Specific DNA sequence amplification in human neuroblastoma cells.

Abstract

Southern blot analysis of a number of EcoRI-digested human neuroblastoma DNAs has revealed the presence of a family of discrete restriction fragments, the majority of which are amplified in most, but not all, of the neuroblastoma cell lines tested. None of these sequences is abundantly present in DNA from other human tumors of different tissue origins, including several either known or presumed to contain amplified DNA. Hence, these sequences appear to be specifically amplified by neuroblastoma cells. Hybridization with metaphase chromosomes in situ has localized these sequences to either the homogeneously staining regions or double-minute chromosomes of different neuroblastoma cell lines, indicating that these chromosomal structures, although present in cell lines established from different patients, share many sequences and may have a common, but as yet unknown, function.

Expression of the amplified domain in human neuroblastoma cells.

“…Recently, we have been able to directly demonstrate by Southern blotting procedures and hybridization in situ with kinetically purified genomic DNA probes that neuroblastoma cells do indeed contain amplified DNA and that their HSRs and DMs harbor these sequences (27). Additionally, our data have also shown that, among several HSR-or DM-containing human neuroblastoma cell lines established from various patients, the great majority amplify a large array of cross-hybridizing fragments in addition to a number of fragments which may be cell line specific (27). In no case, however, have any of the fragments found to be amplified in neuroblastoma cells also been found to be amplified in other nonrelated human ttimor cells known or suspected to contain amplified DNA.…”

“…Preparation of high-molecular-weight DNA and poly(A)+ RNA from cells and tissues was carried out as reported previously (21,(25)(26)(27). DNA and RNA transfer experiments were performed as described by Southern (34) and Thomas (35), respectively, with modifications (21).…”

“…We therefore used probes prepared by Cot fractionation (8) and representative of the amplified DNA from the HSR-containing human neuroblastoma cell line BE(2)-C (27) to screen a partial cDNA library prepared from the polysomal polyadenylated [poly(A)+] RNA of the BE(2)-C cell line. Recombinant plasmids containing inserts hybridizable with components of these heterogeneous amplified DNA probes were then used as probes in Southern blots to demonstrate their homology to amplified neuroblastoma DNA and in Northern EXPRESSION OF AMPLIFIED DNA IN HUMAN TUMOR CELLS 2371 scribed briefly (27) and in detail (5) and include the HSRcontaining lines BE(2)-C and NAP(H), the DM-containing lines SK-N-BE(1), NAP(D), CHP-234, SMS-KAN, SMS-KCN, and SMS-MSN, the ABR-containing line SMS-KANR, and the non-HSR-DM-containing line SH-SY5Y. Nonneuroblastoma cell lines include the fibroblast line WI-38 (16), the retinoblastoma line Y79T (13), the neuroendocrine colon tumor line COLO 321 (29), the small cell lung carcinoma line NCI-H82 (39), the cortical adenocarcinoma and colon adenocarcinoma lines SW-13 (12) and SW-48 (9,12), respectively, and the bladder carcinoma line SW-800 (12).…”

“…Cot 10-300 DNA from neuroblastoma cell line BE(2)-C and from human placenta was prepared by standard hydroxyapatite chromatography as described previously (27). Samples of this DNA were then nick translated (30) with 32P-labeled precursors to a specific activity of 0.5 x 108 to 2.0 x 108 cpm/,lg and, after exhaustive hybridization against cellulose-bound placental DNA to remove highly repeated sequences (8), were used directly to screen partial cDNA libraries.…”

Coordinate amplification of metallothionein I and II genes in cadmium-resistant Chinese hamster cells: implications for mechanisms regulating metallothionein gene expression.

“…Amplification of specific genes or gene families has been demonstrated in response to toxic or metabolic stress (1,4,38,47,48,54,56,57), in neoplastic disorders (9,39), and during development or evolution (7,8,34,52,59). In many studies, gene amplification has been correlated with a proportionate increase in specific mRNA and protein (see references 1, 3, and 48 for review and examples).…”