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1.
PLoS Negl Trop Dis ; 14(5): e0008262, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32469928

RESUMO

Adhesion of T. cruzi trypomastigotes to components of the extracellular matrix (ECM) is an important step in mammalian host cell invasion. We have recently described a significant increase in the tyrosine nitration levels of histones H2A and H4 when trypomastigotes are incubated with components of the ECM. In this work, we used chromatin immunoprecipitation (ChIP) with an anti-nitrotyrosine antibody followed by mass spectrometry to identify nitrated DNA binding proteins in T. cruzi and to detect alterations in nitration levels induced upon parasite incubation with the ECM. Histone H1, H2B, H2A and H3 were detected among the 9 most abundant nitrated DNA binding proteins using this proteomic approach. One nitrated tyrosine residue (Y29) was identified in Histone H2B in the MS/MS spectrum. In addition, we observed a significant increase in the nitration levels of histones H1, H2B, H2A and H4 upon parasite incubation with ECM. Finally, we used ChIP-Seq to map global changes in the DNA binding profile of nitrated proteins. We observed a significant change in the binding pattern of nitrated proteins to DNA after parasite incubation with ECM. This work provides the first global profile of nitrated DNA binding proteins in T. cruzi and additional evidence for modification in the nitration profile of histones upon parasite incubation with ECM. Our data also indicate that the parasite interaction with the ECM induces alterations in chromatin structure, possibly affecting nuclear functions.


Assuntos
Matriz Extracelular/parasitologia , Histonas/análise , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/análise , Trypanosoma cruzi/química , Trypanosoma cruzi/crescimento & desenvolvimento , Imunoprecipitação da Cromatina , Matriz Extracelular/metabolismo , Histonas/metabolismo , Espectrometria de Massas , Nitrosação , Proteômica , Proteínas de Protozoários/metabolismo , Tirosina/análogos & derivados , Tirosina/imunologia
2.
Fertil Steril ; 113(2): 364-373.e2, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32106990

RESUMO

OBJECTIVE: To demonstrate the feasibility of studying exosomes directly from peritoneal fluid, we isolated exosomes from endometriosis patient samples and from controls, and characterized their cargo. DESIGN: Case-control experimental study. SETTING: Academic clinical center. PATIENT (S): Women with and without endometriosis who underwent laparoscopic surgery (n = 28 in total). INTERVENTION (S): None. MAIN OUTCOME MEASURE (S): Concentration of exosomes within peritoneal fluid and protein content of the isolated exosomes. RESULT (S): Peritoneal fluid samples were pooled according to the cycle phase and disease stage to form six experimental groups, from which the exosomes were isolated. Exosomes were successfully isolated from peritoneal fluid in all the study groups. The concentration varied with cycle phase and disease stage. Proteomic analysis showed specific proteins in the exosomes derived from endometriosis patients that were absent in the controls. Five proteins were found exclusively in the endometriosis groups: PRDX1, H2A type 2-C, ANXA2, ITIH4, and the tubulin α-chain. CONCLUSION (S): Exosomes are present in peritoneal fluid. The characterization of endometriosis-specific exosomes opens up new avenues for the diagnosis and investigation of endometriosis.


Assuntos
Líquido Ascítico/química , Endometriose/metabolismo , Exossomos/química , Proteínas/análise , Adulto , Anexina A2/análise , Líquido Ascítico/patologia , Estudos de Casos e Controles , Endometriose/patologia , Exossomos/ultraestrutura , Estudos de Viabilidade , Feminino , Histonas/análise , Humanos , Pessoa de Meia-Idade , Peroxirredoxinas/análise , Proteínas Secretadas Inibidoras de Proteinases/análise , Proteômica , Tubulina (Proteína)/análise , Adulto Jovem
3.
Chemosphere ; 247: 125748, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31954338

RESUMO

INTRODUCTION: Radon-induced biological effects have been studied mainly through epidemiological investigations, and well-controlled in vitro and in vivo experiments. To provide data explaining radon exposure-induced harmful effects in natural environment, exposure assessment under these conditions is needed. The objective of the study was to examine the level of genetic damage assessed with biomarkers of DNA single- and double-strand breaks (SSBs and DSBs) in peripheral blood mononuclear cells obtained from individuals continuously exposed to Rn in homes. Naturally elevated Rn concentrations in homes can be found in the South of Poland, in Kowary city. METHODS: Measurements of expression of phosphorylated histone γH2AX was used as a marker of DNA double strand breaks. To detect DNA single and double-strand breaks and alkali labile sites, the alkaline comet assay was used. Oxidative damage of DNA was evaluated by formamidopyrimidyne (FPG)-modified comet assay. The blood was collected from 94 volunteers living in Kowary. Subjects were grouped according to their status of living in radon concentration ≥100 Bq/m3 (n = 67), and <100 Bq/m3 (n = 27). RESULTS: The statistically significant differences in levels of DNA damage in peripheral lymphocytes assessed with comet assay were found to be associated with levels of radon exposure in indoor air (p = 0.034). DNA damage in the comet assay was significantly correlated with DNA damage assessed with γH2AX staining. CONCLUSIONS: Results of the present study indicate the suitability of alkaline comet assay for the detection of DNA damage in peripheral blood lymphocytes of people environmentally exposed to radon.


