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1.
Anticancer Res ; 40(6): 3239-3246, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32487618

RESUMO

BACKGROUND/AIM: Non-structural maintenance of chromosomes condensin I complex subunit H (NCAPH) is implicated in correct chromosome condensation and segregation during mitosis. However, the functional role of NCAPH in the pathogenesis of non-small-cell lung cancer (NSCLC) remains unclear. The aim of this study was to elucidate the role of NCAPH in NSCLC cells. MATERIALS AND METHODS: A549 and H1299 NSCLC cells were transfected with small-interfering RNA (siRNA) against NCAPH. Subsequently, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony-formation assay and flow cytometry analysis were performed to reveal the role of NCAPH in NSCLC cells. In addition, migration and invasion assay were also performed. RESULTS: NCAPH knockdown inhibited cell proliferation, induced cell-cycle arrest at G2/M phase, and prevented colony formation, migration and invasion by NSCLC cells. CONCLUSION: NCAPH is involved in NSCLC progression and development, and may be a potential therapeutic target for NSCLC treatment.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Ciclo Celular/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Nucleares/metabolismo , Células A549 , Carcinoma Pulmonar de Células não Pequenas/genética , Pontos de Checagem do Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/genética , Invasividade Neoplásica , Proteínas Nucleares/biossíntese , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transfecção , Regulação para Cima
2.
Nat Commun ; 11(1): 2781, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32493900

RESUMO

Mutations disrupting regulatory T (Treg) cell function can cause IPEX and IPEX-related disorders, but whether established disease can be reversed by correcting these mutations is unclear. Treg-specific deletion of the chromatin remodeling factor Brg1 impairs Treg cell activation and causes fatal autoimmunity in mice. Here, we show with a reversible knockout model that re-expression of Brg1, in conjunction with the severe endogenous proinflammatory environment, can convert defective Treg cells into powerful, super-activated Treg cells (SuperTreg cells) that can resolve advanced autoimmunity,  with  Brg1 re-expression in a minor fraction of Treg cells sufficient for the resolution in some cases. SuperTreg cells have enhanced trafficking and regulatory capabilities, but become deactivated as the inflammation subsides, thus avoiding excessive immune suppression. We propose a simple, robust yet safe gene-editing-based therapy for IPEX and IPEX-related disorders that exploits the defective Treg cells and the inflammatory environment pre-existing in the patients.


Assuntos
Diabetes Mellitus Tipo 1/congênito , Diarreia/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Doenças do Sistema Imunitário/congênito , Linfócitos T Reguladores/imunologia , Alelos , Animais , Citocinas/metabolismo , DNA Helicases/deficiência , Diabetes Mellitus Tipo 1/imunologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Doenças do Sistema Imunitário/imunologia , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CXCR3/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Linfócitos T Reguladores/efeitos dos fármacos , Tamoxifeno/farmacologia , Fatores de Transcrição/deficiência
3.
Nucleic Acids Res ; 48(11): 6019-6031, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32379321

RESUMO

ALT tumor cells often contain abundant DNA damage foci at telomeres and rely on the alternative lengthening of telomeres (ALT) mechanism to maintain their telomeres. How the telomere chromatin is regulated and maintained in these cells remains largely unknown. In this study, we present evidence that heterochromatin protein 1 binding protein 3 (HP1BP3) can localize to telomeres and is particularly enriched on telomeres in ALT cells. HP1BP3 inhibition led to preferential growth inhibition of ALT cells, which was accompanied by telomere chromatin decompaction, increased presence of C-circles, more pronounced ALT-associated phenotypes and elongated telomeres. Furthermore, HP1BP3 appeared to participate in regulating telomere histone H3K9me3 epigenetic marks. Taken together, our data suggest that HP1BP3 functions on telomeres to maintain telomere chromatin and represents a novel target for inhibiting ALT cancer cells.


