RESUMO
miR-34a has been identified as a tumor suppressor microRNA (miRNA) involved in the P53 network. Its expression levels correlate to carcinogenesis, which are generally lower in tumor tissue and higher in response to DNA damage. In this study, the response of miR-34a from exposure to genotoxic agents in human lymphoblastoid TK6 cells was evaluated to assess whether the expression of this miRNA could be used as an early indicator for genotoxic damage in mammalian cells. TK6 cells were treated with seven genotoxic agents with different mode-of-actions (cisplatin, N-ethyl-N-nitrosourea, etoposide, mitomycin C, methyl methanesulphonate, taxol, and X-ray radiation) and a non-genetic toxin (usnic acid) at different concentrations for four hours (except for X-rays) and the expression levels of miR-34a were measured 24 h after the beginning of the treatments. The expression levels of miR-34a were significantly increased by these genetic toxins in a dose-dependent manner, while no significant change in miRNA expression was found in the usnic acid-treated cells. These results suggest that miR-34a can respond to genotoxic insults sensitively; thus, miR-34a expression has the potential to be used to evaluate genotoxicity of agents.
Assuntos
Carcinogênese/genética , Dano ao DNA/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Carcinogênese/efeitos dos fármacos , Cisplatino/farmacologia , Cisplatino/toxicidade , Etilnitrosoureia/farmacologia , Etoposídeo/farmacologia , Etoposídeo/toxicidade , Genes Supressores de Tumor , Humanos , Mutagênicos/farmacologia , Mutagênicos/toxicidadeRESUMO
Murine splenic macrophage plays a decisive role in host immunity through phagocytosis against pathogens. It was reported that, macrophages also involves in phagocytosis of some tumour cells upon its activation initiated by certain cytokines produced by other immune cell or by indigenously treated. In this study, we have investigated the killing of leukemic blast cells by macrophages upon stimulated with IL-15 and GM-CSF alone or in combination in ENU challenged leukemic murine model. Along with, the release of TNF-α, IL-12 and IFN-γ by macrophages were assayed by ELISA. NO production by macrophages was also investigated. The molecular expressions like GM-CSF and TLRs were investigated for better understand of macrophage-leukemic cell interaction. Result shows that in disease condition macrophages have poor phagocytic activities which may be due to less release of TNF-α, IL-12 and IFN-γ by macrophages. This impaired phagocytic activity in leukemic mice was increase upon stimulation with IL-15 and GM-CSF.
Assuntos
Etilnitrosoureia/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-15/metabolismo , Leucemia/induzido quimicamente , Leucemia/metabolismo , Macrófagos/metabolismo , Fagócitos/metabolismo , Animais , Células Cultivadas , Citocinas/metabolismo , Interferon gama/metabolismo , Interleucina-12/metabolismo , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Fagócitos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Fagocitose/fisiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
INTRODUCTION: Osimertinib is approved for advanced EGFR-mutated NSCLC, and identification of on-target mechanisms of resistance (i.e., EGFR C797S) to this third-generation EGFR inhibitor are evolving. Whether durable control of subsequently osimertinib-resistant NSCLC with the EGFR-sensitizing mutation (SM)/C797S is possible with first-generation EGFR inhibitors (such as gefitinib or erlotinib) remains underreported, as does the resultant acquired resistance profile. METHODS: We used N-ethyl-N-nitrosourea mutagenesis to determine the profile of EGFR SM/C797S preclinical models exposed to reversible EGFR inhibitors. In addition, we retrospectively probed a case of EGFR SM lung adenocarcinoma treated with first-line osimertinib, followed by second-line erlotinib in the setting of EGFR SM/C797S. RESULTS: Use of N-ethyl-N-nitrosourea mutagenesis against the background of EGFR L858R/C797S in conjunction with administration of gefitinib revealed preferential outgrowth of cells with EGFR L858R/T790M/C797S. A patient with EGFR delE746_T751insV NSCLC was treated with osimertinib with sustained response for 10 months before acquiring EGFR C797S. The patient was subsequently treated with erlotinib, with response for a period of 4 months, but disease progression ensued. Liquid biopsy disclosed EGFR delE746_T751insV with T790M and C797S present in cis. CONCLUSION: EGFR SM NSCLC can acquire resistance to osimertinib through development of the EGFR C797S mutation. In this clinical scenario, the tumor may respond transiently to reversible first-generation EGFR inhibitors (gefitinib or erlotinib), but evolving mechanisms of on-target resistance-in clinical specimens and preclinical systems-indicate that EGFR C797S along with EGFR T790M can evolve. This report adds to the growing understanding of tumor evolution or adaptability to sequential EGFR inhibition and augments support for exploring combination therapies to delay or prevent on-target resistance.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Acrilamidas/administração & dosagem , Alquilantes/farmacologia , Compostos de Anilina/administração & dosagem , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Células Cultivadas , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/administração & dosagem , Etilnitrosoureia/farmacologia , Feminino , Gefitinibe/administração & dosagem , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Pessoa de Meia-Idade , Mutagênese , Mutação , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Estudos Retrospectivos , Resultado do TratamentoRESUMO
