Characterization of N-myc amplification in a human neuroblastoma cell line by clones isolated following the phenol emulsion reassociation technique and by hexagonal field gel electrophoresis.

The N-myc amplification of human neuroblastomas was characterized by the amplified DNA cloned from the cell line MC-NB-1 using the phenol emulsion reassociation technique (PERT). A number of PERT clones exhibiting amplification in this cell line were tested for amplification in other neuroblastoma cell lines. In almost all cell lines examined, only a few clones were co-amplified with N-myc and most of the others were exclusively amplified in a subset of the cell lines. The total aggregate size of the Hind III fragment identified by the PERT clones was approximately 350 kb. Most of the PERT clones were mapped to human chromosome (chr) 2p23-2pter, where the N-myc gene is located. Four types of amplicons, the 100, 420, 480 and 520 kb fragments, shown to be Not I fragments, were identified by hexagonal field gel electrophoresis. Three fragments are ordered in a head-to-tail array, and the remaining fragment is either ordered in a tail-to-head array or something else. Despite the extremely unusual construction of the amplified sequences in this cell line as compared with others, there was a low degree of sequence heterogeneity among the amplicons within this cell line. These observations lead to the idea that the complex rearrangements that give rise to the heterogeneous organization of the amplified sequences among the different cell lines precede the amplification of these sequences.     

Characterization of DNA rearrangements of N-myc gene amplification in three neuroblastoma cell lines by pulsed-field gel electrophoresis

We characterized N-myc gene amplification in three human neuroblastoma cell lines (IMR-32, TGW, GOTO). Rearrangements in long-range regions surrounding amplified N-myc genes were examined by pulsed-field gel electrophoresis. Since rare-cutting enzymes completely digested DNA at the middle of the N-myc gene, we were able to construct a physical map upstream and downstream of the germline N-myc gene, and to obtain information on restriction sites surrounding amplified N-myc genes. This method enables us to envisage the organization of amplified units over a long range. Digestion patterns differed considerably among the germline and the three cell lines, but were simple in each case. We estimated that the minimal distance between neighboring N-myc genes is at least several hundred kilobases. Our data suggest that amplification units contain several DNA fragments derived from ditterent loci, but that they are homogeneous.     

Isolation and structural analysis of a 1.2-megabase N-myc amplicon from a human neuroblastoma.

Oncogene amplification is observed frequently in human cancers, but little is known about the mechanism of gene amplification or the structure of amplified DNA in tumor cells. We have studied the N-myc amplified domain from a representative neuroblastoma cell line, SMS-KAN, and compared the map of the amplicon in this cell line with that seen in normal DNA. The SMS-KAN cell line DNA was cloned into yeast artificial chromosomes (YACs), and clones were identified by screening the YAC library with amplified DNA probes that were obtained previously (B. Zehnbauer, D. Small, G. M. Brodeur, R. Seeger, and B. Vogelstein, Mol. Cell. Biol. 8:522-530, 1988). In addition, YAC clones corresponding to the normal N-myc locus on chromosome 2 were obtained by screening two normal human YAC libraries with these probes, and the restriction maps of the two sets of overlapping YACs were compared. Our results suggest that the amplified domain in this cell line is a approximately 1.2-Mb circular molecule with a head-to-tail configuration, and the physical map of the normal N-myc locus generally is conserved in the amplicon. These results provide a physical map of the amplified domain of a neuroblastoma cell line that has de novo amplification of an oncogene. The head-to-tail organization, the general conservation of the normal physical map in the amplicon, and the extrachromosomal location of the amplified DNA are most consistent with the episome formation-plus-segregation mechanism of gene amplification in these tumors.     

Amplified N-myc in human neuroblastoma cells is often arranged as clustered tandem repeats of differently recombined DNA.

