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1.
PLoS One ; 14(5): e0216081, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31059552

RESUMO

A sensitive biodosimetry tool is required for rapid individualized dose estimation and risk assessment in the case of radiological or nuclear mass casualty scenarios to prioritize exposed humans for immediate medical countermeasures to reduce radiation related injuries or morbidity risks. Unlike the conventional Dicentric Chromosome Assay (DCA), which takes about 3-4 days for radiation dose estimation, cell fusion mediated Premature Chromosome Condensation (PCC) technique in G0 lymphocytes can be rapidly performed for radiation dose assessment within 6-8 hrs of sample receipt by alleviating the need for ex vivo lymphocyte proliferation for 48 hrs. Despite this advantage, the PCC technique has not yet been fully exploited for radiation biodosimetry. Realizing the advantage of G0 PCC technique that can be instantaneously applied to unstimulated lymphocytes, we evaluated the utility of G0 PCC technique in detecting ionizing radiation (IR) induced stable and unstable chromosomal aberrations for biodosimetry purposes. Our study demonstrates that PCC coupled with mFISH and mBAND techniques can efficiently detect both numerical and structural chromosome aberrations at the intra- and inter-chromosomal levels in unstimulated T- and B-lymphocytes. Collectively, we demonstrate that the G0 PCC technique has the potential for development as a biodosimetry tool for detecting unstable chromosome aberrations (chromosome fragments and dicentric chromosomes) for early radiation dose estimation and stable chromosome exchange events (translocations) for retrospective monitoring of individualized health risks in unstimulated lymphocytes.


Assuntos
Aberrações Cromossômicas/efeitos da radiação , Linfócitos/efeitos da radiação , Radiometria/métodos , Animais , Células CHO/efeitos da radiação , Fusão Celular , Centrômero/efeitos da radiação , Cricetulus , Feminino , Raios gama/efeitos adversos , Humanos , Hibridização in Situ Fluorescente , Masculino , Lesões por Radiação/diagnóstico , Lesões por Radiação/genética , Radiação Ionizante , Estudos Retrospectivos , Cariotipagem Espectral/métodos , Telômero/efeitos da radiação , Raios X/efeitos adversos
2.
Curr Protoc Hum Genet ; 99(1): e70, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30215889

RESUMO

Analysis of the organization of the human genome is vital for understanding genetic diversity, human evolution, and disease pathogenesis. A number of approaches, such as multicolor fluorescence in situ hybridization (FISH) assays, cytogenomic microarray (CMA), and next-generation sequencing (NGS) technologies, are available for simultaneous analysis of the entire human genome. Multicolor FISH-based spectral karyotyping (SKY), multiplex FISH (M-FISH), and Rx-FISH may provide rapid identification of interchromosomal and intrachromosomal rearrangements as well as the origin of unidentified extrachromosomal elements. Recent advances in molecular cytogenetics have made it possible to efficiently examine the entire human genome in a single experiment at much higher resolution and specificity using CMA and NGS technologies. Here, we present an overview of the approaches available for genome-wide analyses. © 2018 by John Wiley & Sons, Inc.


Assuntos
Coloração Cromossômica/métodos , Genoma Humano , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hibridização in Situ Fluorescente/métodos , Cariotipagem Espectral/métodos , Humanos
3.
Eur Cell Mater ; 35: 225-241, 2018 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-29683471

