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1.
Anticancer Res ; 39(12): 6547-6553, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31810920

RESUMO

AIM: To evaluate the frequency of loss of mediator of DNA damage checkpoint protein 1 (MDC1) protein expression in endometrial cancer (EC) and to determine whether loss of MDC1 is associated with certain clinicopathological parameters. MATERIALS AND METHODS: MDC1 expression was examined in 426 samples of EC. The nuclear immunoreactivity of the protein was defined as: negative, weak, moderate and strong. RESULTS: Loss of MDC1 expression (defined as negative nuclear staining) was found in 8.9% (38/426) of ECs and was significantly associated with the loss of MRE11 homolog, double-strand break repair nuclease, RAD50 double-strand break repair protein and nibrin complex components. In addition, loss of expression of MDC1 showed a significant correlation with any mismatch repair deficiency, with endometrioid histological subtype and low tumour grading. CONCLUSION: Based on these findings, we suggest that MDC1 loss frequently occurs in ECs with microsatellite instability. Due to deficient homologous recombination DNA repair, MDC1-negative ECs might show an increased sensitivity to poly(ADP-ribose) polymerase-inhibitory therapy.


Assuntos
Proteínas de Ciclo Celular/deficiência , Enzimas Reparadoras do DNA/deficiência , Proteínas de Ligação a DNA/deficiência , Neoplasias do Endométrio/metabolismo , Proteína Homóloga a MRE11/deficiência , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Reparo de Erro de Pareamento de DNA , Neoplasias do Endométrio/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Instabilidade de Microssatélites , Estadiamento de Neoplasias , Análise de Sobrevida , Análise Serial de Tecidos
2.
Gynecol Oncol ; 155(3): 489-498, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31604667

RESUMO

OBJECTIVE: Ovarian clear cell carcinoma (OCCC) is often resistant to conventional, standard chemotherapy using cytotoxic drugs. OCCC harbors a unique genomic feature of frequent (approximately 50%) ARID1A deficiency. The present study was performed to investigate standard chemotherapeutic options suitable for ARID1A-deficient OCCC patients. METHODS: Drugs with selective toxicity to ARID1A-deficient OCCC cells were identified among six cytotoxic drugs used in standard chemotherapy for OCCC by employing multiple ARID1A-knockout cell lines and an OCCC cell line panel. Anti-tumor effects of drug treatment were assessed using a xenograft model. To obtain proof of concept in patients, seven OCCC patients who received single-agent therapy with gemcitabine were identified in a retrospective cohort of 149 OCCC patients. Patient samples and cases were analyzed for association between therapeutic response and ARID1A deficiency. RESULTS: ARID1A-knockout and ARID1A-deficient OCCC cells had selective sensitivity to gemcitabine. IC50 values for gemcitabine of ARID1A-deficient cells were significantly lower than those of ARID1A-proficient cells (p = 0.0001). Growth of OCCC xenografts with ARID1A deficiency was inhibited by administration of gemcitabine, and gemcitabine treatment effectively induced apoptosis in ARID1A-deficient OCCC cells. Three ARID1A-deficient OCCC patients had significantly longer progression-free survival after gemcitabine treatment than four ARID1A-proficient OCCC patients (p = 0.02). An ARID1A-deficient case that was resistant to multiple cytotoxic drugs, including paclitaxel plus carboplatin in the adjuvant and etoposide plus irinotecan in the first-line treatment, exhibited a dramatic response to gemcitabine in the second-line treatment. CONCLUSION: ARID1A-deficient OCCC patients could benefit from gemcitabine treatment in clinical settings.


Assuntos
Adenocarcinoma de Células Claras/tratamento farmacológico , Desoxicitidina/análogos & derivados , Proteínas Nucleares/deficiência , Neoplasias Ovarianas/tratamento farmacológico , Fatores de Transcrição/deficiência , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/metabolismo , Adenocarcinoma de Células Claras/patologia , Adulto , Idoso , Animais , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacologia , Feminino , Técnicas de Inativação de Genes , Células HCT116 , Células HEK293 , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Distribuição Aleatória , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Acta Cytol ; 63(5): 438-444, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31230044

RESUMO

OBJECTIVE: Evidence shows that the switch/sucrose nonfermenting chromatin remodeling complex plays a critical role in DNA repair, cancer progression and dedifferentiation. BRG1 is one of its key catalytic subunits. While the loss of BRG1 expression by immunocytochemistry has been identified in a subset of malignancies arising in various sites with undifferentiated/rhabdoid morphology and poor prognosis, the underlying basis for its loss is unclear. METHODS: A retrospective search was conducted in our cytopathology archive for undifferentiated malignant tumors with rhabdoid phenotype and BRG1 loss. Clinical information was obtained from electronic medical records. Next-generation sequencing was performed following macro-dissection of paraffin-embedded cellblock tissue. RESULTS: Three cases were identified; all presented with widely metastatic disease with no previously diagnosed primary malignancy, and subsequently died within 6 months of initial presentation. Cytologically, the aspirates showed dyshesive and undifferentiated cells with rhabdoid features. Extensive immunocytochemical workup demonstrated immunoreactivity with vimentin only and could not establish a specific lineage. BRG1 expression was absent, while INI1 expression was retained. Two cases harbored deleterious mutations in BRG1/SMARCA4. Pathogenic mutations in TP53 were identified in all tumors. CONCLUSIONS: BRG1 deficiency reflects underlying mutation in SMARCA4 gene in some but not all cases, suggesting that additional mechanisms may be causing BRG1 silencing. Pathogenic mutations in TP53 in all tumors are consistent with their highly aggressive nature. Recognizing the cytomorphology of this group of neoplasms and confirming their BRG1-deficient status by immunocytochemistry not only has prognostic implications, but may also impart potentially therapeutic value in the near future.


