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1.
Dev Cell ; 58(21): 2393-2410.e9, 2023 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-37852252

RESUMO

Centrosomes are the major microtubule-organizing centers in animals and play fundamental roles in many cellular processes. Understanding how their composition varies across diverse cell types and how it is altered in disease are major unresolved questions, yet currently available centrosome isolation protocols are cumbersome and time-consuming, and they lack scalability. Here, we report the development of centrosome affinity capture (CAPture)-mass spectrometry (MS), a powerful one-step purification method to obtain high-resolution centrosome proteomes from mammalian cells. Utilizing a synthetic peptide derived from CCDC61 protein, CAPture specifically isolates intact centrosomes. Importantly, as a bead-based affinity method, it enables rapid sample processing and multiplexing unlike conventional approaches. Our study demonstrates the power of CAPture-MS to elucidate cell-type-dependent heterogeneity in centrosome composition, dissect hierarchical interactions, and identify previously unknown centrosome components. Overall, CAPture-MS represents a transformative tool to unveil temporal, regulatory, cell-type- and tissue-specific changes in centrosome proteomes in health and disease.


Assuntos
Proteoma , Proteômica , Animais , Proteoma/metabolismo , Centrossomo/metabolismo , Centro Organizador dos Microtúbulos , Microtúbulos , Mamíferos
2.
Plant J ; 116(1): 58-68, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37340932

RESUMO

Non-homologous end joining (NHEJ) plays a major role in repairing DNA double-strand breaks and is key to genome stability and editing. The minimal core NHEJ proteins, namely Ku70, Ku80, DNA ligase IV and XRCC4, are conserved, but other factors vary in different eukaryote groups. In plants, the only known NHEJ proteins are the core factors, while the molecular mechanism of plant NHEJ remains unclear. Here, we report a previously unidentified plant ortholog of PAXX, the crystal structure of which showed a similar fold to human 'PAXX'. However, plant PAXX has similar molecular functions to human XLF, by directly interacting with Ku70/80 and XRCC4. This suggests that plant PAXX combines the roles of mammalian PAXX and XLF and that these functions merged into a single protein during evolution. This is consistent with a redundant function of PAXX and XLF in mammals.


Assuntos
Reparo do DNA por Junção de Extremidades , Enzimas Reparadoras do DNA , Animais , Humanos , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA , Mamíferos/genética , Mamíferos/metabolismo
3.
Nucleic Acids Res ; 50(19): 11058-11071, 2022 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-36263813

RESUMO

DNA double strand breaks (DSBs) are induced by external genotoxic agents (ionizing radiation or genotoxins) or by internal processes (recombination intermediates in lymphocytes or by replication errors). The DNA ends induced by these genotoxic processes are often not ligatable, requiring potentially mutagenic end-processing to render ends compatible for ligation by non-homologous end-joining (NHEJ). Using single molecule approaches, Loparo et al. propose that NHEJ fidelity can be maintained by restricting end-processing to a ligation competent short-range NHEJ complex that 'maximizes the fidelity of DNA repair'. These in vitro studies show that although this short-range NHEJ complex requires DNA ligase IV (Lig4), its catalytic activity is dispensable. Here using cellular models, we show that inactive Lig4 robustly promotes DNA repair in living cells. Compared to repair products from wild-type cells, those isolated from cells with inactive Lig4 show a somewhat increased fraction that utilize micro-homology (MH) at the joining site consistent with alternative end-joining (a-EJ). But unlike a-EJ in the absence of NHEJ, a large percentage of joints isolated from cells with inactive Lig4 occur with no MH - thus, clearly distinct from a-EJ. Finally, biochemical assays demonstrate that the inactive Lig4 complex promotes the activity of DNA ligase III (Lig3).


Assuntos
Reparo do DNA por Junção de Extremidades , Reparo do DNA , DNA/genética , Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP/genética , DNA Ligases/genética , DNA Ligases/metabolismo , Biocatálise
4.
Structure ; 30(5): 655-657, 2022 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-35523122

RESUMO

In this issue of Structure, Kantsadi et al. (2022) present the crystal structures of coiled-coil bundles from SAS-6, a core component of the centriole cartwheel, and reveal that two coiled-coil domains interact asymmetrically. This work provides insights into how the polarity of centrioles is established.


