RESUMO
Karyotyping remains an invaluable tool to researchers exploring the cause and consequence of genomic instability in biologic systems. It allows investigators to survey the entire chromosome complement in individual cells and in a single experiment, visualize, and measure different forms or features of instability such as aneuploidy, ongoing chromosomal instability, DNA damage/mis-repair, telomere erosion, chromosome mis-segregation, or defects in cell cycle progression. This chapter describes the combined use of conventional (DAPI-banding) and spectral karyotyping (SKY) to characterize genomic instability in murine cerebellar granule neuron progenitors (CGNPs), using CGNPs with conditional deletion of Atr as a positive control for chromosomal rearrangements. Protocols for preparing slides (metaphase spreads) from fixed cell suspension, DAPI-banding, and spectral karyotyping (SKY) are included. Pertinent aspects of image acquisition and analysis are detailed. These protocols can likely be adapted to other tissue types (murine or human).
Assuntos
Aberrações Cromossômicas , Instabilidade Genômica , Humanos , Camundongos , Animais , Cariotipagem Espectral , Cariotipagem , NeurôniosRESUMO
Karyotype analysis has a great impact on the diagnosis, treatment and prognosis in hematologic neoplasms. The identification and characterization of chromosomes is a challenging process and needs experienced personal. Artificial intelligence provides novel support tools. However, their safe and reliable application in diagnostics needs to be evaluated. Here, we present a novel laboratory approach to identify chromosomes in cancer cells using a convolutional neural network (CNN). The CNN identified the correct chromosome class for 98.8% of chromosomes, which led to a time saving of 42% for the karyotyping workflow. These results demonstrate that the CNN has potential application value in chromosome classification of hematologic neoplasms. This study contributes to the development of an automatic karyotyping platform.
Assuntos
Bandeamento Cromossômico/métodos , Neoplasias Hematológicas/genética , Cariotipagem Espectral/métodos , Algoritmos , Feminino , Humanos , Masculino , Metáfase , Redes Neurais de Computação , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
OBJECTIVE: To evaluate the clinical value of BACs-on-Beads (BoBs) assay in detection of microdeletion and microduplication syndromes. METHODS: A total of 6,814 cases of amniotic fluid cells collected from January 2015 to July 2020 in our hospital were analyzed by chromosomal karyotyping and BoBs assay. Fluorescence in situ hybridization (FISH) or chromosomal microarray analysis (CMA) provided further validation for the cases of microdeletion and microduplication. RESULTS: Thirty microdeletion and microduplication syndromes were identified by BoBs with an incidence of ~1/227, including 22q11.2 microduplication (0.044%, 3/6814), DiGeorge I syndrome (0.044%, 3/6814), 17p11.2 microduplication (0.015%, 1/6814), Smith-Magenis syndrome (0.015%, 1/6814), 17p11.2p11.3 microduplication (0.015%, 1/6814), Williams-Beuren syndrome (0.088%, 6/6814), 7q11.2 microduplication (0.029%, 2/6814), DiGeorge II syndrome (0.015%, 1/6814), 18p11.32p11.21 microduplication (0.015%, 1/6814), Wolf-Hirschhorn syndrome (0.029%, 2/6814), 4p16.3 microduplication (0.015%, 1/6814), Langer-Giedion syndrome (0.015%, 1/6814), Miller-Dieker syndrome (0.015%, 1/6814), Cri du Chat syndrome (0.015%, 1/6814), Xp22.31 microdeletion (0.059%, 4/6814), Prader-Willi syndrome (0.015%, 1/6814). High concordance was obtained between BoBs and FISH or CMA. However, only four cases were detected by chromosomal karyotyping. CONCLUSION: BoBs assay can rapidly detect microdeletion and microduplication syndromes, which compensates the shortcomings of conventional chromosomal karyotyping and greatly improves the efficiency and accuracy of prenatal diagnosis.