N-ethyl-N-nitrosourea (ENU) is highly used in rodent models of tumerogenesis/carcinogenesis. Xenografting human-cancer tissues/cells with estradiol (E2) treatment is also used to generate rodent-models of gynaecological cancers. The altered metabolic-redox environment leading to establishment of pre-tumorigenesis condition and their mechanism are less studied. Here, female Wister rats were treated with these drugs at their pre-tumerogenic dosage (one group ENU single intra-peritoneal dose of 90 mg/kg b.w. and another group were implanted with human breast tumor (stage-IIIB) and fed with 2.5 mg of 17ß-estradiol once in a week for 4 months). After 4 months, animals were sacrificed; their serum and liver tissues were tested. A brief comparison was made with a rat model (regarded as positive control) of toxicity induced by mutagenic environmental pollutant arsenic (0.6 ppm daily/4 weeks). The increase in serum alkaline phosphatase and glutamate-pyruvate transaminase suggests the possible organ toxicity is favoured by the increase in hepatic/systemic free radicals and oxidative stress in all drug application models. But the increase in the serum E2 level as noted in the ELISA data with impairment in the hepatic estrogen sulfotransferase (SULT1E1) protein expression (immuno-blot data) were noticed with interfered hepatic free-thiols only in ENU and xenograft-E2 group compared to arsenic group. It is also evident in the in vitro result from E2/GSH/NAC added hepatic slices with altered antioxidant regulations. Moreover, impairment in hepatic SOD1, catalase and glutathiole peroxidase activities (PAGEzymographic data), especially in the ENU-treated group makes them more vulnerable to the oxidative threat in creating pre-tumerogenic microenvironment. This is evident in the result of their higher DNA-damage and histological abnormalities. The Bioinformatics study revealed an important role of rSULT1E1 in the regulations of E2 metabolism. This study is important for the exploration of the pre-tumerogenic condition by ENU and E2 by impairing SULT1E1 expression and E2 regulations via oxidant-stress signalling. The finding may help to find new therapeutic-targets to treat gynaecological-cancers more effectively.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Estradiol/farmacologia , Etilnitrosoureia/farmacologia , Animais , Antioxidantes/metabolismo , Neoplasias da Mama/metabolismo , Catalase/efeitos dos fármacos , Catalase/metabolismo , Dano ao DNA/efeitos dos fármacos , Estradiol/sangue , Estradiol/metabolismo , Etilnitrosoureia/metabolismo , Feminino , Xenoenxertos , Humanos , Fígado/metabolismo , Oxidantes/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Sulfotransferases/efeitos dos fármacos , Sulfotransferases/genética , Superóxido Dismutase-1/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
The X-linked Pig-a gene encodes an enzyme required for the biosynthesis of glycosyl phosphatidylinositol (GPI) anchors. Pig-a mutant cells fail to synthesize GPI and to express GPI-anchored protein markers (e.g., CD90) on their surface. Marker deficiency serves as a phenotypic indicator of Pig-a mutation in various in vivo assays. Here, we describe an in vitro Pig-a mutation assay in L5178YTk+/- mouse lymphoma cells, in which mutant-phenotype cells are measured by flow cytometry using a fluorescent anti-CD90 antibody. Increased frequencies of CD90-deficient mutants were detected in cells treated with benzo[a]pyrene (B[a]P), N-ethyl-N-nitrosourea (ENU), ethyl methanesulphonate, and 7,12-dimethylbenz[a]anthracene, with near maximum mutant frequencies measured eight days after treatment. The CD90 deficiency in mutant cells quantified by flow cytometry was shown to be due to loss of GPI anchors in a limiting-dilution cloning assay using proaerolysin selection. Individual CD90-deficient cells from cultures treated with ENU, B[a]P, and vehicle were sorted and clonally expanded for molecular analysis of their Pig-a gene. Pig-a mutations with agent-specific signatures were found in nearly all clones that developed from sorted CD90-deficient cells. These results indicate that a Pig-a mutation assay can be successfully conducted in L5178YTk+/- cells. The assay may be useful for mutagenicity screening of environmental agents as well as for testing hypotheses in vitro before committing to in vivo Pig-a assays. Environ. Mol. Mutagen. 59:4-17, 2018. Published 2017. This article is a US Government work and is in the public domain in the USA.