Assuntos
Proteínas de Ciclo Celular , Centríolos , Proteínas de Ciclo Celular/química
5.
Structure ; 28(6): 674-689.e11, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32375023

RESUMO

Centrioles are cylindrical assemblies whose peripheral microtubule array displays a 9-fold rotational symmetry that is established by the scaffolding protein SAS6. Centriole symmetry can be broken by centriole-associated structures, such as the striated fibers in Chlamydomonas that are important for ciliary function. The conserved protein CCDC61/VFL3 is involved in this process, but its exact role is unclear. Here, we show that CCDC61 is a paralog of SAS6. Crystal structures of CCDC61 demonstrate that it contains two homodimerization interfaces that are similar to those found in SAS6, but result in the formation of linear filaments rather than rings. Furthermore, we show that CCDC61 binds microtubules and that residues involved in CCDC61 microtubule binding are important for ciliary function in Chlamydomonas. Together, our findings suggest that CCDC61 and SAS6 functionally diverged from a common ancestor while retaining the ability to scaffold the assembly of basal body-associated structures or centrioles, respectively.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Chlamydomonas/fisiologia , Cílios/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Algas/química , Proteínas de Algas/metabolismo , Linhagem Celular , Chlamydomonas/classificação , Cristalografia por Raios X , Células HEK293 , Humanos , Microtúbulos/metabolismo , Modelos Moleculares , Filogenia , Conformação Proteica , Domínios Proteicos , Multimerização Proteica
6.
J Neuroendovasc Ther ; 14(4): 146-150, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-37520172

RESUMO

Objective: We report a rare case of symptomatic vertebral and posterior inferior cerebellar arteries (VA-PICA) aneurysm-caused ipsilateral hemifacial spasm (HFS) for which coil embolization of the aneurysm with the assistance of abnormal muscle response (AMR) monitoring was effective. Case Presentation: A 62-year-old woman presented with left HFS. Magnetic resonance imaging showed a saccular aneurysm of the left VA-PICA which compressed the seventh cranial nerve at its root exit zone (REZ). Stent-assisted coil embolization resulted in intraoperative disappearance of AMR in the intraoperative electrophysiological study and HFS was relieved temporally. One month after endovascular surgery, HFS slightly occurred again with the re-appearance of the AMR, although there was no recurrence of aneurysm. Thereafter, the frequency of her HFS markedly decreased to once per several days 1 year after the coiling. Conclusion: Although complete disappearance of symptoms was not obtained, it was suggested that coil embolization is one of the therapeutic options for HFS which is caused by aneurysmal compression of REZ and intraoperative AMR is useful for identification of responsible lesions and determination of therapeutic effects.

7.
Prog Biophys Mol Biol ; 147: 26-32, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31014919

RESUMO

DNA double-strand breaks (DSBs), generated by ionizing radiation, reactive oxygen species and DNA replication across nicks, are the most severe DNA damage in eukaryotic cells. Non-Homologous End Joining repairs DNA double-strand breaks directly without a template and so can take place at any point in the cell cycle. Ku70/80 heterodimers rapidly assemble around broken DNA ends, allowing DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase, to be recruited and facilitating synapsis of broken DNA ends. This then provides a stage for end-processing and ligation. Here we review progress leading in 2017 to the medium resolution X-ray structure of DNA-PKcs, a single polypeptide chain of 4128 amino acids. This was followed quickly by chain tracing of cryo-EM structures of DNA-PKcs in complex with Ku and DNA. We discuss how combination of structural information from X-ray and cryo-EM studies can produce a working model for complex multicomponent molecular assemblies such as those found in DNA-double-strand-break repair.


Assuntos
Microscopia Crioeletrônica , Cristalografia por Raios X , Reparo do DNA por Junção de Extremidades , Proteína Quinase Ativada por DNA/química , Proteína Quinase Ativada por DNA/metabolismo , Animais , Humanos , Domínios Proteicos
8.
Nat Struct Mol Biol ; 25(6): 482-487, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29786079

