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.

Can, a putative oncogene associated with myeloid leukemogenesis, may be activated by fusion of its 3' half to different genes: characterization of the set gene.

The translocation (6;9)(p23;q34) in acute nonlymphocytic leukemia results in the formation of a highly consistent dek-can fusion gene. Translocation breakpoints invariably occur in single introns of dek and can, which were named icb-6 and icb-9, respectively. In a case of acute undifferentiated leukemia, a breakpoint was detected in icb-9 of can, whereas no breakpoint could be detected in dek. Genomic and cDNA cloning showed that instead of dek, a different gene was fused to can, which was named set. set encodes transcripts of 2.0 and 2.7 kb that result from the use of alternative polyadenylation sites. Both transcripts contain the open reading frame for a putative SET protein with a predicted molecular mass of 32 kDa. The set-can fusion gene is transcribed into a 5-kb transcript that contains a single open reading frame predicting a 155-kDa chimeric SET-CAN protein. The SET sequence shows homology with the yeast nucleosome assembly protein NAP-I. The only common sequence motif of SET and DEK proteins is an acidic region. SET has a long acidic tail, of which a large part is present in the predicted SET-CAN fusion protein. The set gene is located on chromosome 9q34, centromeric of c-abl. Since a dek-can fusion gene is present in t(6;9) acute myeloid leukemia and a set-can fusion gene was found in a case of acute undifferentiated leukemia, we assume that can may function as an oncogene activated by fusion of its 3' part to dek, set, or perhaps other genes.

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.

Cytogenetic alterations associated with P-glycoprotein- and non-P-glycoprotein-mediated multidrug resistance in SW-1573 human lung tumor cell lines.

Multidrug resistance can be induced in mammalian cells by selection with a single cytotoxic agent. Overproduction of the energy-dependent drug efflux pump P-glycoprotein, encoded by the mdr1 gene, has been identified as the cause of one form of multidrug resistance. The molecular basis of other forms of multidrug resistance is unknown. Doxorubicin selection of the human squamous lung cancer cell line SW-1573 resulted in multidrug-resistant sublines in which a non-P-glycoprotein-mediated form of multidrug resistance precedes mdr1 expression. Here we present a cytogenetic analysis of both non-P-glycoprotein-mediated multidrug-resistant and P-glycoprotein-mediated multidrug-resistant sublines derived from SW-1573. Three independently derived non-P-glycoprotein-mediated multidrug-resistant sublines showed a heterozygous deletion of the short arm of chromosome 2 (p23-pter), whereas alterations of chromosome 7 were present in the P-glycoprotein-mediated multidrug-resistant cell lines. In one series of clonally derived P-glycoprotein-mediated multidrug-resistant sublines, mdr1 overexpression was accompanied by various markers of chromosome 7 with breakpoints at 7q22, the mdr1 gene being known to be located at 7q21.1. Our data suggest that in SW-1573 cells acquisition of non-P-glycoprotein-mediated multidrug resistance is accompanied by a specific deletion or a translocation involving the short arm of chromosome 2, whereas in the emergence of P-glycoprotein-mediated multidrug resistance a rearrangement of the long arm of chromosome 7 is a critical event.

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.

Fiber FISH as a DNA Mapping Tool

Fluorescence in situ hybridization (FISH) applied to metaphase chromosomes provides a mapping resolution of 1 to 3 Mb. FISH applied to interphase nuclei has a resolution of 50 kb and ranges 1-2 Mb. This better resolution is attributed to the higher degree of chromatin decondensation. Here, we describe FISH applied to naked DNA fibers (fiber FISH) and show that with such fully decondensed chromatin a resolution range of at least 1-400 kb can be obtained. Furthermore, we show that DNA fiber FISH provides a mapping tool that is highly supplementary to restriction mapping, because it permits very accurate gap and overlap sizing. Also, DNA fiber FISH provides the means to generate "color bar codes" for disease regions, which can be used to inspect patient DNAs for suspected gene rearrangements.

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.

The development, using poly(Hg-U) in a model system, of a new method to visualize cytochemical hybridization in fluorescence microscopy.