RESUMO

In the development of cell-based medicinal products, it is crucial to guarantee that the application of such an advanced therapy medicinal product (ATMP) is safe for the patients. The consensus of the European regulatory authorities is: "In conclusion, on the basis of the state of art, conventional karyotyping can be considered a valuable and useful technique to analyse chromosomal stability during preclinical studies". 408 chondrocyte samples (84 monolayers and 324 spheroids) from six patients were analysed using trypsin-Giemsa staining, spectral karyotyping and fluorescence in situ hybridisation, to evaluate the genetic stability of chondrocyte samples from non-clinical studies. Single nucleotide polymorphism (SNP) array analysis was performed on chondrocyte spheroids from five of the six donors. Applying this combination of techniques, the genetic analyses performed revealed no significant genetic instability until passage 3 in monolayer cells and interphase cells from spheroid cultures at different time points. Clonal occurrence of polyploid metaphases and endoreduplications were identified associated with prolonged cultivation time. Also, gonosomal losses were observed in chondrocyte spheroids, with increasing passage and duration of the differentiation phase. Interestingly, in one of the donors, chromosomal aberrations that are also described in extraskeletal myxoid chondrosarcoma were identified. The SNP array analysis exhibited chromosomal aberrations in two donors and copy neutral losses of heterozygosity regions in four donors. This study showed the necessity of combined genetic analyses at defined cultivation time points in quality studies within the field of cell therapy.


Assuntos
Corantes Azur/metabolismo , Condrócitos/metabolismo , Bandeamento Cromossômico , Loci Gênicos , Genômica/métodos , Hibridização in Situ Fluorescente , Polimorfismo de Nucleotídeo Único/genética , Cariotipagem Espectral , Idoso , Biópsia , Células Cultivadas , Aberrações Cromossômicas , Cromossomos Humanos/genética , Variações do Número de Cópias de DNA/genética , Endorreduplicação/genética , Feminino , Humanos , Perda de Heterozigosidade/genética , Masculino , Pessoa de Meia-Idade , Poliploidia , Esferoides Celulares/citologia
4.
Cytogenet Genome Res ; 152(3): 122-131, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28898877

RESUMO

Small cell lung cancer (SCLC) is a highly aggressive form of lung cancer. There is an urgent need to develop tools to identify individuals at high risk of developing SCLC. We have previously reported that the cytokinesis-blocked micronucleus (CBMN) assay is a strong predictor of non-small cell lung cancer (NSCLC). Here, we investigate the sensitivity of the CBMN endpoints as predictors of SCLC risk. We conducted the CBMN assay on SCLC patients (n = 216), NSCLC patients (n = 173), and healthy controls (n = 204). Per sample, 1,000 binucleated cells (BN) were scored, and 3 endpoints, micronuclei (BN-MN), nucleoplasmic bridges (BN-NPB), and nuclear buds(BN-BUD), were recorded. Spectral karyotyping was also conducted on SCLC patients (n = 116) and NSCLC patients (n = 137) to identify genomic regions unique to each disease. Significantly higher levels of CBMN endpoints were observed in both cancer groups compared to controls. BN-NPBs were significantly higher among SCLC patients compared to NSCLC patients (p < 0.001). Chromosomes 5 and 17 were associated with BN-MN, and chromosomes 5, 18, 20, and 22 were associated with BN-NPBs in SCLC patients. Given the high frequency of chromosome aberrations observed in SCLC, events such as reinsertion of the micronucleus and chromothripsis may be potential mechanisms for the genetic instability in these patients.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Detecção Precoce de Câncer/métodos , Neoplasias Pulmonares/diagnóstico , Testes para Micronúcleos/métodos , Carcinoma de Pequenas Células do Pulmão/diagnóstico , Cariotipagem Espectral/métodos , Idoso , Biomarcadores Tumorais/sangue , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/genética , Mapeamento Cromossômico , Cromotripsia , Citocinese/genética , Feminino , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Sensibilidade e Especificidade , Carcinoma de Pequenas Células do Pulmão/sangue , Carcinoma de Pequenas Células do Pulmão/genética , Fumar/efeitos adversos
5.
Taiwan J Obstet Gynecol ; 56(3): 394-397, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28600058

