Cryptic terminal chromosome rearrangements in colorectal carcinoma cell lines detected by subtelomeric FISH analysis.

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.

Platinum(II)-based coordination compounds as nucleic acid labeling reagents: synthesis, reactivity, and applications in hybridization assays.

The synthesis, characterization, and molecular interactions of platinum(II) coordination compounds, which contain a distal nonradioactive reporter molecule, with mono- and polynucleotides are described. A [Pt(II)(en)(NH(2)(CH(2))(6)NH-tBoc)Cl](NO(3)) (en=ethylenediamine) entity has been coupled, after removal of the tBoc group, to a number of hapten and fluorophore molecules through succinimide derivatives. The influence of the various tethered reporter groups within these complexes on the reactivity towards guanosine 5'-monophosphate (5'-GMP), as a model for polynucleotide sequences, was investigated to shed light on the use of these reagents in hybridization assays. Reactivity turned out to be strongly dictated by the chemical nature of the distal reporter molecule present. At pH 7.0 the sequence of reactivity is cationic approximately aromatic (stacking) > neutral > anionic; there is approximately an order of magnitude difference between the fastest reacting complex (k=10.2 x 10(-2) M(-1) s(-1)) and the slowest reacting complex (k=0.93 x 10(-2) M(-1) s(-1)) under these conditions. Platination of an oligodeoxynucleotide (30-mer), dsDNA, or an RNA transcript, shows that a Pt/nucleotide ratio between 1:10 and 1:20 (established by using flameless atomic absorption spectroscopy) results in probes with excellent hybridization characteristics. In terms of applicability and detection limits these platinated nucleic acid probes perform equally well compared to conventionally generated nucleic acid probes, that is, through enzymatic incorporation of covalently labeled nucleotide triphosphates. Applications of these reagents to in situ hybridization assays and gene expression profiling on microarrays illustrate the potential of these monofunctional binding platinum triamine compounds.

Insights into the sites of X ray and neutron induced chromosomal aberrations in human lymphocytes using COBRA-MFISH.

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.

Centrosomal abnormalities, multipolar mitoses, and chromosomal instability in head and neck tumours with dysfunctional telomeres.

Carcinomas of the head and neck typically exhibit complex chromosome aberrations but the underlying mutational mechanisms remain obscure. Evaluation of cell division dynamics in low-passage cell lines from three benign and five malignant head and neck tumours revealed a strong positive correlation between multipolarity of the mitotic spindle and the formation of bridges at anaphase in both benign and malignant tumours. Cells exhibiting a high rate of mitotic abnormalities also showed several chromosome termini lacking TTAGGG repeats and a high frequency of dicentric chromosomes. Multicolour karyotyping demonstrated a preferential involvement in structural rearrangements of chromosomes with deficient telomeres. The majority of malignant, mitotically unstable tumours expressed the reverse transcriptase subunit of telomerase. These data indicate that some of the genomic instability in head and neck tumours is initiated by telomere dysfunction, leading to the formation of dicentric chromosomes. These form chromosome bridges at mitosis that could prevent the normal anaphase-telophase transition. In turn, this may cause an accumulation of centrosomes and mitotic multipolarity. Telomerase expression does not confer total stability to the tumour genome but could be crucial for moderating the rate of chromosomal evolution.

The relationship between genetic aberrations as detected by comparative genomic hybridization and vascularization in glioblastoma xenografts.

Angiogenesis is of vital importance for the growth of solid tumors and constitutes a target for anti-cancer therapy. Glioblastomas (GBMs) are histologically characterized by striking microvascular proliferation. The identification of the mechanism of angiogenesis is of major importance for the further development of anti-angiogenic therapy. Tumor angiogenesis might be the result of a combination of local tissue conditions (especially hypoxia) and specific genetic alterations acquired during oncogenesis. In order to investigate the relationship between genetic aberrations and tumor angiogenesis in GBM xenograft lines, the genetic alterations were examined by Comparative Genomic Hybridization (CGH). Two vascular phenotypes of GBM xenografts could be identified: a well vascularized and a poorly vascularized type. In this model, the poorly vascularized type had a larger number of genetic alterations. However, there was no unequivocal correlation between angiogenesis, growth rate and patterns of genetic alterations as detected by CGH.

Mutations in the APC tumour suppressor gene cause chromosomal instability.

