ABSTRACT
A multi-colour fluorescence in situ hybridisation (MFISH) assay has been developed, for simultaneous visualisation of all human chromosomes in 24 different colours. This assay is based on the simultaneous use of combinatorial labelling and ratio labelling, the so called combined binary ratio labelling (COBRA). This technique is used to study the spectra of chromosomal exchanges induced by X ray and neutrons in human lymphocytes. With X rays the dose-effect relationships for both dicentrics and translocations were linear-quadratic, whereas with neutrons these were linear. Among aberrant cells, average estimates of the minimum number of breaks was higher for neutrons than for X rays. Moreover, the induced chromosomal exchange patterns were more complex following neutron irradiation in comparison with X rays. COBRA-MFISH was found to have a greater resolving power over partial labelling for the accurate detection of complex translocations and insertions. With neutrons the frequencies of both were higher than those induced by X rays, and their relative proportions to the total frequencies were independent of dose. These data suggest insertions can be used as the 'signature' of high LET radiation.
Subject(s)
Chromosome Aberrations , DNA/radiation effects , Lymphocytes/radiation effects , Neutrons , X-Rays , Cells, Cultured , Dose-Response Relationship, Radiation , Female , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Lymphocytes/physiology , Middle AgedABSTRACT
Rearrangement of the EWS gene with FLI1 is thought to occur early in the pathogenesis of Ewing's sarcoma family tumors (EFTs) because the chromosomal aberration is pathognomonic for this disease. Recently, adenovirus (Ad) 5 E1A protein has been reported to induce this gene rearrangement in a variety of cell types. This finding, if generally substantiated, not only suggests an etiological role for viral agents in the generation of oncogenic chromosomal aberrations but would also significantly impact the use of adenoviral vectors for gene therapy. In contrast, we now report on the absence of EWS-FLI1 chimeric products from short- and long-term cultures of stably Ad-transformed cells lines and from transiently E1A-expressing cell lines. In addition, we demonstrate the absence of E1A from EFTs. We conclude that there is no role for Ads in EFT pathogenesis. Consequently, evidence for a viral genesis of tumor-specific gene rearrangements is not available.
Subject(s)
Adenovirus E1A Proteins/physiology , Bone Neoplasms/genetics , Oncogene Proteins, Fusion/genetics , Sarcoma, Ewing/genetics , Transcription Factors/genetics , Adenovirus E1A Proteins/genetics , Adenovirus E1A Proteins/metabolism , Blotting, Northern , Blotting, Western , Bone Neoplasms/metabolism , Cell Line , Cell Line, Transformed , DNA, Complementary/genetics , DNA, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Gene Rearrangement , HeLa Cells , Humans , Oncogene Proteins, Fusion/metabolism , Plasmids/genetics , Proto-Oncogene Protein c-fli-1 , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , RNA-Binding Protein EWS , Sarcoma, Ewing/metabolism , Transcription Factors/metabolism , Tumor Cells, CulturedABSTRACT
The broad extension of an existing chemical DNA labeling technique for molecular cytogenetics is described. Called the Universal Linkage System (ULS(TM)), it is based on the capability of monoreactive cisplatin derivatives to react at the N7 position of guanine moieties in DNA. Simple repetitive probes, cosmids, PACs, and chromosome-specific painting probes were labeled by ULS and used in a series of multicolor fluorescence in situ hybridization experiments on interphase and metaphase cells. It is demonstrated that ULS-labeled probes, in general, perform as well as the more conventional enzymatically labeled probes. The advantage of ULS labeling over enzymatic labeling techniques is that it is a fast and simple procedure, and that the labeling can easily be scaled up for bulk probe synthesis. In addition, with ULS labeling it is possible to label degraded DNA, a situation in which enzymatic labeling is known to perform unsatisfactorily.
Subject(s)
Cisplatin/metabolism , Cross-Linking Reagents/metabolism , DNA Probes/metabolism , DNA/metabolism , Guanine/metabolism , In Situ Hybridization, Fluorescence/methods , B-Lymphocytes/pathology , Centromere/genetics , Chromosome Painting/instrumentation , Chromosome Painting/methods , Chromosomes, Human/genetics , Cisplatin/analogs & derivatives , Cisplatin/chemistry , Cosmids/genetics , Cross-Linking Reagents/chemistry , DNA/chemistry , DNA/genetics , DNA Probes/chemistry , DNA Probes/genetics , Fluorescent Dyes/metabolism , Humans , In Situ Hybridization, Fluorescence/instrumentation , Leukemia, Prolymphocytic/genetics , Leukemia, Prolymphocytic/pathology , Male , Mosaicism/genetics , Reproducibility of Results , Sensitivity and Specificity , Time Factors , Translocation, Genetic/genetics , Tumor Cells, CulturedABSTRACT
Fluorescence in situ hybridization on human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments on a single chromosome with an unprecedented resolution. Three nucleic acid probes are used: pUC1.77, p1-79 and the plasmid probe alpha-spectrin. The hybridization signals are very well resolved in the near-field fluorescence images, while the exact location of the probes can be correlated accurately with the chromosome topography as afforded by the shear force image.