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1.
Arthritis Rheumatol ; 76(4): 620-630, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-37975161

RESUMO

OBJECTIVE: The etiopathogenesis of systemic sclerosis (SSc) is unknown. Platelet-derived growth factor receptors (PDGFRs) are overexpressed in patients with SSc. Because PDGFRα is targeted by the adeno-associated virus type 5 (AAV5), we investigated whether AAV5 forms a complex with PDGFRα exposing epitopes that may induce the immune responses to the virus-PDGFRα complex. METHODS: The binding of monomeric human PDGFRα to the AAV5 capsid was analyzed by in silico molecular docking, surface plasmon resonance (SPR), and genome editing of the PDGFRα locus. AAV5 was detected in SSc lungs by in situ hybridization, immunohistochemistry, confocal microscopy, and molecular analysis of bronchoalveolar lavage (BAL) fluid. Immune responses to AAV5 and PDGFRα were evaluated by SPR using SSc monoclonal anti-PDGFRα antibodies and immunoaffinity-purified anti-PDGFRα antibodies from sera of patients with SSc. RESULTS: AAV5 was detected in the BAL fluid of 41 of 66 patients with SSc with interstitial lung disease (62.1%) and in 17 of 66 controls (25.75%) (P < 0.001). In SSc lungs, AAV5 localized in type II pneumocytes and in interstitial cells. A molecular complex formed of spatially contiguous epitopes of the AAV5 capsid and of PDGFRα was identified and characterized. In silico molecular docking analysis and binding to the agonistic anti-PDGFRα antibodies identified spatially contiguous epitopes derived from PDGFRα and AAV5 that interacted with SSc agonistic antibodies to PDGFRα. These peptides were also able to bind total IgG isolated from patients with SSc, not from healthy controls. CONCLUSION: These data link AVV5 with the immune reactivity to endogenous antigens in SSc and provide a novel element in the pathogenesis of SSc.


Assuntos
Doenças Pulmonares Intersticiais , Escleroderma Sistêmico , Humanos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Epitopos , Dependovirus/metabolismo , Autoanticorpos , Simulação de Acoplamento Molecular , Escleroderma Sistêmico/patologia , Peptídeos , Pulmão/patologia
2.
Hematol Oncol ; 41(3): 335-342, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36533316

RESUMO

Although chemotherapy (CHT) exposure is an established cause of telomere attrition, determinants of telomere length (TL) dynamics after chemotherapy are poorly defined. In this study, we analyzed granulocyte telomere dynamics in 34 adult lymphoma patients undergoing first-line CHT. TL was measured by southern blot at each CHT cycle and after 1 year from CHT completion. Median age was 59 yrs (range 22-77). Median number of CHT cycles was 6 (range 3-6). The majority of patients (79%, n = 27) experienced TL shortening following CHT exposure. Mean telomere loss was 673 base pairs (bp) by cycle 6. Telomere shortening was an early event as 87% of the total telomere loss (mean 586 bp) occurred by the end of cycle 3, with no significant recovery after 1 year. A significant correlation was observed between baseline TL and total or fractional telomere loss (p < 0.001), with telomere shortening by cycle 3 observed predominantly in male patients with long telomeres at pre-treatment evaluation. Stratifying the analysis by gender and age only young women (<51 years of age) did not show significant telomere shortening following chemotherapy exposure. These findings indicate that gender and baseline TL are major determinants of TL dynamics following chemotherapy exposure in lymphoma patients.


Assuntos
Linfoma , Adulto , Humanos , Masculino , Feminino , Adulto Jovem , Pessoa de Meia-Idade , Idoso , Linfoma/tratamento farmacológico , Linfoma/genética , Encurtamento do Telômero , Telômero
3.
NAR Genom Bioinform ; 2(4): lqaa096, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33575640

