RESUMO
BACKGROUND: International databases with information on copy number variation of the human genome are an important reference for laboratories using high resolution whole genome screening. Genomic deletions or duplications which have been detected in the healthy population and thus marked as normal copy number variants (CNVs) can be filtered out using these databases when searching for pathogenic copy number changes in patients. However, a potential pitfall of this strategy is that reported normal CNVs often do not elicit further investigation, and thus may remain unrecognised when they are present in a (pathogenic) homozygous state. The impact on disease of CNVs in the homozygous state may thus remain undetected and underestimated. METHODS AND RESULTS: In a patient with syndromic hearing loss, array comparative genomic hybridisation (array CGH) and multiple ligation dependent probe amplification (MLPA) revealed a homozygous deletion on 15q15.3 of a CNV, inherited from hemizygous carrier parents. The deletion is about 90 kilobases and contains four genes including the STRC gene, which is involved in autosomal recessive deafness (DFNB16). By screening healthy control individuals and review of publicly available CNV data we estimated the frequency of hemizygous deletion carriers to be about 1.6%. CONCLUSION: We characterised a homozygous deletion of a CNV region causing syndromic hearing loss by a panel of molecular tools. Together with the estimated frequency of the hemizygous deletion, these results emphasise the role of the 15q15.3 locus in patients with (syndromic) hearing impairment. Furthermore, this case illustrates the importance of not automatically eliminating registered CNVs from further analysis.
Assuntos
Cromossomos Humanos Par 15/genética , Deleção de Genes , Perda Auditiva/genética , Criança , Bandeamento Cromossômico , Hibridização Genômica Comparativa , Dosagem de Genes , Homozigoto , Humanos , Deficiência Intelectual/genética , Masculino , Técnicas de Amplificação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de DNA , SíndromeRESUMO
Cajal bodies (CBs) are subnuclear organelles that contain components of a number of distinct pathways in RNA transcription and RNA processing. CBs have been linked to other subnuclear organelles such as nucleoli, but the reason for the presence of nucleolar proteins such as fibrillarin in CBs remains uncertain. Here, we use full-length fibrillarin and truncated fibrillarin mutants fused to green fluorescent protein (GFP) to demonstrate that specific structural domains of fibrillarin are required for correct intranuclear localization of fibrillarin to nucleoli and CBs. The second spacer domain and carboxy terminal alpha-helix domain in particular appear to target fibrillarin, respectively, to the nucleolar transcription centers and CBs. The presence of the RNP domain seems to be a prerequisite for correct targeting of fibrillarin. Time-lapse confocal microscopy of human cells that stably express fibrillarin-GFP shows that CBs fuse and split, albeit at low frequencies. Recovered fluorescence of fibrillarin-GFP in nucleoli and CBs after photobleaching indicates that it is highly mobile in both organelles (estimated diffusion constant approximately 0.02 microm(2) s(-1)), and has a significantly larger mobile fraction in CBs than in nucleoli.
Assuntos
Nucléolo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Corpos Enovelados/metabolismo , Mutação/genética , Transporte Ativo do Núcleo Celular , Nucléolo Celular/química , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Corpos Enovelados/química , Difusão , Imunofluorescência , Humanos , Cinética , Movimento (Física) , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência/genética , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Microdeletions of 3q29 have previously been reported, but the postulated reciprocal microduplication has only recently been observed. Here, cases from four families, two ascertained in Toronto (Canada) and one each from Edinburgh (UK) and Leiden (Netherlands), carrying microduplications of 3q29 are presented. These families have been characterized by cytogenetic and molecular techniques, and all individuals have been further characterized with genome-wide, high density single nucleotide polymorphism (SNP) arrays run at a single centre (The Centre for Applied Genomics, Toronto). In addition to polymorphic copy-number variants (CNV), all carry duplications of 3q29 ranging in size from 1.9 to 2.4 Mb, encompassing multiple genes and defining a minimum region of overlap of about 1.6 Mb bounded by clusters of segmental duplications that is remarkably similar in location to previously reported 3q29 microdeletions. Consistent with other reports, the phenotype is variable, although developmental delay and significant ophthalmological findings were recurrent, suggesting that dosage sensitivity of genes located within 3q29 is important for eye and CNS development. We also consider CNVs found elsewhere in the genome for their contribution to the phenotype. We conclude by providing preliminary guidelines for management and anticipatory care of families with this microduplication, thereby establishing a standard for CNV reporting.