RESUMO

Repairing DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ) requires multiple proteins to recognize and bind DNA ends, process them for compatibility, and ligate them together. We constructed novel DNA substrates for single-molecule nanomanipulation, allowing us to mechanically detect, probe, and rupture in real-time DSB synapsis by specific human NHEJ components. DNA-PKcs and Ku allow DNA end synapsis on the 100 ms timescale, and the addition of PAXX extends this lifetime to ~2 s. Further addition of XRCC4, XLF and ligase IV results in minute-scale synapsis and leads to robust repair of both strands of the nanomanipulated DNA. The energetic contribution of the different components to synaptic stability is typically on the scale of a few kilocalories per mole. Our results define assembly rules for NHEJ machinery and unveil the importance of weak interactions, rapidly ruptured even at sub-picoNewton forces, in regulating this multicomponent chemomechanical system for genome integrity.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Técnicas Genéticas/instrumentação , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Pareamento Cromossômico , DNA/genética , DNA/metabolismo , DNA Ligase Dependente de ATP/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Enzimas de Restrição do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Autoantígeno Ku/metabolismo , Fosforilação , Células Sf9 , Spodoptera
9.
Inflamm Bowel Dis ; 24(6): 1251-1265, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29669006

RESUMO

Background: Anti-tumor necrosis factor alpha (anti-TNFα) therapy has become the mainstay of therapy for Crohn's disease (CD). However, post-therapy, the recurrence rate is still high. The aim of this study was to dissect the molecular mechanism for recurrence of CD treated with anti-TNFα therapy and investigate novel therapeutic options that could induce complete remission. Methods: We re-analyzed publicly available mucosal gene expression data from CD patients pre- and post-infliximab therapy to extract the transcriptional differences between responders and healthy controls. We used a systematic computational approach based on identified differences to discover novel therapies and validated this prediction through in vitro and in vivo experimentation. Results: We identified a set of 3545 anti-TNFα therapy-untreatable genes (TUGs) that are significantly regulated in intestinal epithelial cells, which remain altered during remission. Pathway enrichment analysis of these genes clearly showed excessive growth state and suppressed terminal differentiation, whereas immune components were clearly resolved. Through in silico screening strategy, we observed that MEK inhibitors were predicted to revert expression of genes dysregulated in infliximab responders. In vitro transcriptome analysis demonstrated that selective MEK1/2 inhibitor significantly normalized reference genes from TUGs. In addition, in vitro functional study proved that MEK1/2 inhibitor facilitated intestinal epithelial differentiation. Finally, using murine colitis model, administration of MEK1/2 inhibitor significantly improved diarrhea and histological score. Conclusions: Our data revealed the abnormalities in anti-TNFα responders' CD colons that would be cause of recurrence of CD. Also, we provided evidence regarding MEK1/2 inhibitor as a potential treatment against CD to achieve sustainable remission.


Assuntos
Doença de Crohn/tratamento farmacológico , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adalimumab , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Células CACO-2 , Colo/patologia , Doença de Crohn/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Infliximab , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Recidiva , Indução de Remissão
10.
J Biol Chem ; 291(47): 24377-24389, 2016 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-27703001

RESUMO

The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PKcs and Artemis for trimming the DNA ends; DNA polymerase µ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). In vivo studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. In vitro studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5' overhangs, and 3' overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed.


Assuntos
Reparo do DNA por Junção de Extremidades , DNA Ligase Dependente de ATP/química , Enzimas Reparadoras do DNA/química , Proteínas de Ligação a DNA/química , Autoantígeno Ku/química , Animais , Linhagem Celular , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Spodoptera
11.
Nihon Rinsho ; 74(4): 655-60, 2016 Apr.
Artigo em Japonês | MEDLINE | ID: mdl-27333755

RESUMO

It is now well recognized that new neurons are continuously generated in the specific regions of the adult brain across all mammalian species including humans. Now the cumulative findings demonstrate that this capacity for neurogenesis at the adult stage is associated with self-repair mechanisms in the damaged brain. Then, one of the main reasons that in vivo growth factor treatment is worth studying is the expectation that such treatment would promote the regeneration of neurons after stroke. Here we show the cumulative evidence that this approach is feasible as one of the brain repair therapies.