Assuntos
Biomarcadores Tumorais/genética , Diferenciação Celular , DNA Helicases/genética , Neoplasias Pulmonares/genética , Mutação , Proteínas Nucleares/genética , Tumor Rabdoide/genética , Neoplasias da Glândula Submandibular/genética , Fatores de Transcrição/genética , Idoso , Biomarcadores Tumorais/deficiência , Biópsia por Agulha Fina , DNA Helicases/deficiência , Análise Mutacional de DNA , Evolução Fatal , Feminino , Predisposição Genética para Doença , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/deficiência , Fenótipo , Valor Preditivo dos Testes , Estudos Retrospectivos , Tumor Rabdoide/enzimologia , Tumor Rabdoide/patologia , Tumor Rabdoide/terapia , Neoplasias da Glândula Submandibular/enzimologia , Neoplasias da Glândula Submandibular/patologia , Neoplasias da Glândula Submandibular/terapia , Fatores de Transcrição/deficiência , Resultado do Tratamento , Proteína Supressora de Tumor p53/genética
4.
Eur J Pharmacol ; 854: 398-405, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31039344

RESUMO

Hemoglobinopathies, such as ß-thalassemia, and sickle cell disease (SCD) are caused by abnormal structure or reduced production of ß-chains and affect millions of people worldwide. Hereditary persistence of fetal hemoglobin (HPFH) is a condition which is naturally occurring and characterized by a considerable elevation of fetal hemoglobin (HbF) in adult red blood cells. Individuals with compound heterozygous ß-thalassemia or SCD and HPFH have milder clinical symptoms. So, HbF reactivation has long been sought as an approach to mitigate the clinical symptoms of ß-thalassemia and SCD. Using CRISPR-Cas9 genome-editing strategy, we deleted a 200bp genomic region within the human erythroid-specific BCL11A (B-cell lymphoma/leukemia 11A) enhancer in KU-812, KG-1, and K562 cell lines. In our study, deletion of 200bp of BCL11A erythroid enhancer including GATAA motif leads to strong induction of γ-hemoglobin expression in K562 cells, but not in KU-812 and KG-1 cells. Altogether, our findings highlight the therapeutic potential of CRISPR-Cas9 as a precision genome editing tool for treating ß-thalassemia. In addition, our data indicate that KU-812 and KG-1 cell lines are not good models for studying HbF reactivation through inactivation of BCL11A silencing pathway.


Assuntos
Sistemas CRISPR-Cas/genética , Proteínas de Transporte/genética , Hemoglobina Fetal/metabolismo , Deleção de Genes , Terapia Genética/métodos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Talassemia beta/terapia , Sequência de Bases , Edição de Genes , Humanos , Células K562 , Proteínas Repressoras , Talassemia beta/genética , Talassemia beta/metabolismo , gama-Globinas/genética
5.
Radiat Res ; 192(2): 135-144, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31141469

RESUMO

Radiotherapy for head and neck cancers can result in extensive damage to the salivary glands, significantly affecting patient quality of life. However, the salivary gland can recover in patients receiving lower doses of radiation. In addition, there is considerable interest in delineating the mechanisms by which stem cells survive radiation exposure and promote tissue regeneration. In this study, we isolated stable radioresistant acinar progenitor cells from the submaxillary gland of the Sprague Dawley rat. Progenitor cells are characterized as c-Kithigh/alpha-amylase+ and are resistant to X rays (≤5 Gy).We further isolated a radiosensitive acinar counterpart, characterized as c-Kitlow/alpha-amylase+, which is effectively killed by exposure to 2 Gy X ray of radiation. Phosphopeptides with homology to the treacle protein (TCOF1) were disproportionately increased in progenitor cells, compared to their radiosensitive counterparts. Silencing of TCOF1 expression (shRNA) radiosensitized progenitor cells, a response conserved in human cells with TCOF1 knockdown. Collectively, these observations indicate that radiation resistance is an intrinsic property of c-Kithigh salivary gland progenitor cells. Since human salivary gland stem cells with c-Kit expression are believed to have enhanced regenerative potencies, our model system provides a stable platform to investigate molecular features associated with c-Kit expression that may contribute to protection or stabilization of the stem cell niche.