Assuntos
Proliferação de Células , Montagem e Desmontagem da Cromatina , Heterocromatina/metabolismo , Histonas/metabolismo , Telômero/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Eucromatina/genética , Eucromatina/metabolismo , Técnicas de Silenciamento de Genes , Heterocromatina/genética , Código das Histonas , Histonas/química , Humanos , Metilação , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Multimerização Proteica , Homeostase do Telômero
4.
J Clin Pathol ; 73(5): 257-260, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32312722

RESUMO

The SMARCA subgroup of genes belong to the SWI1/SNF1 family that are responsible chromatin remodelling and repair. Inactivating mutations in the main SMARCA genes A2 and A4 lead to loss of expression of their respective proteins within the nucleus and, as such have characterised a set of malignancies that are underpinned by SMARCA-deficiency.The morphology of these tumours ranges from small to large epithelioid cells, giant cells and rhabdoid cells. The rhabdoid cells are frequently present in these tumours but are not a sine qua non for the diagnosis. Most of these tumours are undifferentiated or dedifferentiated, high-grade pleomorphic carcinomas. Focally, areas of better differentiation can be seen. The initial description of a SMARCA4-deficient malignancy was the small cell carcinoma of the ovary, hypercalcaemic type. Subsequently, tumours fitting this characteristic morphology and immunophenotype have been described in the lung, thoracic cavity, endometrium and sinonasal tract, gastrointestinal tract and kidney. Immunohistochemical loss of SMARCA2 and SMARCA4 may occur concomitantly or independently of each other.SMARCA-deficient malignant tumours represent a unique subset of tumours with typical morphological and immunohistochemical findings.


Assuntos
Biomarcadores Tumorais/genética , DNA Helicases/genética , Neoplasias/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Biomarcadores Tumorais/deficiência , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/deficiência , DNA Helicases/metabolismo , Humanos , Imuno-Histoquímica , Mutação , Neoplasias/metabolismo , Proteínas Nucleares/deficiência , Fatores de Transcrição/deficiência
5.
Mol Immunol ; 120: 93-100, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32113132

RESUMO

Spontaneous DNA-PKcs deficiencies in animals result in a severe combined immunodeficiency (SCID) phenotype because DNA-PKcs is required to activate Artemis for V(D)J recombination coding end hairpin opening. The impact on signal joint formation in these spontaneous mutant mammals is variable. Genetically engineered DNA-PKcs null mice and cells from them show a >1,000-fold reduction in coding joint formation and minimal reduction in signal joint formation during V(D)J recombination. Does chemical inhibition of DNA-PKcs mimic this phenotype? M3814 (also known as Nedisertib) is a potent DNA-PKcs inhibitor. We find here that M3814 causes a quantitative reduction in coding joint formation relative to signal joint formation. The sequences of signal and coding junctions were within normal limits, though rare coding joints showed novel features. The signal junctions generally did not show evidence of resection into the signal ends that is often seen in cells that have genetic defects in DNA-PKcs. Comparison of the chemical inhibition findings here with the known results for spontaneous and engineered DNA-PKcs mutant mammals is informative for considering pharmacologic small molecule inhibition of DNA-PKcs in various types of neoplasia.


Assuntos
Proteína Quinase Ativada por DNA/antagonistas & inibidores , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Recombinação V(D)J , Animais , Reparo do DNA , Proteína Quinase Ativada por DNA/deficiência , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Endonucleases/deficiência , Endonucleases/genética , Endonucleases/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Camundongos Knockout , Camundongos SCID , Mutação , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Recombinação V(D)J/efeitos dos fármacos
6.
Am J Surg Pathol ; 44(5): 703-710, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31934917

