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pq-COBRA-FISH and premature chromosome condensation; a novel combination in molecular cytogenetics
Abstract
Human chromosomes have been studied for over a century, but it took until the 1950s that the correct human chromosome number was determined. The development of chromosome pretreatment procedures and modification of DNA staining techniques some 20 years later, allowed more precise chromosome identification based on banding patterns. This landmarkadvance in human cytogenetics allowed researchers to address specific clinical and research questions and make rapid progress in understanding the chromosomal basis of many genetic diseases and cancer. Although banding methodologies allow the distinction of individual chromosome pairs and the detection of gross karyotype aberrations, the analysis is heavily dependent on the accumulated experience and sometimes subjective interpretation of cytogeneticists. The low mitotic index, poor growth rate and sub-optimal chromosome morphology, which are hallmarks of tumour metaphase chromosome preparations, make cancer cytogenetics a particularly difficult discipline. Introduction of fluorescent in situ hybridisation (FISH) in combination with recombinant DNA technology opened a new chapter in detection and investigation of chromosomal basis of human genetic diseases in the 1990s. The success of this new discipline, molecular cytogenetics, in gene mapping and precise characterisation of chromosomal abnormalities, hinged on development of nucleic acids modification to incorporate fluorescent reporter molecules into nucleic acid hybridization probes. This thesis contributes to further advancement of molecular cytogenetics. The pq-COBRAFISH method presented in Chapter 2, employs a combination of binary and ratio-labelling to discriminate in one experiment all human chromosome arms, thus enhancing and objectifying the detection of chromosomal abnormalities. In Chapter 3, it has been used to detect chromosomal rearrangements in individuals with abnormal phenotype and normal G-banding patterns. It also aided in identification of a novel cancerous gene alteration by discovering BCL11B, as a new candidate gene in acute myelocytic leukaemia. pq-COBRA-FISH readily resolves complex karyotypes, characteristic of solid tumours. As such it was instrumental to discovery of a novel function of the chromatin remodelling complex, hSNF5, in maintenance of chromosome stability and ploidy (Chapter 4). Although molecular cytogenetics with its sophisticated multicolour FISH technology has allowed significant advancement in cancer and has become an intricate part of diagnosis and research there are still analytical and methodological problems in cancer cytogenetics. Full karyotyping, for example, is still restricted to mitotic cells. Chapter 5 of this thesis presents studies where a potent phosphatase inhibitor, calyculin A, was employed to prematurely condense the chromosomes of non-mitotic cells. This premature chromosome condensation technique or PCC increases the number of analysable chromosome structures compared to classical mitotic arrest, with minimum culture. This greatly improves the capacity of researchers to cytogenetically study solid tumours, where the success of comprehensive karyotyping of biopsy material has always been problematic. The specific morphological characteristics of PCC chromosomes furthermore allow distinction of the G1 and G2 phases of the cell cycle. The possibility to assess (differences in) chromosomal abnormalities of cells in the various cell cycle phases can offer valuable insight into the mechanisms underlying the tumorigenesis processes, including the much debated chromosome instability.
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Cytogenetics is considered one of the most valuable prognostic determinants in acute myeloid leukemia (AML). However, many studies on which this assertion is based were limited by relatively small sample sizes or varying treatment approach, leading to conflicting data regarding the prognostic implications of specific cytogenetic abnormalities. The Medical Research Council (MRC) AML 10 trial, which included children and adults up to 55 years of age, not only affords the opportunity to determine the independent prognostic significance of pretreatment cytogenetics in the context of large patient groups receiving comparable therapy, but also to address their impact on the outcome of subsequent transplantation procedures performed in first complete remission (CR). On the basis of response to induction treatment, relapse risk, and overall survival, three prognostic groups could be defined by cytogenetic abnormalities detected at presentation in comparison with the outcome of patients with normal karyotype. AML associated with t(8;21), t(15;17) or inv(16) predicted a relatively favorable outcome. Whereas in patients lacking these favorable changes, the presence of a complex karyotype, −5, del(5q), −7, or abnormalities of 3q defined a group with relatively poor prognosis. The remaining group of patients including those with 11q23 abnormalities, +8, +21, +22, del(9q), del(7q) or other miscellaneous structural or numerical defects not encompassed by the favorable or adverse risk groups were found to have an intermediate prognosis. The presence of additional cytogenetic abnormalities did not modify the outcome of patients with favorable cytogenetics. Subgroup analysis demonstrated that the three cytogenetically defined prognostic groups retained their predictive value in the context of secondary as well as de novo AML, within the pediatric age group and furthermore were found to be a key determinant of outcome from autologous or allogeneic bone marrow transplantation (BMT) in first CR. This study highlights the importance of diagnostic cytogenetics as an independent prognostic factor in AML, providing the framework for a stratified treatment approach of this disease, which has been adopted in the current MRC AML 12 trial.