Assuntos
Bioensaio/métodos , Linfoma/genética , Proteínas de Membrana/genética , Mutação/genética , Animais , Benzo(a)pireno/farmacologia , Linhagem Celular Tumoral , Metanossulfonato de Etila , Etilnitrosoureia/farmacologia , Citometria de Fluxo/métodos , Camundongos , Mutagênicos/farmacologia , Mutação/efeitos dos fármacos , Antígenos Thy-1/metabolismoRESUMO
ROS1 rearrangement is observed in 1-2% of non-small cell lung cancers (NSCLC). The ROS1 tyrosine kinase inhibitor (TKI) crizotinib has induced marked tumour shrinkage in ROS1-rearranged cancers. However, emergence of acquired resistance to TKI is inevitable within a few years. Previous findings indicate that cabozantinib overcomes secondary mutation-mediated crizotinib-resistance in ROS1-fusion-positive cells. Here we attempted to establish cabozantinib-resistant cells by N-ethyl-N-nitrosourea mutagenesis screening using CD74-ROS1-expressing Ba/F3 cells. Two resistant cell lines with CD74-ROS1 F2004V or F2075C mutations, which are homologous to ALK F1174 or F1245 mutations, survived in the presence of a low dose of ROS1-TKI. Removal of ROS1-TKI from these TKI-addicted cells induced excessive activation of ROS1 tyrosine kinase followed by apoptosis. We succeeded in recapturing the TKI-addicted phenotype using doxycycline-inducible CD74-ROS1 mutant over-expression in Ba/F3 cells, suggesting that excessive ROS1 oncogenic signaling itself induced apoptosis instead of cell growth. Phosphoproteomic analysis and high-throughput inhibitor screening revealed that excessive ROS1 signaling in the TKI-addicted cells phosphorylated or activated apoptosis-related molecules such as FAF1 or p38. Collectively, our findings partly clarify molecular mechanisms of excessive ROS1 oncogenic signaling that mediates paradoxical induction of apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Anilidas/farmacologia , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Etilnitrosoureia/farmacologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imidazóis/farmacologia , Mutagênese Sítio-Dirigida , Fosfopeptídeos/análise , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas em Tandem , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoAssuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Hiperpigmentação/genética , Mutação de Sentido Incorreto , Alelos , Animais , Biópsia por Agulha , Modelos Animais de Doenças , Etilnitrosoureia/farmacologia , Hiperpigmentação/patologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Polimorfismo de Nucleotídeo Único , Distribuição Aleatória , Sensibilidade e EspecificidadeRESUMO
Iron is essential for numerous cellular processes. For diagnostic purposes iron-related parameters in patients are assessed by clinical chemical blood analysis including the analysis of ferritin, transferrin and iron levels. Here, we retrospectively evaluated the use of these parameters in the phenotype-driven Munich N-ethyl-N-nitrosourea mouse mutagenesis project for the generation of novel animal models for human diseases. The clinical chemical blood analysis was carried out on more than 10,700 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the plasma levels of iron-related plasma parameters. We identified animals consistently exhibiting altered plasma ferritin or transferrin values. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of three mutant lines with increased plasma ferritin levels. For two of these lines the causative mutations were identified in the Fth1gene and the Ireb2 gene, respectively. Thus, novel mouse models for the functional analysis of iron homeostasis were established by a phenotype-driven screen for mutant mice.
Assuntos
Etilnitrosoureia/farmacologia , Ferritinas/sangue , Mutagênicos/farmacologia , Animais , Sequência de Bases , Análise Mutacional de DNA , Feminino , Expressão Gênica , Estudos de Associação Genética , Ligação Genética , Testes Genéticos , Ferro/sangue , Masculino , Camundongos Endogâmicos C3H , Mutagênese , Fenótipo , Transferrina/metabolismoRESUMO
Therapy-related acute myeloid leukaemia (t-AML) and therapy-related myelodysplastic syndrome (t-MDS) are well-recognized complications of cytotoxic chemotherapy and/or radiotherapy. There are several features that distinguish t-AML from de novo AML, including a higher incidence of TP53 mutations, abnormalities of chromosomes 5 or 7, complex cytogenetics and a reduced response to chemotherapy. However, it is not clear how prior exposure to cytotoxic therapy influences leukaemogenesis. In particular, the mechanism by which TP53 mutations are selectively enriched in t-AML/t-MDS is unknown. Here, by sequencing the genomes of 22 patients with t-AML, we show that the total number of somatic single-nucleotide variants and the percentage of chemotherapy-related transversions are similar in t-AML and de novo AML, indicating that previous chemotherapy does not induce genome-wide DNA damage. We identified four cases of t-AML/t-MDS in which the exact TP53 mutation found at diagnosis was also present at low frequencies (0.003-0.7%) in mobilized blood leukocytes or bone marrow 3-6 years before the development of t-AML/t-MDS, including two cases in which the relevant TP53 mutation was detected before any chemotherapy. Moreover, functional TP53 mutations were identified in small populations of peripheral blood cells of healthy chemotherapy-naive elderly individuals. Finally, in mouse bone marrow chimaeras containing both wild-type and Tp53(+/-) haematopoietic stem/progenitor cells (HSPCs), the Tp53(+/-) HSPCs preferentially expanded after exposure to chemotherapy. These data suggest that cytotoxic therapy does not directly induce TP53 mutations. Rather, they support a model in which rare HSPCs carrying age-related TP53 mutations are resistant to chemotherapy and expand preferentially after treatment. The early acquisition of TP53 mutations in the founding HSPC clone probably contributes to the frequent cytogenetic abnormalities and poor responses to chemotherapy that are typical of patients with t-AML/t-MDS.