RESUMO

The molecular pathogenesis of poorly differentiated sinonasal carcinoma received significant attention in recent years. As a consequence, several unclassified carcinomas in the morphologic spectrum of sinonasal undifferentiated carcinoma have been reclassified as distinctive genetically defined variants or entities. Among the latter are NUT-rearranged carcinoma and SMARCB1-deficient carcinomas. In this study, we further characterize a rare variant of sinonasal undifferentiated carcinoma-like tumors characterized by inactivation of the SWItch/Sucrose Nonfermentable chromatin remodeler SMARCA4 (BRG1) detectable by immunohistochemistry. Patients were 7 males and 3 females aged 20 to 67 years (median, 44). Tumors originated in the nasal cavity (6), nose and sinuses (2), or at unspecified site (2). Six tumors were initially misdiagnosed as small cell neuroendocrine carcinoma (SCNEC) or large cell neuroendocrine carcinoma (LCNEC). Histologically, the tumors were composed of small basaloid (3 cases) or large epithelioid (7) cells disposed into nests and solid sheets with extensive areas of necrosis. No glands or other differentiating features were noted. Abortive rosettes were seen in 1 case. Immunohistochemistry showed consistent expression of pankeratin and absence of CK5, p63, p16, and NUT in all tumors tested. Other tested markers were variably positive: CK7 (2/6), synaptophysin (9/10; mostly focal and weak), chromogranin-A (4/10; focal), and CD56 (3/5; focal). All tumors showed total loss of SMARCA4 and retained expression of SMARCB1/INI1. Co-loss of SMARCA2 was seen in 1 of 8 cases. Limited data were available on treatment and follow-up. Two patients received surgery (1 also radiotherapy) and 3 received chemotherapy. Metastases (cervical nodes, liver, bone, and lung/mediastinal) were detected in 3 patients; 2 were alive under palliative chemotherapy at 8 and 9 months while 1 died of progressive lung disease at 7 months. Three patients (1 with brain invasion) died soon after diagnosis (1 to 3 mo). In total, 4 of 6 patients (66%) with follow-up died of disease (median, 3 mo). This series characterizes SMARCA4-deficient sinonasal carcinoma as a genetically distinct aggressive entity in the spectrum of undifferentiated sinonasal carcinomas. These variants add to the spectrum of SWItch/Sucrose Nonfermentable-deficient sinonasal carcinomas, at the same time expanding the topographic distribution of SMARCA4-related malignancies.


Assuntos
Biomarcadores Tumorais/deficiência , Carcinoma/química , DNA Helicases/deficiência , Proteínas Nucleares/deficiência , Neoplasias dos Seios Paranasais/química , Fatores de Transcrição/deficiência , Adulto , Idoso , Biomarcadores Tumorais/genética , Carcinoma/genética , Carcinoma/secundário , Carcinoma/terapia , Diferenciação Celular , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neoplasias dos Seios Paranasais/genética , Neoplasias dos Seios Paranasais/patologia , Neoplasias dos Seios Paranasais/terapia , Fatores de Tempo , Resultado do Tratamento , Adulto Jovem
7.
Blood ; 135(9): 610-619, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-31942628

RESUMO

Traditionally, primary immune deficiencies have been defined based on increased susceptibility to recurrent and/or severe infections. However, immune dysregulation, manifesting with autoimmunity or hyperinflammatory disease, has emerged as a common feature. This is especially true in patients affected by combined immune deficiency (CID), a group of disorders caused by genetic defects that impair, but do not completely abolish, T-cell function. Hypomorphic mutations in the recombination activating genes RAG1 and RAG2 represent the prototype of the broad spectrum of clinical and immunological phenotypes associated with CID. The study of patients with RAG deficiency and with other forms of CID has revealed distinct abnormalities in central and peripheral T- and B-cell tolerance as the key mechanisms involved in immune dysregulation. Understanding the pathophysiology of autoimmunity and hyperinflammation in these disorders may also permit more targeted therapeutic interventions.


Assuntos
Imunodeficiência Combinada Severa/imunologia , Autoimunidade/genética , Autoimunidade/imunologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/imunologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Humanos , Proteínas Nucleares/deficiência , Proteínas Nucleares/imunologia , Imunodeficiência Combinada Severa/genética
8.
Bull Cancer ; 107(1): 41-47, 2020 Jan.
Artigo em Francês | MEDLINE | ID: mdl-31916995

