates inflammation and immune defense against pathogens and malignant cells. Although cGAS has been shown to exert antitumor effects in several mouse models harboring transplanted tumor cell lines, its role in tumors arising from endogenous tissues remains unknown. Here, we show that deletion of cGAS in mice exacerbated chemical-induced colitis and colitis-associated colon cancer (CAC). Interestingly, mice lacking cGAS were more susceptible to CAC than those lacking stimulator of interferon genes (STING) or type I interferon receptor under the same conditions. cGAS but not STING is highly expressed in intestinal stem cells. cGAS deficiency led to intestinal stem cell loss and compromised intestinal barrier integrity upon dextran sodium sulfate-induced acute injury. Loss of cGAS exacerbated inflammation, led to activation of STAT3, and accelerated proliferation of intestinal epithelial cells during CAC development. Mice lacking cGAS also accumulated myeloid-derived suppressive cells within the tumor, displayed enhanced Th17 differentiation, but reduced interleukin (IL)-10 production. These results indicate that cGAS plays an important role in controlling CAC development by defending the integrity of the intestinal mucosa.
Assuntos
Neoplasias do Colo/enzimologia , Mucosa Intestinal/enzimologia , Proteínas de Neoplasias/metabolismo , Nucleotidiltransferases/metabolismo , Animais , Neoplasias do Colo/genética , Camundongos , Camundongos Knockout , Células Supressoras Mieloides/enzimologia , Proteínas de Neoplasias/genética , Nucleotidiltransferases/genética , Células-Tronco/enzimologia , Células Th17/enzimologiaRESUMO
Transposable elements or transposons are major players in genetic variability and genome evolution. Aberrant activation of long interspersed element-1 (LINE-1 or L1) retrotransposons is common in human cancers, yet their tumor-type-specific functions are poorly characterized. We identified MPHOSPH8/MPP8, a component of the human silencing hub (HUSH) complex, as an acute myeloid leukemia (AML)-selective dependency by epigenetic regulator-focused CRISPR screening. Although MPP8 is dispensable for steady-state hematopoiesis, MPP8 loss inhibits AML development by reactivating L1s to induce the DNA damage response and cell cycle exit. Activation of endogenous or ectopic L1s mimics the phenotype of MPP8 loss, whereas blocking retrotransposition abrogates MPP8-deficiency-induced phenotypes. Expression of AML oncogenic mutations promotes L1 suppression, and enhanced L1 silencing is associated with poor prognosis in human AML. Hence, while retrotransposons are commonly recognized for their cancer-promoting functions, we describe a tumor-suppressive role for L1 retrotransposons in myeloid leukemia.
Assuntos
Inativação Gênica , Leucemia Mieloide/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Animais , Sistemas CRISPR-Cas/genética , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Epigênese Genética , Regulação Leucêmica da Expressão Gênica , Genoma Humano , Instabilidade Genômica , Hematopoese/genética , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/genéticaRESUMO
In this issue of Cancer Discovery, Cai and colleagues delineate a new mechanism that links cell of origin, the transcription factor EVI1, apoptotic priming, and therapeutic susceptibility in mixed lineage leukemia-rearranged acute myeloid leukemia. These findings establish a cell of origin-dependent program that may be leveraged by therapeutic combinations to overcome drug resistance in chemoresistant leukemias.See related article by Cai et al., p. 1500.
Assuntos
Leucemia Mieloide Aguda , Proteína de Leucina Linfoide-Mieloide , Proteínas de Ligação a DNA , Histona Desmetilases , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Fatores de TranscriçãoRESUMO
There is increasing evidence showing that inflammation is associated with depression in humans. Hesperidin, a natural bioflavonoid, has performed excellent effects on depression. The aim of this research was to investigate the therapeutic eï¬ect of hesperidin on chronic unpredictable mild stress (CUMS)-induced rats. The sucrose preference test (SPT), forced swimming test (FST), and open ï¬eld test (OFT) were performed to measure the depression-related symptoms. The enzyme-linked immunosorbent assay (ELISA) was used to determine the concentrations of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in the prefrontal cortex (PFC) of rats and cellular supernatant. PCR and Western blot were used to monitor the differences of NLRP3, caspase-1, ASC activation in the levels of genes and proteins in the PFC of rats and microglia. The activation of microglia was determined using immunofluorescence staining and flow cytometry assay. Our results show that hesperidin treatment signiï¬cantly relieved depressive like behaviors in CUMS rats. In addition, hesperidin decreased the expression levels of IL-1ß, IL-6, TNF-α, NLRP3, caspase-1, and ASC in the PFC and microglia. This study investigated that hesperidin treatment ameliorated CUMS-induced depression by suppressing microglia and inflammation.