RESUMO

OBJECTIVE: We present prenatal diagnosis and molecular cytogenetic characterization of a small supernumerary marker chromosome (sSMC) derived from chromosome 11. CASE REPORT: A 37-year-old, gravida 3, para 2, woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Amniocentesis revealed a karyotype of 47,XX,+mar[18]/46,XX[4]. The parental karyotypes were normal. Level II ultrasound findings were unremarkable. Array comparative genomic hybridization (aCGH) on the DNA extracted from cultured amniocytes revealed no genomic imbalance. The sSMC was characterized by spectral karyotyping (SKY) using 24-color SKY probes and fluorescence in situ hybridization (FISH) using a whole chromosome paint (wcp) probe and a CEP11 (D11Z1) probe. The result was 47,XX,+mar.ish(11)(SKY+, wcp11+, D11Z1+)[16]/46,XX[4], indicating that the sSMC was derived from chromosome 11. A healthy female baby was delivered at 37 weeks of gestation with no phenotypic abnormalities. The cord blood had a karyotype of 47,XX,+mar[32]/46,XX[8]. Polymorphic DNA marker analysis of the blood excluded uniparental disomy 11. The female infant was normal in growth and psychomotor development during follow-ups at two months of age. CONCLUSION: aCGH, SKY and FISH are useful in prenatal diagnosis of an sSMC derived from the centromeric region of a non-acrocentric chromosome.


Assuntos
Transtornos Cromossômicos/diagnóstico , Cromossomos Humanos Par 11 , Marcadores Genéticos/genética , Mosaicismo , Adulto , Amniocentese , Transtornos Cromossômicos/genética , Hibridização Genômica Comparativa , Feminino , Humanos , Hibridização in Situ Fluorescente , Recém-Nascido , Gravidez , Diagnóstico Pré-Natal/métodos , Cariotipagem Espectral
6.
Oral Oncol ; 69: 1-10, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28559012

RESUMO

OBJECTIVE: The rising incidence of oral tongue squamous cell carcinoma (OTSCC) in patients who have never smoked and the paucity of knowledge of its biological behavior prompted us to develop a new cell line originating from a never-smoker. MATERIALS AND METHODS: Fresh tumor tissue of keratinizing OTSCC was collected from a 44-year-old woman who had never smoked. Serum-free media with a low calcium concentration were used in cell culture, and a multifaceted approach was taken to verify and characterize the cell line, designated UCSF-OT-1109. RESULTS: UCSF-OT-1109 was authenticated by STR DNA fingerprint analysis, presence of an epithelial marker EpCAM, absence of human papilloma virus (HPV) DNA, and SCC-specific microscopic appearance. Sphere-forming assays supported its tumorigenic potential. Spectral karyotype (SKY) analysis revealed numerical and structural chromosomal abnormalities. Whole-exome sequencing (WES) identified 46 non-synonymous and 13 synonymous somatic single-nucleotide polymorphisms (SNPs) and one frameshift deletion in the coding regions. Specifically, mutations of CDKN2A, TP53, SPTBN5, NOTCH2, and FAM136A were found in the databases. Copy number aberration (CNA) analysis revealed that the cell line loses chromosome 3p and 9p, but lacks amplification of 3q and 11q (as does HPV-negative, smoking-unrelated OTSCC). It also exhibits four distinctive focal amplifications in chromosome 19p, containing 131 genes without SNPs. Particularly, 52 genes showed >3- to 4-fold amplification and could be potential oncogenic drivers. CONCLUSION: We have successfully established a novel OTSCC cell line from a never-smoking patient. UCSF-OT-1109 is potentially a robust experimental model of OTSCC in never-smokers.


Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias da Língua/patologia , Adulto , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Meios de Cultura Livres de Soro , Feminino , Humanos , Mutação , Fumar , Cariotipagem Espectral , Neoplasias da Língua/genética
7.
Cytogenet Genome Res ; 151(1): 18-26, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28329743

