Isolation of amplified DNA sequences from IMR-32 human neuroblastoma cells: facilitation by fluorescence-activated flow sorting of metaphase chromosomes.

Abstract

authors request that the following correction be noted. All hybrids described in Fig. 5 contained at least part of the human X chromosome, and the box for each hybrid in the column corresponding to the human X should have been darkened. This clerical error does not alter any conclusions in the paper.Proc. Natt Acad. Sci. USA Vol. 80, pp. A number of human tumors (1-3), in particular neuroblastomas (4-6), exhibit homogeneously staining chromosomal regions (HSRs) and extrachromosomal double minute objects (DMs) which are probably cytological manifestations of amplified genes (see ref. 7 for a review). These H-SRs and DMs are often observed in tumor tissue removed prior to institution of therapy, and it seems reasonable to suggest that they may contain genetic information responsible for some of the phenotypic properties of the tumor cells. This hypothesis can be tested by isolation and characterization of DNA within the amplified chromosomal regions. In one study (8,9), DMs of a rat adrenal tumor cell line were isolated by differential centrifugation and cloned in a A bacteriophage. We have utilized a different approach to isolate sequences specific for the HSRs of a human neuroblastoma cell, exploiting the increased size imparted to metaphase chromosomes by these HSRs.The human neuroblastoma cell line IMR-32, which was established from tissue of a patient who had not been exposed to any chemotherapy (10), constitutes an excellent system for analysis of HSR-specific sequences. IMR-32 cells, which exhibit some karyotypic heterogeneity, possess, in addition to a normal chromosome 1, two abnormal chromosomes 1 in which an HSR is inserted in the short arm (in the middle of band lp3). The HSR-containing chromosome 1 is 50% larger than the normal chromosome 1 (11) and hence is easily separable from it and all other, smaller, human chromosomes by fluorescence-activated sorting of isolated IMR-32 metaphase chromosomes after they have been stained with a DNA-specific fluorochrome such as 33258 Hoechst (12).From the DNA of a few million metaphase chromosomes enriched for the HSR-containing chromosome I by flow sorting, we have prepared recombinant libraries in the Charon phage 21A (13) from which cloned sequences specific for the HSR have been identified both by DNA blotting (14) and by in situ hybridization (15, 16). These cloned sequences provide a unique opportunity to study the composition, formation, and functional significance of the amplified sequences in this and other neuroblastoma cell lines.MATERIAL AND METHODS IMR-32 neuroblastoma cells, originally obtained from the American Type Tissue Culture Collection and maintained in culture for several months, were used in these experiments. Cells were cultured in Ham F10 medium supplemented with 10% fetal calf serum. Cytological metaphase chromosome preparations were stained with quinacrine or chromomycin A3 plus methyl green as described (17).Nonconfluent cells were trapped at metaphase by treatment with Colcemid (45 ng/ml) for 20 hr. Cells were detached by shaking, d...

Co-amplification of a novel gene, NAG, with the N-myc gene in neuroblastoma

“…The N-myc gene spans approximately 7 kb, whereas the size of the N-myc amplicon varies between 350 kb to over 1 Mb (Akiyama et al, 1994;Hiemstra et al, 1994;Kanda et al, 1983). Co-ampli®cation of other¯anking genes, that may in¯uence disease progression, has been suggested (George et al, 1996;Squire et al, 1995).…”

Amplification of oncogene N-myc and the adjacent DNA fragments in human neuroblastoma: Comparison in primary and metastasized tumors and in the established cell line.

“…Clone 8 is a 1.75 kilobase HindIII fragment previously isolated from the HSR of human neuroblastoma cell line IMR-32 (14). G2I is a cloned fragment from a cDNA library of IMR-32 cells and hybridized to EcoRI fragments of 6.6, 5.4, 4.0, 3.2 and 1.5 kilobases (18).…”

“…It is also intriguing that N- Kanda et al myc amplification was observed in diploid-or tetraploid-type neuroblastomas, but not in triploid-type tumors (13,16). Several sequences, which coamplify with N-myc have been isolated from neuroblastoma cell line IMR-32, and all mapped to the short arm of chromosome 2 (14,18).' Tumor DNA analysis, using these coamplified sequences as probe, apparently exhibited variation of amplicon size in the different tumors (300-3,000 kilobases) (25,26).…”

Mapping of the human APOB gene to chromosome 2p and demonstration of a two-allele restriction fragment length polymorphism.

“…One ofthe fibroblast donors was afemale carrier of the reciprocal X/13 translocation t(X;13)(q21-q23::q21-q31) (11). The hybrid cell lines have been characterized for human chromosome complements by isozyme and cytogenetic techniques (13,14) and with cloned DNA probes for each of the human autosomes and the X chromosome (15,16). DNAs prepared from somatic cell hybrids were digested with EcoRI, electrophoresed on 0.7% agarose gels, and subjected to Southern blot analysis.…”