RESUMO

DNA methylation is a stable epigenetic modification, extremely polymorphic and driven by stochastic and deterministic events. Most of the current techniques used to analyse methylated sequences identify methylated cytosines (mCpGs) at a single-nucleotide level and compute the average methylation of CpGs in the population of molecules. Stable epialleles, i.e. CpG strings with the same DNA sequence containing a discrete linear succession of phased methylated/non-methylated CpGs in the same DNA molecule, cannot be identified due to the heterogeneity of the 5'-3' ends of the molecules. Moreover, these are diluted by random unstable methylated CpGs and escape detection. We present here MethCoresProfiler, an R-based tool that provides a simple method to extract and identify combinations of methylated phased CpGs shared by all components of epiallele families in complex DNA populations. The methylated cores are stable over time, evolve by acquiring or losing new methyl sites and, ultimately, display high information content and low stochasticity. We have validated this method by identifying and tracing rare epialleles and their families in synthetic or in vivo complex cell populations derived from mouse brain areas and cells during postnatal differentiation. MethCoresProfiler is written in R language. The software is freely available at https://github.com/84AP/MethCoresProfiler/.

4.
Mol Cancer Res ; 17(2): 628-641, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30242055

RESUMO

The surface glycoprotein THY is a marker of myoepithelial precursor cells, which are basal cells with epithelial-mesenchymal intermediate phenotype originating from the ectoderm. Myoepithelial precursor cells are lost during progression from in situ to invasive carcinoma. To define the functional role of Thy1-positive cells within the myoepithelial population, we tracked Thy1 expression in human breast cancer samples, isolated THY1-positive myoepithelial progenitor cells (CD44+/CD24low/CD90+), and established long-term cultures (parental cells). Parental cells were used to generate a xenograft model to examine Thy1 expression during tumor formation. Post-transplantation cell cultures lost THY1 expression through methylation at the THY1 locus and this is associated with an increase in EGFR and NOTCH1 transcript levels. Thy1-low cells are sensitive to the EGFR/HER2 dual inhibitor lapatinib. High THY1 expression is associated with poorer relapse-free survival in patients with breast cancer. THY1 methylation may track the shift of bipotent progenitors into differentiated cells. Thy1 is a good candidate biomarker in basal-like breast cancer. IMPLICATIONS: Our findings provide evidence that THY1 expression is lost in xenografts due to promoter methylation. Thy1-low cells with increased EGFR and Notch1 expression are responsive to target therapy. Because DNA methylation is often altered in early cancer development, candidate methylation markers may be exploited as biomarkers for basal-like breast cancer.


Assuntos
Células Epiteliais/metabolismo , Receptores de Hialuronatos/metabolismo , Receptor Notch1/metabolismo , Antígenos Thy-1/genética , Animais , Antígeno CD24/metabolismo , Metilação de DNA , Epigênese Genética , Células Epiteliais/patologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Regiões Promotoras Genéticas , Receptor Notch1/genética , Transdução de Sinais/genética , Células-Tronco/metabolismo , Células-Tronco/patologia , Antígenos Thy-1/metabolismo
6.
Sci Data ; 4: 170043, 2017 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-28398335

RESUMO

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles.


Assuntos
Dano ao DNA , Metilação de DNA , Reparo do DNA , Sequência de Bases , Humanos , Sulfitos
8.
Sci Rep ; 6: 33222, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27629060

RESUMO

We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5' and 3' ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells.


Assuntos
Cromatina , Dano ao DNA , Metilação de DNA , Reparo do DNA , Alelos , Histonas/genética , Humanos , Metilação
9.
Front Cell Neurosci ; 10: 146, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27313511

RESUMO

Reactive oxygen species (ROS) are signaling molecules that mediate stress response, apoptosis, DNA damage, gene expression and differentiation. We report here that differentiation of oligodendrocytes (OLs), the myelin forming cells in the CNS, is driven by ROS. To dissect the OL differentiation pathway, we used the cell line MO3-13, which display the molecular and cellular features of OL precursors. These cells exposed 1-4 days to low levels of H2O2 or to the protein kinase C (PKC) activator, phorbol-12-Myristate-13-Acetate (PMA) increased the expression of specific OL differentiation markers: the specific nuclear factor Olig-2, and Myelin Basic Protein (MBP), which was processed and accumulated selectively in membranes. The induction of differentiation genes was associated with the activation of ERK1-2 and phosphorylation of the nuclear cAMP responsive element binding protein 1 (CREB). PKC mediates ROS-induced differentiation because PKC depletion or bis-indolyl-maleimide (BIM), a PKC inhibitor, reversed the induction of differentiation markers by H2O2. H2O2 and PMA increased the expression of membrane-bound NADPH oxidases, NOX3 and NOX5. Selective depletion of these proteins inhibited differentiation induced by PMA. Furthermore, NOX5 silencing down regulated NOX3 mRNA levels, suggesting that ROS produced by NOX5 up-regulate NOX3 expression. These data unravel an elaborate network of ROS-generating enzymes (NOX5 to NOX3) activated by PKC and necessary for differentiation of OLs. Furthermore, NOX3 and NOX5, as inducers of OL differentiation, represent novel targets for therapies of demyelinating diseases, including multiple sclerosis, associated with impairment of OL differentiation.