Human neuroblastoma cells often carry amplified DNA encompassing the gene N-myc. Amplified N-myc has been found localized in "double minutes" in direct tumor cell preparations. In contrast, later passages carried amplified N-myc almost exclusively within a single homogeneously staining chromosomal region located at a chromosomal site different from the normal location of N-myc. We used pulsed field gel electrophoresis to define the structural arrangement of the amplified DNA. Long-range mapping was facilitated by the presence of several sites for rare cutting restriction endonucleases in the 5' region of N-myc. Amplified DNAs of different neuroblastoma cell lines were heterogeneous in size and had undergone recombination at various distances from N-myc. N-myc occupied a central position within the amplified DNA, and in no case was the coding region affected by recombination. Among neuroblastoma cells, varying proportions of amplified DNA (in some instances close to 100%) consisted of multiple tandem arrays of DNA segments ranging in size from 100 to 700 kilobase pairs. Tumor cells with low degrees of amplification revealed regions of amplified DNA in excess of 1,500 kilobase pairs without apparent rearrangement. Our observations, in concert with the cytogenetic findings, suggest a model of gene amplification which involves unscheduled DNA replication, recombination, and formation of extrachromosomal DNA followed by integration into a chromosome and subsequent in situ multiplication. The central position which N-myc occupies within the amplified sequences and the lack of recombination within the coding region of N-mc indicate that N-myc rather than other genetic information provides the selective advantage for retention of the amplified DNA.     

Structure of amplified DNA in different Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

Syrian hamster cell lines selected in multiple steps for resistance to high levels of N-(phosphonacetyl)-L-aspartate (PALA) contain many copies of the gene coding for the pyrimidine pathway enzyme CAD. Approximately 500 kilobases of additional DNA was coamplified with each copy of the CAD gene in several cell lines. To investigate its structure and organization, we cloned ca. 162 kilobases of coamplified DNA from cell line 165-28 and ca. 68 kilobases from cell line B5-4, using a screening method based solely on the greater abundance of amplified sequences in the resistant cells. Individual cloned fragments were then used to probe Southern transfers of genomic DNA from 12 different PALA-resistant mutants and the wild-type parents. A contiguous region of DNA ca. 44 kilobases long which included the CAD gene was amplified in all 12 mutants. However, the fragments cloned from 165-28 which were external to this region were not amplified in any other mutant, and the external fragments cloned from B5-4 were not amplified in two of the mutants. These results suggest that movement or major rearrangement of DNA may have accompanied some of the amplification events. We also found that different fragments were amplified to different degrees within a single mutant cell line. We conclude that the amplified DNA was not comprised of identical, tandemly arranged units. Its structure was much more complex and was different in different mutants. Several restriction fragments containing amplified sequences were found only in the DNA of the mutant cell line from which they were isolated and were not detected in DNA from wild-type cells or from any other mutant cells. These fragments contained novel joints created by rearrangement of the DNA during amplification. The cloned novel fragments hybridized only to normal fragments in every cell line examined, except for the line from which each novel fragment was isolated or the parental population for that line. This result argues that "hot spots" for forming novel joints are rare or nonexistent.     

Clinical significance of gene amplification studied in human neuroblastoma xenografts: relationship with tumor growth rate, chemotherapeutic sensitivities and levels of neuron-specific enolase

Tumor doubling time, sensitivity to chemotherapeutic agents and concentrations of neuron-specific enolase were studied in nine human neuroblastoma xenografts, in which amplifications of N-myc, clones 8 and G21 were known; N-myc was amplified in eight, clone 8 in five and clone G21 in four of these nine xenografts. Tumor doubling time was longest in one xenograft, TNB10, which lacks the amplification of either N-myc or clone 8 or G21, and shortest in TNB1 in which all three DNA sequences are amplified with a DNA rearrangement in clone 8. No correlations were found between genomic amplification of N-myc, clones 8 and G21 and effectiveness of five chemotherapeutic drugs tested, except for cis-platinum. cis-Platinum was found to be effective on all but the one xenograft, TNB10, with the longest tumor doubling time. Concentration of neuron-specific enolase in tumor extract was lowest in TNB1 and correlated with the length of the tumor doubling time.     

Novel DNA rearrangements are associated with dihydrofolate reductase gene amplification

We have employed the technique of chromosome "walking" to determine the structure of 240 kilobases of amplified DNA surrounding the dihydrofolate reductase gene in methotrexate-resistant mouse cell lines. Within this region, we have found numerous DNA rearrangements which occurred during the amplification process. DNA subclones from regions flanking the dihydrofolate reductase gene were also utilized as hybridization probes in other cell lines. Our results show that: 1) amplification-specific DNA rearrangements or junctions are unique to each cell line; 2) within a given cell line, multiple amplification-specific DNA sequence rearrangements are found; 3) the degree of amplification of sequences flanking the dihydrofolate reductase gene shows quantitative variation among and within cell lines; and 4) both the arrangement of amplified sequences as well as the magnitude of gene amplification may vary with prolonged culture even under maintenance selection conditions. These studies indicate that there is no static repetitive unit amplified in these cells. Rather, a dynamic and complex arrangement of the amplified sequences exists which is continually changing.     