Assuntos
Poluentes Radioativos do Ar/toxicidade , Ensaio Cometa/normas , Exposição Ambiental , Linfócitos/efeitos da radiação , Radônio/análise , Ensaio Cometa/métodos , Dano ao DNA/efeitos da radiação , Histonas/análise , Histonas/genética , Humanos , Polônia , Radônio/farmacologia
4.
BMC Bioinformatics ; 21(1): 27, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992200

RESUMO

BACKGROUND: Phosphorylated histone H2AX, also known as γH2AX, forms µm-sized nuclear foci at the sites of DNA double-strand breaks (DSBs) induced by ionizing radiation and other agents. Due to their specificity and sensitivity, γH2AX immunoassays have become the gold standard for studying DSB induction and repair. One of these assays relies on the immunofluorescent staining of γH2AX followed by microscopic imaging and foci counting. During the last years, semi- and fully automated image analysis, capable of fast detection and quantification of γH2AX foci in large datasets of fluorescence images, are gradually replacing the traditional method of manual foci counting. A major drawback of the non-commercial software for foci counting (available so far) is that they are restricted to 2D-image data. In practice, these algorithms are useful for counting the foci located close to the midsection plane of the nucleus, while the out-of-plane foci are neglected. RESULTS: To overcome the limitations of 2D foci counting, we present a freely available ImageJ-based plugin (FocAn) for automated 3D analysis of γH2AX foci in z-image stacks acquired by confocal fluorescence microscopy. The image-stack processing algorithm implemented in FocAn is capable of automatic 3D recognition of individual cell nuclei and γH2AX foci, as well as evaluation of the total foci number per cell nucleus. The FocAn algorithm consists of two parts: nucleus identification and foci detection, each employing specific sequences of auto local thresholding in combination with watershed segmentation techniques. We validated the FocAn algorithm using fluorescence-labeled γH2AX in two glioblastoma cell lines, irradiated with 2 Gy and given up to 24 h post-irradiation for repair. We found that the data obtained with FocAn agreed well with those obtained with an already available software (FoCo) and manual counting. Moreover, FocAn was capable of identifying overlapping foci in 3D space, which ensured accurate foci counting even at high DSB density of up to ~ 200 DSB/nucleus. CONCLUSIONS: FocAn is freely available an open-source 3D foci analyzer. The user-friendly algorithm FocAn requires little supervision and can automatically count the amount of DNA-DSBs, i.e. fluorescence-labeled γH2AX foci, in 3D image stacks acquired by laser-scanning microscopes without additional nuclei staining.


Assuntos
Algoritmos , Reparo do DNA , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Histonas/análise , Histonas/metabolismo , Humanos
5.
Chin Med J (Engl) ; 132(24): 2941-2949, 2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31855962

RESUMO

BACKGROUND: X-linked inhibitor of apoptosis (XIAP) is a vital factor in the anti-apoptosis mechanism of tumors and is highly expressed in renal cell carcinoma (RCC). However, the mechanism through which XIAP regulates DNA damage repair is unknown. This study investigated the regulatory mechanism of XIAP in etoposide-induced apoptosis in two Caki-1 cell lines with high or low XIAP expression. METHODS: The two cell lines were established using RNA interference technology. The differentially expressed proteins in the two cell lines were globally analyzed through an isobaric tags for relative and absolute quantitation-based quantitative proteomics approach. Proteomic analysis revealed 255, 375, 362, and 5 differentially expressed proteins after 0, 0.5, 3, and 12 h of drug stimulation, respectively, between the two cell lines. The identified differentially expressed proteins were involved in numerous biological processes. In addition, the expression of histone proteins (H1.4, H2AX, H3.1, H3.2, and H3.3) was drastically altered, and the effects of XIAP silencing were accompanied by the marked downregulation of H2AX. Protein-protein interactions were assessed and confirmed through immunofluorescence and Western blot analyses. RESULTS: The results suggested that XIAP may act as a vital cell signal regulator that regulates the expression of DNA repair-related proteins, such as H2AX, and influences the DNA repair process. CONCLUSIONS: Given these functions, XIAP may be the decisive factor in determining the sensitivity of RCC cell apoptosis induction in response to chemotherapeutic agents.


Assuntos
Apoptose/efeitos dos fármacos , Carcinoma de Células Renais/tratamento farmacológico , Etoposídeo/farmacologia , Histonas/análise , Neoplasias Renais/tratamento farmacológico , Proteômica/métodos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/fisiologia , Carcinoma de Células Renais/química , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Renais/química , Neoplasias Renais/patologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/análise
6.
Georgian Med News ; (294): 128-131, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31687964