10.
Arthritis Rheumatol ; 68(9): 2263-73, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27111463

RESUMO

OBJECTIVE: To describe a skin-SCID mouse chimeric model of systemic sclerosis (SSc; scleroderma) fibrosis based on engraftment of ex vivo-bioengineered skin using skin cells derived either from scleroderma patients or from healthy donors. METHODS: Three-dimensional bioengineered skin containing human keratinocytes and fibroblasts isolated from skin biopsy specimens from healthy donors or SSc patients was generated ex vivo and then grafted onto the backs of SCID mice. The features of the skin grafts were analyzed by immunohistochemistry, and the functional profile of the graft fibroblasts was defined before and after treatment with IgG from healthy controls or SSc patients. Two procedures were used to investigate the involvement of platelet-derived growth factor receptor (PDGFR): 1) nilotinib, a tyrosine kinase inhibitor, was administered to mice before injection of IgG from SSc patient sera (SSc IgG) into the grafts, and 2) human anti-PDGFR monoclonal antibodies were injected into the grafts. RESULTS: Depending on the type of bioengineered skin grafted, the regenerated human skin exhibited either the typical scleroderma phenotype or the healthy human skin architecture. Treatment of animals carrying healthy donor skin grafts with SSc IgG resulted in the appearance of a bona fide scleroderma phenotype, as confirmed by increased collagen deposition and fibroblast activation markers. Results of the experiments involving administration of nilotinib or monoclonal antibodies confirmed the involvement of PDGFR. CONCLUSION: Our results provide the first in vivo demonstration of the fibrotic properties of anti-PDGFR agonistic antibodies. This bioengineered skin-humanized mouse model can be used to test in vivo the progression of the disease and to monitor response to antifibrotic drugs.


Assuntos
Autoanticorpos/administração & dosagem , Modelos Animais de Doenças , Receptores do Fator de Crescimento Derivado de Plaquetas/imunologia , Esclerodermia Localizada/imunologia , Escleroderma Sistêmico/imunologia , Animais , Fibrose/imunologia , Camundongos , Camundongos SCID , Esclerodermia Localizada/patologia , Escleroderma Sistêmico/patologia , Pele/imunologia
11.
Arthritis Rheumatol ; 67(7): 1891-903, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25808833

RESUMO

OBJECTIVE: To identify the epitopes recognized by autoantibodies targeting platelet-derived growth factor receptor α (PDGFRα) in systemic sclerosis (SSc) and develop novel assays for detection of serum anti-PDGFRα autoantibodies. METHODS: Epstein-Barr virus-immortalized B cells from 1 patient with SSc (designated PAM) were screened for expression of IgG binding to PDGFRα and induction of reactive oxygen species in fibroblasts. The variable regions of anti-PDGFRα IgG were cloned into an IgG expression vector to generate distinct recombinant human monoclonal autoantibodies (mAb), which were characterized by binding and functional assays. The epitopes of anti-PDGFRα recombinant human mAb were defined by molecular docking, surface plasmon resonance binding assays, screening of a conformational peptide library spanning the PDGFRα extracellular domains, and expression analyses of alanine-scanned PDGFRα mutants. Direct or competitive enzyme-linked immunosorbent assays were established to detect all serum anti-PDGFRα autoantibodies or, selectively, the agonistic ones. RESULTS: Three types of anti-PDGFRα recombinant human mAb, with the same VH but distinct VL chains, were generated. Nonagonistic VH PAM-Vκ 13B8 recognized 1 linear epitope, whereas agonistic VH PAM-Vλ 16F4 and VH PAM-Vκ 16F4 recognized 2 distinct conformational epitopes. Serum anti-PDGFRα antibodies were detected in 66 of 70 patients with SSc, 63 of 130 healthy controls, 11 of 26 patients with primary Raynaud's phenomenon (RP), and 13 of 29 patients with systemic lupus erythematosus (SLE). Serum VH PAM-Vκ 16F4-like antibodies were found in 24 of 34 patients with SSc, but not in healthy controls, patients with primary RP, or patients with SLE. Peptides composing the VH PAM-Vκ 16F4 epitope inhibited PDGFRα signaling triggered by serum IgG from SSc patients. CONCLUSION: Agonistic anti-PDGFRα autoantibodies are enriched in SSc sera and recognize specific conformational epitopes that can be used to discriminate agonistic from nonagonistic antibodies and block PDGFRα signaling in patients with SSc.


