RESUMEN
Segregation of mitochondrial DNA (mtDNA) is an important underlying pathogenic factor in mtDNA mutation accumulation in mitochondrial diseases and aging, but the molecular mechanisms of mtDNA segregation are elusive. Lack of high-throughput single-cell mutation load assays lies at the root of the paucity of studies in which, at the single-cell level, mitotic mtDNA segregation patterns have been analyzed. Here we describe development of a novel fluorescence-based, non-gel PCR restriction fragment length polymorphism method for single-cell A3243G mtDNA mutation load measurement. Results correlated very well with a quantitative in situ Padlock/rolling circle amplification-based genotyping method. In view of the throughput and accuracy of both methods for single-cell A3243G mtDNA mutation load determination, we conclude that they are well suited for segregation analysis.
Asunto(s)
ADN Mitocondrial/genética , Separación Celular , Células Cultivadas , Genotipo , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Fluorescente , Mutación , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Temperatura de TransiciónRESUMEN
Apoptosis is fundamental to the regulation of homeostasis of stem cells in vivo. Whereas the pathways underlying the molecular and biochemical details of nuclear breakdown that accompanies apoptosis have been elucidated, the precise nature of nuclear reorganization that precedes the demolition phase is not fully understood. Here, we expressed an inducible caspase-8 in human mesenchymal stem cells, and quantitatively followed the early changes in nuclear organization during apoptosis. We found that caspase-8 induces alteration of the nuclear lamina and a subsequent spatial reorganization of both centromeres, which are shifted towards a peripheral localization, and telomeres, which form aggregates. This nuclear reorganization correlates with caspase-3 sensitivity of lamina proteins, because the expression of lamin mutant constructs with caspase-3 hypersensitivity resulted in a caspase-8-independent appearance of lamina intranuclear structures and telomere aggregates, whereas application of a caspase inhibitor restrains these changes in nuclear reorganization. Notably, upon activation of apoptosis, we observed no initial changes in the spatial organization of the promyelocytic leukemia nuclear bodies (PML-NBs). We suggest that during activation of the caspase-8 pathway changes in the lamina structure precede changes in heterochromatin spatial organization, and the subsequent breakdown of lamina and PML-NB.
Asunto(s)
Caspasa 8/metabolismo , Heterocromatina/metabolismo , Células Madre Mesenquimatosas/metabolismo , Lámina Nuclear/metabolismo , Western Blotting , Caspasa 8/genética , Células Cultivadas , Centrómero/metabolismo , Activación Enzimática , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunohistoquímica , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Lentivirus/genética , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/enzimología , Microscopía Fluorescente , Mutación/genética , Telómero/metabolismoRESUMEN
The presence of tumor cells in bone marrow, peripheral blood and lymph nodes has proven its clinical and prognostic value. Since the frequency of these cells in bone marrow and blood is sometimes as low as 1 per million and due to the fact that for the analysis of lymph nodes many sectioning levels have to be analyzed, automated imaging devices have been suggested as an useful alternative to conventional manual screening of specimens. The aim of this paper is to review the performance of current equipment that is commercially available, based on literature published so far. Requirements for introducing this equipment for routine clinical practice are discussed.
Asunto(s)
Médula Ósea/patología , Diagnóstico por Imagen/instrumentación , Metástasis Linfática/diagnóstico , Metástasis de la Neoplasia/diagnóstico , Células Neoplásicas Circulantes , Humanos , Ganglios Linfáticos/citología , Ganglios Linfáticos/patología , Neoplasias/sangre , Neoplasias/patologíaRESUMEN
PURPOSE: At present, reverse transcription (RT)-PCR against carcino-embryonic antigen mRNA is one of the few research tools for the detection of occult cells in histopathologically assessed negative lymph nodes from patients with colorectal cancer. The aim of this study was to investigate the suitability of supervised low-resolution image analysis of immunohistochemically stained sections as alternative. EXPERIMENTAL DESIGN: Multiple sections (n = 50) of regional lymph nodes from patients with colorectal cancer were immunohistochemically stained and analyzed by applying low-resolution image analysis (flatbed scanning) for semiautomated detection of cytokeratin (CK)-positive stained cells. The sensitivity of this approach was demonstrated for 20 patients with stage II colorectal cancer and compared with RT-PCR regarding the detection of clinically assessed recurrence of disease within 10 years. RESULTS: CK(+) cells were detected in all of the patients (n = 6; 100%) with recurrence, compared with five patients (83%) found positive by carcinoembryonic antigen RT-PCR. From patients (n = 14) who did not develop a recurrence, eight (57%) had positive lymph nodes. In all patients with recurrence, we visually identified at least one group of CK(+) cells (>/==" BORDER="0">2 cells). CONCLUSIONS: Automated image analysis is a promising tool for the detection of occult cells in histopathologically negative nodes. It is potentially more sensitive but less specific for detecting recurrence of disease than conventional histopathology or RT-PCR and is particularly useful for the evaluation of sentinel nodes. Furthermore, it opens new ways for basic research of occult cells based on molecular profiling after laser-microdissection.