Assuntos
Células Acinares/citologia , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Tolerância a Radiação , Células-Tronco/efeitos da radiação , Animais , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Dano ao DNA , Relação Dose-Resposta à Radiação , Técnicas de Silenciamento de Genes , Humanos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fenótipo , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Transporte Proteico/efeitos da radiação , Ratos , Ratos Sprague-Dawley , Células-Tronco/citologia , Células-Tronco/metabolismo
6.
Nucleic Acids Res ; 47(8): 4086-4110, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30986824

RESUMO

Ataxia with oculomotor apraxia type 1 (AOA1) is an early onset progressive spinocerebellar ataxia caused by mutation in aprataxin (APTX). APTX removes 5'-AMP groups from DNA, a product of abortive ligation during DNA repair and replication. APTX deficiency has been suggested to compromise mitochondrial function; however, a detailed characterization of mitochondrial homeostasis in APTX-deficient cells is not available. Here, we show that cells lacking APTX undergo mitochondrial stress and display significant changes in the expression of the mitochondrial inner membrane fusion protein optic atrophy type 1, and components of the oxidative phosphorylation complexes. At the cellular level, APTX deficiency impairs mitochondrial morphology and network formation, and autophagic removal of damaged mitochondria by mitophagy. Thus, our results show that aberrant mitochondrial function is a key component of AOA1 pathology. This work corroborates the emerging evidence that impaired mitochondrial function is a characteristic of an increasing number of genetically diverse neurodegenerative disorders.


Assuntos
Proteínas de Ligação a DNA/genética , GTP Fosfo-Hidrolases/genética , Mitocôndrias/genética , Proteínas Nucleares/genética , Ataxias Espinocerebelares/congênito , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/deficiência , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , GTP Fosfo-Hidrolases/deficiência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Homeostase/genética , Humanos , Linfócitos/metabolismo , Linfócitos/patologia , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Nucleares/deficiência , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação Oxidativa , Transdução de Sinais , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologia
7.
Eur J Pharmacol ; 852: 189-197, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-30876979

RESUMO

The bromodomain and extra-terminal domain (BET) protein BRD4 is emerging as a potential target for cancer therapy. However, BRD4 roles in regulating the stemness of gastric cancer cells are unclear. Here, we demonstrated that BRD4 expression was significantly increased in gastric cancer tissues, cell spheroids, and BRD4 knockdown attenuated the stemness of gastric cancer cells characterized as the decrease of stemness markers expression, capacity of cells spheroids formation and ALDH1 activity. Importantly, BRD4 expression was negatively correlated with overall survival, first progression survival and post progression survival of gastric cancer patients. Mechanistic investigations revealed that miR-216a-3p was the most remarkably upregulated miRNA in response to BRD4 knockdown and Wnt/ß-catenin signaling was necessary for BRD4-mediated promotion on the stemness of gastric cancer cells. Additionally, BRD4 directly bound to the promoter and promoted the methylation level of MIR216A promoter, thus decreasing miR-216a-3p level. Notably, Wnt3a was identified as the direct target of miR-216a-3p in gastric cancer cells. Therefore, our results defined a BRD4/miR-216a-3p/Wnt/ß-catenin pathway in regulating the stemness of gastric cancer cells.


Assuntos
MicroRNAs/genética , Células-Tronco Neoplásicas/patologia , Proteínas Nucleares/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt/genética , Sequência de Bases , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Progressão da Doença , Técnicas de Silenciamento de Genes , Humanos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
8.
PLoS One ; 14(3): e0213383, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30840704

RESUMO

Replicative DNA polymerases are frequently stalled at damaged template strands. Stalled replication forks are restored by the DNA damage tolerance (DDT) pathways, error-prone translesion DNA synthesis (TLS) to cope with excessive DNA damage, and error-free template switching (TS) by homologous DNA recombination. PDIP38 (Pol-delta interacting protein of 38 kDa), also called Pol δ-interacting protein 2 (PolDIP2), physically associates with TLS DNA polymerases, polymerase η (Polη), Polλ, and PrimPol, and activates them in vitro. It remains unclear whether PDIP38 promotes TLS in vivo, since no method allows for measuring individual TLS events in mammalian cells. We disrupted the PDIP38 gene, generating PDIP38-/- cells from the chicken DT40 and human TK6 B cell lines. These PDIP38-/- cells did not show a significant sensitivity to either UV or H2O2, a phenotype not seen in any TLS-polymerase-deficient DT40 or TK6 mutants. DT40 provides a unique opportunity of examining individual TLS and TS events by the nucleotide sequence analysis of the immunoglobulin variable (Ig V) gene as the cells continuously diversify Ig V by TLS (non-templated Ig V hypermutation) and TS (Ig gene conversion) during in vitro culture. PDIP38-/- cells showed a shift in Ig V diversification from TLS to TS. We measured the relative usage of TLS and TS in TK6 cells at a chemically synthesized UV damage (CPD) integrated into genomic DNA. The loss of PDIP38 also caused an increase in the relative usage of TS. The number of UV-induced sister chromatid exchanges, TS events associated with crossover, was increased a few times in PDIP38-/- human and chicken cells. Collectively, the loss of PDIP38 consistently causes a shift in DDT from TLS to TS without enhancing cellular sensitivity to DNA damage. We propose that PDIP38 controls the relative usage of TLS and TS increasing usage of TLS without changing the overall capability of DDT.