Assuntos
Especificidade de Anticorpos/imunologia , Autoanticorpos/imunologia , Epitopos/imunologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/imunologia , Escleroderma Sistêmico/imunologia , Sequência de Aminoácidos , Autoanticorpos/sangue , Autoanticorpos/química , Estudos de Casos e Controles , Colágeno/metabolismo , Mapeamento de Epitopos , Epitopos/química , Feminino , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Conformação Proteica , Doença de Raynaud/sangue , Doença de Raynaud/imunologia , Espécies Reativas de Oxigênio/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/química , Escleroderma Sistêmico/sangue
12.
Arthritis Rheumatol ; 67(6): 1611-22, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25707572

RESUMO

OBJECTIVE: Reactive oxygen species (ROS) contribute to the pathogenesis of fibrosis in systemic sclerosis (SSc; scleroderma), and NADPH oxidase (NOX) is an important source of ROS. Since the role of single NOX isoforms has not been previously investigated in SSc, this study was undertaken to assess the expression of NOX in SSc fibroblasts compared to normal healthy cells and to analyze their role in cell activation. METHODS: Expression of NOX isoforms in dermal fibroblasts from patients with SSc and healthy control subjects was analyzed by real-time polymerase chain reaction, immunoblotting, and immunofluorescence. NOX isoforms were silenced using small interfering RNA. Production of ROS was measured by fluorometry and confocal microscopy. RESULTS: Scleroderma fibroblasts showed up-regulation of NOX-2 and NOX-4 protein and messenger RNA (mRNA) expression. Treatment of the cells with diphenyleneiodonium, a nonselective inhibitor of flavin-containing enzymes, and silencing of NOX2 and NOX4 decreased the production of ROS as well as the expression of type I collagen and α-smooth muscle actin in SSc fibroblasts. ROS generated by NOX-2 and NOX-4 were involved in DNA damage and activation of a DNA repair checkpoint. Incubation of healthy control fibroblasts with platelet-derived growth factor (PDGF) or with IgG isolated from SSc patient serum enhanced the expression of NOX2 and NOX4 mRNA, via ROS, in a time-dependent manner. Treatment with actinomycin D, a transcription inhibitor, reversed the effects of PDGF stimulation but not the effects of SSc IgG. CONCLUSION: Both NOX2 and NOX4 generate ROS in SSc fibroblasts and play a critical role in cell activation and DNA damage. Expression of NOX-2 and NOX-4 in SSc fibroblasts is maintained by a ROS-mediated loop.