RESUMO

Histone modifications represent one of the types of epigenetic changes. Histones, undergo different types of epigenetic modifications, including the phosphorylation of serine residues. pHH3 antibodies specifically detect histon-3 protein, when phosphorylated at 10th and 28th serine residues. Traditionally pHH3 antibodies are used as proliferation marker, as it detects cells in late G2 and M phase. We studied the distribution of phosphor-histon-3 in epithelial tumors of the ovary and its relationship with ER, PR, Ki67, p53 and BCL2. Altogether, we investigated postoperative material from 160 patients. Standard immunohistochemistry was used to detect, phosphohistone-H3 (pHH3), ER, PR, Ki67, p53 and BCL2. The results of our study showed that phosphohistone-H3 expression is negatively associated with the expression of ER and PR expression, as well as with BCL2 expression, on the other hand it positively correlates with Ki67 and mutant p53 (p<0.05). In addition, the expression of phosphohistone-H3 is detected in Ki67 negative cases and its expression is increased along with the increase of malignancy grade. Our study results indicate that PHH3 might be used as an additional marker for the assessment of proliferation and malignancy potential of epithelial tumors of the ovary.


Assuntos
Epigênese Genética , Histonas/análise , Antígeno Ki-67/análise , Índice Mitótico/métodos , Gradação de Tumores/métodos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Fosforilação , Biomarcadores Tumorais , Proliferação de Células , Feminino , Humanos , Imuno-Histoquímica/métodos
7.
Int J Radiat Oncol Biol Phys ; 105(5): 1119-1125, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31425731

RESUMO

PURPOSE: This study seeks to identify biological factors that may yield a therapeutic advantage of proton therapy versus photon therapy. Specifically, we address the role of nonhomologous end-joining (NHEJ) and homologous recombination (HR) in the survival of cells in response to clinical photon and proton beams. METHODS AND MATERIALS: We irradiated HT1080, M059K (DNA-PKcs+/+), and HCC1937 human cancer cell lines and their isogenic counterparts HT1080-shDNA-PKcs, HT1080-shRAD51IND, M059J (DNA-PKcs-/-), and HCC1937-BRCA1 (BRCA1 complemented) to assess cell clonogenic survival and γ-H2AX radiation-induced foci. Cells were irradiated with either clinically relevant photons or 1 of 3 proton linear energy transfer (LET) values. RESULTS: Our results indicate that NHEJ deficiency is more important in dictating cell survival than proton LET. Cells with disrupted HR through BRCA1 mutation showed increased radiosensitivity only for high-LET protons whereas RAD51 depletion showed increased radiosensitivity for both photons and protons. DNA double strand breaks, assessed by γ-H2AX radiation-induced foci, showed greater numbers after 24 hours in cells exposed to higher LET protons. We also observed that NHEJ-deficient cells were unable to repair the vast majority of double strand breaks after 24 hours. CONCLUSIONS: BRCA1 mutation significantly sensitizes cells to protons, but not photons. Loss of NHEJ renders cells hypersensitive to radiation, whereas the relative importance of HR increases with LET across several cell lines. This may be attributable to the more clustered damage induced by higher LET protons, which are harder to repair through NHEJ. This highlights the importance of tumor biology in dictating treatment modality and suggests BRCA1 as a potential biomarker for proton therapy response. Our data also support the use of pharmacologic inhibitors of DNA repair to enhance the sensitivity to different radiation types, although this raises issues for normal tissue toxicity.


Assuntos
Morte Celular/genética , Reparo do DNA por Junção de Extremidades/fisiologia , Genes BRCA1 , Recombinação Homóloga/fisiologia , Transferência Linear de Energia , Fótons , Prótons , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Quebras de DNA de Cadeia Dupla , Inativação Gênica , Histonas/análise , Humanos , Mutação , Rad51 Recombinase/genética , Tolerância a Radiação/genética , Tolerância a Radiação/efeitos da radiação , Fatores de Tempo
8.
Radiother Oncol ; 139: 94-100, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31445839

RESUMO

PURPOSE: a) To investigate if an ex vivo cultured and irradiated tumor biopsy reflects and predicts the radiation response of the corresponding in vivo irradiated tumor measured with the DNA double strand break marker γH2AX foci. MATERIALS AND METHODS: Five human head and neck squamous cell carcinoma (hHNSCC) xenograft models were used. Fine needle biopsies were taken from anesthetized tumor-bearing NMRI nude mice prior to in vivo single dose irradiation (0, 2, 4, or 8 Gy) under ambient blood flow. Biopsies were ex vivo reoxygenated and irradiated with equivalent doses. Tumors and biopsies were fixed 24 h post irradiation, and γH2AX foci were assessed in oxygenated tumor regions. RESULTS: Linear regression analysis showed comparable slopes of the residual γH2AX foci dose-response curves in four out of five hHNSCC models when in vivo and ex vivo cohorts were compared. The slopes from ex vivo biopsies and in vivo tumors could classify the respective tumor model as sensitive or resistant according to the intrinsic radiation sensitivity (TCD50). CONCLUSION: The ability of ex vivo irradiated tumor biopsies to reflect and predict the intrinsic radiation response of in vivo tumors increases the translational potential of the ex vivo γH2AX foci assay as a diagnostic tool for clinical practice.