RESUMO

Multicolor spectral analyses (spectral karyotyping) were performed on mitotic chromosomes of NMRI, CD, and TA mice and on male meiotic chromosomes (diakineses) of NMRI/CD and CD/TA hybrids. All chromosomes, including the various centric (robertsonian) fusions, could be unequivocally identified. Apart from the robertsonian translocations, which were previously detected by conventional banding analyses, no other interchromosomal rearrangements were found in these mice. In both the CD and TA mice, the autosomes 19 and the XY sex chromosomes are not involved in robertsonian translocations. In diakineses of male meiosis of the NMRI/CD hybrid, the 9 expected trivalents were present, whereas in those of the CD/TA hybrids a stable large meiotic multivalent, formed by 15 robertsonian fusion chromosomes and 2 terminally located normal chromosomes, was observed. The specific sequential order of the robertsonian fusion chromosomes found within this meiotic chain was as theoretically predicted. In the majority of diakineses of the NMRI/CD and CD/TA hybrids, the free autosomal bivalent 19 and the XY sex bivalent formed noticeable tight spatial associations.


Assuntos
Meiose/genética , Mitose/genética , Cariotipagem Espectral/métodos , Translocação Genética , Animais , Cromossomos de Mamíferos/genética , Hibridização Genética , Cariótipo , Cariotipagem , Masculino , Camundongos Endogâmicos , Microscopia de Fluorescência , Cromossomo X/genética , Cromossomo Y/genética
8.
J Vis Exp ; (119)2017 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-28117817

RESUMO

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.


Assuntos
Aberrações Cromossômicas , Hibridização in Situ Fluorescente/métodos , Cariotipagem Espectral/métodos , Animais , Coloração Cromossômica , Sondas de DNA , Corantes Fluorescentes , Camundongos , Translocação Genética
9.
Genes Chromosomes Cancer ; 56(3): 199-213, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27750367

RESUMO

Human colorectal carcinomas are defined by a nonrandom distribution of genomic imbalances that are characteristic for this disease. Often, these imbalances affect entire chromosomes. Understanding the role of these aneuploidies for carcinogenesis is of utmost importance. Currently, established transgenic mice do not recapitulate the pathognonomic genome aberration profile of human colorectal carcinomas. We have developed a novel model based on the spontaneous transformation of murine colon epithelial cells. During this process, cells progress through stages of pre-immortalization, immortalization and, finally, transformation, and result in tumors when injected into immunocompromised mice. We analyzed our model for genome and transcriptome alterations using ArrayCGH, spectral karyotyping (SKY), and array based gene expression profiling. ArrayCGH revealed a recurrent pattern of genomic imbalances. These results were confirmed by SKY. Comparing these imbalances with orthologous maps of human chromosomes revealed a remarkable overlap. We observed focal deletions of the tumor suppressor genes Trp53 and Cdkn2a/p16. High-level focal genomic amplification included the locus harboring the oncogene Mdm2, which was confirmed by FISH in the form of double minute chromosomes. Array-based global gene expression revealed distinct differences between the sequential steps of spontaneous transformation. Gene expression changes showed significant similarities with human colorectal carcinomas. Pathways most prominently affected included genes involved in chromosomal instability and in epithelial to mesenchymal transition. Our novel mouse model therefore recapitulates the most prominent genome and transcriptome alterations in human colorectal cancer, and might serve as a valuable tool for understanding the dynamic process of tumorigenesis, and for preclinical drug testing. © 2016 Wiley Periodicals, Inc.


Assuntos
Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/genética , Neoplasias Colorretais/genética , Modelos Animais de Doenças , Genoma , Transcriptoma/genética , Animais , Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Cariotipagem Espectral
11.
Methods Mol Biol ; 1541: 181-187, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27910024

RESUMO

Multicolor fluorescence in situ hybridization (mFISH) approaches are routine applications in tumor as well as clinical cytogenetics nowadays. The first approach when thinking about mFISH is multicolor karyotyping using human whole chromosome paints as probes; this can be achieved by narrow-band filter-based multiplex-FISH (M-FISH) or interferometer/spectroscopy-based spectral karyotyping (SKY). Besides, various FISH-based banding approaches were reported in the literature, including multicolor banding (MCB/mBAND) the latter being evaluated by narrow-band filters, and using specific software. Here, we describe the combined application of multicolor karyotyping and MCB/mBAND for the characterization of simple and complex acquired chromosomal changes in cancer cytogenetics.