Assuntos
Movimento Celular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Células-Tronco Neurais/fisiologia , Acidente Vascular Cerebral/patologia , Animais , Modelos Animais de Doenças , Humanos , Infusões Intraventriculares , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Ratos , Acidente Vascular Cerebral/fisiopatologia , Acidente Vascular Cerebral/terapia
12.
Int J Clin Oncol ; 21(5): 853-861, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27125214

RESUMO

BACKGROUND: We retrospectively investigated the impact on survival of early tumor reduction during definitive radiotherapy for inoperable stage III non-small cell lung cancer (NSCLC) patients, according to their histological subtypes. METHODS: Between November 2006 and December 2012, 152 consecutive patients with inoperable stage III NSCLC who underwent definitive radiotherapy were reviewed retrospectively. Forty-one patients were excluded for not satisfying the inclusion criteria. Forty-five (40.5 %) and 48 (43.2 %) patients were diagnosed with squamous cell carcinoma (SQC) and adenocarcinoma (ADC), respectively. The tumor reduction rate (TRR) was defined as follows: TRR = 1-[gross tumor volume (GTV) on computed tomography at shrinking irradiation field planning]/(GTV on computed tomography at the initial treatment planning). The Cox proportional hazard model was used to identify significant prognostic factors for overall survival (OS) and progression-free survival (PFS). RESULTS: We evaluated 111 patients, with a median follow-up time of 52.2 months in surviving patients. The median TRR was 45.9 %. In all patients, there were significant associations between TRR and PFS (P = 0.036) on multivariate analysis, although TRR had no correlation with OS (P = 0.141). With respect to histological subtype, multivariate analyses revealed that a higher TRR showed significant associations with better OS and PFS in the SQC group (P = 0.013 and 0.040, respectively). In contrast, a higher TRR was associated with poorer OS in the ADC group (P = 0.030); there was no association between TRR and PFS. CONCLUSION: We found that a higher TRR is a promising prognostic factor for better survival and disease control in SQC patients.


Assuntos
Adenocarcinoma/radioterapia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Carcinoma de Células Escamosas/radioterapia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Adenocarcinoma/diagnóstico por imagem , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/patologia , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Pulmonares/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Estudos Retrospectivos , Taxa de Sobrevida , Tomografia Computadorizada por Raios X , Carga Tumoral
13.
Brain Res ; 1636: 118-129, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26845459

RESUMO

In vivo growth factor (GF) treatment is a promising approach to enhance the regenerative capacity of neural stem cells (NSCs) for brain repair. However, how exogenous GFs affect endogenous NSCs is not well understood. This study investigated the impact of intraventricular administration of fibroblast growth factor 2 and epidermal growth factor on NSCs in the subventricular zone of intact adult mice. GFs were administered for various periods (3, 7, 10, and 14 days), and the proliferation and neuronal production of NSCs were assessed during and after GF treatment. We found that proliferation of NSCs and their progeny is markedly augmented during the first 7 days after the initiation of GF treatment. GF treatment for longer periods, however, did not lead to further increases in the NSC pool, but rather attenuated such proliferation and inhibited neurogenesis. As a result, the production of new olfactory bulb neurons was increased in animals treated with GFs for 7 days but decreased in animals treated for 14 days. These results show time-dependent changes in the response of NSCs to exogenous GFs and demonstrate that precise control of the duration of GF treatment is important for significant enhancement of neuronal production by NSCs in vivo for brain repair.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Ventrículos Laterais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Bromodesoxiuridina/metabolismo , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Marcação In Situ das Extremidades Cortadas , Injeções Intraventriculares , Antígeno Ki-67/metabolismo , Ventrículos Laterais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Bulbo Olfatório/efeitos dos fármacos , Bulbo Olfatório/fisiologia , Fosfopiruvato Hidratase/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Canais de Sódio , Fatores de Tempo , Tubulina (Proteína)/metabolismo
14.
Mol Cell ; 61(3): 434-448, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26778126

RESUMO

BRCA1 accumulation at DNA damage sites is an important step for its function in the DNA damage response and in DNA repair. BRCA1-BRCT domains bind to proteins containing the phosphorylated serine-proline-x-phenylalanine (pSPxF) motif including Abraxas, Bach1/FancJ, and CtIP. In this study, we demonstrate that ionizing radiation (IR)-induces ATM-dependent phosphorylation of serine 404 (S404) next to the pSPxF motif. Crystal structures of BRCT/Abraxas show that phosphorylation of S404 is important for extensive interactions through the N-terminal sequence outside the pSPxF motif and leads to formation of a stable dimer. Mutation of S404 leads to deficiency in BRCA1 accumulation at DNA damage sites and cellular sensitivity to IR. In addition, two germline mutations of BRCA1 are found to disrupt the dimer interface and dimer formation. Thus, we demonstrate a mechanism involving IR-induced phosphorylation and dimerization of the BRCT/Abraxas complex for regulating Abraxas-mediated recruitment of BRCA1 in response to IR.