The development, using a model system, of a new method for the detection of cytochemical (in situ) hybrids is described. The method is based on the mercuration of nucleic acids with mercuric acetate. To facilitate hybridization, the acetate ligand is replaced by the CN- ion. In the hybrids formed, the CN- is exchanged for trinitrophenyl(TNP)-glutathione. The TNP-glutathione is subsequently detected by indirect immunofluorescence using anti-TNP antibodies. The feasibility of the approach was investigated using Sepharose- or Sephadex-bound poly(A) and mercurated poly(U). Poly(Hg-U) did hybridize with poly(A)-Sepharose, provided that the acetate ligand was replaced with CN-. The TNP-glutathione hapten thus synthesized bound effectively to mercury-Sepharose but not to amino-Sepharose when the reaction was performed in the dark. Furthermore, binding of TNP-glutathione to Sephadex-bound poly(A) . poly(HG-U) hybrids was detectable with indirect immunofluorescence using anti-TNP antibodies. The fluorescence intensity measured was dependent on the amount of poly(Hg-U) present and on the dilution of the antibody. Nonspecific binding was very low. Calibration of the number of fluorescein molecules found after the complete reaction was performed with fluorescein isothiocyanate-labeled poly(U). It was determined that one fluorochrome molecule per two nucleotides had been obtained, in close agreement with the theoretically expected number. The sensitivity of the method, when applicable to microscopic preparations, is comparable to in situ hybridization with 3H-labeled nucleic acids with a specific activity of 4 x 10(8) dpm/micrograms (two 3H-isotopes per nucleotide) and an exposure time of 1 day. Extension of the method to the cRNA-DNA system and its application to microscopic preparations is under investigation.

Cytochemical hybridization with fluorochrome-labeled RNA. I. Development of a method using nucleic acids bound to agarose beads as a model.

A new procedure to label RNA at the 3'-terminus with a fluorochrome molecule is described. The thiosemicarbazides derived from tetramethylrhodamine isothiocyanate (TRITC) and fluorescein isothiocyanate (FITC) were prepared by reacting these compounds with hydrazine in dimethylsulfoxide (DMSO):pyridine, 99:1 (v/v). They coupled efficiently to the aldehydes generated by periodate oxidation of RNAs. We determined, using Sepharose to which different nucleic acids and proteins had been bound, that the label added no specific binding properties to the RNA, and did not interfere with duplex formation of labeled poly(U) and poly(A). The stability of the fluorochrome-RNA bond under conditions generally used for hybridization was investigated. The bond was found to be unstable at 66 degrees C in 3 x SSC, 0.1% SDS (50% loss within 45 min) but stable for at least 40 hr at 23 degrees C in 70% formamide/3 x SSC. The hybridization characteristics of complementary RNA, both fluorochrome- and 3H-labeled, were investigated using DNA-Sepharose beads as a cytochemical model. Hybridization was measured by scintillation counting of microliter quantities of beads and quantitative fluorescence microscopy of individual Sepharose beads. No influence of the label on the specificity and stability of the hybrids was found. Maximum specific fluorescence was found after hybridization at 23 degrees C in 70% formamide/3 x SSC. These results made possible the successful use of fluorochrome-labeled RNA to perform cytochemical hybridization followed by detection of the hybrids with fluorescence microscopy. This will be described in an accompanying article.

Cytochemical hybridization with fluorochrome-labeled RNA. II. Applications.

The cytochemical detection of specific DNA sequences by hybridization with fluorochrome-labeled RNA and detection of the hybrids by fluorescence microscopy is described. RNAs complementary to the DNA of the kinetoplasts of Crithidia luciliae (an insect trypanosome) or to adenovirus-5 (Ad-5) DNA were labeled with the hydrazine derivative of tetramethylrhodamine isothiocyanate (TRITC). The specificity of the reactions between the complementary RNAs labeled both with 3H and tetramethylrhodamine was studied by cross-hybridization experiments using a model system in which the DNAs were bound to Sepharose beads. The extent of the reaction was measured by scintillation counting of the bead suspensions and quantitative fluorescence microscopy of individual Sepharose beads. The ability of the rhodamine-labeled cRNAs to hybridize and the absence of interference of the fluorochrome label with the specificity of the hybridization reaction was thus demonstrated. After cytochemical hybridization on microscopic preparations of C. luciliae cells the rhodamine-labeled kinetoplast cRNA stains only the kinetoplasts. No fluorescence was observed in the nuclei. After cytochemical hybridization of rhodamine-labeled Ad-5 cRNA with virus infected KB cells a distinct staining pattern in the nuclei was observed. No fluorescence was seen in uninfected cells, or after hybridization with heterologous rhodamine-labeled RNA. The possibilities and limitations of cytochemical hybridization with rhodamine-labeled RNA are discussed.

An evaluation of a new series of fluorescent dUTPs for fluorescence in situ hybridization.