RESUMO

A growing number of studies suggest a tumor suppressor role for the SWI/SNF complex involved in the remodeling of chromatin. Alterations of this complex have been found in many tumors of different origins, with topographic, morphologic and phenotypic diversity. Notably, they define 2 types of thoracic tumors: SMARCA4-deficient non-small cell lung carcinoma and SMARCA4-deficient sarcoma. Some clinical features appear to be common to both, such as intrathoracic localization, smoking exposure, male predominance and poor prognosis. However, the histological distinction between these two entities is sometimes difficult and it is not excluded that these entities belong to the same tumor spectrum with different degrees of differentiation. The therapy of these tumors is not yet codified. These tumors do not seem associated with oncogenic driver mutations allowing the prescription of targeted therapy, but immunotherapy has been shown to be effective in rare reported cases. More specific treatments using EZH2 inhibitors also seem promising in SMARCA4 deficient sarcomas.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Montagem e Desmontagem da Cromatina , DNA Helicases/deficiência , Proteínas de Neoplasias/deficiência , Proteínas Nucleares/deficiência , Sarcoma/genética , Neoplasias Torácicas/genética , Fatores de Transcrição/deficiência , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Quimiorradioterapia , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , Terapia Combinada , Procedimentos Cirúrgicos de Citorredução , DNA Helicases/fisiologia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Neoplasias do Mediastino/genética , Neoplasias do Mediastino/patologia , Neoplasias do Mediastino/terapia , Terapia de Alvo Molecular , Complexos Multiproteicos/efeitos dos fármacos , Complexos Multiproteicos/fisiologia , Invasividade Neoplásica , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/fisiologia , Proteína SMARCB1/fisiologia , Sarcoma/patologia , Sarcoma/terapia , Neoplasias Torácicas/patologia , Neoplasias Torácicas/terapia , Fatores de Transcrição/fisiologia
9.
Gynecol Oncol ; 157(1): 106-114, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31954538

RESUMO

OBJECTIVE: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) and SMARCA4-deficient undifferentiated uterine sarcoma (SMARCA4-DUS) are rare and aggressive tumors, primarily affecting pre- and perimenopausal women. Inactivating SMARCA4 mutations are thought to be the driving molecular events in the majority of these tumors. Here, we report the clinical course of a family with germline SMARCA4 mutation and compare large cohorts of these rare tumor types. METHODS: We extracted clinico-pathological medical record data for the family with germline SMARCA4 mutation. Clinico-genomic data from SCCOHT and SMARCA4-DUS cohorts were retrospectively extracted from the archives of a large CLIA-certified reference molecular laboratory. RESULTS: We identified a single family with an inherited germline SMARCA4 mutation, in which two different family members developed either SCCOHT or SMARCA4-DUS, both of whom died within one year of diagnosis, despite aggressive surgical, chemotherapy and immunotherapy treatment. Retrospective comparative analysis of large SCCOHT (n = 48) and SMARCA4-DUS (n = 17) cohorts revealed that SCCOHT patients were younger (median age: 28.5 vs. 49.0) and more likely to have germline SMARCA4 alterations (37.5% vs. 11.8%) than SMARCA4-DUS patients. CONCLUSIONS: Growing understanding of the role SMARCA4 plays in the pathogenesis of these rare cancers may inform recommended genetic testing and counseling in families with these tumor types.


Assuntos
Carcinoma de Células Pequenas/genética , DNA Helicases/genética , Mutação em Linhagem Germinativa , Hipercalcemia/genética , Proteínas Nucleares/genética , Neoplasias Ovarianas/genética , Sarcoma/genética , Fatores de Transcrição/genética , Neoplasias Uterinas/genética , Adulto , Carcinoma de Células Pequenas/patologia , Carcinoma de Células Pequenas/terapia , Diferenciação Celular/fisiologia , Estudos de Coortes , DNA Helicases/deficiência , Feminino , Humanos , Hipercalcemia/patologia , Hipercalcemia/terapia , Pessoa de Meia-Idade , Proteínas Nucleares/deficiência , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Prognóstico , Estudos Retrospectivos , Sarcoma/patologia , Sarcoma/terapia , Fatores de Transcrição/deficiência , Neoplasias Uterinas/patologia , Neoplasias Uterinas/terapia
10.
Am J Surg Pathol ; 44(2): 263-270, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31567195