Assuntos
Neoplasias de Cabeça e Pescoço/radioterapia , Histonas/análise , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , Animais , Biópsia , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Camundongos , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Anal Chim Acta ; 1080: 116-126, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31409460

RESUMO

Histones participate in epigenetic regulation via dynamic post-translational modifications (PTMs) of histone variants. Comprehensive characterization of histone markers, especially those for the histone variants, could help to decipher the mechanism of epigenetic regulation. However, correctly profiling histone PTMs and its variants using mass spectrometry remains a challenge. Here, we developed an improved, specific and sensitive LC separation in conjunction with a high throughput multiple reaction monitoring combined with stable isotope-labeled internal standards (MRM-SIS) based quantitative method for histone H3 variants in the study of epigenetic regulation in the cell cycle. PTM patterns and the overall abundance of the three main histone H3 variants from Karpas 422 cells were analyzed and quantified simultaneously during different cell stages. The methylation pattern varied between different sites and modification states during the cell cycle. The canonical H3.1 presented regular patterns on K27 and K36, similar to H3.2, albeit differing from variant H3.3. H3.3 K36me2 increased from G1, S to G2 phase, whereas the same marker decreased in both H3.1 and H3.2. This novel discovery inspires more focus and research on the histone variants behavior and function during cell cycle. Moreover, this improved method could be applied to unveil PTMs dynamics of histone variants in several biological processes.


Assuntos
Ciclo Celular/fisiologia , Histonas/análise , Histonas/metabolismo , Acetilação , Sequência de Aminoácidos , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Histonas/química , Humanos , Lisina/química , Espectrometria de Massas/métodos , Metilação , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional
10.
BMC Cancer ; 19(1): 717, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324163

RESUMO

BACKGROUND: Ependymal tumors are pathologically defined intrinsic neoplasms originating in the intracranial compartments or the spinal cord that affect both children and adults. The recently integrated classification of ependymomas based on both histological and molecular characteristics is capable of subgrouping patients with various prognoses. However, the application of histological and molecular markers in Chinese patients with ependymomas has rarely been reported. We aimed to demonstrate the significance of histological characteristics, the v-relavian reticuloendotheliosis viral oncogene homolog A (RELA) fusions and other molecular features in ependymal tumors. METHODS: We reviewed the histological characteristics of ependymal tumors using conventional pathological slides and investigate the RELA fusions and Cylclin D1 (CCND1) amplification by Fluorescence in situ hybridization (FISH) and trimethylation of histone 3 lysine 27 (H3K27me3) expression by immunohistochemistry (IHC) methods. SPSS software was used to analyze the data. RESULTS: We demonstrated that hypercellularity, atypia, microvascular proliferation, necrosis, mitosis, and an elevated Ki-67 index, were tightly associated with an advanced tumor grade. Tumor location, necrosis, mitosis and the Ki-67 index were related to the survival of the ependymomas, but Ki67 was the only independent prognostic factor. Additionally, RELA fusions, mostly presented in pediatric grade III intracranial ependymomas, indicated decreased survival times of patients, and closely related to the patients' age, tumor grade, cellularity, cellular atypia, necrosis and Ki67 index in the intracranial ependymal tumors, whereas reduction of H3K27me3 predicted the worse prognosis in ependymal tumors. CONCLUSIONS: Histological and molecular features facilitate tumor grading and prognostic predictions for ependymal tumors in Chinese patients.


Assuntos
Neoplasias Encefálicas/patologia , Ependimoma/patologia , Histonas/análise , Antígeno Ki-67/análise , Neoplasias da Medula Espinal/patologia , Fator de Transcrição RelA/análise , Adolescente , Adulto , Biomarcadores Tumorais/análise , Neoplasias Encefálicas/cirurgia , Criança , China , Ciclina D1/análise , Ependimoma/cirurgia , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Estimativa de Kaplan-Meier , Masculino , Necrose , Gradação de Tumores , Prognóstico , Neoplasias da Medula Espinal/cirurgia , Adulto Jovem
11.
J Am Soc Mass Spectrom ; 30(12): 2526-2534, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31286445

RESUMO

Histone posttranslational modifications (PTMs) are essential for regulating chromatin and maintaining gene expression throughout cell differentiation. Despite the deep level of understanding of immunophenotypic differentiation pathways in hematopoietic cells, few studies have investigated global levels of histone PTMs required for differentiation and maintenance of these distinct cell types. Here, we describe an approach to couple fluorescence-activated cell sorting (FACS) with targeted mass spectrometry to define global "epi-proteomic" signatures for primary leukocytes. FACS was used to sort closely and distantly related leukocytes from normal human peripheral blood for quantitation of histone PTMs with a multiple reaction monitoring LC-MS/MS method measuring histone PTMs on histones H3 and H4. We validate cell sorting directly into H2SO4 for immediate histone extraction to decrease time and number of steps after FACS to analyze histone PTMs. Relative histone PTM levels vary in T cells across healthy donors, and the majority of PTMs remain stable up to 2 days following initial blood draw. Large differences in the levels of histone PTMs are observed across the mature lymphoid and myeloid lineages, as well as between different types within the same lineage, though no differences are observed in closely related T cell subtypes. The results show a streamlined approach for quantifying global changes in histone PTMs in cell types separated by FACS that is poised for clinical deployment.