Assuntos
Proteína BRCA1/metabolismo , Neoplasias Ósseas/metabolismo , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Dano ao DNA , Osteossarcoma/metabolismo , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/química , Proteína BRCA1/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Núcleo Celular/patologia , Núcleo Celular/efeitos da radiação , Cristalografia por Raios X , Mutação em Linhagem Germinativa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Osteossarcoma/genética , Osteossarcoma/patologia , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Interferência de RNA , Serina , Transfecção
15.
Anticancer Res ; 35(6): 3379-83, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26026098

RESUMO

AIM: The aim of the study was to better characterize the temporal induction of inflammatory cytokines in the serum of patients with prostate cancer (PCa) treated with radiotherapy and to ascertain the influence of hormonal therapy upon those expressions. PATIENTS AND METHODS: Between May 2007 and December 2009, 30 patients with localized PCa were treated with 3-dimensional conformal external-beam radiotherapy. Fifteen patients had received neoadjuvant hormonal therapy using a leuteinizing hormone-releasing hormone (LH-RH) analog for six months prior to radiotherapy. The cytokine levels were collectively measured using a multiplex assay system. RESULTS: Seventeen cytokines were at detectable levels throughout the blood sampling times before and during radiotherapy. Hormonal therapy for six months significantly decreased the serum levels of fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF). The levels of epidermal growth factor (EGF), granulocyte-colony stimulating factor (G-CSF), and interferon-gamma (IFNγ) significantly increased during radiotherapy. Most cytokine levels, except for eotaxin, G-CSF, growth-related oncogene (GRO), transforming growth factor-beta (TGFß)-1 and TGFß2, significantly increased during radiotherapy compared to the levels observed before radiotherapy. CONCLUSION: The present study revealed the influence of hormonal, and of radiation therapy on the proinflammatory cytokine levels in the sera of patients with PCa. In addition, neoadjuvant hormonal therapy amplified the radiation-induced alteration of serum cytokines. Further studies to characterize the mechanism underlying a radiation- or hormone-induced inflammatory state are, therefore, necessary.


Assuntos
Citocinas/sangue , Terapia Neoadjuvante , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/radioterapia , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Hormonais/administração & dosagem , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/sangue , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Hormônio Liberador de Gonadotropina/sangue , Humanos , Interferon gama/sangue , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/sangue , Neoplasias da Próstata/patologia , Radioterapia Conformacional/efeitos adversos , Fator A de Crescimento do Endotélio Vascular/sangue
16.
Am J Hum Genet ; 96(3): 412-24, 2015 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-25728776

RESUMO

Non-homologous end joining (NHEJ) is a key cellular process ensuring genome integrity. Mutations in several components of the NHEJ pathway have been identified, often associated with severe combined immunodeficiency (SCID), consistent with the requirement for NHEJ during V(D)J recombination to ensure diversity of the adaptive immune system. In contrast, we have recently found that biallelic mutations in LIG4 are a common cause of microcephalic primordial dwarfism (MPD), a phenotype characterized by prenatal-onset extreme global growth failure. Here we provide definitive molecular genetic evidence supported by biochemical, cellular, and immunological data for mutations in XRCC4, encoding the obligate binding partner of LIG4, causing MPD. We report the identification of biallelic mutations in XRCC4 in five families. Biochemical and cellular studies demonstrate that these alterations substantially decrease XRCC4 protein levels leading to reduced cellular ligase IV activity. Consequently, NHEJ-dependent repair of ionizing-radiation-induced DNA double-strand breaks is compromised in XRCC4 cells. Similarly, immunoglobulin junctional diversification is impaired in cells. However, immunoglobulin levels are normal, and individuals lack overt signs of immunodeficiency. Additionally, in contrast to individuals with LIG4 mutations, pancytopenia leading to bone marrow failure has not been observed. Hence, alterations that alter different NHEJ proteins give rise to a phenotypic spectrum, from SCID to extreme growth failure, with deficiencies in certain key components of this repair pathway predominantly exhibiting growth deficits, reflecting differential developmental requirements for NHEJ proteins to support growth and immune maturation.