RESUMO

Undifferentiated and dedifferentiated endometrial carcinomas (UDEC) are aggressive uterine tumors which may show loss of expression of SMARCA4 (BRG1) or SMARCB1 (INI-1). The recently described SMARCA4-deficient undifferentiated uterine sarcoma (SDUS) has a morphology which overlaps with UDEC. In this study, we compared clinical, morphologic, immunohistochemical, and molecular characteristics to identify features which differentiate SDUS from UDEC. Cases of SDUS (n=12) were compared with cases of UDEC (n=84, 55 of which were previously published). Immunohistochemistry was performed for p53, mismatch repair proteins, claudin-4, SMARCA4, and SMARCB1. Targeted molecular profiling was performed on 15 cases. Patients with SDUS were significantly younger than those with UDEC (mean 35.8 vs. 61.2 y, P=0.0001). UDEC and SDUS showed morphologic overlap; however, phyllodiform architecture favored a diagnosis of SDUS (36% vs. 0%, P=0.005), while prominent nuclear pleomorphism was only seen in some cases of UDEC (0% vs. 24%, P=0.15). Compared with SDUS, UDEC more frequently showed TP53 mutations (0% vs. 34%, P=0.03), microsatellite instability (0% vs. 44%, P=0.006), and intact SMARCA4 and SMARCB1 (0% vs. 80%); a panel combining these immunohistochemical markers had a sensitivity of 100% and specificity of 92% in distinguishing SDUS and UDEC. Cases of UDEC had mutations in genes associated with endometrial adenocarcinomas (eg, TP53, PTEN, PIK3CA) and occasionally SMARCA4, while SDUS was characterized solely by inactivating mutations in SMARCA4. Disease-specific survival was shorter in SDUS than UDEC (median survival 9 and 36 mo, P=0.01). In conclusion, SDUS occurs in younger patients than UDEC, has a worse prognosis, and in most cases has a distinct molecular and immunohistochemical profile.


Assuntos
Biomarcadores Tumorais/deficiência , DNA Helicases/deficiência , Neoplasias do Endométrio/diagnóstico , Proteínas Nucleares/deficiência , Proteína SMARCB1/deficiência , Sarcoma/diagnóstico , Fatores de Transcrição/deficiência , Neoplasias Uterinas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Diagnóstico Diferencial , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Sarcoma/metabolismo , Sarcoma/patologia , Sensibilidade e Especificidade , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologia
11.
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
12.
Cell Rep ; 29(13): 4471-4481.e6, 2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31875554

RESUMO

During V(D)J recombination, RAG proteins introduce DNA double-strand breaks (DSBs) at recombination signal sequences (RSSs) that contain either 12- or 23-nt spacer regions. Coordinated 12/23 cleavage predicts that DSBs at variable (V) gene segments should equal the level of breakage at joining (J) segments. Contrary to this, here we report abundant RAG-dependent DSBs at multiple Vκ gene segments independent of V-J rearrangement. We find that a large fraction of Vκ gene segments are flanked not only by a bone-fide 12 spacer but also an overlapping, 23-spacer flipped RSS. These compatible pairs of RSSs mediate recombination and deletion inside the Vκ cluster even in the complete absence of Jκ gene segments and support a V(D)J recombination center (RC) independent of the conventional Jκ-centered RC. We propose an improved model of Vκ-Jκ repertoire formation by incorporating these surprisingly frequent, evolutionarily conserved intra-Vκ cluster recombination events.


Assuntos
Linfócitos B/metabolismo , DNA/genética , Região Variável de Imunoglobulina/genética , Recombinação V(D)J/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Sistemas CRISPR-Cas , Células Clonais , DNA/imunologia , Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP/deficiência , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/imunologia , Endonucleases/deficiência , Endonucleases/genética , Endonucleases/imunologia , Feminino , Edição de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Família Multigênica , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Baço/citologia , Baço/imunologia
13.
PLoS Biol ; 17(10): e3000461, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31600191

