RESUMO
One limitation in understanding disease at the cellular level has been the inability to efficiently analyze DNA on a cell-to-cell basis within the natural tissue context. However, DNA analyses at a single-cell resolution should be instrumental for the understanding of cancer cell biology, cancer evolution, for chromosomal mosaic analysis and rare cell events, and should provide otherwise inaccessible information on essential biological processes. Here we present a fluorescence in situ hybridization-based multicolor deconvolution technique for three-dimensional microscopy. We use up to seven different color channels for probe detection, which allows the simultaneous high-resolution localization of multiple point-like sources within a biological specimen with a thickness of up to 30 micro m. In addition, a DNA counterstain is used for volume labeling of the nuclei offering the opportunity for a simultaneous segmentation of nuclei. Furthermore, as the instrumentation consists of a standard fluorescence microscope it represents a low-cost method as compared to confocal microscopy.
Assuntos
Neoplasias/patologia , Centrômero/patologia , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos , Cromossomos Artificiais de Levedura , Sondas de DNA , Humanos , Microscopia/métodos , Neoplasias/genética , Manejo de Espécimes/métodosRESUMO
Alterations in the genetic content of a cell are the underlying cause of many human diseases, including cancers. We have developed a method, called digital karyotyping, that provides quantitative analysis of DNA copy number at high resolution. This approach involves the isolation and enumeration of short sequence tags from specific genomic loci. Analysis of human cancer cells by using this method identified gross chromosomal changes as well as amplifications and deletions, including regions not previously known to be altered. Foreign DNA sequences not present in the normal human genome could also be readily identified. Digital karyotyping provides a broadly applicable means for systematic detection of DNA copy number changes on a genomic scale.
Assuntos
Cariotipagem/métodos , Conversão Análogo-Digital , Biotinilação , Mapeamento Cromossômico , Cromossomos Humanos/genética , Neoplasias Colorretais/genética , Simulação por Computador , Enzimas de Restrição do DNA , DNA de Neoplasias/genética , DNA Viral/análise , DNA Viral/genética , Dosagem de Genes , Marcadores Genéticos , Herpesvirus Humano 4/genética , Humanos , Microesferas , Método de Monte Carlo , Sensibilidade e Especificidade , Estreptavidina , Moldes Genéticos , Células Tumorais CultivadasRESUMO
Chemicals may induce both numerical and structural aberrations. In addition to these chromosomal mutations, chemicals may render cells genetically unstable, which may result in chromosomal instability. For a detailed analysis, sophisticated approaches at single cell resolution are needed. Such approaches have become feasible by recent developments in molecular cytogenetics. In particular, new multicolor fluorescence in situ hybridization (FISH) technologies allow us now to study the effects of chemicals on chromosomes with unprecedented resolution. FISH provides opportunities to analyze the genome in two dimensions, i.e. on metaphase spreads, or in three dimensions, i.e. in interphase nuclei. An arsenal of diverse multicolor FISH approaches has been developed, which allows the analysis of the entire genome with one hybridization on metaphase spreads or the detailed visualization of selected chromosomal regions within intact interphase nuclei. These developments have been complemented by new resources for DNA probes, which have evolved from the human genome project. Here we will review the latest developments and provide some examples in which multicolor FISH technologies were applied to elucidate the effect of chemicals on chromosomes.
Assuntos
Hibridização in Situ Fluorescente/métodos , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Animais , Linhagem Celular , Aberrações Cromossômicas , Cromossomos Humanos/efeitos dos fármacos , Cromossomos Humanos/genética , Feminino , Genoma Humano , Humanos , Cariotipagem , Masculino , CamundongosRESUMO
Bone marrow is a major homing site for circulating epithelial tumor cells. The present study was aimed to assess the proliferative capacity of occult metastatic cells in bone marrow of patients with operable solid tumors especially with regard to their clinical outcome. We obtained bone marrow aspirates from 153 patients with carcinomas of the prostate (n = 46), breast (n = 45), colon (n = 33), and kidney (n = 29). Most of the patients (87%) had primary disease with no clinical signs of overt metastases [tumor-node-metastasis (TNM)-stage UICC (Union Internationale Contre le Cancer) I-III]. After bone marrow was cultured for 21-102 days under special cell culture conditions, viable epithelial cells were detected by cytokeratin staining in 124 patients (81%). The cultured epithelial cells harbored Ki-ras2 mutations and numerical chromosomal aberrations. The highest median number of expanded tumor cells was observed in prostate cancer (2,619 per flask). There was a significant positive correlation between the number of expanded tumor cells and the UICC-stage of the patients (P = 0.03) or the presence of overt metastases (P = 0.04). Moreover, a strong expansion of tumor cells was correlated to an increased rate of cancer-related deaths (P = 0.007) and a reduced survival of the patients (P = 0.006). In conclusion, the majority of cancer patients have viable tumor cells in their bone marrow at primary tumor diagnosis, and the proliferative potential of these cells determines the clinical outcome.