Synthesis of fluorochrome-modified deoxyribonucleotides has been carried out mostly by linking the fluorochrome molecule to the C-5 position of dUTP via an allylamine spacer, similar to the modification of allylamine-dUTP with the haptens biotin and digoxigenin. Recently, a new series of fluorescent nucleotides has been prepared by using an alkynyl bridge between the uracil moiety and the fluorochrome. Here we report the qualitative and quantitative analysis of fluorescence in situ hybridization results obtained on interphase cells and chromosomes with a variety of highly repetitive and single-copy DNA probes that were modified by nick translation with such alkynyl dUTPs. A qualitative comparison was made of the alkynyl dUTPs conjugated to the fluorochromes fluorescein, the cyanine dye Cy3, tetramethylrhodamine, Lissamine and Texas Red. With the exception of tetramethylrhodamine, all fluorochromes performed satisfactorily. The cyanine dye Cy3 provided the highest sensitivity, i.e., cosmid and YAC probes could easily be visualized by conventional fluorescence microscopy. In a quantitative assay, different nick translation conditions were tested using a human chromosome 1 satellite III probe (pUC1.77) and alkynyl dUTPs labeled with fluorescein and Cy3. Using these two nucleotides, FISH signal intensities on interphase nuclei from human lymphocytes were quantitated by digital imaging microscopy. The strongest signals were obtained when during nick translation the ratio between dTTP and fluorescein-dUTP or Cy3-dUTP was 1:5.

A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase-fast red reaction.

We have used naphthol-ASMX-phosphate and Fast Red TR in combination with alkaline phosphatase (APase) to produce fluorescent precipitated reaction products in a non-radioactive in situ hybridization (ISH) method. To obtain optimal and discrete localization of the strongly red fluorescent ISH signals, the enzyme precipitation procedure was optimized. The optimal reaction time and the concentrations of substrate and capture agent were determined. Furthermore, polyvinyl alcohol (PVA) was used to increase the viscosity of the reaction mixture and thus to reduce diffusion of the reaction product. Our results show that the APase-Fast Red detection method has at least the same sensitivity as currently observed in other immunofluorescent detection systems. A single copy DNA sequence of 15.8 KB could be localized with high efficiency in metaphase spreads and in interphase nuclei. Double labeling procedures, in which the FITC- and azo-dye fluorescence are combined, are also feasible. The red fluorescent ISH signals showed hardly any fading as compared with FITC fluorescence on exposure to either light from the mercury-arc lamp or laser light. Therefore, these red fluorescent signals with a virtually permanent character allow a better analysis and three-dimensional localization of such cytochemically detected genomic fractions by means of confocal scanning laser microscopy as compared with the use of FITC, TRITC, or Texas Red as label.

Improved localization of fluorescent tyramides for fluorescence in situ hybridization using dextran sulfate and polyvinyl alcohol.

Recently, a peroxidase-mediated amplification system has been described for immunofluorescence and fluorescence in situ hybridization studies. It is based on the deposition of hapten- or fluorochrome-labeled tyramide molecules. Although providing a significantly increased detection sensitivity compared to conventional procedures, its localization properties are inferior because of free diffusion of intermediate reaction products before they are immobilized. In enzyme cytochemistry, it is well established that improved localization of enzyme activity can be achieved through the addition of viscosity-increasing polymers to the incubation media. In this study we analyzed the effect of different polymers on the localization sharpness and sensitivity of the tyramide-peroxidase reaction in FISH applications. Significantly improved localization of the fluorescent endproduct was observed using dextran sulfate or polyvinylalcohol (PVA) with, respectively, no or little loss of sensitivity.

Chromosomal localization of transfected genes by a combination of hot banding and fluorescence in situ hybridization.

We describe the combination of hot banding with fluorescence in situ hybridization as a rapid and efficient method to identify integration sites of transfected DNA sequences in chromosomes. As a test system we used SW480 EJ2, a clonal cell line obtained after transfection of SW480 with pSV2neoEJ, a plasmid containing a point-mutated, c-Ha-RAS oncogene. Nick-translated probes were compared with random primed-labeled probes to evaluate their relative efficiency in fluorescence in situ hybridization. The fluorescence signals were quantified in interphase nuclei by confocal scanning laser microscopy. Nick-translated probes were found to yield better results. Hot banding followed by fluorescence in situ hybridization localized the integration site of pSV2neoEJ in SW480 EJ2 at the site of a translocation on a marker chromosome Xp+. The combination of fluorescence in situ hybridization and hot banding can be used to (a) rapidly and efficiently analyze integration sites in large numbers of transfectants, (b) assess the clonality of transfected cell lines, and (c) localize the site of integration of transfected genes in the recipient genome.

Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization.

The peroxidase-mediated deposition of hapten- and fluorochrome-labeled tyramides has recently been shown to increase the sensitivity of immunofluorescence and fluorescence in situ hybridization techniques. We have evaluated a number of red, green, and blue fluorescent tyramides for detection of antigens in tissue sections and cytospin preparations and for the detection of hapten- and horseradish peroxidase-labeled probes hybridized in situ to cells and chromosomes. With few exceptions, all fluorescent tyramide-based methods provided a considerable increase in sensitivity compared to conventional immunofluorescence and FISH methods.