RESUMO

Dendritic spine development is crucial for the establishment of excitatory synaptic connectivity and functional neural circuits. Alterations in spine morphology and density have been associated with multiple neurological disorders. Autism candidate gene disconnected-interacting protein homolog 2 A (DIP2A) is known to be involved in acetylated coenzyme A (Ac-CoA) synthesis and is primarily expressed in the brain regions with abundant pyramidal neurons. However, the role of DIP2A in the brain remains largely unknown. In this study, we found that deletion of Dip2a in mice induced defects in spine morphogenesis along with thin postsynaptic density (PSD), and reduced synaptic transmission of pyramidal neurons. We further identified that DIP2A interacted with cortactin, an activity-dependent spine remodeling protein. The binding activity of DIP2A-PXXP motifs (P, proline; X, any residue) with the cortactin-Src homology 3 (SH3) domain was critical for maintaining the level of acetylated cortactin. Furthermore, Dip2a knockout (KO) mice exhibited autism-like behaviors, including excessive repetitive behaviors and defects in social novelty. Importantly, acetylation mimetic cortactin restored the impaired synaptic transmission and ameliorated repetitive behaviors in these mice. Altogether, our findings establish an initial link between DIP2A gene variations in autism spectrum disorder (ASD) and highlight the contribution of synaptic protein acetylation to synaptic processing.


Assuntos
Acetilcoenzima A/genética , Transtorno do Espectro Autista/genética , Cortactina/genética , Espinhas Dendríticas/metabolismo , Morfogênese/genética , Proteínas Nucleares/genética , Processamento de Proteína Pós-Traducional , Acetilcoenzima A/deficiência , Acetilação , Motivos de Aminoácidos , Animais , Animais Recém-Nascidos , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/fisiopatologia , Sítios de Ligação , Cortactina/metabolismo , Espinhas Dendríticas/ultraestrutura , Modelos Animais de Doenças , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Teste de Complementação Genética , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Densidade Pós-Sináptica/metabolismo , Densidade Pós-Sináptica/ultraestrutura , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Células Piramidais/metabolismo , Células Piramidais/ultraestrutura , Transmissão Sináptica
14.
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
15.
PLoS One ; 14(8): e0221512, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31430335

RESUMO

Laminopathies are tissue-selective diseases that affect differently in organ systems. Mutations in nuclear envelopes, emerin (Emd) and lamin A/C (Lmna) genes, cause clinically indistinguishable myopathy called Emery-Dreifuss muscular dystrophy (EDMD) and limb-girdle muscular dystrophy. Several murine models for EDMD have been generated; however, emerin-null (Emd) mice do not show obvious skeletal and cardiac muscle phenotypes, and Lmna H222P/H222P mutant (H222P) mice show only a mild phenotype in skeletal muscle when they already have severe cardiomyopathy. Thus, the underlying molecular mechanism of muscle involvement due to nuclear abnormalities is still unclarified. We generated double mutant (Emd-/-/LmnaH222P/H222P; EH) mice to characterize dystrophic changes and to elucidate interactions between emerin and lamin A/C in skeletal and cardiac muscles. As H222P mice, EH mice grow normally and have breeding productivity. EH mice showed severer muscle involvement compared with that of H222P mice which was an independent of cardiac abnormality at 12 weeks of age. Nuclear abnormalities, reduced muscle fiber size and increased fibrosis were prominent in EH mice. Roles of emerin and lamin A/C in satellite cells function and regeneration of muscle fiber were also evaluated by cardiotoxin-induced muscle injury. Delayed increases in myog and myh3 expression were seen in both H222P and EH mice; however, the expression levels of those genes were similar with control and regenerated muscle fiber size was not different at day 7 after injury. These results indicate that EH mouse is a suitable model for studying skeletal muscle involvement, independent of cardiac function, in laminopathies and an interaction between emerin and lamin A/C in different tissues.