Assuntos
Hibridização in Situ Fluorescente/métodos , Cariotipagem Espectral/métodos , Aberrações Cromossômicas , Bandeamento Cromossômico , Coloração Cromossômica , Biologia Computacional/métodos , Sondas de DNA , Humanos , Processamento de Imagem Assistida por Computador
12.
Lab Invest ; 97(3): 343-351, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27991910

RESUMO

Hereditary renal cell carcinomas (RCCs) are life-threatening disorders not only for the patients but also for their relatives. Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder caused by germline mutations in the folliculin gene (FLCN). The protein product, FLCN, functions as a tumor suppressor, and the affected patients have high risks of developing multiple RCCs. The carcinogenic mechanisms stemming from FLCN dysfunction have been investigated using rodent models and human RCC tissues. However, very limited information has been available about in vitro signaling of human renal cells with genetically mutant FLCN. Herein, we established a new cell line, BHD-F59RSVT, from a BHD patient's chromophobe RCC by transfecting SV40 large T antigen. We investigated FLCN mutations, chromosome profiles, and cytopathologic characteristics of the cell line. BHD-F59RSVT reflected the patient's FLCN germline mutation, a 3-nt deletion in exon 13 (c.1528_1530delGAG). Neither somatic mutation nor loss of heterozygosity of FLCN was detectable. Chromosome 17p11.2 of the FLCN proximal region demonstrated a trimodal pattern. Genome-wide chromosomal analysis revealed a loss of chromosome 16 and mosaic segmental gains in chromosome 7. BHD-F59RSVT cells were positive when immunostained for cytokeratin 7, supporting their origin from distal convoluted tubules. Western blotting analysis demonstrated severely suppressed FLCN expression at the protein level. The collective findings indicate that the established cell line will be suitable for functional analysis of the typical phenotype of BHD-associated RCC with suppressed FLCN expression.


Assuntos
Síndrome de Birt-Hogg-Dubé/genética , Carcinoma de Células Renais/genética , Mutação em Linhagem Germinativa , Neoplasias Renais/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Síndrome de Birt-Hogg-Dubé/complicações , Carcinoma de Células Renais/complicações , Carcinoma de Células Renais/patologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Análise Mutacional de DNA/métodos , Saúde da Família , Feminino , Humanos , Neoplasias Renais/complicações , Neoplasias Renais/patologia , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Linhagem , Proteínas Proto-Oncogênicas/metabolismo , Cariotipagem Espectral/métodos , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/metabolismo
13.
Mutat Res ; 809: 1-15, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27692294

RESUMO

Good cell culture practice and characterization of the cell lines used are of critical importance in in vitro genotoxicity testing. The objective of this initiative was to make continuously available stocks of the characterized isolates of the most frequently used mammalian cell lines in genotoxicity testing anywhere in the world ('IVGT' cell lines). This project was organized under the auspices of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing. First, cell isolates were identified that are as close as possible to the isolate described in the initial publications reporting their use in genotoxicity testing. The depositors of these cell lines managed their characterization and their expansion for preparing continuously available stocks of these cells that are stored at the European Collection of Cell Cultures (ECACC, UK) and the Japanese Collection of Research Bioresources (JCRB, Japan). This publication describes how the four 'IVGT' cell lines, i.e. L5178Y TK+/- 3.7.2C, TK6, CHO-WBL and CHL/IU, were prepared for deposit at the ECACC and JCRB cell banks. Recommendations for handling these cell lines and monitoring their characteristics are also described. The growth characteristics of these cell lines (growth rates and cell cycles), their identity (karyotypes and genetic status) and ranges of background frequencies of select endpoints are also reported to help in the routine practice of genotoxicity testing using these cell lines.