Assuntos
Fibroblastos/metabolismo , Glicoproteínas de Membrana/genética , NADPH Oxidases/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio , Escleroderma Sistêmico/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Feminino , Imunofluorescência , Fluorometria , Inativação Gênica , Humanos , Immunoblotting , Masculino , Glicoproteínas de Membrana/metabolismo , Microscopia Confocal , Pessoa de Meia-Idade , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Escleroderma Sistêmico/metabolismo , Pele/citologia , Pele/metabolismo
13.
Nucleic Acids Res ; 42(17): 11040-55, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25217584

RESUMO

Histone methylation changes and formation of chromatin loops involving enhancers, promoters and 3' end regions of genes have been variously associated with active transcription in eukaryotes. We have studied the effect of activation of the retinoic A receptor, at the RARE-promoter chromatin of CASP9 and CYP26A1 genes, 15 and 45 min following RA exposure, and we found that histone H3 lysines 4 and 9 are demethylated by the lysine-specific demethylase, LSD1 and by the JMJ-domain containing demethylase, D2A. The action of the oxidase (LSD1) and a dioxygenase (JMJD2A) in the presence of Fe++ elicits an oxidation wave that locally modifies the DNA and recruits the enzymes involved in base and nucleotide excision repair (BER and NER). These events are essential for the formation of chromatin loop(s) that juxtapose the RARE element with the 5' transcription start site and the 3' end of the genes. The RARE bound-receptor governs the 5' and 3' end selection and directs the productive transcription cycle of RNA polymerase. These data mechanistically link chromatin loops, histone methylation changes and localized DNA repair with transcription.


Assuntos
Cromatina/química , Código das Histonas , Transcrição Gênica , Tretinoína/farmacologia , Caspase 9/genética , Cromatina/efeitos dos fármacos , Cromatina/enzimologia , Sistema Enzimático do Citocromo P-450/genética , DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Código das Histonas/efeitos dos fármacos , Histona Desmetilases/metabolismo , Histonas/metabolismo , Humanos , Células MCF-7 , Metilação/efeitos dos fármacos , Oxirredução , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Receptores do Ácido Retinoico/metabolismo , Ácido Retinoico 4 Hidroxilase , Transcrição Gênica/efeitos dos fármacos
14.
Sci Signal ; 7(341): ra84, 2014 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-25185156

RESUMO

Systemic sclerosis (SSc) is an autoimmune disease characterized by extensive visceral organ and skin fibrosis. SSc patients have increased production of autoreactive antibodies and Wnt signaling activity. We found that expression of the gene encoding Wnt inhibitor factor 1 (WIF-1) was decreased in fibroblasts from SSc patient biopsies. WIF-1 deficiency in SSc patient cells correlated with increased abundance of the Wnt effector ß-catenin and the production of collagen. Knocking down WIF-1 in normal fibroblasts increased Wnt signaling and collagen production. WIF-1 loss and DNA damage were induced in normal fibroblasts by either SSc patient immunoglobulins or oxidative DNA-damaging agents, such as ultraviolet light, hydrogen peroxide, or bleomycin. The DNA damage checkpoint kinase ataxia telangiectasia mutated (ATM) mediated WIF-1 silencing through the phosphorylation of the transcription factor c-Jun, which in turn activated the expression of the gene encoding activating transcription factor 3 (ATF3). ATF3 and c-Jun were recruited together with histone deacetylase 3 (HDAC3) to the WIF-1 promoter and inhibited WIF-1 expression. Preventing the accumulation of reactive oxygen species or inhibiting the activation of ATM, c-Jun, or HDACs restored WIF-1 expression in cultured SSc patient cells. Trichostatin A, an HDAC inhibitor, prevented WIF-1 loss, ß-catenin induction, and collagen accumulation in an experimental fibrosis model. Our findings suggest that oxidative DNA damage induced by SSc autoreactive antibodies enables Wnt activation that contributes to fibrosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA , Estresse Oxidativo , Proteínas Repressoras/metabolismo , Escleroderma Sistêmico/metabolismo , Proteínas Wnt/metabolismo , Antibióticos Antineoplásicos/química , Biópsia , Bleomicina/química , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Metilação de DNA , Fibroblastos/metabolismo , Fibrose , Inativação Gênica , Humanos , Ácidos Hidroxâmicos/química , Imunoglobulina G/química , Oxigênio/química , Inibidores da Síntese de Proteínas/química , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética
15.
Nucleic Acids Res ; 42(2): 804-21, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24137009

RESUMO

We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15-20 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression.