Assuntos
Lamina Tipo A/genética , Proteínas de Membrana/deficiência , Músculo Esquelético/patologia , Miocárdio/patologia , Proteínas Nucleares/deficiência , Envelhecimento/patologia , Animais , Cardiotoxinas , Núcleo Celular/patologia , Núcleo Celular/ultraestrutura , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Músculo Esquelético/fisiopatologia , Músculo Esquelético/ultraestrutura , Miocárdio/ultraestrutura , Proteínas Nucleares/metabolismo , Fenótipo , Regeneração
16.
Metabolism ; 99: 90-101, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31351995

RESUMO

Moderate or low level hydrogen peroxides has been shown to play an important role in vascular smooth muscle cell (VSMC) function, in which the polymerase DNA-directed interacting protein 2 (Poldip2), functioned as a key regulator of NOX4 activity. In current study, we unexpectedly found that type 2 diabetes mellitus (T2DM) substantially suppresses the hepatic Poldip2 expression, and that the hepatic deficiency of Poldip2 may be correlated with dysregulation of hepatic cholesterol and plasma triglycerides. In cultured hepatocytes, we found that both insulin and leptin may inhibit hepatic expression of Poldip2 under high glucose concentration, but these suppressions were totally abolished under normoglycemic condition. POLDIP2 siRNA knockdown significantly impaired the H2O2 induction by insulin or leptin under normoglycemic condition, contributing the accumulation of cholesterol in cultured liver cells. The in vivo restoration of hepatic Poldip2 expression in T2DM mice remarkably rescued the moderate H2O2 generation in livers versus control mice, resulting in significant amelioration of hepatic cholesterol accumulation and plasma triglyceride levels. Importantly, the moderate induction of H2O2 in livers dramatically improved the hepatic PI3K-C1/AKT signaling or dampened PI3K-C2γ/AKT signaling through suppression of PTEN and PTP1B activities, thereby inhibiting the hepatic expression of HMGCR and SREBP2 for cholesterol synthesis. Moreover, the restitution of hepatic Poldip2 expression in diabetic mice significantly lowered the VLDL-cholesterol production rate, and substantially suppressed PEPCK and G6Pase expressions for gluconeogenesis, thus significantly improving the plasma insulin and glucose levels, and ITT and GTT outcomes in diabetic mice. Our findings suggest that hepatic dysregulation of Poldip2 may contribute to diabetic dyslipidemia and hyperglycemia.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Homeostase , Fígado/metabolismo , Proteínas Mitocondriais/deficiência , Proteínas Nucleares/deficiência , Animais , Células Cultivadas , Dislipidemias/etiologia , Gluconeogênese , Glucose/fisiologia , Hiperglicemia/etiologia , Metabolismo dos Lipídeos , Lipídeos/fisiologia , Fígado/citologia , Camundongos
17.
PLoS One ; 14(7): e0213702, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31291246

RESUMO

Disconnected interacting protein 2 homolog A (DIP2A) is highly expressed in nervous system and respiratory system of developing embryos. However, genes regulated by Dip2a in developing brain and lung have not been systematically studied. Transcriptome of brain and lung in embryonic 19.5 day (E19.5) were compared between wild type and Dip2a-/- mice. An average of 50 million reads per sample was mapped to the reference sequence. A total of 214 DEGs were detected in brain (82 up and 132 down) and 1900 DEGs in lung (1259 up and 641 down). GO enrichment analysis indicated that DEGs in both Brain and Lung were mainly enriched in biological processes 'DNA-templated transcription and Transcription from RNA polymerase II promoter', 'multicellular organism development', 'cell differentiation' and 'apoptotic process'. In addition, COG classification showed that both were mostly involved in 'Replication, Recombination, and Repair', 'Signal transduction and mechanism', 'Translation, Ribosomal structure and Biogenesis' and 'Transcription'. KEGG enrichment analysis showed that brain was mainly enriched in 'Thyroid cancer' pathway whereas lung in 'Complement and Coagulation Cascades' pathway. Transcription factor (TF) annotation analysis identified Zinc finger domain containing (ZF) proteins were mostly regulated in lung and brain. Interestingly, study identified genes Skor2, Gpr3711, Runx1, Erbb3, Frmd7, Fut10, Sox11, Hapln1, Tfap2c and Plxnb3 from brain that play important roles in neuronal cell maturation, differentiation, and survival; genes Hoxa5, Eya1, Errfi1, Sox11, Shh, Igf1, Ccbe1, Crh, Fgf9, Lama5, Pdgfra, Ptn, Rbp4 and Wnt7a from lung are important in lung development. Expression levels of the candidate genes were validated by qRT-PCR. Genome wide transcriptional analysis using wild type and Dip2a knockout mice in brain and lung at embryonic day 19.5 (E19.5) provided a genetic basis of molecular function of these genes.