Assuntos
Técnicas de Cultura de Células/normas , Dano ao DNA/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfoma/tratamento farmacológico , Testes de Mutagenicidade/normas , Mutagênicos/toxicidade , Padrões de Referência , Animais , Células CHO , Células Cultivadas , Cricetulus , Relação Dose-Resposta a Droga , Humanos , Linfócitos/citologia , Linfócitos/metabolismo , Linfoma/metabolismo , Linfoma/patologia , Camundongos , Cariotipagem Espectral , Proteína Supressora de Tumor p53/metabolismo
14.
Anal Bioanal Chem ; 408(21): 5701-5709, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27277813

RESUMO

Current techniques for chromosome analysis need to be improved for rapid, economical identification of complex chromosomal defects by sensitive and selective visualisation. In this paper, we present a straightforward method for characterising unstained human metaphase chromosomes. Backscatter imaging in a dark-field setup combined with visible and short near-infrared spectroscopy is used to monitor morphological differences in the distribution of the chromosomal fine structure in human metaphase chromosomes. The reasons for the scattering centres in the fine structure are explained. Changes in the scattering centres during preparation of the metaphases are discussed. FDTD simulations are presented to substantiate the experimental findings. We show that local scattering features consisting of underlying spectral modulations of higher frequencies associated with a high variety of densely packed chromatin can be represented by their scatter profiles even on a sub-microscopic level. The result is independent of the chromosome preparation and structure size. This analytical method constitutes a rapid, cost-effective and label-free cytogenetic technique which can be used in a standard light microscope. Graphical abstract Hyperspectral backscatter imaging for label-free characterization.


Assuntos
Cromossomos/ultraestrutura , Análise Citogenética/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Cromossomos/química , Humanos , Metáfase , Microscopia/métodos , Imagem Óptica/métodos , Cariotipagem Espectral/métodos
15.
PLoS One ; 11(3): e0149833, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26962861

RESUMO

The genetic profile of human pancreatic cancers harbors considerable heterogeneity, which suggests a possible explanation for the pronounced inefficacy of single therapies in this disease. This observation has led to a belief that custom therapies based on individual tumor profiles are necessary to more effectively treat pancreatic cancer. It has recently been discovered that axon guidance genes are affected by somatic structural variants in up to 25% of human pancreatic cancers. Thus far, however, some of these mutations have only been correlated to survival probability and no function has been assigned to these observed axon guidance gene mutations in pancreatic cancer. In this study we established three novel pancreatic cancer cell lines and performed whole genome sequencing to discover novel mutations in axon guidance genes that may contribute to the cancer phenotype of these cells. We discovered, among other novel somatic variants in axon guidance pathway genes, a novel mutation in the PLXNA1 receptor (c.2587G>A) in newly established cell line SB.06 that mediates oncogenic cues of increased invasion and proliferation in SB.06 cells and increased invasion in 293T cells upon stimulation with the receptor's natural ligand semaphorin 3A compared to wild type PLXNA1 cells. Mutant PLXNA1 signaling was associated with increased Rho-GTPase and p42/p44 MAPK signaling activity and cytoskeletal expansion, but not changes in E-cadherin, vimentin, or metalloproteinase 9 expression levels. Pharmacologic inhibition of the Rho-GTPase family member CDC42 selectively abrogated PLXNA1 c.2587G>A-mediated increased invasion. These findings provide in-vitro confirmation that somatic mutations in axon guidance genes can provide oncogenic gain-of-function signals and may contribute to pancreatic cancer progression.


Assuntos
Axônios/metabolismo , Genoma Humano , Mutação/genética , Proteínas do Tecido Nervoso/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Receptores de Superfície Celular/genética , Idoso , Linhagem Celular Tumoral , Proliferação de Células , Cromossomos Humanos/genética , Análise Mutacional de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Imunofenotipagem , Ligantes , Masculino , Invasividade Neoplásica , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Cariotipagem Espectral , Transfecção , Proteína cdc42 de Ligação ao GTP/metabolismo
17.
Cytogenet Genome Res ; 150(3-4): 287-292, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28249294