Assuntos
Dano ao DNA , Proteínas Nucleares/metabolismo , Animais , Proteínas Aviárias/deficiência , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Linhagem Celular , Galinhas , DNA/biossíntese , DNA/genética , DNA Polimerase beta/deficiência , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , DNA Primase/deficiência , DNA Primase/genética , DNA Primase/metabolismo , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/deficiência , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Técnicas de Inativação de Genes , Genes de Imunoglobulinas , Humanos , Enzimas Multifuncionais/deficiência , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Moldes Genéticos
9.
Cells ; 8(3)2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30871242

RESUMO

Emerin is an essential LEM (LAP2, Emerin, MAN1) domain protein in metazoans and an integral membrane protein associated with inner and outer nuclear membranes. Mutations in the human EMD gene coding for emerin result in the rare genetic disorder: Emery⁻Dreifuss muscular dystrophy type 1 (EDMD1). This disease belongs to a broader group called laminopathies-a heterogeneous group of rare genetic disorders affecting tissues of mesodermal origin. EDMD1 phenotype is characterized by progressive muscle wasting, contractures of the elbow and Achilles tendons, and cardiac conduction defects. Emerin is involved in many cellular and intranuclear processes through interactions with several partners: lamins; barrier-to-autointegration factor (BAF), ß-catenin, actin, and tubulin. Our study demonstrates the presence of the emerin fraction which associates with mitotic spindle microtubules and centrosomes during mitosis and colocalizes during early mitosis with lamin A/C, BAF, and membranes at the mitotic spindle. Transfection studies with cells expressing EGFP-emerin protein demonstrate that the emerin fusion protein fraction also localizes to centrosomes and mitotic spindle microtubules during mitosis. Transient expression of emerin deletion mutants revealed that the resulting phenotypes vary and are mutant dependent. The most frequent phenotypes include aberrant nuclear shape, tubulin network mislocalization, aberrant mitosis, and mislocalization of centrosomes. Emerin deletion mutants demonstrated different chromatin binding capacities in an in vitro nuclear assembly assay and chromatin-binding properties correlated with the strength of phenotypic alteration in transfected cells. Aberrant tubulin staining and microtubule network phenotype appearance depended on the presence of the tubulin binding region in the expressed deletion mutants. We believe that the association with tubulin might help to "deliver" emerin and associated membranes to decondensing chromatin. Preliminary analyses of cells from Polish patients with EDMD1 revealed that for several mutations thought to be null for emerin protein, a truncated emerin protein was present. We infer that the EDMD1 phenotype may be strengthened by the toxicity of truncated emerin expressed in patients with certain nonsense mutations in EMD.


Assuntos
Proteínas de Membrana/metabolismo , Mitose , Distrofia Muscular de Emery-Dreifuss/patologia , Lâmina Nuclear/patologia , Proteínas Nucleares/metabolismo , Anticorpos/metabolismo , Ciclo Celular , Centrossomo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Epitopos/metabolismo , Deleção de Genes , Células HeLa , Humanos , Lamina Tipo A/metabolismo , Proteínas de Membrana/deficiência , Microtúbulos/metabolismo , Proteínas Nucleares/deficiência , Fenótipo , Ligação Proteica , Fuso Acromático/metabolismo , Tubulina (Proteína)/metabolismo
10.
J Biol Chem ; 294(14): 5700-5719, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30733337

RESUMO

The tumor suppressor bridging integrator 1 (BIN1) is a corepressor of the transcription factor E2F1 and inhibits cell-cycle progression. BIN1 also curbs cellular poly(ADP-ribosyl)ation (PARylation) and increases sensitivity of cancer cells to DNA-damaging therapeutic agents such as cisplatin. However, how BIN1 deficiency, a hallmark of advanced cancer cells, increases cisplatin resistance remains elusive. Here, we report that BIN1 inactivates ataxia telangiectasia-mutated (ATM) serine/threonine kinase, particularly when BIN1 binds E2F1. BIN1 + 12A (a cancer-associated BIN1 splicing variant) also inhibited cellular PARylation, but only BIN1 increased cisplatin sensitivity. BIN1 prevented E2F1 from transcriptionally activating the human ATM promoter, whereas BIN1 + 12A did not physically interact with E2F1. Conversely, BIN1 loss significantly increased E2F1-dependent formation of MRE11A/RAD50/NBS1 DNA end-binding protein complex and efficiently promoted ATM autophosphorylation. Even in the absence of dsDNA breaks (DSBs), BIN1 loss promoted ATM-dependent phosphorylation of histone H2A family member X (forming γH2AX, a DSB biomarker) and mediator of DNA damage checkpoint 1 (MDC1, a γH2AX-binding adaptor protein for DSB repair). Of note, even in the presence of transcriptionally active (i.e. proapoptotic) TP53 tumor suppressor, BIN1 loss generally increased cisplatin resistance, which was conversely alleviated by ATM inactivation or E2F1 reduction. However, E2F2 or E2F3 depletion did not recapitulate the cisplatin sensitivity elicited by E2F1 elimination. Our study unveils an E2F1-specific signaling circuit that constitutively activates ATM and provokes cisplatin resistance in BIN1-deficient cancer cells and further reveals that γH2AX emergence may not always reflect DSBs if BIN1 is absent.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Fator de Transcrição E2F1/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/deficiência , Transcrição Genética , Proteínas Supressoras de Tumor/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Reparo do DNA/efeitos dos fármacos , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição E2F1/genética , Histonas/genética , Histonas/metabolismo , Humanos , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Neoplasias/genética , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos
11.
PLoS One ; 14(2): e0211244, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30807587