Assuntos
Cromossomos Humanos/genética , Dosagem de Genes/genética , Duplicação Gênica , Predisposição Genética para Doença/genética , Feminino , Guias como Assunto , Humanos , MasculinoRESUMO
An important application of robotically spotted DNA microarrays is the monitoring of RNA expression levels. A clear limitation of this technology is the relatively large amount of RNA that is required per hybridization as a result of low hybridization efficiency and limiting detection sensitivity provided by conventional fluorescent reporters. We have used a recently introduced luminescent reporter technology, called UPT (up-converting phosphor technology). Down-converting phosphors have been applied before to detect nucleic acids on filters using time-resolved fluorometry. The unique feature of the phosphor particles (size 0.4 microm) used here is that they emit visible light when illuminated with infrared (IR) light (980 nm) as a result of a phenomenon called up-conversion. Because neither support material of microarrays nor biomolecules possess up-conversion properties, an enhanced image contrast is expected when these nonfading phosphor particles are applied to detect nucleic acid hybrids on microarrays. Comparison of the UPT reporter to cyanin 5 (Cy5) in a low-complexity model system showed a two order of maginitude linear relationship between phosphor luminescence and target concentration and resulted in an excellent correlation between the two reporter systems for variable target concentrations (R2 = 0.95). However, UPT proved to be superior in sensitivity, even though a wide-field microscope equipped with a xenon lamp was used. This higher sensitivity was demonstrated by complementary DNA (cDNA) microarray hybridizations using cDNAs for housekeeping genes as probes and complex cDNA as target. These results suggest that a UPT reporter technology in combination with a dedicated IR laser array-scanner holds significant promise for various microarray applications.
Assuntos
Medições Luminescentes , Sondas Moleculares , Ácidos Nucleicos/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Biotinilação , Carbocianinas/metabolismo , Clonagem Molecular , Sondas de DNA/genética , DNA Complementar/genética , Humanos , Raios Infravermelhos , Ácidos Nucleicos/análise , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , RNA Mensageiro/genética , Sensibilidade e Especificidade , Coloração e Rotulagem/métodosRESUMO
BACKGROUND: The underlying causes of mental retardation remain unknown in about half the cases. Recent array-CGH studies demonstrated cryptic imbalances in about 25% of patients previously thought to be chromosomally normal. OBJECTIVE AND METHODS: Array-CGH with approximately 3500 large insert clones spaced at approximately 1 Mb intervals was used to investigate DNA copy number changes in 81 mentally impaired individuals. RESULTS: Imbalances never observed in control chromosomes were detected in 20 patients (25%): seven were de novo, nine were inherited, and four could not have their origin determined. Six other alterations detected by array were disregarded because they were shown by FISH either to hybridise to both homologues similarly in a presumptive deletion (one case) or to involve clones that hybridised to multiple sites (five cases). All de novo imbalances were assumed to be causally related to the abnormal phenotypes. Among the others, a causal relation between the rearrangements and an aberrant phenotype could be inferred in six cases, including two imbalances of the X chromosome, where the associated clinical features segregated as X linked recessive traits. CONCLUSIONS: In all, 13 of 81 patients (16%) were found to have chromosomal imbalances probably related to their clinical features. The clinical significance of the seven remaining imbalances remains unclear. The limited ability to differentiate between inherited copy number variations which cause abnormal phenotypes and rare variants unrelated to clinical alterations currently constitutes a limitation in the use of CGH-microarray for guiding genetic counselling.