Assuntos
Linhagem da Célula/genética , Genes p53/genética , Leucemia Mieloide Aguda/induzido quimicamente , Leucemia Mieloide Aguda/genética , Mutação/genética , Alelos , Animais , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células , Células Clonais , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Etilnitrosoureia/farmacologia , Evolução Molecular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Heterozigoto , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Modelos Genéticos , Mutação/efeitos dos fármacosRESUMO
Chloroethylnitrosoureas (CENUs) are bifunctional alkylating agents widely used for the clinical treatment of cancer. They exert anticancer activity by inducing DNA interstrand cross-links (ICLs) within GC base pairs to form dG-dC cross-links. This lesion inhibits DNA double strand separation during replication and transcription and results in the apoptosis of cancer cells. However, O(6)-alkylguanine DNA alkyltransferase (AGT) repairs the DNA ICLs by removing the alkyl group at the O(6) position of either O(6)-(2-chloroethyl)deoxyguanosine (O(6)-ClEtdGuo) or N1,O(6)-ethanodeoxyguanosine (N1,O(6)-EtdGuo), which are intermediates in the formation of dG-dC cross-links. The action of AGT leads to drug resistance against CENUs. O(6)-Benzylguanine (O(6)-BG) was identified as an effective AGT inhibitor that enhances the antitumor effects of CENUs. In this study, the effect of O(6)-BG on the formation of dG-dC cross-links was investigated by treating human brain glioma SF767 cells with 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosourea (ACNU). The levels of dG-dC cross-link were determined using stable isotope dilution high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results indicated that ACNU induced higher levels of dG-dC cross-link in SF767 cells pretreated with O(6)-BG compared to cells without O(6)-BG pretreatment. The highest dG-dC cross-linking levels were generally observed at 12 h for all drug concentration groups, a result which was consistent with cytotoxicity assay. These results provided direct evidence for the enhancement of dG-dC cross-linking levels caused by the inhibition of AGT by O(6)-BG. These data indicate that dG-dC cross-links may be developed as a biomarker for evaluating the activity of novel O(6)-BG analogues as AGT inhibitors for combination therapy with CENUs.
Assuntos
Alquilantes/farmacologia , Dano ao DNA , Etilnitrosoureia/farmacologia , Guanina/análogos & derivados , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , DNA , Desoxicitidina/metabolismo , Desoxiguanosina/metabolismo , Etilnitrosoureia/análogos & derivados , Glioma , Guanina/farmacologia , Humanos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
BACKGROUND: The DNA damage checkpoint signalling cascade sense damaged DNA and coordinates cell cycle arrest, DNA repair, and/or apoptosis. However, it is still not well understood how the signalling system differentiates between different kinds of DNA damage. N-nitroso-N-ethylurea (NEU), a DNA ethylating agent induces both transversions and transition mutations. METHODS: Immunoblot and comet assays were performed to detect DNA breaks and activation of the canonical checkpoint signalling kinases following NEU damage upto 2 hours. To investigate whether mismatch repair played a role in checkpoint activation, knock-down studies were performed while flow cytometry analysis was done to understand whether the activation of the checkpoint kinases was cell cycle phase specific. Finally, breast epithelial cells were grown as 3-dimensional spheroid cultures to study whether NEU can induce upregulation of vimentin as well as disrupt cell polarity of the breast acini, thus causing transformation of epithelial cells in culture. RESULTS: We report a novel finding that NEU causes activation of major checkpoint signalling kinases, Chk1 and Chk2. This activation is temporally controlled with Chk2 activation preceding Chk1 phosphorylation, and absence of cross talk between the two parallel signalling pathways, ATM and ATR. Damage caused by NEU leads to the temporal formation of both double strand and single strand breaks. Activation of checkpoints following NEU damage is cell cycle phase dependent wherein Chk2 is primarily activated during G2-M phase whilst in S phase, there is immediate Chk1 phosphorylation and delayed Chk2 