RESUMO

The t(11;20)(p15;q11∼12) translocation is a very rare but recurrent cytogenetic aberration that occurs in myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). This translocation was shown to form a fusion gene between NUP98 at 11p15 and TOP1 at 20q12. Here, we describe a new case of de novo AML M2 with t(11;20) which was associated with another balanced translocation. An 81-year-old man was admitted to undergo salvage therapy for relapsed AML. G-banding and spectral karyotyping showed 46,XY,t(2;5)(q33;q31),t(11;20)(p15;q12)[20]. Expression of the NUP98/TOP1 fusion transcript was confirmed: NUP98 exon 13 was in-frame fused with TOP1 exon 8. The reciprocal TOP1/NUP98 fusion transcript was also detected: TOP1 exon 7 was fused with NUP98 exon 14. After achieving hematological complete remission, the karyotype converted to 46,XY,t(2;5)(q33;q31)[19]/46,sl,t(11;20)(p15;q12)[1]. FISH analysis demonstrated that the 5q31 breakpoint of t(2;5) was centromeric to EGR1. In all 10 cases described in the literature, the NUP98 exon 13/TOP1 exon 8 fusion transcript was expressed, indicating that it may be responsible for the pathogenesis of MDS/AML with t(11;20). On the other hand, the TOP1/NUP98 transcript was coexpressed in 4 cases of de novo AML, but not in 3 cases of therapy-related MDS. Thus, this reciprocal fusion may be associated with progression to AML.


Assuntos
Cromossomos Humanos Par 11 , Cromossomos Humanos Par 20 , Cromossomos Humanos Par 2 , Cromossomos Humanos Par 5 , DNA Topoisomerases Tipo I/genética , Leucemia Mieloide Aguda/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Translocação Genética , Idoso , Idoso de 80 Anos ou mais , Humanos , Masculino , Cariotipagem Espectral
18.
Blood ; 127(1): 102-12, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26385350

RESUMO

Somatic hypermutation and class-switch recombination of the immunoglobulin (Ig) genes occur in germinal center (GC) B cells and are initiated through deamination of cytidine to uracil by activation-induced cytidine deaminase (AID). Resulting uracil-guanine mismatches are processed by uracil DNA glycosylase (UNG)-mediated base-excision repair and MSH2-mediated mismatch repair (MMR) to yield mutations and DNA strand lesions. Although off-target AID activity also contributes to oncogenic point mutations and chromosome translocations associated with GC and post-GC B-cell lymphomas, the role of downstream AID-associated DNA repair pathways in the pathogenesis of lymphoma is unknown. Here, we show that simultaneous deficiency of UNG and MSH2 or MSH2 alone causes genomic instability and a shorter latency to the development of BCL6-driven diffuse large B-cell lymphoma (DLBCL) in a murine model. The additional development of several BCL6-independent malignancies in these mice underscores the critical role of MMR in maintaining general genomic stability. In contrast, absence of UNG alone is highly protective and prevents the development of BCL6-driven DLBCL. We further demonstrate that clonal and nonclonal mutations arise within non-Ig AID target genes in the combined absence of UNG and MSH2 and that DNA strand lesions arise in an UNG-dependent manner but are offset by MSH2. These findings lend insight into a complex interplay whereby potentially deleterious UNG activity and general genomic instability are opposed by the protective influence of MSH2, producing a net protective effect that promotes immune diversification while simultaneously attenuating malignant transformation of GC B cells.