Assuntos
Desequilíbrio Alélico/genética , Rearranjo Gênico/genética , Hibridização in Situ Fluorescente , Deficiência Intelectual/genética , Criança , Cromossomos Humanos Par 2/genética , HumanosRESUMO
The ability to probe for the location of DNA sequences in morphologically preserved chromosomes and nuclei by fluorescence in situ hybridization (FISH) provided for cytogenetics a quantum leap forward in resolution and ease of detection of chromosomal aberrations. COBRA-FISH, an acronym for COmbined Binary RAtio-FISH is a multicolor FISH methodology, which enables recognition of all human chromosome arms on the basis of color, thus greatly facilitating cytogenetic analysis. It also permits gene and viral integration site mapping in the context of chromosome arm painting. Here we review the principle, practice and applications of COBRA-FISH.
Assuntos
Hibridização in Situ Fluorescente/métodos , Hibridização in Situ Fluorescente/tendências , Hibridização In Situ/métodos , Hibridização In Situ/tendências , Corantes Fluorescentes , Humanos , Modelos GenéticosRESUMO
Epithelial tumour karyotypes are often difficult to study by standard cytogenetic methods because of poor chromosome preparation quality and the high complexity of their genomic rearrangements. Subtelomeric fluorescence in situ hybridisation (FISH) has proved to be a useful method for detecting cryptic constitutional chromosomal rearrangements but little is known about its usefulness for tumour cytogenetic analysis. Using a combination of chromosome banding, multicolour karyotyping and subtelomeric FISH, five colorectal cancer cell lines were characterised. The resulting data were compared to results from previous studies by comparative genomic hybridisation and spectral karyotyping or multicolour FISH. Subtelomeric FISH made it possible to resolve several highly complex chromosome rearrangements, many of which had not been detected or were incompletely characterised by the other methods. In particular, previously undetected terminal imbalances were found in the two cell lines not showing microsatellite instability.
Assuntos
Quebra Cromossômica , Mapeamento Cromossômico , Neoplasias Colorretais/genética , Telômero/genética , Linhagem Celular Tumoral , Bandeamento Cromossômico , Quebra Cromossômica/genética , Rearranjo Gênico/genética , Humanos , Hibridização in Situ Fluorescente/métodos , CariotipagemRESUMO
AIMS: Presently, in Europe the treatment of node-negative colorectal cancer (CRC) patients consists of surgical resection of the primary tumour without adjuvant systemic therapy. However, up to 30% of these patients will develop disease recurrence. These high-risk patients are possibly identified by occult tumour cell (OTC) assessment in lymph nodes. In this paper, studies on the clinical relevance of OTC in lymph nodes are reviewed. METHODS: A literature search was conducted in the National Library of Medicine by using the keywords colonic, rectal, colorectal, neoplasm, adenocarcinoma, cancer, lymph node, polymerase chain reaction, mRNA, immunohistochemistry, micrometastases and isolated tumour cells. Additional articles were identified by cross-referencing from papers retrieved in the initial search. RESULTS: The upstaging percentages through OTC assessment and the prognostic relevance of OTC in lymph nodes vary among studies, which is related to differences in techniques used to detect OTC. CONCLUSIONS: We conclude that OTC examination techniques should be standardized to illuminate whether OTC in lymph nodes can reliably identify high-risk node-negative patients.
Assuntos
Neoplasias Colorretais/patologia , Linfonodos/patologia , Anticorpos Antineoplásicos/análise , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , DNA de Neoplasias/análise , Humanos , Imuno-Histoquímica , Metástase Linfática , Reação em Cadeia da PolimeraseRESUMO
The association of a particular mitochondrial DNA (mtDNA) mutation with different clinical phenotypes is a well-known feature of mitochondrial diseases. A simple genotype-phenotype correlation has not been found between mutation load and disease expression. Tissue and intercellular mosaicism as well as mtDNA copy number are thought to be responsible for the different clinical phenotypes. As disease expression of mitochondrial tRNA mutations is mostly in postmitotic tissues, studies to elucidate disease mechanisms need to be performed on patient material. Heteroplasmy quantitation and copy number estimation using small patient biopsy samples has not been reported before, mainly due to technical restrictions. In order to resolve this problem, we have developed a robust assay that utilizes Molecular Beacons to accurately quantify heteroplasmy levels and determine mtDNA copy number in small samples carrying the A8344G tRNA(Lys) mutation. It provides the methodological basis to investigate the role of heteroplasmy and mtDNA copy number in determining the clinical phenotypes.