Two forms of genetic instability have been described in colorectal cancer: microsatellite instability and chromosomal instability. Microsatellite instability results from mutations in mismatch repair genes; chromosomal instability is the hallmark of many colorectal cancers, although it is not completely understood at the molecular level. As truncations of the Adenomatous Polyposis Coli (APC) gene are found in most colorectal tumours, we thought that mutations in APC might be responsible for chromosomal instability. To test this hypothesis, we examined mouse embryonic stem (ES) cells homozygous for Min (multiple intestinal neoplasia) or Apc1638T alleles. Here we show that Apc mutant ES cells display extensive chromosome and spindle aberrations, providing genetic evidence for a role of APC in chromosome segregation. Consistent with this, APC accumulates at the kinetochore during mitosis. Apc mutant cells form mitotic spindles with an abundance of microtubules that inefficiently connect with kinetochores. This phenotype is recapitulated by the induced expression of a 253-amino-acid carboxy-terminal fragment of APC in microsatellite unstable colorectal cancer cells. We conclude that loss of APC sequences that lie C-terminal to the beta-catenin regulatory domain contributes to chromosomal instability in colorectal cancer.

Characterization of chromosome aberrations in salivary gland tumors by FISH, including multicolor COBRA-FISH.

Fluorescence in situ hybridization (FISH), including COBRA-FISH, was used to characterize 11 salivary gland tumors that had been investigated by banding analysis. Five cases were pleomorphic adenoma (PA), three were adenoid cystic carcinoma, and one case each was mucoepidermoid carcinoma, carcinoma ex-pleomorphic adenoma (CaPA), and adenocarcinoma. All 11 cases were selected on the basis that they had shown rearrangement of 6q or 9p or had unresolved aberrations after karyotyping. The COBRA-FISH and FISH analyses led to a revised karyotype in all informative cases and made it possible to clarify almost all chromosomal rearrangements occurring in the tumors. Of particular note were the confirmation of the existence of 6q deletions, a common change in salivary gland carcinomas, and the demonstration that a seemingly balanced t(6;9) resulted in del(6q). Other rearrangements that were revealed by FISH included amplification of 12q sequences (MDM2 and CDK4) in one PA. We also investigated the status of the PLAG1 gene in four cases (one PA, one CaPA, one adenoid cystic carcinoma, and one mucoepidermoid carcinoma) with 8q12 rearrangements. Only in the former two cases were the FISH results compatible with intragenic rearrangements. Overall, the results of the study show that, even with good banding quality and in karyotypes of modest complexity, much new information will be gained by supplementing the banding analysis with a multicolor FISH approach, such as COBRA-FISH.

Basic preparative techniques for fluorescence in situ hybridization.

This unit presents protocols for preparing human metaphase chromosome slides from peripheral blood lymphocytes, isolating interphase nuclei from lymphocytes and paraffin-embedded tissues, and preparing DNA fibers. The protocols are designed so that the resulting preparations are amenable to FISH. The methods correspond to a selection of the specimens that can be analyzed with FISH techniques, and the choice of sample preparation method is highly dependent on the molecular cytogenetics question being addressed.

Probe labeling and fluorescence in situ hybridization.

This unit describes in detail basic protocols for probe labeling, denaturing of in situ target DNA, in situ hybridization, and post-hybridization washes. Support protocols for probe labeling cover probe purification and quality control.

Immunocytochemical detection.

This unit provides detailed descriptions for direct and indirect in situ hybridization methods. Protocols are designed for bright-field microscopy using a precipitating chromogenic substrate and for immunofluorescence detection using a hapten-labeled probe. Several alternative procedures are provided together with an excellent discussion of the strategy used for selecting a detection method.

Individuals with abnormal phenotype and normal G-banding karyotype: improvement and limitations in the diagnosis by the use of 24-colour FISH.

The simultaneous identification, by fluorescence in situ hybridisation (FISH), of each chromosome in a distinct colour became feasible a few years ago. The key question in the application of this and many other developments in molecular cytogenetics to clinical situations is whether the results add significant further information that is relevant to the diagnosis. So far, limited data exist regarding how much improvement the technique brings to the diagnosis of phenotypically abnormal individuals in whom no abnormalities have been detected by conventional G-banding analysis. Because of the lack of a conclusive diagnosis, genetic counselling, estimation of recurrence risk and prenatal diagnosis of these individuals and their relatives is problematic. We report a study with 24-colour whole-chromosome painting of 10 familial and 11 isolated cases with abnormal phenotypes and normal G-banding karyotypes. Previously undetected unbalanced translocations were revealed in two cases. The value and current cost-effectiveness of multicolour FISH for cytogenetic diagnosis is discussed.