RESUMO
Cancer cells, including melanoma cells, often metastasize regionally through the lymphatic system before metastasizing systemically through the blood1-4; however, the reason for this is unclear. Here we show that melanoma cells in lymph experience less oxidative stress and form more metastases than melanoma cells in blood. Immunocompromised mice with melanomas derived from patients, and immunocompetent mice with mouse melanomas, had more melanoma cells per microlitre in tumour-draining lymph than in tumour-draining blood. Cells that metastasized through blood, but not those that metastasized through lymph, became dependent on the ferroptosis inhibitor GPX4. Cells that were pretreated with chemical ferroptosis inhibitors formed more metastases than untreated cells after intravenous, but not intralymphatic, injection. We observed multiple differences between lymph fluid and blood plasma that may contribute to decreased oxidative stress and ferroptosis in lymph, including higher levels of glutathione and oleic acid and less free iron in lymph. Oleic acid protected melanoma cells from ferroptosis in an Acsl3-dependent manner and increased their capacity to form metastatic tumours. Melanoma cells from lymph nodes were more resistant to ferroptosis and formed more metastases after intravenous injection than did melanoma cells from subcutaneous tumours. Exposure to the lymphatic environment thus protects melanoma cells from ferroptosis and increases their ability to survive during subsequent metastasis through the blood.
Assuntos
Ferroptose , Linfa/metabolismo , Melanoma/patologia , Metástase Neoplásica/patologia , Animais , Sobrevivência Celular , Coenzima A Ligases/metabolismo , Feminino , Ferroptose/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Ferro/metabolismo , Masculino , Melanoma/sangue , Melanoma/metabolismo , Camundongos , Metástase Neoplásica/tratamento farmacológico , Ácido Oleico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Análise de Componente PrincipalRESUMO
Mutations in protein-coding genes are well established as the basis for human cancer, yet how alterations within noncoding genome, a substantial fraction of which contain cis-regulatory elements (CRE), contribute to cancer pathophysiology remains elusive. Here, we developed an integrative approach to systematically identify and characterize noncoding regulatory variants with functional consequences in human hematopoietic malignancies. Combining targeted resequencing of hematopoietic lineage-associated CREs and mutation discovery, we uncovered 1,836 recurrently mutated CREs containing leukemia-associated noncoding variants. By enhanced CRISPR/dCas9-based CRE perturbation screening and functional analyses, we identified 218 variant-associated oncogenic or tumor-suppressive CREs in human leukemia. Noncoding variants at KRAS and PER2 enhancers reside in proximity to nuclear receptor (NR) binding regions and modulate transcriptional activities in response to NR signaling in leukemia cells. NR binding sites frequently colocalize with noncoding variants across cancer types. Hence, recurrent noncoding variants connect enhancer dysregulation with nuclear receptor signaling in hematopoietic malignancies. SIGNIFICANCE: We describe an integrative approach to identify noncoding variants in human leukemia, and reveal cohorts of variant-associated oncogenic and tumor-suppressive cis-regulatory elements including KRAS and PER2 enhancers. Our findings support a model in which noncoding regulatory variants connect enhancer dysregulation with nuclear receptor signaling to modulate gene programs in hematopoietic malignancies.See related commentary by van Galen, p. 646.This article is highlighted in the In This Issue feature, p. 627.