Assuntos
DNA Mitocondrial/genética , Síndrome MERRF/genética , Reação em Cadeia da Polimerase/métodos , Linhagem Celular , Fluorescência , Dosagem de Genes , Humanos , Síndrome MERRF/patologia , Mutação Puntual , Sensibilidade e EspecificidadeRESUMO
U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Methyl oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.
Assuntos
Sondas RNA , RNA/metabolismo , Animais , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Citomegalovirus/genética , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde , Humanos , Hibridização in Situ Fluorescente , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microinjeções , Microscopia de Fluorescência/métodos , Proteínas Nucleares/genética , Poli A/genética , Poli A/metabolismo , RNA/genética , Sondas RNA/administração & dosagem , Sondas RNA/química , Sondas RNA/genética , RNA Ribossômico 28S/genética , RNA Ribossômico 28S/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Processamento de Serina-Arginina , Células Tumorais CultivadasRESUMO
Progressive chromosomal changes and integration of human papillomavirus (HPV) sequences mark the development of invasive cervical cancer. Chromosomal localization of HPV integration is essential to the study of genomic regions involved in HPV-induced pathogenesis. Yet, the available information about HPV integration loci is still limited, especially with respect to different HPV types. We have established cell lines from five cervical cancers with HPV-16, HPV-45, and HPV-67. We have determined HPV integration sites and karyotype abnormalities by using the multicolor combined binary ratio-fluorescence in situ hybridization method (Tanke et al.) with 24 chromosome-specific paints in combination with full-length HPV DNA probes. All cell lines were cytogenetically abnormal, and exhibited numerical and structural chromosomal deviations. HPV sequences were integrated at various (segments of) chromosomes. Duplicate integration sites were seen in all multiploid cell lines, suggesting that viral integration had preceded chromosomal endoreduplication. HPV-16 was found near the t(3p14.1-14.3;14) breakpoint in cervical squamous cell carcinoma (CSCC)-7 and mainly in episomal form in CSCC-1. HPV-45 was integrated near 3q26-29 in cervical (adeno or adenosquamous) carcinoma (CC)-8 and near 1q21-23 as well as near the t(1q21;22q13) breakpoint in CC-10A and CC-10B variant lines. HPV-67 was localized near the breakpoint of t(3p23-26;13q22-31) in CC-11. Southern blot analysis showed that, except for CSCC-1, the physical state of HPV in the cell lines was the same as in the original tumor lesions. This set of six cervical cancer cell lines included three lines with HPV-45, a major non-Western high-risk HPV type, the first reported HPV-67-positive cell line, and two cell lines with integrated and episomal HPV-16 DNA, respectively. The novel combined binary ratio-fluorescence in situ hybridization technique enabled us to simultaneously map chromosomal rearrangements and HPV integration sites, thereby revealing recurrent integration near translocation junctions for all of these HPV types in the cell lines from three of the five primary tumors. The detection of multiple HPV integration sites at rearranged chromosomes at such high frequency in cervical cancer-derived cells may reflect events that are relevant to the development of cervical cancer.