Differentially painting human chromosome arms with combined binary ratio-labeling fluorescence in situ hybridization.

Recently we developed a novel strategy for differentially painting all 24 human chromosomes. It is termed COBRA-FISH, short for combined binary ratio labeling-fluorescence in situ hybridization. COBRA-FISH is distinct from the pure combinatorial approach in that only 4 instead of 5 fluorophores are needed to achieve color discrimination of 24 targets. Furthermore, multiplicity can be increased to 48 by introduction of a fifth fluorophore. Here we show that color identification by COBRA-FISH of all of the p and q arms of human chromosomes is feasible, and we apply the technique for detecting and elucidating intra- and interchromosomal rearrangements. Compared with 24-color whole chromosome painting FISH, PQ-COBRA-FISH considerably enhances the ability to determine the composition of rearranged chromosomes as demonstrated by the identification of pericentric inversions and isochromosomes as well as the elucidation of the arm identity of chromosomal material involved in complex translocations that occur in solid tumors.

Synthesis and purification of horseradish peroxidase-labeled oligonucleotides for tyramide-based fluorescence in situ hybridization.

A method is presented to conjugate horseradish peroxidase (HRP) to oligodeoxynucleotides for fluorescence in situ hybridization assays employing tyramide signal amplification (TSA). HRP is covalently bound to the oligonucleotide by thiol ether linkage and purified by high-performance liquid chromatography. With TSA detection, a single HRP-labeled oligonucleotide probe is sufficient for in situ detection of clustered DNA repeat sequences with a degree of repetition between 20 and 50.

Chromosomal breakage-fusion-bridge events cause genetic intratumor heterogeneity.

It has long been known that rearrangements of chromosomes through breakage-fusion-bridge (BFB) cycles may cause variability of phenotypic and genetic traits within a cell population. Because intercellular heterogeneity is often found in neoplastic tissues, we investigated the occurrence of BFB events in human solid tumors. Evidence of frequent BFB events was found in malignancies that showed unspecific chromosome aberrations, including ring chromosomes, dicentric chromosomes, and telomeric associations, as well as extensive intratumor heterogeneity in the pattern of structural changes but not in tumors with tumor-specific aberrations and low variability. Fluorescence in situ hybridization analysis demonstrated that chromosomes participating in anaphase bridge formation were involved in a significantly higher number of structural aberrations than other chromosomes. Tumors with BFB events showed a decreased elimination rate of unstable chromosome aberrations after irradiation compared with normal cells and other tumor cells. This result suggests that a combination of mitotically unstable chromosomes and an elevated tolerance to chromosomal damage leads to constant genomic reorganization in many malignancies, thereby providing a flexible genetic system for clonal evolution and progression.

Adenovirus E1A does not induce the Ewing tumor-associated gene fusion EWS-FLI1.

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.

Simultaneous molecular karyotyping and mapping of viral DNA integration sites by 25-color COBRA-FISH.

Combined binary ratio labeling (COBRA) fluorescence in situ hybridization (FISH) allows 24-color FISH karyotyping of human metaphase chromosomes utilizing only four fluorochromes, instead of the five required for combinatorial labeling procedures. Here we show that by introduction of a fifth fluorochrome, COBRA-FISH permits molecular cytogenetic mapping of viral integration sites in complex karyotypes in the context of a 24-color hybridization. We were able to detect a single copy of the human papillomavirus 16 in the SiHa cell line and to confirm the site of integration at 13q21-31. We also demonstrate the gene mapping possibility of 25-color hybridization by detecting a MYC cosmid on normal metaphase chromosomes. The possibility of mapping single-copy probes in the background of 24-color hybridization expands the tools for cytogenetic mapping of DNA sequences and will contribute to the understanding of the role of viral integration and chromosome rearrangement in virus-mediated carcinogenesis.

New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling.

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.

ULS: a versatile method of labeling nucleic acids for FISH based on a monofunctional reaction of cisplatin derivatives with guanine moieties.

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.

Comparative genomic hybridization with lissamine- and fluorescein-labeled nucleotides.

Biotin deoxyuridine 5'-triphosphate (dUTP) and digoxigenin dUTP are the labels most commonly used in comparative genomic hybridization (CGH). The relative infrequent use of direct fluorochrome-labeled nucleotides in CGH is related to the lower sensitivity they provide. Here we report the evaluation of two fluorochrome-conjugated nucleotides that have not been previously used in CGH (lissamine-5-dUTP and fluorescein-N6dATP) and show that this direct label combination performs at least as well as the indirect biotin/digoxigenin pair.