RESUMO

Expulsion of parasitic gastrointestinal nematodes requires diverse effector mechanisms coordinated by a Th2-type response. The evolutionarily conserved JmjC protein; Myc Induced Nuclear Antigen (Mina) has been shown to repress IL4, a key Th2 cytokine, suggesting Mina may negatively regulate nematode expulsion. Here we report that expulsion of the parasitic nematode Trichuris muris was indeed accelerated in Mina deficient mice. Unexpectedly, this was associated not with an elevated Th2- but rather an impaired Th1-type response. Further reciprocal bone marrow chimera and conditional KO experiments demonstrated that retarded parasite expulsion and a normal Th1-type response both required Mina in intestinal epithelial cells (IECs). Transcriptional profiling experiments in IECs revealed anti-microbial α-defensin peptides to be the major target of Mina-dependent retention of worms in infected mice. In vitro exposure to recombinant α-defensin peptides caused cytotoxic damage to whipworms. These results identify a latent IEC-intrinsic anthelmintic pathway actively constrained by Mina and point to α-defensins as important effectors that together with Mina may be attractive therapeutic targets for the control of nematode infection.


Assuntos
Células Epiteliais/metabolismo , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Trichuris/imunologia , Animais , Citocinas/análise , Células Epiteliais/citologia , Intestinos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Neoplasias/deficiência , Proteínas Nucleares/deficiência , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/citologia , Células Th2/imunologia , Células Th2/metabolismo , Transcriptoma , Tricuríase/tratamento farmacológico , Tricuríase/imunologia , Tricuríase/patologia , Trichuris/efeitos dos fármacos , Trichuris/patogenicidade , alfa-Defensinas/genética , alfa-Defensinas/metabolismo
12.
Cancer Cell ; 35(2): 177-190.e8, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30686770

RESUMO

ARID1A encodes an SWI/SNF chromatin-remodeling factor and is frequently mutated in various cancers. This study demonstrates that ARID1A-deficient cancer cells are specifically vulnerable to inhibition of the antioxidant glutathione (GSH) and the glutamate-cysteine ligase synthetase catalytic subunit (GCLC), a rate-limiting enzyme for GSH synthesis. Inhibition of GCLC markedly decreased GSH in ARID1A-deficient cancer cells, leading to apoptotic cell death triggered by excessive amounts of reactive oxygen species. The vulnerability of ARID1A-deficient cancer cells results from low basal levels of GSH due to impaired expression of SLC7A11. The SLC7A11-encoded cystine transporter supplies cells with cysteine, a key source of GSH, and its expression is enhanced by ARID1A-mediated chromatin remodeling. Thus, ARID1A-deficient cancers are susceptible to synthetic lethal targeting of GCLC.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glutamato-Cisteína Ligase/antagonistas & inibidores , Glutationa/metabolismo , Proteínas Nucleares/deficiência , Neoplasias Ovarianas/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Quinuclidinas/farmacologia , Fatores de Transcrição/deficiência , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Feminino , Glutamato-Cisteína Ligase/metabolismo , Células HCT116 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia de Alvo Molecular , Proteínas Nucleares/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Clin Sci (Lond) ; 133(2): 321-334, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30622219

RESUMO

Acute respiratory distress syndrome (ARDS) in a deadly disease that can be brought on by endotoxins such as lipopolysaccharide (LPS). ARDS is characterized by vascular permeability, a severe inflammatory response, lung leukocyte infiltration, and resultant lung edema. Polymerase δ-interacting protein 2 (Poldip2) is a novel regulator of blood-brain barrier permeability; however, its role in regulating lung permeability and vascular inflammation is unknown. Here, the role of Poldip2 in regulating vascular permeability and inflammation in a mouse model of ARDS was assessed. Heterozygous deletion of Poldip2 was found to reduce LPS-induced mortality within 20 h, lung inflammatory signaling, and leukocyte infiltration. Moreover, reduced Poldip2-suppressed LP-induced vascular cell adhesion molecule (VCAM)-1 induction, leukocyte recruitment, and mitochondrial reactive oxygen species (ROS) production in vitro These data indicate that Poldip2 is an important regulator of the debilitating consequences of ARDS, potentially through the regulation of mitochondrial ROS-induced inflammatory signaling. Consequently, inhibition of Poldip2 may be a viable option for therapeutic discovery moving forward.