Assuntos
DNA Viral/genética , Papillomaviridae/genética , Translocação Genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Integração Viral , Adulto , Idoso , Southern Blotting , Técnicas de Cultura de Células/métodos , Feminino , Humanos , Hibridização in Situ Fluorescente , Interfase , Cariotipagem , Pessoa de Meia-Idade , Células Tumorais CultivadasRESUMO
Incidence rates have risen rapidly for esophageal and gastric cardia adenocarcinomas. These cancers, arising at and around the gastroesophageal junction (GEJ), share a poor prognosis. In contrast, there is no consensus with respect to clinical staging resulting in possible adverse effects on treatment and survival. The goal of this study was to provide more insight into the genetic changes underlying esophageal and gastric cardia adenocarcinomas. We have used comparative genomic hybridization for a genetic analysis of 28 adenocarcinomas of the GEJ. Eleven tumors were localized in the distal esophagus and related to Barrett's esophagus, and 10 tumors were situated in the gastric cardia. The remaining seven tumors were located at the junction and could not be classified as either Barrett-related, or gastric cardia. We found alterations in all 28 neoplasms. Gains and losses were distinguished in comparable numbers. Frequent loss (> or = 25% of all tumors) was detected, in decreasing order of frequency, on 4pq (54%), 14q (46%), 18q (43%), 5q (36%), 16q (36%), 9p (29%), 17p (29%), and 21q (29%). Frequent gain (> or = 25% of all tumors) was observed, in decreasing order of frequency, on 20pq (86%), 8q (79%), 7p (61%), 13q (46%), 12q (39%), 15q (39%), 1q (36%), 3q (32%), 5p (32%), 6p (32%), 19q (32%), Xpq (32%), 17q (29%), and 18p (25%). Nearly all patients were male, and loss of chromosome Y was frequently noted (64%). Recurrent high-level amplifications (> 10% of all tumors) were seen at 8q23-24.1, 15q25, 17q12-21, and 19q13.1. Minimal overlapping regions could be determined at multiple locations (candidate genes are in parentheses): minimal regions of overlap for deletions were assigned to 3p14 (FHIT, RCA1), 5q14-21 (APC, MCC), 9p21 (MTS1/CDKN2), 14q31-32.1 (TSHR), 16q23, 18q21 (DCC, P15) and 21q21. Minimal overlapping amplified sites could be seen at 5p14 (MLVI2), 6p12-21.1 (NRASL3), 7p12 (EGFR), 8q23-24.1 (MYC), 12q21.1, 15q25 (IGF1R), 17q12-21 (ERBB2/HER2-neu), 19q13.1 (TGFB1, BCL3, AKT2), 20p12 (PCNA), 20q12-13 (MYBL2, PTPN1), and Xq25. The distribution of the imbalances revealed similar genetic patterns in the three GEJ tumor groups. However, loss of 14q31-32.1 occurred significantly more frequent in Barrett-related adenocarcinomas of the distal esophagus, than in gastric cardia cancers (P = 0.02). The unclassified, "pure junction" group displayed an intermediate position, suggesting that these may be in part gastric cardia tumors, whereas the others may be related to (short-segment) Barrett's esophagus. In conclusion, this study has, fist, provided a detailed comparative genomic hybridization-map of GEJ adenocarcinomas documenting new genetic changes, as well as candidate genes involved. Second, genetic divergence was revealed in this poorly understood group of cancers.
Assuntos
Adenocarcinoma/genética , Esôfago de Barrett/genética , Deleção Cromossômica , Cromossomos Humanos Par 4 , Neoplasias Esofágicas/genética , Junção Esofagogástrica , Neoplasias Gástricas/genética , Adenocarcinoma/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Esôfago de Barrett/diagnóstico , Cárdia , Diagnóstico Diferencial , Neoplasias Esofágicas/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico , Neoplasias Gástricas/diagnósticoRESUMO
AIMS: Sentinel node mapping (SNM) has been introduced in colorectal cancer (CRC) to improve staging by facilitating occult tumour cell (OTC) assessment in lymph nodes that are most likely to be tumour-positive. In this paper, studies on the feasibility and reliability of SNM in CRC are reviewed. METHODS: A literature search was conducted in the National Library of Medicine by using the keywords colonic, rectal, colorectal, neoplasm, adenocarcinoma, cancer and sentinel. Additional articles were identified by cross-referencing from papers retrieved in the initial search. RESULTS: There is a large variation in identification rates and false-negative rates mainly due to the learning curve effect, differences in SNM technique and tumour stage. CONCLUSIONS: We conclude that SNM in CRC is technically feasible. Standardization of SNM procedures is mandatory to resolve the debate on the reliability of sentinel node status for predicting the tumour status of all lymph nodes. Only then can adjuvant treatment of patients upstaged by OTC detection in sentinel nodes be justified.