Chromosomes usually condense during mitosis, but condensation may be uncoupled from mitotic events under certain circumstances. This phenomenon is known as premature chromosome condensation (PCC). Here we show that 100 nM okadaic acid or calyculin A can induce PCC at any time during the cell cycle of several types of mammalian cells; G1-, S- and G2-PCC were observed. Both okadaic acid and calyculin A are specific inhibitors of type 1 and type 2A protein serine/threonine phosphatases, suggesting that these phosphatases play a central role in regulating chromosome condensation.
We have used fluorescein-11-dUTP in a nick-translation format to produce fluoresceinated human nucleic acid probes. After
In situ hybridization of fluoresceinated DNAs to human metaphase chromosomes, the detection sensitivity was found to be 50 -100 kb.
The feasibility and the increase In detection sensitivity of microscopic imaging of In situ hybridized, fluoresceinated DNA with an integrating solid state camera for rapid cosmld mapping is illustrated. Combination
of fluoresceinated DNA with biotinated and dlgoxigenlnated DNAs allowed easy performance of triple fluorescence In situ hybridization. The potential of these techniques for DNA mapping, cytogenetics and biological doslmetry is briefly discussed.
The SWI/SNF family of chromatin-remodeling complexes facilitates
gene expression by helping transcription factors gain access to their
targets in chromatin. SWI/SNF and Rsc are distinctive members of this
family from yeast. They have similar protein components and catalytic
activities but differ in biological function. Rsc is required for cell
cycle progression through mitosis, whereas SWI/SNF is not. Human
complexes of this family have also been identified, which have often
been considered related to yeast SWI/SNF. However, all human subunits
identified to date are equally similar to components of both SWI/SNF
and Rsc, leaving open the possibility that some or all of the human
complexes are rather related to Rsc. Here, we present evidence that the
previously identified human SWI/SNF-B complex is indeed of the Rsc
type. It contains six components conserved in both Rsc and SWI/SNF.
Importantly, it has a unique subunit, BAF180, that harbors a
distinctive set of structural motifs characteristic of three components
of Rsc. Of the two mammalian ATPases known to be related to those in
the yeast complexes, human SWI/SNF-B contains only the homolog that
functions like Rsc during cell growth. Immunofluorescence studies with
a BAF180 antibody revealed that SWI/SNF-B localizes at the
kinetochores of chromosomes during mitosis. Our data suggest that
SWI/SNF-B and Rsc represent a novel subfamily of chromatin-remodeling
complexes conserved from yeast to human, and could participate in cell
division at kinetochores of mitotic chromosomes.
This report describes the use of fluorescence in situ hybridization for chromosome classification and detection of chromosome aberrations. Biotin-labeled DNA was hybridized to target chromosomes and subsequently rendered fluorescent by successive treatments with fluorescein-labeled avidin and biotinylated anti-avidin antibody. Human chromosomes in human-hamster hybrid cell lines were intensely and uniformly stained in metaphase spreads and interphase nuclei when human genomic DNA was used as a probe. Interspecies translocations were detected easily at metaphase. The human-specific fluorescence intensity from cell nuclei and chromosomes was proportional to the amount of target human DNA. Human Y chromosomes were fluorescently stained in metaphase and interphase nuclei by using a 0.8-kilobase DNA probe specific for the Y chromosome. Cells from males were 40 times brighter than those from females. Both Y chromosomal domains were visible in most interphase nuclei of XYY amniocytes. Human 28S ribosomal RNA genes on metaphase chromosomes were distinctly stained by using a 1.5-kilobase DNA probe.
RSC is an essential 15 protein nucleosome-remodeling complex from S. cerevisiae. We have identified two closely related RSC members, Rsc1 and Rsc2. Biochemical analysis revealed Rsc1 and Rsc2 in distinct complexes, defining two forms of RSC. Genetic analysis has shown that Rsc1 and Rsc2 possess shared and unique functions. Rsc1 and Rsc2 each contain two bromodomains, a bromo-adjacent homology (BAH) domain, and an AT hook. One of the bromodomains, the BAH domain, and the AT hook are each essential for Rsc1 and Rsc2 functions, although they are not required for assembly into RSC complexes. Therefore, these domains are required for RSC function. Additional genetic analysis provides further evidence that RSC function is related to transcriptional control.
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. Genes Chromosomes Cancer 28:92–97, 2000. © 2000 Wiley-Liss, Inc.