Characterization of N-myc amplification in a human neuroblastoma cell line by clones isolated following the phenol emulsion reassociation technique and by hexagonal field gel electrophoresis

The N-myc amplification of human neuroblastomas was characterized by the amplified DNA cloned from the cell line MC-NB-1 using the phenol emulsion reassociation technique (PERT). A number of PERT clones exhibiting amplification in this cell line were tested for amplification in other neuroblastoma cell lines. In almost all cell lines examined, only a few clones were co-amplified with N-myc and most of the others were exclusively amplified in a subset of the cell lines. The total aggregate size of theHind III fragment identified by the PERT clones was approximately 350 kb. Most of the PERT clones were mapped to human chromosome (chr) 2p23-2pter, where the N-myc gene is located. Four types of amplicons, the 100, 420, 480 and 520 kb fragments, shown to beNot I fragments, were identified by hexagonal field gel electrophoresis. Three fragments are ordered in a head-to-tail array, and the remaining fragment is either ordered in a tail-to-head array or something else. Despite the extremely unusual construction of the amplified sequences in this cell line as compared with others, there was a low degree of sequence heterogeneity among the amplicons within this cell line. These observations lead to the idea that the complex rearrangements that give rise to the heterogeneous organization of the amplified sequences among the different cell lines precede the amplification of these sequences.     

Amplified DNA sequences in cancers

Amplification of genes other than known oncogenes was analyzed using an in-gel DNA renaturation method, in which a mixture of restriction fragments of radioactively labelled tracer DNA and unlabelled driver DNA was electrophoresed and amplified DNA fragments were visualized after two cycles of denaturation and renaturation in the gel. Different DNA fragments were found to be amplified more than 400 fold in NB1, a neuroblastoma cell line, in Y79, a retinoblastoma cell line and in H69, a small cell lung carcinoma cell line, in addition to 120 to 160-fold amplification of N-myc gene in these three cell lines.     

Cloning and physical mapping of DNA sequences encompassing a region in N-myc amplicons of a human neuroblastoma cell line.

Cloning and physical mapping of DNA sequences encompassing N-myc amplicons of a human neuroblastoma cell line were done. A number of lambda phage clones within this region were isolated using the probes prepared by the phenol emulsion reassociation technique. Based on the restriction mapping, they were integrated into 8 contigs with sizes of 25-60 kb which, in total, encompassed a 330 kb region. Several amplicons, 100, 420, 480 and 520 kb in size as a Notl fragment, were identified using hexagonal field gel electrophoresis, and the contigs were assigned in these Notl fragments. The region encompassed by the contigs was equivalent to some 60-80% of the amplicons identified as a Notl fragment. In order to compare the amplified regions flanking the N-myc gene among the cell lines, the phage clones to cover the whole contigs were used for hybridization as a probe. The results showed that the portions of the whole contigs ranging 18-45% were also amplified in the cell lines examined. These results allowed us to identified the 'rearranged sites' which were rather evenly distributed, one at every 40 kb, through the contigs. These observations lead to the idea that an amplified DNA domain is constructed after the multiple rearrangements and then increases in number, finally resulting in the formation of subsets of amplicons with sequence homogeneity.     

Retinoic acid treatment of human neuroblastoma cells is associated with decreased N-myc expression

Cells from human neuroectodermal tumors (retinoblastoma and neuroblastoma) and from neuroblastoma cell lines express a gene, N-myc, which is frequently amplified in these tumors. We report here that N-myc mRNA content is markedly decreased in cells of a neuroblastoma cell line (LA-N-5) following differentiation induced with retinoic acid. Exposure of the cells to retinoic acid induced morphologic changes consistent with neuronal differentiation, and led to a 75% decrease in expression of N-myc mRNA. These results suggest that N-myc expression is intimately related to an undifferentiated phenotype in neuroblastoma cells, and support other studies which relate N-myc expression to the malignant phenotype in neuroblastoma tumors.     

Excision of N-myc from chromosome 2 in human neuroblastoma cells containing amplified N-myc sequences.