Assuntos
Metilação de DNA , Reparo de DNA por Recombinação , Transcrição Gênica , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ciclo Celular/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Metiltransferase 3A , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases
16.
PLoS One ; 8(2): e56592, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23451058

RESUMO

MicroRNAs (miRNAs) have been recognized as significantly involved in prostate cancer (PCa). Since androgen receptor (AR) plays a central role in PCa carcinogenesis and progression, it is imperative to systematically elucidate the causal association between AR and miRNAs, focusing on the molecular mechanisms by which miRNAs mediate AR signalling. In this study, we performed a series of time-course microarrays to observe the dynamic genome-wide expressions of mRNAs and miRNAs in parallel in hormone-sensitive prostate cancer LNCaP cells stimulated by androgen. Accordingly, we introduced Response Score to identify AR target miRNAs, as well as Modulation Score to identify miRNA target mRNAs. Based on theoretical identification and experimental validation, novel mechanisms addressing cell viability in PCa were unravelled for 3 miRNAs newly recognized as AR targets. (1) miR-19a is directly up-regulated by AR, and represses SUZ12, RAB13, SC4MOL, PSAP and ABCA1, respectively. (2) miR-27a is directly up-regulated by AR, and represses ABCA1 and PDS5B. (3) miR-133b is directly up-regulated by AR, and represses CDC2L5, PTPRK, RB1CC1, and CPNE3, respectively. Moreover, we found miR-133b is essential to PCa cell survival. Our study gives certain clues on miRNAs mediated AR signalling to cell viability by influencing critical pathways, especially by breaking through androgen's growth restriction effect on normal prostate tissue.


Assuntos
Androgênios/farmacologia , MicroRNAs/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Algoritmos , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Imunoprecipitação da Cromatina , Procedimentos Clínicos , Humanos , Masculino , Modelos Teóricos , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
17.
Rheumatology (Oxford) ; 52(1): 143-54, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23175568

RESUMO

OBJECTIVES: A higher incidence of cancer in scleroderma patients compared with the general population has been suggested by several observational studies, reporting, however, different estimates. Therefore, we aimed to perform a systematic review and meta-analysis to definitely assess this association. METHODS: We searched MEDLINE and Embase for all original articles of observational studies on cancer incidence in scleroderma patients without language restriction published up to December 2011. Two independent authors reviewed all titles/abstracts and retrieved detailed full-text of potentially relevant articles to identify studies according to predefined selection criteria. Summary estimates were derived using random-effects model and reported as relative risk (RR). Publication bias was evaluated by trim and fill analysis. RESULTS: From articles initially identified, 16 original studies, involving more than 7000 patients, were included in the present review. Compared with the general population, the summary RR to develop all invasive cancers in scleroderma patients was 1.75 (95% CI 1.41, 2.18). The results for selected cancer sites indicated a strong association with lung cancer (RR 4.35; 95% CI 2.08, 9.09), and a significant increased risk also for haematological neoplasms (RR 2.24; 95% CI 1.53, 3.29). The relation with breast cancer, suggested in some previous epidemiological studies, was not confirmed (RR 1.05; 95% CI 0.86, 1.29). CONCLUSION: The present meta-analysis, the first on scleroderma and cancer risk, provides definite estimates on the association between scleroderma and cancer.


Assuntos
Neoplasias/epidemiologia , Escleroderma Sistêmico/epidemiologia , Comorbidade , Humanos , Incidência , Risco
18.
Int J Oncol ; 40(5): 1627-35, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22366926