Assuntos
Proteínas de Ligação a DNA/genética , Nanismo Hipofisário/genética , Nanismo/genética , Microcefalia/genética , Mutação , Alelos , Sequência de Aminoácidos , Criança , Pré-Escolar , Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Ligases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Campo Pulsado , Exoma , Fácies , Feminino , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Fenótipo , Conformação Proteica , Imunodeficiência Combinada Severa/genética
17.
Science ; 347(6218): 185-188, 2015 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-25574025

RESUMO

XRCC4 and XLF are two structurally related proteins that function in DNA double-strand break (DSB) repair. Here, we identify human PAXX (PAralog of XRCC4 and XLF, also called C9orf142) as a new XRCC4 superfamily member and show that its crystal structure resembles that of XRCC4. PAXX interacts directly with the DSB-repair protein Ku and is recruited to DNA-damage sites in cells. Using RNA interference and CRISPR-Cas9 to generate PAXX(-/-) cells, we demonstrate that PAXX functions with XRCC4 and XLF to mediate DSB repair and cell survival in response to DSB-inducing agents. Finally, we reveal that PAXX promotes Ku-dependent DNA ligation in vitro and assembly of core nonhomologous end-joining (NHEJ) factors on damaged chromatin in cells. These findings identify PAXX as a new component of the NHEJ machinery.


Assuntos
Antígenos Nucleares/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Linhagem Celular Tumoral , Cristalografia por Raios X , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Autoantígeno Ku , Estrutura Secundária de Proteína , Interferência de RNA
18.
DNA Repair (Amst) ; 17: 98-109, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24636752

RESUMO

Non-homologous end joining (NHEJ) repairs DNA double-strand breaks generated by DNA damage and also those occurring in V(D)J recombination in immunoglobulin and T cell receptor production in the immune system. In NHEJ DNA-PKcs assembles with Ku heterodimer on the DNA ends at double-strand breaks, in order to bring the broken ends together and to assemble other proteins, including DNA ligase IV (LigIV), required for DNA repair. Here we focus on structural aspects of the interactions of LigIV with XRCC4, XLF, Artemis and DNA involved in the bridging and end-joining steps of NHEJ. We begin with a discussion of the role of XLF, which interacts with Ku and forms a hetero-filament with XRCC4; this likely forms a scaffold bridging the DNA ends. We then review the well-defined interaction of XRCC4 with LigIV, and discuss the possibility of this complex interrupting the filament formation, so positioning the ligase at the correct positions close to the broken ends. We also describe the interactions of LigIV with Artemis, the nuclease that prepares the ends for ligation and also interacts with DNA-PK. Lastly we review the likely affects of Mendelian mutations on these multiprotein assemblies and their impacts on the form of inherited disease.


Assuntos
Reparo do DNA por Junção de Extremidades , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Sítios de Ligação , Quebras de DNA de Cadeia Dupla , DNA Ligases/química , DNA Ligases/metabolismo , Proteína Quinase Ativada por DNA/química , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Endonucleases , Humanos , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica
19.
Neurol Med Chir (Tokyo) ; 54(6): 435-40, 2014 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-24670312