Assuntos
Permeabilidade Capilar , Células Endoteliais/metabolismo , Pulmão/irrigação sanguínea , Proteínas Mitocondriais/deficiência , Proteínas Nucleares/deficiência , Edema Pulmonar/prevenção & controle , Síndrome do Desconforto Respiratório do Adulto/metabolismo , Vasculite/prevenção & controle , Animais , Adesão Celular , Técnicas de Cocultura , Citocinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/patologia , Feminino , Humanos , Leucócitos/metabolismo , Leucócitos/patologia , Masculino , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Edema Pulmonar/genética , Edema Pulmonar/metabolismo , Edema Pulmonar/patologia , Espécies Reativas de Oxigênio/metabolismo , Síndrome do Desconforto Respiratório do Adulto/genética , Síndrome do Desconforto Respiratório do Adulto/patologia , Transdução de Sinais , Células THP-1 , Molécula 1 de Adesão de Célula Vascular/metabolismo , Vasculite/genética , Vasculite/metabolismo , Vasculite/patologia
14.
DNA Repair (Amst) ; 74: 70-79, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30606609

RESUMO

DSBs are harmful lesions produced through endogenous metabolism or by exogenous agents such as ionizing radiation, that can trigger genomic rearrangements. We have recently shown that exposure to 2 Gy of X-rays has opposite effects on the induction of Shh-dependent MB in NHEJ- and HR-deficient Ptch1+/- mice. In the current study we provide a comprehensive link on the role of HR/NHEJ at low doses (0.042 and 0.25 Gy) from the early molecular changes through DNA damage processing, up to the late consequences of their inactivation on tumorigenesis. Our data indicate a prominent role for HR in genome stability, by preventing spontaneous and radiation-induced oncogenic damage in neural precursors of the cerebellum, the cell of origin of MB. Instead, loss of DNA-PKcs function increased DSBs and apoptosis in neural precursors of the developing cerebellum, leading to killing of tumor initiating cells, and suppression of MB tumorigenesis in DNA-PKcs-/-/Ptch1+/- mice. Pathway analysis demonstrates that DNA-PKcs genetic inactivation confers a remarkable radiation hypersensitivity, as even extremely low radiation doses may deregulate many DDR genes, also triggering p53 pathway activation and cell cycle arrest. Finally, by showing that DNA-PKcs inhibition by NU7441 radiosensitizes human MB cells, our in vitro findings suggest the inclusion of MB in the list of tumors beneficiating from the combination of radiotherapy and DNA-PKcs targeting, holding promise for clinical translation.


Assuntos
Neoplasias Cerebelares/genética , Reparo do DNA/efeitos da radiação , Meduloblastoma/genética , Neoplasias Induzidas por Radiação/genética , Receptor Patched-1/deficiência , Receptor Patched-1/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/efeitos da radiação , Linhagem Celular Tumoral , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/terapia , Dano ao DNA , Reparo do DNA por Junção de Extremidades/efeitos da radiação , DNA Helicases/genética , Proteína Quinase Ativada por DNA/deficiência , Proteínas de Ligação a DNA/deficiência , Relação Dose-Resposta à Radiação , Recombinação Homóloga/efeitos da radiação , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Meduloblastoma/terapia , Camundongos , Terapia de Alvo Molecular , Mutação , Neoplasias Induzidas por Radiação/metabolismo , Neoplasias Induzidas por Radiação/patologia , Neoplasias Induzidas por Radiação/terapia , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Risco , Raios X/efeitos adversos
15.
Biochem Biophys Res Commun ; 508(2): 536-542, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30509497

RESUMO

WNT/ß-catenin signaling plays pivotal roles in mammary development and tumorigenesis; and aberrant activation of this pathway is frequently observed in human breast cancer, correlating with poor outcome. However, the mechanisms underlying WNT-driven mammary tumorigenesis remain incompletely understood. Here, we used mouse mammary tumor virus (MMTV)-Wnt1 transgenic mice, which develop aggressive mammary adenocarcinomas, to examine whether Limb-Bud-and-Heart (LBH) - a WNT/ß-catenin target transcription co-factor overexpressed in human triple-negative breast cancers with WNT pathway hyperactivation, contributes to WNT-induced tumorigenesis. We found LBH is specifically overexpressed in basal epithelial tumor cells of MMTV-Wnt1 mammary tumors reminiscent of its basal cell-restricted expression in the normal postnatal mammary gland. To determine the role of LBH in mammary tumorigenesis, we crossed MMTV-Wnt1 mice with basal epithelial-specific Keratin 14/K14-Cre;LbhloxP knockout mice. Mammary glands from virgin LBH-deficient MMTV-Wnt1 mice exhibited reduced hyperplasia, cell proliferation and increased apoptosis. Importantly, LBH inactivation in mammary epithelium significantly delayed tumor onset in MMTV-Wnt1 transgenic mice, with a median tumor-free survival of 32.5 weeks compared to 22.5 weeks in control LBH wild type MMTV-Wnt1 mice (p < 0.05). This data provides the first evidence that LBH plays an essential role in WNT-induced mammary tumorigenesis by promoting hyperplastic growth and tumor formation.