response. Surprisingly, the mismatch repair system does not play a role in checkpoint activation, at doses and duration of NEU used in the experiments. Interestingly, NEU caused disruption of the well-formed polarised spheroid archithecture and upregulation of vimentin in three-dimensional breast acini cultures of non-malignant breast epithelial cells upon NEU treatment indicating NEU to have the potential to cause early transformation in the cells. CONCLUSION: NEU causes damage in mammalian cells in the form of double strand and single strand breaks that temporally activate the major checkpoint signalling kinases without the occurrence of cross-talk between the pathways. NEU also appear to cause transformation in three-dimensional spheroid cultures.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Quinase do Ponto de Checagem 2/biossíntese , Proteínas Quinases/biossíntese , Proteínas Mutadas de Ataxia Telangiectasia/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Etilnitrosoureia/farmacologia , Humanos , Fosforilação , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Natural regulatory T (nTreg) cells generated in the thymus are essential throughout life for the maintenance of T-cell homeostasis and the prevention of autoimmunity. T-cell receptor (TCR)/CD28-mediated activation of nuclear factor-κB and (J)un (N)-terminal kinase pathways is known to play a key role in nTreg cell development but many of the predicted molecular interactions are based on extrapolations from non-Treg cell TCR stimulation with non-physiological ligands. For the first time, we provide strong genetic evidence of a scaffold function for the Caspase Recruitment Domain (CARD) of the TCR signalling protein CARD-MAGUK1 (CARMA1) in nTreg cell development in vivo. We report two, new, N-ethyl-N-nitrosourea-derived mutant mice, Vulpo and Zerda, with a profound block in the development of nTreg cells in the thymus as well as impaired inducible Treg cell differentiation in the periphery. Despite independent heritage, both mutants harbour different point mutations in the CARD of the CARMA1 protein. Mutations in vulpo and zerda do not affect expression levels of CARMA1 but still impair signalling through the TCR due to defective downstream Bcl-10 recruitment by the mutated CARD of CARMA1. Phenotypic differences observed between Vulpo and Zerda mutants suggest a role for the CARD of CARMA1 independent of Bcl-10 activation of downstream pathways. We conclude that our forward genetic approach demonstrates a critical role for the CARD function of CARMA1 in Treg cell development in vivo.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Diferenciação Celular , Etilnitrosoureia/farmacologia , Mutagênicos/farmacologia , Mutação , Linfócitos T Reguladores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Autoimunidade/genética , Proteína 10 de Linfoma CCL de Células B , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Células Cultivadas , Genótipo , Hereditariedade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fenótipo , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Timócitos/imunologia , Timócitos/metabolismo , Timo/imunologia , Timo/metabolismoRESUMO
This study was carried out to investigate possible protection effect of 1-ethyl-3-[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)]-1-nitrosourea (SLENU), synthesized in our laboratory, against oxidative liver injuries induced in mice treated by antitumor drugs: doxorubicin (DOX), bleomycin (BLM), or gamma irradiation (R). Specifically, alterations in some biomarkers of oxidative stress, such as lipid peroxidation products measured as malondialdehyde (MDA) levels and activities of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), were studied in liver homogenates isolated from tumor bearing C57 black mice after i.p. treatment with solutions of DOX (60 mg/kg), BLM (60 mg/kg), or after total body gamma-irradiation with a single dose of 5 Gy. The same biomarkers were also measured after i.p. pretreatment of mice with SLENU (100 mg/kg). Statistical significant increased MDA levels and SOD and CAT enzymes activities were found in the liver homogenates of tumor bearing mice after alone treatment with DOX or gamma-irradiation compared to the control mice, while these parameters were insignificantly increased after BLM administration compared to the same controls.
