It has long been considered that genetic instability is an integral component of human neoplasia1–3. In a small fraction of tumours, mismatch repair deficiency leads to a microsatellite instability at the nucleotide sequence level4,5. In other tumours, an abnormal chromosome number (aneuploidy) has suggested an instability, but the nature and magnitude of the postulated instability is a matter of conjecture. We show here that colorectal tumours without microsatellite instability exhibit a striking defect in chromosome segregation, resulting in gains or losses in excess of 10 –2 per chromosome per generation. This form of chromosomal instability reflected a continuing cellular defect that persisted throughout the lifetime of the tumour cell and was not simply related to chromosome number. While microsatellite instability is a recessive trait6,7, chromosomal instability appeared to be dominant. These data indicate that persistent genetic instability may be critical for the development of all colorectal cancers, and that such instability can arise through two distinct pathways.

Genetic instability in colorectal cancers

Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9

A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported. DNA inserts from a single chromosomal library are labeled with biotin and partially preannealed with a titrated amount of total human genomic DNA prior to hybridization with cellular or chromosomal preparations. The cross-hybridization of repetitive sequences to nontargeted chromosomes can be markedly suppressed under appropriate preannealing conditions. The remaining single-stranded DNA is hybridized to specimens of interest and detected with fluorescent or enzymelabeled avidin conjugates following post-hybridization washes. DNA inserts from recombinant libraries for chromosomes 1, 4, 7, 8, 13, 14, 18, 20, 21, 22, and X were assessed for their ability to decorate specifically their cognate chromosome; most libraries proved to be highly specific. Quantitative densitometric analyses indicated that the ratio of specific to nonspecific hybridization signal under optimal preannealing conditions was at least 8:1. Interphase nuclei showed a cohesive territorial organization of chromosomal domains, and laserscanning confocal fluorescence microscopy was used to aid the 3-D visualization of these domains. This method should be useful for both karyotypic studies and for the analysis of chromosome topography in interphase cells.

https://epub.ub.uni-muenchen.de/9279/1/cremer_thomas_9279.pdf

Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries

Studies of higher-order chromatin arrangements are an essential part of ongoing attempts to explore changes in epigenome structure and their functional implications during development and cell differentiation. However, the extent and cell-type-specificity of three-dimensional (3D) chromosome arrangements has remained controversial. In order to overcome technical limitations of previous studies, we have developed tools that allow the quantitative 3D positional mapping of all chromosomes simultaneously. We present unequivocal evidence for a probabilistic 3D order of prometaphase chromosomes, as well as of chromosome territories (CTs) in nuclei of quiescent (G0) and cycling (early S-phase) human diploid fibroblasts (46, XY). Radial distance measurements showed a probabilistic, highly nonrandom correlation with chromosome size: small chromosomes-independently of their gene density-were distributed significantly closer to the center of the nucleus or prometaphase rosette, while large chromosomes were located closer to the nuclear or rosette rim. This arrangement was independently confirmed in both human fibroblast and amniotic fluid cell nuclei. Notably, these cell types exhibit flat-ellipsoidal cell nuclei, in contrast to the spherical nuclei of lymphocytes and several other human cell types, for which we and others previously demonstrated gene-density-correlated radial 3D CT arrangements. Modeling of 3D CT arrangements suggests that cell-type-specific differences in radial CT arrangements are not solely due to geometrical constraints that result from nuclear shape differences. We also found gene-density-correlated arrangements of higher-order chromatin shared by all human cell types studied so far. Chromatin domains, which are gene-poor, form a layer beneath the nuclear envelope, while gene-dense chromatin is enriched in the nuclear interior. We discuss the possible functional implications of this finding.

/pdf/three-dimensional-maps-of-all-chromosomes-in-human-male-2cdmfxnlw0.pdf

Three-Dimensional Maps of All Chromosomes in Human Male Fibroblast Nuclei and Prometaphase Rosettes

Precise coordination of the S and M phases of the eukaryotic cell cycle is critical not only for normal cell division, but also for effective growth arrest under conditions of stress. When damaged, a cell must communicate signals to both the mitotic and DNA synthesis machineries so that a mitotic block is not followed by an extra S phase, or vice versa. The biochemical mechanisms regulating this coordination, termed checkpoints, have been identified in lower eukaryotes, but are largely unknown in mammalian cells. Here we show that p21 WAF1/CIP1, the prototype inhibitor of cyclin-dependent kinases (CDKs), is required for this coordination in human cells. In the absence of p21, DNA-damaged cells arrest in a G2-like state, but then undergo additional S phases without intervening normal mitoses. They thereby acquire grossly deformed, polyploid nuclei and subsequently die through apoptosis. Perhaps not by coincidence, the DNA-damaging agents that can cause S/M uncoupling are used in the clinic to kill cancer cells preferentially.

Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21

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 cosmid mapping is illustrated. Combination of fluoresceinated DNA with biotinated and digoxigeninated DNAs allowed easy performance of triple fluorescence in situ hybridization. The potential of these techniques for DNA mapping, cytogenetics and biological dosimetry is briefly discussed.

In situ hybridization with fluoresceinated DNA

On the DNA content and ploidy of trypanosomes.

Detection of chromosome aberrations in interphase tumor nuclei by nonradioactive in situ hybridization.

The development and application of a procedure for interphase cytogenetics on brain tumor material is described. Nuclei isolated from freshly removed brain tumor tissue were investigated for chromosomal aberrations by nonradioactive in situ hybridization with a panel of chromosome-specific probes. The panel consisted of nine satellite DMA probes specific for the centromeric regions of chromosomes 1, 6, 7, 10, 11, 17, 18, X, and Y. For each probe, the number of hybridization signals per cell was determined in 200 nuclei. It was inferred from the hybridization results that in 11 gliomas (seven astrocytomas grade II-IV, three oligodendrogliomas, and one ependymoma) the numerical aberrations were gains of chromosomes 1 (once), 7 (twice), 10 (once), 11 (twice), and X (twice); losses of chromosomes 1 (once), 10 (twice), 17 (twice), and Y (once); and complete tetraploidy (once). Among the 18 investigated meningiomas monosomy 18 and trisomy 17 were observed once and twice, respectively. An additional hybridization with a cosmid probe for the BCR gene on 22q 11 indicated monosomy 22q in 11 meningiomas. These results show the value of interphase cytogenetics for the analysis of solid tumors for which it is relatively difficult to obtain sufficient metaphases of good quality for conventional cytogenetics.

Interphase cytogenetics of brain tumors.

Triple fluorescence in situ hybridization with a plasmid DNA library from sorted human chromosomes 8 in combination with bacteriophage clones flanking the breakpoint in 8q24 of the Burkitt lymphoma cell line Jl was used for the specific delineation of this breakpoint in individual tumor cells. With this approach, tumor-specific breakpoints in translocation chromosomes can be detected at all stages of the cell cycle with high specificity.

https://epub.ub.uni-muenchen.de/9354/1/cremer_thomas_9354.pdf

Specific metaphase and interphase detection of the breakpoint region in 8q24 of Burkitt lymphoma cells by triple-color fluorescence in situ hybridization

Mels van der Ploeg

Papers

In situ hybridization with fluoresceinated DNA

On the DNA content and ploidy of trypanosomes.

Detection of chromosome aberrations in interphase tumor nuclei by nonradioactive in situ hybridization.

Interphase cytogenetics of brain tumors.

Specific metaphase and interphase detection of the breakpoint region in 8q24 of Burkitt lymphoma cells by triple-color fluorescence in situ hybridization