Assuntos
Carcinogênese/induzido quimicamente , Hiperplasia/prevenção & controle , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Animais/prevenção & controle , Proteínas Nucleares/deficiência , Animais , Proteínas de Ciclo Celular , Feminino , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Transgênicos , Proteína Wnt1/genética
16.
Neuron ; 101(1): 103-118.e5, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30503644

RESUMO

Stromalin, a cohesin complex protein, was recently identified as a novel memory suppressor gene, but its mechanism remained unknown. Here, we show that Stromalin functions as a negative regulator of synaptic vesicle (SV) pool size in Drosophila neurons. Stromalin knockdown in dopamine neurons during a critical developmental period enhances learning and increases SV pool size without altering the number of dopamine neurons, their axons, or synapses. The developmental effect of Stromalin knockdown persists into adulthood, leading to strengthened synaptic connections and enhanced olfactory memory acquisition in adult flies. Correcting the SV content in dopamine neuron axon terminals by impairing anterograde SV trafficking motor protein Unc104/KIF1A rescues the enhanced-learning phenotype in Stromalin knockdown flies. Our results identify a new mechanism for memory suppression and reveal that the size of the SV pool is controlled genetically and independent from other aspects of neuron structure and function through Stromalin.


Assuntos
Proteínas de Drosophila/deficiência , Memória/fisiologia , Proteínas Nucleares/deficiência , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/ultraestrutura , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Proteínas Nucleares/genética , Sinapses/genética , Sinapses/metabolismo , Sinapses/ultraestrutura , Vesículas Sinápticas/genética
17.
Lab Invest ; 99(3): 387-398, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30237457

RESUMO

Polymerase delta-interacting protein 2 (Poldip2) is a multi-functional protein with numerous roles in the vasculature, including the regulation of cell apoptosis and migration, as well as extracellular matrix deposition; however, its role in VSMC proliferation and neointimal formation is unknown. In this study, we investigated the role of Poldip2 in intraluminal wire-injury induced neointima formation and proliferation of vascular smooth muscle cells in vitro and in vivo. Poldip2 expression was observed in the intima and media of human atherosclerotic arteries, where it colocalized with proliferating cell nuclear antigen (PCNA). Wire injury of femoral arteries of Poldip2+/+ mice induced robust neointimal formation after 2 weeks, which was impaired in Poldip2+/‒ mice. PCNA expression was significantly reduced and expression of the cell cycle inhibitor p21 was significantly increased in wire-injured arteries of Poldip2+/‒ animals compared to wild-type controls. No difference was observed in apoptosis. Downregulation of Poldip2 in rat aortic smooth muscle cells significantly reduced serum-induced proliferation and PCNA expression, but upregulated p21 expression. Downregulation of p21 using siRNA reversed the inhibition of proliferation induced by knockdown of Poldip2. These results indicate that Poldip2 plays a critical role in the proliferation of VSMCs.


Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proliferação de Células/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas Mitocondriais/deficiência , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Neointima/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/deficiência , Antígeno Nuclear de Célula em Proliferação/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proliferação de Células/genética , Regulação para Baixo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Neointima/patologia , Neointima/prevenção & controle , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/genética , Ratos , Superóxidos/metabolismo
18.
Am J Surg Pathol ; 43(4): 455-465, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30451731

RESUMO

SMARCA4-deficient thoracic sarcoma (SMARCA4-DTS) is a recently described entity with an aggressive clinical course and specific genetic alterations of the BAF chromatin remodeling complex. In the present study, we reviewed the clinical and pathologic features of 30 cases of SMARCA4-DTS, discussed its main differential diagnoses and the challenging diagnostic scenarios that the average pathologist may face. In addition, we tested the specificity of the "SMARCA4-DTS immunohistochemical signature" (co-loss of SMARCA4 and SMARCA2 with overexpression of SOX2) in a large cohort of intrathoracic malignancies. Patients ranged from 28 to 90 years of age (median: 48 y), with a marked male predominance (male:female=9:1) and they were usually smokers. Tumors were generally large compressive masses located in the mediastinum (n=13), pleura (n=5), lung (n=2) or in 2 or more of these topographies (n=10). Treatment strategies were varied, including 1 case treated with EZH2 inhibitors. Median overall survival was 6 months. Histologically, tumors were poorly differentiated frequently showing rhabdoid features. A subset of cases showed a focal myxoid stroma (7%, n=2/30) and rare cases displayed a previously unreported pattern simulating desmoplastic small round cell tumors (7%, n=2/30). Making a diagnosis was challenging when dealing with biopsy material from massively necrotic tumors and in this setting the expression of SOX2, CD34, and SALL4 proved useful. All tested cases displayed concomitant loss of SMARCA4 and SMARCA2 and most tumors expressed epithelial markers (Pan-keratin or EMA) (n=29/30), SOX2 (n=26/27), and CD34 (n=17/27). SMARCB1 expression was retained in all cases (23/23). SALL4 and Claudin-4 were expressed in a subset of cases (n=7/21 and 2/19, respectively). TTF-1 and P63 were focally expressed in 1 case each. P40 and NUT were not expressed (0/23 and 0/20, respectively) The SMARCA4-DTS immunohistochemical signature was both sensitive and specific, with only a subset of small cell carcinoma of the ovary hypercalcemic type showing overlapping phenotypes. Our study confirms and expands the specific features of SMARCA4-DTS, emphasizing the fact that they can be straightforwardly identified by pathologists.