Assuntos
Citometria de Fluxo/métodos , Código das Histonas , Leucócitos/citologia , Espectrometria de Massas em Tandem/métodos , Células Cultivadas , Cromatografia Líquida/métodos , Histonas/análise , Humanos , Leucócitos/química
12.
Int J Surg Pathol ; 27(7): 706-712, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31146625

RESUMO

Background. Risk of progressive disease of gastrointestinal stromal tumors (GISTs) relies on mitotic index, size, and location of the tumor. However, manual mitotic counting on hematoxylin and eosin-stained slides (MMC-HE) is inefficient with low reproducibility. Manual count of phospho-histone H3 (MC-PHH3)-positive cells on immunohistochemical stained slides has been shown to have comparable reliability with MMC-HE. This study aims to confirm the reliability of MC-PHH3 in GISTs compared with MMC-HE and then to further compare MC-PHH3 with computer-assisted image analysis of PHH3-positive cells (Comp-PHH3). Methods. The study included 119 patients with GISTs. PHH3 stains were performed. MC-PHH3 was assessed as counts/5 mm2 high-power fields. Whole slide images were captured and the tumor area with greatest mitotic activity was manually identified. The PHH3-positive cells were automatically counted in 0.5 mm2 using Ventana Virtuoso software. Results. MMC-HE ranged from 0 to 157/5 mm2. MC-PHH3 ranged from 0 to 35.6/5 mm2. Comp-PHH3 ranged from 0 to 66/0.5 mm2. Interclass correlation coefficient (ICC) indicates good agreement between the 3 pathologists for MC-PHH3 (ICC = 0.74, P = .42). There is a strong correlation between MMC-HE and MC-PHH3. The Spearman correlation coefficient was 0.63 (P < .0001). Lin's concordance further indicated a moderate diagnostic agreement between MC-PHH3 and Comp-PHH3. Conclusion. MC-PHH3 is proposed as a superior alternative to MMC-HE with potential application in GIST reporting and prognostication. Furthermore, Comp-PHH3 may be a valid alternative to MC-PHH3.


Assuntos
Tumores do Estroma Gastrointestinal/diagnóstico , Histonas/análise , Processamento de Imagem Assistida por Computador/métodos , Índice Mitótico/métodos , Imagem Molecular/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Contagem de Células/instrumentação , Contagem de Células/métodos , Corantes/química , Amarelo de Eosina-(YS)/química , Feminino , Tumores do Estroma Gastrointestinal/patologia , Tumores do Estroma Gastrointestinal/cirurgia , Hematoxilina/química , Histonas/metabolismo , Humanos , Processamento de Imagem Assistida por Computador/instrumentação , Masculino , Pessoa de Meia-Idade , Imagem Molecular/instrumentação , Fosforilação , Prognóstico , Reprodutibilidade dos Testes , Medição de Risco/métodos , Software , Coloração e Rotulagem/métodos , Fixação de Tecidos
13.
Am J Surg Pathol ; 43(10): 1323-1330, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31107719

RESUMO

Spindle cell rhabdomyosarcoma (RMS) is an aggressive sarcoma type with a predilection for the head and neck and frequent transactivating MYOD1 mutations. Malignant peripheral nerve sheath tumors (MPNST) show heterologous (most often rhabdomyoblastic) differentiation in 10% of cases; such tumors have been referred to as malignant "Triton" tumors. MPNST frequently harbors inactivating mutations in SUZ12 or EED, resulting in PRC2 dysfunction and loss of histone H3 lysine 27 trimethylation (H3K27me3), most often seen in sporadic and radiation-associated, high-grade tumors; immunohistochemistry (IHC) for H3K27me3 is a useful diagnostic marker. We recently encountered a tumor showing H3K27me3 loss but with otherwise typical features of spindle cell RMS. The purpose of this study was to evaluate H3K27me3 in spindle cell RMS and further investigate putative spindle cell RMS with loss of H3K27me3. IHC for H3K27me3 was performed on 50 tumors diagnosed as spindle cell RMS. Targeted sequencing of all exonic and selected intronic regions of ~450 genes was performed on the tumors with H3K27me3 loss using hybrid capture with a custom probe set and massively parallel (next-generation) sequencing (NGS). Of the 50 patients, 32 were male and 18 were female with a median age of 33 years (range, 6 wk to 77 y). Tumors most often involved head and neck (N=23), extremities/limb girdles (N=11), and trunk wall (N=5). Three cases (6%) showed loss of H3K27me3; based on all available evidence, we believe at least 2 of these cases in fact represent MPNST with complete heterologous rhabdomyoblastic differentiation: a deep-seated groin mass in a 76-year-old female and a paratesticular mass in a 22-year-old male (neither of whom had a history or signs of type 1 neurofibromatosis). The tumors showed similar histologic appearances: fascicular architecture, marked nuclear atypia, eosinophilic cytoplasm, and a high mitotic rate; rhabdomyoblasts were not apparent. One tumor showed focal areas with scant myxoid stroma and alternating hypocellularity and hypercellularity. By IHC, the tumors showed diffuse staining for desmin, myogenin, and MyoD1, whereas S100 protein and SOX10 were negative. NGS on 2 tumors revealed (1) 2-copy deletion of NF1, CDKN2A, and SUZ12 and a TP53 mutation with arm-level loss of 17p; and (2) 2-copy deletion of CDKN2A and an NF1 mutation with loss of 17q11, findings characteristic of MPNST. NGS on the third tumor showed no distinctive alterations. MPNST may occasionally show complete heterologous rhabdomyoblastic differentiation without histologic evidence of residual conventional MPNST, closely mimicking spindle cell RMS. IHC for H3K27me3 reliably distinguishes MPNST from spindle cell RMS.