Assuntos
Deleção Cromossômica , Duplicação Cromossômica , Diagnóstico Pré-Natal/métodos , Cariotipagem Espectral/métodos , Adulto , Líquido Amniótico/química , Cromossomos Artificiais Bacterianos/genética , Feminino , Idade Gestacional , Humanos , Masculino , Idade Materna , Análise em Microsséries , GravidezRESUMO
Acute myeloid leukemia (AML) with an inv(16)(p13q22) or t(16;16)(p13;q22) chromosomal abnormality represents one of the most common subtypes of de novo cases. These chromosomal rearrangements result in multiple CBFB-MYH11 fusion transcripts, with type-A being the most frequent. We here describe a unique case of de novo AML-M1, with inv(16)(p13q22), leading to an unusual CBFB-MYH11 fusion transcript, and der(7)t(7;11)(q31;q21). The fusion transcript involves a CBFB exon 5 with a breakpoint at nucleotide 754, an insertion of a 13-bp sequence of CBFB intron 5 at the fusion point, and the MYH11 exon 27 with a breakpoint at nucleotide 3464. To our knowledge, this CBFB-MYH11 fusion transcript has never been reported previously. The clinical characteristics of the present case are in line with previous reports suggesting that rare CBFB-MYH11 fusion transcripts lead to aberrant characteristics such as an atypical cytomorphology and additional cytogenetic abnormalities.
Assuntos
Cromossomos Humanos Par 16/genética , Rearranjo Gênico , Leucemia Mieloide Aguda/genética , Proteínas de Fusão Oncogênica/genética , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Medula Óssea/patologia , Feminino , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Cariotipagem Espectral , Resultado do TratamentoRESUMO
The human kidney embryonic 293 cell line (293 cells) is extensively used in biomedical and pharmaceutical research. These cells exhibit a number of numerical and structural chromosomal anomalies. However, the breakpoints responsible for these structural chromosomal rearrangements have not been comprehensively characterized. In addition, it is not known whether chromosomes with structural rearrangement are more sensitive to external toxic agents, such as ionizing radiation. We used G-banding, spectral karyotyping (SKY), and locus- and region-specific fluorescence in situ hybridization (FISH) probes designed in our lab or obtained from commercial vendor to address this gap. Our G-banding analysis revealed that the chromosome number varies from 66 to 71, with multiple rearrangements and partial additions and deletions. SKY analysis confirmed 3 consistent rearrangements, two simple and one complex in nature. Multicolor FISH analysis identified an array of breakpoints responsible for locus- and region-specific translocations. Finally, SKY analysis revealed that radio-sensitivity of structurally rearranged chromosomes is dependent on radiation dose. These findings will advance our knowledge in 293 cell biology and will enrich the understanding of radiation biology studies.
Assuntos
Pontos de Quebra do Cromossomo/efeitos da radiação , Translocação Genética/efeitos da radiação , Aberrações Cromossômicas , Bandeamento Cromossômico , Coloração Cromossômica , Citogenética , Rearranjo Gênico/efeitos da radiação , Células HEK293 , Humanos , Tolerância a Radiação/genética , Cariotipagem EspectralRESUMO
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 adversosRESUMO
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 , HumanosRESUMO
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/citologiaRESUMO
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 adversosRESUMO
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 EspectralRESUMO
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/genéticaRESUMO
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/genéticaRESUMO
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 GenéticaAssuntos
Linfoma Difuso de Grandes Células B/diagnóstico por imagem , Neoplasias Vasculares/diagnóstico por imagem , Feminino , Humanos , Linfoma Difuso de Grandes Células B/genética , Imageamento por Ressonância Magnética/métodos , Pessoa de Meia-Idade , Cariotipagem Espectral/métodos , Neoplasias Vasculares/genéticaRESUMO
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 EspectralRESUMO
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 ComputadorRESUMO
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/metabolismoRESUMO
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/metabolismoRESUMO
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/métodosRESUMO
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.