RESUMO

Phosphoinositide-3-OH kinase (PI3K) signalling regulates various cellular processes, including cell survival, growth, proliferation and motility, and is among the most frequently mutated pathways in cancer. Although the involvement of p85αPI3K SH2 domain in signal transduction has been extensively studied, the function of the SH3 domain at the N-terminus remains elusive. A serine (at codon 83) adjacent to the N-terminal SH3 domain in the PI3K regulatory subunit p85αPI3K that is phosphorylated by protein kinase A (PKA) in vivo and in vitro has been identified. Virtually all receptors binding p85αPI3K can cooperate with cAMP-PKA signals via phosphorylation of p85αPI3KSer83. To analyse the role of p85αPI3KSer83 in retinoic acid (RA) and cAMP signalling, in MCF7 cells, we used p85αPI3K mutated forms, in which Ser83 has been substituted with alanine (p85A) to prevent phosphorylation or with aspartic acid (p85D) to mimic the phosphorylated residue. We demonstrated that p85αPI3KSer83 is crucial for the synergistic enhancement of RARα/p85αPI3K binding induced by cAMP/RA co-treatment in MCF7 cells. Growth curves, colorimetric MTT assay and cell cycle analysis demonstrated that phosphorylation of p85αPI3KSer83 plays an important role in the control of MCF7 cell proliferation and in RA-induced inhibition of proliferation. Wound healing and transwell experiments demonstrated that p85αPI3KSer83 was also essential both for the control of migratory behaviour and for the reduction of motility induced by RA. This study points to p85αPI3KSer83 as the physical link between different pathways (cAMP-PKA, RA and FAK), and as an important regulator of MCF7 cell proliferation and migration.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/enzimologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Tretinoína/farmacologia , Alanina , Animais , Ácido Aspártico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Bovinos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Classe Ia de Fosfatidilinositol 3-Quinase/química , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Feminino , Humanos , Mutação , Invasividade Neoplásica , Fosforilação , Receptores do Ácido Retinoico/efeitos dos fármacos , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Serina , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Domínios de Homologia de src
19.
PLoS One ; 5(11): e14003, 2010 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-21103368

RESUMO

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal illness whose pathogenesis remains poorly understood. Recent evidence suggests oxidative stress as a key player in the establishment/progression of lung fibrosis in animal models and possibly in human IPF. The aim of the present study was to characterize the cellular phenotype of fibroblasts derived from IPF patients and identify underlying molecular mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: We first analyzed the baseline differentiation features and growth ability of primary lung fibroblasts derived from 7 histology proven IPF patients and 4 control subjects at different culture passages. Then, we focused on the redox state and related molecular pathways of IPF fibroblasts and investigated the impact of oxidative stress in the establishment of the IPF phenotype. IPF fibroblasts were differentiated into alpha-smooth muscle actin (SMA)-positive myofibroblasts, displayed a pro-fibrotic phenotype as expressing type-I collagen, and proliferated lower than controls cells. The IPF phenotype was inducible upon oxidative stress in control cells and was sensitive to ROS scavenging. IPF fibroblasts also contained large excess of reactive oxygen species (ROS) due to the activation of an NADPH oxidase-like system, displayed higher levels of tyrosine phosphorylated proteins and were more resistant to oxidative-stress induced cell death. Interestingly, the IPF traits disappeared with time in culture, indicating a transient effect of the initial trigger. CONCLUSIONS/SIGNIFICANCE: Robust expression of α-SMA and type-I collagen, high and uniformly-distributed ROS levels, resistance to oxidative-stress induced cell death and constitutive activation of tyrosine kinase(s) signalling are distinctive features of the IPF phenotype. We suggest that this phenotype can be used as a model to identify the initial trigger of IPF.


Assuntos
Diferenciação Celular , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Actinas/metabolismo , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Fibrose Pulmonar Idiopática/patologia , Pessoa de Meia-Idade , Músculo Liso/química , Oxidantes/farmacologia , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
20.
Sci Transl Med ; 2(23): 23ps13, 2010 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-20375002

RESUMO

In this issue of Science Translational Medicine, a report by Loeys et al. on mutations in the fibrillin-1 gene in patients with skin fibrosis (stiff skin) adds a new piece of information on a connective tissue disorder that resembles systemic sclerosis, an autoimmune disease characterized by skin fibrosis and visceral organ involvement. Here, we discuss the implications of these findings, as well as new opportunities for targeted therapy for fibrosis.


Assuntos
Dermatopatias/patologia , Pele/patologia , Animais , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibrilina-1 , Fibrilinas , Fibrose/tratamento farmacológico , Fibrose/genética , Fibrose/patologia , Humanos , Inflamação/genética , Inflamação/patologia , Síndrome de Marfan/genética , Síndrome de Marfan/patologia , Camundongos , Proteínas dos Microfilamentos/genética , Dermatopatias/tratamento farmacológico , Dermatopatias/genética , Proteínas Smad/metabolismo
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