Assuntos
Diferenciação Celular , Neurofibrossarcoma/patologia , Rabdomiossarcoma/patologia , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Criança , Pré-Escolar , Diagnóstico Diferencial , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/análise , Humanos , Imuno-Histoquímica , Lactente , Masculino , Pessoa de Meia-Idade , Mutação , Neurofibrossarcoma/química , Neurofibrossarcoma/genética , Valor Preditivo dos Testes , Rabdomiossarcoma/química , Rabdomiossarcoma/genética , Adulto Jovem
14.
Radiother Oncol ; 137: 24-31, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31048234

RESUMO

BACKGROUND AND PURPOSE: Predictive biomarkers can be instrumental to treatment individualisation of cancer patients and improve therapy outcome. Residual γH2AX foci represent a promising biomarker to predict tumour radiosensitivity. In this pre-clinical study, the slope of the dose-response curve was evaluated for its predictive relevance in head and neck squamous cell carcinoma xenografts (HNSCC). Additionally, the feasibility of the translated assay was tested in a clinical setting in patient derived HNSCC samples, and associations between residual γH2AX foci and clinical parameters were analysed. MATERIALS AND METHODS: Seven HNSCC xenografts models (FaDu, SAS, SKX, UT-SCC-5, UT-SCC-14, UT-SCC-45, XF354) were used. Tumour bearing NMRI nude mice were randomly distributed to five treatment arms (0-8 Gy). Residual γH2AX foci (24 h post irradiation) were counted by visual scoring in a micromilieu dependent manner (assessed with BrdU and pimonidazole). The local tumour control values measured as TCD50 (tumour control dose 50%) have previously been published. Patient derived HNSCC biopsies were cultivated ex vivo for 24 h including 4 h of pimonidazole and BrdU treatment, subsequently irradiated with 0-8 Gy and fixed after 24 h. RESULTS: In the pre-clinical study, the dose-response curve slopes negatively correlated with the tumour control dose after fractionated irradiation (TCD50,fx, R2 = 0.63, p = 0.032) and after single dose irradiation under homogeneous hypoxia (TCD50,SD,clamp, R2 = 0.66, p = 0.027). The γH2AX assay in clinical HNSCC samples showed a dose-response relationship, with the values of the slopes ranging from 0.099 Gy-1 to 0.920 Gy-1 (coefficient of variation = 52.8%). Slopes derived from patients were in the same ranges as the sensitive, moderate and resistant models of the pre-clinical study. Statistical analysis revealed a significant negative correlation between the slope and the patients' age (R2 = 0.65, p = 0.001). CONCLUSION: These results further support the promise of the slope of the residual γH2AX foci dose-response as a biomarker for radiosensitivity. In the clinical samples, the variation in the slopes reveals patients' specific repair capacities, which could hold potential value for treatment individualisation.


Assuntos
Neoplasias de Cabeça e Pescoço/radioterapia , Histonas/análise , Tolerância a Radiação , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , Animais , Feminino , Humanos , Masculino , Camundongos , Nitroimidazóis/uso terapêutico , Dosagem Radioterapêutica , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Methods Mol Biol ; 1989: 193-215, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31077107

RESUMO

The regulated proliferation of cells is a critical factor in tumor progression, antineoplastic therapies, immune system regulation, and the cellular developmental of multicellular organisms. While measurement of cell cycle state by fluorescent flow cytometry is well established, mass cytometry allows the cell cycle to be measured along with large numbers of other antigens enabling characterization of the complex interactions between the cell cycle and wide variety of cellular processes. This method describes the use of mass cytometry for the analysis of cell cycle state for cells from three different sources: in vitro cultured cell lines, ex vivo human blood or bone marrow, and in vivo labeling of murine tissues. The method utilizes incorporation of 5-Iodo-2'-deoxyuridine (IdU), combined with measurement of phosphorylated retinoblastoma protein (pRb), Cyclin B1, and phosphorylated Histone H3 (pHH3). These measurements can be integrated into a gating strategy that enables clear separation of all five phases of the cell cycle.