Assuntos
Neoplasias Hematológicas/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , HumanosRESUMO
The molecular mechanism of evaluating 17ß-estradiol (E2)-induced toxicity in female Daphnia magna has not been determined. In this study, the transcriptome of D. magna was analyzed after exposure to three different concentrations (0, 10, and 100 ng L-1) of E2 at 3, 6, and 12 h. The results showed 351-17,221 significantly up-regulated and 505-10,282 significantly down-regulated genes (P < 0.05). Overall, the selected largest 10,282 (10 ng L-1vs control at 12 h) down-regulated and 17,221 (100 vs 10 ng L-1) up-regulated genes were identified; following annotation, pathways in cancer and RNA transport were found to be enriched according to the interaction network. Among all completed comparisons, KEGG pathways related to the immune system, cancer, disease infection, and active compound metabolism were identified by short time series expression miner analysis. A different set of genes fluctuated in a "U"-shaped pattern over time and at different concentrations of E2, whereas some genes associated with disintoxication showed a reverse "U"-shaped response as E2 administration was increased. These results suggest that E2 exposure caused transcriptional changes in the immune system, disintoxication, disease prevention, and the protein degradation pathway.
Assuntos
Daphnia , Exposição Ambiental , Estradiol , Transcriptoma , Animais , Daphnia/efeitos dos fármacos , Estradiol/toxicidade , Feminino , Transcriptoma/efeitos dos fármacos , Poluentes Químicos da Água/toxicidadeRESUMO
Tissue-specific gene expression requires coordinated control of gene-proximal and -distal cis-regulatory elements (CREs), yet functional analysis of gene-distal CREs such as enhancers remains challenging. Here we describe CRISPR/dCas9-based enhancer-targeting epigenetic editing systems, enCRISPRa and enCRISPRi, for efficient analysis of enhancer function in situ and in vivo. Using dual effectors capable of re-writing enhancer-associated chromatin modifications, we show that enCRISPRa and enCRISPRi modulate gene transcription by remodeling local epigenetic landscapes at sgRNA-targeted enhancers and associated genes. Comparing with existing methods, the improved systems display more robust perturbations of enhancer activity and gene transcription with minimal off-targets. Allele-specific targeting of enCRISPRa to oncogenic TAL1 super-enhancer modulates TAL1 expression and cancer progression in xenotransplants. Single or multi-loci perturbations of lineage-specific enhancers using an enCRISPRi knock-in mouse establish in vivo evidence for lineage-restricted essentiality of developmental enhancers during hematopoiesis. Hence, enhancer-targeting CRISPR epigenetic editing provides opportunities for interrogating enhancer function in native biological contexts.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Elementos Facilitadores Genéticos , Epigênese Genética , Edição de Genes/métodos , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Feminino , Células HEK293 , Hematopoese/genética , Humanos , Células Jurkat , Células K562 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Neoplasias/genética , RNA Guia de Cinetoplastídeos/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genéticaRESUMO
Metastasis requires cancer cells to undergo metabolic changes that are poorly understood1-3. Here we show that metabolic differences among melanoma cells confer differences in metastatic potential as a result of differences in the function of the MCT1 transporter. In vivo isotope tracing analysis in patient-derived xenografts revealed differences in nutrient handling between efficiently and inefficiently metastasizing melanomas, with circulating lactate being a more prominent source of tumour lactate in efficient metastasizers. Efficient metastasizers had higher levels of MCT1, and inhibition of MCT1 reduced lactate uptake. MCT1 inhibition had little effect on the growth of primary subcutaneous tumours, but resulted in depletion of circulating melanoma cells and reduced the metastatic disease burden in patient-derived xenografts and in mouse melanomas. In addition, inhibition of MCT1 suppressed the oxidative pentose phosphate pathway and increased levels of reactive oxygen species. Antioxidants blocked the effects of MCT1 inhibition on metastasis. MCT1high and MCT1-/low cells from the same melanomas had similar capacities to form subcutaneous tumours, but MCT1high cells formed more metastases after intravenous injection. Metabolic differences among cancer cells thus confer differences in metastatic potential as metastasizing cells depend on MCT1 to manage oxidative stress.