Detection of the Mbcr/abl translocation in chronic myeloid leukemia by fluorescence in situ hybridization: comparison with conventional cytogenetics and implications for minimal residual disease detection.

The correlation between the detection of the Philadelphia chromosome by conventional cytogenetics and the identification of Mbcr/abl translocation by fluorescence in situ hybridization (FISH) in both metaphase and interphase cells is prospectively analyzed in a group of 21 chronic myeloid leukemia (CML) patients. To gain insight into the sensitivity and specificity of the detection of the bcr/abl translocation by FISH, a group of 10 healthy volunteers was also studied. Our results show that for the detection of bcr/abl translocation in CML patients, FISH is more sensitive than conventional cytogenetics because it detects significantly higher proportions of cells carrying the translocation both in metaphase (P < .0002) and interphase nuclei (P < .003). Moreover, in the metaphases of the controls analyzed, no bcr/abl+ chromosome was detected that makes the colocalization of bcr and abl signals in the CML patients highly specific. Conversely, in control interphase nuclei, a small proportion of cells (ranging between 0% and 3%, mean value of 1.7% +/- 0.9%) displaying colocalization of both signals is usually detected. This limits, at least for the moment, the routine use of FISH for the detection of minimal residual disease in CML patients at levels lower than 10(-1).

Partial trisomy of 3q detected by chromosome painting in a case of juvenile chronic myelomonocytic leukemia.

Juvenile chronic myelomonocytic leukemia (JCMMoL) is a rare disease with no specific type of chromosome aberration yet delineated. We report a 2-year-old boy who had in his leukemic bone marrow (BM) and peripheral blood (PB) cells the 46,XY,der(15)t(3;15)(q13.1;q26) karyotype. Phytohemagglutinin (PHA)-stimulated lymphocytes of peripheral blood had a normal 46,XY karyotype. The origin of the duplicated part of 3q was proved by fluorescence in situ hybridization (FISH) with the pHSR(sat III 15p) DNA probe and a chromosome 3-specific DNA library (i.e., chromosome painting). The chromosome finding in our case provides further proof of the close relationship between the rearrangement in region 3q13-->3q26 and the pathogenesis of acute myeloid leukemia (AML). Our patient has transformed into erythroleukemia [M6 according to the French-American-British (FAB) classification] during the course of the disease.

Three-way and two-way rearrangements involving chromosomes 10, 2, 5 and 5, 2 in two marker chromosomes of a human melanoma cell line.

Two marker chromosomes (mar1 and mar2), provided with two closely spaced heterochromatic bands, were observed in the 14932 cell line established from a human metastatic melanoma. Fluorescence in situ hybridization (FISH) with the alphoid sequence p82H common to all human centromeres showed strong signals over the double C-bands of mar1 and mar2. These were recognized by a chromosome 2-specific alphoid probe, although chromosome in situ suppression (CISS) hybridization with a chromosome 2 library failed to reveal any painting along mar1 and mar2. The centromere of mar1 was identified by a chromosome 10-specific alphoid sequence and the marker chromosome was decorated from pter to a region proximal to the interpolated C-band by a chromosome 10 library. The centromere of mar2 could not be recognized by any chromosome-specific alphoid probe, but the whole mar2 was decorated by a chromosome 5 library. This library also painted the distal q arm of mar1, which was not painted by the chromosome 10 library, as well as a small band proximal to the double C-band. Identification of the two marker chromosomes reveals their common origin and indicates a role for chromosomes 2, 5 and 10 in the genesis and/or progression of the 14932 melanoma. Alteration to the chromosome-specific alphoid sequence in the centromere of mar2 provides evidence for rearrangement of constitutive heterochromatin alphoid sequences in human tumours.

A cytogenetic follow-up study of the victims of a radiation accident in Goiania (Brazil).

A radiation accident involving a cesium-137 therapy source occurred in Goiania (Brazil) in September 1987, in which more than 50 individuals were exposed to moderate to high doses (0.2-7 Gy) of gamma-radiation. A cytogenetic technique (i.e., frequencies of dicentrics and rings in peripheral lymphocytes) was employed to estimate the absorbed radiation dose. The follow-up study extending over more than 1 year indicated a decline in the frequencies of dicentrics in the lymphocytes. Using chromosome-specific biotinylated library probes for chromosomes 1, 2, 8 and 19, we studied the frequencies of chromosomal translocations and deletions and the incidence of aneuploidy in the lymphocytes of exposed individuals. In some individuals there was a significant increase in the frequency of translocations and aneuploidy. In other experiments, in which the frequencies of HPRT mutations were determined in lymphocytes using the BrdU-labeling method, some individuals showed an increase (from about 2- to 50-fold) in mutant frequencies.

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.