Assuntos
DNA Helicases/deficiência , Proteínas Nucleares/deficiência , Sarcoma/diagnóstico , Sarcoma/patologia , Neoplasias Torácicas/diagnóstico , Neoplasias Torácicas/patologia , Fatores de Transcrição/deficiência , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , DNA Helicases/genética , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Sarcoma/genética , Neoplasias Torácicas/genética , Fatores de Transcrição/genética
19.
DNA Repair (Amst) ; 73: 7-16, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30409670

RESUMO

DNA-PKcs deficiency has been studied in numerous animal models and cell culture systems. In previous studies of kinase inactivating mutations in cell culture systems, ablation of DNA-PK's catalytic activity results in a cell phenotype that is virtually indistinguishable from that ascribed to complete loss of the enzyme. However, a recent compelling study demonstrates a remarkably more severe phenotype in mice harboring a targeted disruption of DNA-PK's ATP binding site as compared to DNA-PKcs deficient mice. Here we investigate the mechanism for these divergent results. We find that kinase inactivating DNA-PKcs mutants markedly radiosensitize immortalized DNA-PKcs deficient cells, but have no substantial effects on transformed DNA-PKcs deficient cells. Since the non-homologous end joining mechanism likely functions similarly in all of these cell strains, it seems unlikely that kinase inactive DNA-PK could impair the end joining mechanism in some cell types, but not in others. In fact, we observed no significant differences in either episomal or chromosomal end joining assays in cells expressing kinase inactivated DNA-PKcs versus no DNA-PKcs. Several potential explanations could explain these data including a non-catalytic role for DNA-PKcs in promoting cell death, or alteration of gene expression by loss of DNA-PKcs as opposed to inhibition of its catalytic activity. Finally, controversy exists as to whether DNA-PKcs autophosphorylates or is the target of other PIKKs; we present data demonstrating that DNA-PK primarily autophosphorylates.


Assuntos
Proteína Quinase Ativada por DNA/deficiência , Proteínas de Ligação a DNA/deficiência , Proteínas Nucleares/deficiência , Fenótipo , Trifosfato de Adenosina/metabolismo , Animais , Quebras de DNA de Cadeia Dupla , Células HCT116 , Humanos , Camundongos , Fosforilação
20.
Vet Pathol ; 56(2): 322-331, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30381013

RESUMO

Lipin-1 ( Lpin1)-deficient lipodystrophic mice have scant and immature adipocytes and develop transient fatty liver early in life. Unlike normal mice, these mice cannot rely on stored triglycerides to generate adenosine triphosphate (ATP) from the ß-oxidation of fatty acids during periods of fasting. To compensate, these mice store much higher amounts of glycogen in skeletal muscle and liver than wild-type mice in order to support energy needs during periods of fasting. Our studies demonstrated that there are phenotypic changes in skeletal muscle fibers that reflect an adaptation to this unique metabolic situation. The phenotype of skeletal muscle (soleus, gastrocnemius, plantaris, and extensor digitorum longus [EDL]) from Lpin1-/- was evaluated using various methods including immunohistochemistry for myosin heavy chains (Myh) 1, 2, 2a, 2b, and 2x; enzyme histochemistry for myosin ATPase, cytochrome-c oxidase (COX), and succinyl dehydrogenase (SDH); periodic acid-Schiff; and transmission electron microscopy. Fiber-type changes in the soleus muscle of Lpin1-/- mice were prominent and included decreased Myh1 expression with concomitant increases in Myh2 expression and myosin-ATPase activity; this change was associated with an increase in the presence of Myh1/2a or Myh1/2x hybrid fibers. Alterations in mitochondrial enzyme activity (COX and SDH) were apparent in the myofibers in the soleus, gastrocnemius, plantaris, and EDL muscles. Electron microscopy revealed increases in the subsarcolemmal mitochondrial mass in the muscles of Lpin1-/- mice. These data demonstrate that lipin-1 deficiency results in phenotypic fiber-specific modulation of skeletal muscle necessary for compensatory fuel utilization adaptations in lipodystrophy.


Assuntos
Lipodistrofia/patologia , Músculo Esquelético/patologia , Proteínas Nucleares/deficiência , Fosfatidato Fosfatase/deficiência , Animais , Modelos Animais de Doenças , Feminino , Lipodistrofia/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Rápida/patologia , Fibras Musculares de Contração Rápida/ultraestrutura , Fibras Musculares de Contração Lenta/metabolismo , Fibras Musculares de Contração Lenta/patologia , Fibras Musculares de Contração Lenta/ultraestrutura , Músculo Esquelético/ultraestrutura , Proteínas Nucleares/genética , Fenótipo , Fosfatidato Fosfatase/genética
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