RESUMO

There are still few studies of low-dose recombinant tissue plasminogen activator (rtPA) therapy (0.6 mg/kg) for acute ischemic stroke (AIS) patients ≥ 80 years old, though most strokes occur in elderly people. The safety and effectiveness of this form of thrombolysis without endovascular therapy were evaluated in AIS patients ≥ 80 years old at our hospital. The data were collected from August 2006 to April 2010, before approval of the Mechanical Embolus Removal in Cerebral Ischemia (MERCI) retriever in Japan. Intravenous rtPA was administered to patients within 3 hours of stroke onset. The incidence of intracerebral hemorrhage (ICH), the recanalization rate of the occluded artery, and the modified Rankin Scale (mRS) score 3 months after stroke were examined. The patients who received rtPA therapy were stratified into two age groups: a younger group (< 80 years) and an older group (≥ 80 years). Of the 87 patients who received rtPA therapy, 17 (19.5%) were ≥ 80 years old. The incidence of symptomatic ICH was not significantly different between the younger (4.3%) and older (0%) groups (p = 0.61). The recanalization rate of the occluded artery was not significantly different between the younger (54%) and older (50%) groups (p = 0.78). The rate of an mRS score of 0-2, 3 months after stroke was significantly higher in the younger (44.3%) than in the older group (11.8%) (p = 0.013). Low-dose rtPA therapy appears to be as safe and feasible for AIS patients ≥ 80 years old as it is for younger people. This therapy should not be withheld because of age.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Fibrinolíticos/uso terapêutico , Terapia Trombolítica/estatística & dados numéricos , Ativador de Plasminogênio Tecidual/uso terapêutico , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Doenças Assintomáticas , Dano Encefálico Crônico/epidemiologia , Dano Encefálico Crônico/etiologia , Isquemia Encefálica/epidemiologia , Causas de Morte , Hemorragia Cerebral/induzido quimicamente , Hemorragia Cerebral/epidemiologia , Relação Dose-Resposta a Droga , Avaliação de Medicamentos , Feminino , Fibrinolíticos/administração & dosagem , Fibrinolíticos/efeitos adversos , Humanos , Incidência , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Neuroimagem , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/uso terapêutico , Reperfusão , Estudos Retrospectivos , Índice de Gravidade de Doença , Análise de Sobrevida , Terapia Trombolítica/efeitos adversos , Ativador de Plasminogênio Tecidual/administração & dosagem , Ativador de Plasminogênio Tecidual/efeitos adversos , Resultado do Tratamento
20.
Int J Radiat Oncol Biol Phys ; 87(4): 738-46, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24064322

RESUMO

PURPOSE: To determine whether volume-based parameters on pretreatment (18)F-fluorodeoxyglucose positron emission tomography/computed tomography in breast cancer patients treated with mastectomy without adjuvant radiation therapy are predictive of recurrence. METHODS AND MATERIALS: We retrospectively analyzed 93 patients with 1 to 3 positive axillary nodes after surgery, who were studied with (18)F-fluorodeoxyglucose positron emission tomography/computed tomography for initial staging. We evaluated the relationship between positron emission tomography parameters, including the maximum standardized uptake value, metabolic tumor volume (MTV), and total lesion glycolysis (TLG), and clinical outcomes. RESULTS: The median follow-up duration was 45 months. Recurrence was observed in 11 patients. Metabolic tumor volume and TLG were significantly related to tumor size, number of involved nodes, nodal ratio, nuclear grade, estrogen receptor (ER) status, and triple negativity (TN) (all P values were <.05). In receiver operating characteristic curve analysis, MTV and TLG showed better predictive performance than tumor size, ER status, or TN (area under the curve: 0.85, 0.86, 0.79, 0.74, and 0.74, respectively). On multivariate analysis, MTV was an independent prognostic factor of locoregional recurrence-free survival (hazard ratio 34.42, 95% confidence interval 3.94-882.71, P=.0008) and disease-free survival (DFS) (hazard ratio 13.92, 95% confidence interval 2.65-103.78, P=.0018). The 3-year DFS rate was 93.8% for the lower MTV group (<53.1; n=85) and 25.0% for the higher MTV group (≥53.1; n=8; P<.0001, log-rank test). The 3-year DFS rate for patients with both ER-positive status and MTV<53.1 was 98.2%; and for those with ER-negative status and MTV≥53.1 it was 25.0% (P<.0001). CONCLUSIONS: Volume-based parameters improve recurrence prediction in postmastectomy breast cancer patients with 1 to 3 positive nodes. The addition of MTV to ER status or TN has potential benefits to identify a subgroup at higher risk for recurrence.


Assuntos
Neoplasias da Mama/diagnóstico por imagem , Fluordesoxiglucose F18 , Linfonodos/diagnóstico por imagem , Imagem Multimodal/métodos , Recidiva Local de Neoplasia/diagnóstico por imagem , Compostos Radiofarmacêuticos , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Antineoplásicos/uso terapêutico , Axila , Neoplasias da Mama/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Intervalo Livre de Doença , Feminino , Fluordesoxiglucose F18/farmacocinética , Glicólise , Humanos , Metástase Linfática , Mastectomia , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons/métodos , Cuidados Pós-Operatórios , Valor Preditivo dos Testes , Prognóstico , Curva ROC , Compostos Radiofarmacêuticos/farmacocinética , Receptores de Estrogênio , Medição de Risco , Tomografia Computadorizada por Raios X/métodos , Neoplasias de Mama Triplo Negativas/química , Neoplasias de Mama Triplo Negativas/diagnóstico por imagem , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/terapia , Carga Tumoral
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