Assuntos
Antineoplásicos/toxicidade , Etilnitrosoureia/análogos & derivados , Fígado/efeitos dos fármacos , Fígado/metabolismo , Alanina Transaminase/sangue , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Aspartato Aminotransferases/sangue , Bleomicina/toxicidade , Catalase/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Doxorrubicina/toxicidade , Etilnitrosoureia/química , Etilnitrosoureia/farmacologia , Raios gama/efeitos adversos , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos da radiação , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Nitrogênio/metabolismo , Marcadores de Spin , Superóxido Dismutase/metabolismoRESUMO
The pathogenesis of bone marrow failure in myelodysplastic syndromes (MDS) is an unresolved mystery. MDS causes peripheral blood cytopenias and increased bone marrow cellularity. This apparent paradox has been interpreted as a sign of intramedullary destruction of a substantial portion of the developing hematopoietic cells by apoptosis. The present study aimed to delineate the exact mechanistic relationship between the bone marrow hypercellularity and the accelerated apoptosis in an N-ethyl-N-nitrosourea (ENU)-induced experimental MDS mouse model. The observations made so far clarify the quantitative and qualitative changes that occur in the bone marrow microenvironment through cell cycle analysis, especially involving the telomerase reverse transcriptase (TERT) and p53 expression patterns. The survival fate of the bone marrow cells were observed by measuring the expression level of some intracellular protein molecules like apoptosis signal-regulating kinase 1 (ASK-1), c-Jun N-terminal kinase (JNK), and cleaved caspase-3 of the extrinsic pathway toward apoptosis. We found myelodysplasia damage occurs within one or more multipotent progenitor populations resulting in uncontrolled cellular proliferation within the MDS bone marrow. Then, due to homeostatic balance, this high cellular burden is minimized by activating the apoptosis pathway. As a result, the peripheral blood suffers cellular deprivation. This study can throw some light on the mechanism of disease progression and also help to reveal the paradoxical nature of the disease.
Assuntos
Apoptose , Células da Medula Óssea/metabolismo , Hemoglobinúria Paroxística/patologia , Síndromes Mielodisplásicas/patologia , Anemia Aplástica , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/patologia , Doenças da Medula Óssea , Transtornos da Insuficiência da Medula Óssea , Caspase 3/metabolismo , Ciclo Celular , Proliferação de Células , Sobrevivência Celular , Etilnitrosoureia/farmacologia , Feminino , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/metabolismo , Hemoglobinúria Paroxística/induzido quimicamente , Hemoglobinúria Paroxística/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase Quinase 5/biossíntese , MAP Quinase Quinase Quinase 5/metabolismo , Masculino , Camundongos , Síndromes Mielodisplásicas/induzido quimicamente , Síndromes Mielodisplásicas/metabolismo , Telomerase/biossíntese , Telomerase/metabolismo , Encurtamento do Telômero , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Receptor fas/biossíntese , Receptor fas/metabolismoRESUMO
Cellular stress responses consist of a complex network of pathways and linked processes that, when perturbed, are postulated to have roles in the pathogenesis of various human diseases. To assess the impact of environmental insults upon this network, we developed a novel stress response resolution (SRR) assay for investigation of cellular stress resolution outcomes and the effects of environmental agents and conditions thereupon. SRR assay-based criteria identified three distinct groups of surviving cell clones, including those resembling parental cells, those showing Hprt/HPRT mutations, and a third type, "Phenotype-altered" clones, that occurred predominantly in cells pretreated with a chemical mutagen, was heterogeneous in nature, and expressed significant alterations in cell morphology and/or function compared with parental cells. Further evaluation of Phenotype-altered clones found evidence of various alterations that resembled epithelial-to-mesenchymal transition, phenotype switching, checkpoint dysfunction, senescence barrier bypass, and/or epigenetic reprogramming. Phenotype-altered clones were found to occur spontaneously in a cell line with a mutator phenotype, to represent the major surviving clone type in a variation of the SRR assay, and to be tumorigenic in nude mice. Assessment of SRR assay final results showed that pretreatment with a chemical mutagen induced significant changes in cellular stress response prosurvival capacity, in damage avoidance versus damage tolerance stress resolution outcomes, and in the damage burden in the final surviving cell populations. Taken together, these results support the conclusion that use of the SRR assay can provide novel insights into the role of environmental insults in the pathogenesis of cancer and other human diseases.