Assuntos
Melanoma/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Simportadores/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Melanoma/genética , Melanoma/secundário , Camundongos , Transportadores de Ácidos Monocarboxílicos/genética , Estresse Oxidativo , Simportadores/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Epigenetic gene regulation and metabolism are highly intertwined, yet little is known about whether altered epigenetics influence cellular metabolism during cancer progression. Here, we show that EZH2 and NRASG12D mutations cooperatively induce progression of myeloproliferative neoplasms to highly penetrant, transplantable, and lethal myeloid leukemias in mice. EZH1, an EZH2 homolog, is indispensable for EZH2-deficient leukemia-initiating cells and constitutes an epigenetic vulnerability. BCAT1, which catalyzes the reversible transamination of branched-chain amino acids (BCAA), is repressed by EZH2 in normal hematopoiesis and aberrantly activated in EZH2-deficient myeloid neoplasms in mice and humans. BCAT1 reactivation cooperates with NRASG12D to sustain intracellular BCAA pools, resulting in enhanced mTOR signaling in EZH2-deficient leukemia cells. Genetic and pharmacologic inhibition of BCAT1 selectively impairs EZH2-deficient leukemia-initiating cells and constitutes a metabolic vulnerability. Hence, epigenetic alterations rewire intracellular metabolism during leukemic transformation, causing epigenetic and metabolic vulnerabilities in cancer-initiating cells. SIGNIFICANCE: EZH2 inactivation and oncogenic NRAS cooperate to induce leukemic transformation of myeloproliferative neoplasms by activating BCAT1 to enhance BCAA metabolism and mTOR signaling. We uncover a mechanism by which epigenetic alterations rewire metabolism during cancer progression, causing epigenetic and metabolic liabilities in cancer-initiating cells that may be exploited as potential therapeutics.See related commentary by Li and Melnick, p. 1158.This article is highlighted in the In This Issue feature, p. 1143.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/genética , GTP Fosfo-Hidrolases/genética , Leucemia/patologia , Proteínas de Membrana/genética , Transtornos Mieloproliferativos/genética , Transaminases/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Animais , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Humanos , Leucemia/genética , Leucemia/metabolismo , Camundongos , Mutação , Transtornos Mieloproliferativos/complicações , Transtornos Mieloproliferativos/metabolismo , Transplante de Neoplasias , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
The hormone 17ß-estradiol (E2) can be found in rivers, effluents, and even drinking water. Researches have demonstrated that E2 affects various metabolic pathways through gene activation and may cause reproductive toxicity in fish. Therefore, the aim of this study was to evaluate E2-induced toxicity via testicular transcriptome of zebrafish (Danio rerio) exposed to different concentrations (10â¯ngâ¯L-1, and 100â¯ngâ¯L-1) of E2. A total of >600 significant differentially expressed genes (DEGs) were enriched among the three treatments. Short time-series expression miner analysis revealed five KEGG pathways including drug metabolism, other enzymes, calcium signaling pathway, ECM-receptor interaction, gap junction, and cell adhesion molecules. Twenty genes were selected to verify the accuracy of RNA-Seq. Other reported genes related to sex differentiation, development, energy metabolism, and other processes were found. One set of genes significantly increased/decreased/fluctuated over time, especially 12â¯h after E2 exposure. Genes associated with ovaries (zp3c), and development (bmp15, gdf9, and sycp2l) were significantly upregulated with increasing E2 concentration. E2 and testosterone was significantly decreased by 10 (except for T) and 100â¯ngâ¯L-1 E2 exposure at 12â¯h. The current study demonstrated that sex differentiation, development, energy metabolism, immunity, and ribosome biogenesis in male zebrafish were all significantly affected by 17ß-estradiol exposure through transcriptional alterations.
Assuntos
Exposição Ambiental , Estradiol/metabolismo , Estradiol/farmacologia , Testículo/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Animais , Masculino , Reprodução/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologiaRESUMO
Drug resistance remains the key problem in cancer treatment. It is now accepted that each myeloma patient harbors multiple subclones and subclone dominance may change over time. The coexistence of multiple subclones with high or low chromosomal instability (CIN) signature causes heterogeneity and drug resistance with consequent disease relapse. In this study, using a tandem affinity purification-mass spectrometry (TAP-MS) technique, we found that NEK2, a CIN gene, was bound to the deubiquitinase USP7. Binding to USP7 prevented NEK2 ubiquitination resulting in NEK2 stabilization. Increased NEK2 kinase levels activated the canonical NF-κB signaling pathway through the PP1α/AKT axis. Newly diagnosed myeloma patients with activated NF-κB signaling through increased NEK2 activity had poorer event-free and overall survivals based on multiple independent clinical cohorts. We also found that NEK2 activated heparanase, a secreted enzyme, responsible for bone destruction in an NF-κB-dependent manner. Intriguingly, both NEK2 and USP7 inhibitors showed great efficacy in inhibiting myeloma cell growth and overcoming NEK2-induced and -acquired drug resistance in xenograft myeloma mouse models.
