Rapid interphase and metaphase assessment of specific chromosomal changes in neuroectodermal tumor cells by in situ hybridization with chemically modified DNA probes

Abstract

Repeated DNAs from the constitutive heterochromatin of human chromosomes 1 and 18 were used as probes in nonradioactive in situ hybridization experiments to define specific numerical and structural chromosome aberrations in three human glioma cell lines and one neuroblastoma cell line. The number of spots detected in interphase nuclei of these tumor cell lines and in normal diploid nuclei correlated well with metaphase counts of chromosomes specifically labeled by in situ hybridization. Rapid and reliable assessments of aneuploid chromosome numbers in tumor lines in double hybridization experiments were achieved, and rare cells with bizarre phenotype and chromosome constitution could be evaluated in a given tumor cell population. Even with suboptimal or rare chromosome spreads specific chromosome aberrations were delineated. As more extensive probe sets become available this approach will become increasingly powerful for uncovering various genetic alterations and their progression in tumor cells. 0 1988 Academic Press. Inc.Specific chromosome aberrations are thought to be important in the development of malignant human gliomas. However, several groups have noted that adequate chromosome analyses of both primary cultures and of solid glioma samples are problematic [ 1, 21. These difficulties are generally encountered in all cytogenetic analyses of solid tumors; many tumor preparations do not contain sufficient numbers of well-spread and banded metaphase cells, and a large number of mitotic cells within solid tumors are inaccessible for analysis. With such small samples of mitotic cells it is difficult to determine if a chromosomal pattern is representative of the whole tumor cell population. Thus specific numerical and structural aberrations in a given tumor type are difficult to confirm on a broad scale, and rare aberrant cells with special malignant propensities may also be overlooked. Furthermore the analysis of complex chromosome changes in very aneuploid cells requires the detailed attention of highly skilled observers. Even when these time consuming analyses are performed by experienced cytogeneticists, it is difficult to pinpoint chromosomal breakpoints and segments involved in duplication, deletion, or translocation [l].We are attempting to develop procedures and DNA probe sets for visualization of specific numerical and structural aberrations in tumor cells. The detection of chromosome abnormalities in interphase nuclei, an approach termed "interphase cytogenetics"[3], appears to be a useful addition to the classical metaphase analysis. The examination of numerical aberrations in interphase nuclei can yield ' TO whom reprint requests should be addressed 199

Rapid generation of chromosome-specific alphoid DNA probes using the polymerase chain reaction

“…In situ hybridization and washing procedures were performed essentially as described previously (Cremer et al 1988b) with the following modifications. Between 5 to 20rig biotinylated alphoid PCR-amplified DNA was applied per slide using 24 • 50 mm cover glasses.…”

“…Probe detection with avidin conjugated with fluorescein-isothiocyanate (FITC) was carried out as described previously (Cremer et al 1988b). Signals were amplified otlce according to the protocol of Pinkel et al (1986).…”

“…In clinical cytogenetics, chromosome-specific alphoid probes have become an important aid for the detection of specific numerical chromosome aberrations and for the definition of marker chromosomes (Cremer et al 1986(Cremer et al , 1988bHopman et al 1989;Anastasi et al 1990;Poddighe et al 1991). Recently, Koch et al (1989) have used the polymerase chain reaction (PCR) for the rapid generation of alphoid DNA probes directly from somatic hybrid cells containing the human chromosomes X and 11.…”

Characterization of two marker chromosomes in a patient with acute nonlymphocytic leukemia by two-color fluorescence in situ hybridization

“…Fluorescence in situ hybridization (FISH) with DNA libraries from sorted human chromosomes and regional DNA probes has improved identification of marker chromosome [1][2][3][4][5][6][7]. Recently, we showed the usefulness of DNA probes generated by Alu polymerase chain reaction (PCR} of yeast artificial chromosome (YAC) clones [8] for detection of structural chromosome aberrations in patients with leukemia and myelodysplastic syndromes (MDS) at any stage of the cell cycle [9].…”

Chromosome Aberration Detection with Hybridized DNA Probes: Digital Image Analysis and Slit Scan Flow Cytometry

“…The rationale of this new approach is the use of hybridization techniques to visualize specific chromosomes and chromosome subregions (Durnam et al, 1985;Manuelidis et al, 1985;Schardin et al, 1985;Cremer et al, 1986Cremer et al, , 1988aPinkel et al, 1986;Emmerich et al, 1989). To exemplify the method, chromosomes from Chinese hamster X human hybrid cell lines are used.…”