Assuntos
Células Epiteliais/efeitos dos fármacos , Mutagênicos/toxicidade , Estresse Fisiológico/efeitos dos fármacos , Anfotericina B/farmacologia , Anfotericina B/toxicidade , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Relação Dose-Resposta a Droga , Células Epiteliais/fisiologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Etilnitrosoureia/farmacologia , Etilnitrosoureia/toxicidade , Nucleotídeos de Guanina/farmacologia , Nucleotídeos de Guanina/toxicidade , Humanos , Hipoxantina Fosforribosiltransferase/genética , Lamivudina/farmacologia , Lamivudina/toxicidade , Camundongos , Mutagênicos/farmacologia , Mutação/efeitos dos fármacos , Fenótipo , Tionucleotídeos/farmacologia , Tionucleotídeos/toxicidade , Testes de Toxicidade , Zidovudina/farmacologia , Zidovudina/toxicidadeRESUMO
The events or factors that lead from normal cell function to conditions and diseases such as aging or cancer reflect complex interactions between cells and their environment. Cellular stress responses, a group of processes involved in homeostasis and adaptation to environmental change, contribute to cell survival under stress and can be resolved with damage avoidance or damage tolerance outcomes. To investigate the impact of environmental agents/conditions upon cellular stress response outcomes in epithelium, a novel quantitative assay, the "stress response resolution" (SRR) assay, was developed. The SRR assay consists of pretreatment with a test agent or vehicle followed later by a calibrated stress conditions exposure step (here, using 6-thioguanine). Pilot studies conducted with a spontaneously-immortalized murine mammary epithelial cell line pretreated with vehicle or 20 µg N-ethyl-N-nitrososurea/ml medium for 1 hr, or two hTERT-immortalized human bronchial epithelial cell lines pretreated with vehicle or 100 µM zidovudine/lamivudine for 12 days, found minimal alterations in cell morphology, survival, or cell function through 2 weeks post-exposure. However, when these pretreatments were followed 2 weeks later by exposure to calibrated stress conditions of limited duration (for 4 days), significant alterations in stress resolution were observed in pretreated cells compared with vehicle-treated control cells, with decreased damage avoidance survival outcomes in all cell lines and increased damage tolerance outcomes in two of three cell lines. These pilot study results suggest that sub-cytotoxic pretreatments with chemical mutagens have long-term adverse impact upon the ability of cells to resolve subsequent exposure to environmental stressors.
Assuntos
Células Epiteliais/efeitos dos fármacos , Mutagênicos/toxicidade , Estresse Fisiológico/efeitos dos fármacos , Testes de Toxicidade , Anfotericina B/farmacologia , Anfotericina B/toxicidade , Animais , Calibragem , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Exposição Ambiental , Poluentes Ambientais/farmacologia , Poluentes Ambientais/toxicidade , Células Epiteliais/fisiologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Etilnitrosoureia/farmacologia , Etilnitrosoureia/toxicidade , Nucleotídeos de Guanina/farmacologia , Nucleotídeos de Guanina/toxicidade , Humanos , Lamivudina/farmacologia , Lamivudina/toxicidade , Camundongos , Mutagênicos/farmacologia , Tionucleotídeos/farmacologia , Tionucleotídeos/toxicidade , Zidovudina/farmacologia , Zidovudina/toxicidadeRESUMO
Gliomas are the most common primary brain tumors in adults and have a poor prognosis. Galectin-3 is a ß-galactosidase-binding lectin which is important in pre-mRNA splicing, regulation of cell proliferation, cell adhesion and apoptosis. Although galectin-3 has been shown as a glioma related marker and expression of galectin-3 has been reported to correlate with WHO grade in human gliomas, expression of galectin-3 in early neoplastic lesions such as early neoplastic proliferation (ENP) and microtumor is still far from fully understood. In the present study, expression of galectin-3 in ethylnitrosourea-induced rat gliomas including preneoplastic and neoplastic lesions was examined by immunohistochemistry for galectin-3, Iba-1 (a specific microglial cell marker), GFAP (a specific astrocyte cell marker), and conventional hematoxylin and eosin staining (for morphological observation). The results showed that exact location of ENP was detected clearly by galectin-3 immunohistochemistry whereas normal brain tissues were negative. In ENP and microtumor, galectin-3 was expressed in neoplastic astrocytic cells but rarely in microglia. In malignant glioma, however, galectin-3 was expressed in both neoplastic astrocytic cells and microglia. This suggests that galectin-3 is activated in microglia and macrophages according to the progression of glioma. Galectin-3 was not expressed in oligodendrocytic cells. Our results indicate that galectin-3 is a good specific marker indicating the early stage of glioma tumorigenesis.