Assuntos
Neoplasias do Colo/patologia , Neoplasias Retais/patologia , Biópsia de Linfonodo Sentinela , Estudos de Viabilidade , Humanos , Linfonodos/patologia , Metástase Linfática/patologia , Estadiamento de Neoplasias , Reprodutibilidade dos TestesRESUMO
Malignant cells from 24 cases of hairy cell leukemia were studied by in situ hybridization for evidence of selective aneuploidy using alphoid and satellite probes specific for 16 human chromosomes. Based on these data, hairy cell leukemia appears to be diploid for the chromosomes studied and is a malignancy which displays the phenomenon of pairing of the centromere and p arm of chromosome 15 during interphase.
Assuntos
Leucemia de Células Pilosas/genética , Centrômero , Aberrações Cromossômicas , Cromossomos Humanos Par 15 , Diploide , Humanos , Hibridização In Situ , Interfase , Leucemia de Células Pilosas/patologiaRESUMO
Twenty-seven samples (cell cultures prepared for routine cytogenetics) of leukemia patients with known cytogenetic abnormalities were stained by in situ hybridization for interphase cytogenetics with centromere specific probes for chromosome Nos 4, 6, 7, 8, 9, 12, 17, 18, X and Y. The number of hybridization domains per nucleus was quantified using a semi-automated system developed in our laboratory. Results of this automated counting procedure (with and without verification of the counting results by the operator) were compared with conventional cytogenetic data and with visual scoring of the number of hybridization dots. The findings show that the system is capable of analysing 1000 cell nuclei in less than 30 min, including the necessary verification of the results by the operator. Automated counting and visual scoring were in good agreement. Conventional cytogenetics and interphase cytogenetics agreed in only 50% of the cases, confirming other studies showing that conventional cytogenetic results are not always representative for the majority of the cell population.
Assuntos
Hibridização in Situ Fluorescente/métodos , Leucemia/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Automação , Pré-Escolar , Feminino , Humanos , Interfase , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos TestesRESUMO
Cell sorting by flow cytometry usually involves preliminary staining with fluorescent dyes or reagents (antibodies or probes) that interact specifically with cellular constituents. Passage through a focused beam of light enables cells to be sorted on the basis of their light-scattering or fluorescence characteristics. Integration with other techniques and refinement of labelling specificity is enabling high-speed sorting to be developed for an expanding range of both analytical and preparative applications--including isolation of specific cells for PCR amplification, establishing high-expressing cell clones and chromosome sorting.
Assuntos
Separação Celular/métodos , Citometria de Fluxo/métodos , Biotecnologia , Separação Celular/instrumentação , Estudos de Avaliação como Assunto , Citometria de Fluxo/instrumentação , Corantes Fluorescentes , Humanos , Luz , Magnetismo , Espalhamento de RadiaçãoRESUMO
We used the single cell gel electrophoresis assay (comet assay) to study ultraviolet B (UVB)-induced DNA damage in pigment cells. This assay detects DNA damage, mainly DNA strand breaks and alkali labile sites in the DNA molecule. We studied the effect of biologically relevant doses (comparable to 2-3 MED (minimal erythemal dose) for in vivo irradiated full-thickness skin) of monochromatic UVB light of 302 nm on cultured melanocytes derived from foreskin, common melanocytic nevi, and dysplastic nevi. We were able to demonstrate a linear dose-response relationship between UV dose and the migration coefficient of the comet tail in all three types of pigment cells. Nevus cells originating from dysplastic nevi showed the highest sensitivity to UVB irradiation: 65% higher induction of DNA damage compared to the induction in foreskin melanocytes. Common melanocytic nevus cells were most resistant and showed a 30% lower induction of DNA damage in comparison to foreskin melanocytes. Differences in chromatin structure and cell cycle profile may influence the results of the comet assay. Control experiments with x-ray irradiation, which is well known to produce direct DNA strand breaks via radical formation, revealed only small differences between the three types of melanocytic cells. It is unlikely, therefore, that intrinsic nuclear characteristics may account for the observed differences.