Assuntos
Ciclo Celular , Ciclina B1/análise , Citometria de Fluxo/métodos , Histonas/análise , Espectrometria de Massas/métodos , Proteína do Retinoblastoma/análise , Coloração e Rotulagem/métodos , Animais , Células da Medula Óssea/metabolismo , Humanos , Idoxuridina/análogos & derivados , Idoxuridina/metabolismo , Camundongos , Fosforilação
16.
Int J Radiat Oncol Biol Phys ; 105(1): 193-205, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31085283

RESUMO

PURPOSE: Epidermal cells are positioned on the body surface and thus risk being exposed to genotoxic stress, including ionizing radiation (IR), ultraviolet rays, and chemical compounds. The biological effect of IR on the skin tissue is a significant problem for medical applications such as radiation therapy. Keratinocyte stem cells and progenitors are at risk for IR-dependent tumorigenesis during radiation therapy for cancer treatment. To elucidate the molecular mechanism of genome stability in epidermal cells, we derived skin keratinocytes from human-induced pluripotent stem cells (iPSCs) and analyzed their DNA damage response (DDR). METHODS AND MATERIALS: Skin keratinocytes were derived from iPSCs and designated as first- (P1), second- (P2), and third- (P3) passage cells to compare the differentiation states of DDR. After 2 Gy gamma-ray exposure, cells were immunostained with DNA double-strand break markers γ-H2AX/53BP1 and cell senescence markers p16/p21 for DDR analysis. DDR protein expression level, cell survival, and apoptosis were analyzed by western blotting, WST-8 assay and TUNEL assay, respectively. DDR of constructed 3D organoid modeling was also analyzed. RESULTS: P1, P2, and P3 keratinocytes were characterized with keratinocyte markers keratin 14 and p63 using immunofluorescence, and all cells were positive to both markers. Derived keratinocytes showed high expression of integrin α6 and CD71 (real-time (qRT)-PCR ratio: iPSCs: integrin α6: 1.12, CD71: 1.25, P1: integrin α6: 7.80, CD71: 0.43, P2: integrin α6: 5.53, CD71: 0.48), suggesting that P1 and P2 keratinocytes have potential as keratinocyte progenitors. Meanwhile, P3 keratinocytes showed low expression of integrin α6 and CD71 (qRT-PCR ratio: P3: integrin α6: 0.55, CD71: 0.10), suggesting differentiated keratinocytes. After IR exposure, the P1 and P2 keratinocytes showed an increase in DNA repair activity by a γ-H2AX/53BP1 focus assay (P1: γ-H2AX: 28.0%, 53BP1: 17.0%, P2: γ-H2AX: 37.7%, 53BP1: 28.3%) but not in P3 keratinocytes (P3: γ-H2AX: 74.7%, 53BP1: 63.7%) compared with iPSCs (γ-H2AX: 57.0%, 53BP1: 55.0%). Furthermore, in derived keratinocytes, expression of the cellular senescence markers p16 and p21 were increased compared with iPSCs (P16: non irradiated, iPSCs: 0%, P1: 12.5%, P2: 14.5%, P3: 29.7%, IR, iPSCs: 0%, P1: 19.5%, P2: 34.8%, P3: 64.5%). DDR protein expression, cellular sensitivity, and apoptosis activity decreased in derived keratinocytes compared with iPSCs. CONCLUSIONS: We have demonstrated the derivation of keratinocytes from iPSCs and their characterization of differentiated states and DDR. Derived keratinocytes showed progenitors like character as a result of DDR. These results suggest that derived keratinocytes are useful tools for analyzing the effects of IR, such as DDR on the skin tissue from radiation therapy for cancer.


Assuntos
Senescência Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Células-Tronco Pluripotentes Induzidas/citologia , Queratinócitos/efeitos da radiação , Antígenos CD/metabolismo , Antígenos de Superfície/metabolismo , Biomarcadores/análise , Diferenciação Celular , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Raios gama , Histonas/análise , Humanos , Queratina-14/análise , Queratinócitos/química , Queratinócitos/fisiologia , Proteínas de Membrana/análise , Receptores da Transferrina/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/análise
17.
Dev Dyn ; 248(6): 488-500, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30939211

RESUMO

BACKGROUND: Male germ cells are unique because they express a substantial number of variants of the general DNA binding proteins, known as histones, yet the biological significance of these variants is still unknown. In the present study, we aimed to address the expression pattern of the testis-specific histone H2B variant (TH2B) and the testis-specific histone H2A variant (TH2A) within the neonatal mouse testis. RESULTS: We demonstrate that TH2B and TH2A are present in a testis-enriched for undifferentiated spermatogonia. Co-localization studies with an undifferentiated marker, ZBTB16, revealed that TH2B and ZBTB16 co-localize in the neonatal testis. Upon the appearance of the primary spermatocytes, TH2B no longer co-localized with the ZBTB16 positive spermatogonia but were instead detected within the differentiating spermatogonia. This pattern of expression where TH2B and ZBTB16 no longer co-localize was maintained in the adult testis. CONCLUSION: These findings are in contrast to previous studies, which demonstrated that TH2B and TH2A were found only in adult spermatocytes. Our data are in support of a switch in the expression of these variants following the first round of spermatogonial differentiation. These studies reinforce current understandings that spermatogonia within the neonatal mouse testis are inherently different from those residing within the adult testis.