Assuntos
Neoplasias Encefálicas/complicações , Galectina 3/metabolismo , Glioma/complicações , Lesões Pré-Cancerosas/etiologia , Lesões Pré-Cancerosas/metabolismo , Alquilantes/farmacologia , Animais , Neoplasias Encefálicas/induzido quimicamente , Neoplasias Encefálicas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Etilnitrosoureia/farmacologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Glioma/induzido quimicamente , Glioma/metabolismo , Humanos , Proteínas dos Microfilamentos/metabolismo , Ratos , Ratos WistarRESUMO
Very low doses of ionizing radiation, 5 to 100 mGy, can induce adaptive responses characterized by elevation in cell survival and reduction in micronuclei formation. Utilizing these end points, RKO human colon carcinoma and transformed mouse embryo fibroblasts (MEF), wild-type or knockout cells missing TNF receptors 1 and 2 (TNFR1(-)R2(-)), and C57BL/6 and TNFR1(-)R2(-) knockout mice, we demonstrate that intact TNF signaling is required for induction of elevated manganese superoxide dismutase (SOD2) activity (P < 0.001) and the subsequent expression of these SOD2-mediated adaptive responses when cells are challenged at a later time with 2 Gy. In contrast, amifostine's free thiol form WR1065 can directly activate NF-κB giving rise to elevated SOD2 activity 24 h later and induce an adaptive response in both MEF wild-type and TNF signaling defective TNFR1(-)R2(-) cells. Transfection of cells with SOD2 siRNA completely abolishes both the elevation in SOD2 activity and expression of the adaptive responses. These results were confirmed in vivo using a micronucleus assay in splenocytes derived from C57BL/6 and TNFR1(-)R2(-) knockout mice that were exposed to 100 mGy or 400 mg/kg amifostine 24 h prior to exposure to a 2 Gy whole-body dose. A dose of 100 mGy also conferred enhanced protection to C57BL/6 mice exposed 24 h later to 100 mg/kg of N-Ethyl-N-nitrosourea (ENU). While very low radiation doses require an intact TNF signaling process to induce a SOD2-mediated adaptive response, amifostine can induce a similar adaptive response in both TNF receptor competent and knockout cells, respectively.
Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/efeitos da radiação , Superóxido Dismutase/metabolismo , Alquilantes/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Relação Dose-Resposta à Radiação , Etilnitrosoureia/farmacologia , Feminino , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/efeitos da radiação , Humanos , Mercaptoetilaminas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiaçãoRESUMO
BACKGROUND: Understanding the early relationship between brain tumor cells and their environment could lead to more sensitive biomarkers and new therapeutic strategies. We have been using a rodent model of neurocarcinogenesis in which all animals develop brain tumors by six months of age to establish two early landmarks in glioma development: the appearance of a nestin(+) cell at thirty days of age and the appearance of cellular hyperplasia between 60 and 120 days of age. We now report an assessment of the CSF proteome to determine the changes in protein composition that occur during this period. MATERIALS AND METHODS: Nestin(+) cell clusters and microtumors were assessed in 63 ethylnitrosourea-exposed rats on 30, 60, and 90 days of age. CSF was obtained from the cisterna magna from 101 exposed and control rats at 30, 60, and 90 days and then analyzed using mass spectrometry. Differentially expressed peaks were isolated and identified. RESULTS: Nestin(+) cells were noted in all ethylnitrosourea-exposed rats assessed pathologically. Small microtumors were noted in 0%, 18%, and 67% of 30-, 60-, and 90-day old rats, respectively (p<0.05, Chi square). False Discovery Rate analysis of peak intensities showed that the number of true discoveries with p<0.05 increased markedly with increasing age. Isolation and identification of highly differentially detected proteins at 90 days of age revealed increases in albumin and a fragment of α1 macroglobulin and alterations in glutathionylated transthyretin. CONCLUSIONS: The presence of increased albumin, fragments of cerebrospinal fluid proteins, and glutathione breakdown in temporal association with the development of cellular hyperplasia, suggests that, similar to many other systemic cancers, inflammation and oxidative stress is playing an important early role in the host's response to brain tumor development and may be involved in affecting the early growth of brain tumor.
Assuntos
Neoplasias Encefálicas/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioma/líquido cefalorraquidiano , Proteínas de Filamentos Intermediários/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Animais , Biomarcadores/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/metabolismo , Modelos Animais de Doenças , Etilnitrosoureia/farmacologia , Glioma/metabolismo , Glutationa/metabolismo , Nestina , Proteoma , Proteômica/métodos , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Copper influences the pathogenesis of prion disease, but whether it is beneficial or detrimental remains controversial. Copper homeostasis is also essential for normal physiology, as highlighted by the spectrum of diseases caused by disruption of the copper transporting enzymes ATP7A and ATP7B. Here, by using a forward genetics approach in mice, we describe the isolation of three alleles of Atp7a, each with different phenotypic consequences. The mildest of the three, Atp7a(brown), was insufficient to cause lethality in hemizygotes or mottling of the coat in heterozygotes, but did lead to coat hypopigmentation and reduced copper content in the brains of hemizygous males. When challenged with Rocky Mountain Laboratory scrapie, the onset of prion disease was delayed in Atp7a(brown) mice, and significantly less proteinase-resistant prion protein was found in the brains of moribund Atp7a(brown) mice compared with WT littermates. Our results establish that ATP7A-mediated copper homeostasis is important for the formation of pathogenic proteinase-resistant prion protein.