Assuntos
Dano ao DNA , Síndrome do Nevo Displásico/genética , Melanócitos/efeitos da radiação , Nevo Pigmentado/genética , Neoplasias Cutâneas/genética , Raios Ultravioleta , Movimento Celular , DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Síndrome do Nevo Displásico/patologia , Eletroforese/métodos , Humanos , Masculino , Melanócitos/fisiologia , Nevo Pigmentado/patologia , Pênis , Neoplasias Cutâneas/patologiaRESUMO
Multicolour in situ hybridisation (MFISH) is increasingly applied to karyotyping and detection of chromosomal abnormalities. So far 27 colour analyses have been described using fluorescently labelled chromosome painting probes in a so-called combinatorial approach. In this paper a new strategy is presented to use efficiently the currently available number of spectrally separated fluorophores in order to increase the multiplicity of MFISH. We introduce the principle of COBRA (COmbined Binary RAtio labelling), which is based on the simultaneous use of combinatorial labelling and ratio labelling. Human chromosome painting in 24 colours is accomplished using four fluorophores only. Three fluorophores are used pair wise for ratio labelling of a set of 12 chromosome painting probes. The second set of 12 probes is labelled identically but is also given a binary label (fourth fluorophore). The COBRA method is demonstrated on normal human chromosomes and on a lymphoma (JVM) cell line, using probes enzymatically labelled with fluorescein, lissamine and cy5 as primary fluorophores, and diethylaminocoumarin (DEAC), a blue dye, as combinatorial fourth label to demonstrate incorporated digoxigenin. In addition, the principle was tested using chemical labelling. The first set of 12 painting probes was therefore labelled by ULS (Universal Linkage System), using DEAC, cy3 and cy5 as primary labels, and the second set was labelled similarly, but also contained a digoxigenin-ULS label, which was indirectly stained with fluorescein. Subsequently, a mathematical analysis is presented and methods are indicated for achieving an MFISH multiplicity of 48, 96 or even higher using existing technology.
Assuntos
Hibridização in Situ Fluorescente/métodos , Cromossomos Humanos , HumanosRESUMO
Propidium iodide (PI) and diamidinophenylindol (DAPI) were compared with ethidium bromide (EB) in cytotoxicity tests. Double blind studies showed similar results with the 3 dyes in terms of percentages of dead (fluorescing) cells. The dyes were also investigated for their potential combination with the immunofluorescent membrane markers FITC and TRITC. Both EB-FITC and PI-FITC are suitable for conventional HLA-DR typing procedures. Because of its spectral properties, the DAPI-TRITC combination is promising for automated read out of DR-typing tests by image analysis systems.
Assuntos
Imunofluorescência , Corantes Fluorescentes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Testes Imunológicos de Citotoxicidade/métodos , Etídio/farmacologia , Antígenos HLA-DR , Antígenos de Histocompatibilidade Classe II/análise , Humanos , Indóis/farmacologia , Linfócitos/metabolismo , Propídio/farmacologiaRESUMO
A method is described for the identification of leucocytes in permanent cytocentrifuge rosette preparations made with the fluorescent methyl green pyronin-SITS (MPS) staining technique. The percentages of rosettes in stained slides agree well with the results obtained with the conventional haemocytometer method. Application of the MPS staining to cytocentrifuge preparations permits reliable morphological differentiation between free and rosetting leucocytes, and should be particularly valuable in studies on lymphocyte subpopulations in patients.