Amplification of one of three growth-stimulating myc genes is a common method by which many tumor types gain a proliferative advantage. In metastatic human neuroblastoma, the amplification of the N-myc locus, located on chromosome 2, is a dominant feature of this usually fatal pediatric cancer. Of the many models proposed to explain this amplification, all incorporate as the initial step either disproportionate overreplication of the chromosomal site or recombination across a loop structure. The original locus is retained within the chromosome in the overreplication models but is excised in the recombination models. To test these models, we have used somatic cell hybrids to separate and analyze the chromosomes 2 from a neuroblastoma cell line containing in vivo amplified N-myc. Our results demonstrate that N-myc is excised from one of the chromosomes, suggesting that deletion is a requisite part of gene amplification in a naturally occurring system.     

Amplification of the N-myc oncogene in an adenocarcinoma of the lung

c-myc oncogene is the most extensively studied member of the myc gene family, which now consists of three characterized members, namely the c-myc, N-myc, and L-myc genes. Deregulation owing to amplification and/or rearrangements of the c-myc gene have been described in a variety of human malignancies. Several neuroblastomas have amplifications of the N-myc genes. The c-myc, N-myc, or L-myc oncogenes are also found amplified in different cell lines from small cell carcinomas of the lung. In this study, we have examined the c-myc, N-myc, and c-erbB oncogenes in 34 clinical and autopsy tumor specimens representing various histopathological types of human lung cancer, including nine small cell lung cancers. A 30-fold amplification of the N-myc gene was found in a tumor histopathologically and histochemically verified as a typical adenocarcinoma. No amplifications of the c-myc or c-erbB oncogenes were seen in any of the tumors. In the DNA of one small cell carcinoma, an extra c-myc and N-myc cross-hybridizing restriction fragment was observed, possibly owing to an amplification of a yet uncharacterized myc-related gene.     

Similar 150-kilobase DNA sequences are amplified in independently derived methotrexate-resistant Chinese hamster cells.

We have isolated overlapping recombinant cosmids that represent 150 kilobases of contiguous DNA sequence from the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). This sequence includes the 25-kilobase dihydrofolate reductase gene and an origin of DNA synthesis. Eight cosmids that span this domain have been utilized as radioactive hybridization probes to analyze the similarities among the dihydrofolate reductase amplicons in four independently derived methotrexate-resistant Chinese hamster cell lines. We have observed no significant differences among the four cell lines within the 150-kilobase DNA sequence that we have examined, except for polymorphisms that result from the amplification of one or the other of two possible alleles of the dihydrofolate reductase domain. We also show that the restriction patterns of the amplicons in these four resistant cell lines are virtually identical to that of the corresponding, unamplified sequence in drug-susceptible parental cells. Furthermore, measurements of the relative copy numbers of fragments from widely separated regions of the amplicon suggest that all fragments in this 150-kilobase region may be amplified in unison. Our data show that in methotrexate-resistant Chinese hamster cells, the amplified unit is large relative to the dihydrofolate reductase gene itself. Furthermore, within the 150-kilobase amplified consensus sequence that we have examined, significant rearrangements do not seem to occur during the amplification process.     

Amplification and novel locations of endogenous mouse mammary tumor virus genomes in mouse T-cell lymphomas.

Endogenous mouse mammary tumor virus genomes are amplified and located in novel cell DNA sequences in many mouse T-cell lymphomas. Transplanted tumors recovered from the same mouse strain and shown to be of independent origin by chromosomal analysis, by the presence of JH immunoglobulin gene rearrangements, or by the integration patterns of exogenous Moloney MuLV genomes frequently showed similar patterns of novel mouse mammary tumor virus-containing cell DNA fragments. This process of amplification and relocation can occur within a limited number of cell generations and in C57BL/6 mice does not lead to the synthesis of mature virus-encoded proteins. In some instances, amplified mouse mammary tumor virus genomes contained novel restriction cleavage sites in the gag-pol region. The restricted time course of occurrence, lack of synthesis of mature virion proteins, and apparent site specificity indicate that this process of retrovirus amplification differs significantly from virus replication after exogenous infection.     

Structural features of the amplified N-myc oncogene detected by micrococcal nuclease digestion of neuroblastoma cell nuclei

The structure of chromatin containing amplified N-myc in neuroblastoma and retinoblastoma cells was investigated using micrococcal nuclease digestion of isolated nuclei. The size distribution of DNA fragments containing N-myc, produced by micrococcal nuclease digestion of nuclei, was determined and compared to that of DNA containing the structural gene for dihydrofolate reductase. A perturbation of the native structure of chromatin containing N-myc was evident from the association of N-myc with more extensively digested DNA when compared with chromatin containing dihydrofolate reductase.