Assuntos
Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Animais , Bortezomib/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Técnicas de Silenciamento de Genes , Glucuronidase/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Modelos Biológicos , Mieloma Múltiplo/patologia , NF-kappa B/metabolismo , Quinases Relacionadas a NIMA/antagonistas & inibidores , Prognóstico , Ligação Proteica , Estabilidade Proteica , Transdução de Sinais , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Peptidase 7 Específica de Ubiquitina/genética , Peptidase 7 Específica de Ubiquitina/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pharmacological doses (> 1mM) of ascorbate (a.k.a., vitamin C) have been shown to selectively kill cancer cells through a mechanism that is dependent on the generation of H2O2 at doses that are safely achievable in humans using intravenous administration. The process by which ascorbate oxidizes to form H2O2 is thought to be mediated catalytically by redox active metal ions such as iron (Fe). Because intravenous iron sucrose is often administered to colon cancer patients to help mitigate anemia, the current study assessed the ability of pharmacological ascorbate to kill colon cancer cells in the presence and absence of iron sucrose. In vitro survival assays showed that 10mM ascorbate exposure (2h) clonogenically inactivated 40-80% of exponentially growing colon cancer cell lines (HCT116 and HT29). When the H2O2 scavenging enzyme, catalase, was added to the media, or conditionally over-expressed using a doxycycline inducible vector, the toxicity of pharmacological ascorbate was significantly blunted. When colon cancer cells were treated in the presence or absence of 250µM iron sucrose, then rinsed, and treated with 10mM ascorbate, the cells demonstrated increased levels of labile iron that resulted in significantly increased clonogenic cell killing, compared to pharmacological ascorbate alone. Interestingly, when colon cancer cells were treated with iron sucrose for 1h and then 10mM ascorbate was added to the media in the continued presence of iron sucrose, there was no enhancement of toxicity despite similar increases in intracellular labile iron. The combination of iron chelators, deferoxamine and diethylenetriaminepentaacetic acid, significantly inhibited the toxicity of either ascorbate alone or ascorbate following iron sucrose. These observations support the hypothesis that increasing intracellular labile iron pools, using iron sucrose, can be used to increase the toxicity of pharmacological ascorbate in human colon cancer cells by a mechanism involving increased generation of H2O2.
Assuntos
Ácido Ascórbico/toxicidade , Compostos Férricos/farmacologia , Ácido Glucárico/farmacologia , Ferro/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Catalase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Desferroxamina/farmacologia , Óxido de Ferro Sacarado , Células HCT116 , Células HT29 , Humanos , Peróxido de Hidrogênio/metabolismo , Quelantes de Ferro/farmacologiaRESUMO
Advances in genomic profiling present new challenges of explaining how changes in DNA and RNA are translated into proteins linking genotype to phenotype. Here we compare the genome-scale proteomic and transcriptomic changes in human primary haematopoietic stem/progenitor cells and erythroid progenitors, and uncover pathways related to mitochondrial biogenesis enhanced through post-transcriptional regulation. Mitochondrial factors including TFAM and PHB2 are selectively regulated through protein translation during erythroid specification. Depletion of TFAM in erythroid cells alters intracellular metabolism, leading to elevated histone acetylation, deregulated gene expression, and defective mitochondria and erythropoiesis. Mechanistically, mTORC1 signalling is enhanced to promote translation of mitochondria-associated transcripts through TOP-like motifs. Genetic and pharmacological perturbation of mitochondria or mTORC1 specifically impairs erythropoiesis in vitro and in vivo. Our studies support a mechanism for post-transcriptional control of erythroid mitochondria and may have direct relevance to haematologic defects associated with mitochondrial diseases and ageing.