Assuntos
Encéfalo/metabolismo , Pulmão/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animais , Encéfalo/embriologia , Diferenciação Celular/genética , Sobrevivência Celular/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Pulmão/embriologia , Camundongos , Camundongos Knockout , Anotação de Sequência Molecular , Neurônios/citologia , Neurônios/metabolismo , Proteínas Nucleares/deficiência , Análise de Sequência de RNA , Fatores de Transcrição/genética
18.
PLoS One ; 14(7): e0220199, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31329649

RESUMO

Thy28, also known as ThyN1, is a highly conserved nuclear protein. We previously showed that in a chicken mature B cell line, Thy28 binds to the promoter of the gene encoding Pax5, a transcription factor essential for B cell development, and positively regulates its expression. Here, we generated a Thy28-deficient mouse line to analyze its potential role in B cell development in mice. Thy28-deficient mice showed normal development of B cells, and the expression of Pax5 was comparable between wild-type and Thy28-deficient primary B cells. Thus, species-specific mechanisms regulate Pax5 expression and B cell development.


Assuntos
Linfócitos B/metabolismo , Proteínas Nucleares/deficiência , Fator de Transcrição PAX5/genética , Animais , Linfócitos B/citologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Fator de Transcrição PAX5/metabolismo
19.
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
20.
Neurochem Int ; 129: 104488, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31202882

RESUMO

Aversive conditions elicit anxiety responses that prepare the organism to an eventual threat. Nonetheless, prolonged anxiety is a pathological condition associated with various neuropsychiatric disorders. Here, we evaluated whether the conspecific alarm substance (CAS), a chemical cue that elicits aversion, influences anxiety-like behaviors and modulates brain oxidative stress-related parameters in wild-type (WT) and leopard (leo) zebrafish following a repeated exposure protocol. CAS exposure was performed for 5 min, once daily for 7 consecutive days. In the 8th day, animals were tested in the light/dark and novel tank tests and their brains were further dissected for biochemical analyses. CAS chronically induced anxiogenic-like states in WT and leo populations when their behaviors were analyzed in the light/dark and novel tank tests. CAS also increased catalase (CAT) and glutathione S-transferase (GST) activities, as well as non-protein thiol (NPSH) content in WT and leo, but only leo had increased thiobarbituric reactive substance (TBARS) levels in the brain. At baseline conditions, leo was more 'anxious' when compared to WT, displaying lower CAT activity and carbonylated protein (CP) levels. Overall, CAS chronically triggers anxiety-like behavior in zebrafish populations, which may be associated with changes in oxidative stress-related parameters. Furthermore, the use of different zebrafish populations may serve as an interesting tool in future research aiming to investigate the neurobehavioral bases of neuropsychiatric disorders in vertebrates.


Assuntos
Ansiedade/fisiopatologia , Aprendizagem da Esquiva/fisiologia , Encéfalo/fisiopatologia , Comportamento Exploratório/fisiologia , Medo/fisiologia , Reação de Congelamento Cataléptica/fisiologia , Estresse Oxidativo , Peixe-Zebra/fisiologia , Animais , Ansiedade/induzido quimicamente , Ansiedade/genética , Aprendizagem da Esquiva/efeitos dos fármacos , Encéfalo/metabolismo , Catalase/análise , Comportamento Exploratório/efeitos dos fármacos , Medo/efeitos dos fármacos , Feminino , Reação de Congelamento Cataléptica/efeitos dos fármacos , Glutationa Transferase/análise , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Feromônios/farmacologia , Carbonilação Proteica/efeitos dos fármacos , Compostos de Sulfidrila/análise , Superóxido Dismutase/análise , Extratos de Tecidos/farmacologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/análise , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia
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