Assuntos
Variação Genética , Histonas/genética , Espermatogênese , Testículo/química , Animais , Animais Recém-Nascidos , Histonas/análise , Masculino , Camundongos , Espermatócitos/química
18.
Nat Commun ; 10(1): 1055, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30837475

RESUMO

Lysine acetylation is a reversible posttranslational modification that occurs at thousands of sites on human proteins. However, the stoichiometry of acetylation remains poorly characterized, and is important for understanding acetylation-dependent mechanisms of protein regulation. Here we provide accurate, validated measurements of acetylation stoichiometry at 6829 sites on 2535 proteins in human cervical cancer (HeLa) cells. Most acetylation occurs at very low stoichiometry (median 0.02%), whereas high stoichiometry acetylation (>1%) occurs on nuclear proteins involved in gene transcription and on acetyltransferases. Analysis of acetylation copy numbers show that histones harbor the majority of acetylated lysine residues in human cells. Class I deacetylases target a greater proportion of high stoichiometry acetylation compared to SIRT1 and HDAC6. The acetyltransferases CBP and p300 catalyze a majority (65%) of high stoichiometry acetylation. This resource dataset provides valuable information for evaluating the impact of individual acetylation sites on protein function and for building accurate mechanistic models.


Assuntos
Histona Acetiltransferases/metabolismo , Histona Desacetilases/metabolismo , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Acetilação , Conjuntos de Dados como Assunto , Células HeLa , Histonas/análise , Humanos , Lisina/metabolismo , Proteoma/análise , Proteoma/metabolismo , Software , Estatísticas não Paramétricas
19.
Am J Clin Pathol ; 151(6): 542-550, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30788495

RESUMO

OBJECTIVES: To determine the utility of phosphohistone H3 (PHH3) mitotic count (MC) in grading follicular lymphoma (FL). METHODS: FL cases were identified (2005-2017). Three hematopathologists recorded their average Ki-67 proliferation index, MC/high-power field (hpf) using PHH3 and H&E stains, and number of centroblasts/hpf. Results were assessed for correlations and interobserver variability. RESULTS: Forty-three cases of FL were studied. PHH3 MC resulted in the strongest correlation to grade (r = 0.701, P < .0001) compared with Ki-67 proliferation index (PI) (r = 0.681, P < .0001) and H&E MC (r = 0.536, P = .0002) and the strongest linear relationship to centroblast count (R2 = 0.453). Agreement among pathologists was strongest for PHH3 (intraclass correlation coefficient [ICC] = 0.86) followed by Ki-67 PI (ICC = 0.85) and H&E MC (ICC = 0.78). CONCLUSIONS: PHH3 correlates best to histologic grade and has less interobserver variability compared with Ki-67 PI and H&E MC. These results support using PHH3 as an adjunct in FL grading.


Assuntos
Histonas/análise , Antígeno Ki-67/análise , Linfoma Folicular/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Índice Mitótico , Gradação de Tumores , Variações Dependentes do Observador , Adulto Jovem
20.
Dig Dis Sci ; 64(8): 2147-2157, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30788686

RESUMO

BACKGROUND: Histone methylation, as an essential pattern of posttranslational modifications, contributes to multiple cancer-related biological processes. Dysregulation of histone methylation is now considered a biomarker for cancer prognosis. AIMS: This study investigated and evaluated the potential role of four histone lysine trimethylation markers as biomarkers for esophageal squamous cell carcinoma (ESCC) prognosis. METHODS: Tissue arrays were made from 135 paraffin-embedded ESCC samples and examined for histone markers by immunohistochemistry, and 10 pairs of cancer and noncancerous mucosa tissues from ESCC patients were investigated with Western blot. Chi-squared test, Kaplan-Meier analysis with log-rank test, and Cox proportional hazard trend analyses were performed to assess the prognostic values of the markers. RESULTS: Histone 3 lysine 4 trimethylation (H3K4me3), histone 3 lysine 9 trimethylation (H3K9me3), and histone 4 lysine 20 trimethylation (H4K20me3), but not histone 3 lysine 36 trimethylation (H3K36me3), showed stronger immunostaining signals in tumor tissues than in the corresponding adjacent non-neoplastic mucosa tissues. The expression patterns of H3K36me3, H3K9me3, and H4K20me3 correlated with tumor infiltrating depth, lymph node involvement, and pTNM stage. Low-scoring H3K9me3 and H4K20me3 predicted better prognosis, while H3K36me3 manifested the opposite trend. Poor prognosis occurred in ESCC patients with expression patterns of high levels of H3K9me3, high levels of H4K20me3, and low levels of H3K36me3 expression. CONCLUSIONS: H3K9me3, H4K20me3, and H3K36me3 showed a close relationship with clinical features and were considered independent risk factors for survival of ESCC patients. The combination of H3K9me3, H4K20me3, and H3K36me3 expression, rather than the expression of a single histone marker, is believed to further enhance evaluations of ESCC prognosis and management.


Assuntos
Biomarcadores Tumorais/análise , Metilação de DNA , Epigênese Genética , Neoplasias Esofágicas/química , Carcinoma de Células Escamosas do Esôfago/química , Histonas/análise , Processamento de Proteína Pós-Traducional , Adulto , Idoso , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Humanos , Imuno-Histoquímica , Lisina , Masculino , Metilação , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Análise Serial de Tecidos
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