Assuntos
Transformação Celular Neoplásica/patologia , Citidina Desaminase/metabolismo , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Proteína 2 Homóloga a MutS/fisiologia , Uracila-DNA Glicosidase/fisiologia , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/genética , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Centro Germinativo , Técnicas Imunoenzimáticas , Switching de Imunoglobulina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Proteínas Proto-Oncogênicas c-bcl-6 , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Hipermutação Somática de Imunoglobulina/genética , Cariotipagem Espectral , Células Tumorais Cultivadas
19.
Int J Hematol ; 103(2): 196-201, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26676804

RESUMO

We performed cytogenetic and molecular cytogenetic analyses of a primary cutaneous CD8-positive aggressive epidermotropic cytotoxic T-cell lymphoma, a rare type of primary cutaneous T-cell lymphoma. G-banded analysis at initial diagnosis and recurrence revealed complex karyotype and clonal evolution reflecting genomic instability that parallels the aggressive clinical course observed. Spectral karyotyping revealed numerous structural abnormalities. SNP array-based analysis of an initial diagnostic sample revealed numerous gains and losses of chromosomal material, including loss of short arm of the chromosome 17, to which TP53 is mapped. The molecular cytogenetics and array data of this case suggest genomic instability, particularly chromosomal instability and haploinsufficiency for TP53, the latter possibly giving rise to alteration of p14ARF-Mdm2-p53 tumor suppressor protein pathway, likely to be associated with unfavorable clinical course.


Assuntos
Antígenos CD8 , Análise Citogenética , Linfoma Cutâneo de Células T/genética , Criança , Cromossomos Humanos Par 17/genética , Feminino , Instabilidade Genômica , Humanos , Polimorfismo de Nucleotídeo Único , Cariotipagem Espectral , Proteína Supressora de Tumor p53/genética
20.
PLoS One ; 10(11): e0139663, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26599546

RESUMO

Primary mediastinal B-Cell lymphoma (PMBL) is a recently defined entity comprising ~2-10% non-Hodgkin lymphomas (NHL). Unlike most NHL subtypes, PMBL lacks recurrent gene rearrangements to serve as biomarkers or betray target genes. While druggable, late chemotherapeutic complications warrant the search for new targets and models. Well characterized tumor cell lines provide unlimited material to serve as preclinical resources for verifiable analyses directed at the discovery of new biomarkers and pathological targets using high throughput microarray technologies. The same cells may then be used to seek intelligent therapies directed at clinically validated targets. Four cell lines have emerged as potential PMBL models: FARAGE, KARPAS-1106P, MEDB-1 and U-2940. Transcriptionally, PMBL cell lines cluster near c(lassical)-HL and B-NHL examples showing they are related but separate entities. Here we document genomic alterations therein, by cytogenetics and high density oligonucleotide/SNP microarrays and parse their impact by integrated global expression profiling. PMBL cell lines were distinguished by moderate chromosome rearrangement levels undercutting cHL, while lacking oncogene translocations seen in B-NHL. In total 61 deletions were shared by two or more cell lines, together with 12 amplifications (≥4x) and 72 homozygous regions. Integrated genomic and transcriptional profiling showed deletions to be the most important class of chromosome rearrangement. Lesions were mapped to several loci associated with PMBL, e.g. 2p15 (REL/COMMD1), 9p24 (JAK2, CD274), 16p13 (SOCS1, LITAF, CIITA); plus new or tenuously associated loci: 2p16 (MSH6), 6q23 (TNFAIP3), 9p22 (CDKN2A/B), 20p12 (PTPN1). Discrete homozygous regions sometimes substituted focal deletions accompanied by gene silencing implying a role for epigenetic or mutational inactivation. Genomic amplifications increasing gene expression or gene-activating rearrangements were respectively rare or absent. Our findings highlight biallelic deletions as a major class of chromosomal lesion in PMBL cell lines, while endorsing the latter as preclinical models for hunting and testing new biomarkers and actionable targets.


Assuntos
Genoma Humano , Linfoma de Células B/genética , Neoplasias do Mediastino/genética , Idoso , Linhagem Celular Tumoral , Análise por Conglomerados , Hibridização Genômica Comparativa , Análise Citogenética , Variações do Número de Cópias de DNA/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Loci Gênicos , Humanos , Hibridização in Situ Fluorescente , Perda de Heterozigosidade/genética , Polimorfismo de Nucleotídeo Único/genética , Análise de Componente Principal , Cariotipagem Espectral , Transcrição Genética
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